Amelioration of biotic stress by using rhizobacteria for sustainable crop produce

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Rhizobiome

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Rhizobiome
Ecology, Management and
Application
Microbiome Research in Plants and Soil
Edited by
Javid A. Parray
Government Degree College Eidgah, Srinagar,
Jammu and Kashmir, India
Nowsheen Shameem
SP College Srinagar, Cluster University Srinagar,
Jammu and Kashmir, India
Dilfuza Egamberdieva
Institute of Fundamental and Applied Research,
National Research University, Tashkent, Uzbekistan
R.Z. Sayyed
Professor and Head, Department of Microbiology,
PSGVPM’S ASC College, SHAHADA

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Contents
List of Contributors............................................................................... xvii
CHAPTER 1 Diversity of various symbiotic associations
between microbes and host plants........................... 1
Bisma Farooq, Asma Nazir, Shahnaz Anjum,
Madeha Farooq and Mohammad Umer Farooq
1Introduction ........................................................................ 1
2Categories of symbionts .. ...................................................... 4
3Plant supplement carriers for arbuscular mycorrhizal
beneficial interaction............................................................. 5
3.1 Plant factors in rhizo nodule beneficial interaction................6
3.2 Bacterial elements in RN beneficial interaction ....................6
4Inescapable herbivore-symbiont ventures into sap-taking
care of specialties................................................................. 7
5Dynamic capability of the coordinated bug microorganism
amino corrosive digestion.....................................................10
References.............................................................................11
Further reading.......................................................................16
CHAPTER 2 Amelioration of biotic stress by using
rhizobacteria for sustainable crop produce.............19
Fadime Karabulut, Tahir Khan, Nusrat Shafi and
Javid A. Parray
1Introduction .......................................................................19
1.1 Effects of biotic stresses on plants ................................... 20
1.2 PGPRs as a growth enhancer .......................................... 21
2PGPRs systemic effects on the functioning and physiology of
plant .................................................................................23
2.1 PGPRs effect on plant nutrition....................................... 23
2.2 PGPRs effect on plant transcriptome ................................ 23
2.3 PGPRs effect on plant metabolome.................................. 24
3The effect of plant and rhizobacteria interaction on
secondary metabolites..........................................................25
4Impact of plant growth and development regulators on root
architecture ........................................................................27
5Stimulating the defense reaction of rhizobacteria in plants .........28
5.1 Direct mechanisms........................................................ 28
5.2 Indirect mechanisms...................................................... 28
6Plant defense with biocontrol agents.......................................30
7Conclusions and future perspectives .......................................32
v

References.............................................................................33
Further reading.......................................................................41
CHAPTER 3 Microorganisms as salient tools in achieving
ecosystem approaches...........................................43
Sneha P. Nair and Renitta Jobby
1Introduction .......................................................................43
2Impact of human and other interactions on ecosystem
and climate change..............................................................44
2.1 Climate change affecting ecosystem................................. 44
3Soil microbes .....................................................................45
3.1 Importance of soil microbes............................................46
3.2 Nature and composition .. ............................................... 46
4Impact of different class of microbes on climate change ............47
4.1 Marine biome............................................................... 47
4.2 Terrestrial biome........................................................... 49
5Ecosystem approaches .........................................................50
5.1 The need for ecosystem approaches ................................. 50
6Microbes as the tools for achieving ecosystem approaches .........50
6.1 Microbes for carbon sequestration ................................... 51
6.2 Microbes to reduce methane emissions ............................. 51
6.3 Nitrous oxide mitigation ................................................ 52
6.4 Microbes as a sustainable fuel......................................... 52
7Conclusion.........................................................................52
References.............................................................................53
CHAPTER 4 Role of rhizobacterial volatile compounds in
increasing plant tolerance to biotic and abiotic
stresses.................................................................61
Devendra Kumar, Archana T. S., Vipul Kumar,
Shivam Singh, Kartik Sawant, Rafakat Hussain and
Gagan Kumar
1Introduction......................................................................61
2Volatile organic compounds ................................................62
3Volatile organic compounds producing rhizobacteria ...............63
4Different type of volatile organic compound ..........................64
5Phytopathogens targeted by PGPR VOCs ..............................67
6Antibiosis of volatile organic compound ...............................68
7Alcohol compounds...........................................................68
8Ketone and aldehyde compounds .........................................69
9Alkanes and alkenes compounds..........................................70
vi Contents

10 Sulfur compounds .............................................................70
11 Volatile organic compound against abiotic stress... ..................71
11.1 Increased salt tolerance .............................................. 71
12 Defense against water loss . .................................................72
13 Enhancement of sulfur acquisition.. ......................................74
14 Optimization of iron homeostasis .........................................74
References.............................................................................76
CHAPTER 5 Bioremediation potential of rhizosphere
microbesdcurrent perspectives.............................81
Mehreen Shah and Sirajuddin Ahmed
1Introduction .......................................................................81
2Bioremediation ...................................................................82
2.1 Microorganisms used for bioremediation........................... 82
3Techniques employed in bioremediation..................................83
3.1 Rhizoremediation.......................................................... 83
3.2 Rhizoremediation of heavy metals ................................... 84
4Plant bacteria interactions in rhizoremediation .........................85
4.1 Colonization of root ...................................................... 85
4.2 Regulation of gene expression by roots............................. 85
4.3 Signal exchange/communication in the rhizosphere ............ 87
5Rhizoremediation of PET and PLA plastics .............................87
6Conclusions .......................................................................89
References.............................................................................90
Further reading.......................................................................93
CHAPTER 6 Plant growth promoting rhizobacteria (PGPR): an
overview for sustainable agriculture and
development..........................................................95
Harikrishna Naik Lavudi, Parameshwar Jakinala,
Shiva Kumar J, Nani Babu B, M. Srinivas and
Madhumohan Rao Katika
1Introduction .......................................................................95
2Rhizosphere .. .....................................................................95
2.1 Plant-microbial interactions ............................................ 96
2.2 Importance of agriculture . .............................................. 98
2.3 Improper use of fertilizers .............................................. 99
2.4 Lack of adoption of biofertilizers................................... 100
2.5 Institutional constraints................................................ 100
2.6 Socioeconomic constraints............................................ 100
3Chemical fertilizers .. .........................................................100
Contents vii

3.1 Nitrogen, phosphorus, and potassium in Indian
agriculture ................................................................. 101
3.2 Nitrogen.................................................................... 101
3.3 Phosphate.................................................................. 102
3.4 Potassium.................................................................. 102
4Biofertilizers ....................................................................102
4.1 Plant growth promoting rhizobacteria (PGPR)................ 104
4.2 Microorganisms with PGPR........................................ 107
4.3 The importance and applications of PGPR..................... 107
4.4 Mechanisms of PGPR ................................................ 108
4.5 Biological nitrogen fixation......................................... 108
4.6 Increase in growth..................................................... 109
4.7 Phosphate solubilization ............................................. 109
4.8 Microbial antagonism .. .............................................. 110
4.9 Pesticide-specific biosurfactants................................... 112
4.10 HCH degradation ...................................................... 113
5Other applications .. ...........................................................113
6Conclusion.......................................................................115
References...........................................................................115
Further reading.....................................................................125
CHAPTER 7 Rhizospheric microbiome: organization and
bioinformatics studies....... ................................... 127
Archana T. S., Devendra Kumar, Vipul Kumar,
Shivam Singh, Nakishuka Bitaisha Shukuru and
Gagan Kumar
1Introduction .....................................................................127
2Bioinformatics... ...............................................................128
3Bioinformatics impact on genomics......................................129
4Bioinformatic tools............................................................130
5Bioinformatic resources and platforms for plant microbes
interaction study ...............................................................131
6Proteomics.......................................................................133
6.1 Experimental methodologies......................................... 133
6.2 Computational proteomics............................................ 133
6.3 Metaproteomics in plant microbialeassociated studies . ..... 134
7Recent and new approaches to study plant-microbe
interactions ......................................................................135
8Conclusion.......................................................................137
References...........................................................................137
viii Contents

CHAPTER 8 Microbiome biodiversitydcurrent advancement
and applications ....... ........................................... 143
Shital Shinde, Swapnil Kadam, Vipul Patel and
Sharav Desai
1Introduction....................................................................143
2Rhizosphere microbiota....................................................144
3Ecology.........................................................................145
4Rhizosphere microbiome assembly and its impact on plant
growth...........................................................................146
5Rhizosphere differentials that affect the microbial community
assembly........................................................................147
6Plant growth variationsdmicrobiome assembly and root
metabolism .................................................................... 148
6.1 Abiotic and biologic stresses alter root exudation . ...........149
6.2 Plant growth and disease resistance...............................150
7Biochemical mediators in plant growth promoting
microorganisms...............................................................151
8Plant disease-resisting microorganisms (PDRM) ...................152
8.1 Disease-suppressive soils.............................................152
8.2 Biosynthesis of antimicrobial compounds.......................153
9Management of rhizosphere microbiota............................... 154
9.1 Management of plant growth under drought stress and
rhizosphere microbiota................................................154
9.2 Role of plants in rhizosphere development .....................154
9.3 Improved soil and plant production through rhizosphere
management..............................................................155
10 Holobiont-based control of rhizospheric biota ......................156
11 Impact of plant-friendly, plant-pathogenic, and
human-pathogenic microbes..............................................156
12 Conclusion and future outlook........................................... 158
References...........................................................................158
Further reading.....................................................................164
CHAPTER 9 Multifunctional growth-promoting microbial
consortium-based biofertilizers and their
techno-commercial feasibility for sustainable
agriculture........................................... ................ 167
Deepak Kumar, Sanjay K. Singh, Santosh K. Arya,
Deepti Srivastava, Vishnu D. Rajput and Raja Husain
1Introduction .....................................................................167
2Beneficial microbes as active ingredients of microbial
consortia..........................................................................168
Contents ix

2.1 Nitrogen fixation microorganisms .................................. 169
2.2 Phosphate solubilizing microorganisms........................... 170
2.3 Potassium solubilizing microorganisms........................... 171
2.4 Zinc solubilizing microorganisms .................................. 171
2.5 Sulfur oxidizing microorganisms ................................... 172
2.6 Plant growth promoting rhizobacteria (PGPR) ................. 173
3Microbial consortia............................................................174
3.1 Bacterial consortia ...................................................... 174
3.2 Fungal consortia .. ....................................................... 176
3.3 Fungal-bacterial consortia ............................................ 177
4Carrier materials for microbial consortia ...............................178
5Regulatory framework for commercialization of microbial
consortium biofertilizers.....................................................180
6Multifunctional plant growth-promoting attributes of
microbial consortia on different crops...................................182
7Challenges and constraints with microbial consortiaebased
biofertilizers.....................................................................193
7.1 Biological constraints .................................................. 193
7.2 Technical constraints ................................................... 193
7.3 Quality control constraints............................................ 194
7.4 Biofertilizer carrier constraints ...................................... 194
7.5 Field-level constraints.................................................. 194
7.6 Regulatory constraints ................................................. 195
8Conclusion and future perspectives.......................................195
References...........................................................................196
CHAPTER 10 Nutrition and cultivation strategies of core
rhizosphere microorganisms............................... 209
Hetvi Naik, Komal A. Chandarana, Harshida A. Gamit,
Sapna Chandwani and Natarajan Amaresan
1Introduction....................................................................209
2Members of rhizomicrobiome............................................211
3Bacteria.........................................................................212
4Fungi ............................................................................ 212
5Others .. .........................................................................213
6Why rhizospheric microbiome is important? . .......................213
7Nutritional strategies for beneficial rhizospheric microbes ...... 215
8Cultivation strategies for beneficial rhizospheric microbes......215
9Azospirillum .. ................................................................215
10 Azotobacter....................................................................216
11 Bacillus .........................................................................217
xContents

12 Enterobacter...................................................................217
13 Frankia..........................................................................218
14 Klebsiella.......................................................................218
15 Methylobacterium .. .........................................................219
16 Pseudomonas..................................................................219
17 Rhizobium .. ...................................................................220
18 Streptomyces..................................................................220
19 Aspergillus.....................................................................221
20 Metarhizium...................................................................221
21 Penicillium.....................................................................222
22 Trichoderma...................................................................222
23 Conclusion... ..................................................................222
References...........................................................................223
CHAPTER 11 Bioengineering of rhizobiome toward
sustainable agricultural production .............. ...... 233
Bal Krishna, Rakesh Kumar, Hansraj Hans,
Ashutosh Kumar, Banshidhar, Talekar Nilesh Suryakant,
Harmeet Singh Janeja, Birender Singh
and Dharm Nath Kamat
1Introduction .....................................................................233
2Bioengineering .................................................................235
3Why rhizosphere engineering for sustainable agriculture? ........236
3.1 Plant-based rhizosphere engineering............................... 237
3.2 Microbiome-based rhizosphere engineering ..................... 238
3.3 Soil-based rhizosphere engineering ................................ 240
4Rhizosphere engineering for abiotic .....................................241
5Rhizosphere engineering for biotic stress...............................244
6Conclusions and future outlook ...........................................250
References...........................................................................251
CHAPTER 12 Bioinformatics study to unravel the role of
rhizobiome to biologically control the
pathogens in vegetables..................................... 267
Vanya Bawa, Meghna Upadhyay and Sheetal Verma
1Introduction .....................................................................267
1.1 Potential spatial effect ................................................. 269
1.2 Microbiome on the molecular level................................ 270
1.3 Databases and methods for sequence classification ........... 272
1.4 Sequencing protocols and data processing ....................... 273
1.5 Omics to metagenomic approaches ................................ 275
Contents xi

2Conclusion.......................................................................277
References...........................................................................278
CHAPTER 13 Azospirillumda free-living nitrogen-fixing
bacterium .............................................. ............ 285
M.D. Jehani, Shivam Singh, Archana T. S.,
Devendra Kumar and Gagan Kumar
1Diazotrophic (nitrogen-fixing) population..............................286
1.1 Systems of associative diazotrophs................................. 286
2Modes of action................................................................288
2.1 Mechanisms for promoting plant growth......................... 288
3Measurement/quantification ................................................291
3.1 Assay for C
2
H
2
reduction............................................. 291
3.2 Measurement of N
2
-fixation with
15
N
2
gas ...................... 292
3.3 Isotope
15
N dilution .................................................... 293
3.4 N-fixed using a different approach ................................. 293
4The nonsymbiotic N
2
-fixation-related factors ......................... 294
4.1 Oxygen ..................................................................... 294
4.2 The presence of C....................................................... 295
4.3 Aggregates of soil....................................................... 295
4.4 Temperature............................................................... 295
4.5 Moisture.................................................................... 296
4.6 Supplemental nutrition and minerals .............................. 296
4.7 Managing techniques................................................... 297
5Plants and other creatures actualized nitrogen from
diazotrophs ......................................................................297
6Extending the utility of nonsymbiotic N
2
-fixation .. .................298
6.1 Inoculation ................................................................ 298
6.2 Conserving consistent N (reducing N losses) ................... 298
7Conclusion.......................................................................299
References...........................................................................300
CHAPTER 14 Plant-microbe interactions: different perspectives
in promoting plant growth and health.................. 309
Belur Satyan Kumudini, Sunita Mahadik,
Amrisha Srivastava and Savita Veeranagouda Patil
1Introduction .....................................................................309
2Plant-microbe interactions: a dynamic association...................310
2.1 Plant-beneficial fungi................................................... 310
2.2 Plant-beneficial oomycetes .. ......................................... 313
2.3 Plant-bacteria interactions ............................................ 313
xii Contents

2.4 Plant beneficial bacteria ............................................... 314
3Plant-microbe interactions in enhancing plant growth
and health........................................................................314
3.1 Phytohormone production ............................................ 316
3.2 ACC deaminase activity............................................... 316
3.3 Phosphate (P) solubilization.......................................... 317
3.4 Siderophore production................................................ 317
4Perspectives on plant productivity in a different scenario .........318
5Future prospects................................................................319
References...........................................................................319
CHAPTER 15 Recent advances in discovery of new drugs
from plants-associated microbes ..................... ... 329
Sharav Desai, Vipul Patel and Neha Hajare
1Introduction .....................................................................329
1.1 Actinobacteria............................................................ 331
1.2 Algae........................................................................ 331
1.3 Endophytes................................................................ 333
1.4 Future outlook............................................................ 336
References...........................................................................337
Further reading.....................................................................343
CHAPTER 16 Plant health: Feedback effect of root exudates
and rhizobiome interactions ............... ................ 345
Shrikrishna Bhagat, Pranali Shete and Ashish Jain
1Introduction....................................................................345
2Rhizosphere and rhizobiome: a dynamic system ................... 346
2.1 Rhizobiome: microbial collection in the rhizosphere ........347
2.2 Communication in the rhizome ....................................348
3Role of rhizobiome in plant health .....................................348
4Rhizobiome contributes to limiting nutrient acquisition.......... 348
4.1 Rhizobiome produce plant growth hormones ..................350
4.2 Rhizobiome as biocontrol agent ...................................351
5Root exudates: the spray of chemical signals........................352
6Root exudation transport mechanism .. ................................ 352
7Factors affecting root exudate profile..................................358
8Root exudates and rhizobiome: synergistic influence
on plant health................................................................359
Contents xiii

8.1 Relation with nitrogen-fixing bacteria............................359
8.2 Relation with PGPR .. .................................................359
8.3 Root exudates: role in limiting nutrient and mineral
acquisition................................................................360
8.4 Root exudates: mystical ingredient of plant defense .........361
9Future viewpoints............................................................361
10 Summary .. .....................................................................363
References...........................................................................363
CHAPTER 17 Ecotypic adaptation of plants and the role of
microbiota in ameliorating the environmental
extremes using contemporary approaches........... 377
Mohan Singh Rana, Jyoti Ranjan Rath,
Chejarla Venkatesh Reddy, Sangay Pelzang,
Rahul G. Shelke and Smit Patel
1Introduction .....................................................................377
2Plant ecotype and the associated microbiota ..........................379
3Secondary metabolites associated with microbiota . .................382
4Mechanism of action (nutritional absorption and plant
health).............................................................................383
5Nutritional absorptions by bacteria.......................................385
6Nutritional absorptions by fungi........................................... 386
7Role of the microbiota in amelioration of environmental
extremes..........................................................................387
7.1 Disease and pathogens................................................. 387
8Conclusion and future prospect............................................394
References...........................................................................394
CHAPTER 18 Ecological and structural attributes of soil
rhizobiome improving plant growth under
environmental stress. ......................................... 403
Ali Reza Mirzaei, Bahman Fazeli-Nasab and
Moharram Valizadeh
1Introduction....................................................................403
2Drought stress.................................................................404
3Salinity stress ................................................................. 405
4Abscisic acid hormone (ABA)...........................................405
5Properties and potential of plant growth promoting
rhizobacteria (PGPR) .. .....................................................407
6Siderophores...................................................................410
7Phosphate solubilization ...................................................410
xiv Contents

8Nitrogen fixation.............................................................410
9Auxins, cytokinins, gibberellins .........................................411
10 ACC Deaminase..............................................................411
11 Effectiveness of PGPR in hydrocarbons and heavy metals
contaminated soils...........................................................411
12 PGPR to face salinization and drought facing the abiotic
stresses..........................................................................412
12.1 Drought and salinity pressure .....................................412
13 Water phytodepurationdconstructed wetlands (CW). ............413
References...........................................................................414
CHAPTER 19 Modulation of rhizosphere microbial populations
using Trichoderma-based biostimulants for
management of plant diseases ........................... 421
Efath Shahnaz, Saba Banday, Ali Anwar,
M.N. Mughal, G.H. Mir, Qadrul Nisa, Gazala Gulzar,
Atufa Ashraf and Diksha Banal
1Introduction .....................................................................421
2Improvement of soil nutrient uptake ..................................... 424
3Adaptation under different climatic conditions .. .....................424
4Response to plant pathogens ............................................... 425
5Trichoderma-based biostimulants......................................... 426
6Conclusion.......................................................................426
References...........................................................................426
CHAPTER 20 Multiomics analysis of rhizosphere and plant
health................................... ............................. 433
Tulasi Korra, Thiru Narayanan Perumal and
Uday Kumar Thera
1Introduction .....................................................................433
2Rhizospheric microbe metabolomics..................................... 434
3Metabolomics uses ............................................................435
4Metabolomics challenges....................................................435
4.1 Metagenome .............................................................. 435
5Multiomics investigation on an agroecosystem demonstrates
organic nitrogen’s function in increasing crop output...............436
5.1 General rhizosphere-omics challenges ............................ 436
6NGS of rhizospheric microbes.............................................439
6.1 NGS difficulties.......................................................... 439
7Mass spectrometry (MS) ....................................................440
8Rhizospheric metaproteomics..............................................440
9Conclusions .....................................................................440
References...........................................................................441
Contents xv

CHAPTER 21 Chemical profiling of metabolites of
Bacillus species: A case study ......................... .. 445
Aurelio Ortiz and Estibaliz Sansinenea
1Introduction .....................................................................445
2Chemical extraction and isolation of natural products..............446
3Antifungals .. ....................................................................447
4Antibacterials ...................................................................450
5Plant growth promoting compounds .....................................452
6Conclusions .....................................................................453
References...........................................................................453
CHAPTER 22 Achievements of Professor Hiltner vis a vis the
contributions toward rhizosphere science........... 457
Aparna Baban Gunjal
1Introduction .....................................................................457
2Rhizosphere science ..........................................................457
3Lorenz Hiltner..................................................................459
4Rhizosphere soil ...............................................................460
5Beneficial microorganisms near the rhizosphere region ............460
6Group of microorganisms near the rhizosphere region .............460
6.1 Bacteria .................................................................... 460
6.2 Actinobacteria............................................................ 460
6.3 Protozoa.................................................................... 460
7Factors affecting microbial population in the rhizosphere
region .............................................................................461
7.1 Soil type and its moisture............................................. 461
7.2 Soil amendments and fertilizers..................................... 461
7.3 Rhizosphere pH. ......................................................... 461
7.4 Proximity of root with soil .. ......................................... 461
7.5 Plant species .. ............................................................ 461
7.6 Root exudates ............................................................ 461
8Conclusion.......................................................................461
References...........................................................................462
Index...................................................................................................463
xvi Contents

List of contributors
Sirajuddin Ahmed
Department of Environmental Science and Engineering, Jamia Millia Islamia
(Central University), New Delhi, Delhi, India
Natarajan Amaresan
C. G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Bardoli, Gujarat,
India
Shahnaz Anjum
Department of Agriculture, Lovely Professional University, Phagwara, Punjab,
India
Ali Anwar
Division of Plant Pathology, Sher-e-Kashmir University of Agricultural Sciences &
Technology of Kashmir, Shalimar, Jammu & Kashmir, India
Santosh K. Arya
R&D Division, Nextnode Bioscience Pvt Ltd, Opposite GEB Office, Kadi, Gujarat,
India
Atufa Ashraf
Division of Plant Pathology, Sher-e-Kashmir University of Agricultural Sciences &
Technology of Kashmir, Shalimar, Jammu & Kashmir, India
Nani Babu B
Mobile Virology Research and Diagnostics Laboratory (MVRDL) BSL 3&2, ESIC
Medical College & Hospital, Hyderabad, Telangana, India
Diksha Banal
Division of Plant Pathology, Sher-e-Kashmir University of Agricultural Sciences &
Technology of Kashmir, Shalimar, Jammu & Kashmir, India
Saba Banday
Division of Plant Pathology, Sher-e-Kashmir University of Agricultural Sciences &
Technology of Kashmir, Shalimar, Jammu & Kashmir, India
Banshidhar
Department of Genetics and Plant Breeding, School of Agriculture, Lovely
Professional University, Jalandhar, Phagwara, Punjab, India
Vanya Bawa
Department of Botany, Central University of Jammu, Jammu, Jammu & Kashmir,
India
Shrikrishna Bhagat
Dept. of Microbiology, Smt. C.H.M. College Ulhasnagar, University of Mumbai,
Mumbai, Maharashtra, India
xvii

Komal A. Chandarana
C. G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Bardoli, Gujarat,
India
Sapna Chandwani
C. G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Bardoli, Gujarat,
India
Sharav Desai
Sanjivani College of Pharmaceutical Education and Research, Kopargaon,
Maharashtra, India
Bisma Farooq
Department of Biomedical Engineering, Sathyabama Institute of Science and
Technology, Chennai, Tamil Nadu, India
Madeha Farooq
Department of Microbiology, Asian University, Imphal, Manipur, India
Mohammad Umer Farooq
Department of Agriculture, Lovely Professional University, Phagwara, Punjab,
India
Bahman Fazeli-Nasab
Department of Agronomy and Plant Breeding, Agriculture Institute, Research
Institute of Zabol, Zabol, Sistan and Baluchestan, Iran
Harshida A. Gamit
C. G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Bardoli, Gujarat,
India
Gazala Gulzar
Division of Plant Pathology, Sher-e-Kashmir University of Agricultural Sciences &
Technology of Kashmir, Shalimar, Jammu & Kashmir, India
Aparna Baban Gunjal
Department of Microbiology, Dr. D.Y. Patil, Arts, Commerce & Science College,
Pimpri, Pune, Maharashtra, India
Neha Hajare
Sanjivani College of Pharmaceutical Education and Research, Kopargaon,
Maharashtra, India
Hansraj Hans
Division of Crop Research, ICAR Research Complex for Eastern Region, Patna,
Bihar, India
Raja Husain
Department of Agriculture, Himalayan University, Itanagar, Arunachal Pradesh,
India
xviii List of contributors

Rafakat Hussain
Department of Plant Science, College of Plant Science and Technology,
Huazhong Agricultural University, Wuhan, China
Shiva Kumar J
Mobile Virology Research and Diagnostics Laboratory (MVRDL) BSL 3&2, ESIC
Medical College & Hospital, Hyderabad, Telangana, India; Department of
Microbiology, Osmania University, Hyderabad, Telangana, India
Ashish Jain
Dept. of Microbiology, Smt. C.H.M. College Ulhasnagar, University of Mumbai,
Mumbai, Maharashtra, India
Parameshwar Jakinala
Mobile Virology Research and Diagnostics Laboratory (MVRDL) BSL 3&2, ESIC
Medical College & Hospital, Hyderabad, Telangana, India; Department of
Microbiology, Osmania University, Hyderabad, Telangana, India
Harmeet Singh Janeja
Department of Genetics and Plant Breeding, School of Agriculture, Lovely
Professional University, Jalandhar, Phagwara, Punjab, India
M.D. Jehani
Department of Plant Pathology, School of Agriculture, Lovely Professional
University, Phagwara, Punjab, India
Renitta Jobby
Amity Institute of Biotechnology, Amity University Maharashtra, Navi Mumbai,
Maharashtra, India; Amity Centre of Excellence in Astrobiology, Amity University
Maharashtra, Navi Mumbai, Maharashtra, India
Swapnil Kadam
Sanjivani College of Pharmaceutical Education and Research, Kopargaon,
Maharashtra, India
Dharm Nath Kamat
Sugarcane Research Institute, Department of Plant Breeding and Genetics, Dr.
Rajendra Prasad Central Agricultural University, Samastipur, Bihar, India
Fadime Karabulut
Department of Biology, Firat University, Elazıg, Turkey
Madhumohan Rao Katika
Mobile Virology Research and Diagnostics Laboratory (MVRDL) BSL 3&2, ESIC
Medical College & Hospital, Hyderabad, Telangana, India
Tahir Khan
Yunnan Herbal Laboratory, School of Ecology and Environmental Sciences,
Yunnan University, Kunming, Yunnan, China
List of contributors xix

Tulasi Korra
Mycology and Plant Pathology, Banaras Hindu University, Institute of Agricultural
Sciences, Varanasi, Uttar Pradesh, India
Bal Krishna
Department of Genetics and Plant Breeding, School of Agriculture, Lovely
Professional University, Jalandhar, Phagwara, Punjab, India
Ashutosh Kumar
Department of Genetics and Plant Breeding, School of Agriculture, Lovely
Professional University, Jalandhar, Phagwara, Punjab, India
Deepak Kumar
R&D Division, Nextnode Bioscience Pvt Ltd, Opposite GEB Office, Kadi, Gujarat,
India
Devendra Kumar
Department of Plant Pathology, School of Agriculture, Lovely Professional
University, Phagwara, Punjab, India
Gagan Kumar
Krishi Vigyan Kendra, Narkatiaganj, Dr. Rajendra Prasad Central Agricultural
University, Samastipur, Bihar, India
Rakesh Kumar
Division of Crop Research, ICAR Research Complex for Eastern Region, Patna,
Bihar, India
Vipul Kumar
Department of Plant Pathology, School of Agriculture, Lovely Professional
University, Phagwara, Punjab, India
Belur Satyan Kumudini
Department of Biotechnology, School of Sciences, JAIN (Deemed-to-be
University), Bengaluru, Karnataka, India
Harikrishna Naik Lavudi
Mobile Virology Research and Diagnostics Laboratory (MVRDL) BSL 3&2, ESIC
Medical College & Hospital, Hyderabad, Telangana, India
Sunita Mahadik
Department of Biotechnology, School of Sciences, JAIN (Deemed-to-be
University), Bengaluru, Karnataka, India
G.H. Mir
Division of Plant Pathology, Sher-e-Kashmir University of Agricultural Sciences &
Technology of Kashmir, Shalimar, Jammu & Kashmir, India
xx List of contributors

Ali Reza Mirzaei
Department of Agronomy and Plant Breeding, Faculty of Agriculture and Natural
Resources, University of Mohaghegh Ardabili, Ardabil, Iran
M.N. Mughal
Division of Plant Pathology, Sher-e-Kashmir University of Agricultural Sciences &
Technology of Kashmir, Shalimar, Jammu & Kashmir, India
Hetvi Naik
C. G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Bardoli, Gujarat,
India
Asma Nazir
Department of Clinical Biochemistry, Kashmir University, Srinagar, Jammu and
Kashmir, India
Qadrul Nisa
Division of Plant Pathology, Sher-e-Kashmir University of Agricultural Sciences &
Technology of Kashmir, Shalimar, Jammu & Kashmir, India
Aurelio Ortiz
Facultad de Ciencias Quı
´micas, Beneme
´rita Universidad Auto
´noma de Puebla,
Puebla, Pue, Me
´xico
Sneha P. Nair
Amity Institute of Biotechnology, Amity University Maharashtra, Navi Mumbai,
Maharashtra, India
Javid A. Parray
Government Degree College, Eidgah, Srinagar, Jammu and Kashmir, India
Vipul Patel
Sanjivani College of Pharmaceutical Education and Research, Kopargaon,
Maharashtra, India
Smit Patel
Department of Biotechnology, Amity University, Kant Kalwar, Kant, Rajasthan,
India
Savita Veeranagouda Patil
Department of Biotechnology, School of Sciences, JAIN (Deemed-to-be
University), Bengaluru, Karnataka, India
Sangay Pelzang
Department of Life Sciences and Biotechnology, South Asian University, New
Delhi, India
List of contributors xxi

Thiru Narayanan Perumal
Mycology and Plant Pathology, Banaras Hindu University, Institute of Agricultural
Sciences, Varanasi, Uttar Pradesh, India
Vishnu D. Rajput
Academy of Biology and Biotechnology, Southern Federal University, Rostov-on-
Don, Rostov Oblast, Russia
Mohan Singh Rana
Department of Environment and Life Sciences, Sherubtse College, Royal
University of Bhutan, Kanglung, Bhutan
Jyoti Ranjan Rath
Department of Plant Sciences, School of Life Sciences, University of Hyderabad,
Gachibowli, Hyderabad, Telangana, India
Chejarla Venkatesh Reddy
Department of Civil Engineering, National Institute of Technology, Farmagudi,
Ponda, Goa, India
Estibaliz Sansinenea
Facultad de Ciencias Quı
´micas, Beneme
´rita Universidad Auto
´noma de Puebla,
Puebla, Pue, Me
´xico
Kartik Sawant
Department of Plant Pathology, School of Agriculture, Lovely Professional
University, Phagwara, Punjab, India
Nusrat Shafi
Department of Chemistry, GDC Eidgah, Srinagar, Jammu and Kashmir, India
Mehreen Shah
Department of Environmental Science and Engineering, Jamia Millia Islamia
(Central University), New Delhi, Delhi, India
Efath Shahnaz
Dryland Agriculture Research Station, SKUAST-Kashmir, Rangreth, Jammu and
Kashmir, India
Rahul G. Shelke
Pepthera Laboratories Pvt Ltd., DL-3, Atal Incubation Center, Gujarat
Technological University, Chandkheda, Ahmedabad, Gujarat, India
Pranali Shete
Dept. of Microbiology, Smt. C.H.M. College Ulhasnagar, University of Mumbai,
Mumbai, Maharashtra, India
Shital Shinde
Sanjivani College of Pharmaceutical Education and Research, Kopargaon,
Maharashtra, India
xxii List of contributors

Nakishuka Bitaisha Shukuru
Department of Plant Pathology, School of Agriculture, Lovely Professional
University, Phagwara, Punjab, India
Sanjay K. Singh
Biodiversity and Palaeobiology, Agharkar Research Institute, Pune, Maharashtra,
India
Shivam Singh
Krishi Vigyan Kendra-Baghpat, S.V.P University of Agriculture and Technology,
Meerut, Uttar Pradesh, India
Birender Singh
Department of Plant Breeding and Genetics, Bihar Agricultural University,
Bhagalpur, Bihar, India
M. Srinivas
Mobile Virology Research and Diagnostics Laboratory (MVRDL) BSL 3&2, ESIC
Medical College & Hospital, Hyderabad, Telangana, India
Amrisha Srivastava
Department of Biotechnology, School of Sciences, JAIN (Deemed-to-be
University), Bengaluru, Karnataka, India
Deepti Srivastava
Integral Institute of Agricultural Science and Technology, Integral University,
Lucknow, Uttar Pradesh, India
Talekar Nilesh Suryakant
Department of Genetics and Plant Breeding, School of Agriculture, Lovely
Professional University, Jalandhar, Phagwara, Punjab, India
Archana T. S.
Department of Plant Pathology, School of Agriculture, Lovely Professional
University, Phagwara, Punjab, India
Uday Kumar Thera
Department of Plant Science and Landscape Architecture, University of
Maryland, College Park, MD, United States
Meghna Upadhyay
Department of Botany, Central University of Jammu, Jammu, Jammu & Kashmir,
India
Moharram Valizadeh
Research Center of Medicinal Plants, University of Sistan and Baluchestan,
Zahedan, Sistan and Baluchestan, Iran
Sheetal Verma
Department of Botany, Central University of Jammu, Jammu, Jammu & Kashmir,
India
List of contributors xxiii

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Diversity of various
symbiotic associations
between microbes and
host plants 1
Bisma Farooq
1
, Asma Nazir
2
, Shahnaz Anjum
4
, Madeha Farooq
3
,
Mohammad Umer Farooq
4
1
Department of Biomedical Engineering, Sathyabama Institute of Science and Technology,
Chennai, Tamil Nadu, India;
2
Department of Clinical Biochemistry, Kashmir University, Srinagar,
Jammu and Kashmir, India;
3
Department of Microbiology, Asian University, Imphal, Manipur,
India;
4
Department of Agriculture, Lovely Professional University, Phagwara, Punjab, India
1.Introduction
Herbivory, characterized here as benefiting from live plant tissues, is a developmen-
tally determined method of taking care of bugs (Labandeira &Sepkoski, 1993).
Transformative advances to herbivory, explicitly herbivory on angiosperms, have
been trailed by sped-up paces of bug species expansion. Herbivorous bugs are
without a doubt phenomenally different, as estimated both by species numbers
and by their shifted ways of life (Futuyma &Agrawal, 2009). This macroevolu-
tionary design suggests that herbivory is fruitful in taking care of mode, however
solely after key obstacles are survived.
Even though plants address the biggest wellspring of biomass in earthbound
frameworks, their tissues are generally made out of unmanageable atoms like cellu-
lose, hemicellulose, gelatin, and lignin that are troublesome or difficult to process for
most creatures (Watanabe &Tokuda, 2010). Moreover, the degrees of nitrogen
compared with carbon will generally be low when contrasted with the nourishing
necessities of creatures and display significant variety among various plant tissues,
development stages, and species. Nutritive quality is frequently brought down
further because many plants contain mixtures, for example, protease inhibitors
that inactivate stomach-related catalysts in the bug stomach, forestalling processing
and retention. At long last, plant tissues are bound with a wide assortment of other
harmful allelochemicals that differ broadly among plant taxa and in a method of ac-
tivity on their objective. There is lot of literature available on the numerous morpho-
logical, metabolic, and social variations of bugs that empower herbivory and on the
transformations of plants for staying away from adverse consequences of bug her-
bivory (Schoonhoven et al., 2005).
CHAPTER
1
Rhizobiome. https://doi.org/10.1016/B978-0-443-16030-1.00006-7
Copyright ©2023 Elsevier Inc. All rights reserved.

More specialists have started to see the value in the potential for a relationship
with cooperative organisms to extend the environmental scope of eukaryotic hosts
and creatures specifically (McFall-Ngai et al., 2013). Symbionts, characterized as
organisms that have structure determined, harmless relationships with plants, are
normal in bugs by and large and in herbivorous bugs specifically. Microorganisms
offer abilities for integrating supplements, processing obstinate plant polymers,
and killing plant poisons. The likelihood that advantageous affiliations could shape
associations with plants has been proposed by various analysts (Felton &Tumlinson,
2008;Janson et al., 2008;Clark et al., 2010;Frago et al., 2012).
The effect of symbionts on whether bugs can involve plants as food and on which
plants is utilized. The essential justification for expecting symbionts to have the op-
tion to help bugs in taking advantage of plants is that microorganisms, including mi-
crobes and growths, have numerous utilitarian abilities completely missing from
creatures. These incorporate biosynthetic abilities for all-around required metabo-
lites, for example, nutrients and a few amino acids, and catabolic capacities, for
example, cellulolytic action and the creation of numerous poisonous particles.
Thus, they present a huge new tool stash with the potential to work with herbivory
as a way of life. Then again, symbiont securing by hosts might be untrustworthy, or
tissue confinement of symbionts may restrict their commitments to explicit host ca-
pabilities. Investigations of herbivore-plant collaborations range from various de-
grees of examination, from robotic investigations of how specific bugs digest
explicit plant tissues to developmental and populace investigations. The utilization
of new genomic information and subatomic methods to deduce symbiont jobs in
deciding skills to defeat deterrents to benefiting from specific plant tissues or species
is emphasized. This assessment fundamentally includes considering explicit systems
through which advantageous microorganisms could empower the utilization of spe-
cific plant tissues or plant species.
The connection between plant and microbial variety gives experiences into the
biological drivers of local area design and capability (Allen et al., 2013;Duhamel
et al., 2019). Even though dirt microbial gatherings differ in their practical associa-
tions with plants, they can advance plant local area variety through various systems.
For instance, harmonious mutualists can advance plant local area variety by expand-
ing supplement accessibility or working with specialty dividing (Van Der Heijde and
van der Heijden, 2006;Bever et al., 2015;Van Der Putten, 2017), while soil micro-
organisms can taint and kill plant seedlings straightforwardly, or stifle useful micro-
bial cooperations with the plant has, which both add to the endurance of
heterospecifics and lead to higher plant variety (Mangan et al., 2010;Liu et al.,
2016b). Plants act as the primary providers of energy for decomposers, symbionts,
and microorganisms, subsequently overseeing the specialty space for various soil or-
ganisms to persevere (Berg and Smalla, 2009;Bulgarelli et al., 2013;Steinauer et al.,
2015). Hence, the variety of plant local area and soil microbial local area has for
quite some time been thought to be firmly related (Falkowski et al., 2008). Notwith-
standing, the current data about the plant-microbial relationship at the worldwide
2CHAPTER 1 Diversity of various symbiotic associations

scale is inconsistent or uncertain (Tedersoo et al., 2014;Prober et al., 2015;Ochoa-
Hueso et al., 2018).
As indicated by broad biogeographic speculations, we could anticipate that dirt
microbial variety should follow the example of vascular plant variety, which de-
clines with expanding scope and rise. Notwithstanding, studies have uncovered con-
flicting outcomes. Along latitudinal inclinations, both tropical backwoods at the low
scope (Mueller et al., 2007;Tedersoo et al., 2014) and mild woodlands at midlatitude
(Fierer et al., 2012;Shi et al., 2014) contain the most elevated soil microbial variety.
Conversely, with a couple of exemptions (Bahram et al., 2018), most examinations
track down no expansion in soil microbial variety from polar to tropical areas (Fen-
chel and Finlay, 2004;Fuhrman, 2009;Fierer et al., 2012;Hendershot et al., 2017).
In like manner, soil microbial variety designs fluctuate broadly along height slopes,
where monotonic downfalls (Bryant et al., 2008), unimodal examples (Liu et al.,
2016a), and no connections (Fierer et al., 2012;Shen et al., 2014) with expanding
height have all been accounted for. As an outcome, studies intending to inspect
the connection between a plant variety and soil microbial variety across biogeo-
graphic scales see as feeble (Bryant et al., 2008;Prober et al., 2015), or even no re-
lationships (Lanze
´n et al., 2016;Cameron et al., 2019). These clashing outcomes
recommend that the connection between soil microbial variety and plant variety
could contrast across biomes and spatial scales.
A few elements might make sense of the sensational variety in the connection
between microbial variety and plant variety. The plant-microbial relationship can
shift among various microbial scientific classifications. For instance, given a site-
level examination, the lavishness of certain bacterial taxa, like nitrogen-fixing
microscopic organisms, was more firmly connected with plant variety than were
other scientific classifications (Liang et al., 2016;Zhou et al., 2016). The examina-
tion of soil microbial and plant networks is frequently led at various spatial scales,
going from nearby destinations (Go
¨mo
¨ryova
´et al., 2009;Navarro Cano et al., 2014)
to expansive geographic locales (Fierer et al., 2012;Zhou et al., 2016), with the end
goal that changeability in abiotic factors like soil pH (Lauber et al., 2009;Shen et al.,
2013;Siles and Margesin, 2016), all out natural carbon (Calbrix et al., 2007;Dimi-
triu and Grayston, 2010;Ding et al., 2015), and mean yearly temperature (Luo et al.,
2014;Zhou et al., 2016) among locales might additionally entangle the plant-
microbial relationship. Subsequently, a far-reaching examination utilizing informa-
tion across scientific classifications and spatial scales is expected to evaluate the
connection between a plant variety and a microbial variety.
The information from 84 examinations has been reviewed that included north of
3900 examples to decide the connection between a plant variety and soil microbial
variety at a worldwide scale. The following factors include designated scientific
categorization, microbial reaction type in variety (lavishness, overflow, variety
file, and arrangement), microbial assessment strategy, examining degree (scope
and rise cover range), biome type, soil type, and ecological elements (e.g., soil
pH, all-out natural carbon, precipitation, and temperature) to evaluate the examples
1. Introduction 3

of the plant-microbial connection and factors making sense of their variety were also
explored.
The following points were inferred -(1) Are plant and microbial varieties
emphatically corresponding at the worldwide scale? In light of the useful job of
soil organisms as the fundamental decomposer of plant-determined substrates, we
anticipate a positive connection between them. (2) Are predicated plant-related
taxa, for example, mycorrhizal growths, more unequivocally corresponded with
plant variety than other taxa? The cooperative organisms, which connect straightfor-
wardly with plants, may empower a more grounded relationship. (3) Which environ-
ments truly do establish variety and microbial variety show a higher relationship?
Tropical backwoods ought to have a higher plant-microbial relationship, taking
into account the uncommon high biodiversity and the solid communication between
plant local area and parasitic microorganisms (Bagchi et al., 2014;Sarmiento et al.,
2017). (4) Which ecological variables decide the plant and microbial connections in
biodiversity across scientific classifications and spatial scales? We anticipate that
dirt pH should be one of the main natural variables at the worldwide scale, and
the jobs of other soil characteristic impacts fluctuate among scientific classifications
and spatial scales.
2.Categories of symbionts
The symbionts were characterized extensively to incorporate any organisms that
have a persevering relationship with their hosts and that don’t cause pathogenic
side effects. Bug symbionts can be partitioned into three general classifications
because of their area compared with the bug body.
Firstly some symbionts are intracellular or housed inside the body pit and are
gone between ages through eggs, bringing about a maternal legacy. Heritable, gener-
ally intracellular, symbionts are found in an assortment of bug gatherings, which
frequently develop specific cells (bacteriocytes) for lodging them.
Second, organisms possess the guts of most creatures including most bugs,
residing inside the stomach lumen where they structure networks of fluctuating in-
tricacy in various bug species.
Third, microorganisms routinely colonize food before it is ingested and may in-
fluence its creation. These ecological symbionts are significant in bugs that feed on a
put-away food asset inside a home are confined to taking care of regions like a nerve
or exhibition.
The likely elements of microbial symbionts in working with or confining the uti-
lization of host plants are obliged by their area and by the carelessness of their rela-
tionship with have ancestries. For instance, heritable symbionts structure long-haul
relationships which have heredities and are accordingly unmistakably situated to
contribute developmentally huge abilities; however, they are frequently bound to
the cytoplasm of particular host cells and in this manner unfit to discharge quality
items straightforwardly into the stomach lumen or into oral emissions, where they
4CHAPTER 1 Diversity of various symbiotic associations

could support absorption, detoxification, or alteration of host plant acknowledgment.
Stomach symbionts or ecological symbionts are better situated for contributing these
capabilities, yet their transmission among individual hosts might be less reliable in
some bug life cycles, so their commitments could frequently be excessively temper-
amental to work with the development of host plant range. Intracellular symbionts
can contribute little particles to have digestion, however, these commitments will
rely upon having the ability for shipping these atoms across films and all through
the bug body.
Specifically, genomes of heritable symbionts commonly go through broad qual-
ity misfortune over their drawn-out advancement and don’t integrate novel qualities,
prompting lessening metabolic abilities (McCutcheon and Moran, 2012). Assuming
herbivorous bugs need to answer both present moment and long haul changes in the
nourishing quality, plant acknowledgment, or poisonousness of plant tissues that
they ingest, long haul commits symbionts with decreased genomes will probably
not be going to give the fundamental oddity. Conversely, most facultative heritable
symbionts, as well as stomach and natural symbionts show dynamic genomes,
including continuous addition and loss of useful qualities, genome modifications,
and versatile components, processes that are ordinary of most bacterial heredities.
3.Plant supplement carriers for arbuscular mycorrhizal
beneficial interaction
AM parasites retain minerals, including phosphate and nitrogen, using extraradical
hyphae and supply them to the plant, perhaps through exceptionally expanded struc-
tures inside root cells known as arbuscules (Harrison, 1998;Parniske, 2008). The
arbuscules are encompassed in plant-determined films, the peri arbuscular layer.
To retain phosphate, mycorrhiza-actuated phosphate carrier qualities, like MtPT4,
are dominatingly or only up-controlled in plant root cells containing arbuscules.
A portion of these qualities has been demonstrated to be expected for AM advanta-
geous interaction as well as the obtaining of phosphate conveyed by the AM parasite
(Javot et al., 2007). Curiously, notwithstanding phosphate, AM growths move signif-
icant measures of nitrogen to the host plants (Javot et al., 2007). Nitrogen sources,
for example, ammonium and amino acids are moved to the plant using parasitic hy-
phae. A few ammonium carriers (AMTs) have been distinguished in plants, and it
will be fascinating to decide their exact articulation designs and subcellular area.
Kobae et al. report in this issue that GmAMT4.1 distinguished from the soybean
genome dataset shows explicit articulation in arbusculated cortical cells. Addition-
ally, they show that GmAMT4.1 is limited on the branch spaces of periarbuscular
layers, demonstrating that arbuscular branches are dynamic locales for ammonium
transport (Kobae et al., 2010).
3. Plant supplement carriers 5

3.1 Plant factors in rhizo nodule beneficial interaction
RN beneficial interaction in vegetables includes explicit acknowledgment and post-
undeveloped improvement of a nitrogen-fixing organ, the root knob. Contrasted with
our broad information on the bacterial elements expected for RN beneficial interac-
tion, for example, gesture factors, lipo- and exopolysaccharides (Jones et al., 2007),
little is realized about the harmonious variables regarding the plant. Subatomic he-
reditary qualities and genomics of two model vegetables, Lotus japonicus, and Med-
icago truncatula, have made a critical commitment to the ID of various plant factors
and comprehension of the subatomic premise of knob inception, rhizobial contam-
ination, organogenesis, nitrogen obsession, senescence, and input guideline
(Oldroyd &Downie, 2008;Kouchi et al., 2010). Among these peculiarities, knob
senescence, in particular the guideline of knob life expectancy is an unfamiliar
area that however would acquire significance when our point is to accomplish
long-haul nitrogen-fixing action in vegetables. For knob capability, carbon is given
for the most part as sucrose got from photosynthesis and moved using the phloem.
These creators show that the MtATB2 records happen in and around the vascular tis-
sue of knobs and roots and in the knob summit (D’haeseleer et al., 2010).
Leguminous plants strictly control knob numbers, since nodulation and nitrogen
obsession are energy channels on the host. To keep up with the harmonious offset
with rhizobia, plants have developed negative input frameworks known as autoregu-
lation of nodulation (AON). AON includes significant distance flagging through
shoot-root correspondence and is intervened by CLAVATA1-like receptor kinases
like L. japonicus HAR1, Glycine max NARK and M. truncatula SUNN (Ferguson
et al., 2010;Oka-Kira &Kawaguchi, 2006). In L. japonicus, uniting tests have
shown HAR1 and KLAVIER capability in the shoot to limit the knob number while
TML capabilities are seen in the root. In this issue, Yoshida et al. report the qualities
of an original root-directed hypernodulating freak in L. japonicus named Bounty.
Not at all like TML, Bounty seems to act autonomously of HAR1-intervened signif-
icant distance control of nodulation and intercedes nitrate flagging (Yoshida et al.,
2010). Consequently, the Bounty is important for analyzing the complicated snare
of negative administrative frameworks in nodulation.
NSP1 and NSP2, which encode a GRAS family record factor, are expected for
knob inception and go about as parts of the Gesture factor flagging pathway down-
stream of Ca
2þ
spiking (Kouchi et al., 2010;Oldroyd &Downie, 2008). The homo-
logs are generally rationed in nonleguminous plants, however their capabilities are
obscure.
3.2 Bacterial elements in RN beneficial interaction
In light of the feeling of flavonoids oozing from vegetable roots into the soil, rhizobia
blend flagging particles that are answerable for knob arrangement. These flagging
atoms, named gesture factors, have been recognized as lipochito-oligosaccharides
embellished with different compound replacements (Spaink, 1995). Gesture factors
6CHAPTER 1 Diversity of various symbiotic associations

are surely a critical trigger for vegetable cooperative flagging and knob organogen-
esis (Kouchi et al., 2010). In any case, other rhizobial frameworks, for example, exo-
polysaccharide discharge (Skorupska et al., 2006), ethylene biosynthesis guideline
(Gresshoff et al., 2009;Okazaki et al., 2004), protein emission frameworks (Deakin
&Broughton, 2009) and BacA (LeVier et al., 2000) are frequently expected for the
foundation of advantageous interaction with vegetables, presumably because they
are engaged with bacterial delivery into the host cytoplasm and bacteroid turn of
events.
BacA protein, a cytoplasmic layer protein, giving protection from peptide anti-
toxins, was the principal bacterial element distinguished as fundamental for bacte-
roid improvement in Sinorhizobium meliloti (LeVier et al., 2000). Curiously, a
homolog of BacA in Brucella abortus, a creature microorganism, is expected for
compelling endurance in murine macrophages (LeVier et al., 2000). In this excep-
tional issue, Maruya and Saeki (2010) analyze the physiological elements of the
BacA homolog in Mesorhizobium loti. From the investigation of a bacA freak,
they observed that BacA is unnecessary for M. loti advantageous interaction with
L. japonica. Notwithstanding, the M. loti bacA quality somewhat reestablished
the beneficial interaction flawed aggregate of an S. meliloti bacA freak by hereditary
complementation. These outcomes bring up the issue of why BacA isn’t needed for
beneficial interaction with M. loti; however, it is expected with S. meliloti. As of late,
other vegetable rhizobium accomplices were inspected for their BacA prerequisite
for advantageous interaction (Karumakaran et al., 2010). One captivating clarifica-
tion is that BacA is solely expected in galegoid vegetables creating defensin-type
antimicrobial peptides (NCR peptides).
4.Inescapable herbivore-symbiont ventures into
sap-taking care of specialties
The focal premise of these commit symbioses is supplement provisioning, as uncov-
ered by both genomic and trial studies (Moran et al., 2009;Hansen and Moran,
2011). Creatures benefiting from plants are for the most part restricted by accessible
nitrogen (Schoonhoven et al., 2005), and nitrogen fixations are regularly two to mul-
tiple times lower in phloem and xylem, separately, contrasted and leaves. Moreover,
xylem and phloem sap contain lopsided profiles of amino acids and are particularly
lacking in the purported fundamental amino acids, that is to say, those that can’t be
orchestrated by creatures. Sap-taking care of bugs conquers these wholesome re-
strictions by a partner with intracellular, heritable microscopic organisms that
arrange fundamental amino acids (Baumann, 2005). Given the wholesome neces-
sities of bugs, the metabolic abilities of microbes, and the confined supplement con-
tent of xylem and phloem sap, it is nothing unexpected that sustenance-provisioning
symbionts are common among sap-taking care of bug taxa.
4. Inescapable herbivore-symbiont ventures 7

In these old commit symbioses, an outcome of the drawn-out maternal transmis-
sion and coming about clonality of symbionts is that their genomes are decreased in
size and quality number and show fast succession development and an A þT predis-
position in nucleotide base piece (McCutcheon and Moran, 2012).
Despite the loss of up to 90% of hereditary qualities, these commit symbiont ge-
nomes to hold qualities for fundamental amino corrosive pathways yet have lost
most qualities for unnecessary amino corrosive biosynthesis, predictable with their
dietary jobs (Moran et al., 2009;McCutcheon and Moran, 2012). While a couple of
qualities encoding proteins in specific fundamental amino corrosive pathways are
now and then missing, qualities present in the bug have or potentially other cohap-
pening commit symbionts seem to give these missing catalysts. For instance, cohap-
pening commit symbionts in six autonomously developed symbiont frameworks
encode catalysts that supplement chemicals or pathways missing from their accom-
plice symbiont (McCutcheon and Moran, 2010;McCutcheon and von Dohlen, 2011;
Sloan and Moran, 2012a).
Notwithstanding this genomic information, trial results accessible for the aphid-
Buchnera framework likewise support a job of commit symbionts in amino corrosive
provisioning (Akman Gu
¨ndu
¨z and Douglas, 2009;Macdonald et al., 2011). Buch-
nera involves trivial amino acids as substrates for the development of fundamental
amino acids. In particular, Buchnera may productively utilize glutamate orchestrated
by bug enzymatic apparatus (GOGAT cycle), which reuses squander smelling salts,
to support this nitrogen-restricted mutualism (Hansen and Moran, 2011), albeit the
degree of such reusing isn’t yet clear (Macdonald et al., 2012).
In pea aphids (Acyrthosiphon pisum), Buchnera encodes qualities engaged with
riboflavin creation. Baumannia, one of the symbionts of the sharpshooter Homalo-
disca vitripennis (Cicadellidae: Cicadellinae), holds 84 qualities anticipated to
empower the development of a few B-nutrients. Baumannia was the first commit
symbiont sequenced from xylem-taking care of host, and the presence of these qual-
ities was at first conjectured to mirror the shortfall of these mixtures from the xylem
sap diet of sharpshooters. Notwithstanding, resulting genome groupings of symbi-
onts of xylem feeders need a large number of these qualities (McCutcheon and
Moran, 2012). Carotenoids likewise seem, by all accounts, to be helpful or require
supplements for phloem-taking care of bugs, and phloem misses the mark on com-
pounds, which are fat-dissolvable. As of late, it has become obvious that phloem-
taking care of bugs has freely advanced instruments for creating their carotenoids,
now and again through the joining of unfamiliar qualities straightforwardly into
the bug genome. On account of whiteflies, carotenoid biosynthetic qualities are un-
blemished in the genome of their commit symbiont (Portiera), regardless of its very
diminished genome (358 kb) (Sloan and Moran, 2012b).
In aggregate, heritable, intracellular symbionts have been basic in empowering
bugs to possess and rule environmental specialties in which fundamental amino
acids are restricting, for example, dietary specialization on xylem and phloem sap
(Baumann, 2005).
8CHAPTER 1 Diversity of various symbiotic associations

Similar examinations of genomes from various commit symbiont heredities of
assorted hemipteran taxa uncover joined designs in quality misfortune and mainte-
nance (Moran et al., 2009;McCutcheon and Moran, 2012). While analyzing symbi-
onts across sap-taking care of hemipteran species, with single or accomplice
symbionts, a similar by and large example is obvious: symbionts can arrange
most or all fundamental amino acids in every bug species. This similarity mirrors
the known lack of fundamental amino acids in both phloem sap and xylem sap across
assorted plants. Since the single or matched symbionts encode most of the funda-
mental amino corrosive pathways and because plants are by and large comparable
in amino acids accessible in phloem and xylem, symbionts generally seem to
have a restricted impact on plant expansiveness. In some bug species, numerous
types of heritable symbionts have been sequenced; these cases incorporate Buchnera
of aphids, Carsonella ruddii of psyllids, and Sulcia muelleri of auchenorrhyncha sap
feeders. In every one of these cases, comparable quality substance and request have
been kept up with among strains, particularly for fundamental amino corrosive
biosynthesis, regardless of millions of long periods of disparity in isolated bug he-
redities of bug has benefited from unique host plants (Degnan et al., 2011;Sloan and
Moran, 2012a;McCutcheon and Moran, 2012).
Although there are wide likenesses across plants in sap synthesis and heritable
symbionts in supplement provisioning abilities, plant species in all actuality do
show some variety in sap organization, and a few symbionts need qualities for
various strides in amino corrosive biosynthesis. This variety raises the likelihood
that symbiont biosynthetic abilities to some degree compel plant broadness. In a
few cases, qualities that encode most of the methionine, tryptophan, histidine, or
arginine pathways have been lost.
Specifically, pathways for the sulfur-containing amino acids methionine and
cysteine, and sulfur absorption through sulfate decrease, are not encoded in 70%
of symbionts from phloem feeders and are likewise missing from the little genome
of the sole symbiont of mesophyll-taking care of scale bug (Sabree et al., 2013).
These misfortunes present a problem since creatures don’t encode catalysts to lessen
sulfur to fuse into the fundamental amino acids methionine and cysteine, notwith-
standing other fundamental sulfur-containing metabolites. Conversely, most
xylem-taking care of bugs concentrated to date have symbionts that hold the most
strides in the pathways for methionine biosynthesis and sulfur osmosis. Possibly, xy-
lem sap needs adequate decreased sulfur or sulfur-containing natural particles and
subsequently forces a more rigid necessity that symbionts absorb inorganic sulfur.
This contrast between xylem and phloem feeders could mirror the way that a signif-
icant type of diminished sulfur, S-methyl-methionine, is moved basically in the
phloem not xylem, particularly when sulfur is decreased in mature leaves. Be that
as it may, the site of sulfur decrease fluctuates relying upon natural circumstances
and plant species (Tan et al., 2010). For instance, in vegetables like a pea (Pisum sat-
ivum), sulfur decrease happens in the roots, and S-methyl-methionine is then moved
from the xylem to the phloem (Buchner et al., 2004). In aggregate, it isn’t yet clear
why certain commit symbionts hold or lose pathways for methionine, sulfur
4. Inescapable herbivore-symbiont ventures 9

decrease, and cysteine. The maintenance and loss of these pathways are related to
the method of decreased sulfur movement in the plant and to the bug host’s capacity
to absorb this diminished sulfur into methionine and cysteine.
Notwithstanding the boundless loss of the sulfate osmosis pathway from phloem
feeders, a few other amino corrosive biosynthetic pathways are sometimes missing.
For instance, pathways for tryptophan, histidine, and arginine biosynthesis are
missing from Carsonella, the dietary symbiont of psyllids (Nakabachi et al.,
2006;Sloan and Moran, 2012a). In some psyllid species, for example, Heteropsylla
cubana and Ctenarytaina eucalypti, the tryptophan as well as arginine pathways are
encoded in genomes of symbionts (Sloan and Moran, 2012a). Nonetheless, symbi-
onts are deficient in some psyllid species, and how these psyllids procure histidine
and tryptophan isn’t clear (Nakabachi et al., 2006;Sloan and Moran, 2012a).
Possibly a few psyllids, for example, nerve-shaping types of Pachypsylla, get
improved sustenance through control of the host plant. As referenced over, a larger
part of sequenced genomes of symbionts of phloem-taking care of bugs likewise
need qualities for arginine biosynthesis, explicitly for the middle metabolite orni-
thine; by and by, most hold qualities encoding the catalysts that catalyze the re-
sponses for arginine blend from ornithine, recommending that bug and
additionally plant-inferred ornithine is expected for arginine biosynthesis.
All in all, commit symbionts seem to lose a few qualities inside fundamental
amino corrosive pathways if these amino acids or their intermediates can be provi-
sioned by the bug or ectosymbiont or straightforwardly from the eating regimen.
Loss of symbiont qualities is irreversible because long-haul symbiont genomes
don’t acquire qualities through even exchange (Degnan et al., 2011; Lamelas
et al., 2011). At last, such quality misfortune in symbionts may compel bugs to a
particular host plant that is improved, specifically supplements, except if these
can be gotten from symbionts as well as bug-encoded proteins. Possibly this can
prompt expanding quantities of committed relationships with symbionts that arrange
different required supplements, as found in the symbionts of Auchenorrhyncha
(McCutcheon and Moran, 2012). It is muddled whether symbiont gain or substitu-
tions can increase plant broadness.
5.Dynamic capability of the coordinated bug
microorganism amino corrosive digestion
The bug’s biosynthetic capacities, and their guideline, should be incorporated with
those of supplement provisioning symbionts. This joining of bug and symbiont
metabolic abilities could influence dietary healthful necessities and hence bugs
have plant range. For instance, in one pea aphid-Buchnera genealogy, a frameshift
transformation was identified in argC, one of the five qualities expected for the
biosynthesis of ornithine, a transitional in arginine biosynthesis (Vogel and Moran,
2010). This aphid clone brought about wellness costs when it benefited from a fake
10 CHAPTER 1 Diversity of various symbiotic associations

eating regimen lacking arginine (Vogel and Moran, 2010). This arginine reliance
was credited not exclusively to Buchnera’s frameshift in argC, yet additionally to
the bug having encoded factors given hereditary crosses (Vogel and Moran,
2010). This perception fits with the way that ornithine might be either present in
the phloem sap or delivered by the host bug, for instance, through the protein orni-
thine aminotransferase, which interconverts ornithine and glutamate and which is
upregulated in pea aphid bacteriocytes (Hansen and Moran, 2011). Accordingly,
in this model, the vehicle of ornithine into Buchnera cells might be basic, and this
transport is probably going to be reliant upon having encoded items, as carrier qual-
ities are generally missing from Buchnera genomes and are available in the aphid
genome and upregulated in bacteriocytes (Hansen and Moran, 2011;Poliakov
et al., 2011). Accordingly, notwithstanding little variety in which amino corrosive
pathways are encoded by symbionts, fundamental amino corrosive prerequisites
might change because the variety in the bug has a genotype. For instance, arginine
reliance might be affected by variety among pea aphid genotypes influencing capac-
ities for proficiently shipping ornithine into Buchnera cells, while in aphid species in
which Buchnera has lost the ornithine pathway, the aphid might have the option to
ship ornithine to Buchnera productively.
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16 CHAPTER 1 Diversity of various symbiotic associations

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Further reading 17

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18 CHAPTER 1 Diversity of various symbiotic associations

Amelioration of biotic
stress by using
rhizobacteria for
sustainable crop produce 2
Fadime Karabulut
1
, Tahir Khan
2
, Nusrat Shafi
3
, Javid A. Parray
4
1
Department of Biology, Firat University, Elazıg, Turkey;
2
Yunnan Herbal Laboratory, School of
Ecology and Environmental Sciences, Yunnan University, Kunming, Yunnan, China;
3
Department
of Chemistry, GDC Eidgah, Srinagar, Jammu and Kashmir, India;
4
Government Degree College,
Eidgah, Srinagar, Jammu and Kashmir, India
1.Introduction
Agricultural products, besides being the basic food and livelihood of people, are also
the main raw material source of industries. Due to climate changes and newly
emerging diseases and pests, the yield of agricultural products must be increased
by 70% to meet the nutritional needs of approximately 9.8 billion people worldwide
by 2050 (FAO, 2017, p. 163). It has been found that biotic factors are responsible for
yield losses ranging from 31% to 42% in cultivated plants grown in various coun-
tries. One of the main ways to improve and increase agricultural production is to
reduce these stress-related losses. Chemical control has been one of the widely
preferred methods for many years in the fight against plant diseases in agricultural
production (Koike &Gordon, 2015). In order to reduce the use of chemicals in the
fight against diseases in recent years, new alternative methods have become criti-
cally important due to all these negative effects (Koike &Gordon, 2015).
Plants have the ability to grow throughout their lives, thanks to the constantly
active apical meristems at the bud and root tips. Growth is the growth of vegetative
organs as well as an irreversible increase in the dry matter accumulation of the plant.
In order for cells to grow, macromolecule synthesis must proceed more quickly than
degradation. As a result, growth happens. Growth is also defined as the structural and
functional changes that take place in various plant parts as they develop and grow.
Events like volume expansion, cell division, and tissue and organ differentiation
in plants also demonstrate its development.
The balance of life is greatly influenced by microorganisms. In situ identification
of microorganisms is now possible thanks to the development of molecular tech-
niques like metagenomics; however, little is understood about the ecological func-
tions performed by microbial communities (Berendsen et al., 2012). Microbial
components in the plant microbiota have important functions that support plant
CHAPTER
19
Rhizobiome. https://doi.org/10.1016/B978-0-443-16030-1.00008-0
Copyright ©2023 Elsevier Inc. All rights reserved.

growth and health (Brader et al., 2017). Rhizospheredis known as a place where
effective and important interactions occur between plants, soil, and microfauna.
Its effect between plant roots and the microorganisms living in it increases as it ap-
proaches the root surface. Plants exhibit many interactions involving various ecolog-
ical situations (competitive, exploitative, neutral, compensatory, mutual) with
microbes living in the rhizosphere, phyllosphere, and endosphere. There are studies
on plant-microorganism interaction, and these studies are mostly focused on miti-
gating pathogenic effects such as infection (Zhang et al., 2013) and reducing stress
conditions. It is also characterized by positive ecological interactions. Gene expres-
sion and functionality of plant-rhizobacteria interactions are provided improvements
in crop growth and quality, and knowledge that plants can benefit from rhizobacteria
(Hacquard, 2016). Most rhizobacteria are able to decompose organic matter in the
soil, cellulose, hemicellulose, other polysaccharides, hydrocarbons, and lignin de-
rivatives into receivable form. In addition, it transforms the nitrogen, sulfur, and
some minerals (Fe
þ3
,Fe
þ2
, etc.) in the soil into useable forms, enabling the plant
nutrients necessary for the plant to become useful. In addition, some of the Plant
Growth and Development Regulators (PGPRs) applied to different crops have
been commercialized (Hardoim et al., 2015;Reinhold-Hurek et al., 2015).
1.1 Effects of biotic stresses on plants
The negative effects of environmental conditions in the control of growth and devel-
opment of plants reveal the concept of stress in the plant. These stresses not only
reduce agricultural productivity but also prevent different land use in agricultural ac-
tivities. Plant species do, however, exhibit morphological, anatomical, and meta-
bolic responses. In this case, multiple stress factors provide the structural and
functional shaping of the plants. The term stress is also defined as “the change
caused by one or more environmental and biological factors in physiological events”
or briefly “the potential to cause harm” and it has been stated that the damage can be
observed both with the decrease in growth and yield and with the death of all or a
part of the plant. Stress resilience can be defined as an organism’s ability to survive
under adverse conditions. While a plant can be resistant to stress as a whole, some-
times only certain parts of the plant are resistant to stress (Larcher, 1995). For
example, seeds and resting buds are resistant to stress, while meristems and succu-
lent organs are susceptible. Similar to this, during the plant’s period of development
and growth, the degree of its resistance to any stress factor may change. Young seed-
lings, for instance, are more vulnerable to stress. Depending on the level of stress,
how long it lasts, and the kinds of tissues and organs exposed to the stress, plants
respond differently. In addition, the responses of different cultivars or genotypes
of the same species to the same stress may be different. Stress can cause loss of qual-
ity and quantity in the product (decrease in product quality and quantity), death of
the plant or its organs, and significant physiological and metabolic changes. The de-
gree of genetic environment adaptation of the plant affects the damage brought on by
stress factors. The goal of biotechnological applications is to create plant varieties
20 CHAPTER 2 Amelioration of biotic stress

that are resistant to stress and to stop potential nutritional issues from developing in
the future. Plants have two ways of dealing with stress: either they stop the effects of
stress factors with preventive mechanisms they have developed, or they continue to
live by coping with them with tolerance mechanisms (Larcher, 1995). The excess
amount of stress in the soil reduces the plant’s transpiration, respiration, taking up
water from the ground, and root development. Reduced photosynthesis, inability
to utilize nitrate, and consequently slowed protein synthesis result from the presence
of these conditions (Do
¨larslan &Gu
¨l, 2012). Biotic stresses are caused by animals,
weeds, insects, microorganisms, weeds, and pathogens.
Plants under stress can prevent the negative effects of stress with the mechanisms
they have developed, or they can survive by fighting stress with their resistance
mechanisms. Free radicals that increase as a result of stress cause damage to mac-
romolecules such as protein and nucleic acid by peroxidation of membrane lipids
and disrupt the cell wall and membrane structure (Sreenivasulu et al., 1999). There
are many enzymes in plants (as antioxidants) against oxygen radicals formed as a
result of stress. Superoxide dismutase (SOD), one of them, is produced by plants
to neutralize the harmful effects of the oxygen radical superoxide (O
2

), and
hydrogen peroxide (H
2
O
2
) is formed as a result of SOD activity. Some enzymes
then convert toxic H
2
O
2
, which is created as a result of SOD activity, to H
2
O and
O
2
(Dionisio-Sese &Tobita, 1998;Hernandez et al., 1995). Growth, nutrient uptake,
seedling vigor, and rice yield are just a few of the activities of rhizobial inoculants
(Biswas et al., 2000). Disease suppression is another benefit of Plant Growth and
Development Regulator (PGPR), which is achieved through the induction of sys-
temic resistance (ISR) and competition for nutrients in the local area. PGPR pro-
vides biological control with the formation of systemic resistance to prevent some
plant diseases. Induced systemic resistance (ISR) occurs in plants when certain types
of PGPR are used on young plants or seeds. Systemic resistance that has been
induced is an event that is brought on by numerous biotic stimuli. Additionally,
plants have the capacity to acquire generalized resistance to pathogen invasion
(Udayashankar et al., 2011).
1.2 PGPRs as a growth enhancer
PGPR increases plant growth and engages in other activities to make plants more
resistant to pathogens of all kinds (Zakry et al., 2012). These include nutrition fix-
ation, the prevention of diseases through the production of enzymes, biotic stress
neutralization, and volatile organic compounds. In particular, different types of
PGPR show different effects depending on the host plant (Garcı
´a-Fraile et al.,
2015). Biotic elements consist of many elements that affect plant genotypes, defense
systems, developmental periods, and microbial communities (Vacheron et al., 2013).
Indole acetic acid, an auxin derivative, can be produced by different bacterial spe-
cies. Sphingomonas hizobium,Mycobacterium,Azospirillum,Burkholderia spp.
and Microbacterium bacteria species are given as examples (Uma Maheshwari
et al., 2013). In many studies, it has been determined that PGPR applications
1. Introduction 21

have a significant effect on the hormone content of cabbage seedlings. The levels of
IAA, gibberellic acid, and salicylic acid rose after PGPR vaccination.
P. agglomerans was found to have the highest concentrations of salicylic acid,
IAA, and RK-92 gibberellic acid. The control group was found to have the highest
level of abscisic acid (Turan et al., 2014). Species considered PGPR are Pseudo-
monas putida, Pseudomonas aeruginosa, Enterobacter asburiae, Paenibacillus pol-
ymyxa, Azotobacter chroococcum, Mesorhizobium ciceri, Klebsiella oxytoca,
Rhizobium leguminosarum, and Stenotrophomonas maltophilia. The hormones pro-
duced by these bacteria are ethylene, kinetin, auxins, and gibberellins, which are
very important for root growth (Ahemad &Kibret, 2014).
Plants under biotic stress produce excessive amounts of 1-aminocyclopropane-1-
carboxylic acid (ACC), a precursor for the biosynthesis of ethylene. Many plant
physiological processes are regulated by the phytohormone ethylene. With the in-
crease of concentration in this phytohormone application, plant growth decreases,
causing senescence (Fig. 2.1). The enzyme ACC deaminase is produced by PGPR
and plays a role in ethylene homeostasis by converting the ethylene precursor
ACC into ketobutyrate and ammonia. As a result, the amount of ethylene produced
by plants is reduced. An enzyme called ACC deaminase has been identified in Alca-
ligenes, Achromobacter, Acidovorax, Klebsiella, Enterobacter, Pseudomonas, Meth-
ylobacterium, Variovorax, and Rhizobium from rhizobacteria (Chandran et al.,
2021). PGPR induces transcription of an enzyme necessary for the biosynthesis of
ethylene, which in turn causes plant cells to elongate. Adenosine monophosphate
(AMP) is synthesized from S-adenosyl methionine by the enzyme ACC synthase.
The ACC enzyme then changes it into ethylene. Plants exposed to stress induce
the ACC synthase enzyme that occurs in the roots. With an increase in ACC syn-
thase, the bacterial enzyme ACC deaminase is broken down. Due to its ability to
FIGURE 2.1
Degradation of the ethylene precursor ACC (Chandran et al., 2021).
22 CHAPTER 2 Amelioration of biotic stress

boost plant growth and stress tolerance via ACC deaminase activity, PGPR repre-
sents an important biotechnological advancement toward achieving sustainable
agriculture.
2.PGPRs systemic effects on the functioning and
physiology of plant
PGPRs can change the physiology and function of plants by affecting different tis-
sues of the plant, apart from colonization in the root part. PGPR increases the ab-
sorption of nutrients from the root part of the plant. Additionally, some PGPR
cause specific systemic reactions that are brought on by a variety of unidentified
signaling pathways. Plant transcriptome and metabolomics studies have shown
that PGPR has an impact on metabolite accumulation and gene expression in plants.
Thus, it is stated that the systemic effect of PGPR will be better understood.
Developing new bioprotectants, bioinoculants, and bioenhancers is a key compo-
nent of comprehensive microorganism research because they can deliver essential
metabolites like osmoprotectants, biosurfactants, phytohormone precursors, antiox-
idant enzymes, and nutrients. In addition to biotic stress-resistant bacteria, con-
sortium use in agriculture plays an important role, as various bacteria not only
help manage biotic stress but also provide resistance to phytopathogens. To better
explain the interactions between various rhizobacteria and plant systems, rhizobac-
teria should make greater use of inoculants. It will thus make it possible to increase
plants’ growth and tolerance for a variety of biotic stresses. However, research needs
to be done to develop specific bioformulations that help plants grow under stress by
carefully examining the gene expression level of stress-tolerant rhizobacteria and the
characteristics of multifunctional PGPR (Wo o &Pepe, 2018).
2.1 PGPRs effect on plant nutrition
PGPRs promote root growth by inducing nutrient absorption in plant nutrition (Man-
telin &Touraine, 2004). PGPR coordinates multiple pathways to increase nutrient
uptake and stimulate plant growth. Its solubility in phosphate is one of PGPR’s
most significant effects on plant nutrition. As a result, fertilizers applied to soils
accumulate large amounts of phosphorus over time. Plants use only a small amount
of the accumulated phosphorus. In plants, mono and dibasic phosphate are self-
absorbed. Microorganisms must mineralize and dissolve organic and insoluble phos-
phate (Ramaekers et al., 2010). Bacteria that can dissolve phosphate in insoluble
forms are Pseudomonas, Bacillus, and Rhizobium (Richardson et al., 2009).
2.2 PGPRs effect on plant transcriptome
Investigations were conducted into how gene expression affected PGPR applications
in plants. Five hundred and twenty genes were upregulated as a result of grafting the
2. PGPRs systemic effects on the functioning and physiology of plant 23

Pseudomonas putida strain onto Arabidopsis leaves. These genes participate in a va-
riety of metabolic functions, ABA and Ca signaling, Induced Systemic Resistance
(ISR), and chemical synthesis (Srivastava et al., 2012). N was obtained by nitrogen
fixation by applying Azospirillum brasilense Sp245, Cultivar IR42, and IAC 4440
bacterial strains to two rice cultivars with two opposite capacities. In this context,
it was determined that the expression of more ethylene receptors was also observed
(Vargas et al., 2012). All transcripts of ethylene receptors are essential for a healthy
symbiotic relationship between plants and bacteria (Miche
´et al., 2006). Inoculation
of Herbaspirillum seropedicae induced expression of ethylene and auxin sensitive
genes in rice as well as suppression of defense-related proteins PBZ1 and thionines
(Brusamarello-Santos et al., 2012). There is increased resistance to bacterial path-
ogen infections in plants that have been treated with the biocontrol PGPR. Jasmo-
nate and ethylene hormone sensitivity are necessary for rhizobacteria-mediated
ISR applied to Arabidopsis plant. The expression of 97 genes in roots changed
significantly as a result of Pseudomonas fluorescens WCS417r (Verhagen et al.,
2004). Bacterial-infected Arabidopsis plant shoots were found to have higher levels
of transcripts related to defense (Van de Mortel et al., 2012). P. fluorescens SS101
ISR has been shown to send out stronger salicylic acid signals than other hormones
(Van de Mortel et al., 2012). Glucosinolates and camalexin are also crucial players in
the Induced Systemic Resistance (ISR). It has been found that defense-related tran-
scripts are increased in wheat treated with P. fluorescens (Maketon et al., 2012).
These interactions suggest that PGPR and plant and helpful microorganism coordi-
nation, which is mutually important, influences plant immunology.
2.3 PGPRs effect on plant metabolome
Both under stressed and unstressed conditions, PGPR-induced metabolic changes
can be seen in the metabolite content of plants. When the roles of PGPRs in the syn-
thesis of their metabolites are changed, changes are also observed in the activity of
PGPRs plant root enzymes (Shaw et al., 2006). The amount of carbon compounds
leached from plant roots and into the soil was found to be greater for some of the
Azospirillum strains. This increase in rate was observed to be one-third of the carbon
compound released into the soil (Heulin et al., 1987). Some microbe-made chemi-
cals, like DAPG and phenazines, have even been shown to boost plant amino acid
production (Phillips et al., 2004). For example, Rhizobacteria Chryseobacterium
balustinum differs in the output of flavonoids from the roots of the soybean plant
(Dardanelli et al., 2010). The efflux of flavonoids released in the root parts of plants
belonging to the Fabaceae family can be influenced by PGPR (Dardanelli et al.,
2010) or Azospirillum (Burdman et al., 1996). A significant elevation in malate
and amino acid levels was found in rice plant shoots after Herbaspirillum seropedi-
cae was applied to the rice plant roots (Curzi et al., 2008). In other studies, secondary
metabolite changes were observed. It has been reported that the amount of isofla-
vones increased in the soybean plant to which different types of PGPR were applied
(Ramos-Solano et al., 2010). In addition, plants with medicinal pharmacological
24 CHAPTER 2 Amelioration of biotic stress

properties applied PGPR showed a significant increase in the amounts of alkaloids
and terpenoids (Bharti et al., 2013). When Azospirillum strains were applied to
maize cultivars (Walker et al., 2011) and two rice cultivars (Chamam et al.,
2013), secondary metabolite profiles were found to change. These plant metabolic
changes suggest that they vary depending on the Azospirillum strain-cultivar com-
bination (Chamam et al., 2013). When Azospirillum, Rhizophagus, Pseudomonas
strains or all three strains were applied to the roots of maize plant 8 (Walker
et al., 2012), differences in root secondary metabolites were detected. These differ-
ences depend on the type of microorganisms applied and the degree of fertilization.
PGPRs can help plants recover from stress. In rice roots, the concentration of glycine
betaine increased after being exposed to Pseudomonas pseudoalcaligenes (Bharti
et al., 2013). Bacillus subtilis GB03 applied to the root part of the Arabidopsis plant
resulted in a rise in the amount of glycine betaine (Zhang et al., 2010). In order to
mitigate the negative effects of cold stress on the grapevine plant, the roots could
be sprayed with water containing Burkholderia phytofirmans PsJN (Ait Barka
et al., 2006). Genes related to the cold are expressed in conjunction with this, and
defense is boosted (Theocharis et al., 2012). Bacterification is increased twice the
amount of soluble sugar and starch content. In addition, sugars with low-
temperature tolerance appear to be higher in bacteria-treated seedlings (Fernandez
et al., 2012).
3.The effect of plant and rhizobacteria interaction on
secondary metabolites
Herbal metabolites are divided into two as primary and secondary metabolites ac-
cording to the functions of chemical compounds and the character of their biosyn-
thetic pathways. Primary metabolites are synthesized in almost the same way
among living things and perform basic tasks for life. Secondary metabolites are
organic substances that help plants adapt to their surroundings, participate in repro-
duction, and play significant roles in their biotic and abiotic defense (Alvarez, 2014).
These compounds are more complex than primary metabolites (Bartwal et al.,
2012). In the field of biology, Kossel was the first person to use the term “secondary
metabolite” (1891) (Bourgaud et al., 2001). To produce secondary metabolite mol-
ecules in an environmentally friendly manner, it is essential to develop novel
methods. Due to their chemical makeup, secondary metabolites are typically catego-
rized into three distinct partsdalkaloids, terpene-steroids, and phenolics. It has been
claimed that plants only contain very small amounts of secondary metabolites pro-
duced in specialized cells at specific stages of development or under stress (Alvarez,
2014). Researchers wonder about the causes and mechanisms of change in the
amount of these secondary metabolites.
When the fungal-plant interaction is examined, it is seen that the amount of phos-
phorus in the leaf increases and changes in the leaf metabolome occur in Poaceae,
Plantaginaceae, and Fabaceae family plants inoculated with Rhizophagus irregularis
3. The effect of plant and rhizobacteria interaction 25

(Schweiger et al., 2014). Recent studies in this field have sought to answer the ques-
tion of whether bioactive phytochemicals are produced by the plant itself or as a
result of interactions with beneficial organisms in its tissues. These studies were car-
ried out recently. A combination of inducing elements derived from both plants and
fungi has been found to increase the accumulation of secondary metabolites in both
plants and fungi, respectively (Engels et al., 2008).
When the bacteria-plant interaction was examined, it was stated that inoculation
of basil plant (Ocimum basilicum) rich in secondary metabolites with Bacillus sub-
tilis (GB03) greatly increased the synthesis of terpene (from a-terpineol and
eugenol) (Banchio et al., 2009). A large increase in monoterpenes (thymol, carva-
crol, sabinen hydrate and terpinene) has been reported after inoculation of Italian
thyme roots with soil bacteria (A. brasilense and P. fluorescens)(Banchio et al.,
2010). A large amount of root formation occurs when Agrobacterium rhizogenes
transfers the auxin synthesis genes (role genes) found in the bacterial DNA in its
plasmid to the host tissue (Ambros et al., 1986). In the meantime, it has been
mentioned that the induction of disease-resistant genes causes an increase in second-
ary metabolite expression (Flores et al., 1999). Because of the ways in which gene
expressions are regulated in plant-microorganism interaction, researchers aim to
observe secondary metaboliteerelated gene expression changes; they discovered
that the inoculation of moths (Catharanthus roseus) caused an increase in the expres-
sion of genes in these plants that are involved in the production of secondary metab-
olites with bacterial strains (Pseudomonas sp., Curvularia sp.) (Singh et al., 2020).
Plant soil is not only a food source, but also an ecosystem in which the soil
contains fungi, bacteria, animals, and protists. These ecosystem processes, such as
nutrient recycling, the nitrogen cycle, the carbon cycle, and soil formation, depend
on these soil microorganisms (Rillig &Mummey, 2006). The microbiota in the
rhizosphere influences the interactions of bacteria, fungi (Arbuscular Mycorrhizal
Fungi: AMF), viruses, oomycetes, and other similar structures with the plant root
(Schweiger et al., 2014;Singh et al., 2020).
There are different claims about the origin of secondary metabolism in plants
(Karuppusamy, 2009). These claims suggest that plants or endophytic fungi, where
plants and rhizosphere microbes associate with defense-related signaling pathways
to produce secondary metabolites. In studies of biosynthetic pathways in which
radiolabeled primary amino acids were used, it was discovered that plants and endo-
phytic fungi have metabolic pathways for the production of secondary metabolites
that are similar to one another but distinct from one another (Zhang et al., 2009).
In plants, the quality of their secondary metabolites is primarily affected by biotic
factors found in the rhizosphere. Microbial diversity in the rhizosphere is essential
for improving aromatic plants’ medicinal properties. When comparing microbial di-
versity in the rhizosphere to that of bulk soil, as well as the large differences between
studies, it becomes challenging to provide a general description of the rhizosphere
microbiome. In the plant-microorganism interaction, besides increasing the toler-
ance of abiotic and biotic stresses of the host plants, it transforms the organic mol-
ecules into the form that the plant can take; regulates the rhizosphere soil; increases
26 CHAPTER 2 Amelioration of biotic stress

the mobilization of some molecules; provides carbohydrates, lipids, amino acids,
phytohormones, metabolites, etc., to the host plants (Karabulut et al., 2021;
Fazeli-Nasab et al., 2022). This dual relationship is important because it provides
the molecule. In addition, some amino acids, carbohydrates, lipids, and terpenoids
that are not secreted by plants in plant-microbial interaction need to be investigated
in detail by the microbes giving or taking them to plants, the underlying mecha-
nisms, the change in plant gene expressions, and the receptors through which this
signaling is received (C¸ etiz &Memon, 2021).
4.Impact of plant growth and development regulators on
root architecture
In order to better understand how PGPR works, researchers examine its host plant
and strain in detail. Also, PGPRs do not function alone. Synergistic or antagonistic
effects are seen when different populations of PGPR interact with a host plant. Rhi-
zobacteria strains that promote plant growth include different taxonomic groups that
coexist in soil (Almario et al., 2013). Selected bacterial isolates are crucial in deter-
mining their taxonomic identity for advantages on plant development (Upadhyay
et al., 2009). Populations of PGPR performing the same function make up the func-
tional group (plant growth promotion, ISR, nitrogen fixation, etc.). Before employ-
ing functional group methods, the selected genes must be documented. According to
the genetic makeup of the PGPR-plant interaction, coexisting PGPR strains have
two effects that are in opposition to one another. First, populations of PGPR showing
synergistic effect, PGPR function is greater than that of a single strain. With more
functions, the plant’s nutritional needs increase (Spaepen et al., 2007). The imple-
mentation of the PGPR function should also take into account regulatory effects
(Prigent-Combaret et al., 2008). There are significant interactions between different
types of PGPR in the rhizosphere in a particular region. Some of the PGPR func-
tional groups show inhibitory and competitive (Couillerot et al., 2011) and positive
signaling (Combes-Meynet et al., 2011). The effectiveness of PGPR is influenced by
the way these interactions modify spatial colonization patterns on roots (Couillerot
et al., 2011).
Rhizobial colonization occurs at the root tip of a plant and initiates nodule devel-
opment (Desbrosses &Stougaard, 2011). Since their roots can colonize with plants
and use their beneficial properties, PGPRs must possess beneficial properties
(Combes-Meynet et al., 2011). Root architecture is influenced by PGPRs, which
act on plant hormones. To ensure the viability and production of products, PGPR en-
gages in a variety of soil-related activities (Gupta et al., 2015). PGPR is in compe-
tition while colonizing the root portion. At the same time, these rhizobacteria are
involved in phosphate solubility, root growth, regulation of surface area (Ahemad
&Khan, 2012), nitrogen fixation (Glick, 2012), production of siderophores
(Jahanian et al., 2012), 1-amino-cyclopropane-1-carboxylate (ACC) deaminase,
and hydrogen cyanide (Xie et al., 2016)dvarious mechanisms by increasing plant
4. Impact of plant growth and development regulators on root architecture 27

growth. PGPRs in this context can also cause plants to produce phytohormones. Phy-
tohormones are organic substances that can affect, slow, or speed up plant growth
even when present in minute amounts (Damam et al., 2016;Sureshbabu et al.,
2016). Exogenous hormones, hormone extracts, or synthetic hormones can all be
used to control plant growth. Phytohormones are very important in regulating
nutrient absorption depending on climatic conditions and soil type. Primary root
development typically stops expanding while lateral roots and root hairs multiply.
PGPRs provide stress resistance in host root colonization through synthesis of
growth metabolites. Thus, a great increase in crop yield is observed. It can also pro-
vide root repair and stress resistance.
5.Stimulating the defense reaction of rhizobacteria in
plants
PGPR’s direct and indirect processes have previously been tested with and without
the presence of a number of biotic stressors (Khatoon et al., 2020;Morales-Ceden
˜o
et al., 2021;Phour et al., 2020;Ullah et al., 2019). However, we will go through a
few of them briefly here.
5.1 Direct mechanisms
It includes facilitating nutrient uptake such as mineralization (Glick, 2012) and
phosphate solubility, a process also in mycorrhizae, in promoting direct plant nutri-
tion and growth. Siderophores produced by PGPR can bind or dissolve iron in the
rhizosphere. It is possible for rhizobia and other PGPRs to convert atmospheric
N
2
into the essential plant nutrient ammonia (Peralta et al., 2004). Producing hor-
mones like cytokinin, salicylic acid, gibberellin, brassinosteroids, abscisic acid,
jasmonate, and indole acetic acid is another direct method of promoting plant growth
(Munne
´-Bosch &Mu
¨ller, 2013). Some PGPR species can create many hormones,
with the impact of each hormone being determined by its endogenous concentration
in the plant. For instance, many PGPR produce the enzyme ACC deaminase and
phytohormones, which lower plant ethylene levels (Glick, 2004). In the presence
or absence of different types of biotic stress, the interaction between the hormones
produced by the plant and those produced by PGPR can regulate various develop-
mental stages (Kumar et al., 2020;Wu et al., 2020).
5.2 Indirect mechanisms
The function of potential pathogens is inhibited and the plant defense system is stim-
ulated by PGPRs, which can also indirectly increase plant development. Given that
they do not require the use of chemical biocides, the indirect mechanisms of PGPR
are very intriguing for field application (Khatoon et al., 2020). A more thorough bio-
inocula may benefit from the inclusion of direct or indirect mechanisms of PGPB
28 CHAPTER 2 Amelioration of biotic stress

that promote plant growth (Herna
´ndez-Leo
´n et al., 2015). If a bacterial strain pos-
sesses both types of mechanisms and interacts favorably with rhizospheric microor-
ganisms, its beneficial effect on the plant might be synergistic. As a result,
understanding the molecular processes of microbial interactions is of great interest.
A group of lytic enzymes are produced by some PGPR, oomycetes, and pathogenic
fungi and break down the cell walls of these organisms. Oomycetes and fungi have a
dynamic cellular structure essential for their viability, pathogenicity, and cell
morphogenesis (Bowman &Free, 2006). PGPR produces the lytic enzymes chiti-
nase, glucanase, protease, lipase, and cellulase activity. Fungal cell walls include
chitin, protein, cellulose, and beta-D-glucans, whereas oomycetes predominantly
have alpha-D-mannans, beta-D-glucans, and quintin, with cellulose in little quanti-
ties (Inglis &Kawchuk, 2002).
The majority of oomycetes are able to produce hyphae, and this ability is a
defining characteristic of the group. Oomycetes are plant diseases that include obli-
gate biotrophs, white rusts, hemibiotrophs, and necrotrophs (Dodds et al., 2009).
These pathogens cause significant harm to crops; therefore, preventing disease
development through PGPR is critical for the long-term viability of agricultural eco-
systems (Morales-Ceden
˜o et al., 2021). Lytic enzymes produced by PGPR can break
down the protein matrix and chitin that make up the eggshells of plant-parasitic nem-
atodes. Since nematodes cause 12.3% of global losses every year, mainly in impov-
erished countries (Gamalero &Glick, 2020), following activity may be of significant
relevance for designing a novel biofertilizer/biopesticidedthe production of anti-
biotic molecules, as well as whether or not they are diffusible, is a major indirect
pathway (e.g., pyrrolnitrin, phenazines, pyoluteorin, 2,4-diacetyl phloroglucinol,
etc.) or volatile (e.g., hydrogen cyanide, dimethyl hexadecyl amine, dimethyl disul-
fide, etc.) (Glick, 2012).
Territorial expansion of plant areas that phytopathogens would typically occupy
and the creation of chelating substances like siderophores by reducing the availabil-
ity of iron to the potentially dangerous microbial community, are additional indirect
antagonism mechanisms (Dowling &O’Gara, 1994;Kloepper et al., 1980). Some of
the chemicals and enzymes listed above (for example, b-glucans, chitin, and pyover-
dine) can also operate as ISR triggers in plants (Choudhary &Johri, 2009;Sarma
et al., 2015). Plants produce excessive levels of ethylene when they are stressed
by numerous biotic causes. Inducing abscission, chlorosis, and senescence in plants,
this stress hormone can exacerbate the negative effects of infections (Dubois et al.,
2018;Etesami &Glick, 2020).
This is why the secretion of the enzyme ACC deaminase by particular PGPB can
help reduce ethylene levels in stressed plants, thereby facilitating their recovery.
This enzyme converts ACC, a form of the ethylene precursor, into a-ketobutyrate
and ammonia (Orozco-Mosqueda et al., 2020). Ethylene synthesis is decreased
when ACC, a precursor to ethylene, is present at lower levels, which eliminates a
stress signal from the plant. Plants under stress from abiotic factors can reduce their
ethylene production with the help of ACC deaminase and other substances like
trehalose (Pac¸o et al., 2020).
5. Stimulating the defense reaction of rhizobacteria in plants 29

Plants possess antioxidant defense mechanisms that help them deal with the
oxidative stress and limited production of reactive oxygen species (Chandran
et al., 2021). Both enzymatic and nonenzymatic antioxidant defense mechanisms
are present in plants (Table 2.1). Inoculation with PGPR has been shown to activate
antioxidant defense mechanisms in plants, thereby reducing oxidative damage
caused by a variety of abiotic stimuli. Plants with PGPR inoculation have higher
antioxidant levels. Root inoculation of two PGPR strains, Bacillus amyloliquefa-
ciens GB03 and Pseudomonas fluorescens WCS417r, on Mentha piperita growing
under stress resulted in elevated peroxidase and superoxide dismutase enzyme activ-
ity. By hydrolyzing hydrogen peroxide to oxygen and water, Bacillus licheniformis
(FMCH001) injection of plants increased CAT activity in the roots, which counter-
acts the ROS (Chandran et al., 2021).
6.Plant defense with biocontrol agents
Nutrients for plant growth come from the diverse ecosystem in soil, which includes
bacteria, protists, fungi, and animals living in active/coordinated groups. Many
plant-associated soil/root microorganisms have developed exploitative, competitive,
or neutral interactions with plants; the phytomicrobiome and the plant comprise the
holobiont. Researchers have recently begun to look at the idea of using beneficial
rhizobacteria to both alleviate pathogenic effects and promote plant development.
Plant roots host different microorganisms. Therefore, the next step is to determine
which rhizobacteria will benefit the rhizobacteria mixture or which rhizobacteria
will be most beneficial to the host plant. Microorganisms that naturally occur on
plants and promote plant growth have many uses, one of which is the prevention
of disease. Plant species have different rhizosphere microbiomes, and plants influ-
ence the makeup of their microbiomes. Microbes have an effect on plant health
and phenotypic plasticity because of their long-term coevolution with plants.
They do this by regulating plant growth and defense responses. In the rhizosphere,
you’ll find a diverse community of microbes that collectively produce an abundance
of plant growth regulators (PGPR). Members of the phytomicrobiome that are bene-
ficial to plants include the bacterial population that lives on the surface of roots, in
the rhizosphere, or between stem cells and stem cortex cells. Since they first colo-
nized terrestrial environments, PGPR and related plants have coevolved, resulting
in synergistic interactions with their host plants.
A great number of articles have been published surrounding research into the
mechanisms, impacts, and possibilities for the accomplished practice of PGPR to
agricultural plant production in controlled environments. This is critical for devel-
oping more broad biological control strategies, which must consider field circum-
stances. Because the environmental conditions are controlled in greenhouse
production systems, it is very simple to distribute PGPR; at the same time, many po-
tential BCE strains have been identified and distributed. Several isolates, including
Bacillus subtilis,Bacillus amyloliquefaciens, and Pseudomonas stutzeri were
30 CHAPTER 2 Amelioration of biotic stress

Table 2.1 Plant growth-promoting mechanisms and PGPR (Kumawat et al.,
2022).
Plant PGPR
Plant growth
promoting
mechanism(s) References
Soybean Bradyrhizobium
japonicum USDA110,
Bacillus firmus SW5,
Pseudomonas putida
TSAU1
Antioxidant enzyme
production; alternation
in root architecture
El-Esawi et al. (2018)
Soybean Pseudomonas putida H-
2e3
Production of salicylic
acid, ABA, gibberellins,
and jasmonic acid
Kang et al. (2014)
Soybean Pseudomonas simiae Phosphate
solubilization;
production
siderophore; and IAA
Vaishnav et al. (2016)
Soybean Pseudomonas
aeruginosa LSE-2,
Bradyrhizobium sp.
LSBR-3, Leclercia
adecarboxylata LSE-1
Production of ACC
deaminase, IAA, and
siderophores;
phosphate
solubilization;
production of exo-
polysaccharide; and
development of biofilm
Kumawat, Sharma,
Singh et al. (2019),
Kumawat, Sharma,
Sirari, et al. (2019)
Cucumber Rhodopseudomonas
palustris G5
IAA and 5-
aminolevulinic acid
synthesis; potassium
solubilization and
phosphate; N fixation
Ge and Zhang (2019)
Cucumber Variovorax paradoxus,
Bacillus megaterium,
Pseudomonas
fluorescens
Production of
siderophore,
exopolysaccharide,
ACC deaminase, and
IAA
Nadeem et al. (2016)
Tomato Pseudomonas migulae
8R6, Pseudomonas
fluorescens YsS6
ACC deaminase
production
Ali et al. (2014)
Tomato Streptomyces sp.
PGPA39
IAA and ACC
deaminase production;
phosphate
solubilization
Palaniyandi et al.
(2014)
Rice Pantoea agglomerans
KL
Ammonia production;
IAA, ACC deaminase,
phosphate
solubilisation, and
exopolysaccharide
Bhise and Dandge
(2019)
Continued
6. Plant defense with biocontrol agents 31

discovered and found to be successful in root colonization and to inhibit the disease
Phytophthora capsici during cucumber plant development because the environ-
mental conditions are controlled in greenhouse production systems and these strains
are effective in greenhouse experiments. For instance, Bacillus spp. have become
important pathogen suppressors in the field, and Pseudomonas fluorescens have
been hailed as promising biocontrol agents. Several isolates were discovered and
demonstrated to be effective in root colonization and controlling the disease Phy-
tophthora capsici during cucumber plant development. These isolates included Ba-
cillus amyloliquefaciens, Bacillus subtilis, and Pseudomonas stutzeri.
Protein phosphorylation is one of the main mechanisms involved in interactions
between plants and pathogens, plant defense, and induced resistance (Xing et al.,
2002). Many phosphatases and protein kinases discovered link signaling to plant de-
fense responses. Proteomics and genomics will continue to significantly impact
research on phosphorylation in these interactions as well as uncovering new compo-
nents in plant-pathogen interactions (Xing et al., 2002).
7.Conclusions and future perspectives
The presence of PGPR in high concentrations in the rhizosphere, or the area sur-
rounding the root, has been highlighted in this section, and it is free bacteria that
have beneficial importance for agriculture and plants. Additionally, it has been thor-
oughly explained how some PGPRs work through indirect or direct mechanisms that
boost the production of metabolites, antibiotics, enzymes, bioactive factors, and
Table 2.1 Plant growth-promoting mechanisms and PGPR (Kumawat et al.,
2022).dcont’d
Plant PGPR
Plant growth
promoting
mechanism(s) References
Rice Curtobacterium albidum
SRV4
ACC deaminase, N
fixation, HCN
production,
exopolysaccharide,
and IAA
Vimal et al. (2019)
Rice Enterobacter sp. P23
Phosphate
ACC deaminase,
siderophore, IAA, and
HCN production;
solubilization
Sarkar et al. (2018)
Wheat Enterobacter cloacae
ZNP-3
Production of IAA,
HCN, and ACC
deaminases;
phosphate
solubilization
Singh et al. (2017)
32 CHAPTER 2 Amelioration of biotic stress

growth stimulants to encourage plant growth and development. In conclusion, some
PGPRs are useful rhizobacteria used as biofertilizers, low cost, renewable, and envi-
ronmentally friendly fertilizers that can be used to increase agricultural crop produc-
tivity while reducing the long-term application of inorganic fertilizers, improve soil
health, and facilitate nutrient availability. In order to boost agricultural output, the
ecosystem should not be harmed. PGPR applied in agriculture is crucial for biolog-
ical remediation, crop quality, ecosystem functioning, and biocontrol. Therefore, it
is common knowledge that making the most of PGPR in agriculture will be highly
effective and will become a renewable tool for sustainable agriculture. The ecolog-
ical manageability and diverse uses of PGPRs in agro-trade are model frameworks
that can yield new bioactive chemicals and hereditary compounds. PGPR’s means of
action, colonization capacity, reasonable diversity, definition, and implementation
can be credible parts of sustainable rural management and can foster future progress.
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Gimenez, E., Salinas, M., & Manzano-Agugliaro, F. (2018). Worldwide research on plant de-
fense against biotic stresses as improvement for sustainable agriculture. Sustainability,
10(2), 391. https://doi.org/10.3390/su10020391
Hashem, A., Mohammed Alarjani, K., Almutariri, K. F., Parray, J. A., Sharma, S. K.,
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J. A. Parray, N. Shameem, E. F. Abd-Allah, & M. Y. Mir (Eds.), Core microbiome:
Improving crop quality and productivity (1st ed.). John Wiley and Sons Ltd.
Hashem, A., Shameem, N., Parray, J. A., & Abd-Allah, El. F. (2022). Mycorrhizal strategy for
the management of hazardous chromium contaminants. In J. A. Parray, N. Shameem,
E. F. Abd-Allah, & M. Y. Mir (Eds.), Core microbiome: Improving crop quality and pro-
ductivity (1st ed.). John Wiley and Sons Ltd.
Karabulut, F., Parray, J. A., & Mir, M. Y. (2021). Emerging trends for harnessing plant metab-
olome and microbiome for sustainable food Production. MicroEnvironer, 1(1), 33e53.
https://doi.org/10.54458/mev.v1i01.6672
Mir, M. Y., Hamd, S., & Parray, J. A. (2022). Phyllosphere microbiomes: implications and
ecofunctional diversity. Microbial Diversity and Ecology in Hotspots.https://doi.org/
10.1016/B978-0-323-90148-2.00005-5
Parray, J. A., Ali, U., Mir, M. Y., & Shameem, N. (2021). A high throughputs and consistent
method for the sampling and isolation of Endophytic bacteria allied to high altitude the
medicinal plant Arnebia benthamii (Wall ex. G. Don). Microbes and Environments,
1(1), 1e6. https://doi.org/10.54458/mev.v1i01.6668
42 CHAPTER 2 Amelioration of biotic stress

Microorganisms as salient
tools in achieving
ecosystem approaches 3
Sneha P. Nair
1
, Renitta Jobby
1,2
1
Amity Institute of Biotechnology, Amity University Maharashtra, Navi Mumbai, Maharashtra,
India;
2
Amity Centre of Excellence in Astrobiology, Amity University Maharashtra, Navi Mumbai,
Maharashtra, India
1.Introduction
Ecosystem is the integrated system or a community where both living and nonliving
things coexist. The term was initially coined by Arthur G. Tansley. It is the dynamic
system where living organisms interact with themselves and with the physical envi-
ronment that affects the properties of each other. Natural ecosystem is the one which
is essential and vital to maintain life on this planet.
An ecosystem that is durable and able to preserve its structure and function in the
face of external stress is one that is healthy (Constanza &Michael, 1999). Following
several conferences and discussions, it was determined that health of the ecosystem
is a subjective matter, and it varies progressively as our knowledge of the natural
world advances, making it inappropriate to use as a scientific basis for environmental
management (Lancaster, 2000). The majority of normative concepts such as biolog-
ical diversity, ecological restoration, ecological sustainability, etc., are enshrined in
both domestic and international law and policy (Calicott et al., 1999). The Rio
Declaration on Environment and Development uses ecosystem health to create a
global plan for managing ecosystems.
Humans benefit from ecosystems in a variety of ways, such as through market-
able goods like pharmaceuticals, recreational activities like camping, and ecosystem
services that include erosion control and water purification. Due to the growing hu-
man population that has led to both habitat degradation, water and air pollution, the
regions are coming under more and more threat, despite the crucial functions that
ecosystems serve. Increasing greenhouse gas concentrations in the atmosphere are
a new concern, and they are causing global climate change and have added to these
stresses (Ecosystems and Global Climate Change - Center for Climate and Energy
SolutionsCenter for Climate and Energy Solutions, n.d.).
CHAPTER
43
Rhizobiome. https://doi.org/10.1016/B978-0-443-16030-1.00012-2
Copyright ©2023 Elsevier Inc. All rights reserved.

2.Impact of human and other interactions on ecosystem
and climate change
Changes in the hydrosphere and atmosphere have a significant impact on the
biosphere, the thin layer of life on Earth that provides the favorable environment
for human cultures (Malhi et al., 2020).
The distribution of ecosystem, species range, and processes on Earth are funda-
mentally controlled by the climate. The effects of climate change will also be felt on
how ecosystems work, particularly, how the herbivores, plants, carnivores, and soil
dwelling organisms that constitute the biological components of ecosystems trans-
port energy and chemicals (Ecosystems and Global Climate Change - Center for
Climate and Energy SolutionsCenter for Climate and Energy Solutions, n.d.).
The ecosystem’s composition and operation are affected by climate change and it
further affects the individual relationships between species and how they interact
with other living organisms (Raposa, 2011). Different countries get affected by
climate change in a different way and the ways by which they mitigate also varies
considerably, thereby making climate change one of the biggest challenges to the
mankind (Klingelho
¨fer et al., 2020). Ecosystem process is affected by both the
changes in the environmental conditions such as alterations to biological ecosystems
and global warming (Chapin et al., 1997;Hooper et al., 2005;Smith et al., 2000).
Terrestrial ecosystems are a major contributor to these feedbacks on the climate
because they emit and absorb greenhouse gases and also store a substantial amount
of soil carbon and vegetation. Some of the major greenhouse gases are carbon diox-
ide, nitrous oxide, and methane which remain a persistant problem (Schimel et al.,
1994).
2.1 Climate change affecting ecosystem
Climate of a region is basically the long-term shift in the weather that is typical, such
as rainfall, etc., and climate change refers to that phenomenon where periodic alter-
nation’s in the climate is caused by the natural forces such as biological, geological
factors and even due to the moving or shifting of continents and these are most
importantly in combination with the human activities According to some data,
from 10,000 years ago or about, with the development of agriculture, human soci-
eties are experiencing climate change. Due to CO
2
’s significant contribution to
global warming, there are growing concerns about the quantity of atmospheric
CO
2
(Sun et al., 2021). Some of the key ecosystem level properties that affect the
climate change are as follows.
2.1.1 Role of primary producers in the climate change
Foundation of most food webs are the primary producers and other photosynthetic
organisms that play a role in producing oxygen and control the key mechanisms
like carbon sequestration and cycling (Raposa, 2011). Modifications to main produc-
tion at higher trophic levels are likely to be magnified (Chust et al. 2014;Lefort
44 CHAPTER 3 Microorganisms as salient tools in achieving

et al., 2015;Stock et al., 2014). There are many components to change in climate on
which production depends such as increase in the growth of vegetation due to
increased atmospheric carbon dioxide (Norby et al., 2002); air pollution and lack
of nutrients limit the growth (Norby et al., 2010;Oren et al., 2001;Pan et al.,
2009). Climate change’s effects on marine primary production such as phyto-
plankton trap CO
2
that enters the ocean and penetrates to the seafloor. Growth rates
of phytoplankton have an impact on fisheries production as well as CO
2
intake from
the ocean and organic carbon export (Tyrrell, Link, &Moustahfid, 2011).
2.1.2 Interaction between species
Different species react differently to changes in the climate and atmosphere. The
composition and structure of ecosystems can be drastically altered by variations
in species interactions, and as a result, the consequences may be much more signif-
icant than those of a changed climate (Hughes, 2012). Trophic levels higher are pro-
jected to be more vulnerable to climate change; therefore, rates of encounter by
predators are likely to shift (Abbott et al., 2014;Daly &Brodeur, 2015;Dell
et al., 2014). The geographic adjustment or disruption of species interaction net-
works as a result of climate change will have diverse effects on the guild’s species
and likely result in an increase in competition and/or intraguild predation (Brambilla
et al., 2020).
2.1.3 Extreme events causing climate change
Increasing temperatures and changed precipitation patterns cause more severe
droughts and wildfires, which influence the ecosystems’ structure and function;
especially those that are forested. The tree growth is reduced and mortality is
increased (Peters et al., 2015). Drought reduces trees’ defenses, making them
more vulnerable to other disturbances like insects, viruses, and invasive species
and wildfires (Trottier et al., 2017;Littell et al., 2016;Logan, 2004;Weed et al.,
2013).
3.Soil microbes
Soil acts as a critical and dynamic part in both managed and natural ecosystems and
as regulating center for the bulk of ecosystem functions. It provides substrate to
various human activities. The microclimatic features exhibit a high level of physical
and chemical diversity at tiny sizes, and the phonologies of its species favor the cre-
ation and preservation of an extremely large quantity of niches. The soil environ-
ment is home to a complex and diversified biological community (Tiedje et al.,
2001;Ettema, 2002). A significant amount of genetic diversity comprises among mi-
crobes present in the soil and the impact of these soil microbes is little known despite
their abundance (Whitman et al., 1998). These microbes have a major influence on
various ecosystem functions like soil formation, carbon cycling, nutrient accumula-
tion, etc. (van der Heijden, Bardgett, &van Straalen, 2008).
3. Soil microbes 45

3.1 Importance of soil microbes
Soil microbes are essential for soil fertility. The health of soil depends on both biotic
and abiotic factors and also on various interactions within the system. These inter-
actions significantly affect microbial activity and support a number of essential func-
tions (Frąc, Hannula, Marta, &Ma1gorzata, 2018).
Most important soil processes, including the biotransformation of nutrients and
organic materials, are carried out by bacteria and other soil microorganisms. Both
soil physicochemical processes and ecological interactions have an impact on their
behavior. The essential nutrient and patterns of energy flow that ensure forest pro-
duction, biodiversity maintenance, and control climate stability are mediated by
soil, a crucial component of forest ecosystems (Hartmann et al., 2012).
Despite the latter’s greater diversity, larger soil creatures (i.e., macrofauna) have
gotten significantly more attention than the former, as only about 0.1% of microbial
taxa out of numerous fungi and bacteria prevalent in fertile surface soil have been
grown in order to better comprehend their metabolic role. Therefore, with the appli-
cation of molecular techniques, this trend has seen a change (Lynch et al., 2004).
According to DNA-based techniques, soil typically contains between 10
9
and 10
10
microorganisms per gram that could represent thousands of bacterial species
(Gans et al., 2005). Due to the ongoing development of high-throughput molecular
technologies, microbial ecologists can now define the taxonomic, phylogenetic, and
functional diversity of soil microbial communities (Fierer et al., 2012).
3.2 Nature and composition
Diverse soils are home to billions of distinct creatures, millions of species, ranging
from larger organisms like ants and earthworms to small microorganisms like bac-
teria, archaea, fungi, and protists (Ko
¨berl et al., 2020). Natural and anthropogenic
disturbances have a direct impact on the makeup of soil microorganisms. For
example, the variety of ectomycorrhizal fungi might be decreased in boreal forests
due to wood exploitation (Hartmann et al., 2012) and the relative biomass decompo-
sition of genes in the soil (Cardenas et al., 2015).
The biomass, activity, and makeup of the microbial community gets altered as
plant production levels and variety increase (Zak et al., 2003). The kind of soil
and its heterogeneity determine the overall number of microorganisms, their compo-
sition, and their abundance. The biological activity of microorganisms is affected by
the chemical and physical characteristics of soils (Gagelidze et al., 2018).
The term “community of microorganisms” describes functionally and actively
interacting structured populations rather than merely coincidental associations.
These interactions are consistent and difficult for external variables to affect. During
the microbial biocenosis of the soil, a vast number of genera and species, including
members of the genera, were present. More significant are Pseudomonas, Bacillus,
and Rhodococcus (Gagelidze et al., 2018). Ammonification of proteins and urea and
also destruction of phosphoric organic compounds is done by Bacillus.
46 CHAPTER 3 Microorganisms as salient tools in achieving

Pseudomonas actively participates in organic substance’s mineralization and
thereby plays a key role in reduction of nitrates to molecular nitrogen; Rhodococcus
degrades organic and mineral substances, including the components of humus, as
part of its self-remediation of soil (Gagelidze et al., 2018).
4.Impact of different class of microbes on climate change
Each environment on Earth that is habitable to macroscopic species is inhabited by
microorganisms, and in some settings, such as the deep below and “extreme” envi-
ronments, they are the only life forms (Cavicchioli et al., 2019). Microbes play a vi-
tal role in climate change; they generate three main gases that account for 98% of
global warmingdcarbon dioxide, nitrous oxide, and methane. Their current rate
is attributed to the variations in the activities of humans resulting in bacteria having
greater access to nitrogen and carbon and transforming to these products (Tiedje
et al., 2022a). Microbes are necessary for halting climate change, yet they are rarely
the focus of research on the issue, and their needs are not considered while formu-
lating policies (Cavicchioli et al., 2019).
4.1 Marine biome
Three-fourth of the Earth’s surface is covered by marine regions that include the
mangroves, coral reefs, and coastal estuaries all of which lead to the open ocean.
In the marine environment, dominance is taken over by the archaea, bacteria, and
eukarya that belong to the three domains of life. The temperature ranges from
that of polar areas to deep sea hydrothermal vents of more than 100C. The salinity
is nearly uniform about 3.5%. Microorganisms may live and develop in this environ-
ment, either by existing independently of other living things or by cooperating with
them. It is said that 98% of marine primary productivity is from microorganisms
(Sogin et al., 2006) that play key role in the marine food webs, carbon and energy
cycles.
Due to the varied and versatile biochemistry that their genetic materials have
encoded, the microbial life has the ability to flourish in all aspects of the marine
environment, from surface sea ice to deep ocean vents. The makeup of microbial
communities should be far more diverse than the stated estimates of a few thousand
distinct kinds of microorganisms per liter of seawater due to the billions of years that
marine bacteria have been there and developed (Sogin et al., 2006). There are sessile
benthic creatures, thriving in diverse marine environments including shallow trop-
ical polar reefs, temperate polar reefs that contribute to diversity and biomass
(Dayton, 1989,Hentschel et al., 2012). As per estimations made by marine biolo-
gists using statistical projections, there are presently expected to be near to a million
species in the world, with roughly 226,000 marine eukaryotes currently known
(Appeltans et al., 2012).
4. Impact of different class of microbes on climate change 47

4.1.1 Effect of climate change on marine biome
The phototrophic microorganisms use sun’s energy and the marine life present in the
deeper zones uses organic and inorganic chemicals for energy (Jørgensen &Boetius,
2007). The global ocean has absorbed 93% of the extra energy brought on by human-
caused greenhouse gas emissions, which has resulted in an increase in sea surface
temperature of nearly 1C since the turn of the 20th century (Poloczanska et al.,
2016). Both the abiotic and biotic compartments of marine ecosystems get affected
by the global climate change depending upon the taxa, functional groups, and
oceanic regions (Poloczanska et al., 2016;Thackerey et al., 2016). There is also a
decrease in dissolved oxygen concentration due to the anthropogenic factors causing
both chemical and physical change (Andrews et al., 2013), ocean circulation getting
altered (Cai et al., 2005;Wu et al., 2012).
Ocean acidification is predicted to decrease calcification in marine calcifiers like
corals and coccolithophores in addition to influencing a range of other processes
including development and reproduction (Kroeker et al., 2013). There are many fac-
tors that influence the response to environmental changes like habitat, physiological
tolerance, species generation time. Given that many marine organisms have a disper-
sive planktonic stage and that different life stages may inhabit various environments,
marine animals frequently have complicated life cycles with each stage having a
unique exposure to and sensitivity to climate change (Rijnsdorp et al., 2009).
4.1.2 Effect of microorganisms on climate change
Marine phytoplankton, which is distributed over a larger surface area as compared to
the terrestrial biome, is subjected to less seasonal change, and has a higher turnover
rate than trees, perform half of the CO
2
fixation (Behrenfeld, 2014). Consequently,
phytoplankton reacts to changes in the climate quickly on a global scale. The strat-
ification of the ocean is facilitated by an increase in temperature, sunlight, and fresh-
water supplies to the surface waters. This limitation on nutrient transfer from deep to
surface waters lowers primary production (Behrenfeld, 2014;Behrenfeld et al.,
2006,2017). Microbial primary generation makes a significant contribution to
CO
2
sequestration in maritime environments. Additionally, marine microbes recycle
nutrients for the marine food chain while simultaneously releasing CO
2
into the at-
mosphere (Cavicchoili et al., 2019).
Diatoms when compared with other phytoplankton groups have the fastest sink-
ing rates, and they export around 40% of the particulate carbon to deep waters (Boyd
et al., 2019;Tre
´guer, 2018). Although seafloor methanogens and methanotrophs
play a critical role in the generation and consumption of CH
4
, it is unclear how
much of an impact they have on the atmospheric flow of this greenhouse gas
(Boetius &Wenzho
¨fer, 2013).
Aerosols have an impact on cloud formation, which affects precipitation and sun-
light irradiation, although it’s unclear how much or how (Rosenfeld et al., 2019).
Phytoplankton emits dimethyl sulfide and it’s derivate sulfate promotes cloud forma-
tion (Charlson et al., 1987;Sanchez et al., 2018). Microbial activity controls the
amount of carbon that is emitted as CO
2
and CH
4
after remineralization; their effects
48 CHAPTER 3 Microorganisms as salient tools in achieving

on microbial communities must be assessed. The amount of vegetated coastal hab-
itats has decreased by 25%e50% over the last 50 years due to human activities,
particularly anthropogenic change in climate, and the number of aquatic predators
has decreased by up to 90% (Atwood et al., 2015;Duarte et al., 2013;Myers &
Worm, 2003).
4.2 Terrestrial biome
The overall prevalence of microbes in terrestrial environment is 1 x 10
29
, which is
comparable to the number altogether in marine habitats (Flemming &Wuertz,
2019). The availability of macronutrients, such as nitrogen and phosphorus, that
determine soil microbial productivity indirectly affects how much of organic carbon
is stored and then released into the atmosphere as well as how much of it is retained
in plants and soils (Singh et al., 2010;Bardgett &van der Putten, 2014). Terrestrial
plants account for half of net world primary output; hence, it is predicted that soils
contain 2000 billion tonnes of organic carbon, which is greater than the sum of the
carbon reservoirs in the atmosphere and plants (Singh et al., 2010).
Through photosynthesis, plants take carbon dioxide and generate organic mate-
rial that fuels terrestrial ecosystems. On the other hand, autotrophic respiration in
plants (60PgC per year) and microbes heterotrophic respiration (60PgC per year) re-
turn the atmosphere’s carbon dioxide (Ballantyne et al., 2017;Singh et al., 2010).
4.2.1 Effect of climate change on terrestrial biome
Plant diversity is influenced by soil microbial diversity, which is crucial for main-
taining ecosystem processes like carbon cycling (Delgado-Baquerizo et al., 2016;
Jing et al., 2015). Significant alterations in fungal and bacterial communities were
also observed in forest soils with an average yearly temperature range of greater
than 20C.
Plants produce organic matter that powers terrestrial ecosystems and utilizes
photosynthesis to take CO
2
from the atmosphere. Because plants provide less carbon
to rhizosphere microorganisms in nutrient-rich forests, microbial respiration may be
reduced there (for example, as root exudates) (Ho
¨gberg et al., 2001). Fifty percent of
fixed carbon released by the plants into the soil is available for microbial growth
(Clemmensen et al., 2013; Keiluweit et al., 2015; Tang &Riley, 2015). Change in
climate affects microbial communities and their activities both directly and indi-
rectly through a number of interconnected elements, including temperature, precip-
itation, soil characteristics, and plant input.
4.2.2 Effect of terrestrial microbes on climate change
In peatlands, water saturation limits oxygen exchange, hinders microbial decompo-
sition, promotes the growth of anaerobes, and leads to the production of CO
2
and
CH
4
(Bragazza et al., 2013;Hodgkins et al., 2018). Increases in water-saturated soils
brought on by permafrost melting encourage methanogens’ anaerobic CH
4
synthesis
4. Impact of different class of microbes on climate change 49

and the creation of CO
2
by a variety of microorganisms (Knoblauch et al., 2018).
When these methanogens become active in melting permafrost, as was discovered
during a 7-year laboratory investigation of CO
2
and CH
4
production under anoxic
circumstances, they produce an equal quantity of carbon dioxide and methane. Addi-
tionally, it was predicted that carbon emissions from anoxic habitats will have a
higher impact on climate change in comparison to emissions from oxic conditions
by this century’s end (Knoblauch et al., 2018).
5.Ecosystem approaches
The ecosystem approach encourages conservation and sustainable usage while man-
aging land, water, and life resources fairly. The standard definition suggested by
Convention of Biological diversity is: “a strategy for the integrated management
of land, water and living resources that promotes conservation and sustainable use
in an equitable way.” (Morand &Claire, 2018).
5.1 The need for ecosystem approaches
To tackle issues such as inadequate environmental conditions, depleting resources,
and growing environmental needs.
To make clear the ecosystem services, such as health advantages, that don’t have
a commercial value.
Promote efficiency and cooperation among various public plans and initiatives.
Encourage people to consider how their decisions will affect future generations
and people outside of their immediate community.
6.Microbes as the tools for achieving ecosystem
approaches
The situation has gotten worse according to a new analysis from the Intergovern-
mental Panel on Climate Change (IPCC), with 3.3 billion people on Earth now
considered to be highly vulnerable to climate change. Additionally, the report notes
that the ecosystems and humans are becoming more vulnerable to climate risks as a
result of present unsustainable growth trends.
As discussed earlier, microbes generate and consume three principal gasesd
carbon dioxide (CO
2
), nitrous oxide (NO), and methane (CH
4
) account for 98%
of the rise in global warming (Tiedje et al., 2022). When provided appropriate con-
ditions, microbes can consume these three gases as resources: nitrifiers, cyanobac-
teria, algae, and methanotrophy (methane oxidizers) are all examples of photo- or
chemoautotrophic growth.
50 CHAPTER 3 Microorganisms as salient tools in achieving

6.1 Microbes for carbon sequestration
The largest carbon reservoir is considered to be in the soil. Through plant photosyn-
thesis and animal decay, carbon is naturally fixed in the soil as carbon dioxide. Car-
bon sequestration is a mechanism for absorbing atmospheric carbon dioxide and
storing more soil organic carbon. Since atmospheric CO
2
makes up 76% of all
greenhouse gas emissions, there is a considerable interest in lowering it in order
to address one of the major problems that human society is now facing (Rossi,
Olguı
´n, Diels, &De Philippis, 2015). The links between microbial biomass, commu-
nity structure, and byproducts of microorganisms, and other soil characteristics
including quality, distribution of size of pore, mineralogy of clay and collective dy-
namics control the contribution of microbes to carbon sequestration (Six et al.,
2006).
6.1.1 Ocean microbial carbon pump (MCP)
Carbon cycling in the ocean is basically known as MCP. This process results in the
long-term carbon stored in the dissolved phase of the water column. It could operate
as a modulating element in the transfer of carbon among the Earth’s surface reser-
voirs, which would affect climate change (Sexton et al., 2011). While Particles of
Matter (POM) make up the majority of the primary production, some fixed carbon
is discharged as DOM (Dissolved Organic Matter) into the water.
While POM is sinking, attached bacteria hydrolyze a major portion of it into
DOM that contributes to the MCP, while some Recalcitrant Dissolved Oxygen Mat-
ter (RDOM) molecules are scavenged by POM and enter the biological pump.
RDOM remains throughout the water column in contrast to the POM flux’s exponen-
tial attenuation along with water depth (Jiao &Azam, 2011).
There are few researchers focusing on employing a carbon sequestering strategy
using microbial inoculants to counteract climate change, despite their shown poten-
tial to absorb carbon dioxide from the atmosphere into the soil. To find out how
effective this method may be at reducing climate change, further research and effort
are thus needed.
6.2 Microbes to reduce methane emissions
To make sure that methanogenesis rates do not outpace rates of oxidation of
methane, monitoring and current models are required. In addition to producing
biomass of methanotrophs that may be put into landfill cover soil, aboveground
methanotrophic bioreactors have the potential to transform recovered methane
into useful goods like single-cell protein or bioplastic (Tiedje et al., 2022).
By eliminating the methane produced by methanogens and geothermally, meth-
anotrophs play an important role in the world’s carbon cycle. The mechanism
involved is due to the presence of certain unique enzymes such as methane mono-
oxygenases and soluble methane monooxygenases that mediate the oxidation of
methane to methanol (Kaupper et al., 2021). They have been widely used as a bio-
catalyst to remove halogenated hydrocarbons from soil and subterranean water and
6. Microbes as the tools for achieving ecosystem approaches 51

reduce methane’s impact as a greenhouse gas. Under environmentally benign con-
ditions, the generation of fuels, chemicals, and other biomaterials can employ meth-
anotrophs as a biocatalyst (Kalyuzhnaya et al., 2013).
It has also been proven that with the usage of methanotrophic biofilters, active
methane removal was observed and thus can be used for treatment of gases with
high levels of methane, such as landfill gases and animal stall exhausts.
6.3 Nitrous oxide mitigation
N
2
O is a strong and persistent greenhouse gas (GHG) having a 298-times greater
global warming potential than CO
2
and the ability to deplete the stratospheric ozone
layer (Yoro &Daramola, 2020). The extensive use of mineral nitrogen in agriculture
and the continued use of legumes as cover and major crops, they release nitrogen at
the end of their life cycles, agriculture is responsible for around 60% of the world’s
N
2
O output (Avnery et al., 2011;Davidson, 2009).
One way to mitigate is using dissolved N
2
O to oxidize biogas methane by
releasing it into the gas phase and cooxidizing it with oxygen. In reality and in prac-
tice, feed scheme optimization, process optimization, and aeration control are the
key mitigating strategies (Duan et al., 2021).
6.4 Microbes as a sustainable fuel
Fossil fuels act as the major energy source but due to which there is more amount of
greenhouse gas released that contributes to climate change. Carbon dioxide is used
as an energy source by photosynthetic organisms, such as cyanobacteria and micro-
algae, to produce biofuels (Nozzi, Oliver, &Shota, 2013). The fastest-growing in-
dustry, transportation, consumes around 27% of primary energy. Therefore, the
transportation sector can be considered as a promising target for reduction in green-
house gas emissions. On an industrial scale, bioethanol and biodiesel are used.
Today, the usage of bio-based alcohols as solvents or fundamental chemicals is
once more being discussed due to growing crude oil costs and a rise in political un-
rest in countries that produce oil. Through diversification, reduced reliance on a
small number of key energy sources, generation of CO
2
-neutral energy, and the
retention of excess gross domestic product and economic power within the nation,
political independence is supported by the production of chemicals and fuels from
locally cultivated plant material (Antoni et al., 2007).
7.Conclusion
Microbes play a very important role as they create the majority of the gases that ac-
count for 98% of global warming, which plays a crucial role in climate change. Due
to the diverse microorganisms present in both marine and terrestrial environments,
the effect of each organism on climate change is different and microbes interact with
52 CHAPTER 3 Microorganisms as salient tools in achieving

each other in order to give a cumulative result. Microbes even though play a role in
contributing to the emissions of gases leading to warming of atmosphere, they
sequester carbon, using them for sustainable fuel, mitigate the emissions of nitrous
oxide and methane which are released mainly due to anthropogenic factors.
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Role of rhizobacterial
volatile compounds in
increasing plant tolerance
to biotic and abiotic
stresses
4
Devendra Kumar
1
, Archana T. S.
1
, Vipul Kumar
1
, Shivam Singh
2
, Kartik Sawant
1
,
Rafakat Hussain
3
, Gagan Kumar
4
1
Department of Plant Pathology, School of Agriculture, Lovely Professional University, Phagwara,
Punjab, India;
2
Krishi Vigyan Kendra-Baghpat, S.V.P University of Agriculture and Technology,
Meerut, Uttar Pradesh, India;
3
Department of Plant Science, College of Plant Science and
Technology, Huazhong Agricultural University, Wuhan, China;
4
Krishi Vigyan Kendra,
Narkatiaganj, Dr. Rajendra Prasad Central Agricultural University, Samastipur, Bihar, India
1.Introduction
A significant number of microorganisms, including bacteria, viruses, and fungi, have
been found in soil; only a small number of them are harmful to plants while the ma-
jority of soil-dwelling or soil-invading microorganisms are helpful to crops. Rhizo-
bacteria are helpful microorganisms that are found in the endosphere of plants as
well as in the phyllosphere and rhizosphere of their roots. The term “beneficial rhi-
zobacteria” refers to a number of species of bacteria from the rhizobacterial majority
that are known to encourage plant growth. Using beneficial rhizobacteria can
encourage plant growth in a number of different ways like direct growth-
promoting techniques such as nitrogen (N) fixation, phytohormones synthesis, and
phosphorus solubilization.
Rhizobacteria are essential for improving soil health, bioremediation, and stress
management in both biotic and abiotic environments. There were a number of indi-
rect mechanisms at work during root colonization, such as biocontrol activity, the
expression of stress-relieving genes, the production of secondary metabolites, and
the emission of volatile organic compounds by rhizobacteria. Several potential spe-
cies, such as Bacillus spp., several Pseudomonas,Enterobacter spp., Lysobacter
spp., Serratia, and Burkholderia spp., have proven superior plant growth promoters
and rhizobacteria. They have been genetically developed to live in the soil beneficial
rhizobacteria.
Many plants respond to volatile organic compounds (VOCs) by activating host de-
fense mechanisms and effectively promoting plant growth. Low-molecular-weight
CHAPTER
61
Rhizobiome. https://doi.org/10.1016/B978-0-443-16030-1.00002-X
Copyright ©2023 Elsevier Inc. All rights reserved.

chemicals from the rhizosphere, which surround microorganisms that colonize roots,
release specific VOCs. With the aid of gas chromatographic analysis, numerous low
molecular weight chemicals, such as the growth-promoting volatile 2R, 3R-
butanediol, have been discovered from complicated bacterial emissions of various
prominent bacterial strains (Ryu et al., 2004). Later, it was discovered that the rate
at which P. c h l o r o ra p h i s O6 elicits systemically induced resistance against E.caroto-
vora in tobacco is exactly proportional to the generation of 2R,3R-butanediol (Han
et al., 2006). Acetoin (3-hydroxy-2 butanone), generated by several Bacillus subtilis,
as well as Bacillus amyloliquefaciens, and some strains of Streptomyces, is another
volatile made by rhizobacteria (Farag et al., 2006).
In addition to manage the spread of pathogens in the leaves, they also safeguard
the plant by lessening the severity of disease. However, due to their low potency and
evaporative nature in comparison to other chemical pesticides, they face some sig-
nificant obstacles when applied in the field. Nevertheless, several volatile chemicals
have been used successfully in the field to prevent plant disease.
In-depth research is currently being done on signaling pathways to understand
how microbial volatiles affect a variety of cellular processes, such as plant growth
and development, pathogen-mediated defense, and several biotic and abiotic stress
adaptation (Cho et al., 2008).
Rhizobacteria from many genera reside in a plant’s rhizosphere. Several species
of bacteria from the rhizobacterial majority are known to promote plant growth and
are referred to be “beneficial rhizobacteria.” The adoption of advantageous rhizobac-
teria can promote plant growth in a variety of ways.
Growth stimulation should be accomplished directly through processes such as
various nutrient cycling as well as solubilization, plant hormone synthesis, and
bioactive material modulation. Additionally, various indirect mechanisms have
been identified during root colonization, including biocontrol activities, the regula-
tion of stress-relieving genes, the creation of secondary metabolites, and the produc-
tion of volatile organic compounds by advantageous rhizobacteria. By genetically
advancing the helpful rhizobacteria that live in soil, a number of promising species,
including several species of Bacillus, various Pseudomonas,Enterobacter spp.,
Lysobacter spp., Serratia spp., and Burkholderia, have demonstrated remarkable
growth promotion and plant defense features toward sustainable agriculture. Bacilli
are thought to be the main forces behind the various biostimulation and several
biocontrol mechanisms that promote plant growth though antibiosis, competition,
parasitism, and induced resistance. Therefore, advantageous rhizobacteria have
the natural potential to maintain plant nourishment.
2.Volatile organic compounds
Volatile organic compounds (VOCs) are naturally occurring substances in the atmo-
sphere that have an impact on secondary organic aerosol generation and tropo-
spheric ozone production. These effects have a global impact on climate, human
62 CHAPTER 4 Rhizobacterial volatile compounds and tolerance

health, and other biological processes (Andreae &Crutzen, 1997). Species from all
spheres of life contribute about 90% of the total amount of atmospheric VOCs
(Audrain et al., 2015).
Biogenic VOCs primarily serve as enemy or ally signals for inter- and intraspe-
cies communications across kingdoms, among many other activities (Davis et al.,
2013). Despite their long-known existence, it has only been in the last several de-
cades that their impact on ecology at the subcellular, organismal, and population
levels has received significant attention (Zhang &Ryu, 2013).
A recurring theme in many papers that are producing ground-breaking new in-
sights into the chemical variety and function of VOCs is the continual influx of
new findings based on metabolite profiling and functional analysis. According to
a recently published comprehensive database that draws data from papers that
have been published, PGPRs and other microbial species as a whole emit about
1000 VOCs (Lemfack et al., 2014). The database’s VOCs are divided into various
key elemental chemical classes, allowing users to browse and search for a particular
VOC based on either class or chemical name, species of origin, activities, and spe-
cific chemical formula. Numerous studies now unequivocally demonstrate that
VOCs form the basis of both advantageous and harmful plantemicrobe interactions
(Effmert et al., 2012).
Microbial VOCs have gained attention recently because to their possible appli-
cations in industry and agriculture as well as their involvement in airborne commu-
nications. Fascinating new discoveries in the field of VOCs in the not-too-distant
future given the prospect of the development and simple availability of various
analytical tools with greater compound separation capabilities combined with
powerful bioinformatics tools, as well as various genetic, genomics, proteomics,
metabolomics, and large-scale functional phenomics tools.
These studies should not only clarify the role of VOCs but also offer fresh ideas
for the creation of agrochemicals that are environmentally friendly, cost-effective,
and safe for human health and the environment. In-depth discussions and inventories
of VOCs produced by plants, animals, and a variety of microorganisms have recently
been published in several publications (Davis et al., 2013)(Fig. 4.1).
3.Volatile organic compounds producing rhizobacteria
Numerous rhizobacterial species, both Gram-positive and Gram-negative, generate
VOCs (Farag et al., 2013). It makes sense that the majority of these research used
PGPRs with antibacterial activity that was fairly well described. The majority of
these investigations had first centered on the whole volatile blends created by these
bacteria’s antibacterial properties. However, numerous additional research have
used GCeMS to profile VOCs. Various species of Bacillus such as B. subtilis,
B. amyloliquefaciens,B. cereus,Paenibacillus spp., Pseudomonas spp., Burkholde-
ria ambifaria,Streptomyces spp., Anthrobacter, and Serratia spp. are some of the
major PGPRs for which VOCs have been characterized (Table 4.1).
3. Volatile organic compounds producing rhizobacteria 63

Some strains of plant pathogens, such as Xanthomonas campestris,Pectobacte-
rium carotovorum,Pseudomonas phaseolicola,Pseudomonas syringae, and Agro-
bacterium tumefaciens, have been reported to emit volatile compounds in addition
to PGPR. Vesicatoria have been shown to release VOCs that have antifungal prop-
erties that are harmful to plant fungus (Kai et al., 2009). Numerous VOCs released
by PGPRs are likewise released by plants when they are harmed by herbivores (Mat-
sui, 2006). Some of these VOCs, like 2,3-butanediol, hexenal, and 1-octene-3-ol, are
also produced by fungi that help plants grow, indicating that they might have an old
evolutionary history and broad cytotoxicity (Kishimoto et al., 2007).
4.Different type of volatile organic compound
Numerous investigations have noted PGPR-related antibacterial and plant growth-
promoting actions using the split-plate or double-plate approach. Collectively, these
investigations have shown that the majority of PGPRs create a mixture of chemically
different molecules in various ratios. In general, simple hydrocarbons such alcohols,
ketones, aldehydes, substances containing sulfur, alkanes, alkenes, ethers, and
organic acids make up the VOCs of PGPRs.
Additionally, a number of other derivative substances that belong to various
chemical categories have also been found in PGPRs. But for a number of reasons,
it is believed that this diversity is vastly underestimated. First, factors that affect
growth, such as media type, temperature, length of growth, and aeration, have a sig-
nificant impact on how microorganisms metabolise food (Farag et al., 2013). Since
FIGURE 4.1
Volatile organic compounds against biotic and abiotic stress.
64 CHAPTER 4 Rhizobacterial volatile compounds and tolerance

Table 4.1 List of microbial volatile compounds with target pathogenic microbes.
Bacteria Compound Targeted pathogen Targeted cite References
Pseudomonas P2
strain
Dimethyl disulfide, dimethyl
trisulfide
Rhizoctonia solani Inhibit mycelial
growth
Elkahoui et al. (2014)
Streptomyces
alboflavus
2-methylisoborneol Fusarium moniliforme,Aspergillus
flavus, A. ochraceus, A. niger and
Penicillium citrinum
Inhibit sporulation
and mycelial growth
Wang et al. (2013)
B. subtilis B2g,
B. cepacia 1S18,
P. fluorescens
L13-6-12
Panagrellus redivivus,
Bursaphelencus xylophilus
Inhibit growth Vespermann et al.
(2007) and Tarkka &
Piechulla (2007)
Streptomyces griseus Carvacrol, dimethyl, sulfoxide
(DMSO), cyclohexanol,
naphthalene
Penicillium chrysogenum and
Botrytis cinerea
Inhibited spore
germination and
mycelium growth
Danaei et al. (2014)
Bacillus subtilis A. alternata,
C. oxysporum,
F oxysporum,
P. lilacinus,
P. variotii, and P. afertile
Deformation of
mycelial, hyphal, and
conidial structures
Chaurasiaa et al.
(2014)
Saccharomyces
cerevisiae
Six VOC including ethanol
and ester compounds
Sclerotinia sclerotiorum Inhibited mycelial
growth
Fialho et al. (2011)
Pseudomonas
fluorescens and
Serratia plymuthica
Volatile compound Agrobacteriumtumefaciens and
A. vitis
Bacteriostatic effects Dandurishvili et al.
(2011)
Lysobacter
enzymogenes
ISE13
2,4-Di- tertbutylphenol Colletotrichumacutatum,
Phytophthora capsici
Inhibited spore
germination and
mycelium growth
Sang and Kim (2011)
Continued
4. Different type of volatile organic compound 65

Table 4.1 List of microbial volatile compounds with target pathogenic microbes.dcont’d
Bacteria Compound Targeted pathogen Targeted cite References
Pseudomonas
chlororaphis,
P. corrugate
Cyclohexanol,
2-ethyl-1-hexanol
Sclerotinia sclerotiorum Inhibition of mycelial
growth
Fernando et al.
(2005)
Bacterial and fungal volatile-mediated-induced systemic resistance in plants
Mushroom 1-octen-3-ol allo-ocimene
and a C-6 aldehyde
Botrytis cinerea Arabidopsis thaliana Kishimoto et al.
(2007)
66 CHAPTER 4 Rhizobacterial volatile compounds and tolerance

analyses in the majority of studies conducted to date are typically constrained to a
small number of highly artificial growth conditions, such as synthetic media, growth
temperature, and lighting, the spectrum of VOC obtained under these conditions
may differ from that obtained under natural conditions. Second, present sampling
and detection techniques are insufficient for capturing and resolving the entire spec-
trum of VOCs.
The use of a variety of complementary analytical methods could overcome these
technical constraints. Finally, PGPRs that can be cultivated on synthetic media have
only been included in VOC profiling to this point. Many novel chemicals still need to
be found because the diversity of bacteria in soil is many orders of magnitude greater
than what has been studied thus far. Conditions as close to natural as feasible would
be needed to tap into these PGPRs. Inorganic profiling is typically limited to a few
well-known chemicals, such as HCN, [NH4], and H
2
S, in addition to organic vola-
tiles (Farag et al., 2013). These substances have been found in numerous PGPRs, and
their function is related to either plant growth promotion or general growth inhibi-
tion (Audrain et al., 2015).
5.Phytopathogens targeted by PGPR VOCs
VOCs are likely to have an effect on ecological systems both above and below
ground because of how easily they can diffuse through soil and air in their gaseous
state. This idea is supported by the fact that VOCs are known to prevent both foliar
and soil-borne diseases. Their activity has been documented against bacteria, oomy-
cetes, such as Phytophthora infestans and several Pythium species, and true fungi,
such as Alternaria spp., Botrytis spp., Colletotrichum spp., Fusarium oxysporum,
Sclerotinia rolfsii,Verticillium spp., and Rhizoctonia solani. Similar to this, some
volatiles, particularly dimethyl disulfide (DMDS), which contains sulfur, inhibits
multiple life stages of different phytopathogenic nematodes like Meloidogyne
incognita.
Some VOCs also exhibit herbicidal and insecticidal properties in addition to their
ability to kill bacteria, fungi, oomycetes, and nematodes (Coosemans, 2005). In
many fungal infections, VOCs have been found to impair mycelial development
and spore germination at the anatomical level. In some instances, it has been docu-
mented that VOCs have an impact on additional epidemiological parameters, such as
spore generation (Popova et al., 2014). Researchers are unable to make broad gen-
eralizations about the specificity of VOCs produced with the data at hand. These
kinds of investigations are crucial since great specificity may be required in some
circumstances, such as when managing only pathogenic fungi and excluding host
plants and their beneficial mycorrhizae.
In other circumstances, it would be advantageous to have a blend of VOCs with
broad-spectrum activity against weeds and soil-borne plant diseases for preplanting
therapy in a way quite similar to methyl bromide. Numerous research that examined
how various PGPRs affected various pathogens imply that target pathogens exhibit
5. Phytopathogens targeted by PGPR VOCs 67

variation in their response to VOCs; however, they do not explicitly address this sub-
ject. According to a summary of the findings from these research, some fungal spe-
cies are severely inhibited by VOCs from various PGPRs, but not always (Hunziker
et al., 2015).
VOCs from the same PGPR species sometimes inhibited one fungus but not
another, while in other instances, VOCs from distinct PGPR species more power-
fully inhibited the same fungi. Recent research with over 32 PGPR strains from
various rhizobacterial genera revealed that P. infestans responded much more
strongly to VOCs from specific Pseudomonas spp. than that of fungi like
R. solani as well as Fusarium oxysporum. Similar variations can be found in Pseu-
domonas species, some of which exhibit little activity against P. infestans. HCN pro-
duction and other compounds, particularly ketones like 1-undecene, were found at
high concentrations in the headspace of antioomycete strains. It is unknown whether
any of the known VOCs display specificity, while combinations of VOCs from
different PGPRs (Hunziker et al., 2015).
DMDS, 1-octen-3-ol, benzothiazole, 1-undecene, several ketones, and citro-
nellol are just a few of the prominently detected VOCs that exhibit broad-
spectrum activity against species from various kingdoms, including prokaryotes,
fungi, oomycetes, algae, insects, nematodes, and plants (Zhao et al., 2011). It is
crucial to evaluate numerous VOCs both singly and in combination, ideally in the
same experimental setting, utilizing a battery of target species from various nema-
todes, bacteria, fungi, and bacteria.
6.Antibiosis of volatile organic compound
Antibiosis is defined as the reversible or irreversible suppression of a live organism
by various specific or nonspecific metabolites which have been produced by a
PGPR. The great majority of PGPR-produced VOCs have been investigated for their
antibacterial activity against bacteria, fungus, nematodes, insects, and occasionally
against plants and algae. Numerous PGPRs and other phytopathogens have been
documented to exhibit antibiosis brought on by total VOCs. Similar to that,
numerous individual VOCs have also been reported to be active. The sorts of
VOCs that exhibit antibiosis roughly correspond to those that were found in the
PGPRs’ headspace.
They generally consist of molecules containing sulfur as well as alcohols, ke-
tones, aldehydes, alkanes, alkenes, and aldehydes. Some substances display antibi-
osis and ISR activity, indicating that they have a multifaceted mechanism of action
mentioned below.
7.Alcohol compounds
Alcohols of various sorts were found in the PGPRs’ headspace. Alcohols as a group
showed broad-spectrum activity against fungi and bacteria. One of the most
68 CHAPTER 4 Rhizobacterial volatile compounds and tolerance

prevalent alcohols in a variety of PGPRs was 2,3-butanediol, which has also under-
gone substantial in vitro analysis. The enzyme acetoin reductase/2,3-butanediol de-
hydrogenase converts acetoin into 2,3-butanediol in yeast and various bacterial
species, including B. subtilis (Xiao &Xu, 2007).
According to Zhang et al. (2014), this reaction is reversible with alkaline condi-
tions favoring oxidation (2,3-butanediol to acetoin) and acidic or neutral conditions
favoring reductase activity (acetoin to 2,3-butanediol to 2,3-butanediol to 2,3-
butanediol to 2,3-butanediol to activity). Since acetoin exhibits antibiosis as well,
this could broaden the bioactivity zones’ pH range, which varies greatly in agricul-
tural soils. Antibiosis is defined as the reversible or irreversible suppression of a live
organism by specific or nonspecific metabolites produced by a PGPR for the pur-
poses of the discussion that follows.
The great majority of PGPR-produced VOCs have been investigated for their
antibacterial activity against bacteria, fungi, nematodes, insects, and occasionally
against plants and algae. Numerous PGPRs and other phytopathogens have been
documented to exhibit antibiosis brought on by total VOCs. Similar to that,
numerous individual VOCs have also been reported to be active. The sorts of
VOCs that exhibit antibiosis roughly correspond to those that were found in the
PGPR’s headspace. They generally consist of molecules containing sulfur as well
as alcohols, ketones, aldehydes, alkanes, alkenes, and aldehydes. Some substances
display antibiosis and ISR activity, indicating that they have multifaceted mecha-
nisms of action.
8.Ketone and aldehyde compounds
When numerous different PGPRs are cultivated in vitro, many ketones are
frequently found in the headspace of those cells. Both the SPME and the CLSA pro-
cedures are used to extract them (Popova et al., 2014). They have similar antidiverse
fungal and bacterial plant pathogen activity to alcohols. Acetoin stands out among
the ketones because it not only exhibits antibiosis but is also known to cause ISR.
It is unknown if other ketones that exhibit antibiosis also exhibit ISR action.
A-acetolactate decarboxylase and diacetyl-acetoin reductase can both be used to
make acetoin from a-acetolactate or from diacetyl (Xiao &Xu, 2007). A-
acetolactate also yields another ketone, 2,3-butanedione, indicating that several ke-
tones may be synthesized in concert to broaden their range of activity. The
sequenced genomes of various PGPRs contain genes that code for enzymes in the
biosynthetic pathways of several additional ketones in addition to the well-studied
acetoin biosynthesis genes found in Bacillus species These genes can be cloned
and further used to manipulate the biosynthesis of their respective ketones for the
best generation and improvement of PGPR bioactivity.
8. Ketone and aldehyde compounds 69

9.Alkanes and alkenes compounds
A variety of PGPRs emit a number of long-chain hydrocarbons. Tridecane and dime-
thylhexadecylamine (DMHDA) are arguably the two most well-known compounds
that have been demonstrated to exhibit antimicrobial action against fungi and bac-
teria. Different Pseudomonas spp. were also found to produce a number of alkenes
in various ratios. Due to their ability to suppress the growth of numerous fungi,
several bacteria, different cyanobacterium species, several genera of nematodes,
and Drosophila at comparable micromolar concentrations, antimicrobial investiga-
tions revealed that these substances are general inhibitors (Popova et al., 2014).
Some of these substances demonstrated more potent activity against one type of or-
ganism than another, indicating a degree of selectivity. For instance, 1-undecene
strongly inhibited P. infestans (Hunziker et al., 2015), Caenorhabditis elegans,
and Drosophila, but not R. solani and Agrobacterium, suggesting that it may have
effect against oomycetes, nematodes, and insects (Popova et al., 2014). One of
the most notable VOCs with anti-Phytophthora action was 1-undecene, which was
discovered via a thorough screening of rhizosphere and phyllosphere PGPRs. It sup-
pressed the growth of P. infestans’ mycelium, the generation of sporangia, and the
release of zoospores with various dose-dependent ways. It is currently unknown
how 1-undecene employs its broad-spectrum activity in contradiction of diverse
P. infestans life stages on a molecular level; solving this problem would be highly
helpful for the development of this molecule for use in agriculture.
10.Sulfur compounds
Many sulfur-containing chemicals, particularly DMDS, dimethyl trisulfide, and
dimethyl sulfide, as well as benzothiazole to a lesser extent, are regularly found in
the GC-profiles of VOCs of a wide range of various PGPRs. They exhibit wide-
spread antibiosis against bacterial and fungal pathogens together. Similar to the ma-
jority of other VOCs, the precise mechanism of action of sulfur-containing VOCs is
unknown; nevertheless, it has been shown that DMDS interferes with the operation
of mitochondria and potassium channels in insects (Dugravot et al., 2003). Similar to
this, the production mechanisms for these sulfur compounds in PGPRs are unknown,
but based on research on lactobacillus species and truffles, they may involve the
pathways for the catabolism of methionine.
Methanethiol, which is produced by the enzyme L-methioninase from the amino
acid L-methionine, or 4-methylthio-2-oxobutanoic acid can be demethiolated to pro-
duce them spontaneously (Liu et al., 2013). Immobilized fungal L-methioninase can
be used to increase the generation of methanethiol, hence offering a more effective
synthesis platform. A generalization concerning the antibiosis nature of VOCs
cannot be made because there has not been many research sought to determine if
VOCs are biostatic or biocide in their mode of action.
70 CHAPTER 4 Rhizobacterial volatile compounds and tolerance

Serratia plymuthica strain IC1270 and Pseudomonas chlororaphis 205 and Pseu-
domonas fluorescens B-4117 were reported to have bacteriostatic action (Popova
et al., 2014). All Pseudomonas and Serratia strains examined acted as biocides
against cyanobacteria, but only S. plymuthica strain IC1270s VOCs killed larvae
of a worm. The three fungi R. solani, Helminthsporium sativum, and
S. sclerotiorum were resistant to the activity of both Pseudomonas sp. and Serratia
sp. VOCs since they resumed growth after being exposed to no-VOC conditions.
However, activities of total VOCs produced by four different Pseudomonas species,
P. chlororaphis, P. fluorescens, P. corrugata, and P. aurantiaca, against
S. sclerotiorum were fungicidal in another study because transfer of mycelial plugs
or sclerotia from the VOC exposed plate to fresh plate did not resume growth (Fer-
nando et al., 2005). In this investigation, Pseudomonas sp.-produced benzothiazole,
cyclohexanol, n-decanal, dimethyl trisulfide, 2-ethyl 1-hexanol, and nonanal signif-
icantly inhibited S. sclerotiorum, although individual compounds’ fungicidal or
fungistatic efficacy was not examined.
To find out whether the most potent volatiles revealed in these investigations are
biostatic or biocidal, it would be very fascinating to undertake a thorough investiga-
tion against a battery of various fungal, bacterial, and nematode species. This type of
research will also address the question of individual VOC specificity, which has
either not been fully explored or has produced rather inconsistent results. Numerous
diverse beneficial creatures interact with one another in their natural habitat. It is
conceivable that VOCs could be involved in these interactions, and as a result,
VOCs produced by helpful PGPRs could be harmful to other PGPRs or helpful or-
ganisms like mycorrhizae.
Future research should examine the effects of VOCs on beneficial organisms
prior to their development and use in disease-management programs because several
VOCs exhibit general inhibition against a variety of organisms, including those that
are helpful like Trichoderma spp. (Hernandez-Leon et al., 2015).
11.Volatile organic compound against abiotic stress
11.1 Increased salt tolerance
For plant cells, too much sodium (Na
þ
) causes ionic and osmotic stressors that
restrict plant growth and lower agricultural yields. Several PGPR strains have
been found to produce salt tolerance in plants, including Bacillus amyloliquefaciens
GB03 (formerly known as Bacillus subtilis GB03) displaying VOC-mediated ef-
fects. Related to salt-stressed plants without VOC treatment, Arabidopsis plants
treated with GB03 VOCs produced more biomass and accumulated less Na
þ
(Zhang
et al., 2008).
The fact that wild-type (WT) plants exhibited this VOC-induced stress tolerance
but not the hkt1 null mutant suggests that HKT1 plays a critical role in mediating the
salt stress tolerance caused by GB03 VOCs. Na
þ
is excluded from leaves by
11. Volatile organic compound against abiotic stress 71

Arabidopsis HKT1, a Na
þ
transporter that is expressed in the xylem parenchyma.
Because GB03 VOCs transcriptionally upregulate HKT1 in shoots and simulta-
neously downregulate HKT1 in roots, they are thought to increase HKT1-
dependent shoot-to-root Na
þ
recirculation, which in turn reduces Na
þ
buildup in
Arabidopsis shoots under salinity stress (Zhang et al., 2008).
Although the exact mechanism by which GB03 VOCs control HKT1 transcrip-
tion is yet unknown, the organ-specific patterns seem to be essential for auxin-
mediated growth promotion as well as VOC-induced salt tolerance. Na
þ
levels
were reduced by around 50% overall by GB03 VOCs in WT Arabidopsis plants,
indicating either decreased Na
þ
absorption, increased Na
þ
exudation, or both. It’s
interesting to note that GB03 only decreased 15% plant Na
þ
levels in the Arabidop-
sis sos3 mutant. The H
þ
/Na
þ
antiporter SOS1, which regulates root Na
þ
exudation
and long-distance Na
þ
transport in plants, needs SOS3 to be posttranscriptionally
activated (Shi et al., 2000).
As a result, the decreased buildup of Na
þ
in VOC-treated plants is probably due
to SOS3-dependent Na
þ
exudation, which is a component of the integrated regula-
tion of Na
þ
homeostasis. Additionally, according to GB03 VOCs also induce the
rhizosphere to become acidic, creating a proton gradient that may help the roots’
ability to export Na
þ
through the action of SOS1.
Plants change their endogenous metabolism in response to salinity to deal with
the osmotic stress brought on by the excessive buildup of Na
þ
. Recently, it was
discovered that Pseudomonas simiae strain AU volatile emissions boosted the accu-
mulation of proline, which protects cells from osmotic stress and decreased root Na
þ
levels, leading to the development of PGPR-induced salt tolerance in soybean plants.
Plants treated with AU VOCs displayed greater levels of the vegetative storage pro-
tein (VSP) and numerous other proteins that are known to support plant growth un-
der stress conditions, which is consistent with the development of induced systemic
tolerance under salt (Vaishnav et al., 2015).
12.Defense against water loss
Dehydration is a common risk for plants that are experiencing osmotic stress from
salinity, dryness, or cold. Plants under dehydration stress may accumulate more
osmoprotectants, which can improve cellular osmotic pressure by lowering the
free water potential of cells and preventing water loss as well as stabilizing proteins
and membrane structures. Arabidopsis exposed to GB03 VOCs accumulated more
choline and glycine betaine under osmotic stress than plants not treated with
VOCs. Important osmoprotectants that give plants dehydration resistance include
choline and glycine betaine (Rhodes &Hanson, 1993).
Plants treated with GB03 VOCs or inoculated with GB03 showed improved
resistance to dehydration stress, which was consistent with the elevated osmoprotec-
tant levels. Because VOC treatment increased the level of PEAMT transcripts and
because genetic dysfunction of PEAMT prevented VOC induction of dehydration
72 CHAPTER 4 Rhizobacterial volatile compounds and tolerance

tolerance, it was hypothesized that PEAMT, an essential enzyme in the biosynthesis
pathway of choline and glycine betaine, played a significant role in mediating VOC-
induced plant tolerance to dehydration (Zhang et al., 2010). 2,3-Butanediol, which is
included in GB03 VOCs encourages plant development and increased disease resis-
tance (Ryu et al., 2004).
2,3-Butanediol can also be identified in the VOCs of some other PGPR strains,
such as Pseudomonas chlororaphis strain O6, a bacterium that can cause induced
systemic resistance in plants, in addition to being present in GB03 VOCs. Increased
stress tolerance in Arabidopsis plants treated to 2,3-butanediol or P. chlororaphis O6
under drought conditions was definitely caused by increased stomatal closure and
decreased water loss (Cho et al., 2008). Applying P. chlororaphis O6 or 2,3-
butanediol to mutants with defects in different hormone signaling pathways revealed
that the induced drought tolerance is controlled by a number of traditional hormones,
including ethylene, salicylic acid, and jasmonic acid. Additionally, free SA levels
dramatically increased in plants exposed to 2,3-butanediol or P.chlororaphis O6 sug-
gesting that SA plays a major role in the induction of drought resistance (Cho et al.,
2008).
In a follow-up investigation, it was discovered that 2,3-butanediol increased
plant synthesis of nitric oxide and hydrogen peroxide, while chemically altering
NO accumulation decreased it. These findings showed a crucial function for NO
signaling in the drought tolerance caused by 2,3-butanediol. 2,3-Butanediol stimu-
lated plant survival under drought stress (Cho et al., 2013). Under situations of dehy-
dration, the phytohormone abscisic acid (ABA) is known to regulate plant stress
responses. But given that osmotic stress increased ABA to comparable levels in
plants exposed to GB03 VOCs and those that weren’t, it suggests that the improved
osmoprotection of GB03 VOC-treated plants is unrelated to ABA, or at least to ABA
production (Zhang et al., 2014). Further evidence that ABA is not the cause of
PGPR-induced plant drought tolerance comes from findings that Arabidopsis and
cucumber plants treated with PGPR accumulated less ABA than control plants
(Cho et al., 2008). However, considering the intricate interplay between ABA,
NO, SA, and hydrogen peroxide signaling pathways in plants, an indirect role for
ABA in such PGPR-triggered abiotic stress tolerance cannot be entirely ruled out
(Denance
´et al., 2013). Increased antioxidant responses at the levels of enzyme ac-
tivity and metabolite accumulation can also contribute to PGPR-induced drought
tolerance, as was seen in wheat infected with Bacillus safensis strain W10 and
Ochrobactrum pseudogrignonense strain IP8 (Chakraborty et al., 2013). In potato
plants treated with PGPR, higher proline accumulation and ROS-scavenging
enzyme gene expression were seen. These plants also showed increased tolerance
to a variety of abiotic stimuli, such as salt, drought, and heavy metal toxicity (Guru-
rani et al., 2013). It would be beneficial to ascertain whether volatile emissions sup-
port plants under dehydration stress’s antioxidative mechanisms.
Exopolysaccharides produced by some PGPR strains, including Pseudomonas
aeruginosa strain Pa2, help the bacteria regulate soil moisture levels and increase
plants’ tolerance to drought (Naseem &Bano, 2014). Acetic acid is one bacterial
12. Defense against water loss 73

VOC that can cause the development of biofilms, which are primarily composed of
exopolysaccharides (Chen et al., 2015). Since exopolysaccharide synthesis is medi-
ated by several PGPR VOCs, it is conceivable that these compounds may indirectly
boost plant drought tolerance.
13.Enhancement of sulfur acquisition
The macronutrient sulfur (S), which is a component of numerous primary metabo-
lites including the amino acids cysteine as well as methionine, is crucial for plant
survival. Plants that are lacking in S experience suppressed photosynthesis and a
basic metabolism that is disrupted (Burke et al., 1986). Plants can assimilate S
from S-containing molecules in the air, including some volatile chemicals that are
released by soil microorganisms; however, plants primarily obtain S by root uptake
of SO
4
2 from soil, dimethyl disulfide (DMDS), volatile S-containing chemical that
is frequently generated by several bacteria and fungi in soil (Kanchiswamy et al.,
2015). Nicotiana attenuata plants’ natural symbiont Bacillus sp. strain B55, which
emits DMDS, prevented plant growth retardation brought on by S deficiency (Mel-
dau et al., 2013). By including radiolabeled 35S to the bacterial growth media, it was
possible to show that bacteria-emitted S was incorporated into plant proteins. In
addition to finding DMDS in several Bacillus sp. B55 VOCs, for two reasons, the
scientists chose to credit DMDS rather than S-methyl pentanethioate for producing
the majority of the S nutrition produced by Bacillus sp. B55 VOCs. First, it was
discovered that S methyl pentanethioate was present only in trace amounts, whereas
DMDS was a significant component of the volatile emissions. Second, synthetic
DMDS was more effective than natural VOC blends at saving N. attenuata plants
with S-starvation phenotypes.
Because the sulfur in SO4 2 is oxidative, biological digestion of the sulfur neces-
sitates an energy-intensive reduction process (Takahashi et al., 2012). Sulfur is
chemically decreased in DMDS. Therefore, it seems that DMDS may aid plants
avoid using energy to reduce sulfate in addition to providing S. DMDS supplemen-
tation dramatically reduced the expression of S assimilation genes as well as methi-
onine biosynthesis and recycling, supporting this notion. B55 VOCs and other
S-containing volatile chemicals, like dimethyl sulfide and dimethyl trisulfide,
have been found in different microbial VOC blends in high concentrations
(Kanchiswamy et al., 2015).
14.Optimization of iron homeostasis
A redox potential is produced by the change from ferrous iron (Fe
2þ
) to ferric iron
(Fe
3þ
), which is crucial for electron transfer reactions like photosynthesis. Chlorosis
of the leaves and significant photochemical impairment result from Fe deficiency.
74 CHAPTER 4 Rhizobacterial volatile compounds and tolerance

While nongraminaceous monocots and dicots employ plasma membrane ferric
reductase to reduce Fe
3þ
and then transfer Fe
2þ
into roots, graminaceous monocots
and dicots create siderophores that improve Fe
3þ
mobility in soil and allow for direct
uptake of Fe
3þ
without reduction (Curie &Briat, 2003).
When exposed to GB03 VOCs, which don’t include any known siderophores,
Arabidopsis showed increased Fe uptake (Farag et al., 2006). In the presence of
Fe-sufficient growth conditions, plants exposed to GB03 VOCs exhibited typical
Fe deficiency responses, such as transcriptional upregulation of the root Fe
3þ
reduc-
tase gene FRO2 and the Fe
2þ
transporter gene IRT1, increases in FRO2 enzyme ac-
tivity, and acidification of the rhizosphere (Zhang et al., 2008).
As a result, plants exposed to VOCs had higher Fe levels, which is consistent
with the presence of more Fe-rich photosynthetic machinery. Because VOC was un-
able to induce IRT1 or FRO2 in the fit1 knockout mutant, IRT1 and FRO2 are
required for GB03 VOC-triggered gene induction. In the fit1 mutant, VOC treatment
did not improve iron absorption or photosynthesis.
It is still unclear how VOC-treated plants begin to exhibit inducible iron-deficient
reactions. One potential is that a need for extra iron may emerge from increased
photosynthesis and/or VOC-induced leaf cell growth. The identity of the compo-
nent(s) in GB03 VOCs that causes plant iron shortage reactions is also unknown.
On the other hand, the rhizosphere acidification that is directly brought on by
VOC exposure may be explained by acid components like diethyl acetic acid (Farag
et al., 2006).
Although PGPR VOCs have been demonstrated to benefit plants by directly pro-
moting growth, inducing resistance to biotic stress, and increasing tolerance to
abiotic stress (Bitas et al., 2013), the majority of the data pertaining to these positive
effects have been obtained in artificial environments. A central partition of Iplates,
which are commonly used in current investigations using PGPR VOCs, isolates
plants from bacteria while allowing bacterial VOCs to spread throughout the plate.
However, in natural conditions, roots would be the principal receptors of PGPR
VOCs that spread through rhizosphere soil pores. This experimental setup appears
to favor the sensing of volatile chemicals by leaves.
I-plate use raises another issue since, in addition to generating volatile chemicals,
soil microorganisms can secrete nonvolatile substances (Glick, 1999), which may be
ingested by plant roots and obstruct plant responses to VOCs. The fact that the same
PGPR strain of VOCs may affect plant development and stress tolerance differently
depending on the type of growing media and the bacterium’s population density adds
another layer of complexity to the research of PGPR VOCs’ effects on plants. A rise
in the bacterial population in the same area may change the VOC profiles, produce
more harmful substances or higher concentrations of already-present poisonous sub-
stances. Alternately, the VOC component that promotes plant development may
build up to such high levels that it has a negative impact on plant growth. For
instance, indole enhanced plant development at low concentrations but killed plants
at high concentrations (Blom et al., 2011). For plants treated with DMDS, both
growth promotion and growth inhibition have been noted. Beneficial PGPR VOCs
14. Optimization of iron homeostasis 75

may potentially turn inhibitory due to changes in the environmental factors that
affect plant growth. The FIT1 and IRT1 genes are activated by GB03 VOCs, which
improve plant iron absorption and photosynthesis (Zhang et al., 2009). IRT1 trans-
ports cadmium and other metal ions into roots in addition to carrying iron. There-
fore, it is conceivable that GB03 VOCs could make plants under cadmium stress
more poisonous to the metal.
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Bioremediation potential of
rhizosphere microbes—
current perspectives 5
Mehreen Shah, Sirajuddin Ahmed
Department of Environmental Science and Engineering, Jamia Millia Islamia (Central University),
New Delhi, Delhi, India
1.Introduction
Rhizoremediation is a form of bioremediation that explains the remediation that
takes place in the soil zone surrounding the root (Erickson et al., 1995) due to the
catalytic activity and the secretions by the rhizobacteria colonizing the root (Kuiper
et al., 2004). The symbiotic association of roots and bacteria helps in the biodegra-
dation of toxic compounds such as PHA (poyhydroxyl alkanoates). Siderophores,
phytohormones, and hydrogen cyanide produced by rhizobacteria help in the chela-
tion of heavy metals, breakdown of toxic compounds, and lysis of harmful patho-
gens (Roskova et al., 2022). Rhizoremediation takes place via various
mechanisms most commonly identified as immobilization and biosorption of the
pollutant (Gadd, 2000). Bacillus subtilis, Brevibacterium halotolerans, Bacillus
pumilus and Pseudomonas pseudoalcaligenes are able to affect rhizoremediation
of metal contaminated soil by accumulation of metal (Abou-Shanab et al., 2007).
Rhizoremediation is an attractive solution as the ability to be resistant to the pollut-
ants in the soil, such as anthracene or heavy metals, is conferred to the plant thus
providing great economic benefit to the agricultural sector. Cook &Hesterberg
(2013) have shown that rhizoremediation emerges as a popular way to deal with
the problem of remediation of PAH-contaminated soil. Rhizobacteria that are help-
ful usually belong to the genus of Bacillus, Sphingomonas, Mycobacterium, Pseudo-
monas,Rhodococcus, etc. (Bisht et al., 2015). Rhizoremediation is mainly
incorporated while we practice degradation, attenuation immobilization, and/or
detoxification of a plethora of chemical/toxic waste and physically hazardous con-
taminants from the surrounding site of pollution through the powerful action of the
microorganisms. The main principle is to degrade and convert or transform the pol-
lutants to lesser toxic forms. Rhizobacteria are very hardy and can survive in a
plethora of ranges such as highly saline conditions (Kushneria, Halomonas, and Ba-
cillus are halophyte rhizospheric microbes that confer saline-resistance to alfalfa
plants by inoculation of roots) (Kearl et al., 2019). These characteristic qualities
of microbes that are beneficial for bioremediation are their high adaptability and
their ability to utilize carbon as their food source.
CHAPTER
81
Rhizobiome. https://doi.org/10.1016/B978-0-443-16030-1.00019-5
Copyright ©2023 Elsevier Inc. All rights reserved.

2.Bioremediation
Bioremediation emerges as an attractive and viable tool as it employs the use of mi-
croorganisms to reduce the organic content of the garbage or contamination. These
microorganisms are much easier to reclaim and attenuate once their role to mitigate
the contamination has been fulfilled, to remove chemicals from the site of pollution,
hence bioremediation makes the job of municipal corporations much easier. Biore-
mediation is mainly incorporated while we practice degradation, attenuation immo-
bilization, and/or detoxification of a plethora of chemical/toxic waste and physically
hazardous contaminants from the surrounding site of pollution through the powerful
action of the microorganisms. The main principle is to degrade and convert or trans-
form the pollutants into lesser toxic forms (Verma &Jaiswal, 2016). The process of
bioremediation can be carried out in ex situ and in situ manner, depending on several
factors, which include the cost of remediation, characteristics of the contaminated
site, type, and concentration of hazard to be remediated. According to these param-
eters a bioremediation technique is then finally shortlisted. Various engineered meth-
odologies are employed which are a brainchild of technological engineering using
machines that are juxtaposed with biotechnology, incorporating microorganisms
whose potential to degrade pollutants is harnessed in various facilities that are engi-
neered to help these microorganisms degrade pollutants at their utmost optimum
peak and potential in a controlled facility.
2.1 Microorganisms used for bioremediation
Microorganisms are extremely small in size and those employed for bioremediation
by environmental scientists cannot be seen with the naked eye. They through their
beneficial action help restore the ecological balance. There is a conversion into
harmless byproducts from the precursor polluted medium with the help of bacteria,
yeast, fungi, or algae. Microbes are very hardy and can survive in a plethora of
ranges such as highly saline conditions; they can live and replicate in extremely
hot and cold temperatures as well as in the presence of hazardous compounds. These
characteristic qualities of microbes are beneficial for bioremediation namely their
high adaptability and their ability to utilize carbon as their food source. Bioremedi-
ation processes can be carried out by different species of microbes in different envi-
ronmental situations and conditions. The microorganisms used are Achromobacter,
Arthrobacter, Alcaligenes, Bacillus, Corynebacterium, Pseudomonas, Mycobacte-
rium, Xanthobacter, Flavobacterium, Nitrosomonas, etc.
The microbes which are used in bioremediation can be categorized as.
(1) Aerobic: aerobic bacteria degrade complex organic compounds in the presence
of oxygen such as Pseudomonas, Sphingomonas, Nocardia, Flavobacterium,
Acinetobacter, Rhodococcus, and Mycobacterium. These microbes can
degrade pesticides and insecticides, various hydrocarbons, alkanes, and poly-
aromatic compounds.
82 CHAPTER 5 Bioremediation potential of rhizosphere

(2) Anaerobic: anaerobic bacteria degrade pollutants in the absence of oxygen.
Anaerobic bacteria are used for the bioremediation of chlorinated aromatic
compounds, dechlorination of trichloroethylene and chloroform, poly-
chlorinated biphenyls, and other compounds. They generate methane gas as a
byproduct which can be then used as biofuel.
3.Techniques employed in bioremediation
Bioremediation techniques can be broadly classified into two categories based on the
mode of application, that is, ex situ and in situ. Nature of contaminants, depth and
amount of pollution, type of environment of pollutant generation, location, labora-
tory conditions, and facilities, are the selection criteria that are considered priorly
before selecting any bioremediation technique. Performance and success are based
on oxygen and nutrient concentrations, the physiochemical nature of the site, types
of pollutants, temperature, pH, and other factors that determine the efficiency of
bioremediation processes (El Fantroussi et al., 2005).
3.1 Rhizoremediation
Rhizoremediation is a subset of bioremediation that specifically focus on the reme-
diation effected in the soil zone surrounding the root (Erickson et al., 1995) due to
the catalytic activity and the secretions by the rhizobacteria colonizing the root
(Kuiper et al., 2004). The rhizosphere is a symbiotic association between the root
of the host plant and the rhizobacteria, and they secrete hydrolytic enzymes, bio-
surfactants, and chemicals that help in the effective breakdown of toxic compounds
in the soil (Anderson et al., 1993;Anderson &Coats, 1995). The xenobiotics are uti-
lized by the rhizobium for its substrate hence allowing the process of rhizoremedia-
tion to take place (Kamaludeen &Ramasamy, 2008). The very first case of
rhizoremediation to be studied was by Researchers of microbiology in 1994 which
have shown that rhizobium diversity is a function of the root exude of the plant, the
host plant species, and soil conditions.
Transposon-mediated ISMoB gene transfer is one mechanism by which rhizor-
emediation bacteria can be transferred via gene transfer hence allowing the decon-
tamination of polluted sites. Hong et al. (2011) showed that siderophores, organic
acids, and ACC deaminase synthesized by the rhizobacteria Gordonia sp. S2RP-
17 was effective in the remediation of sites contaminated by diesel spills by inocu-
lating seeds of Zea mays.Wu et al. (2006) showed that expression of a metal-binding
peptide (EC20) in the rhizobacteria named Pseudomonas putida 06909 helped in the
uptake of Cd and also reduced toxic effects on the host plant sunflower whose roots
were inoculated with the rhizobacteria. Alami et al. (2000) showed that nitrogen up-
take was improved when Rhizobium sp. was inoculated into the roots of the sun-
flower plant. Rhizoremediation takes place via various mechanisms most
commonly identified as immobilization and biosorption of the pollutant (Gadd,
3. Techniques employed in bioremediation 83

2000). Phytochelatins (PCs) and metallothioneins (MTs) help in the immobilization
of the contaminants. Through uptake by roots the pollutant especially a volatile-
organic pollutant may be then volatilized by the plant (phytovolatilization)
(Fig. 5.1)(Limmer &Burken, 2016).
3.2 Rhizoremediation of heavy metals
Some plants possess unique ranges of mechanisms that enable their resilience and
hardiness to grow in soil that is contaminated with a range of metals including heavy
metals. These plants possess microbes in their rhizosphere that help confer them
resistance to the harmful effects of the metals in the soil (Jiang et al., 2008). The
presence of heavy metals in soil can cause inhibition of microbial activity (Obbard
et al., 1994), and decline in microbial population (Abou-Shanab et al., 2005).
Several plant growth promoting rhizobacteria are found to colonize roots and also
free living in the soil that not only assist in the growth of plants but also accumulate
metals so plants are not affected by them. Barley was inoculated with rhizobacteria
to confer resistance from Pb and Cd (Belimov et al., 2004). Some plant species are
named hyperaccumulators as they grow in heavy metalecontaminated soil and sim-
ply accumulate the metals without suffering any adverse impact on their health
(Baker &Brooks, 1989). Rhizosphere supports the Agrostis tenuis for As uptake
significantly in contaminated soil through the aid of its rhizosphere microbiota.
Ni-contaminated soil showed plants that possessed rhizospheric association with
the bacteria Pseudomonas, Cupriavidus, and Bacillus (Pal et al., 2007). In Cu-
contaminated soil, Phragmites sp. were observed in rhizome association with the
FIGURE 5.1
A diagrammatic representation of rhizoremediation in the root sphere.
84 CHAPTER 5 Bioremediation potential of rhizosphere

tolerant rhizobacteria. The rhizobacteria exude exopolymers that help in the solubi-
lization of the Cu (Kunito et al., 2001). Bioremediation of heavy metals such as Pb
and Cu was achieved by Kumar &Fulekar (2018) by using grass species Cenchrus
ciliaris inoculated with rhizospheric bacteria and fungi. Siderophores secreted by
the rhizobium helps in mobilizing and solubilizing the metal from the soil and pre-
vent deleterious effect on the plant (Roskova et al., 2022). Phytochelators help in
chelating the metal from the soil solution and include phytochelatins, phytin, metal-
lothioneins, and low molecular weight organic acids.
Arbuscular mycorrhizal (AM) fungi are found in association with roots of plants
in heavily polluted soils, rampant with heavy metals which not only aided in plant
growth but affected metal uptake through phytoextraction (Wang et al., 2007).
Brevundimonas sp. KR013, Kluyvera ascorbata SUD165, Rhizobium leguminosa-
rum bv. trifolii NZP561, Pseudomonas fluorescens CR3, Brevundimonas sp.
KR013, etc.,are some PGPR that are useful for bioremediation (Zhuang et al.,
2007)(Table 5.1 and Fig. 5.2.)
4.Plant bacteria interactions in rhizoremediation
All rhizoremediation processes occur in the small space between the roots and the
surrounding soil. Three processes occur, firstly the seed is inoculated, that is, intro-
duced to the rhizomicrobes, then the rhizobacteria penetrate the root. Then by a se-
ries of catabolic processes, the pollutant is broken down (Kuiper et al., 2004).
4.1 Colonization of root
The bacteria enter into the system of the plants through mainly two mechanisms.
Firstly, the seed may be inoculated with the rhizobacteria. Secondly, the plant
may be simply exposed to the rhizobacteria. The root exudes chemicals called che-
moeffectors, which consist of hundreds of complex compounds that affect a chemo-
tactic movement and proliferate the bacteria into the root, leading to colonization
(Feng et al., 2021). The rhizosphere shows a gigantic capacity of carrying up to
10
11
microbial cells per gram of root (Egamberdieva et al., 2008).
4.2 Regulation of gene expression by roots
Bharti et al. (2016) elucidated through wheat grown in saline soil, which was inoc-
ulated with rhizobacteria, that conferred its resilience to halo-soil conditions with a
few observations. A gene that confers salinity resistance was expressed called TaST.
Certain pathways that were related to genes (SOS1 and SOS4) were modulated in
wheat shoots and root systems that were priorly inoculated with plant growth pro-
moting rhizobacteria.
4. Plant bacteria interactions in rhizoremediation 85

Table 5.1 Bioremediation of metals by rhizosphere along with the effect on plant.
Metal Plant associated Activity in the rhizosphere Results observed in plant References
TCE
and
heavy
metals
Helianthus annus Engineered rhizobacteria Pseudomonas
putida 06909 inoculated with metal binding
peptide EC20
Cd phytotoxicity declined by 40%
Increased accumulation of Cd in the
plant root
Wu et al.
(2006)
Cd Arabidopsis sinicus Recombinant Mesorhizobium
huakuii sub sp. Rengei inoculated with metal
binding peptide B3
Cd accumulation in nodules
increased by 1.5 fold
Sriprang
et al. (2003)
As Pteris vittata An increased amount of P as Mycorrhizae
increased transporters at hyphae level for As
uptake
Increase in shoot biomass and
accumulation of As
Al Agely et al.
(2005)
Cd Trifolium repens Brevibacillus sp. and AM fungus inoculated
together
Cd uptake increases by w36% Vivas et al.
(2003)
Cai et al.
(2004)
Ni Lycopersiconesculentum
Alyssum murale
ACC deaminase activity and siderophore
production
Ni is solubilized by rhizobacteria
Reduction in toxicity toward plant
species
Inside the shoot, Ni uptake was
seen by w31%
Burd et al.
(1998)
Abou-
Shanab R
et al. (2003)
Se Brassica juncea L Se is volatilized into harmless forms such as
dimethyl selenide
w34 of Se is now unavailable by
bioaccumulation and the plant is
unharmed
De Souza
et al. (1999)
Pb
Zn
Cu
Zea mays Rhizobacteria Brevibacterium halotolerans is
inoculated
Zn (4 g/kg), Pb(0.2 g/kg), and Cu
(2 g/kg) were found translocated in
shoots
Abou-
Shanab et al.
(2007)
86 CHAPTER 5 Bioremediation potential of rhizosphere

4.3 Signal exchange/communication in the rhizosphere
An effective exchange of signals between the rhizobacteria and the root of the host is
evident in the rhizosphere. Among the varying nature of the signal, they can be
exchanged about a stressor which may be abiotic (salinity, drought, etc.) or biotic
(such as a potential infestation). Quorum sensing (QS) signaling molecules help
in cell-to-cell signaling (Hong et al., 2011). These signals help direct the production
of certain enzymes during stress, conjugation, attack pathogens and suppress growth
temporarily, and aid the survival of the host plant (Newton &Fray, 2004). For Gram-
negative rhizobacteria, the primary QS molecules are N-Acyl Homoserine Lactones
(AHLs) (Ferluga et al., 2008)(Table 5.2).
5.Rhizoremediation of PET and PLA plastics
Polylactide (PLA) and polyethylene terephthalate (PET) are some common plastics
used in industry (Wadey, 2003). PET is made up of aliphatic carbons and two con-
stituents namely terephthalic acid and ethylene glycol. It can take up to 16e48 years
for PET to naturally decompose in the soil. Hence using rhizoremediation can help
in speeding up this process (Mu
¨ller et al., 2001). PET was degraded by Rhizobacteria
Arthrobacter sulfonivorans, Laccaria laccata found from the soil of host plant Salix
viminali (Janczak et al., 2020). Yoshida et al. (2016) achieved complete biodegrada-
tion of PET from the rhizosphere of Ideonella sakaiensis isolated from soil in just
6 months. Serratia plymuthica IV-11-34 strain was isolated from the rhizosphere
of soil and showed a biofilm formation and degradation of PET. One hundred
fifty-five genes were identified in genomic sequencing, which is associated with
FIGURE 5.2
Mechanism of rhizoremediation of heavy metals by the rhizosphere.
5. Rhizoremediation of PET and PLA plastics 87

Table 5.2 Rhizosphere remediating variety of contaminants and impact on plants.
Plant Rhizosphere microbe Pollutant targetted Result References
T. aestivum Mycorrhiza
Funneliformis
geosporum
Zn Improved in the
photosynthesizing ability of the
plant
Abu-Elsaoud
et al. (2017)
Prosopis juliflora
L.
Rhizobium strain Fly ash and heavy
metals
Increment in photosynthetic
pigment and nitrate reductase
activity
Rai et al.
(2004)
Sedum alfredii Pseudomonas sp. DDT-1 DDT and its metabolite
and Cd
Reduction in levels of DDT by
53.6% and of Cd by 31.1%
Zhu et al.
(2012)
Brassica napus Pantoea sp. FC 1 Phenol and Cr Reduced Cr(IV) to Cr(III) Ontan
˜on
et al. (2014)
Usage of phenol as a carbon
source
Lotus
corniculatus L.
and Oenothera
biennis L.
26 endophytes belonging to genera
Rhizobium,Pseudomonas,
Stenotrophomonas, and Rhodococcus
Hydrocarbon Degradation of petroleum Pawlik et al.
(2017)
Trifolium repens AMF Glomusmosseae PAH Reduced levels of pollutants Levyal et al.
(2001)
Brassica Napus P.fluorescence G10, Microbacterium sp.
G16
Lead Increased uptake of Pb in
shoots
Sheng et al.
(2008)
Medicago Sativa P. aeruginosa Petroleum
hydrocarbon and
heavy metal
contamination
Decrease in metal
concentration. 68% removal of
PAH
Agnello et al.
(2016)
Robinia
pseudoacacia
AMF Pb Pb uptake in root and shoot Yang et al.
(2016)
Festuca
arundinacea
Bacillus licheniformis, Bacillus mojavensis PAH Reduced levels of naphthalene,
phenanthrene, and dibenzo
[a,h] anthracene
Eskandary
et al. (2017)
88 CHAPTER 5 Bioremediation potential of rhizosphere

xenobiotic remediation hence affirming the potential of bioengineered rhizobacteria
for plastic remediation (Dąbrowska et al., 2021). The reclamation of microplastic-
contaminated barren soil through inoculation of seeds with PET-degrading rhizobac-
teria and plants showed an improvement in growth along with degradation of PET
and PLA in the soil after inoculation with strains of Laccaria laccata in the rhizo-
sphere of Brassica napus plants. Farzi et al. (2019) used common rhizosphere-
inhabiting bacteria Streptomyces sp. from soil and used this source to achieve
68.8% weight loss of PET (212 mm) in just 18 days. The rhizosphere exudes
amylase, cellulase, lipase, and several organic acids that act as sources of hydrolytic
enzymes that affect the breakdown of contaminants in the soil. Since plastic is a
rampant problem and a consequence of rapid urbanization, it is hence essential to
devise newer ways to degrade these. So rhizoremediation appears as an attractive
and viable option to achieve this end. Using rhizoremediation we can degrade these
plastics hence saving land sites from being converted into dumpyards.
6.Conclusions
Pollution is a well-known problem since it is difficult to reverse. Our ecofriendly as-
pirations are hampered by a lack of alternatives. To attain a safe environment, we
must have safe soil that is free of chemicals and toxins. Once in the soil, various
xenobiotic and recalcitrant substances are damaging, poisonous, and extremely diffi-
cult to remove. When they enter the food chain, they cause developmental and
fertility problems, causing species to diminish. Rhizoremediation, because of its
high efficiency, appears as a great alternative to traditional remediation approaches.
The plant, together with its associated root bacteria, contributes to improved soil
quality. Root-associated bacteria absorb contaminants and heavy metals and aid in
photodegradation. Plants exhale chemicals that boost the density of hydrocarbon-
degrading enzymes in soil. Plants exude compounds through their roots which
increase the density of hydrocarbon-degrading enzymes in soil that help in the
effective lysis, immobilization, and breakdown of contaminants. The symbiotic
relationship between the root and bacteria assists in the biodegradation of hazardous
chemicals such as PHA (poyhydroxyl alkanoates). Heavy metals are also
immobilized and chelated, preventing their negative impact on plants. Rhizobacteria
produce siderophores, phytohormones, and hydrogen cyanide, which aid in the
chelation of heavy metals, the breakdown of toxic compounds, and the lysis of
harmful pathogens. Thus, rhizoremediation aids in the opening of new
avenues and frontiers in our collective purpose to combat environmental pollution
and achieve a safer environment for the benefit of the plant, animal, and human
health.
6. Conclusions 89

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Zhuang, X., Chen, J., Shim, H., & Bai, Z. (2007). New advances in plant growth-promoting
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Daane, L. L., Harjono, I., Zylstra, G. J., & Haggblom, M. M. (2001). Isolation and character-
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94 CHAPTER 5 Bioremediation potential of rhizosphere

Plant growth promoting
rhizobacteria (PGPR): an
overview for sustainable
agriculture and
development
6
Harikrishna Naik Lavudi
1
, Parameshwar Jakinala
1,2
, Shiva Kumar J
1,2
,
Nani Babu B
1
, M. Srinivas
1
, Madhumohan Rao Katika
1
1
Mobile Virology Research and Diagnostics Laboratory (MVRDL) BSL 3&2, ESIC Medical College
&Hospital, Hyderabad, Telangana, India;
2
Department of Microbiology, Osmania University,
Hyderabad, Telangana, India
1.Introduction
Protection of plants from pathogens through induced systemic resistance (ISR) has
been the thrust area of research since the late 19th century. This phenomenon (ISR)
has been successfully demonstrated in a variety of host-pathogen interactions
(Choudhary et al., 2007;Jonathan et al., 2006). Currently, plant growth promoting
rhizobacteria (PGPR)-mediated ISR has received considerable attention, as biolog-
ical approaches to manage pests and diseases are more ecologically sustainable than
the use of synthetic chemicals. Some Bacillus spp. are known to promote plant
growth either directly by producing hormones or indirectly by producing antimicro-
bial substances which act against pathogens. They are involved in induced resistance
in plants. Similarly, several chemicals are also known to induce resistance in the host
plants against pathogen attack. Besides phenolic compounds, there are several other
plant metabolites like pathogenesis-related (PR) proteins, which have been found to
be associated with the induction of resistance in the host as many of them are found
to be antifungal (Savci, 2012).
2.Rhizosphere
The rhizosphere is the narrow area of soil specially motivated through the founda-
tion system (Dobbelaere et al., 2003;Walker et al., 2003). The rhizosphere is popu-
lated by a huge variety of microorganisms. The bacteria colonizing this place are
referred to as rhizobacteria (Schroth &Hancock, 1982). The term “rhizobacteria”
implies a collection of rhizosphere microorganisms in a position of colonizing the
CHAPTER
95
Rhizobiome. https://doi.org/10.1016/B978-0-443-16030-1.00017-1
Copyright ©2023 Elsevier Inc. All rights reserved.

basis surroundings (Kloepper, Wei, &Tuzun, 1991). The rhizosphere is rich in nu-
trients. This takes place because of the buildup of various plant exudates, which
include amino acids and sugars, which provide a rich supply of power and nutrients
for bacteria. This situation is supported by the truth that the range of bacteria inside
the rhizospheric area is commonly 10 to 100 instances better than that within the
bulk soil (Gray &Smith, 2005;Weller &Thomashow, 1994).
The rhizosphere is the place across the root and has excessive nutrient availabil-
ity. This is because of the loss of as a great deal as 40% of plant photosynthates from
the roots (Lynch &Whipps, 1991). As a result, this area contains large and lively
microbial populations that could exert beneficial, impartial, or unfavorable effects
on the growth of plant. The rhizospheric microbial populations play a vital role in
maintaining root health, nutrient uptake, and environmental stress tolerance (Bowen
&Rovira, 1999;Cook, 2002). These microorganisms are important components of
management practices to acquire higher crop yield.
Plant roots play a primary function in supplying water and nutrient uptake. Plant
roots secrete wide type of compounds that promotes soil microbial groups, called as
root exudates, that enable in promoting the plant-microbe interactions and inhibiting
the growth of the plant species (Nardi et al., 2000). These exudates might also act as
attractants or repellants and their composition depends upon the physiological popu-
larity and species of vegetation. The fine quality and quantity of root exudates are
encouraged by way of microbial interest in the rhizosphere, which influences rooting
styles and the supply of available vitamins to plants. These exudates are metabolized
by microbes as C and N assets, and the ensuing molecules are used by the plants
(Kang et al., 2010).
Indeed, carbon fluxes are crucial determinants of rhizosphere function. Root
exudation contributes approximately 5%e21% of photosynthetically constant car-
bon transported to the rhizosphere (Marschner, 1995). The soil, the rhizoplane,
and the basis are unique components interacting in the rhizosphere, of which the
rhizosphere is the area of soil encouraged by way of roots through the release of sub-
strates that affect microbial hobby. The surface of the foundation to which the soil
particles adhere strongly is described as rhizoplane. When the microorganisms colo-
nize the rhizoplane or root tissues, it is called root colonization, while rhizosphere
colonization refers to the colonization of the adjoining volume of soil underneath
the impact of the root (Kloepper et al., 1991;Kloepper, 1994;Barea et al., 2005).
Thus, the rhizosphere can be described as any extent of soil especially influenced
by means of plant roots and in association with roots hairs, and plant-produced ma-
terials (Dessaux et al., 2009).
2.1 Plant-microbial interactions
Because the plant rhizosphere has high availability of vitamins, numerous soil mi-
croorganisms pick it to be their ecological area of interest. Growth promotion
may be due to different mechanisms that include manufacturing of phytohormones
within the rhizosphere and different PGP sports (Arshad et al., 1993;Glick, 1995).
96 CHAPTER 6 Plant growth promoting rhizobacteria (PGPR)

Depending at the association of PGPR with their host plant, they may be of two
types: intracellular PGPR and extracellular PGPR. Intracellular PGPR are those
positioned in the specialized nodular systems of root cells and extracellular
PGPR, the ones that exist within the rhizosphere, at the rhizoplane or in the areas
between the cells of root cortex (Martinez-Viveros et al., 2010). Intracellular
PGPR includes endophytes including Allorhizobium (de Lajudie et al., 1998a), Azo-
rhizobium caulinodans (Dreyfus et al., 1988), Bradyrhizobium japonicum (Guerinot
&Chelm, 1984), Mesorhizobium chacoense (Peix et al., 2001), Mesorhizobium plu-
riforium (de Lajudie et al., 1998b), Rhizobium ciceri (Nour et al., 1994), R. etli
(Segovia et al., 1993), R. fredii (Scholla &Elkan, 1984), R. galegae (Lindstrom,
1989) and frankia species each of that could symbiotically restore atmospheric ni-
trogen with the better plants (Verma et al., 2010). Examples of extracellular
PGPR are Agrobacterium, Arthrobacter, Azotobacter, Azospirillum, Bacillus, Fla-
vobacterium, Micrococcus, Pseudomonas, Serratia.
There may be root-root, root-insect, and root-microbe interactions, which lead to
extra production of plant exudates. The community structures of the rhizobacteria
had been studied via programs including PCR and DGGE which showed changes
in plant-microbe interactions (Herschkovitz et al., 2005).
According to Whipps (2001), interactions among the rhizobacteria and devel-
oping plants can be impartial, poor, or positive. Most plant-related rhizobacteria
are commensals and set up interaction and not using a seen effect at the host plant’s
growth and physiology (Beattie, 2006). Negative interaction is one in which phyto-
pathogenic rhizobacteria produce bad effect on plant increase and physiology by
means of manufacturing of poisonous materials together with hydrogen cyanide
or ethylene whereas in effective interaction, the rhizobacteria leads to plant growth
enhancement through diverse mechanisms. Direct mechanisms consist of nitrogen
fixation, nutrient solubilization, and so forth, whereas, indirect mechanisms include
aggressive exclusion of phytopathogens or the removal of phytotoxic substances
(Bashan &de-Bashan, 2010).
Three distinctive areas are diagnosed, wherein plant-microbe interactions take
location. These areas are: phyllosphere, endosphere, and rhizosphere. Phyllo-
sphere relates to the aerial parts of the flowers and endosphere with inner shipping
machine. Rhizosphere can be described as that soil location which is basically
stimulated by using the plant roots and plant-produced fabric. It also can be defined
as that vicinity of soil used by plant roots, extending some millimeters from the
basis surface and which is a good deal richer in microorganisms than surrounding
bulk soil (Bringhurstetal.,2001;Hiltner, 1904). Plant exudates within the rhizo-
sphere are predominant source of power and nutrients and due to the rich availabil-
ity of vitamins, microbial populations are higher in this area (Martinez-Viveros
et al., 2010;Haas and De
´fago, 2005). Microorganisms present within the rhizo-
sphere play a critical position in nutrient cycling (Cardoso &Freitas, 1992).
Most important step closer to use of micro organisms as PGPR is root colonization.
Bacillus spp. and Pseudomonas sp. are exquisite rhizosphere colonizing bacteria
2. Rhizosphere 97

(Steenhoudt &Vanderleyden, 2000;Trivedietal.,2005). To be a successful PGPR,
strain needs to be able to compete in colonization, plant boom stimulation, and
biocontrol (Kevin Vessey, 2003;Weller et al., 2002). Rhizosphere colonization
has been nicely documented in vegetation together with potato, wheat, grasses,
maize, and cucumber (Cakmakci et al., 2006). One mechanism for rhizosphere
colonization is siderophore manufacturing. Few examples include Bradyrhizobium
japonicum,R. leguminosarum, and S. meliloti (Carson et al., 2000;El-Tarabily &
Sivasithamparam, 2006).
Whichever mechanism is used by PGPR for growth enhancement of plant, root
colonization is essential (Glick, 1995). Actinomycetes, a chief aspect of rhizosphere
microbiota, are crucial sources of different antimicrobial metabolites. They help in
nutrient cycling as nicely in plant boom-merchandising (Elliot &Lynch, 1995;
Halder et al., 1991;Merzaeva &Shirokikh, 2006;Terkina et al., 2006). Antagonistic
activity of endophytic Streptomyces griseorubiginosus against Fusarium oxysporum
f. Sp. Cubense has been recorded (Cao et al., 2004). There have been reports of rhi-
zospheric Streptomycetes as biocontrol agent of Fusarium and Armillaria pine rot
and as PGPR of Pinus taeda (de Vasconcellos &Cardoso, 2009). Potential biocon-
trol PGPR among actinomycetes (Gomes et al., 2000;Sousa et al., 2008) are Micro-
monospora sp., Streptomyces spp., Streptosporangium sp., and Thermobifida sp.,
against root pathogenic fungi (Franco-Correa et al., 2010). In a study performed
on endophytic actinomycetes found in neem and tulsi leaves for PGPR tendencies,
actinomycete isolate A7 (Streptomyces sp. Mrinalini7) confirmed enormous PGPR
interest. Isolate A7 changed into inoculated model tomato plant for evaluation of
its potential to sell seed germination and plant boom and great biomass production
of tomato become recorded (Nair &Padmavathy, 2014). Fig. 6.1 shows the develop-
ment of PGPR-assisted phytoremediation technology, plant microbial interaction in
sustainable agriculture.
2.2 Importance of agriculture
India is one of the fastest growing countries in terms of population and economics,
sitting at a population of 1239.96 million (2017) and growing at 10%e14% annually
(from 2011 to 2017). The combination of people living in poverty and the recent
economic growth of India has led to the co-emergence of two types of malnutrition:
undernutrition and overnutrition (Glick, 2014). The urgency thus is to break the
yield plateau for rapid gains in food grains production to maintain pace with the pop-
ulation growth. It is estimated that the population of the country would be touching
nearly 1400 million by 2025 AD and the food grain output requirement in 2021e22
would be around 307.31 million tonnes, which is almost double of the present pro-
duction. Hence, the main challenge is to attain self-sufficiency in food grains pro-
duction to meet the increasing demand of protein and ensuring the environmental
security by way of checking the degradation of soil due to intensive cultivation of
high input demanding crops.
98 CHAPTER 6 Plant growth promoting rhizobacteria (PGPR)

2.3 Improper use of fertilizers
The fertilizers application is required to increase agricultural profitability, at the same
time a balanced fertilization has to be applied for maintaining soil health for sustainable
use. Imbalanced use of fertilizers has already distorted soil fertility and deteriorated soil
health in India (Santhi et al., 2011). Accordingly, much attention has to be given to the
integrated use of organic and mineral nutrition to the soil for meeting the economic
needs of farmers as well as for sustainability in terms of soil fertility and productivity.
Degradation of soil health has also been reported due to long-term improper use of fer-
tilizers and nutrients. Although, the overall nutrient use of N:P
2
O
5
:K
2
O is 4:2:1 is
considered ideal for Indian soils, the present use ratio of 6.8:2.8:1 is far off the
mark. This imbalance of nutrient use has resulted in wide gap between crop removal
and fertilizer application. Declining soil fertility and mismanagement of plant nutrients
have made this task more difficult. Balanced NPK fertilization has received consider-
able attention in India (Gosh et al., 2004;Hegde &Babu, 2004;Prasad et al., 2004).
FIGURE 6.1
Schematic diagram of plant microbial interactions in the rhizosphere (Bramhachari et al.,
2017).
2. Rhizosphere 99

Food grains are grown with a tendency that not much of fertilizer application is
required for the crop. Though the recommended dose in Telangana, India, was
15e25 kg of nitrogen and 20e50 kg of superphosphate per hectare in food grains,
the actual application of chemical fertilizer, on an average, is found to be 6.4 kg
in lentil, 8.13 kg in black gram, and 12.04 kg in chickpea. For the growth and devel-
opment of root nodules, phosphorous is absolutely necessary and application of
40 kg P
2
O
5
per hectare has been recommended. Use of fertilizers, especially in
kharif food grains, even in the study period was low. With the withdrawal of sub-
sidies on fertilizers resulting in a hike in the prices, the use of fertilizers is expected
to decline. Soil testing helps the farmers to use fertilizers according to needs of crop;
it balances soil and applied nutrients from inorganic as well as organic sources to
balance nutrition of crops and maintenance of good soil health.
2.4 Lack of adoption of biofertilizers
Though efforts to popularize Rhizobium inoculants have been going on for a long
time and several public and private sector units are manufacturing them, the adop-
tion of these biofertilizers is found to be very negligible. Sustained use of Rhizobium
inoculants in the long run thus seems to be difficult.
2.5 Institutional constraints
The final testing ground of any technology is the farmer’s field. Technology transfer
on institutional level is not appropriate as the efforts are not directed toward human
resource development. Trainings, frontline demonstrations, and on-farm trials
should be conducted and a wide area should be covered. Dissemination of informa-
tion and awareness of benefits of food grains crops, Rhizobium inoculants, high
yielding and resistant varieties among the farmers should be ensured. Strict quality
control standards need to be enforced in the manufacture and sale of the inoculants
and a system should be developed in which research institutes should be involved for
regular monitoring of the performance of different technologies.
2.6 Socioeconomic constraints
Farmers generally grow food grains on marginal lands. The risk associated with food
grains production is the major factor that affects farmer psychology. There is hardly
any visible technological change in food grains farming in the country since a long
time. This clearly shows that technological stagnation has been primarily responsible
for the backwardness of food grains in the country in the food grains production scenario.
3.Chemical fertilizers
India is predominantly an agricultural country with 65% of its people depending on
agriculture. Agriculture in India, until the middle of the 20th century, depended
100 CHAPTER 6 Plant growth promoting rhizobacteria (PGPR)

mostly on organic manure (Jaga and Patel, 2012). With the introduction of modern
high yielding varieties and development of irrigation facilities during 1960s, con-
sumption of chemical fertilizer increased markedly. In 1950e51, consumption of ni-
trogen fertilizer in the country was only 0.06 million tons which increased to 10.8
million tons in 2000e01. An increase in consumption of phosphorous fertilizer
has also increased with 0.01 million tons in 1950e51 to 1.8 million tons in
2000e01. However, use of potassium fertilizer has been very low with almost nil
in 1950e51 to only 0.81 million tons in 2000e01. Along with increase in the con-
sumption of fertilizer, agricultural production has increased considerably from
1950e51 to 2000e01 (Pathak et al., 2006;Tomich et al., 1995). Nutrient budgeting
is a useful tool in determining present and future productivity of agricultural land as
well as undesirable effects of nutrients on environment. Budgets of nitrogen, phos-
phorous, and potassium were calculated for India during 2000e01, taking into
consideration the inputs of inorganic fertilizer, animal manure, compost, green
manure, leguminous fixation, nonleguminous fixation, crop residues, rain and irriga-
tion water and outputs of crop uptake and losses through leaching, volatilization, and
denitrification (Pathak et al., 2010). Inorganic fertilizer was the dominant source
contributing 64% of nitrogen and 78% of phosphorous inputs in Indian agriculture,
whereas, potassium input through inorganic fertilizer was 26%. Removal of nitro-
gen, phosphorous, and potassium by major agricultural crops in the country are
7.7, 1.3, and 7.5 million tons, respectively. There are positive balance of nitrogen
(1.4 million tons) and phosphorous (1.0 million tons) and a negative balance of po-
tassium (3.3 million tons). It was projected that nitrogen, phosphorous, and potas-
sium requirement by Indian agriculture would be 9.78, 1.57 and 9.52 million tons,
respectively, to meet the food demand of 1.3 billion people by 2020.
3.1 Nitrogen, phosphorus, and potassium in Indian agriculture
One of the important reasons for the poor yield of Agriculture is the negligence of
fertilizer requirement aspect. As a matter of fact this crop is not manured at all. To
produce one ton of food grains about 56 kg N, 5 kg P, and 22 kg K are required
(Bhattacharyya &Jha, 2012). However, this is heavy feeder of the soil nutrients;
hence care should be taken to ensure that the crop does not suffer from lack of nu-
trients. Regular monitoring of soil fertility level is a must and recommendations of
fertilizer application should be made on the basis of soil test values.
3.2 Nitrogen
Nitrogenous fertilizers: It comprises calcium, ammonium sulfate, sodium nitrate,
urea, etc. Nitrogenous fertilizer influences crop growth in many ways. It encourages
the development of foliage, imparts a green color to leaves. In case of cereals, it
tends to produce lumpiness in seeds and it tends to produce succulence or tenderness
in the plant. But if used in large quantity nitrogen may prove harmful to the crop for
it may increase resistance to disease, and it may lower its quality. It may weaken the
stems and cause lodging in cereals (Baruah et al., 2012).
3. Chemical fertilizers 101

3.3 Phosphate
Phosphate fertilizers are given to soil in the form of phosphorous, which is derived
from various sources like bones and rock phosphates. When powdered rock phos-
phate is applied to the soil, phosphoric acid becomes readily available. Phosphorous
helps the growth of plants in many ways, e.g., it hastens maturity of crop, it encour-
ages root development, decreases the ratio of straw to grain in cereals, it strengthens
stems and reduces the tendency to lodge in cereals, it increases the resistance to dis-
eases and it improves the quality of crops. Phosphorous balances or offsets the harm-
ful effects of excessive nitrogen and even it are applied in excessive quantities it
produces no bad effect on the crop (Samreen &Kausar, 2019).
3.4 Potassium
Potassium fertilizers are given to the soil in the form of potassium chloride and po-
tassium sulfate. These fertilizers help the transference of food materials from one
part of the plant to another, they provide the needed green color to the leaves,
tend to increase plumpness in grains, and have a balancing effect between the first
two types of fertilizers. Its presence in large quantities in the soil produces no detri-
mental effect on the crop. There is a wrong notation that sustainable ecofriendly
agriculture can be practiced using organic and biofertilizers and excluding chemical
fertilizers. The nutrient needs are so large that no single source can deliver the goods.
In our country, inorganic fertilizers are largely imported from other countries
which lead to huge outflow of foreign exchange. To meet the increased productivity
of 40 tonnes per hectare of vegetables, the fertilizer demand would be 533,122
tonnes of N, 319,518 tonnes of P
2
O
5
and 399,620 tonnes of K20 amounting to
9.63% of the total fertilizer consumption in our country (Som Dutt, 1999, p. 2).
Moreover the chemical fertilizers are not available at affordable prices to the farmers
in the need of the hour. The use of chemical fertilizers and off-farm inputs in inten-
sive agriculture continuously and injudiciously has resulted in its dependency on
crop yield, imbalance of nutrients in soil, deterioration of soil health, and adverse
effect of soil physiochemical properties (Ninawe, 1994). Further, it was stated by
several workers that the increased use of fertilizer may lead to health hazards, envi-
ronmental pollution, apart from soil erosion. So, it is imperative to look for alterna-
tive farming practices.
4.Biofertilizers
Biofertilizers are the basic inputs of nutrients for sustainable and organic farming.
Microbial strain which has more PGPR activities should be isolated from different
ecological zones and commercialized. Nowadays the liquid biofertilizers are more in
demand than the carrier-based biofertilizers. Therefore, the production of liquid bio-
fertilizers should be encouraged. Only 0.1% PGPR strains are formulated as
102 CHAPTER 6 Plant growth promoting rhizobacteria (PGPR)

commercialized till date (Devendra Singh et al., 2021). The demand for bio-
fertilizers is very high but production is very less. So, we should work in the field
of microbial formulation to develop biofertilizers according to agroecological zones
to provide chemical-free food grains for human beings.
The world population has grown by about four billions since the beginning of the
green revolution. Experts and planners believe that without further green revolution
the world would face famine and malnutrition of greater severity. Therefore, at pre-
sent the most challenging issue is to increase the production of food grains from the
rapidly shrinking per capita agricultural land (Muraleedharan et al., 2010). While
agriculture output increased as a result of the green revolution, the energy input to
produce a crop has increased faster; as a result the ratio of crops produced to energy
input has decreased over time, and loss of sustainability has appeared in terms of
production (Ansari et al., 2015). Green revolution techniques also heavily rely on
chemicals, fertilizers, pesticides, and herbicides. The quest of food security has
totally transformed the modern agriculture (Usha and Kanimozhi, 2011). The over-
exploitation of available natural resources without giving a thought over the possible
consequences has worsened the situation and advancement made in the crop produc-
tion and other related disciplines has caused environmental degradation in terms of
poor soil health, mineral imbalance in soil, and contamination of soil and food with
toxic residues of agrochemicals (Kumaresan &Reetha, 2011). Public concern over
the use of pesticides in agriculture and their effects on the environment continue to
increase and are compounded by perceiving risks associated with genetic modifica-
tion of plants that have resulted in a desire for a more environmentally sustainable
approach to agriculture, horticulture, forestry, and related industries. Globally, these
views have resulted in greater restrictions on use of chemicals and pesticides. The
development of resistance to chemical pesticides and concern over their deleterious
effects on environmental and human safety have provided a strong impetus for
consideration of microbial control agents for use in integrated management of pests
and pathogens. Recent researches have been carried out with greater systematic
approach and practical utility and have demonstrated potential of biological control
in the present agriculture. In present scenario where world population is increasing,
there is a dire need to increase the agricultural produce. To attain this, farmers or
growers are dependent on chemical fertilizers, insecticides, or fungicides, which
have helped to provide food security to the increasing population of the world.
Excessive use of chemical fertilizers and xenobiotic compounds not only affects
the health of the soil but also causes certain level of toxicity to the produce (Savci,
2012). However, these synthetic compounds dominate the market. Variety of
research questions remains to be fully answered about the nature of biological con-
trol and means of most effective management under present production conditions
(Louws et al., 1999;McSpadden Gardner &Weller, 2001). The inoculation of bene-
ficial microorganisms into the soil, which improves plant growth and productivity, is
called biofertilizing. Research during the past few decades has led to the identifica-
tion of certain biological organisms and their products that could potentially be used
as fertilizer sources. This strategy of fertilizing the soil with biological sources has
4. Biofertilizers 103

been widely accepted and recognized as a viable alternative to the application of
chemical fertilizers. Among the different groups of plants, some are used as effective
fertilizer sources for enriching the soil with carbon, nitrogen, phosphorous, and other
minerals. Nitrogen is one of the basic requirements for the growth, productivity, and
yield of plants. Thomas-Stanford (1926) was one of the first to recognize competi-
tion between saprophytic and pathogenic organisms for nutrients at the site of initial
infection as a form of biological control of plant disease. As time progressed various
bacteria, fungi, mycorrhizae, etc., have been tested for their ability to suppress plant
diseases. Awareness about practicing cultivation using biofertilizer has been created
among farmers by practical demonstrations by the government and also by the
nongovernmental organizations (NGOs). The concept of sustainable agriculture
has been recently developed and it involves the successful management of resources
to satisfy the changing human needs for maintaining and enhancing the quality of the
environment and for conserving resources (Brar et al., 2012;Pindi &Satyanarayana,
2012). Thorough knowledge about beneficial microbes has now been obtained with
the help of in-depth research and advancement of technology.
4.1 Plant growth promoting rhizobacteria (PGPR)
Bacteria that are present in the soil colonize plant roots and enhance plant growth by
different mechanisms are referred to as plant growthepromoting rhizobacteria
(PGPR) (Dutta &Podile, 2010). These PGPR are highly diverse bacteria present
in the soil; here we focus on rhizobacteria as biocontrol agents. Their effects can
occur via local antagonism to soil-borne pathogens or by induction of systemic resis-
tance against pathogens throughout the entire plant. Several substances produced by
antagonistic rhizobacteria have been related to pathogen control and indirect promo-
tion of growth in many plants, such as siderophores and antibiotics. Induced sys-
temic resistance (ISR) in plants resembles pathogen-induced systemic acquired
resistance (SAR) under conditions where the inducing bacteria and the challenging
pathogen remain spatially separated. Both types of induced resistance render unin-
fected plant parts more resistant to pathogens in several plant species. Rhizobacteria
induce resistance through the salicylic acidedependent SAR pathway, or require jas-
monic acid and ethylene perception from the plant for ISR. Rhizobacteria belonging
to the genera Pseudomonas and Bacillus are well known for their antagonistic effects
and their ability to trigger ISR. Resistance-inducing and antagonistic rhizobacteria
might be useful in formulating new inoculants with combinations of different mech-
anisms of action, leading to a more efficient use for biocontrol strategies to improve
cropping systems.
PGPR colonizes in the rhizosphere, i.e., the narrow zone of soil immediately sur-
rounding the root system is much richer in bacteria than the surrounding bulk soil
(Adesemoye &Kloepper, 2009). This phenomenon was termed as rhizosphere effect
which was caused by the fact that a substantial amount of the carbon fixed by the
104 CHAPTER 6 Plant growth promoting rhizobacteria (PGPR)

plant, 5%e21% (Armstrong et al., 2009), was secreted, mainly as root exudate. The
concentration of bacteria in the rhizosphere was reported 10 to 1000 times higher
than that in bulk soil. This type of microbial colonization of the plant root surface
was observed in patches along the root, ultimately covering w15%e40% of the total
plant root surface (Watt et al., 2005). The density and structure of the microbes on
the root surface were reported as dependent on nutrient availability and physico-
chemical variations throughout the root surface.
The process of root colonization was observed under the influence of various pa-
rameters such as bacterial traits, several other biotic and abiotic factors, and root ex-
udates. The composition of root exudate was mainly affected by stage of plant
development (Loper et al., 2008) and serves as a food source and chemoattractant
for microbes which then attach to the root surface and form microcolonies. By
the secretion of root exudates, the roots regulate the soil microbial community in
their immediate vicinity to cope with herbivores, encourage beneficial symbioses,
change the chemical and physical properties of the soil, and inhibit the growth of
competing plant species (Lugtenberg &Kamilova, 2009). The compounds secreted
by plant roots serve important role as chemical attractants and repellants in the
rhizosphere (Bais et al., 2006). On the other hand, it is essential trait for a PGPR
strain to be good competitor for nutrients and niches (Kamilova et al., 2005). The
poor rhizoplane colonization by PGPR was reported to reduce the ability of bacteria
to be an effective biocontrol agent (Knief et al., 2011). Mostly the bacterial coloni-
zation and attachment was reported at the junction of epidermal cell, root hairs, axial
groves, cap cells and sites of emerging lateral roots (Danhorn &Fuqua, 2007).
Further, rhizobacteria could eventually grow into larger biofilms consisting of mul-
tiple layers of bacteria (Quadriya et al., 2018). Even in many cases the effectiveness
of rhizobacteria was reported in a density-dependent manner (Rudrappa et al., 2008).
Sometimes, rhizobacteria may depend on other microbes for nutrient sources as one
microbe may convert plant exudates into a form that can be used by another microbe.
Thus, rhizosphere was reported as a versatile and dynamic ecological environment
of intense plant-microbe interactions and harnessing essential micro and macronu-
trients affecting plant growth (Mayak et al., 2004).
Based on the consequences on plant growth, bacteria related to the plants can be
classified into useful, deleterious, and impartial (Dobbelaere et al., 2003). Different
bacterial genera are concerned in some of biotic activities of the soil atmosphere, as
a result making it extra dynamic in terms of nutrient availability and sustainable
agriculture, which are essential additives of soils (Chandler et al., 2008). They
help inside the stimulation of plant increase through nutrient mobilization in soils
and manufacturing of different plant boom regulators rendering protection against
phytopathogens either through controlling or inhibiting them. This improves the
shape of soil and enables in bioremediation of polluted soils. They achieve this
with the aid of sequestering toxic heavy steel species and degradation of xenobiotics
which include insecticides (Ahemad, 2012;Ahemad &Malik, 2011;Braud et al.,
2009;Hayat et al., 2010;Rajkumar et al., 2010). The rhizobacteria are extra green
inside the reworking, mobilizing, and solubilizing nutrients and consequently are the
4. Biofertilizers 105

fundamental driving forces for recycling of nutrients present in the soil leading to
increased fertility of soil (Glick, 2012;Hayat et al., 2010).
The rhizosphere, representing the thin layer of soil surrounding the roots of the
plant and the soil adhering to the roots, supports huge lively agencies of bacteria
referred to as plant boom promoting rhizobacteria. When rhizobacteria are intro-
duced as inoculants into the plant, it confers a positive and beneficial effect on
the plant boom (Kloepper &Schroth, 1978). Efficient root colonization, potential
to live to tell the tale and compete, and plant boom advertising are few vital charac-
teristics to be recognized as an effective plant increase promoting rhizobacteria
(Kloepper, 1994). PGPR rapidly colonize the rhizosphere and promote suppression
of soil-borne pathogens at the basis floor and stimulate plant growth (Bloemberg &
Lugtenberg, 2001). Among PGPR, fluorescent Pseudomonas spp. is taken into
consideration to be the maximum promising organism. They assist inside the
biocontrol of various plant diseases and convey secondary metabolites inclusive
of antibiotics, volatile compound, phytohormones, and siderophores. Their potential
to sell plant increase is specifically because of the manufacturing of antibiotics,
indole acetic acid, and siderophores. The genera of PGPR include Acetobacter,Azo-
spirillum,Azotobacter,Bacillus,Burkholderia,Paenibacillus,Pseudomonas, and
few participants of the Enterobacteriaceae. Enormous research in the region is the
direct use of microorganisms for plant boom promoting and plant pest control.
The preliminary step in the pathogenesis of soil-borne microorganisms is the capa-
bility to colonize the rhizosphere and is crucial for the microbial inoculants for use
as biofertilizers, phytostimulators, biocontrol retailers, and bioremediators. Pseudo-
monas spp. is often used as version root colonizing microorganism (Lugtenberg
et al., 2001). The rhizosphere is defined as the sector of soil in which the microflora
are motivated through the foundation (Hiltner, 1904).
Among so many specific bacterial genera identified as PGPR, bacillus and pseu-
domonas spp. are major (Podile &Kishore, 2006). PGPR and their institutions with
flowers are exploited commercially for boom promotion in plants for achieving sus-
tainable agriculture and such associations have been studied in barley, canola, cu-
cumber, wheat, oat, peas, tomatoes, and wheat (Gray &Smith, 2005;Podile &
Kishore, 2006).
Currently, there may be a sturdy emphasis to discover organic techniques for
development of crop manufacturing following integrated plant nutrient control de-
vice. Different symbiotic and nonsymbiotic rhizobacteria are actually being used
worldwide as bioinoculants for plant growth promotion and improvement under
numerous stresses like heavy metals, insecticides, herbicides, and fungicides (Ahe-
mad &Khan, 2010;Wani &Khan, 2010;Ma et al., 2011a,b). Among symbiotic
PGPR are Rhizobium, Bradyrhizobium, Mesorhizobium whereas Pseudomonas, Ba-
cillus, Klebsiella, Azotobacter, Azospirillum, Azomonas are non-symbiotic PGPR.
Although, the mechanisms of PGPR are not yet absolutely recognized, those houses
help in augmenting plant increase and improvement (Khan et al., 2009;Zaidi et al.,
2009).
106 CHAPTER 6 Plant growth promoting rhizobacteria (PGPR)

4.2 Microorganisms with PGPR
Many plant-associated microbes have been studied, which can be used in place of
chemical fertilizers and that can also confer biocontrol properties. These are termed
as Plant Growth Promoting Rhizobacteria. These encompass several genera
including Azotobacter, Arthrobacter, Bacillus, Clostridium, Hydrogenophaga,
Enterobacter, Serratia, Burkholderia Azospirillum, etc. (Chaudhary et al., 2011;
Lugtenberg &Kamilova, 2009)(Fig. 6.2). Using these diversified organisms, crop
productivity had increased to an appreciable measure (ICAR report, 2006e07).
To further increase the capabilities of these microorganisms, there is a need to study
their interactions with host plants and to genetically modify these organisms for
improved plant growth promoting activities (Zaidi et al., 2015).
4.3 The importance and applications of PGPR
The term “rhizobacteria” was delivered through Kloepper and Schroth (1978) to
those soil bacteria, which were able to colonize the roots of vegetation, and pro-
moted their boom with the aid of lowering the plant disorder occurrence. The
term “PGPR” became additionally given by way of Kloepper et al. (1991) to explain
useful soil bacteria that colonize the plant roots and facilitate in enhancing their
boom. PGPR are the part of rhizospheric biota that helps within the increase of flora
through diverse mechanisms either directly or circuitously. Plant boom selling rhi-
zobacteria has innate genetic ability for the management of agricultural practices
(Cook, 2002). The boom of plant life in agricultural soils is influenced by diverse
biotic and abiotic factors. Many exclusive bodily and chemical methods had been
used by the growers for the management of soil surroundings for the improvement
of crop yields. But application of microbial merchandise for this reason is a less not
unusual education.
FIGURE 6.2 Image of different PGPRs.
4. Biofertilizers 107

PGPR gives an environmentally sustainable approach for enhancing crop
manufacturing and health. With the utility of molecular equipment, it’s far viable
to manipulate the rhizosphere effectively, which can result in new products with pro-
gressed effectiveness. As a consequence, some of PGPR, e.g., Bacillus subtilis A13
(Turner &Backman, 1991), B. licheniformis CECT5106, B. pumilus CECT5105
(Probanza et al., 2002); Enterobacter cloacae UW4 and CAL2 (Li et al., 2018;
Shah et al., 1998), and others like P. fluorescens Pf-five, P. fluorescens 2-seventy
nine, P. fluorescens CHA0 (Wang et al., 2000), Pseudomonas putida GR12-2
were recognized (Jacobson, 1993). Bacteria, specifically pseudomonas and bacilli
have been discovered in the rhizosphere of diverse leguminous crops. These bacteria
efficiently colonize the roots and suppress soil-borne phytopathogens (Parmar &
Dadarwal, 1999).
The interactions between PGPR and rhizobia may be synergistic or antagonistic.
The beneficial effects of these interactions can be exploited for increasing the bio-
logical nitrogen fixation and crop yield (Dubey, 1996). Due to the harmful effects
of artificial fertilizers on the environment and their high cost, there has been increase
in the use of beneficial soil microorganisms such as PGPR for sustainable agriculture
all around the world. PGPR are considered as efficient biofertilizers for sustainable
agriculture thereby improving crop yields.
4.4 Mechanisms of PGPR
PGPR are able to directly enhance plant growth by mechanisms such as atmospheric
nitrogen fixation that is transferred to the plant (biofertilizers), siderophore produc-
tion (antifungal activity), solubilization of minerals such as phosphorus, and phyto-
hormones synthesis like auxins, cytokinins, and ethylene synthesis (biostimulants),
synthesis of antifungal metabolites (bioprotectants) or induction of systemic resis-
tance (Kloepper &Beauchamp, 1992;Glick, 1995;Frankenberger &Arshad,
1995;Bloemberg &Lugtenberg, 2001).
PGPR strains may use one or more direct or indirect mechanisms in the rhizo-
sphere. Few PGPR strains, when inoculated on the seed before planting, may estab-
lish themselves on the roots of the crop which is a very common way for reduction of
damping-off (Pythium ultimum) among crops. Bacteria in the genera Bacillus, Strep-
tomyces, Pseudomonas, Burkholderia, and Agrobacterium are the biological control
agents predominantly studied (Kloepper, 1992). Commercialized PGPR organism,
Bacillus subtilis, has biocontrol potential against variety of pathogenic fungi
(Boland &Kuykendall, 1998).
4.5 Biological nitrogen fixation
Biological nitrogen fixation plays an important role in sustainable agriculture,
because it reduces the need for exogenous fertilizer while providing carbon, nitro-
gen, phosphorus, and other nutrients for producing protein-rich foods. Therefore,
the agriculture system excessively depends on fertilizer application. Nitrogen is
108 CHAPTER 6 Plant growth promoting rhizobacteria (PGPR)

one of the essential, common nutrient elements for restraining crop yield (Passricha
et al., 2020).
Nitrogen (N) is the principal plant nutrient. Major portion of nitrogen (78%) is
present in unavailable form in the atmosphere. Leaching of minerals and losses
due to rains might be the reasons for the limited availability of this vital nutrient.
Biological nitrogen fixation or diazotrophy is the conversion of atmospheric N to
NH
3
by nitrogen fixing microorganisms using nitrogenase enzyme. The nif genes
are responsible for nitrogen fixation, occurs in symbiotic as well as free living sys-
tems (Kim &Rees, 1994). In case of rhizobium, symbiotic activation of these genes
depends on low concentration of oxygen. This is regulated by fixfix-genes, common
in symbiotic and free living nitrogen-fixing bacteria (Dean &Jacobson, 1992;Kim
&Rees, 1994). The nitrogen-fixing microorganisms are widely distributed in nature
(Raymond et al., 2004). The ability to fix nitrogen is widespread among prokaryotes
with representatives in both bacteria and archaea (Dekas et al., 2009). They are eco-
friendly, low cost, and an alternate source for chemical fertilizers (Ladha et al.,
1997).
4.6 Increase in growth
Plant weight of tuber-treated potatoes increased by 80% on average by midseason
and emergence increases of 10%e40% resulted for canola when seeds were coated
with PGPR before planting. Yield increases between 10% and 20% with PGPR ap-
plications have been documented for several agricultural crops (Kloepper et al.,
1991).
Alagawadi and Gaur (1992) reported combined inoculation of sorghum with
A. brasilense and phosphate solubilization bacteria; P. striata or B. polymyxa signif-
icantly increased grain yield and dry matter content, N and P uptake as compared
with single inoculation of individual organisms. The stimulatory effects of these
PGPR strains on the yield and growth of these crops were attributed to the ability
to fix nitrogen, phytohormone synthesis, and mineral solubilization (Cakmakci
et al., 2007;Karlidag et al., 2007;Kevin Vessey, 2003). For C. roseus, P. fluorescens
is known to enhance biomass yield and ajmalicine alkaloid content under water-
deficient stress (Jaleel et al., 2007). The higher N, P, and K content in PGPR com-
bination treatment may have resulted from the nitrogen fixation and P-solubilizing
ability of these strains. The role of PGPR strains in increase of the plant nutrient el-
ements has been discussed (Attia et al., 2020;Aslantas et al., 2007;Cakmakci et al.,
2007;Karlidag et al., 2007).
4.7 Phosphate solubilization
The development of soil fertility is one of the maximum commonplace techniques to
increase agricultural production. Biological N fixation performs an essential func-
tion in improving the soil fertility. Phosphorus (P), second best to nitrogen nutrient
in requirement for flora, is an important macronutrient for biological growth and
4. Biofertilizers 109

development. Major portion of phosphorous in soil exists as nonutilizable insoluble
phosphates that flowers can’t take up without delay (Pradhan &Sukla, 2006). Micro-
organisms can also solubilize insoluble inorganic P of soil making it available to the
flowers. This capacity of a few microorganisms is pretty crucial for yield enhance-
ment in plants (Chen et al., 2006;Rodriguez et al., 2006). Such rhizobacterial lines
may additionally act as efficient growth promoting retailers in agricultural plants
(Chaiharn et al., 2008).
There has been an lot of research as hobby for enhancing plant production with
the aid of microorganisms capable of solubilizing mineral phosphates leading to
extended P availability. Enhanced phosphate availability to rice has been attributed
to the PGPR’s capability to solubilize triggered phosphates, promoting plant in-
crease below field condition (Verma et al., 2001). Enhanced phosphorus uptake
with the aid of flowers is stated by using the use of PSB as inoculants (Chen
et al., 2006;Igual et al., 2001). Phosphate solubilizing microorganisms (PSM)
together with bacteria have provided a biotechnological solution in sustainable agri-
culture to meet the P demands of plant life. The most green phosphate solubilizers
among microorganisms belong to genera Bacillus, Rhizobium, and Pseudomonas.
Among fungi, Aspergillus and Penicillium are acknowledged to be efficient phos-
phate solubilizers. It is now viable to manipulate our agriculture machine in a
more sustainable manner due to extended information concerning the mechanisms,
colonizing competencies, and commercial packages of such useful microorganisms
(Zaidi et al., 2009).
4.8 Microbial antagonism
Biocontrol agents can be described as bacteria that aid in reducing plant disease inci-
dence/severity. On the other hand, antagonists are those bacteria that exhibit antag-
onistic activity toward a pathogen (Beattie, 2006). Bacterial antagonistic activities
include synthesis of hydrolytic enzymes (chitinases, glucanases, proteases, and li-
pases) that lyse pathogenic fungal cells, compete for nutrients, and colonize niches
at the root surface, regulate plant ethylene levels through the ACC-deaminase
enzyme for stress resistance, and produce siderophores and antibiotics (Glick &
Bashan, 1997;Van Loon, 2007).
Rhizobacteria are suitable candidates for use as biocontrol agents. These rhizo-
bacteria inhabit the rhizosphere and through their antagonistic activity protect the
plants before and during primary infection of roots caused by diverse plant patho-
gens. PGPR are native to soil and plant rhizosphere and possess the ability to control
or inhibit a broad spectrum of bacterial, fungal, and nematode diseases and because
of their contribution toward plant growth and protection against pathogens, the use
of PGPR has increased all over the world. This application of PGPR holds a great
significance for agriculture for biocontrol of plant pathogens and biofertilization
(Siddiqui, 2005, pp. 111e142). Multiple PGP traits were found to be exhibited by
bacterial strains isolated from Lolium perenne rhizosphere that may act as suitable
PGPR and biocontrol agents (Shoebitz et al., 2009). Suppression of phytopathogens
110 CHAPTER 6 Plant growth promoting rhizobacteria (PGPR)

by PGPR leading to enhanced plant growth may occur through different mechanisms
such as production of antibiotics, fungal wall-lysing enzymes, or hydrogen cyanide.
Pseudomonas-mediated antagonistic microbe-microbe interactions aid in the
biocontrol of phytopathogenic fungi in the rhizosphere contributing in enhanced
plant growth and survival (Winding et al., 2004). According to Costa et al.
(2007), antagonistic interactions involves synthesis of various antibiotics like 2, 4-
DAPG, pyoluteocin, etc. (Figs. 6.3 and 6.4).
Plants, like other organisms, have evolved to associate with a variety of mi-
crobes. While most of these are neutral commensals, some are relevant to plants
via pathogenesis, growth-promotion or disease-resistance. The two latter benefits
to plants, sometime, are provided by a group of bacteria that effectively colonize
plant roots, often referred to as plant growth promoting bacteria (PGPR) (Glick,
2012). Plant growth promoting rhizobacteria (PGPR) shows an important role in
the sustainable agriculture industry. The increasing demand for crop production
with a significant reduction of synthetic chemical fertilizers and pesticides use is
a big challenge nowadays. The use of PGPR has been proven to be an environmen-
tally sound way of increasing crop yields by facilitating plant growth through either
a direct or indirect mechanism (Nakkeeran et al., 2005). The mechanisms of PGPR
include regulating hormonal and nutritional balance, inducing resistance against
plant pathogens, and solubilizing nutrients for easy uptake by plants. In addition,
PGPR show synergistic and antagonistic interactions with microorganisms within
the rhizosphere and beyond in bulk soil, which indirectly boosts plant growth rate
(Bashan et al., 2014). There are many bacteria species that act as PGPR, described
FIGURE 6.3 Showing the PGPR promote plant growth in different ways (Garcia-Fraile et al.,
2013).
4. Biofertilizers 111

in the literature as successful for improving plant growth. PGPR are not only asso-
ciated with the root to exert beneficial effects on plant development but also have
positive effects on controlling phytopathogenic microorganisms. Therefore, PGPR
serve as one of the active ingredients in biofertilizer formulation (Martinez-
Viveros et al., 2010). Based on the interactions with plants, PGPR can be separated
into symbiotic bacteria, whereby they live inside plants and exchange metabolites
with them directly, and free-living rhizobacteria, which live outside plant cells.
The working mechanisms of PGPR can also be separated into direct and indirect
ones (Gouda et al., 2018).
The direct mechanisms are biofertilization, stimulation of root growth, rhizore-
mediation, and plant stress control. On the other hand, the mechanism of biological
control by which rhizobacteria are involved as plant growth promotion indirectly is
by reducing the impact of diseases, which include antibiosis, induction of systemic
resistance, and competition for nutrients and niches (Dey et al., 2004). Plant growth
is influenced by a variety of stresses due to the soil environment, which is a major
constraint for sustainable agricultural production. These stresses can be classified
into two groups, biotic and abiotic. Biotic refers to the stresses due to plant patho-
gens and pests such as viruses, fungi, bacteria, nematodes, insects, etc., while abiotic
is stresses due to the content of heavy metal in soils, drought, nutrient deficiency,
salinity, and temperature (Chaudhary et al., 2011).
4.9 Pesticide-specific biosurfactants
Due to biodegradative property of biosurfactants, they are ideally suited for envi-
ronmental applications, especially for removal of the pesticidesdan important
FIGURE 6.4 Schematic representation of PGPR applications.
112 CHAPTER 6 Plant growth promoting rhizobacteria (PGPR)

step in bioremediation. The noteworthy feature being the increasing interest shown
by the various researchers on: (i) degradation of pesticides, (ii) production and
exploitation of BS for the removal of pesticides from the environment, and (iii)
postulates on the possible replacement of synthetic surfactants with the bio-
surfactants in the pesticide formulation and clean-up (Martins and Martins,
2018;Jakinala et al., 2019).
4.10 HCH degradation
Hexa-chlorocyclohexane (HCH) is still the highest ranking pesticide used in India
and many other countries. Of the eight known isomers of HCH, the alpha-form con-
stitutes more than 70% of the technical product, which is not only noninsecticidal
but also a suspected carcinogen. The use of technical HCH, which is a mixture of
isomers, will continue in the Indian market because of their all-time availability
with good insecticidal efficiency and at a price which is 10e12 times less than
that of the pure gamma HCH (Lindane). It is pertinent to note that the environment
burden of already-dumped HCH continues to pose threat to all forms of life. The
poor solubility is one of the limiting factors in the microbial degradation of
alpha-HCH. Presence of six chlorines in the molecule is another factor that renders
HCH lipophilic and persistent in the biosphere (Das &Chandran, 2010). Even
though several reports are available on biodegradation of specific isomers of HCH
in animals, plants, soil, and microbial systems, literature on metabolism of alpha-
HCH by microorganisms is limited. Furthermore, the exact mechanism of transloca-
tion of HCH to the site of destruction and degradation of alpha-HCH in bacteria is
not well understood (Vandana et al., 2014a,b,c).
5.Other applications
By virtue of properties of biodegradability, substrate specificity, chemical and func-
tional diversity, and rapid/controlled inactivation, biosurfactants (BS) are gaining
importance in various industries like agriculture, food, medical, textiles, petrochem-
icals, etc (Archana et al., 2019,Nageshwar et al., 2022,Zargar et al., 2023). BS from
some other bacterial taxa may be of public health concern. Methyl rhamnolipids
from Pseudomonas aeroginosa have cytotoxic effects. Lipo polyglycans from my-
coplasmas show endotoxic properties, potentially inducing procoagulant activity
in human leukocytes. The toxicity and antigenic properties of mycobacterial glyco-
lipids, produced by pathogenic mycobacteria such as M. avium intracellure,
M. scrofulaceum, and M. fortulitum, which are habitats of water polluted with indus-
trial and domestic residues, are well known. The varied uses of BS also imply scope
for MS, and the need to strengthen the research in this emerging area (Parhi et al.,
2016).
5. Other applications 113

Use of chemical pesticides or fungicides is at present the main option of disease
control, which has caused serious threat to the environment as well as human health.
Therefore, a disease controlling strategy creating least ecological threat and at the
same time providing economical and reliable method of disease control is highly
desirable. A potential principle for chemically and/or microbial mediated disease
control could be based on agents that would induce systemic and/or local resistance
responses. The availability of a long-lasting, broad spectrum, stable solution to dis-
ease control using systemic inducers would undoubtedly have enormous positive
impact on food production and ecological safety and this is also one of the most
prominent needs of the day. Therefore, further experiments are needed to explore
the mechanism(s) of plant-pathogen interactions, signaling and SAR responses,
local defense mechanisms, and receptors for the chemicals or electrical signals in
plants (Fig. 6.5)(Silva et al., 2014).
The commercial formulations of these microorganisms available in the market
are needed to be evaluated for their performance and for compatibility with effica-
cious fungicides under field condition in order to devise an effective integrated treat-
ment of biopesticide and a much lower dose of fungicide to achieve ecologically
sustainable and economically feasible control of diseases of plants.
FIGURE 6.5 Role of PGPR in different applications in the plants (Gouda et al., 2018).
114 CHAPTER 6 Plant growth promoting rhizobacteria (PGPR)

6.Conclusion
The ability of PGPR to produce several substances to promote the growth and path-
ogen control in many plants could be of significant agronomic importance and center
of the research debate for a long time. Resistance-inducing and antagonistic PGPR
might be useful and an excellent alternative of environmentally friendly biological
control of plant disease and it can be improving the crops in a most profitable way.
These PGPR will require a systematic strategy designed to fully utilize all these
beneficial factors, applying different mechanisms of action to increase crop yields.
Different microbial strains which have more PGPR activities should be isolated and
commercialized from different agroecological zones. Biofertilizers are considered
an ecologically excellent alternative to chemical fertilizer. Nowadays more focus
is on liquid biofertilizers than the carrier-based biofertilizers. We need to encourage
the production and implementaion of liquid biofertilizers in the field. Till date there
are only <0.2% PGPR strains formulated are commercialized, very less work has
been done in the formulation of microbial strains for biofertilizers rather than chem-
ical fertilizers. Farmers are not benefited yet because formulation of biofertilizers
has not been carried forward. Now the biofertilizer demand is very high due to
limited production. So, we should work in the field of PGPR-based microbial formu-
lation to develop biofertilizers and should make easily available for farmers and
need to take in largescale production for the benefit of farmers and for sustainable
agriculture and development.
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122 CHAPTER 6 Plant growth promoting rhizobacteria (PGPR)

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Biotechnology, 25, 305e314.
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Lorenzo Segovia, J., Young, P. W., & Martinez-Romero, E. (1993). Reclassification of Amer-
ican Rhizobium leguminosarum Biovar Phaseoli Type I Strains as Rhizobium etli sp. nov.
International Journal of Systemic and Evolutionary Microbiology, 43(2), 374e377.
https://doi.org/10.1099/00207713-43-2-374
Pieterse, C. M. J., Van Pelt, J. A., Van Wees, S. C. M., Ton, J., Leon-Kloosterziel, K. M.,
Keurentjes, J. J. B., Verhagen, B. W. M., Knoester, M., Van der Sluis, I.,
Bakker, P. A. H. M., & Van Loon, L. C. (2001). Rhizobacteria-mediated induced systemic
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107,51e61, 2001.
Further reading 125

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Rhizospheric microbiome:
organization and
bioinformatics studies 7
Archana T. S.
1
, Devendra Kumar
1
, Vipul Kumar
1
, Shivam Singh
2
,
Nakishuka Bitaisha Shukuru
1
, Gagan Kumar
3
1
Department of Plant Pathology, School of Agriculture, Lovely Professional University, Phagwara,
Punjab, India;
2
Krishi Vigyan Kendra-Baghpat, S.V.P University of Agriculture and Technology,
Meerut, Uttar Pradesh, India;
3
Krishi Vigyan Kendra, Narkatiaganj, Dr. Rajendra Prasad Central
Agricultural University, Samastipur, Bihar, India
1.Introduction
Rhizosphere microbes containing oomycetes, fungi, and bacteria that make up a
plant’s second genome are closely associated with the development and health of
plants (Berendsen et al., 2012;Cai et al., 2017;Mueller &Sachs, 2015;Wu et al.,
2018). Rhizobia and mycorrhiza are two examples of typical functional groups.
Over past decades, fungi and the pathogenic microbes of rhizosphere microorgan-
isms that affect plant health and growth have received extensive research (Dardanelli
et al., 2011;Mendes et al., 2013;Tedersoo et al., 2020), but in the relationship be-
tween rhizosphere microbial organisms and other plants, the understanding of com-
munities is lacking (Berendsen et al., 2012;Tedersoo et al., 2020). The various
environmental conditions created by soil texture favor a variety of microorganisms
living together in harmonies such as archaea, viruses, bacteria, oomycetes, fungi,
and protists, all of which engage in intricate interactions with one another forming
a networked trophic interaction.
Microorganisms in the rhizosphere can either be healthy for the host plant or un-
healthy (Yu et al., 2019). The pathogenic microbes, such as those found in soil,
decrease plant growth, decrease yield, and endanger agricultural production, which
has been extensively investigated for years (Yin et al., 2021). However, advanta-
geous microbes such as mutualistic microbes by increasing nutrient levels can
encourage plant growth, increase tolerance, increase availability, and produce plant
hormones as well as abiotic stresses (Fig. 7.1)(Haney et al., 2015;Jacoby et al.,
2017;Rolli et al., 2015;Yin et al., 2021).
CHAPTER
127
Rhizobiome. https://doi.org/10.1016/B978-0-443-16030-1.00016-X
Copyright ©2023 Elsevier Inc. All rights reserved.

2.Bioinformatics
An interdisciplinary field called bioinformatics creates techniques and various soft-
ware tools for comprehending biological data, especially when the datasets are
extremely big and complicated. To analyse and interpret biological data, the inter-
disciplinary field of science known as bioinformatics brings together computer sci-
ence, biology, chemistry, physics, information engineering, mathematics, and
statistics. Biological queries have been analysed in silico using computational and
statistical methods and bioinformatics. Both biological studies that assimilate com-
puter programming into their methodology and specialized analysis “pipelines,”
mainly in the field of genomics, are included in bioinformatics.
Single nucleotide polymorphisms and candidate gene identification are frequent
applications of bioinformatics (SNPs). Such identification is frequently done to
comprehend the various genetic underpinnings of disease, particular adaptations,
desirable traits (especially in agricultural species), or population-level variations.
FIGURE 7.1
Soil rhizosphere and microbe interaction.
128 CHAPTER 7 Rhizospheric microbiome

Proteomics, a less formal term for bioinformatics, is the study of the organizing prin-
ciples found in nucleic acid and protein sequences.
Large amounts of raw data are used to extract useful results using image and
signal processing. They support the sequencing and annotation of genomes and their
observed mutations in genetics. They contribute to the organization and querying of
biological data through the text mining of biological literature and the creation of
biological and gene ontologies. They also contribute to the study of the regulation
and expression of genes and proteins. The comparison, analysis, and interpretation
of genetic as well as genomic data to understand the evolutionary aspects of molec-
ular biology have been made easier with the aid of bioinformatics tools. They aid in
the analysis and cataloging of the biological networks and pathways that are a
crucial component of systems biology on a more integrative level.
DNA sequences of organisms have been sequenced, decoded, and stored in da-
tabases ever since the Phage-X174 was sequenced in 1977. This sequence data is
analysed to identify and understand regulatory sequences as well as structural mo-
tifs, repetitive sequences, RNA genes, genes encoding proteins, and regulatory se-
quences. Assessment of genes within a species or different species can illustrate
resemblances between protein functions, or relations between species (the use of
molecular systematics to construct phylogenetic trees). Computer programs such
as basic alignment search tool (BLAST) are used regularly to search sequences
from more than 260,000 organisms, containing over 190 billion nucleotides.
Fig. 7.2 explains various steps involved in genome sequencing steps.
3.Bioinformatics impact on genomics
The worldwide human genome project and a private genomic company’s work
resulted in the announcement that the entire human genome had been mapped.
But in recent years, many other organisms’ complete genome sequences have
been completed, which has amazed the scientific community. A major accomplish-
ment for bioinformatics is the analysis of the newly emerging genomic sequence
data. In 1995, the Haemophilus influenzae was sequenced using a novel method
FIGURE 7.2
Steps involved in genome sequencing steps.
3. Bioinformatics impact on genomics 129

for randomly sequencing the entire genome (the so-called “shot gun technique”).
This was the primary fully sequenced genome of a free-living organism. Soon after,
the genomes of other bacteria, including Mycoplasma genitalium and Mycobacte-
rium tuberculosis, were sequenced. Later, the genome of the bacterium that causes
the plague, Yersinia pestis, was sequenced.
Saccharomyces cerevisiae, a yeast, had the first eukaryotic genome sequenced
and annotated, and then came the genomes of other eukaryotic species like Caeno-
rhabditis elegans, a worm, Drosophila melanogaster, a fruit fly, and Arabidopsis
thaliana (mustard weed).
Several other species are currently sequenced by both private and public
sequencing initiatives, including zebrafish, pufferfish, mice, rats, and nonhuman pri-
mates. The information learnt from these sequence data will have a significant
impact on how to understand biology and medicine. Comparative genomic and pro-
teomic research will soon allow us to identify every human gene and fully compre-
hend each one’s function.
4.Bioinformatic tools
A bioinformatician’s primary resources are the internet and computer software.
Sequence analysis of DNA and proteins by means of various online databases and
programs is a fundamental activity. Anyone with internet access and access to rele-
vant websites, including clinicians and molecular biologists, can now freely learn
the structure of biological molecules like nucleic acids and proteins by using simple
bioinformatic tools. This does not mean that everyone can handle and analyse raw
genomic data with ease. Expert bioinformaticians now use sophisticated software
programs to retrieve, sort, analyse, predict, and store DNA and protein sequence
data as the field of bioinformatics develops.
Bioinformaticians are employed by large businesses like pharmaceutical firms to
perform and sustain the complex bioinformatic requirements of these industries.
Most biomedical laboratories need their in-house bioinformaticians due to the
growing need for constant input from bioinformatic experts.
Basic Local Alignment Search Tool (BLAST) is one of the most straightforward
and well-known search engines and can be found at www.ncbi.nlm.nih.gov/BLAST/.
This algorithm software enables the comparison of an unknown DNA or amino acid
sequence with hundreds or thousands of sequences from humans or other organisms
until a match is found. It is also capable of searching databases for genes with similar
nucleotide structures. Thus, similar sequences that could be homologs of the query
sequence are found using databases of known sequences. According to homology,
sequences may be related by their divergence from a common ancestor or by having
similar functional characteristics. When a newly discovered sequence (referred to as
the query sequence) is used to search a database, local alignment between the query
sequence and any related sequences in the database takes place.
130 CHAPTER 7 Rhizospheric microbiome

Bioinformatics is now used for a wide variety of other significant tasks in addi-
tion to the analysis of genome sequence data, such as the analysis of gene variation
and expression, the analysis and prediction of gene and protein structure and func-
tion, the prediction and detection of gene regulatory networks, simulation environ-
ments for whole-cell modeling, complex modeling of gene regulatory dynamics and
networks, and the presentation and analysis of molecular pathways to reverse dis-
ease. Designing primers short oligonucleotide sequences required for DNA amplifi-
cation in polymerase chain reaction experiments and predicting the function of gene
products are two examples of straightforward bioinformatic tasks that are useful to
clinical researchers, albeit on a smaller scale.
5.Bioinformatic resources and platforms for plant microbes
interaction study
Numerous online resources offer software tools, instructions, protocols, and guide-
lines for processing, statistical analysis, and visualizing metabolomic data. Plat-
forms that concentrate on biomarker discovery and classification, like the
Metabolic Workbench (Sud et al., 2016), XCMS for MS-based data (Gowda
et al., 2014), or MetaboAnalyst (Xia et al., 2015), offer a wealth of resources to
this end. Plant-specific databases, such as PlantSEED, which provides annotation
and model data for 10 plant genomes (Seaver et al., 2014), or Gramene/Plant Reac-
tome, a free and open-source, curated plant pathway database portal (Naithani et al.,
2017;Tello-Ruiz et al., 2018) can be used to annotate plant genomes and build meta-
bolic models. The Plant Metabolic Network, with the PlantCyc database containing
1200 pathways in more than 350 plant species as of version 12.0, is another enor-
mous resource for plant metabolic networks (Schlapfer et al., 2017). Overall, com-
plete annotation of plant metabolomes has not yet been accomplished, despite
ongoing advancements in nontargeted metabolomics (Viant et al., 2017).
The molecular features of the rhizosphere microbiome were mapped by Oyser-
man et al. (2022) as quantitative traits of a diverse hybrid population of wild and
domesticated tomatoes. A genetic basis for variance of different rhizobacterial lin-
eages is suggested by gene content analysis of prioritized tomato quantitative trait
loci. This analysis includes a Streptomyces-associated 6.31 Mbp region harboring
tomato domestication sweeps and encodes, among other things, the iron regulator
FIT and the water channel aquaporin SlTIP2.3.
Bacterial genes are involved in the various metabolisms of plant
polysaccharidesdiron, sulfur, trehalose, and vitamins within metagenome-
assembled genomes. Streptomyces and Cellvibrio genetic variation associates with
particular tomato QTLs. Putative plant and reciprocal rhizobacterial traits underly-
ing microbiome assembly by combining “microbiomics” and quantitative plant ge-
netics, paving the way for future research on the relationship between the plant and
the microbiome breeding programs.
5. Bioinformatic resources and platforms for plant microbes interaction 131

Rhizosphere microbiomes of wheat and chickpea were homogenous (65%e87%
similarity) in the presence of decaying root (DR) systems, they were heterogeneous
(3%e24% similarity) where DR was disrupted by tillage. This suggests that the
detritussphere microbiome controls the composition and function of the rhizosphere
microbiome to a greater extent than plant type. When the rhizosphere and detritus-
sphere microbiomes interact in the presence of DR, the rhizosphere microbiome
significantly degrades plant root exudates, and genes linked to membrane trans-
porters, carbohydrate, and amino acid metabolism are enriched (Zhou et al., 2020).
In terrestrial ecosystems, the rhizosphere microbiota is crucial for plant growth
and proper health. Numerous investigations have been conducted to characterize the
microbial communities in various soil types as well as their rhizospheric interac-
tions. Ma et al. (2020) sequenced the 16S bacterial rRNA genes and the fungal
ITS regions from the rhizosphere microbiota using high-throughput amplicon tech-
nology and a number of bioinformatics and statistical tools. It was later discovered
that a number of widespread bacterial and fungal communities were very much
crucial for the survival of the healthy plant growth.
Finally, using variance analysis, the soil-preferred microbiota was found and
further characterized at the genus level. Rich and varied microbial communities
were found in the thin layer of soil that adheres to the roots. Different soil types
have similar dominant microbial phyla compositions, but the size of individual mi-
crobial community varies greatly. In particular, as the soil ecosystem shifts from clay
to sandy, the abundance of Firmicutes rises from 9.69% to 61.66%, which can not
only promote the breakdown of complex plant materials but also be in charge of
pathogen disinfestation.
Understanding the rhizospheric ecology at the molecular level has been acceler-
ated by the current development in high-throughput sequencing technologies, the
advent of the omics era. A number of bioinformatics tools have been introduced
by the hybrid multiomics approach and are being used to reveal complex microbial
interactions in the active rhizospheric zone. Metagenomic analysis applications
aided in the understanding of structural and functional diversity. In addition, unlike
the majority of the literature on plant-microbe studies that is currently available, the
significance of hybrid multiomics methods examined through computational pro-
gramming and various bioinformatics tools. Rhizomicrobiome bioengineering
would be made easier by the combined strength of multiomics techniques and
related bioinformatics (Pal &Sengupta, 2022).
Sun et al. (2022) discovered the syntrophic cooperation between the inoculant
(Bacillus velezensis SQR9) and the native, plant-friendly Pseudomonas stutzeri in
the cucumber rhizosphere using bioinformatic, genetic, transcriptomic, and metab-
olomic analyses. The synergistic interaction between these two species is highly
environment-dependent; syntrophic cooperation only became apparent in a static
niche that was rich in nutrients, such as pellicle biofilm and the rhizosphere. The
findings showed that syntrophic cooperation involves pathways for the biosynthesis
of branched-chain amino acids (BCAAs). Additionally, various metabolic profiling
as well as genome-scale metabolic modeling revealed metabolic facilitation among
132 CHAPTER 7 Rhizospheric microbiome

the bacterial strains. Bacillus biofilm matrix components were additionally crucial
for the interaction. The two-species consortium had an important impact on plant
growth and salt stress reduction.
Sen et al. (2022) evaluated six soil samples from the Darjeeling hills’ Alnus
rhizosphere and nonrhizosphere were compared using a 16S rRNA amplicon anal-
ysis. Through the MG-RAST web server, bioinformatics analyses were carried
out. Thirty-two core bacterial genera were found in both the rhizosphere and the
nonrhizosphere, according to the findings. Contrary to nonrhizospheric or bulk
soil, Alnus rhizospheric soil samples had a higher concentration of nitrogen-fixing
taxa. Nitrogen-fixing microorganisms like cyanobacteria and frankia may contribute
significantly to the healthy growth and development of these plants. They are essen-
tial for boosting the soil’s nitrogen content through nitrogen fixation, gradually
boosting soil fertility, and assisting Alnus in developing properly through various
stages of succession.
6.Proteomics
The study of proteins in a microbial community from an environmental sample is
known as metaproteomics. Metaproteomics, which reflects the structure, dynamics,
and metabolic activities of microbial communities, provides direct evidence for pro-
teins, posttranslational modifications, protein-protein interactions, and protein turn-
over (Hettich et al., 2013). Metaproteomics typically makes use of techniques from
mass spectrometry (MS)-based proteomics.
6.1 Experimental methodologies
For large-scale, high-throughput experiments to identify and quantify (characterize)
thousands of microbial proteins, MS-based proteomics is a potent analytical tech-
nique. Top-down and bottom-up approaches to analyze intact proteins or peptides
from synthetic proteolytic digestion, respectively, can be distinguished in MS-
based proteomics. The more popular bottom-up strategy will be the main focus of
this review. Sample lysis, protein extraction, protein separation, proteolytic digest,
peptide fractionation, and MS analysis are the principal experimental steps, in
that order (Siggins et al., 2012).
6.2 Computational proteomics
Millions of spectra are easily produced by MS analysis in a large-scale bottom-up
experiment, necessitating automated mass spectral interpretation. Spectrum prepro-
cessing, peptide identification, quantification (e.g., label-free), protein grouping, and
in a metaproteomic context LCA analysis, e.g., UniPept (Mesuere et al., 2018) and
Megan, are significant steps in the computational workflow (Huson et al., 2007).
Metaproteomics relies heavily on peptide identification to infer the majority of a
6. Proteomics 133

microbial sample’s components. Database searching, de novo sequencing, such as
PEAKS (Ma et al., 2003), and spectral library searching, such as SpectraST (Lam
et al., 2007), are some of the most widely used methods to assign a peptide sequence
to a spectrum.
A protein sequence database is in silico digested and fragmented during database
searching to produce theoretical spectra that can be compared to experimental
spectra. Although the majority of protein sequence databases use gene predictions
from primary genome assemblies, they are constructed from a variety of omic sour-
ces. Accordingly, the content in reference databases like UniProtKB (Pundir et al.,
2017), RefSeq (O’Leary et al., 2016), or Ensembl is significantly influenced by the
genome’s quality and its assembly (Zerbino et al., 2018). A database in proteomics
must strike a balance between three factors: complexity for statistical validation
down the line, completeness to identify the majority of constituents, and size to
manage sensitivity and processing time (Zerbino et al., 2018).
Different methods add to current reference databases or create unique databases
that take the microbial communities into account to address those aspects of meta-
proteomics. To create sample-specific, custom protein sequence databases, the pro-
teogenomics field uses metatranscriptome or metagenome data (Nesvizhskii, 2014).
This is particularly helpful for nonmodel organisms that lack a reference genome
database or to find novel proteins that are not included in a reference database. To
analyse MS data without extensive prior genome annotation, even draught metage-
nomes are sufficient (Armengaud et al., 2014). Many short read assembly algorithms
in metatranscriptomics and metagenomics use de Brujin graphs as their main data
structure to infer primary assemblies.
To match MS spectra and create a more extensive database of putative proteins:
The loss of sensitivity brought on by an increase in the number of databases or the
size of databases is a frequent problem for metaproteomics and proteogenomics
(Jagtap et al., 2013). The creation of a smaller database from a “survey” search in
two steps and database clustering before searching are two techniques for reducing
the size of databases (Marx et al., 2013).
6.3 Metaproteomics in plant microbialeassociated studies
For example, in studies of the plant microbiome, metaproteomics was used to assess
the bacterial communities in the phyllosphere of different tree species in a pristine
Atlantic Forest (Lambais et al., 2017), to examine the reaction of the plant PGPB
Bacillus amyloliquefaciens FZB42 to the presence of plant root exudates (Kierul
et al., 2015), and to compare the differences in soil protein abundance.
Despite its successful application, the plant microbiome’s use of metaproteomics
is constrained by the lower protein expression of plant microbial samples and the
paucity of database data (Levy et al., 2018).
134 CHAPTER 7 Rhizospheric microbiome

7.Recent and new approaches to study plant-microbe
interactions
The recent development of high-throughput sequencing in conjunction with a vari-
ety of “omics” techniques enables researchers to identify host interactions and
microbiome structure and dynamics at a previously unheard-of level. In-depth
knowledge about the identity and relative abundance of the microbial partners of
plants is made available by modern sequencing techniques. The cultivation of micro-
bial isolates is not required because sequences are generated directly from the envi-
ronmental sample (Epstein, 2013;Hug et al., 2016). Although the freedom provided
by sequencing technology can lead to a flood of data, this must be mitigated by
choosing an experimental strategy and sequencing methodology that are pertinent
to the particular scientific question at hand. When selecting a specific sequencing
method, care should be taken to carefully consider the types of expected biases
and errors.
To better understand the makeup, structure, and spatial distribution of microbial
communities in the environment, high-throughput sequencing of marker gene ampli-
cons is increasingly used in plant microbiome studies (Knief et al., 2012). Amplicon
sequencing has the advantage of being able to target single groups of microbes (such
as Bacteria, Archaea), or even functional genes (DsrA, AmoA, etc.) with extreme
specificity (Herbold et al., 2015).
Amplicon sequencing’s high specificity makes it possible to use it to positively
identify even rare organisms, but because of its sensitivity, it is also vulnerable to
contamination (Glassing et al., 2016). For any experiment that heavily relies on
amplicon sequencing, it is crucial to include both positive (known mock commu-
nities) and negative controls (reagent and extraction blanks).
Shotgun metagenomics can “bin” the data into draught genome sequences, but it
is less accurate than amplicon sequencing at detecting the presence of rare organ-
isms (Poretsky et al., 2014). Amplicon sequencing can also “bin” data into a
more accurate abundance measurement. These make it possible to link taxonomic
identity to vital plant processes like nitrogen fixation or to ascertain whether symbi-
onts may be able to “communicate” with plants via secretion systems or effectors
(Eichinger et al., 2016). Other high-throughput molecular methods, such as tran-
scriptomics, proteomics, and metabolomics, can be used to supplement metage-
nomic approaches.
Metatranscriptomics are well established in the study of the human microbiome
(Bashiardes et al., 2016) and can be used as a model for application in the study of
the plant microbiome. The most recent review of RNA-seq data analysis is the best
practices (Conesa et al., 2016). Metaproteomic data can be used to identify post-
translational modifications, frameshifts, and offer insights into entire microbial com-
munities in plants, in addition to providing evidence for protein expression and
quantification (Nesvizhskii, 2014). Fig. 7.3 explains the flowchart outlining steps
taken in a typical metagenome analysis.
7. Recent and new approaches to study plant-microbe interactions 135

By examining plant metabolomes, researchers can learn about primary and sec-
ondary plant metabolites that may interact with the host’s microbiome (plant solute
transport) as well as surface exudates secreted by phylloplane and rhizoplane on the
outside (van Dam &Bouwmeester, 2016).
Understanding the functions of microbes and how they interact with plants has
greatly benefited from bioinformatic analysis (Cha et al., 2016;Koberl et al.,
2013;Marasco et al., 2012;Spence et al., 2014). For instance, metagenomic data
FIGURE 7.3
Flowchart outlining steps taken in a typical metagenome analysis.
136 CHAPTER 7 Rhizospheric microbiome

analysis was initially used to determine that Pseudomonas spp. was to blame for
sugar beet affliction in soil that was resistant to Rhizoctonia solani (Mendes et al.,
2011). Testing computational predictions in the lab or on the job, however, is
frequently not an easy task. Computational modeling (Scheuring &Yu, 2012) and
synthetic community experiments combined with multiomics are recent efforts to-
ward engineered plant microbiomes (Vorholt et al., 2017).
8.Conclusion
The ecological significance of enlisting particular rhizosphere microorganisms for
plants against pathogen invasion has been made clear by high-throughput rhizo-
sphere microbiome profiling and perturbation experiments.
These developments have increased our understanding of the interactions be-
tween plants and microbes, and more study in this area will significantly advance
one crucial idea, utilizing the rhizosphere microbiome in disease resistance. But
there are still a number of crucial issues that need to be resolved.
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Microbiome biodiversity—
current advancement and
applications 8
Shital Shinde, Swapnil Kadam, Vipul Patel, Sharav Desai
Sanjivani College of Pharmaceutical Education and Research, Kopargaon, Maharashtra, India
1.Introduction
In the past ten years, there have been fascinating developments in our understanding
of microbes from various ecologies, such as complex which take place beneath the
surface of water. These occur at constructive margins, subduction zones, and within
tectonic plates due to hotspots (Danovaro et al., 2017), hardy survivors in the Ata-
cama desert (an ecology similar to Mars) (Schulze-Makuch et al., 2018), and “huge
phages” explored in a variety of ecosystems (Thompson et al., 2017). Focused ini-
tiatives, such as the Tara Expeditions and Earth Microbiome project (Moss et al.,
2020) that utilize extensive sampling and next-generation sequencing (NGS) have
greatly increased the number of recognized microorganisms. It is sobering to realize
that, despite our best efforts, we are only sampling a tiny portion of the 1012 micro-
biological species forecast is a statement about a future event or data to exist at this
time (Chen et al., 2019) and an even smaller portion of the organisms that have ever
existed and will exist in the future. Despite how important these efforts are to
furthering our knowledge, they are still incredibly insignificant compared to the
vast majority of organisms. We predict that these unique microbes will teach us
new things about the laws of life and biocatalyzed chemistry in the coming decade,
for instance, methods to put together whole genomes. The ability to monitor mobile
genetic elements (Durrant et al., 2020;Moss et al., 2020) and situated or controlled
by factors outside the chromosome extrachromosomal inheritance elements (Beitel
et al., 2014) might ultimately enable us to fully understand the processes of genetic
material interchange and evolution. In the same direction that CRISPR/Cas systems
have revolutionized molecular biology, this will also give us insight into novel clas-
ses of enzymes that communicate with nucleic acids in essential processes like DNA
repair, epigenetic modifications, and recombination and have translational utility in
genome engineering. While novel species may now be explored because of techno-
logical advancements, Escherichia coli and other well-studied model organisms are
now becoming better understood. For illustration, improvements in biochemical,
scientific computation and microscopy-based tools have made it possible for us to
examine the kind and degree of subcellular structure in this well-known organism,
leading to fascinating new fundamental discoveries. Unbelievably many
CHAPTER
143
Rhizobiome. https://doi.org/10.1016/B978-0-443-16030-1.00021-3
Copyright ©2023 Elsevier Inc. All rights reserved.

microorganisms that can achieve cell densities substantially higher than the number
of plant cells inhabit plants. Additionally, there are many more microbial genes in
the rhizosphere than plant genes. Numerous studies have shown that various micro-
organisms associated with plants can have a significant impact on seedling from a
seed, seedling vigor, primary plant growth and improvement, nutrition, diseases,
and production. The collective populations of microorganisms associated with
plants are referred to as the plant microbiome or as the plants’ second genome, in
keeping with nomenclature used for bacteria inhabiting the human body. In this
sense, plants may be thought of as supraorganisms that depend on their microbiota
in part for the particular features and activities. In consequence, plants release the
carbon they have fixed through photosynthetic processes into the spermosphere,
phyllosphere, rhizosphere, and mycorrhizosphere, which are their immediate
surroundings.
2.Rhizosphere microbiota
The microbiome may be summarized as a collection of microbial genomes in a
particular environment. The rhizosphere, a region of intense biological activity
that surrounds the root system, is home to numerous intercourses among plants
and microbes that have a significant negative influence on the performance of the
host. As a function, maintaining homeostasis in the soil ecosystem depends on the
variety of rhizosphere bacteria. Numerous species with advantageous characteristics
that influence plant development and robustness make up the microbes population
that inhabit the rhizosphere. These species include soil-borne plant diseases in addi-
tion to nitrogen-fixing bacteria, growth-promoting rhizobacteria (PGPR), biocontrol
agents, and plant mycorrhizas fungi. Diverse quantities and kinds of organic chem-
icals released by plant roots are having an impact on this complex microbial
ecosystem. Root exudates differ among plant species, shift along the root axis,
and change in response to disease assault, nutritional condition, and stress. Through
the activation or repression of members of the rhizosphere community, the sub-
stances released by the roots can influence the microbiome’s structure. As a result,
the kind and volume of root exudates influence the enrichment of particular micro-
bial communities. The soil type is another important factor influencing the rhizo-
sphere microbiome structure, together with the genotype of the plant and its
developmental stage (Grunert et al., 2019). The variety and abundance of bacteria
in the rhizosphere are also influenced by nutrition, carbon content, soil pH, biogeog-
raphy, and microbial interactions (Qiao et al., 2017). Given that the microbiome may
affect plant growth at various developmental stages, it is crucial to comprehend that
interactions between the host plant and its microbiome play a key role in health and
production. The connection of a number of variables and microorganisms that re-
sults in favorable conditions for plant health must be considered when investigating
the microbiome’s ability to support plant development. The application of microbial
consortiums, the use of management techniques that enrich the microbial
144 CHAPTER 8 Microbiome biodiversity

community with advantageous functions, or plant breeding strategies that take the
microbiome into consideration are just a few examples of how this knowledge
can be useful in the agricultural context (Compant et al., 2019). When the
complexity of microbiome-mediated protection is understood, sustainable solutions
for ensuring healthy production systems that take into account interactions between
the microbiome and the soil, plants, and environment may be developed. Thus, the
host plant’s genetic characteristics (abiotic parameters), pathogens, and the soil
microbiome (biotic environmental variables) all have a role in determining the
plant’s health condition. According to a recent study (Carrio
´n et al., 2019),
disease-suppressive soil contains beneficial microbial taxa that act as a protective
layer of microbial defense not only in the rhizosphere of plants but also in the
root endosphere. These findings show how some bacterial families are more abun-
dant in the endosphere, where they provide crucial plant defense mechanisms that
function as a second microbial layer of plant protection.
3.Ecology
The living network of plant roots is encircled by the rhizosphere, which is situated
under the soil’s surface and is a hub of biologic, biochemical, and physical activity,
is the intersection of the soil habitat. This environment is distinct from bulk soil by
complex fine-scale gradients in substrate availability, water potential, and redox
state, which also limit the distribution and activity of the incredibly varied rhizo-
sphere biota (Voss-Fels et al., 2018). Along with plant roots, this site is home to
groups of animals, fungi, bacteria, protists, and archaea. The actions of each popu-
lation affect those of the others on orders-of-magnitude of time and space scales
(Cook et al., 2020). The way that this biota trade and transform resources like
organic carbon, mineral nutrients, and water, which function as universal biological
currencies, dictates the pathways of energy flow and moulds community structure
and ecosystem characteristics. Through processes including quorum sensing and
the creation of phytohormone mimics, information is also conveyed among rhizo-
sphere dwellers. Beyond the rhizosphere itself, the effects of rhizosphere activity
may be seen in patterns of community organization, ecosystem processes, and soil
formation that are consistent with time and place across the landscape. Addressing
contemporary environmental concerns in both natural and managed ecological pro-
cesses across the world requires an understanding of soil’s biological, physical, and
chemical activity. Although understanding of above-ground processes has improved,
understanding of below-ground function has lagged behind (Adl et al., 2019). The
fields of agronomy, mycology, plant physiology, and microbiology have all long
been areas of interest in rhizosphere ecology. Understanding soil’s physical and
chemical characteristics, plant biology, and the activity and structure of microorgan-
isms and soil organisms is necessary for modern rhizosphere research, which is
multidisciplinary by necessity (Ourry et al., 2018). Rhizosphere-focused syntheses
reflect this multidisciplinary character and also highlight the practical significance
3. Ecology 145

of rhizosphere research for the management of natural and agricultural ecosystems.
In more recent works, specialized areas of rhizosphere biology have been explored,
frequently using reductionist methods to look at the nature and purpose of particular
types of interactions (Starr et al., 2021). First, numerous chapters provide a deeper
grasp of the glacial-interglacial and solid characteristics of the rhizosphere environs
and habitat as well as a correlate with comprehension of the impact of rhizosphere
ecology on the course of soil genesis. Under soil evolution, rhizospheres are basic
operators of considerable soil hydrological alteration and overall soil enlargement,
so several that nearly all soil might be shown as rhizosphere soil of varying ages.
The analysis of the patterns and effects of phylogeny and effective diversity in the
rhizosphere is a second focus. For instance, functional biodiversity associated
with mycorrhizal evolutionary ancestry has significant consequences for broad,
biogeographic specimen in under-ground function even at global scales. The depen-
dence of the mycorrhizal symbiotic partners on freeliving saprotrophs for fossiliza-
tion of nutrients from natural matter suggests a biogeographic gradient from
grasslands to tundra. Saprophytic capacities are minimum among plural mycor-
rhizae and maximal among cricoid mycorrhizae. It highlights the need of most rele-
vant biodiversity in the rhizosphere in order to produce agriculture systems that are
self-sufficient and require less fertilizer inputs. They emphasize that, prior to the
heavy-input, mechanized agriculture practiced today, plants and the rhizosphere
biota they were associated with evolved together in the functioning rhizosphere sys-
tem, only to be conceptually and physically separated when tillage, fertilizer, and
pest control inputs were used. These opposing and conflicting viewpoints imply
that the confluence of reductionist and integrative ecosystems methods will produce
the most potent discernment into the aspect of rhizosphere ecology. Greater scale
principle, a biogeochemical or biogeographical setting, or ancient context can be
used to help direct the target and interpolation of mechanistic investigations because
the rhizosphere’s enormous organismal diversity makes it impossible to examine all
of the potential interactions and mechanistic controllers in detail. Therefore,
research addressing the function of larger-scale or higher-order ecological systems
can more easily take into account the possible consequences of recently revealed
fine-scale rhizosphere patterns and processes. Recent discoveries that shed light
on the rhizosphere are discussed below (Sokol et al., 2022).
4.Rhizosphere microbiome assembly and its impact on
plant growth
In the soil that adheres tightly to the roots of tellurian plants, such as the rhizosphere
and the root microbiota, there is a very complex microbiota community (designed as
endophyte and epiphyte). One of the most dynamic interfaces on Earth is the rhizo-
sphere, which is the soil next to roots at a distance of millimeters. It offers a niche for
interactions between plant roots and microorganisms (Zou et al., 2021). In the rhizo-
sphere environment, connections between microbes and plants that are mediated by
146 CHAPTER 8 Microbiome biodiversity

biochemistry are now understood to be widespread. The two elements of plant-
microbe interactions are.
(a) Numerous elements, including biotic elements like diseases and insect pests, as
well as abiotic elements like soil types, climate, and root exudates, have an
impact on the microbial communities that live in the plant rhizosphere (Sasse
et al., 2018). Root phenotypes, plant species (Schmidt et al., 2019), genotypes,
and developmental phases are all discussed in detail in the following sections
(Jin et al., 2018;Cotton et al., 2019).
(b) By supplying bioavailable mineral nutrients and producing phytohormones
(Zhang et al., 2019), rhizosphere microbiota, also known as plant growth-
beneficial rhizosphere bacteria, cause a feedback control on plant development
and disease resistance (PGBR).
Over the last century, microbial products have been used in agricultural produc-
tion, but as new analytical and omics tools are developed, biochemical mechanisms
that drove the coevolution between beneficial microbiota and plant hosts are now
progressively emerging (Lu et al., 2018;Thomashow et al., 2019). In comparison
to traditional agrochemicals, microbial products made from PGBR are viewed as po-
tential biofertilizers, biopesticides, and biostimulators, since they are more “green”
and sustainable. The number of microbial items utilized as agricultural fertilizers or
crop management equipment has increased to about 400 (Finkel et al., 2017). How-
ever, more innovative and effective microbial products are required to bridge the gap
between functional research and actual PGBR applications.
5.Rhizosphere differentials that affect the microbial
community assembly
The “rhizosphere effect,” an occurrence where the microbial population in the rhizo-
sphere is different from the suburb in large soils, suggests that plant roots draw
certain microorganisms from bulk soil into the rhizosphere and collect them there.
More and more data suggested that the rhizosphere contains species-specific micro-
bial communities. Utilizing 16S rRNA gene amplicon sequencing, a study of the
microorganism populations in the rhizosphere of several types of herbaceous plants
revealed that each plant species evaluated had unique operational taxonomic units
(OTUs) that acted similarly to every plant species rhizosphere (Finkel et al.,
2017). Although the influence of the plant genotypes on the microbiome assembly
is fairly limited, different genotypes within the same species can also create unique
rhizosphere microbial communities (Brisson et al., 2022). However, our results show
that host genetics influence plant microbiome assembly. Numerous studies demon-
strate that plant development phases, soil types, and root morphologies all influence
the rhizosphere microbial populations (Brisson et al., 2022). Additionally, recently,
the impact of plant pests or diseases on the development of rhizosphere microbiomes
was highlighted: While the western corn insect-resistant, a pest maize louses, pushes
5. Rhizosphere differentials that affect the microbial community assembly 147

particular microbial taxa (such as Acinetobacter lactucae, Hoyosellaceae, and Aero-
microbium) in the rhizosphere, the false mildew pathogen inoculating on Arabis
thaliana leaves modifies the rhizosphere microbial community. Unlike other influ-
ences, the plant itself has the ability to produce signals that involve unknown low
molecular weight chemicals to affect the rhizosphere microbiome. The well-
known signaling mechanism known as quorum sensing (QS) hinges on how the
rhizobacteria cell density controls the gene expression of microbial physiological
activities. QS enriches a large number of plant-associated bacteria in plant-
associated settings and is necessary for QS to control a number of critical activities,
including rhizosphere competition, conjugation, and biofilm maturation. Although
the precise pattern of compound action is unclear, QS may be affected by
QS-mimicking exudates from plants, suggesting that plants have some hidden mech-
anisms by which they might impact the rhizosphere bacteria. Overall, the aforemen-
tioned elements have a significant role in defining the makeup of the microbial
community in the rhizosphere, but there are still other crucial but unidentified ele-
ments that play a role in determining the microbiome composition. Because of
this, we still have a limited grasp of how biotic and abiotic variables influence the
composition of the rhizosphere community (Massalha et al., 2017).
6.Plant growth variationsdmicrobiome assembly and root
metabolism
The rhizosphere microbiome is shaped by the development, aging, and multiplica-
tion of plants. For instance, plant roots discharge root cap border cells into the rhizo-
sphere as a form of rhizodeposition to increase the rhizospheric impact and to attract
particular microbes. According to Vicre
´et al. (2005),A. thaliana root caps produce
borderlike cells to encourage the growth of Rhizobium sp. YAS34. In addition to pri-
mary and secondary metabolites including sugars, organic acids, amino acids, muci-
lage, and so on, root exudates also include around 10% of photosynthetically fixed
carbon and about 15% of all plant nitrogen. Plant roots with a sophisticated trans-
membrane system emit these compounds (Canarini et al., 2019). It is important to
note that different plant species produce different amounts and types of root exu-
dates (Lu et al., 2019). For example, in order to attract B. amyloliquefaciens
SQR9 and create a unique biofilm, cucumber roots released citric acid.
A. thaliana’s particular root segment allows Bacillus subtilis to invade thanks to a
number of amino acids in the root exudate. The organic acids and sugars in the
exudate from tomato roots encourage the colonization of Pseudomonas, an anti-
fungal organism. The genotype and physiological state of the host influence exuda-
tions. For example, various genotypes have varied amounts of glucosinolates,
flavonoids, and phenylpropanoids, and these exudates are crucial in determining
the sort of rhizosphere microbiome that exists and used benzoxazinoid (BX) muta-
tion (Huang et al., 2019).
148 CHAPTER 8 Microbiome biodiversity

Knockout mutants are employed to further illustrate this viewpoint. Exudation
varies according to the phases of plant development. It examined the abundances
of four phyla in root exudates from the thaliana plant at several developmental
time periods, including seedling, vegetative, bolting, and flowering. Significant re-
lationships exist between the four phyla and the previously stated root exudate
chemicals. The rhizosphere microbiome is really regulated by plant species, geno-
types, and developmental stages in addition to their diurnal cycle, which contradicts
the conventional wisdom that it is comparatively stable over a short period of time.
The circadian clock functions as a “pushing hands” mechanism for the physiological
processes that occur in plants throughout the day. Kudjordjie et al. (2019) showed
that a broken circadian clock has a significant influence on the microbiome of
A. thaliana, especially the uncommon species; however, the study did not identify
the primary causes of rhizosphere microbiota alteration. In contrast to the composi-
tion of organic matter in the rhizosphere, which significantly varies between light
and dark cycle samples, root exudates are measured in accordance with the biolog-
ical clock’s pattern and part of them are verified within a diurnal mucociliary pattern
to test the potential factors involved in the diurnal rhythms driving root-associated
grassroots shifts. Additionally, A. thaliana’s bacterial communities differed dramat-
ically during the cycle of light and darkness, and a 13% relative abundance of
family-level communities was discovered to exhibit a circadian rhythm. Similar to
the abundance of certain rhizosphere bacteria in fodder, this is not circadian but is
higher at predawn than at postdawn, these oscillations in gene expression were
compatible with the changes in glucose and amino acid metabolism in the times
of day and night (Baraniya et al., 2018).
6.1 Abiotic and biologic stresses alter root exudation
Abiotic and biotic stressors are frequent challenges for plants growing in compli-
cated environments. The abiotic and biotic factors that affect the rhizosphere micro-
biota must be studied.
(i) Nutritional stress promotes the formation of the root microbiome. Although
there is a lot of phosphorus (P) in the biosphere, only inanimate orthophos-
phate can be used by plants. When phosphate stress occurs, phosphate star-
vation responses (PSRs) are activated, which control a large number of
genesda process that produces both main and secondary metabolites (organic
acids, glucosinolates), as well as the makeup of the root microbiome
(ii) Rhizosphere communities are altered by pesticide residues and other envi-
ronmental pollutants. Atrazine inoculation of soil considerably enriched three
OTUs: Halobacillus uncultured, Bacillus decolorationis, and Cesiribacter sp.
JJ02, indicating a direct influence (Xu et al., 2018). Furthermore, glyphosate
applied to leaf surfaces can be transferred by maize to soils, greatly increasing
the quantity of Fusarium on maize roots. Notably, pesticides can modify plant
exudation, which can change the microbiota in the rhizosphere. For instance,
6. Plant growth variationsdmicrobiome assembly and root metabolism 149

after being treated with diclofop-methyl, rice seedlings greatly enhanced
the root exudation of amino acids, organic acids, and fatty acids. This altered
the abundance and variety of microorganisms in the rhizosphere and raised the
relative abundance of the Massilia and Anderseniella taxa. Other environ-
mental pollutants, that is Ag nanoparticles, plastic film residues (Chen et al.,
2017), antibiotics (Qian, Zhu, et al., 2018), chiral pesticides, and single-walled
carbon nanotubes, also affected the soil microbial population (Liu et al., 2020).
(iii) Infection by plant pathogens modifies the rhizosphere community. Inoculating
A. thaliana leaves with Pseudomonas syringae pv tomato alters the root
exudation patterns of plants by enriching the rhizosphere significantly with the
Roseiflexus genus and increasing the exudation of amino acids, nucleotides,
and long-chain organic acids while lowering the exudation of sugars, alcohols,
and short-chain organic acids. This alteration in exudation patterns attracts
more advantageous rhizosphere microorganisms, enhancing A. thaliana’s
ability to fend against above-ground diseases (Qian, Ke, et al., 2018). Carex
arenaria is a well-known example because, following a Fusarium culmorum
infection, its root emits a specific collection of volatile organic compounds
into the rhizosphere, attracting a unique rhizosphere community that is distinct
from the rhizosphere of wholesome plants(Schulz-Bohm et al., 2018;Yuan
et al., 2018). Salicylic acid A. thaliana mutants develop certain bacterial taxa
in the root microbiome in response to pathogen infection, presumably as a
result of SA mutants altering the root exudate profiles. Mutants of A. thaliana
have distinct root microbiomes and root exudates, which include less orni-
thine, tryptophan, and asparagine but more Enterobacteriaceae in the myc2
rhizosphere and Bacillus,Lysinibacillus, and Streptomyces in the med25
rhizosphere, respectively. These investigations demonstrated a strong associ-
ation between the rhizosphere microbiota and the plant mechanism of defense.
Overall, the rhizosphere microbiome assembly must depend on the connec-
tions between roots and the microbiota that are mediated by biochemistry. The
precise mechanisms by which the rhizosphere microbial colony is assembled
continue to be unknown, and it is particularly unknown how particular plant
root indication and exudates newest to the microbiome to the plant’s rhizo-
sphere. Future investigation will be necessary to pinpoint the particular pro-
cess through which plants control microbiome assembly. Notably, we must not
overlook the microbial community’s advantageous roles in the environment.
6.2 Plant growth and disease resistance
One of the most effective strategies for enhancing agricultural sustainability and
reducing the use of dangerous pesticides is biological control. In studies, concen-
trated on using root-associated microbiomes to enhance plant defense against ail-
ments, pests, and abiotic stresses as well as to encouraging plant growth and
boost output.
150 CHAPTER 8 Microbiome biodiversity

7.Biochemical mediators in plant growth promoting
microorganisms
Beneficial rhizobacteria can affect plant development through a number of different
molecular processes once they have become established in the plant rhizosphere. We
looked at the molecular processes by which PGPM controls physiological responses
in plants to encourage plant development. PGPM facilitated mineral nutrient uptake
by controlling various plant physiological activities. One of the key components for
plant development is nitrogen. Despite being a significant source of N
2
, most plants
cannot directly use N
2
. Few plants are capable of converting atmospheric N
2
to
ammonia through intricate metabolic mechanism. As an illustration, consider the
nitrogenase enzyme system found in nitrogen-fixing bacteria, which converts nitro-
gen into ammonium by the following reaction: N
2
þ8Hþþ16 ATP
2NH
3
þH
2
þ16ADP þ16Pi. The term for this procedure is biological nitrogen
fixing (BNF). BNF is a prominent component of the rhizobia-legume symbiosis
and might be used in sustainable farming systems to replace artificial N fertilizers.
BNF fixes around 21,011 kg of nitrogen yearly, which is nearly 3/4 of the total
amount of nitrogen required worldwide for plant development. Since their introduc-
tion decades ago, 79 Rhizobia inoculants, which are generated from bacteria that fix
nitrogen, have greatly improved crop output (Nascimento et al., 2019). For example,
the usage of the rhizobacteria Pseudomonas protegens Pf-5 X940 and Pseudomonas
stutzeri A1501 promotes the availability of nitrogen and increases the yield of wheat
seeds. Exogenous rhizobium injection boosts association among the rhizosphere
microbiome and modifies the core microorganisms of soybean, in addition to pro-
moting plant development (Zhong et al., 2019). These results offer compelling proof
that the rhizosphere microbiome may be modified by adding particular microbial in-
oculants. More recent advances in analytical techniques and microbiome genome
sequencing technology have helped to create more efficient bacterial inoculants
(Waltz, 2017). The intricate interactions between rhizobia, native microorganisms,
and rhizobia inoculants prevent the effective colonization of these exogenous
rhizobia inoculants into the rhizosphere; consequently, it is not always successful
to use these bacterial inoculants directly as biofertilizer. Therefore, the fundamental
research objective for the foreseeable future will be to discover novel strategies for
increasing inoculant establishment. Its success hinges on our ability to specify an
essential group of physiological processes, which is essential for rhizosphere colo-
nization, using future genome technology and clear molecular pathways. P is yet
another vital nutrient for agriculture. After separating the relevant biochemical path-
ways, it is critical to create novel methods to mineralize or solubilize phosphorus
brought on by the separation of advantageous rhizosphere microorganisms. Through
the excess of malate, phytase, and chitinase, it was able to extract five rhizospheric
actinobacteria from wheat plants, promoting the development and biomass of the
plant. Bautista-Cruz et al. (2019) discovered that Pseudomonas luteola and Bacillus
sp.in particular had a synergistic impact on promoting the development of Agave
7. Biochemical mediators in plant growth promoting microorganisms 151

angustifolia plants. Arbuscular mycorrhizal fungi are a common PGPM that in-
crease plant P and other nutrient absorption by expanding hyphae. In addition, a
number of other variables, such as fungal species and plant root shape, affect P ab-
sorption through mycorrhizal fungus. Another element that is necessary for all plants
to thrive and reproduce is iron. Despite the fact that iron is also plentiful in soil,
plants only get it in the form of iron Fe
3þ
oxides. Fe
3þ
oxides have a poor bioavail-
ability for plants, which is unfortunate. As a result, plants have created a variety of
paths to improve iron absorption. First, dicots and nongraminaceous monocots
recover iron by increasing the availability of iron by root exudation of protons
and organic acids. Second, some grass roots may emit molecules that chelate iron,
such as mugineic acid, so that it can be bound and absorbed by plants. Third, the
“iron-deficiency response,” a complex chemical process, stimulates A. thaliana roots
to absorb iron in alkaline soils. Similarly, in iron-limited environments, microbes
also produce siderophores. Instead of “cooperative companionship,” there was a
complicated fight for iron involving microorganisms and plants in the rhizosphere.
Please take note that siderophores released by rhizosphere microorganisms indi-
rectly aid in the stimulation of plant development. In most cases, soil-derived pyo-
verdine is produced by fluorescent pseudomonads to absorb iron and stop the growth
of Fusarium oxysporum, a kind of fungal plant disease, thereby encouraging plant
growth. Similar to this, Bacillus spp. boosts iron absorption by colonizing or modi-
fying the biofilm on cucumber roots (Xu et al., 2019). Additionally, Pseudomonas
simiae WCS417 increases the quantity of Fe as well as the fresh weight of
A. thaliana shoots and produces the “Fe deficiency response” in the A. thaliana
root by activating the marker genes MYB72 and IRT1 (Verbon et al., 2019). Rhizo-
sphere microorganisms can compete with plants for nutrients, but they also possess
characteristics that encourage plant development. The necessity of microorganism
adaptation as required to deal with the constrained supply of nutrients in the rhizo-
sphere is amply demonstrated by these results. To further understand how rhizo-
sphere microorganism interactions affect plant growth promotion, more in-depth
investigations are required (Xiao et al., 2019).
8.Plant disease-resisting microorganisms (PDRM)
An ecologically sustainable strategy for reducing plant diseases is to use helpful mi-
croorganisms. Studies on the value of helpful bacteria, particularly those that help
plants fight illness, are becoming more and more common.
8.1 Disease-suppressive soils
Plant diseases significantly reduce agricultural output. Interactions between patho-
gens and plants have raised a lot of concerns. However, pathogen infection pathways
are frequently overlooked by researchers. In the intense rhizosphere soil, microor-
ganisms contend for scarce nutrients derived by plants. Before viruses may
152 CHAPTER 8 Microbiome biodiversity

successfully infect plant tissues, they must successfully live in the soil and multiply
to a sufficient quantity. A pathogen’s ability to spread is hampered by the general
sickness protection provided by the microbiota in polluted soils, which is present
in all soils and depends on the activities and competitiveness of the entire microbial
population. A pathogen’s ability to spread is hampered by the general sickness pro-
tection provided by the microbiota in polluted soils, which is present in all soils and
depends on the activities and competitiveness of the entire microbial population. By
altering the activity of microbial communities, organic matter amendment can
improve the overall suppression of illness. According to study, the cultivars of the
bean Phaseolus vulgaris that are resistant to the soil-borne fungal disease Fusarium
oxysporum have unique rhizosphere microbiomes. These particular microbiomes
are Oxalobacteriaceae, Burkholderiaceae, and Sphingobacteriaceae which benefit
numerous antifungal genes, according to 16S rRNA and metagenome analyses
(Mendes et al., 2018). The “specific suppression” impact of the particular rhizo-
sphere microbiomes for soil-borne diseases can be transmitted between soils.
8.2 Biosynthesis of antimicrobial compounds
In the rhizosphere, bacteria produce a large number of antibiotic substances. These
chemicals have a suppressive impact on harmful microbes, which is a crucial direct
inhibitory technique for fending off plant diseases. Here, we focus primarily on the
substances released by PDRM that have a role in plants’ resistance to pathogens that
are found in the soil. The main types of substances that suppress the development of
infections are antibiotics and related substances (Mhlongo et al., 2018). The chem-
icals produced by the Bacillus and Pseudomonas genera as extracted from a healthy
plant rhizosphere have been the subject of several researches (Hashem et al., 2019).
Model Gram-positive bacteria Bacillus subtilis has been used as a biopesticide and
has the ability to mold the biofilm on plant roots. The production of antibacterial
substances is necessary for B. subtilis’ biological control action. For instance, the
lytic enzymes laminarase, cellulase, and protease generated by the B. subtilis strain
330-2 may break down the cell walls of pathogenic fungi and prevent the growth of
Rhizoctonia solani. In order to combat F. oxysporum, the B. amyloliquefaciens L3
strain may also produce the volatile antifungal substances 2-nonanone and 2-
heptanone. Significantly, different B. subtilis strains are reputed to create a variety
of antibiotics. Fengycin, surfactin, and iturin are examples of the important class
of antimicrobials known as lipopeptide antibiotics. To suppress Fusarium oxyspo-
rum and Rosellinia necatrix,B. subtilis strains PCL1612 isolated from a healthy av-
ocado rhizosphere release large amounts of Iturin-A, which benefits from Iturin A’s
broad range of fungus inhibition. Fengycin, which is generated by the B. mojavensis
RRC101 strain and is reliable against Fusarium verticillioides, may be an indirect
antagonist since surfactin stimulates host immunological responses. Nanotechnol-
ogy’s application in agriculture has recently drawn more interest. Among other
things, nanoparticles enable the regulated release of nutrients, and nanopesticides
are now being developed. It’s interesting to note that B. subtilis covered with
8. Plant disease-resisting microorganisms (PDRM) 153

nanoparticles on its exterior not only inhibits fungal growth but also promotes plant
growth, opening up new opportunities for the development of biopesticides (Wu
et al., 2019). Together, antibiotic compounds are crucial in preventing and treating
B. subtilis illness. Due to the diversity of antimicrobial chemicals, however, the
study of their function and variety is constrained in this section (Ababaf et al.,
2021;Zhang et al., 2021). Despite this, we are certain that these antimicrobial com-
pounds have a great potential for producing biopesticides. Further thought should be
given to the idea of nanobiopesticides (Marzaini &Mohd-Aris, 2021). Through at
least two methods, the microbiome of the plant rhizosphere can improve plants’
resilience to diseases: Pathogens are suppressed by the rhizosphere microbiome
either through the production of antimicrobial chemicals or through competition
within an ecological niche (Marzaini &Mohd-Aris, 2021).
9.Management of rhizosphere microbiota
9.1 Management of plant growth under drought stress and
rhizosphere microbiota
There is an urgent need to redefine agriculture from an integrative perspective since
drought creates a complicated global picture (Garreaud et al., 2020). It entails the
exploration of new water sources, the use of tolerant crops and genotypes, enhanced
irrigation systems, and other crucial but underutilized options including biotechno-
logical techniques that can improve water usage efficiency (Malhi, Kaur, &Kaushik,
2021;Gerten et al., 2020). A substantial body of research has shown that some
strains of the three main microbial rhizosphere groups arbuscular mycorrhizal fungi,
yeasts, and bacteria can increase the drought tolerance of their host plants by pos-
sessing a variety of PGP (plant growth-promoting) traits (Santiba
´n
˜ez Quezada,
2017). With this background, it is conceivable to hypothesize that provided their
co-inoculation does not result in antagonistic reactions, the co-use of different
PGP microorganisms might cause positive interactions or additive beneficial effects
on their host plants. Until now, single omics methods like genomics, metabolomics,
or proteomics have only been used to examine these impacts in part (Santander et al.,
2017). The application of multiomics techniques to find interactions between PGP
and host plants, however, has a knowledge deficit. This strategy must be the next
step in the research of how soil, plants, and microorganisms interact. The combined
utilization and drive to boost crop yields, enhancing production methods to meet the
rising global need for food by using multiomics techniques to deeply understand the
processes that take place in plants associated with microbes (Table 8.1)(Rojas et al.,
2019).
9.2 Role of plants in rhizosphere development
The species diversity integrates the way of visualizing and directs them toward the
root and other phytoconstituents that can be used for plant metabolism and
154 CHAPTER 8 Microbiome biodiversity

physiological demands (Roberts, 2022). The rootstock performs the most proactive
role in constructing the soil and purpose of this analysis environment (Gurikar et al.,
2022;Kaur &Vishnu, 2022). The species diversity contributes to the layout of the
rhizosphere by having more low and high cell biology mass carbohydrates through
the roots of the plants. These compounds serve as a feed ingredient for microbial
populations, which in turn affects the rhizosphere’s physiology and signaling. The
rhizospheric shape is formed by the features of microbial colonization inside the
root and rhizobial, the characteristics and quantity of substances published by
the root, plant, and microorganisms (Barnawal et al., 2019).
9.3 Improved soil and plant production through rhizosphere
management
Rhizobial management is the effective management of plants, soil, and microbes
with the goal of improving soil health, plant productivity, and nutrient utilization ef-
ficiency. Because of this, the green revolution cannot assist in further increasing food
production, and our reliance on it results in a degradation in the quality of our soil
and crop yield. Therefore, we must concentrate on crop, soil, and biological manage-
ment techniques. To obtain the greatest benefits, these can be categorized under
distinct management strategies and utilized at various levels. By modifying certain
biological communities and enhancing rhizospheric soil’s overall biodiversity, these
management techniques benefit us (Song et al., 2019). The following are all manage-
ment categories:
I. Crop management
In order to make the most effective use of soil and plant resources, crop man-
agement takes into account both simple, varied plant community management
and complicated, individual plant-based management. It is possible to enhance
each plant’s root system for increased rhizospheric activity (Drobek et al.,
2019).
II. Soil control
The objectives of soil management systems include increasing tilth, weed-free
conditions, seedbed preparation, changing the biotic potential of the soil, and
Table 8.1 The impact of soil biota on soil, natural and plant functioning.
Purpose By improving the soil biota Via enhanced soil biota
Soil development Advance
Reclusion of carbon Enhance Enhance
Plant nutrient guff Expanded
Lost nutrient Decrement
Biological invasion Decrement Decrement
9. Management of rhizosphere microbiota 155

decreasing financial wealth. Tillage techniques can interfere with the life of the
soil. Furthermore, by encouraging the degradation and mineralization of nat-
ural and inorganic sources, it may offer a chance to increase the efficiency of
nutritional utilization (Drobek et al., 2019).
III. Microbial control
Throughout the solubilization and mobilisation of natural and inorganic nutrient
sources and assistance in supplying them to plant roots, such as growth regu-
lators and VAM, the soil biological population aids in improving plant per-
formance. Crop productivity has been positively impacted by biotic community
inoculation of the soil and seedlings such as the rhizobia spp. inoculation of
legumes provided a chance for plants to use less exogenous nitrogen because of
nitrogen-fixing (Mhatre et al., 2019).
10.Holobiont-based control of rhizospheric biota
Studies have demonstrated that the soil biota with plants may work together to form
the rhizosphere. It’s crucial to breed the species composition for scientific purposes
in order to increase rhizobial variability with a focus on agricultural plants (Cesaro
et al., 2021). The objectives of agriculture can be met by integrating plant and rhizo-
bial biota behavior with various breeding techniques. For instance, root exudation
and the transfer of carbon to the rhizosphere serve as a source of energy for root sym-
biotic organisms. Crops that excrete additional carbon are able to boost the activity
of the rhizospheric biota. However, a specific plant microbiome offers a chance to
change plant characteristics, control illnesses, and plant blooming period, among
other things (Faure et al., 2018). Consider a Bacillus spp. genetically modified to
increase the quantity and concentration of plant hormones by modifying the
nitrogen-fixing process (Sevellec et al., 2018). A combination three-strain consortia
that includes better nitrogen consultants like Bacillus spp., Pseudomonas,
Rhizobium,orBradyrhizobium may offer excellent chances for a diversified and
comprising rhizospheric physiological health (Pita et al., 2018;Simon et al., 2019).
11.Impact of plant-friendly, plant-pathogenic, and
human-pathogenic microbes
Although the role played by the rhizosphere microbiome in the operation of plant
ecosystems has long been understood, traditional efforts to elucidate these roles
have been shown to be insufficient, and nothing is known about the overwhelming
majority of rhizosphere organisms. Therefore, combining established methods with
cutting-edge next-generation sequencing tools to evaluate organismal or community
ecology and physiology will result in new understandings of the microbial life in the
rhizosphere. It will be possible to determine if and how plants attract and promote
156 CHAPTER 8 Microbiome biodiversity

helpful organisms by identifying the exudates, signals, and important participants in
the rhizosphere microbiome. Improved crop protection and the discovery of various
as-yet-unknown soil microorganisms, functions, and genes for a variety of applica-
tions are possible benefits of unraveling the rhizosphere microbiome. We also need
to figure out how to stop human pathogens from multiplying in plant habitats to the
point where they start to harm people. Therefore, it is crucial to preserve human
health to have a better knowledge of the variables and cues that allow human infec-
tions to find an appropriate niche on plant surfaces. Different and complementary
approaches that redirect the rhizosphere microbiome in favor of bacteria that inhibit
diseases from germination, growth, attachment, and invasion of the root tissue
should be developed in order to keep plant and human infections under control.
One strategy is to start plant breeding initiatives aimed at elucidating the molecular
underpinnings of interactions between plant lines and advantageous rhizosphere
microbiome inhabitants. A good framework for this is provided by the earliest inves-
tigations on tomato line QTL mapping for features that enable beneficial rhizobac-
teria. It should be possible to determine if contemporary plant breeding can choose
for plant features that are necessary for harboring helpful microbes when combined
with in-depth investigation of the rhizosphere microbiomes of wild relatives of
commercially significant food crops. The identification of novel rhizosphere bacte-
ria, genes, and features that may be used for various purposes will probably result
from this “back to the roots” strategy. Therefore “core microbiome” to fight soil-
borne pathogens in various agroecosystems in order to lessen the effects of plant ill-
nesses will be a better option. Similar to the idea of the core microbiome in the field
of human microbiology (Huse et al., 2012;Smith et al., 1999;Turnbaugh et al.,
2009), we define the core rhizosphere microbiome in terms of plant health as the
collection of bacteria required to successfully protect plants from soil-borne dis-
eases. It is unknown how many microbes should make up the core microbiome,
and it is also unclear how many features are necessary to adequately defend plants
from diseases. There may be some functional redundancy among the core micro-
biome’s constituents since several rhizosphere microbes share a number of antago-
nistic properties (Gill et al., 2006;Juhas et al., 2011;Kinkel et al., 2011;Moya et al.,
2009). One may create a “minimal microbiome” in this regard. Similar to the idea of
the minimal genome, the minimal microbiome would have the bare minimum of mi-
crobial characteristics required to provide a certain ecosystem service, in this
instance defense of plants against soil-borne illnesses. One may contend that various
diseases on various crops require a unique collection of hostile bacteria (Kinkel
et al., 2011). This is probably true for the many classes of oomycetes, nematodes,
fungi, bacteria, and other soil-borne plant pathogens. However, it could be possible
to construct a core microbiome for each of these pathogen families individually. This
assumption is supported by a number of observations, including the fact that studies
on naturally disease-suppressive soils have identified common actors, identical
mechanisms, and genes in the suppressiveness of soils to various fungal pathogens
(Weller et al., 2002). The foundation for choosing possible candidate members of the
core microbiome and setting their initial densities to jumpstart disease
11. Impact of plant-friendly, plant-pathogenic, and human-pathogenic 157

suppressiveness will come from knowledge of the changes in microbial community
makeup and activity throughout this transition period. Additionally, a core micro-
biome may be created to do the additional tasks described that promote and maintain
plant health and growth. In keeping with the work of Burke et al. (2011), we suggest
that the core microbiomes be assembled more from a functional standpoint than just
based on taxonomic classification.
12.Conclusion and future outlook
All ecosystems have microbiomes, which are made up of various microbial popula-
tions. Microbiome disruption causes unfavorable phenotypes in the hosts, leading to
illnesses and other conditions and upsetting the harmony of the surrounding ecosys-
tems. The rhizosphere serves as a hub for interactions between plants and microbes
that affect plant health. Research on the root-associated microbiome has grown
rapidly, but many important topics remain unresolved, such as how to identify mi-
croorganisms that are vital for plant function and the methods by which roots
form their microbial communities. It is necessary to estimate microbiome diversity
and composition using scientific techniques that take into consideration the under-
lying functional and geographical variability of individual roots within a root system
in order to respond to these issues.
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˚gren, G. I.,
164 CHAPTER 8 Microbiome biodiversity

Weih, M., Amaral, F., Abelho, M., Amtmann, A., Armengaud, P., Asner, G. P.,
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nisms developed by plants and microorganisms against pesticides. Microbes and
Environments, 1(2), 63e77. https://doi.org/10.54458/mev.v1i02.6674
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olome and microbiome for sustainable food Production. MicroEnvironer, 1(1), 33e53.
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Borntraeger.
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Further reading 165

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Multifunctional growth-
promoting microbial
consortium-based
biofertilizers and their
techno-commercial
feasibility for sustainable
agriculture
9
Deepak Kumar
1
, Sanjay K. Singh
2
, Santosh K. Arya
1
, Deepti Srivastava
3
,
Vishnu D. Rajput
4
, Raja Husain
5
1
R&D Division, Nextnode Bioscience Pvt Ltd, Opposite GEB Office, Kadi, Gujarat, India;
2
Biodiversity and Palaeobiology, Agharkar Research Institute, Pune, Maharashtra, India;
3
Integral
Institute of Agricultural Science and Technology, Integral University, Lucknow, Uttar Pradesh,
India;
4
Academy of Biology and Biotechnology, Southern Federal University, Rostov-on-Don,
Rostov Oblast, Russia;
5
Department of Agriculture, Himalayan University, Itanagar,
Arunachal Pradesh, India
1.Introduction
Over the past few decades, substantial amounts of chemical fertilizers have been
applied to arable areas in an effort to prevent global food shortages and increase
crop productivity (Lin et al., 2019;Yan &Gong, 2010). However, the overuse of
chemical fertilizers has resulted in a number of problems, including substantial
soil deterioration; nitrogen leaching; soil compaction; a decline in soil organic mat-
ter; environmental contamination; soil carbon loss; and negative effects on human
health. Additionally, the impact of chemical fertilizers on agricultural output has
been waning over time (Horrigan et al., 2002;Nkoa, 2014;Savci, 2012;Sun
et al., 2015). A promising substitute for conventional fertilizers in agricultural pro-
duction is the use of microbial inoculants. Microbial inoculants can reduce the need
for chemical fertilizers without impacting on the plants’ needs for nitrogen, phos-
phorus, potassium, and other minerals (Aziz et al., 2019). Microbial inoculants
are crucial elements of organic agricultural practices and they provide a healthy
growth environment for plants and soils that is sustainable for upcoming growing
seasons. The size of the world market for biofertilizers was estimated at USD
1.80 billion in 2021. Market analysts predict that it will increase by 12.04%
CHAPTER
167
Rhizobiome. https://doi.org/10.1016/B978-0-443-16030-1.00010-9
Copyright ©2023 Elsevier Inc. All rights reserved.

CAGR from 2022 to 2029, from USD 2.02 billion to USD 4.47 billion (Fortune
Business Insight, 2022). In the current scenario, the main driver behind the con-
sumption of a huge demand for microbial inoculants or biofertilizers across the
globe is increasing the area of organic agriculture and organic foods. Because of
consumer perception of safe and healthy foods following the pandemic COVID-
19 incidence, demand for organic food products has significantly increased. This
consumer behavior persists after the COVID-19 pandemic and affects the food ser-
vice industry.
Although there are many ways to address sustainable agriculture and feed people
while minimizing environmental impacts, it has been widely reported that encour-
aging agricultural practices that increase biodiversity and the makeup of soil micro-
organisms, like organic or agro-ecological agriculture, represents a significant
alternative for obtaining high-quality food, a healthy environment, and establishing
environmental good governance aspects (Eyhorn et al., 2019). The resilience of
agro-ecosystems to climate change is increased by increased microbial biodiversity,
which stabilizes their functioning (Isbell et al., 2015). The understanding and knowl-
edge of the distribution and composition of microbial communities in relation to
various geographic areas and over a range of time intervals (such as seasonal fluc-
tuations) is essential for maximizing the enormous potential of the soil microbiome
(Delgado-Baquerizo et al., 2016). This fundamental confidentiality of microbial po-
tential in terms of growth promoting activities, mineral solubilization, toxic residue
reclamation, and biocontrol activity can be exploited in commercial formulations of
microbes as microbial consortiumebased biofertilizer (Aguilar-Paredes et al.,
2020). Microbial inoculants, or biofertilizers, are low-cost, sustainable sources of
plant nutrients that can be used in addition to chemical fertilizers. Seed treatment,
soil application, and foliar spraying of plants are frequently used as delivery strate-
gies for biofertilizers. By providing vital nutrients like nitrogen, phosphorus, potas-
sium, and zinc, they aid in the growth of plants. Thus, in order to reap the many
benefits of microbial inoculants in agricultural systems and, indirectly, in human
health, it is vital to comprehend the processes that define the types, composition,
and abundance of these inoculants. This chapter attempts to describe the key traits
of microbial consortia, formulations, delivery mechanisms, and beneficial impacts
on crops and the regulatory framework in order to support sustainable agriculture.
2.Beneficial microbes as active ingredients of microbial
consortia
Microbial inoculants, also known as biofertilizers, are substances that contain live
bacteria, fungi, and algae to encourage plant growth and boost agricultural output
(Yosefi et al., 2011). Beneficial microbes are found in biofertilizers, which enhance
the chemical and biological properties of soil by solubilizing phosphate and fixing
nitrogen, or cellulolytic activity. They occupy the rhizosphere regardless of whether
they are applied to seeds, plant surfaces, roots, or soil. The biological activity of
168 CHAPTER 9 Microbial consortium-based biofertilizers

microbes improves nutrient bioavailability, encourages plant growth, and expands
the soil microflora (Babalola, 2010;Umesha et al., 2018). Broad spectrum microbes
like Rhizobium, Azospirillum, Azotobacter, phosphorus-solubilizing bacteria (PSB),
vesicular-arbuscular mycorrhiza (VAM), and blueegreen algae (BGA) are incorpo-
rated as active ingredients of biofertilizers and commercialized on the market
(Seenivasagan &Babalola, 2021). The enzymatic activity of plant growth promoting
microbes (PGPMs) for crop development carries out competition strategies and
antagonism activity, like phytoparasites and inhibition of phytohormones, and it
also assists plants in coping with stress caused by toxic metal contamination and pol-
lutants (Saharan &Nehra, 2011;Tak et al., 2013). In order to achieve sustainable
agriculture, PGPMs are crucial. They boost soil fertility, encourage microbial diver-
sity and interaction with other beneficial microbes, hinder the growth and infectious
action of potential pathogens, and generally preserve the sustainability of the sys-
tems. They also raise the yield of certain crops (Abhilash et al., 2016;Santoyo
et al., 2021). The diversity of microbes exhibits multifunctional plant growthe
promoting attributes that are specific to genetic makeup and ecological parame-
ters. There are diverse groups of microbes that show potential abilities with regard to
nitrogen fixation, mineral solubilization, phytohormone production, bioremediation
of heavy metals, and biocontrol activities (Fig. 9.1).
2.1 Nitrogen fixation microorganisms
Nitrogen fixation is a complex process that uses a lot of energy. One method of trans-
forming elemental nitrogen into a form that a plant can use is through biological
FIGURE 9.1
The strategy for producing microbial consortium-based biofertilizers for desired benefits.
2. Beneficial microbes as active ingredients of microbial consortia 169

nitrogen fixation. Nitrogen-fixing microorganisms convert organic molecules into
innocuous nitrogen dioxide (Bakulin et al., 2007;Rosenblueth et al., 2018). Nitrogen
fixers, or N
2
fixing species, are employed as active ingredients in biofertilizers as
they contain living microbial inoculant groups. These microbial inoculants aid in ni-
trogen fixation by transforming atmospheric nitrogen into a form that plants can use,
such as Rhizobium, Azotobacter, Azospirillum, BlueeGreen Algae (BGA), and
Azolla. Legume plants have bacteria from the rhizobia species Rhizobium, Sinorhi-
zobium, Bradyrhizobium, Azorhizobium, and Mesorhizobium living in their root
nodules (Gonzalez et al., 2005;Seenivasagan &Babalola, 2021). When rhizobial
culture is applied in the field, rhizobial symbiosis takes place, improving crop output
by up to 20% and increasing the production of pulse crops by up to 15e20 kg N/ha
(Dubey, 2006, p. 732). By nature, Azotobacter sp. plays a significant role in the ni-
trogen cycle by fixing nitrogen. Azotobacter synthesizes many phytohormones, viz.
indole acetic acid (IAA), gibberellins (GA), cytokinins (CK), vitamins including
thiamine and riboflavin, as well as other compounds (Gauri et al., 2012;Sahoo
et al., 2013). Azospirillum is a Gram-negative, free-living, aerobic, and motile bac-
terium that can flourish in flood-prone environments and assist diverse elements of
plant development and growth (Sahoo et al., 2014). Azospirillum-inoculated plants
demonstrated greater water and mineral uptake, which improves production
(Gonzalez et al., 2005). Moreover, cyanobacteria, such as Tolypothrix, Scytonema,
Plectonema, Aulosira, Anabaena, Aulosira, and Nostoc, are typically used as
nitrogen-fixing bioinoculants and are essential for significant N
2
fixation on Earth
(Abdel-Lateif et al., 2012;Roy &Srivastava, 2013).
2.2 Phosphate solubilizing microorganisms
One of the most prevalent metallic elements in the Earth’s crust is phosphorus,
which can be found in soils both in organic and inorganic forms (Gyaneshwar
et al., 2002). The main characteristics linked with phosphorous nutrition include
its critical function in metabolic activities like energy transfer, photosynthesis, nitro-
gen fixation in legumes, crop quality, signal transduction, and resistance to plant dis-
eases (Khan et al., 2014). Phosphorus is absorbed or used by plants in inorganic form
(i.e., in orthophosphate: HPO
4
2
, and H
2
PO
4
(Hinsinger, 2001). Moreover, the
structural element phosphorus, which is also found in numerous coenzymes, phos-
phoproteins, and phospholipids, also contributes to the genetic information, or
“DNA,” found in all lifeforms (Ingle &Padole, 2017). Several heterotrophic micro-
organisms excrete organic acids that solubilized P, chelated cationic ligands of P
ions, and released the P straight into solution (Seenivasagan &Babalola, 2021).
Since the 1950s, biofertilizer has been created using these phosphate solubilizing
bacteria (PSB). By assimilating soluble P, microbial inoculants inhibit it from
adhering to surfaces or being fixed, respectively (Ingle &Padole, 2017). The break-
down, mineralization, and release of nutrients are only a few of the ways these mi-
crobes have an impact on soil fertility. Phosphate solubilizing microorganisms
increase the availability of previously unavailable P in readily available forms to
170 CHAPTER 9 Microbial consortium-based biofertilizers

the plants (Chen et al., 2006;Ingle &Padole, 2017). Pseudomonas and Bacillus spe-
cies are the most common microbes that are used as phosphate-solubilizing micro-
bial inoculants. The other reported microbes belong to Xanthomonas, Burkholderia,
Serratia, Phyllobacterium, Arthrobacter, Klebsiella, Beijerinckia, Rhodococcus,
Enterobacter, Flavobacterium, Chryseobacterium, PantoeaVibrio proteolyticus,
Delftia sp., Azotobacter, Erwinia, Xanthobacter agilis, Gordonia, Microbacterium
and Rhizobium ((Ingle &Padole, 2017;Seenivasagan &Babalola, 2021). Some
fungi, such as Penicillium, Aspergillus, Trichoderma, Fusarium, and Chaetomium,
are also good phosphate solubilizers and suitable for phosphate solubilizing in acidic
soil, which is a limitation for phosphate solubilizing bacteria (Kaushik et al., 2019).
2.3 Potassium solubilizing microorganisms
One of the most widely absorbed and necessary macronutrient, potassium (K), is
crucial for plant development and metabolism. A tiny amount of the potassium is
accessible to plants because it is available in a form that is immediately absorbed
by plant roots, whereas the soil K reserves are typically vast, but a major amount
of it is present in the form of insoluble K minerals. Numerous bacteria change inac-
cessible forms of K into forms that are available. As a result, the potassium solubi-
lizing bacteria (KSB) have a promising strategy to increase the availability of
potassium in soils, play a significant role in potassium-limited soils, and decrease
the use of chemical fertilizers based on potassium (Jain et al., 2022). Potassium sol-
ubilizing bacteria (KSB), in different types of potassium-rich soil, secrete organic
acids that convert K into readily available forms for plants and boost the soil’s
fertility status. Several saprophytic bacteria and fungi, including Acidithiobacillus
ferrooxidans,Bacillus edaphicus,Bacillus mucilaginosus,Bacillus circulans,Fra-
teuria aurantia,Burkholderia sp. Paenibacillus sp., Aspergillus sp., Penicillum
sp., Fusarium sp., and Aspergillus terreus, are used in K-solubilization as microbial
fertilizer. There are many sources of unavailable potassium minerals, such as mica,
orthoclase, feldspar, muscovite, illite, biotite, and feldspar, which are present in the
soil in a permanent form that the plant cannot directly absorb but can be readily used
in an available form through the application of potassium-solubilizing microor-
ganism (Meena et al., 2016;Sun et al., 2020).
2.4 Zinc solubilizing microorganisms
A crucial element for optimal plant growth is zinc. Microorganisms that solubilize
zinc are potential substitutes for zinc supplements because they transform applied
inorganic zinc into useable forms. Although plants can absorb zinc as a divalent
cation, the amount of soluble zinc in soil solutions is quite small (Kamran et al.,
2017). Minerals and insoluble aggregates make up the remaining zinc. One of the
most common micronutrient deficiencies, zinc insufficiency, is brought on by the
absence of zinc in soil (Alloway, 2008;Kamran et al., 2017). Various microorgan-
isms are reported as zinc solubilizers; when inoculated into plants, they showed
2. Beneficial microbes as active ingredients of microbial consortia 171

improved growth, yield, and zinc content. Some of the examples are Pseudomonas
sp., Bacillus sp., Gluconacetobacter diazotrophicus,Burkholderia cenocepacia,
Serratia liquefaciens, Bacillus thuringiensis. Pseudomonas fluorescence and Azo-
spirillum (Abaid-Ullah et al., 2015;Hussain et al., 2015;Kamran et al., 2017;Pawar
et al., 2015;Yadav et al., 2020). Aspergillus niger,Penicillium luteum, and Gluco-
nacetobacter diazotrophicus all produce gluconic acids and their Acidithiobacillus
(At.) thiooxidans and thermophilic iron’s facultative oxidizers derivatives, 2- and
2,5-keto-derivatives, that are used to solubilize zinc compounds (Saravanan et al.,
2007). Iron-oxidizing bacteria, Acidithiobacillus (At.) thiooxidans and thermophilic
iron’s facultative oxidizers have a remarkable capacity to dissolve zinc from sulfide
ore (Bosecker, 1997). It is known that certain fungi, including Penicillium,Asper-
gillus, and arbuscular mycorrhizal fungi (AMF), as well as other microorganisms,
can solubilize zinc. The possibility of using these zinc-solubilizing microbes as bio-
fertilizers for zinc biofortification in crops to counteract zinc deficiency in the hu-
man population exists (Yadav et al., 2020).
2.5 Sulfur oxidizing microorganisms
As a component of the essential amino acids cystine, methionine, and cysteine, sul-
fur are some of the most crucial plant nutrients after N, P, and K, notably in pulse and
oilseed crops (Chaudhary et al., 2019). The majority of sulfur is absorbed as sulfate,
which is then converted to sulfide to generate other essential compounds. The ma-
jority of the sulfur in soils comes from sulfide minerals found in parent materials,
plant and animal remnants, or from the addition of elemental S from outside sources.
The significant sulfide-bearing minerals found in rocks and soils include iron pyrite,
sphalerite, gypsum, epsomite, chalcopyrite, anhydrite, galena, and arsenopyrite. The
processes of mineralization, mobilization, immobilization, oxidation, and reduction
are used to cycle sulfur between its principally organic and inorganic forms (Jamal
et al., 2010). S-deficient plants exhibit symptoms similar to those of nitrogen-
deficient plants, such as light green or yellow leaves. The most noticeable signs
of a sulfur shortage in leguminous and oilseed crops are typically young foliage
with pale chlorotic leaves and high red tints at the leaf margins, decreased nodula-
tion, stifled growth, inadequate branching, a weak, fragile stem, etc. (Chaudhary
et al., 2019). Sulfur oxidizing bacteria (SOB) play a significant role in the biogeo-
chemical cycle of sulfur-based compounds in the environment. Bacillus sp., Entero-
bacter sp., Serratia sp., and Thiobacillus sp. are the genera that are used as SOB
inoculum for sulfur mineralization in the soil. The genus Thiobacillus among the
SOB in soil is crucial for biological S oxidation. Sulfur oxidising bacteria (SOB)
accelerate the formation of sulfates and improve the oxidation of sulfur, making it
more readily available to plants (Zainab et al., 2019). There are two distinct cate-
gories of metabolically active groups: obligate chemolithotrophic bacteria, which
can only use oxidizable S compounds (CO
2
as a carbon source), and heterotrophic
microorganisms, which may also adhere to the chemolithotrophic way of nutrition
(Chaudhary, 2018). Obligate chemolithotrophs are typically represented by the
172 CHAPTER 9 Microbial consortium-based biofertilizers

bacteria Thiobacillus thioparus,Thiobacillus neapolitanus,Thiobacillus thiooxi-
dans (extreme acidophiles), and Thiobacillus denitrificans (denitrifiers), whereas,
Thiobacillus novellus, Thiobacillus acidophilus, Thiobacillus aquaesulis, Paracoc-
cus denitrificans, Xanthobacter tagetidis, Thiobacillus intermedius, Thiomicrospira
thyasirae, Thiosphaera pantotropha, and P. versutus are the most prevalent exam-
ples of heterotrophs in this category (Chaudhary et al., 2019;Kuenen &Beudeker,
1982). Some fungi, including Aspergillus sp., Penicillium sp., Alternaria tenius, Epi-
coccum nigrum, Scolecobasidium constrictum, Myrothecium cinctum, and Aureoba-
sidium pullulans, oxidise elemental sulfur and thiosulphate (Seenivasagan &
Babalola, 2021;Shinde et al., 1996).
2.6 Plant growth promoting rhizobacteria (PGPR)
An important group of helpful, root-colonizing bacteria called plant growth promot-
ing rhizobacteria (PGPR) are present in both the bulk soil and the rhizosphere of
plants. They interact with the soil microbiota in both positive and negative ways
and carry out a variety of ecologically significant functions. By improving abiotic
and biotic stress tolerance and boosting host plants’ nutrition, they encourage plant
growth. PGPRs are viewed as a safe substitute for harmful chemical fertilizers
because of their active growth-endorsing capabilities (Basu et al., 2021). Different
PGPR strains can improve seedling emergence, boost nodulation in legumes, display
biocontrol, improve resistance to foliar diseases, and increase crop yields (Kalam
et al., 2020;Swarnalakshmi et al., 2020;Vejan et al., 2016). These helpful bacterial
groups provide protection for plants and aid in the promotion of plant growth
through a variety of mechanisms of action, such as root colonization, favorable ef-
fects on plant physiology and growth, biofertilization, induction of systemic resis-
tance, and biocontrol of phytopathogens (Basu et al., 2021). Direct and indirect
pathways functioning inside and outside of the plant, respectively, have historically
been used to categorize the ways in which PGPRs support plant development (Glick,
2012;Goswami et al., 2016). Phytohormones, such as auxins, gibberellins, cytoki-
nins, abscisic acid, and ethylene as well as fixed nitrogen and other phytonutrients
that solubilized the soil’s minerals (like K, P, Fe, Zn, and many other essential mi-
croelements) are examples of direct modes of PGPR action. Phytohormones can also
be used to stimulate plant growth and development (Gouda et al., 2018;Kalam et al.,
2020;Parray et al., 2016). Whereas the indirect effects of PGPRs on plant health are
suppressing deleterious phytopathogens through parasitism, inducing systemic
resistance, competing with phytopathogens for nutrients within the rhizosphere
niche, and synthesizing antagonistic molecules (like antibiotics, siderophores,
hydrogen cyanide, antimicrobial metabolites) and lytic enzymes (such as proteases,
glucanases, and chitinases) in host plants against the broad range of foliar and root
pathogens (Berg et al., 2017;Islam et al., 2016;Meena et al., 2020;Sayyed et al.,
2019). There are many reported PGPRs genera, which include many members
from Azotobacter, Arthrobacter, Azospirillum, Acinetobacter, Allorhizobium, Agro-
bacterium, Aeromonas, Azorhizobium, Azoarcus, Burkholderia, Bacillus,
2. Beneficial microbes as active ingredients of microbial consortia 173

Bradyrhizobium, Chromobacterium, Caulobacter, Delftia, Frankia, Enterobacter,
Flavobacterium, Klebsiella, Mesorhizobium, Gluconacetobacter, Paenibacillus,
Micrococcus, Pseudomonas, Rhizobium, Pantoea, Streptomyces, Serratia, Thioba-
cillus, and many others (Basu et al., 2021;Seenivasagan &Babalola, 2021;Kalam
et al., 2020;Parray et al., 2016;Ahemad &Kibret, 2014,Table 9.1).
3.Microbial consortia
The term “microbial consortium” refers to a collection of various microorganisms
with the capacity to cooperate in a community. In other words, two or more bacterial
or microbial groups that are symbiotically coexisting in a community are known as a
microbial consortium. Occasionally, consortiums may be both endo- and ectosym-
biotic (Madigan et al., 2019). Johannes Reinke first proposed the idea of a “con-
sortium” in 1872, while the term “symbiosis” was first used in 1877 before being
further developed (Kull, 2010;Reinke, 1872). Different types of bacteria may be
included in bioinoculants based on microbial consortia, whereas other bioinoculants
may contain both fungi and useful bacteria. A variety of advantages for the plant
should result from the application of various PGPM species with various modes
of action, including direct stimulation of growth and health as well as increases in
output. It would also be anticipated that pathogen-related illnesses would decline
(Behera et al., 2020;Brada
´
cova
´et al., 2020). On the basis of the types of microbes
(bacteria, fungi, or both) used in making consortia, microbial consortia can be clas-
sified into three major categories, namely (i) bacterial consortia, (ii) fungal consor-
tia, and (ii) fungal-bacterial consortia.
3.1 Bacterial consortia
Many microbial inoculants have been commercialized in recent years as a result of
thorough research into the effects of various PGPB strains on plants. The creation of
bacterial consortia has attracted interest as a viable method for sustainable crop pro-
duction, since it can boost the beneficial properties demonstrated by these bacteria.
In an additive or synergistic interaction, two or more suitable bacteria from
different species form a bacterial consortium (Panwar et al., 2014;Santoyo et al.,
2021;Sarma et al., 2015). In some conditions, a combination of various strains
from the same species might display improved behavior and also be regarded as
a consortium. Because they span a wide range of plant growth promotion and bio-
logical control mechanisms, bacterial consortia have been found to enhance favor-
able features in plants compared to individual strains (Ju et al., 2019). The
employment of these consortia is a workable method for reducing crop pests, phyto-
pathogenic infections, pests, salinity, nitrogen uptake, and drought (Joshi et al.,
2020;Nawaz et al., 2020;Rana et al., 2012;Rodrı
´guez et al., 2019;Santoyo
et al., 2021). Additionally, some bacterial consortia have the ability to fix nitrogen,
convert inaccessible nutrients into assimilable forms, chelate iron, and produce
174 CHAPTER 9 Microbial consortium-based biofertilizers

Table 9.1 Plant growthepromoting rhizobacteria (PGPR) and associated
growth-promoting substances (acids).
Microorganisms
Growth promoting
substances (acids) References
Bacillus megaterium BHUPSB14,
Bacillus pumilus,B. licheniformis,
B. subtilis, and Paenibacillus
polymyxa
Ethylene, auxins, gibberellins,
ACC deaminase, and
cytokinin
Conway et al.
(2022),
Seenivasagan and
Babalola (2021),
Nascimento et al.
(2020),
Swarnalakshmi
et al. (2020),
Olanrewaju et al.
(2017)
Koulman et al.
(2012),
Harrington et al.
(2012), and Perrig
et al. (2007).
Pseudomonas fluorescens
BHUPSB06, Pseudomonas
tabaci,Pseudomonas syringae,
P. putida, P. corrugata,
P. aeruginosa, P. fluorescens
G20-18, and P. cepacia
Indole-3-acetic acid,
ethylene, ACC deaminase,
and cytokinin
Rhizobium tropici,Rhizobium
leguminosarum,Rhizobium
phaseoli, and Rhizobium etli
Cytokinin, HCN and indole-3-
acetic acid (IAA)
Azospirillum lipoferum,
Azospirillum brasilense,
Azospirillum halopraeferens,
Azospirillum largimobile,
Azospirillum irakense and
Azospirillum doebereiner
Gibberellic acid (GA3), indole-
3-acetic acid, abscisic acid
(ABA), zeatin, and ethylene
Rhizobacterial isolates Gibberellic acid (GA3), indole-
3-acetic acid (IAA)
Azotobacter chroococcum,
Aeromonas veronii, Azospirillum
amazonense, Agrobacterium sp.,
Comamonas acidovorans, Serratia
marcescens, Serratia
marcescens, Klebsiella oxytoca,
Bacillus subtilis, Mesorhizobium
cicero, Erwinia
herbicola, Enterobacter asburiae,
and Rhizobium sp.
Indole-3-acetic acid
Pantoea herbicola and Pantoea
agglomerans
Auxin and IAA
Enterobacter cloacae and
Alcaligenes piechaudii
ACC deaminase, and indole-
3-acetic acid
Gluconobacter albidus,
Gluconobacter diazotrophicus,
Gluconobacter cerevisiae,
Gluconobacter cerinus, and
Gluconobacter asaii
Indole-3-acetic acid,
gibberellin GA1, and GA3
Variovorax boronicumulans and
Variovorax paradoxus
ACC deaminase, and auxin
(IAA)
3. Microbial consortia 175

phytohormones, all of which are crucial for maintaining the health and quality of
the soil and can reduce the detrimental effects of some conventional, unsustainable
agricultural practices (Gosal &Kaur, 2017). Bacterial consortia can be classified as
either simple or complex. This difference in bacterial consortia is based on a mass
production or fermentation strategy in which different strains are cultivated sepa-
rately or in combination with other species or strains in a medium appropriate
for all PGPB species (Bashan et al., 2020). The efficacy of bacterial interaction
in any consortium depends on (i) taxonomic identification of strains, (ii) a proper
description of the consortium and how it was prepared, (iii) a full description of
physiological and sensitive parameters and recommended dosage for application,
and (iv) the recommended population of each bacterial strain in the consortium,
which affects the inoculated consortium’s performance (Dı
´az et al., 2020;Santoyo
et al., 2021). Moreover, the success of any bacterial consortia under field conditions
depends on the type and functions of the strains, adaptation to climatic conditions,
survival ability, and persistence in the soil after inoculation (Gosal &Kaur, 2017;
Verbruggen et al., 2013).
3.2 Fungal consortia
The fungal consortium is widely used but less frequently acknowledged in the liter-
ature. The mycorrhizal fungi (AMF, VAM), which are root obligatory biotrophs
capable of establishing mutualistic symbiosis with >80% of vascular plant species,
provide the greatest example. These fungi include Gigaspora, Rhizophagus
(Glomus), Funneliformis,andLaccaria (Pringle et al., 2009;Rouphael et al.,
2015). They participate in carbon exchange and increase the plant’s ability to
absorb nutrients and water, thereby minimising the damaging impacts of biotic
and abiotic stressors. Mycorrhizal fungi (AMF, VAM) are known to promote
salinity tolerance by employing several mechanisms, such as enhancing water
use efficiency and nutrient acquisition by producing plant growth hormones and
regulators, improving photosynthetic rate, balancing ionic equilibrium, and produc-
ing antioxidants (Evelin et al., 2019;Montesinos-Navarro et al., 2012;Sagar et al.,
2021). Trichoderma is yet another fungal inoculant that is a key component of many
commercially available agricultural products around the world, has a variety of ad-
vantageous effects on plants, and is widely applied in integrated and biological pest
management (Woo &Pepe, 2018,Woo et al., 2014;Lorito and Woo, 2015). Many
Trichoderma strains are effective microbial biological control agents (MBCA) of
different plant diseases. Initially, it was believed that the biopesticidal activity
was the main advantage, but soon it became clear that these MBCA were also useful
as biofertilizers, biostimulants, and bioenhancers of crop resilience to both biotic
and abiotic challenges (Fontenelle et al., 2011;Lorito &Woo, 2015). In reality,
research suggested that a genuine symbiotic relationship might be the cause of
the plant growth promoting effect (Studholme et al., 2013;Woo &Pepe, 2018). Tri -
choderma may, under some conditions, induce a state of alertness in the plant (i.e.,
priming), resulting in a prepared response to pathogen attack that eventually
176 CHAPTER 9 Microbial consortium-based biofertilizers

foresees the emergence of an “induced systemic resistance” (ISR) and/or “systemic
acquired resistance” (SAR) (Hossain et al., 2017;Manganiello et al., 2018). Over
250 metabolic products are produced by Trichoderma species, including peptides,
enzymes that break down cell walls, secondary metabolites, and other proteins.
Many of these substances have bioactive properties that can influence how plants
react to other microorganisms by enhancing defensive mechanisms and promoting
plant growth and development, particularly at the root level (Lombardi et al., 2018;
Ruocco et al., 2015). Various combinations or consortia of different strains, metab-
olites, and mixes of bioactive substances, coming from Trichoderma as well as
other microorganisms or plants, have been shown to have synergistic effects on
biocontrol, suggesting a wealth of potential for creating a new generation of bio-
stimulants (Wo o &Pepe, 2018).
3.3 Fungal-bacterial consortia
An emerging area of research that uses the methods of contemporary microbial ecol-
ogy is the study of fungal-bacterial interactions. The mycorrhizosphere has previ-
ously been the focus of research, but during the past 10 years, there has been
increased attention on the function of bacteria in other fungal habitats (Grube &
Berg, 2009;Sagar et al., 2021). For the growth and development of most plants,
the cohabitation of AMF and PGPR in the rhizosphere is particularly advantageous.
Positive interactions between mycorrhizal fungi and PGPR as fungal-bacterial con-
sortia encourage each other’s growth, which eventually helps the plant. This syner-
gistic impact benefits both the plant and the mycorrhizal fungi. By influencing root
colonization and nutrient uptake, for instance, PGPR improved AMF growth and
survival. Moreover, plants exposed to a saline environment showed evidence of
the synergistic interactions between AMF and PGPR (Baradar et al., 2015;Lee
et al., 2015). The growth of various crop plants was positively impacted by the com-
bined treatment of AMF and PGPR. These include increased production of organic
acids, soluble sugars, antioxidant enzymes, and metabolites for scavenging ROS, as
well as decreased Na þlevels in plants under salt stress. Additionally, a fungal-
bacterial consortium (AMF and PGPR) improved plant performance in a saline
environment, which is mediated through activation of ABA-signaling, sodium ion
channels, and salt excessively sensitive (SOS) pathways (Hassani et al., 2014;Sagar
et al., 2021). Rhizobium and AMF were coinjected into alfalfa under salt stress, and
this significantly improved the nitrogenase activity, leghemoglobin, IAA synthesis,
nodulation, yield, and nutrient uptake (Ashrafi et al., 2014). Certain AMF species
synthesize metabolites (like volatile molecules, i.e., ethylene, nonvolatile metabo-
lites, and organic acids) that attract particular bacteria (Younesi &Moradi, 2014).
Likewise, some bacteria that are well-known to encourage AMF colonization are
known as mycorrhiza-helper bacteria (MHB). Phosphates are soluble in soil due
to PGPR, and AMF’s efficient colonization increases soil phosphate absorption
(Nanjundappa et al., 2019). AMF and PGPR combined in consortium boost the
host immune response and provide tolerance against abiotic and biotic stresses
3. Microbial consortia 177

(Hunter et al., 2019). The AMF (Rhizophagus irregularis) was coinjected with either
Bacillus amyloliquefaciens or Pseudomonas sp.to decrease the phytopathogen
Fusarium oxysporum-induced disease symptoms and boost root and shoot dry
weight in tomato plants (Yusran et al., 2009). In comparison to plants inoculated
with just one of the microorganisms, soybean plants coinoculated with AMF
(Glomus mosseae) and Bradyrhizobium sp. showed greater resistance in the presence
of the phytopathogen fungus Cylindrocladium parasiticum. This shows that AMF
and PGPB may work well together to create a successful biocontrol approach for
certain significant field crops (Gao et al., 2012). Although the impacts on the local
rhizosphere microbiota have not been properly clarified, the various studies on Tri-
choderma and Azotobacter imply that these fungi and bacteria might be functionally
synergetic in a fungal-bacterial consortium. Additionally, a Trichoderma-
Azotobacter consortium can be developed as genuinely efficient and dependable
plant products with a combination of inorganic substances, botanicals, polymers,
seaweeds, and other animal-derived substances as carriers (Fiorentino et al., 2018;
Ventorino et al., 2018).
4.Carrier materials for microbial consortia
Bioinoculants are mixtures of advantageous microbes that have been produced with
suitable and effective carrier materials. Microbial inoculation is a time-honored tech-
nique for crop improvement, but in the field, single-strain inoculations have unpre-
dictable effects due to their low capacity to respond to changing environmental
factors. Microbial consortia have shown that employing mixed inoculants of diverse
microbes could improve the adaptability of bacterial inoculants (Khan et al., 2017;
Vassilev et al., 2015). Due to various environmental constraints, including careless
handling that causes cell dispersal and the short storage life of the liquid inoculum,
the application of a microbial inoculum to the rhizosphere under natural circum-
stances may fail to enhance crop growth. Farmers have chosen to use biofertilizers
less frequently than other techniques as a result of these difficulties. Therefore, spe-
cific substances, known as carriers, are required for the effective application of inoc-
ulants. These carriers must have the ability to stimulate microbial activity and have a
longer shelf life with formulation (Zafar-ul-Hye et al., 2019). Due to their simplicity
of use and ability to be stored for an extended period of time, carrier-based microbial
inoculants are very effective (El-Fattah et al., 2013). The cost and availability of the
carrier(s) are the major factors to consider. As a result, when choosing a carrier ma-
terial, it is important to take into account the following factors: the carrier material
must be readily available, affordable, nontoxic to microbes, physically and chemi-
cally stable, promote plant growth, be recyclable and free of pollutants, be simple
to manage, have a high moisture holding capacity, and have a good buffering capacity
(Pacheco-Aguirre et al., 2017). Depending on their origin, carriers can be either
organic (such as biogas slurry, charcoal, crushed corn cobs, compost, peat, etc.) or
inorganic (such as lignite, perlite, zeolite, talc, etc.) (Sohaib et al., 2020). Vegetable
178 CHAPTER 9 Microbial consortium-based biofertilizers

oils, broth cultures, oil-in-water suspensions, and minerals are just a few examples of
the various liquid inoculants that can be used as carriers (Table 9.2). It is necessary for
the carrier material to be safe for both the microbial inoculants and the plants them-
selves (Seenivasagan &Babalola, 2021).
A variety of microbial consortium biofertilizers are produced by various manu-
facturers, depending on their physical makeup and the carrier materials they use.
These include dried cultures, broth cultures, and inoculants based on agar. Inocula-
tion success in tropical legumes appears to be more likely with the use of new bio-
fertilizer production techniques like (i) precoated seeds (e.g., Prillcoare, New
Zealand), (ii) freeze-dried inoculants (e.g., BAIF, IARI, India), (iii) granular inocu-
lant (e.g., Nitragin, USA), (iv) pelleting form (Pelinoc of Nitragin, USA), (v) Rhizo-
bium paste (e.g., KALO Inc., USA), and (vi) polyacrylamide-entrapped rhizobia
(e.g., Agrosoke) (BioFIT,https://www.bio-fit.eu/).
On the basis of the carrier material used and their physical nature, there are two
types of microbial consortium: (1) solid carrierebased microbial consortium, and
(2) liquid microbial consortium. In order to improve soil fertility, microbial inocu-
lums are now delivered as carrier-based microbial inoculants. The term “carrier”
Table 9.2 Types of carrier materials that are used for microbial consortium
biofertilizer.
Category of carrier
materials Types of materials Reference(s)
Biopolymer Xanthan gum, guar gum, alginate, agar-
agar
Seenivasagan
and Babalola
(2021),
Sohaib et al.
(2020),
Bashan and de
Bashan (2005)
Lyophilized microbial
cultures
Preservatives (glycerol, mineral oil,
vegetable oil, polyvinylpyrrolidone,
Arginine, mannitol etc.), and culture media
(powder and liquid)
Soils Clays, coal, charcoal, bentonite,
pressmud, peat, lignite, and l eonardite
Inert materials Vermiculite, powdered rock phosphate,
talc powder, perlite, calcium sulfate,
carrageenan, polysaccharide-like alginate,
and polyacrylamide
Waste materials or
decomposed
agricultural waste
Coconut shell powder (cocopeat), paddy
husk, farmyard manure, wheat bran, plant
debris, teak leaf powder, husk powder,
pressmud, black ash, spent mushroom
compost, city compost, vermicompost,
food industry waste, soybean meal, de-
oiled cake powder of neem, jatropha,
karanja, and castor, black ash, wood ash,
fly ash, and decomposed fruit and
vegetable wastes
4. Carrier materials for microbial consortia 179

refers to a medium that can hold the microorganisms in large enough quantities and
maintain their viability under particular conditions while being simple to provide to
farmers. The creation of high-quality biofertilizers requires the use of suitable car-
rier materials. Whereas, a liquid microbial consortium is a formulation that contains
the desired microorganisms in their dormant state, along with the nutrients and in-
gredients that promote the formation of dormant spores or cysts for greater resis-
tance to stress and a longer shelf life. Upon entering the soil, the dormant forms
of spores germinate to form a new set of active cells that consume the carbon supply
in the soil or via root exudates and proliferate. Liquid formulation technology, which
offers greater benefits because of its longer shelf life and lower contamination than
solid carrierebased microbial formulations as an alternative to conventional carrier-
based microbial formulations, has been developed by many organizations, like
TNAU in Coimbatore, IARI in New Delhi, and ICAR-IIHR in Bangalore, respec-
tively (BioFIT,https://www.bio-fit.eu/).
5.Regulatory framework for commercialization of
microbial consortium biofertilizers
Microbial consortia (especially bacterial consortia) are commercialized in India in
two categories: (i) carrier-based consortia and (ii) liquid consortia. Both types of
bacterial consortia are regulated under the Schedule-III [(clause 2(h) and (q)] of Fer-
tilizer (Control) Order, 1985. The Fertilizer (Control) Order came into force in 1957
to regulate the sale, price, and quality of fertilizers. Since then and till 1985, nearly
70 amendments were made to the Order. As per the Fertilizer (Control) Order 1985,
the State Government may, by notification in the Official Gazette, appoint regis-
tering authorities for the purpose of this Order and may, in such notification, define
the limits of the local area within which such registering authority shall exercise his
jurisdiction. As per the Fertilizer (Control) Order 1985, the Controller has the au-
thority to grant a certificate of registration on Form “B” within 30 days of the receipt
of an application to any person who applies for it under clause 8. Every certificate
issued under clause 9 and every authorization letter issued under clause 8 is valid
for 3 years from the date of issue unless renewed, suspended, or canceled. Each
manufacturer has to get a “no objection certificate” (NOC) from the state’s Direc-
torate of Agriculture for fertilizer manufacturing by submitting the complete set
of documents as per the Fertilizer Control Order, 1985 guidelines (Table 9.3). More-
over, for retail and wholesale licenses of fertilizer sales, manufacturers must also
register their agro-inputs with the state authorities, respectively. The specifications
of both types of bacterial consortia (carrier and liquid) under the Fertilizer (Control)
Order, 1985, are as follows.
The Environmental Protection Agency (EPA) in the United States is in charge of
controlling pesticides to make sure they don’t have any negative impacts on people
or the environment. All regulatory actions pertaining to biological inoculants or bio-
pesticides are handled by the EPA’s Biopesticides and Pollution Prevention Division
180 CHAPTER 9 Microbial consortium-based biofertilizers

Table 9.3 Specifications of carrier-based and liquid bacterial consortia under Schedule-III of the Fertilizer (Control) Order
1985 (As amended upto, 2019, https://www.faidelhi.org/).
Carrier-based bacterial consortia
(i) Base Carrier based in form of moist granules or powder
(ii) Viable count CFU (min.) in mixture of any two or maximum three of following microorganisms:
CFU (min.) Rhizobium or Azospirillum or azotobacter:110
7
per g
CFU (min.) phosphate solubilizing bacteria (PSB): 1 10
7
per g
CFU (min.) potassium solubilizing bacteria (KSB): 1 10
7
per g
(iii) Particle size of carrier moist powder All carrier material shall pass through 0.15e0.212 mm IS sieve
(iv) Total viable count of all the microorganisms in
the product
CFU (min.): 5 10
7
cells/g of carrier or powder
(v) Moisture percent by weight (maximum) in case
of carrier based
30%e40%
(vi) Contamination No contamination at 10
4
dilution for granule/carrier-based inoculants
(vii) Efficiency character
•Azotobacter sp.
•Azospirillum sp.
•Rhizobium
•PSB
•KMB
•The Azotobacter strain should be capable of fixing at least 10 mg of N (Nitrogen)
fixation/g of carbon source
•The Azospirillum strain should be capable of fixing at least 10 mg of N (Nitrogen)
fixation/g of malate applied
•Nodulation test should be positive
•minimum 5 mm zone of solubilization on PSB media having at least 3 mm
thickness
•minimum 5 mm zone of solubilization on KSB media having at least 3 mm
thickness
Liquid bacterial consortia
(i) Individual viable count in liquid-based consortia CFU (min.) in mixture of any two or more of following microorganisms:
CFU (min.) Rhizobium or Azospirillum or azotobacter:110
8
per g
CFU (min.) phosphate solubilizing bacteria (PSB): 1 10
8
per g
CFU (min.) potassium solubilizing bacteria (KSB): 1 10
8
per g
(ii) Total viable count of all the microorganisms in
the product
CFU (min.): 5 10
8
cells per ml of liquid based
(iii) Contamination No contamination at any dilution
(iv) pH 5.0e7.0
(v) Efficiency character
•Azotobacter sp.
•Azospirillum sp.
•Rhizobium
•PSB
•KMB
•the Azotobacter strain should be capable of fixing at least 10 mg of N (nitrogen)
fixation/g of carbon source
•The Azospirillum strain should be capable of fixing at least 10 mg of N (nitrogen)
fixation/g of malate applied
•Nodulation test should be positive
•minimum 5 mm zone of solubilization on PSB media having at least 3 mm
thickness
•minimum 5 mm zone of solubilization on KSB media having at least 3 mm
thickness
5. Regulatory framework for commercialization of microbial consortium 181

(BPPD). Biopesticides are recognized as a regulatory category and are controlled us-
ing a distinct set of data criteria than for conventional chemicals. The EPA normally
classifies biopesticides as “reduced-risk pesticides,” meaning they pose a lower
danger than traditional pesticides. In the European Union, a dual system exists in
which the European Food Safety Authority (EFSA) evaluates the active ingredients
used in plant protection products and member states evaluate and authorize the
goods at the national level. The framework Regulation (EC) No. 1107/2009 primar-
ily governs plant protection goods. Regulation (EC) No. 396/2005 covers every
aspect of the legal restrictions on the amount of pesticide residue that can be present
in food and feed. This rule also includes guidelines for government oversight of
pesticide residues that may result from their usage for plant protection in foods
with both plant and animal origins (Teicher, 2018).
Microbial consortium-based biofertilizers are very popularly commercialized
worldwide and actively used by farmers, especially in organic and good agricultural
practices (GAP) production systems. Public and private sector organizations are
actively engaged in the mass production of liquid and carrier-based microbial con-
sortium. For example, the Indian Institute of Horticultural Research (ICAR-IIHR),
Bangalore, developed the liquid microbial consortium with the brand name “Arka
Microbial Consortia" using Azotobacter tropicalis, Pseudomonas taiwanensis, and
Bacillus aryabhattai (https://www.iihr.res.in/). Similarly, BASF Corporation,
USA, developed a cutting-edge technology of bacterial consortia of Rhizobium sp.
and Bacillus subtilis under the brand name Nodulator Duo SCG, in which both bac-
terial strains make biofilm on the surface of the carrier that strengthens roots for
nodulation and shields roots from soil stresses in peas. The other successful brands
of microbial consortium are available on the market and are critically discussed in
Table 9.4 and presented in Fig. 9.2.
6.Multifunctional plant growth-promoting attributes of
microbial consortia on different crops
Climate change, changes in land use, and an increase in agrochemicals all have the
potential to negatively impact microbial interactions in agroecosystems. Stress con-
ditions (biotic and abiotic) can affect a plant’s physiology, biochemistry, gene regu-
lation, and microbiological and physicochemical characteristics of the soil, which
can significantly reduce productivity and yield (Goswami &Deka, 2020;Vimal
et al., 2017). Therefore, inoculating plants with microbial consortia might lessen
the detrimental effects of biotic or abiotic adverse conditions on crops. Beneficial
bacteria can support the plant’s inherent resistance to these difficulties (Santoyo
et al., 2021;Vimal et al., 2017). However, novel approaches are needed to investi-
gate the interactions between a microbial consortium and plants under abiotic and
biotic stress conditions in order to identify potential stress-tolerant or resistant mi-
crobial inoculants to improve plant development and disease resistance. Microbial
inoculants are used as plant biostimulants to enhance crop yield and the nutritional
182 CHAPTER 9 Microbial consortium-based biofertilizers

Table 9.4 Some of the selected microbial consortiumebased biofertilizers available on the global market.
Type of
microbial
consortium
Species involved in
microbial consortium Product name
Producing
company
Country
of origin Mode of action Reference
Bacterial
consortium
Azotobacter chroococcum
Pseudomonas striata
Frateuria aurantia
Jaswonder Shri Ram
Solvent
Extractions
Pvt. Ltd.
India The bacterial consortium
produces phytohormones in
the rhizosphere, which
increases seed germination,
root proliferation, shoot
number, and growth.
https://neemplus.
com/
Azotobacter sp.,
Acetobacter sp.,
Rhizobium sp.
Utkarsh
microbes
Utkarsh
Agrochem
Pvt. Ltd.
India This consortium supports the
plant rhizosphere system
through the assimilation of
nitrogen. It is a special
combination of protective
and nutritive microorganisms
of nitrogen-fixing bacteria.
https://utkarshagro.
com/
Rhizobium sp.,
Azotobacter sp.,
phospho-bacteria, and
potash solubilizing
bacteriaphospho-bacteria,
and potash solubilizing
bacteria
IFFCO Liquid
Consortia
Biofertilizer
Indian
Farmers
Fertiliser
Cooperative
Limited
(IFFCO)
India It provides more nitrogen,
phosphorus, and potassium
available to the crops.
https://www.
iffcobazar.in/
Azotobacter tropicalis
(NAIMCC-B-01336)
Pseudomonas taiwanensis
(NAIMCC-B-01337)
Bacillus aryabhattai
(NAIMCC-B-01335)
Arka Microbial
Consortium
Indian
Institute of
Horticultural
Research
(IIHR)
India Through solubilizing the
unavailable macro and
microminerals in their
available form, this
consortium is very effective in
early seed germination,
increasing seed vigor, and
reducing N and P fertilizer
requirements by 25%e30%.
https://www.iihr.res.
in/
Continued
6. Multifunctional plant growth-promoting attributes of microbial consortia 183

Table 9.4 Some of the selected microbial consortiumebased biofertilizers available on the global market.dcont’d
Type of
microbial
consortium
Species involved in
microbial consortium Product name
Producing
company
Country
of origin Mode of action Reference
Azotobacter tropicalis
Pseudomonas taiwanensis
Bacillus aryabhattai
NexBio-NPK Nextnode
Bioscience
Pvt. Ltd.
India This consortium is helpful in
solubilizing the unavailable
phosphorus, potash, and
iron through releasing the
organic acids and increasing
the availability of these
minerals to the crops. It also
has a positive effect on seed
germination.
http://www.
nextnodebioscience.
com/
Azotobacterchroococcum
Pseudomonas striata
Frateuria aurantia
Parle Gold
N.P.K. Special
Parle Biocare
LLP
India It offers macro-and
micronutrients, increasing
crop production in the
process. Utilizing Bio-NPK
lessens plant illnesses and
pest attacks.
https://parlebio.com/
Azotobacter sp.,
Pseudomonas sp., and
Frateuria aurantia
Premium EMC IPL
Biologicals
India It prevents the spread of
pathogenic microorganisms
and controls diseases.
Vitamins, hormones,
enzymes, antioxidants, and
antibiotics are just a few of
the bioactive compounds it
produces that can either
directly or indirectly improve
plant growth and defense.
https://www.
iplbiologicals.com/
184 CHAPTER 9 Microbial consortium-based biofertilizers

Azotobacter sp.,
Bacillus sp., and Frateuria
sp.
Consort-NPK T. Stanes and
Company
Limited
India It transforms inaccessible
forms of soil nutrients into
accessible forms. With
increased nutrient flow into
the plants, it improves root
multiplication and shoot
growth.
https://tstanes.com/
Rhizobium sp.,Bacillus
subtilis (biofilm)
Nodulator Duo
SCG
BASF
corporation
USA It is a cutting-edge inoculant
that combines a highly active
Rhizobium strain with a
Bacillus subtilis surface
biofilm that strengthens
roots. This inoculum
improves nodulation and
shields roots from soil
stressors, allowing plants to
focus more energy on
growth.
https://agriculture.
basf.us/
Rhodopseudomonas
Palustris,
Lactobacillus Casei
Lactobacillus Plantarum,,
Saccharomyces cerevisiae
Microm Indogulf
BioAg
USA
Canada
It affects the microbial
environment in a way that
favors the growth of
beneficial microorganisms.
By utilising fermentation, this
fosters an environment
where microbes can
contribute positively to plant
development, plant quality,
and soil fertility.
https://www.
indogulfbioag.com/
Rhodopseudomonas
Palustris,
Lactobacillus Casei,
Lactobacillus Plantarum,
Saccharomyces cerevisiae
Micro-manna Indogulf
BioAg
USA
Canada
It affects the microbial
environment in a way that
favors the growth of
beneficial microorganisms.
https://www.
indogulfbioag.com/
Continued
6. Multifunctional plant growth-promoting attributes of microbial consortia 185

Table 9.4 Some of the selected microbial consortiumebased biofertilizers available on the global market.dcont’d
Type of
microbial
consortium
Species involved in
microbial consortium Product name
Producing
company
Country
of origin Mode of action Reference
Fungal-
bacterial
consortium
Penicilium bilaii,
Rhizobactria
Recover PO4 Brett-Young
Seeds
Canada It serves as a phosphate
solubilizer and also fixes
nitrogen through nodulation.
The active component
improves early vigour, boosts
root growth, and increases
leaf surface area by making
soil and fertilier phosphate
more available to plants.
https://brettyoung.
ca/
Mycorrhiza fungi,
Rhizobacteria
Multi-Bio Indogulf
BioAg
USA
Canada
The mycorrhizal fungal and
rhizobacterial consortium
gives the plant’s root all the
goodness. It also provides all
the vital nutrients the plant
needs to develop strongly
and healthily.
https://www.
indogulfbioag.com/
Penicillium bilaiae,
Bradyrhizobium japonicum
TagTeam Novozymes USA This consortium aids in the
release of phosphate that
has been bonded to soil and
fertilizer. Moreover, it also
helps in increasing its
availability to plants and
promoting nodulation for
increased nitrogen fixation
capability.
https://biosolutions.
novozymes.com/
186 CHAPTER 9 Microbial consortium-based biofertilizers

Actinomycetes,
rhizobacteria, and
thermotolerant fungi
Bannari Bio
decomposer
Bannari
Amman
Sugars
Limited
India This consortium converts the
lignin and cellulose from farm
waste to quickly transform
them into nutrient-rich,
pathogen-free organic
fertilizer.
https://www.
bannarianfu.com/
Fungal
consortium
Consortium of mycorrhizal
fungi
RISEHoP
mycorrhiza HM
RISEHoP Canada It effectively joins the plant’s
root system and creates a
vast underground network of
interconnected filaments. It
also enhances nutrient
uptake, marketable yields,
and crop quality overall.
https://risehop.com/
Consortium of endo-
mycorrhizal fungi
UpLoad ENDO-
mycorrhiza
RISEHoP Canada
USA
Potent endo-mycorrhizae
found in UpLoadÒboost
mineral solubilization, crop
performance, and economic
value.
https://risehop.com/
Aspergillus chevalieri
(NAIMCCeSFe0094)
Aspergillus quadrilineatus
(NAIMCCeSFe0095)
AsperPhos Nextnode
Bioscience
Pvt. Ltd.
India Strong phosphate
solubilizing consortia that
support seed germination,
mineral solubilization, and
crop yield.
https://neemplus.
com/
Trichoderma harzianum
Trichoderma viridae
Ampelomyces quisqualis
FungFree Utkarsh
Agrochem
Pvt. Ltd.
India Crop plants are protected
from soil- and air-borne
diseases by FungFree. It is
also a potent treatment for
diseases that affect all types
of plantations and crops.
https://utkarshagro.
com/
6. Multifunctional plant growth-promoting attributes of microbial consortia 187

value of agrifood products. They are frequently incorporated into agricultural man-
agement techniques meant to boost output, reduce the use of chemicals, and restore
the natural homeostasis in agro-ecosystems (Wo o &Pepe, 2018,Table 9.5).
Microbial consortia are typically more effective and efficient at inducing plant
defense than a single microbial inoculant. Therefore, there is a lot of room to use
these mechanisms for regulating yield and reducing illnesses in a related crop, espe-
cially in light of the capacity of several microbes in a consortium to trigger pathways
relevant to resistance or biocontrol (de Vrieze et al., 2018;Sharma et al., 2018). In
this context, Singh et al. (2014) assessed the performance of a microbial consortium
developed with Trichoderma harzianum THU0816, Pseudomonas aeruginosa
PHU094, and Mesorhizobium sp., RL091, three compatible rhizosphere microorgan-
isms that are effective at accelerating plant development and releasing phenolic acid
from Sclerotium rolfsii-infected chickpea.
In this experiment, the treated plants acquired increased concentrations of a num-
ber of phenolic compounds, including ferulic acid, syringic acid, myricetin, and
quercetin. Bacillus subtilis CRN-16 and Pseudomonas putida CRN-09 were tested
for their synergistic interaction in Vigna radiata (mung bean), where the microbial
consortium increased the level of scavenging enzymes like polyphenol oxidase
(PPO), peroxidase (PO), b-1,3 glucanase, chitinase, and phenylalanine ammonia-
lyase (PAL) and resulted in the expression of systemic resistance against the plant
pathogen fungus Macrophomina phaseolina (Sharma et al., 2018). Moreover, De
Vrieze et al. (2018) used three different potato varieties in a leaf disc assay to test
the efficacy of nine different Pseudomonas strains against the phytopathogenic fun-
gus Phytophthora infestans, both singly and in consortium. According to the find-
ings, two bacterial strains, P. fluorescens S49 and P. frederiksbergensis S19,
together proved to be very successful at containing the pathogen. Similar findings
were made regarding the effects of a bacterial consortium (Azospirillum sp. and
P. aeruginosa) on plant yield and the suppression of root rot disease brought on
by Rhizoctonia bataticola in cotton plants under both half and full doses of synthetic
FIGURE 9.2
Some of the commercial microbial consortiaebased biofertilizers available on the market.
188 CHAPTER 9 Microbial consortium-based biofertilizers

Table 9.5 Beneficial effects of different plant growthepromoting microbes as consortium in crop production.
Microbial species
Type of
microbial
consortium Crop/plant
Mode of action and beneficial
effects Reference
Pseudomonas fluorescens (PGPR)
Actinomycetes, and Arthrobacter
Bacterial
consortium
Soybean
(Glycine max)
- Significantly increased the
amounts of available nitrogen (N),
phosphorus (P) and potassium (K)
- Enhanced the crop yield, microbial
populations, and available soil
nutrients
Yaduwanshi
et al. (2021)
Bacillus subtilis,Pseudomonas aeruginosa,
Klebsiella pneumoniae, and Citrobacter
youngae
Bacterial
consortium
Tomato
(Lycopersicon
esculentum Mill)
- Phosphate solubilization
- Phytohormones (IAA, ammonia,
and hydrogen cyanide) production
- Enhanced the stem girth and
seedling height
- Increased the leaf area and
number of leaves
Oluwambe
and
Kofoworola
(2016)
Pseudomonas grimontii,Sphingobacterium
kitahiroshimense,
Microbacterium oxydans
Bacterial
consortium
Oilseed rape
(Brassica Napus
L.)
- Significantly growth in oilseed
rape, chlorophyll content index,
shoot length, and number of leaves
- Increased nutrient availability,
- Resistance to biotic stress (fungal
pathogen) suppression and abiotic
stress
- Tolerance of plants to salt stress
Brzezinska
et al. (2021)
Azospirillum brasilense Sp7, Pseudomonas
putida KT2440, Acinetobacte r sp. EMM02,
and Sphingomonas sp. OF178A
Bacterial
consortium
Maize
Zea mays L.
- The bacterial consortium provided
a safe alternative to chemical
fertilization and decreased the
chemical fertilization
- Increased the crop yield
Molina-
Romero et al.
(2021)
Continued
6. Multifunctional plant growth-promoting attributes of microbial consortia 189

Table 9.5 Beneficial effects of different plant growthepromoting microbes as consortium in crop production.dcont’d
Microbial species
Type of
microbial
consortium Crop/plant
Mode of action and beneficial
effects Reference
Actinomycetes,
Arthrobacte r, and P. fluorescens (PGPR)
Bacterial
consortium
Soybean
(Glycine max)
- Significantly improved plant
growth attributes (biomass, plant
height, chlorophyll content and
yield), and nutrient content (N, P, K)
- Increased nodulation in treated
plants
Yaduwanshi
et al. (2019)
Azotobacter chroococcum, Bacillus
megaterium,Pseudomonas fluorescens,
Bacillus subtilis,
Trichoderma harzianum
Bacterial-
fungal
consortium
Cabbage
(Brassicaceae
oleraceae var.
capitata Linn
- Antibacterial and antifungal
activities
- More biocontrol efficacy and least
disease incidence
- Enhanced total biomass, seedling
vigor
Sudharani
et al. (2014)
PGPR (Acinetobacter sp., Rahnella aquatilis),
rhizobia (Ensifer meliloti RhOF4, E. meliloti
RhOF155), and AMF (Glomus sp., Sclerocystis
sp.)
Bacterial-
fungal
consortium
Faba bean (Vicia
faba L.)
Wheat (Triticum
durum L.)
- Mineral solubilization and
improved the available N, P, K, Ca,
and Na contents in shoots
- Enhanced the contents of proteins
and sugar
- Improved the growth parameters
(dry weights of roots and shoots,
number of leaves) in both crops
- Significantly improved the
productivity of both plants
presented by the weight and
number of bean pods and wheat
spikes
Raklami et al.
(2019)
190 CHAPTER 9 Microbial consortium-based biofertilizers

Rhizoglomus irregulare (AMF), Pseudomonas
putida (PSB)
Bacterial-
fungal
consortium
Zea mays L.,
cultivar sincere
- Increased the mineral
solubilization
- Improved the phosphate use
efficiency and enhanced the maize
productivity
Pacheco
et al. (2021)
Coprinopsis cinerea LA2,
Cyathus stercoreus ITCC3745
Fungal
consortium
Rice (Oryza
sativa)
- Improved the microbial
communities and activity of soil
hydrolytic enzymes
- Degradation of crop residue
- Improved the soil organic carbon
- Increase the grain yield of the
wheat crop
Kumar et al.
(2022)
Beauveria bassiana,
Metarhizium anisopliae,
Pochonia chlamydosporia,Purpureocillium
lilacinum, and Trichoderma asperella
Fungal
consortium
Soybean, corn,
and sugarcane
- Induced root growth and stress
o(auxins, abscisic, salicylic, and
jasmonic acids)
- Mineral solubilization and soil
resource acquisition
- Promoting greater absorption of
water and nutrients
Farias et al.
(2018)
Daldinia eschscholtzii,Sarocladium oryzae,
Rhizoctonia oryzae, Penicillium allahabadense,
and Aspergillus foetidus
Fungal
consortium
Cucumber
(Cucumis
sativus)
- Phytohormones (IAA, gibberellins)
production
- Siderophore producing and
phosphate-solubilizing ability or
mineral solubilization
- Successfully increased root
growth, fresh weight, and plant
height growth
Syamsia et al.
(2021)
6. Multifunctional plant growth-promoting attributes of microbial consortia 191

fertilization (Marimuthu et al., 2013). Similarly, Krestini et al. (2020) investigated
the microbial consortium’s (Trichoderma harzianum, Bacillus subtilis, A. chroococ-
cum, and Pseudomonas cepacia) best features for growth and yield quality, along
with its impact on the emergence of the wilt disease brought on by Fusarium oxy-
sporum Schelecht in garlic cultivation. According to the results, the usage of
150 g of the microbial consortium resulted in the greatest value for high plant param-
eters (leaves, yield weight, and tuber diameter). When compared to control manage-
ment, the adoption of a microbial consortium 40 days after planting can prevent
fusarium disease by 14.7%e41.17%.
The oxidative damage caused by free radicals created in a plant’s cells, which
attack essential biological structures like DNA and cellular membranes, can impede
a plant’s growth in response to abiotic conditions (Kumar et al., 2016;Santoyo et al.,
2021). Catalase and peroxidase are two antioxidant enzymes that can neutralize
these reactive chemicals and save the cells from harm (Kumar &Verma, 2018).
Many bacterial species have the capacity to increase the activity of these scavenging
enzymes as well as synthesize protective compounds like trehalose that also aid in
enhancing the capacity of plants to respond to abiotic stress (Glick &Glick, 2015).
Heidari and Golpayegani (2012) used a bacterial consortium consisting of Azospir-
illum brasilense,Bacillus lentus, and Pseudomonas sp. under water stress condi-
tions, which showed an increase in chlorophyll content and antioxidant activity.
Similarly, on commercial hardwood cuttings of the Populus nigra clone OP-367 x
Populus deltoides, the influence of a consortium made up of 10 bacterial strains
was examined. Following a month of being subjected to water stress, there was a sig-
nificant promotion of growth and improvement in foliar physiology in response to
bacterial endophytic colonization (Khan et al., 2016). According to Saikia et al.
(2018), the inoculation of a group of three ACC-deaminase-producing rhizobacteria
(Pseudomonas sp. RJ15, Ochrobactrum pseudogrignonense RJ12, and Bacillus sub-
tilis RJ46) gradually improved the seed germination rate, shoot length, root length,
and dry weight of the legumes Pisum sativum and Vigna mungo under drought
conditions.
As opposed to the impacts of either organism alone, the benefits that both organ-
isms bring to a plant are increased by the combination of AMF and PGPB, which
leads to an increase in plant growth promotion (Barea et al., 2005;Santoyo et al.,
2021). Dual inoculation of a fungal-bacterial consortium (Bacillus sp. and
G. mosseae) leads to better outcomes in terms of the accumulation of nitrogen, phos-
phorus, and potassium in AMF Pelargonium graveolens and lettuce than each single
inoculation (Alam et al., 2011;Vivas et al., 2003). The consortia of Trichoderma sp.
and Bradyrhizobium sp. also improved the overall shoot and root dry weight of
cowpea plants and shielded them from Rhizoctonia solani infection (Junior et al.,
2015).
192 CHAPTER 9 Microbial consortium-based biofertilizers

7.Challenges and constraints with microbial
consortiaebased biofertilizers
There is growing interest in using microbial-based products as biofertilizers in
organic farming in the wake of the COVID-19 pandemic. However, there are a num-
ber of difficulties with their utilization when going from the lab to the field. These
bioinoculants have been used experimentally on crop plants like grains and legumes
(Basu et al., 2021). An initial laboratory screening is necessary for creating a new
microbial consortium that can function as an efficient bioinoculant. This screening
depends on particular direct and indirect processes of PGPRs that promote plant
growth. Primary screening of isolated axenic culture plants for PGPR characteristics
alone does not ensure effective plant growth promotion in the field. Furthermore,
pure culture isolates with fewer in vitro growth-promoting behaviors may have
different plant growth promotion strategies. Due to our incomplete understanding
of these pathways, such isolates are challenging to screen for under normal circum-
stances. As a result, because of their inferior in vitro performance, beneficial strains
exhibiting these pathways may get thrown out (Basu et al., 2021;Cardinale et al.,
2015). The following significant issues and numerous difficulties and restrictions
must be resolved in order to produce and use microbial consortium biofertilizers
on a large scale.
7.1 Biological constraints
It is challenging in and of itself to choose specific microbial strain(s) for the produc-
tion of microbial consortium biofertilizer. The strain(s) should be broad in their host
range and not overly selective or targeted to specific crops. Their selectivity is one of
the key challenges that limit them. Unlike plant growthepromoting microorganisms
(PGPM), conventional agrochemicals have a tendency to affect the entire resident
microbiota. However, because other microorganisms are present in the field, the
quality and effectiveness of these PGPM invariably fluctuate. Potential microbial
isolates should be chosen based on how well they function in the field on different
varieties of crops and in various soil types and environmental settings (Meena et al.,
2020). The microbial strains must have the capability of successfully replacing the
native and ineffective strains and must not compete with other rhizosphere-dwelling,
advantageous microorganisms (Mahajan &Gupta, 2009). Selected microbes must
be capable of sufficiently colonizing the roots of the host plant, providing a suitable
rhizosphere for plant growth, and boosting the bioavailability of N, P, K, and antag-
onistic characteristics when acting as microbial consortium biofertilizers. These
methods of selecting microbes in microbial consortiums produce efficient and
long-lasting results in the field (Vejan et al., 2016).
7.2 Technical constraints
The shelf life of a biofertilizer is among the major difficulties encountered in the
development and commercialization of an efficient microbial strain (Zandi &
Basu, 2016). Biofertilizers with a limited shelf life run the risk of being recycled
7. Challenges and constraints 193

if they are not used or sold before they go bad, which would cause the marketing
company to incur a net financial loss. Biofertilizers must be handled with additional
caution during storage and shipping since they contain living microbial cells. Tech-
nical limitations include the possibility of product degradation due to a shorter shelf
life or unexpected mutations developing during the fermentation process for mass
production or during storage. The mutations cause a serious issue that raises the pro-
duction cost and reduces the bioinoculant’s quality, resulting in a net decrease in its
efficacy. The broad use of bioinoculants is significantly constrained by the inade-
quate regional availability of strains that are particular to soil (Basu et al., 2021;
Mahajan &Gupta, 2009).
7.3 Quality control constraints
Quality control is the most crucial feature that growers seek in a biofertilizer. Living
microorganisms have an extremely limited shelf life because they are natural prod-
ucts. Any microbial-based product’s failure in the field could be attributed to the
availability of subpar or fake goods. There are no quality checks for biofertilizers
currently available. Establishing quality control criteria for biofertilizers is therefore
very important in order to demonstrate the effectiveness of these fertilisers in boost-
ing plant development in the fields (Mahajan &Gupta, 2009;Meena et al., 2020).
7.4 Biofertilizer carrier constraints
The limited shelf life of the microbial inoculants necessitates the use of a suitable
carrier for the biofertilizer when applied in the field. As a result, one of the major
barriers to its widespread use in fields is a lack of a suitable carrier. Peat, charcoal,
lignite, and other suitable carriers are used in the mass production of biofertilizer.
Due to the fact that the majority of these carriers are not available in developing na-
tions like India, they once again present technical challenges. These carriers are
insufficiently plentiful and have a desirable property. Only charcoal can be used
as a forming agent because it is easily accessible on the Indian market. Moreover,
in the case of peat, the problem is that it has a shelf life of less than 6 months, which
makes it the least ideal carrier out of those that are accessible. The commercial bio-
fertilizer should have a number of other qualities to demonstrate its effectiveness as a
carrier-based formulation. It should be inexpensive, have a high concentration of
organic matter and water-holding ability, and have a longer period of organism
retention. For a biofertilizer to be of high quality, it must be nearly sterile, have
no moisture, be toxic-free, be non-polluting, and have a pH that is nearly neutral
(Backer et al., 2018;Bashan et al., 2014;Basu et al., 2021).
7.5 Field-level constraints
Since the inoculum requires time to develop its concentration and root colonization,
the response of crops to an applied microbial inoculant is very sluggish and
194 CHAPTER 9 Microbial consortium-based biofertilizers

occasionally fruitless. As a consequence, farmers embrace biofertilizers only to a
limited extent. In field application, inoculation methods and inoculant purity are
both crucial factors. Because of the dangerous aftereffects of synthetic pesticides
and current unfavorable abiotic circumstances, biofertilizer efficiency is reduced
(Parnell et al., 2016). Environmental stress, such as salt and drought in some regions,
is another significant factor in the decline of the biological activity of a microbial
inoculant. The soil’s acidity and alkalinity, the use of pesticides, and excessive con-
centrations of nitrate in the soil restrict the ability of the bioinoculants to fix nitrogen
or mineral solubilization, resulting in poor performance of the bioinoculants in the
fields (Arora et al., 2010;Basu et al., 2021). Moreover, various soils have dangerous
levels of heavy metals such as Hg, Cd, and Cr, as well as a lack of other essential
minerals like Cu, P, Co, and Mo, which lower the bioactivity of microbial-based fer-
tilizers (Bhardwaj et al., 2014;Ndeddy Aka &Babalola, 2016). The performance of
the field application of a microbial consortium depends on the climatic conditions
necessary for certain types of cultivated crops. As a result, the microbial strain
that is used to produce a microbial consortium should be as region-specific as
possible in order to perform well in the field (Mahanty et al., 2017).
7.6 Regulatory constraints
The difficulties in product registration and patent applications are examples of reg-
ulatory restrictions. The laws are frequently inconsistent and change between
different countries and regions. Additionally, the regulatory procedures are quite
complicated, and although the costs vary, they are typically on the higher side.
The documentation requirements for product registration are similarly rigorous
and difficult. In India, the fungal-based consortia are not approved by the regulatory
authority for field application, whereas it is very complicated for microbial product
registration or approval for field application in the European Union because of a dual
system that exists under the European Food Safety Authority (EFSA). There is no
uniform, standardised legal or regulatory definition available for “plant bio-
stimulant” and “microbial consortium” worldwide that can be used as a coordinated
uniform policy for harmonization of the microbial products trade. Each nation has its
own rules and guidelines for how to respond in its own language, and the licensing
authority may additionally ask for even more information that causes an extra
burden on the manufacturer (Basu et al., 2021;Teicher, 2018;Timmusk et al., 2017).
8.Conclusion and future perspectives
After the incidence of pandemic COVID-19, the area of organic agriculture has
increased worldwide because of the consumer demand for healthy, fresh, and safe
food products. Therefore, the demand for organic inputs (such as microbial inocu-
lants) as an alternative to chemical fertilizers and pesticides also increased in sus-
tainable or organic production systems. Microbes can utilize a variety of carbon
8. Conclusion and future perspectives 195

sources, according to a microbial consortium. It gives microbial resilience in the
face of environmental stresses. A consortium of microbes can carry out intricate
tasks that are challenging for a single microbe to complete. Technology development
has made it possible to comprehend microbial interconnection and produce consor-
tia. Many types of beneficial microbes (like nitrogen-fixing microbes, phosphate-
solubilizing microbes, zinc-solubilizing microbes, PGPRs, siderophore-producing
microbes, and sulfur-dissolving microbes) are used in developing microbial con-
sortium as per the requirements of the soil and environmental conditions. Now, re-
searches can expand the boundaries of biotechnology from pure cultures to mixed
cultures as a microbial consortium at commercial level. Microbial consortiums
with multifunctional plant growth-promoting attributes and phytopathogen-
controlling potential have also been tested on several crops and showed significant
results on the growth, yield, and performance of the crops under stress conditions.
Microbial consortiums are divided into three types based on their structure, modes
of interaction, and functions: (i) bacterial consortiums, (ii) fungal consortiums, and
(iii) fungal-bacterial consortiums that are commercialized worldwide for nutrient
management, disease management, and insect pest management. On the global mar-
ket, there are several formulations that combine a variety of microorganisms,
including nematodes, bacteria, viruses, fungi, and algae (Ram et al., 2022). Howev-
er, many limitations and constraints are also associated with successful production
of microbial consortium-based biofertilizers, like selection of a potential microbial
strain, the right carrier material for longer storage, standard operating procedures
(SOPs) for mass multiplication, quality analysis of the finished product, and regula-
tory issues for marketing and trade globally. Single-cell genomics (SCG), microflui-
dics, fluorescence imaging, and membrane separation are some of the current
advances in the study of microbial interactions and functions. In addition, a number
of cutting-edge technologies can be employed for community profiling to identify
microbial strains for consortium development. These advanced tools include ampli-
fied ribosomal DNA restriction analysis (ARDRA), PCR-DGGE, and terminal re-
striction fragment-length polymorphism (T-RFLP) (Bhatia et al., 2018).
Furthermore, a uniformly harmonized policy for the commercialization of microbial
consortiums is required for nations to gain unrestricted access to global markets.
This standardized system would benefit new businesses all over the world producing
microbial-based bio-inputs.
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208 CHAPTER 9 Microbial consortium-based biofertilizers

Nutrition and cultivation
strategies of core
rhizosphere
microorganisms 10
Hetvi Naik, Komal A. Chandarana, Harshida A. Gamit, Sapna Chandwani,
Natarajan Amaresan
C. G. Bhakta Institute of Biotechnology, Uka Tarsadia University, Bardoli, Gujarat, India
1.Introduction
The Greek word “rhiza” means the root (Hartmann &Zimmer, 1994). The German
scientist of plant physiology Lorenz Hiltner in 1904 first termed “rhizosphere” to
describe plant-root interface. Hiltner described that they surround a plant root
inhabited by various microbes, including bacteria, fungi, algae, and actinomycetes.
There are three main parts in the rhizosphere. They are endorhizosphere, rhizoplane,
and ectorhizosphere. The endorhizosphere involves segments of the cortex and
endodermis, that cations and microbes can inhabit. The rhizoplane is the portion be-
tween the endorhizosphere and ectorhizosphere, which is directly adjacent to the
root, including root and mucilage. The outermost zone of the rhizosphere is the ecto-
rhizosphere. Rhizospheric microbes include bacteria, fungi, algae, arthropods, pro-
tozoa, archaea, and viruses. The bacteria found in the rhizosphere include
Azospirillum, Bacillus, Streptomyces, Flavobacterium, Enterobacter, Bradyrhi-
zobium, Rhodococcus, Klebsiella, Azotobacter, Pseudomonas, Alcaligenes, Meso-
rhizobium, and Arthrobacter (Malviya et al., 2021). These microbes are part of
the complex food chain and utilize the large amounts of nutrients released by plants
such as rhizodeposits (Mendes et al., 2013). Rhizodeposits are defined as all material
lost from plant roots, including exudates, lysates, and gases such as CO
2
and
ethylene (Cheng &Gershenson, 2007). Rhizodeposits are rich in nitrogen and car-
bon elements and can positively stimulate the augmentation of rhizomicrobiomes.
Plants grown under high CO
2
conditions mostly show increased growth, total rhizo-
sphere respiration, and rhizodeposition. As the quantity of rhizodeposits is directly
proportional to the plant carbon balance, they are a great source of nutrients for rhi-
zospheric microbes (Cheng &Gershenson, 2007). Fig. 10.1 shows the nutrients
derived from rhizodeposits.
The roots are an important plant organ. Rhizomicrobiomes receive nutrients
from plant root exudates (Balasubramanian et al., 2020). The root exudates not
only attract microorganisms but also suppress the growth of microbes that are
CHAPTER
209
Rhizobiome. https://doi.org/10.1016/B978-0-443-16030-1.00018-3
Copyright ©2023 Elsevier Inc. All rights reserved.

unfavorable for plant growth. The effects of specific root exudates on rhizospheric
microbes are shown in Table 10.1. Primary and secondary metabolites are released
into the soil by plants through the root tip via facilitated diffusion (Canarini et al.,
2019). The environmental sense of the surroundings allows the plant to decide
when to secrete metabolites (2000). The primary metabolites released from plants
consist of organic acids, sugars, and amino acids (Badri &Vivanco, 2009). Second-
ary metabolites mostly consist of a variety of small molecules that are abundant in
the rhizosphere of plants and are used in different ways to benefit rhizomicrobiomes
and plants (Weston &Mathesius, 2014).
The rhizosphere is a microecosystem that surrounds plant roots, and is highly
affected by root exudate chemicals. These effects vary from root to root, and from
root to microbes. Stimulation by abiotic stress and biotic factors also affects the
exudation of chemicals from the root system (Aulakh et al., 2001). The chemical
composition of the root exudates consists of primary and secondary metabolites.
Chemicals exuded from plant roots have specific functions that are beneficial for
plants as well as rhizomicrobiomes. These functions include making low-level soil
nutrients such as phosphorous available to plants, accommodation to support micro-
bial populations in the rhizosphere, including endophytic and rhizobial bacteria,
mycorrhizal fungi, and the absorption and accumulation of heavy metals (Badri &
Vivanco, 2009;Bouwmeester et al., 2007;Faure et al., 2009). Several factors can in-
fluence the rhizospheric microbiome. The structure of plants, nature of plants, rhizo-
deposits, developmental stage of plants, and other root exudes dictate the surrounding
rhizospheric microbial population (Chaparro et al., 2014;Dey et al., 2012).
FIGURE 10.1
Rhizodeposits: nutrients for soil microbes.
210 CHAPTER 10 Nutrition and cultivation strategies

Each plant species supports a different group of rhizospheric microbes. The same
plants species, but with different genotypes, exhibit different groups of rhizospheric
microbes. Previous studies have shown that plant richness and functional diversity
positively impact microbial functional diversity (Goswami et al., 2021).
2.Members of rhizomicrobiome
Microbial communities in the rhizosphere can have mutualistic relationships that
benefit each other. Microbial populations in the rhizosphere can be classified as bac-
teria, fungi, protozoa, actinomycetes, and nematodes. Among these fungi and bac-
teria are the most extensively studied, and the population of bacteria is higher
than that of another microflora (Khan et al. 2020).
Table 10.1 Effect of specific root exudates on rhizobacterial activities.
Root exudates Microbes Function References
Organic acids Paenibacillus
polymyxa
Colonization in watermelon
plant roots
Ling et al.
(2011)
L- Malic acid Bacillus subtilis Biofilm development and
colonization on tomato and
Arabidopsis plant roots
Chen et al.
(2012)
Benzoxazinoids Pseudomonas
putida
Bacterial cell chemoattraction
to maize roots
Neal et al.
(2012)
Arabinogalactan Bacillus subtilis Biofilm development on
Arabidopsis
Beauregard
et al. (2013)
Citric acid Bacillus
amyloliquefaciens
Biofilm development and
colonization on cucumber
roots
Zhang et al.
(2014)
Flavonoids Rhizobium, Frankia,
and arbuscular
mycorrhizal fungi
Chemoattractant Huang et al.
(2014)
Abdel-Lateif
et al. (2014)
Strigolactones Mycorrhiza Activation of mitochondria
thus stimulation of AMF
(Huang et al.,
2014)
(Besserer
et al., 2006)
Humic acid Pseudomonas
putida
Bacterial chemotaxis Jimenez-
Sanchez
et al. (2015)
Amino acids
(mixture)
Bacillus subtilis Chemotaxis toward
Arabidopsis roots
Allard-
Massicotte
et al. (2016)
2. Members of rhizomicrobiome 211

3.Bacteria
Bacteria can adapt to any situation in the surrounding environment owing to their
easy metabolism and fast growth. The bacterial community in the rhizosphere is
largely dependent on the developmental stage of the plants and the root exudates
of diverse plant species (Olanrewaju et al., 2019). The most prominent bacterial com-
munities in the rhizosphere are Pseudomonas, Bacillus, Acinetobacter, Rhizobium,
Azospirillum, Azotobacter, and Agrobacterium. Depending on their location, there
are two types of PGPR: intracellular plant growth promoting rhizobacteria (iPGPR)
and extracellular plant growth promoting rhizobacteria (ePGPR). Extracellular PGPR
are most conspicuously found in the rhizosphere and root cortex (Khan et al., 2020).
In contrast, iPGPR are present inside the plant cells and cause nodule formation.
Rhizobium and Frankia are the most prominent examples of iPGPR that aid in nitro-
gen fixation. PGPRs are classified as rhizoremediators, bioprotectants (biocontrol
agents and biopesticides), phytostimulators, and biofertilizers (Vejan et al., 2016).
4.Fungi
Fungal diversity is predominant in all the soil types. The diversity of fungi in the
rhizospheric soil is determined by monitoring the presence of fruiting bodies
(Frąc et al., 2018). Rhizosphere microbial communities inherit both symbiotic and
pathogenic fungi (Khan et al. 2020). The fungi present in the soil and associated
with plants are known as mycorrhizae. Mycorrhizae are directly associated with
plants, and their association is based on plants that provide essential organic com-
pounds as well as carbohydrates to fungi; in turn, fungi help plants to take up nutri-
ents (Suleiman et al., 2015). Three types of mycorrhizae are present in the soil,
which include endomycorrhiza, ectomycorrhiza, and ectendomycorrhiza. Endomy-
corrhizae (arbuscular mycorrhizae) are the most important mycorrhizae present in
the soil. They are also called vesicular-arbuscular mycorrhizae (VAM) (Linderman,
2015). VAM transports mineral nutrients and water from the soil to plants, and host
plants provide carbon compounds to the fungi. Therefore, this fungus has a signif-
icant effect on plant function and form. Depending on host plants, other mycorrhizae
include Ericoid mycorrhizae, Arbutoid mycorrhizae, Monotropoid mycorrhizae, and
Orchidaceous mycorrhizae (Barman et al., 2016).
The fungal community of the rhizospheric soil also acts as a biocontrol agent.
Such rhizospheric soil fungi significantly contribute to plant protection. Some exam-
ples of this include Trichoderma spp. These fungi compete with plant pathogens
through various mechanisms such as parasitism, induced resistance, antibiosis,
and competition (Zhang et al., 2016). Other species of Trichoderma also help to
enhance plant growth by producing growth-regulating substances and facilitating
nutrient uptake by solubilizing them (Li et al., 2015). The rhizospheric yeast Saccha-
romyces unispora and Candida steatolytica have inhibitory effects against the plant
pathogenic fungus Fusarium oxysporum, which causes wilt disease in tomato and
212 CHAPTER 10 Nutrition and cultivation strategies

kidney bean. Verticillium biguttatum isolated from rhizospheric soil also shows
antagonistic activity against Rhizoctonia solani, which has wide host range and is
responsible for major economic loss worldwide (Lahlali et al., 2007).
Oomycetes are also known as water molds, which are responsible for some plant
diseases such as seedling blights, foliar blights, etc. (Fry &Gru
¨nwald, 2010). Phy-
tophthora and Pythium are the predominant species of oomycetes present in rhizo-
spheric soil. It has been reported that the oomycete Pythium oligandrum in the
tomato rhizosphere produces tryptamine, which is an auxinlike compound, and oli-
gandrin, a glycoprotein elicitor (Vallance et al., 2009). In plants, tryptamine in
minuscule amount acts as an important substrate for the plant hormone IAA
(Kousara et al., 2017). Oomycetes act through antibiosis, competition for nutrients,
and mycoparasitism, and also promote plant growth (Rey et al., 2008). The nutri-
tional requirements of each community are different and their interactions with
plants differ. Their nutritional requirements also depend on the nature, species,
and growth stage of the host plant, and the presence of other microbial community
members of the rhizosphere.
5.Others
Bacteria and fungi cover a major portion of the biomass in rhizomicrobiomes, but
there are also other members that reside in rhizomicrobiomes. These members
include protozoans, nematodes, and oomycetes. Nematodes are free-living worms
that are known for their pathogenic activities in plants. In some cases, they also
possess biocontrol activities that control the growth of other pathogens present in
the rhizosphere (Kenney &Eleftherianos, 2016). Nematodes from the genera Steiner-
nema anong and Heterorhabditis act as biocontrol agents by restricting the growth of
different pathogens in the rhizosphere, and can be used for pest management (Khan
et al. 2020). The secondary metabolites released as root exudates attract the nema-
todes and act as selection criteria for entomopathogenic nematodes (EPN) compared
to pathogenic nematodes (Hiltpold et al., 2015). Other members reside in the rhizo-
sphere other than bacteria and fungi; nematodes and protozoa that feed on microbes
for food supply. The presence of protozoa supplies the nitrogen to plants as they have
limited access to nitrogen and enhance the release of organic matter. Protozoa in
combination with AMF enhance the nutrient supply to plants. Protozoa also facilitate
the translocation of photosynthates from their source to the roots (Koller et al., 2013).
6.Why rhizospheric microbiome is important?
Currently, chemical fertilizers are used to supply nutrients to plants in the soil. How-
ever, the cons of chemical fertilizers such as price, availability, and their effects on
the environment are well known, and the modern world is switching toward natural
fertilizers (Majeed et al., 2015). The application of microbial inoculants or PGPR for
6. Why rhizospheric microbiome is important? 213

plant growth promotion (PGP) and enhancement of crop production is widely
accepted for sustainable agriculture (Nasrin &Rahman, 2007). PGPR are soil bac-
teria that colonize the rhizosphere and surround the plant roots (Kumar et al., 2014).
The PGP properties of these microbes are attributed to their primary and second-
ary metabolites produced during their growth. Several studies have been conducted
to understand the mechanisms by which PGPR benefits the host plants. These
includes
(i) Their ability to produce phytohormones or growth regulators such as gibber-
ellins, indole acetic acid (IAA), and cytokinins (Glick, 1995;Marques et al.,
2010).
(ii) Mineralization of nutrients or organic phosphate and solubilization of inorganic
phosphate (Glick, 1995;Jeon et al., 2003).
(iii) Their ability to enhance asymbiotic N
2
fixation (Khan, 2005;¸
Sahin et al.,
2004).
The rhizosphere microbial community differs from the microbial community in
bulk soil because plant roots employ and accumulate specific microbes in the rhizo-
sphere (Hein et al., 2008). Plant growth promoting microbes (PGPM) promote
nutrient uptake by regulating various physiological activities of plants (Qu et al.,
2020). PGPM from rhizospheric soil also helps to increase yield by regulating the
diversity of rhizomicrobiomes. PGPM also enhance the abiotic stress tolerance in
plants. Such microbes also help in the induction of the plant immune system by pro-
ducing antimicrobial compounds such as surfactin, iturin, fengycin, 2-nonanone,
and 2-heptanone (Cazorla et al., 2007;Wu et al., 2019).
PGPM not only helps plants in their development but is also beneficial for soil.
PGPM helps to solubilize minerals (Kalyanasundaram et al., 2021). PGPM exploits
root exudates and metabolizes them to produce organic acids (Goswami et al.,
2014). These organic acids functions as ca
2þ
chelators and release phosphates.
The solubilization of aluminum (Al) and iron (Fe) occurs via proton release, thus
decreasing the charge of the adsorbing surface and assisting the sorption of nega-
tively charged P ions (Kalyanasundaram et al., 2021). In terms of K ions, the bac-
teria released by K include Bacillus mucilaginosus, B. circulans,andPseudomonas
spp. Other PGPM such as Enterobacter cloacae, Klebsiella variicola, and Pantoea
agglomerans help in the release of silicon (Si) and Al (Zhang &Kong, 2014).
PGPM also produce signaling chemicals that bridge the communication between
plants and microbes. Plants produce primary metabolites such as proteins and
carbohydrates, and secondary metabolites such as phenolic compounds and phyto-
hormones act as signal compounds for plant-microbe interactions and microbe-
microbe interactions (Engelmoer et al., 2014). The production of hormones by
rhizomicrobiomes benefits plants by altering root elongation (Kalyanasundaram
et al., 2021). PGPM also performs antagonistic actions against various pathogens
through several mechanisms such as lytic enzyme production, ISR, detoxification
and degradation of virulence factors, production of antibiotics, and hydrogen cya-
nide (Ambrosini et al., 2012). PGPM also induces systemic resistance and enhances
214 CHAPTER 10 Nutrition and cultivation strategies

the tolerance of plants against various abiotic stresses such as heavy metals,
salinity, and moisture (Kalyanasundaram et al., 2021).
7.Nutritional strategies for beneficial rhizospheric
microbes
Rhizosphere microbes derive nutrients from plant root exudates. Plant root exudates
and other substances released from plants may serve as criteria for microorganism
selection (Berendsen et al., 2012;Walker et al., 2003). In plants, lignocellulose
also acts to select bacteria from the soil (Bulgarelli et al., 2012). Phenol from the
root extracts has also been found to play a crucial role in conditioning bacterial com-
munities (Lo
´pez-Guerrero et al., 2013). Seeds are also a great source of nutrients for
plant-associated bacteria, and contain large amounts of phytate (Lott et al., 2000).
Phylae are the primary storage forms of both phosphate and inositol (Kumar
et al., 2010). Rhizospheric microbes derive sulfur from germinated seedlings
(Snoeck et al., 2003). In order to isolate the beneficial rhizospheric microbes, the nu-
trients provided to them play a crucial role in the isolation process. Nutrients such as
carbon sources, sugars, and amino acids may vary among microbes. Thus, it is
important to determine the requirements for specific rhizospheric microbes.
8.Cultivation strategies for beneficial rhizospheric
microbes
After nutrition, it is important to understand the cultivation of rhizospheric mi-
crobes. As the nutritional requirements of rhizospheric microbes vary, the cultivation
methods, media, and conditions also vary and largely depend on the nature of the
microbe. Cultivation provides appropriate environmental conditions and nutritional
sources. After cultivation, pure cultures are obtained and used for various purposes.
Fig. 10.2 shows the general method for cultivating rhizospheric microbes. Nutri-
tional requirements and cultivation strategies for beneficial rhizospheric microbes
are discussed below.
9.Azospirillum
Azospirillum spp. have been known for many years as PGPR. Azospirillum spp. are
Gram-negative, free-living nitrogen-fixing bacteria that possess versatile C- and
N-metabolism. This makes them well established and adapted to the competitive
environment of the rhizosphere (Steenhoudt &Vanderleyden, 2000). Azospirillum
spp. utilize nitrate, ammonium, nitrite, molecular nitrogen, and amino acids as N
sources (Hartmann &Zimmer, 1994). Under harsh conditions such as nutrient lim-
itation and desiccation they can be converted into enlarged cystlike forms (Lamm &
Neyra, 1981). This cyst formation is caused by the formation of an outer layer of
9. Azospirillum 215

polysaccharides. This coat is made by the accumulation of poly b-hydroxybutyrate
granules, which are used as a C and energy source under stress and starvation con-
ditions (Tal &Okon, 1985;Tal et al., 1990).
Azospirillum beneficial traits includes their ability to be used as biofertilizers and
their ability to induce plant stress tolerance to salinity and water stress (Pedraza
et al., 2020). New fabian broth (NFb) and NFb semi-solid medium is used for the
isolation and cultivation of Azospirillum spp. The basic NFb medium contains malic
acid as a carbon source, dipotassium phosphate, calcium chloride, and sodium chlo-
ride, and other salts provide buffering effect and inorganic salts provide nutrients
(Baldani et al., 2014).
10.Azotobacter
Azotobacter spp. are Gram-negative, aerobic, and heterotrophic bacteria. They are
nonsymbiotic nitrogen fixers capable of operating under normal atmospheric partial
FIGURE 10.2
Cultivation of rhizospheric microbes.
216 CHAPTER 10 Nutrition and cultivation strategies

pressure of oxygen. Azotobacter spp. has the highest metabolic rate among other mi-
crobes (Jensen, 1954). Azotobacter spp. are heterotrophic and require carbon and
other nutrients. Carbohydrates, organic acids, and sugars fulfill the carbon require-
ment. Azotobacter receives energy from redox reactions, and uses organic com-
pounds as electron donors. The metabolic capability of Azotobacter is to fix
nitrogen by conversion to ammonia. Three different nitrogenase enzymes are used
for nitrogen fixation by Azotobacter and are of interest for their role in agriculture
(Patil et al., 2020).
Azotobacter is important for sustainable agriculture because it possesses PGP
properties and can act as a biocontrol agent against various phytopathogens. The
current isolation and cultivation of Azotobacter includes the use of N-free solid me-
dia such as Norris medium (Norris, 1959), Burk medium (Wilson &Knight, 1952),
LG medium (Lipman, 1904), and Ashby’s medium (Ashby, 1907). All these media
are somewhat similar and vary only in minerals, micronutrients, macronutrients, and
carbon sources. The optimum temperature for the growth of Azotobacter is
27e30C. Azotobacter spp. are mannitol positive; therefore, the medium used for
isolation, cultivation, and identification involves mannitol as a carbon source (Patil
et al., 2020).
11.Bacillus
Bacillus spp. are Gram-positive heterotrophic bacteria that derive their nutrition
from the environment. Bacillus spp. can degrade organic matter under nitrate-
reducing conditions. They use organic carbons such as glucose, which can serve
as electron donors and carbon sources for their growth and development. Most
importantly, they derive their nutrients in the rhizosphere from root exudates. The
nutrients present in the root exudates of dicotyledonous plants are organic acids,
sugars, and amino acids (Bais et al., 2006).
The genus Bacillus is important in agroecology because of its plant growth-
promoting properties, and biocontrol functions. Thus, the cultivation of such
microbes is important. Several selective media are available for different Bacillus
species. Here, we have included standard as well as some selective media used
for the isolation and cultivation of Bacillus spp. Standard media includes nutrient
agar, glucose mineral base agar, and trypticase soy agar. Other media for enrichment
and cultivation includes soil extract agar, Schaeffer’s sporulation agar, anaerobic
culture agar, citrate, and propionate utilization medium (Borriss, 2020).
12.Enterobacter
Enterobacter spp. are Gram-negative, facultative anaerobes. Enterobacter spp.
ferment a wide range of carbohydrates and produce several toxins and virulence fac-
tors. Enterobacter spp. reduce nitrates to nitrites. Enterobacter spp. also ferments
12. Enterobacter 217

sugars to produce lactic acid. Glucose, gluconate, glycerol, and lactose are carbon
and energy sources for fermentation (Ve
´ron &Minor, 1975).
Enterobacter spp. are widely dispersed in soil, water, plants, skin, and feces.
They are cultivated on standard media because they do not have any selective media
for cultivation. They use different sugars and sugar derivatives as carbon sources.
Certain species of Enterobacter have been isolated on yeast extract-mannitol agar
(Khalifa et al., 2016). Other media used includes nutrient agar, tryptic soy agar,
and blood agar. For E. sakazakii, a new chromogenic medium named Druggan-
Forsythe-Iversen agar involves the use of the a-glucosidase enzyme (Iversen
et al., 2004).
13.Frankia
Frankia are nitrogen-fixing, Gram-positive bacteria that are able to form actinorhizal
(root nodules formed by Frankia) symbiosis (Verghese &Misra, 2002). Frankia are
usually chemoorganotrophic (Zaı
¨d et al., 2003). In actinorhizal symbiosis, C
4
dicar-
boxylates derived from the metabolism of sucrose in host cells serve as a carbon
source for Frankia strains. Frankia can also utilize a number of fatty acids residues
from Tween and fatty acids as the sole source of carbon. However, they cannot uti-
lize glucose as the carbon source. In the case of N source, Frankia strains use amino
acids and NH
4
þ
(Blom, 1982). No selective media for Frankia spp. have been re-
ported yet, but some simple media such as defined propionate media (DPM) (Baker
&O’Keefe, 1984) and complex media such as the QMod medium of Lalonde and
Calvert (Diem et al., 1982) are used.
14.Klebsiella
Klebsiella spp. are Gram-negative, nonmotile bacteria that possess polysaccharide-
based capsules. The ideal growth temperature and pH are about 35e37C and 7.2
respectively. Klebsiella spp.have no specific growth requirements. They are facul-
tative anaerobes that utilize ammonia as a nitrogen source and citrate and glucose as
carbon sources (Strettoti et al., 1984).
Klebsiella spp. can grow readily on routine media such as blood agar, tryptic
casein soy agar, nutrient agar, bromocresol purple lactose agar, MacConkey agar,
Drigalski agar, eosin-methylene blue agar, and bromothymol blue agar. Klebsiella
strains also grow in minimum medium with ammonium ions or nitrate as a nitrogen
and carbon source. They can also grow on meat extract semi-solid agar at room tem-
perature. Some of them may require growth factors such as arginine, adenine, orni-
thine, and uracil. The selective media used for Klebsiella are based on MacConkey
agar. In this medium lactose is replaced by inositol, and carbenicillin is also added.
This medium is named MacConkey-inositol-carbenicillin agar (Bagley &Seidler,
1978). Wong et al. (1985) also suggested that minimal medium in which the nitrogen
218 CHAPTER 10 Nutrition and cultivation strategies

source is potassium nitrate and lactose is the carbon source. Bruce et al. (1981) com-
bined Koser citrate and raffinose, which serve as carbon sources and ornithine for
low pH.
15.Methylobacterium
Methylobacterium are Gram-negative, strictly aerobic bacteria that only grow in the
presence of single carbon compounds such as methylamine, methanol, methane, and
other methylated compounds. Methylobacterium also uses sulfur as an energy source
(Dourado et al., 2015). Methylobacterium uses methanethiol, dimethyl sulfide, and
volatile carbon compounds as sources of carbon, which is the reason for the survival
of Methylobacterium in the human mouth and feet. This group of bacteria uses
methyl dehydrogenase (MDH) to oxidize methanol to formaldehyde and then
metabolize to formate (Jinal et al., 2020).
Minimal agar medium supplemented with 1% methanol is usually used for the
isolation and cultivation of Methylobacterium because it uses single carbon-
containing compounds as a carbon source. Other media included MacConkey
agar, heart infusion agar, nutrient agar, buffered charcoal yeast extract agar, sheep
blood agar, tryptic soy broth, and peptone yeast agar. They can grow between 5C
and 37C and are slow-growing microbes that require 3e4 days for clear detection
(Lee et al., 2004). Selective media such as methanol mineral salt, minimal medium
agar, and ammonium mineral salt (AMS) are used to isolate Methylobacterium.
These mediums are supplemented with methanol and cycloheximide (Anda et al.,
2011). Delaney et al. (2013) also developed Methylobacterium PIPES medium
(MP) to cultivate genetically modified M. extorquens. This medium is supplemented
with citrate-chelated trace metal solution containing other metals.
16.Pseudomonas
The genus Pseudomonas are aerobic, Gram-negative, rod-shaped, and motile bacte-
ria. They utilize polyalcohols, amino acids, and organic acids as carbon sources.
They are chemoorganotrophic microbes that are catalase-positive (Palleroni,
2005). They also utilize ammonia and acetate as nitrogen and carbon sources,
respectively.
Pseudomonas spp. are used in agriculture and as bioscrubbers to degrade diverse
organic compounds in polluted waters and air. Some of these are human pathogens,
but some are also beneficial. They can grow on simple media containing protein hy-
drolyzate, magnesium chloride, potassium sulfate, and agar. Nonselective agar such
as blood agar, calcium caseinate agar, tributyrin agar, and King’s B agar are also
used for the cultivation of Pseudomonas spp.The selective media for Pseudomonas
contains nalidixic acid, cetrimide, cephaloridine, pimaricin, penicillin G, and mala-
chite green GSP agar, and HiFluoro Pseudomonas agar (Meyer et al., 2002).
16. Pseudomonas 219

17.Rhizobium
Rhizobium spp. are aerobic, chemoorganotrophic organisms that have oxidative
metabolism. Rhizobia require organic carbon to generate energy in the form of
ATP within the tricarboxylic acid (TCA) cycle. The TCA cycle of the host plant
is a source of organic carbon for rhizobia in the root nodules (Andersen, 2020).
Rhizobium spp. also require phosphate for their growth. In one study conducted
by Beck and Munns (1984), low levels of phosphate reduced the growth rate of
Rhizobium spp.and even stopped the growth of bacteria. Low phosphate levels
are critical for the growth and development of Rhizobium spp. Rhizobium spp.
also possess the ability to store and utilize phosphate for subsequent growth. This
trait of Rhizobium spp. is largely strain-dependent. Some Rhizobium spp. strains
require thiamine and pantothenate for growth (Sullivan et al., 2001;Watson et al.,
2001). In the case of nitrogen source, Rhizobium spp.utilize ammonium, urea, ni-
trates, amino acids, and salts (Verma et al., 2020;Patel, 2021).
Symbiotic nitrogen-fixing Rhizobium spp. play a vital role in plant growth pro-
motion (PGP) activity by secreting growth hormones such as indole acetic acid
(IAA), which is important for root nodule formation. They are also able to solubilize
phosphate, which is advantageous for plants and reduces fertilizer cost (Verma et al.,
2020). Other advantages of Rhizobium include increased tolerance of plants to
abiotic stress, siderophore production, and biocontrol activities. Thus, the cultivation
of Rhizobium is important. The optimum temperature for the growth and develop-
ment of rhizobia is 25e30C. Growth on sugar and alcohol-containing media results
in acidification of the media. The C requirement is much higher than that of N. The
use of specific anionic or cationic resins in purified media components as well as
metal chelators such as ethylene diamine tetra acetic acid (EDTA) and nitrile triac-
etate for the removal of residual nutrient contaminants from media is essential
(Abreu et al., 2012). At present, for the isolation of Rhizobium spp. medium includes
mannitol, which is an energy source, nitrogen is supplied in the form of yeast extract,
and the pH and osmotic changes are well buffered with phosphate and sodium chlo-
ride salts (Rao, 1995).
18.Streptomyces
The genus Streptomyces accounts for 50% of the total soil population of Actinobac-
teria (Sathya et al., 2017). The bacteria belonging to this genus are Gram-positive,
have high G þC content ranging from 69 to 78 mol %, and are aerobic (Korn-
Wend is ch &Kutzner, 1992). As their secondary metabolites are bioactive, they are
used in agriculture and human medicine (Watve et al., 2001). Therefore, isolation
and cultivation are important. Bacteria belonging to the Streptomyces genus do not
require growth factors or vitamins. They require only inorganic nitrogen sources,
organic carbon sources, and mineral salts (Lee &Demain, 1997). There are selective
media available for the Streptomyces such as lindenbein medium modified by
220 CHAPTER 10 Nutrition and cultivation strategies

Benedict (Porter et al., 1960). Other media include actinomycete isolation agar (AIA),
Czapek Dox agar, starch casein agar, and yeast malt glucose agar (M6) (Hayakawa
et al., 1991). Kenknight and Munaier agar (KMA) is also one of the mediums used
for the isolation and maintenance of Streptomyces (Gopalakrishnan et al., 2011).
19.Aspergillus
The genus Aspergillus has high economic and social impact. They produce the most
toxic mycotoxin, aflatoxins, and thus, have economic impact. When dead, decaying
matter is targeted, this type of nutrition is called saprophytic. The energy and nutri-
tional uptake of Aspergillus varies depending on the habitat. These compounds
include proteins, starches, fats, cellulose, other sugars, and amino acids (Pimenta
et al., 2020).
The Aspergillus genus includes section Flavi, which includes historically impor-
tant species with conidia of yellowishegreen to brown color. The Aspergillus differ-
entiation medium is used to differentiate the fungi (Aspergillus flavus) from others.
Chloramphenicol and dicholoran are added to the medium to restrict the growth of
molds and bacteria. Yeast extract and peptone provide the nitrogen, B complex
vitamins, and amino acids. Ferric ammonium citrate gives a characteristic yellowe
orange pigment to fungi, which aids in identification (Pitt et al., 1983). Other media
used for Aspergillus spp. includes rose Bengal agar, Czapek-Dox agar, potato
dextrose agar, inhibitory mold agar, and modified rose Bengal agar.
20.Metarhizium
The fungi belonging to this genus are known as entomopathogenic fungi. Such fungi
act as pathogens for insects and kill or disable them. As they are natural biocontrol
agents and provide plants with protection against resistant to salt stress (Khan et al.,
2012), trigger plant growth, increase biomass (Elena et al., 2011) They are environ-
mentally safe, and considered as one of the important microbes to deeply understand
and cultivate (Patel, 2020). Metarhizium spp. colonize the roots of host plants. Some
species are nonpathogenic to insects that are rare and closely related to the lizard
pathogens Metarhizium viride and Metarhizium granulomatis. Metarhizium spp.
use carbohydrate-degrading enzymes to degrade and feed on plant materials.
Because they are root colonizers, they also gain nutrients from root exudates and
become endophytes (St Leger and Wang, 2020). Isolation and cultivation of Meta-
rhizium spp. from soil samples is carried out on selective media containing
dodine-based in which oatmeal agar and Czapek, potato dextrose agar, and yeast
extract (PDAY) are used as the base. These media are developed to eliminate
contaminating microbes by using dodine fungicides (Rangel et al., 2010). Another
medium such as oatmeal agar medium containing cetyl trimethyl ammonium bro-
mide is also used to isolate and cultivate Metarhizium spp. (Patel, 2021).
20. Metarhizium 221

21.Penicillium
Penicillium is a common fungi present in the rhizosphere that have a significant
impact on human life. They can grow even under harsh conditions such as very
high/low temperatures and extremely high sugar/salt concentrations. Penicillium
are parasites. They obtain their nutrition from decaying vegetation or from parasitic
relationships with other organisms. They also form with lichens and receive nutri-
tion through them (Visagie et al., 2014).
Penicillium spp.are widespread and have been isolated from soil, decaying food,
wood, and air. They can also be isolated from the building materials in water-
damaged environments. They are usually grown on Czapek Dox agar with 2%
malt extract. The selective medium was also developed by Frisvad (1983), in which
the base medium was yeast extract agar containing sucrose, chloramphenicol, and
chlortetracycline. Potato dextrose agar with slight modifications are also used for
the Penicillium cultivation.
22.Trichoderma
Trichoderma involves a number of species and are significant group of microbes,
which have impact on plants as well as human. They also show biocontrol activities
against various phytopathogens, and thus provide protection to plants. Trichoderma
usually feed on plants and other fungal substrates. The best carbon sources reported
for Trichoderma spp. are fructose, dextrose, xylose, ribose, cellobiose, mannose, and
galactose. N sources include L-glutamic acid, casamino acids, and L-alanine
(Danielson &Davey, 1973).
Trichoderma are soil-borne fungi that can easily grow in all types of soil, manure,
and decaying wood. Different types of selective media are used for the isolation of
Trichoderma.Trichoderma selective medium (TSM) and Trichoderma medium E
(TME) are selective media used for the isolation of Trichoderma spp. (Papavizas
&Lumsden, 1982). Modified versions of TSM were also developed by Askew &
Laing (1993) and McLean et al. (2005) namely TSM þP and TSM-LU, respec-
tively. Bastos (2001) reported that the optimum temperature for Trichoderma growth
is 20e30C and maximum biomass was achieved at pH between 5.5 and 7.5, in
liquid media containing molasses-yeast extract, molasses-maize steep liquor, potato
molasses, starch-maize steep liquor, and potato-maize steep liquor. For the biomass
production of T. viride PDA and Czapak’s Dox media are most suitable (Gupta et al.,
2003). Other media used for Trichoderma spp. include malt extract agar (MEA), spe-
cial nutrient agar (SNA), oatmeal agar (OMA), cellulose agar (CAM).
23.Conclusion
Soil dwelling microorganisms are economically important and possess beneficial
qualities that protect plants from phytopathogens, increase plant tolerance to stress,
222 CHAPTER 10 Nutrition and cultivation strategies

trigger plant growth, and beneficial for soil texture and fertility. Thus, the beneficial
effects of rhizospheric microbes are not only limited to plants, but also extend to soil.
Soil is a habitat for largely unexploited microbes for plant growth promotion and
sustainable agriculture. Thus, the study of natural habitats, their nutritional require-
ments, and the isolation and cultivation of these important microbes are crucial. The
study of rhizosphere ecology is essential for better understanding the functional re-
lationships among soil, microbes, and plants. In addition, future research work
should focus on meta-omics to utilize beneficial enzymes and genes of unculturable
microbes.
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Bioengineering of
rhizobiome toward
sustainable agricultural
production 11
Bal Krishna
1
, Rakesh Kumar
2
, Hansraj Hans
2
, Ashutosh Kumar
1
, Banshidhar
1
,
Talekar Nilesh Suryakant
1
, Harmeet Singh Janeja
1
, Birender Singh
3
,
Dharm Nath Kamat
4
1
Department of Genetics and Plant Breeding, School of Agriculture, Lovely Professional
University, Jalandhar, Phagwara, Punjab, India;
2
Division of Crop Research, ICAR Research
Complex for Eastern Region, Patna, Bihar, India;
3
Department of Plant Breeding and Genetics,
Bihar Agricultural University, Bhagalpur, Bihar, India;
4
Sugarcane Research Institute, Department
of Plant Breeding and Genetics, Dr. Rajendra Prasad Central Agricultural University, Samastipur,
Bihar, India
1.Introduction
The widespread use of agrochemicals adversely affects the human health as well as
deteriorates soil biota. It causes environmental pollution with agrochemical
residues, burning of the fossil fuels, and, emission of greenhouse gases (Cha
´vez-
Dulanto et al., 2021;Masson-Delmotte et al., 2021). Physical, chemical, and biolog-
ical compounds form complex dynamics of soil systems that are a part of lithosphere
(Del Carmen Orozco-Mosqueda et al., 2019;Lehmann et al., 2020). Biotic and
abiotic stresses, viz., pests, diseases, drought, and extreme temperatures brought
by the climate change impact the plants and reduce crop yields. Plant growth and
development are antagonistically impacted due to stress (Khan et al., 2020;Phour
&Sindhu, 2022). Abiotic stress also degrades the physiochemical properties of
the soil and abundance of microbes, which reduces crop yield and crop productivity
(Chandran et al., 2021;Goswami &Deka, 2020). The agricultural policy is focusing
on sustainable production systems and emphasizes the use of beneficial soil mi-
crobes existing in rhizosphere, which stimulates growth and development and plays
an important role in mitigating the harmful effects of stress on crop plants (Basu
et al., 2021;Chandran et al., 2021;Cherif-Silini et al., 2021). Plant protection and
resistance are influenced by the selective aggregation of microbial communities un-
der abiotic and biotic challenges through the inherited impacts and soil-plant feed-
back in subsequent generations (Hakim et al., 2021;Kostenko &Bezemer, 2020).
Inherently healthy soil and its good quality are vital for agricultural production sys-
tems (Karimi et al., 2020). The term “sustainability” is a buzzword, especially
CHAPTER
233
Rhizobiome. https://doi.org/10.1016/B978-0-443-16030-1.00011-0
Copyright ©2023 Elsevier Inc. All rights reserved.

considering the present scenario of the environmental change and biodiversity sta-
tus. As a result of increased consciousness and restricted use of agrochemicals, there
is an incentive to adopt more sustainable agricultural practices (Andreolli et al.,
2021;Dries et al., 2021). Ecological health, monetary suitability, and social and
financial decency are three important goals of the sustainable agricultural production
system, characterized by a systematic approach to managing the natural and human
resources (Zucca et al., 2009). Biodiversity conservation and ecological resilience
are greatly aided by combined contribution of plants and rhizobiome (Haldar &Sen-
gupta, 2017). Soil surrounds the root of the plant, known as rhizosphere and it is
crucial to growth and development of plants. The rhizosphere, a region of soil
that is significantly affected and bound to root of crop plants, is formed as expansion
of plant roots (Bakker et al., 2020;CarmenOrozco-Mosqueda, 2022). The rhizo-
sphere is a small area enclosed by plant roots and a potential hotspot for fungi, bac-
teria, algae, and nematodes. The rhizosphere microbiome, otherwise called
rhizomicrobiome refers to the entire microbial community that exists in rhizosphere
and varies from the bacteriological community of adjacent soils (Chandran et al.,
2021;Kumar &Dubey, 2020). The rhizosphere microbiome alludes to a group of
micro-organisms that interact with plant roots. The way of behaving of the soil mi-
crobial networks, especially those in the rhizosphere, has a profound effect on the
productivity of agricultural systems (Bakker et al., 2012). It is critical to recognize
that the rhizobiome can be present and engaged in a variety of interactions with
plants, making it a vital point of contact (CarmenOrozco-Mosqueda, 2022;Pollak
&Cordero, 2020). Plant root exudates serve as indicators to microbial populations
and support their growth and development (Chandran et al., 2021;Meena et al.,
2020;Zehra et al., 2021). To select an effective bioinoculation agent, it is very
important to find useful services in rhizobiome that have gainful associations and
the capacity to cooperate with the arrangement (Orozco-Mosqueda et al., 2021;
Vasseur-Coronado et al., 2021). Plant microbiomes provide feasible ways to boost
crop production and rationalize agricultural practices (Dries et al., 2021;Li et al.,
2020). Some soil bacteria, called plant growth promoting rhizobacteria (PGPR),
colonize the surface of the root system and increase plant growth and development
(Bhat et al., 1952;Chandran et al., 2021). This PGPR can be parasitic, saprophytic,
free-living, or symbiotic. It may be categorized into two groups: external plant
growth promoting rhizobacteria (PGPR) and intracellular plant growth promoting
rhizobacteria (Figueiredo et al., 2010;Martı
´nez-Viveros et al., 2010). Free-living
rhizobacteria, like Pseudomonas,Agrobacterium,Bacillus,Erwinia,Micrococcus,
and Serratia are incorporated in external plant growth promoting rhizobacteria
(Gray &Smith, 2005). Endophytic symbiotic bacteria called intracellular plant
growth promoting rhizobacteria are often found in root cells in nodular structures
like Rhizobium,Frankia, and Mesorhizobium (Verma et al., 2010). Actinomycetes
predominate in the rhizosphere, viz., Streptomyces sp., Strepto-sporangium sp.,
Streptomyces sp., and Thermobifida sp. It is believed to stimulate plant growth
and control root-related fungal diseases (Merzaeva &Shirokikh, 2006). The
PGPR advances plant growth through the production of volatile organic compounds,
234 CHAPTER 11 Bioengineering of rhizobiome toward sustainable

phytohormones, nitrogen fixation, and phosphate decomposition (Chandran et al.,
2020;Mukesh Meena et al., 2017). It also reduces the plant stress caused by biotic
and abiotic factors by promoting nutrient uptake osmolyte accumulation and
increasing antioxidant production and metabolism (Chandran et al., 2021;Ha-
Tran et al., 2021;Kumar et al., 2018,2019). PGPRs have a critical impact on biore-
mediation by detoxifying xenobiotics, heavy metals, and pesticides (Shukla et al.,
2011;Tak et al., 2013).
However, since genetically modified organisms (GMOs) are not widely
accepted, two new research strategies for sustainable agricultural practices are gain-
ing more attention: plant engineering and rhizosphere engineering. Models include
sustainable food, fiber, and energy production, biodiversity conservation, overseeing
water assets, and mitigation of environmental change (Dessaux et al., 2016;Haldar
&Sengupta, 2017). Plant engineering studies the extent to which breeding programs
can improve genotype of crop plant. Rhizosphere engineering is the study and opti-
mization of plant rhizosphere. For example, some fertilizers or microbial inoculants
might modify microbial community of rhizosphere. This change attempts to
improve nutrient absorption or trigger a plant’s immune response and is often
applied in agriculture (Dries et al., 2021;Taye et al., 2020). This present chapter
summarizes the global effort by shedding more emphasis on mechanistic and
ecological understanding, particularly rhizosphere engineering, which provides a
benefit for impending agricultural sustainability.
2.Bioengineering
The global population is projected to reach w10 billion, by 2050 (United Nations,
The Future of Food and Agriculture, 2017), and there will be w50% rise in demand
for agricultural products over 2013. Population expansion and rising earnings will
cause a continued increase in demand for food, fiber, fuel, and other amenities.
The strain on environment and natural resources to meet these increasing demands
is causing resource scarcity and environmental catastrophe. The only solution to this
issue is said to be sustainable agricultural diversification and intensification (Greg-
ory et al., 2013). Maximizing agricultural output from existing lands, while reducing
environmental stress, is the aim of sustainable intensification. An integrative strategy
fusing biological research and community ecology is required to accomplish this
(Haldar &Sengupta, 2017;Reynolds et al., 2014). In coming decades, as demand
rises, bioengineering will play an important role in expansion and sustainability
of world’s food supply (Kahn, 2015). In recognition of complementary roles that mi-
croorganisms and plants play, we shall use general word “bioengineering” to refer to
any one or more of a variety of biologically based effects that change properties of
soil profile (Verboom &Pate, 2006). Bioengineering and agricultural engineering
are rapidly developing disciplines that utilize the concepts of biology and physical
science to address the issues with agriculture and environment. These engineers
create machinery and systems that raise agricultural production systems and food
2. Bioengineering 235

safety. In addition, they manage and conserve agricultural resources like soil, water,
air, and energy. Conservation of soil surfaces from erosion, soil stabilization, and
better drainage abilities are some of most typical application of bioengineering. Syn-
thetic microbiomes for promoting plant growth, and biotic and abiotic stress toler-
ance or regulation are among many areas of bioengineering in agricultural fields,
and they offer a special possibility (Ahkami et al., 2017). The development of
biotechnology offered a new strategy for overcoming those challenges in agricul-
tural crop productivity (Ye &Gao, 2015). Biopesticides, genetically modified crops,
fertilizers derived from biological waste, and inclusion of biochar in fertilizers are
only a few examples of effective methods to lower environmental costs and improve
the sustainability of agricultural production. Even though many different strains of
bacteria have been found to have advantageous benefits, engineering sustainable
synthetic microbes is a difficult task (Ahkami et al., 2017). Rhizosphere engineering,
however, is taking lead and making the most attention including increasing crop
resistance and warding off disease.
3.Why rhizosphere engineering for sustainable
agriculture?
Rossbach et al. (1994) put up the idea of generating a biased rhizosphere based on
genetically modified plants and their metabolism to produce the particular exudates
that could only be metabolized by the beneficial microbes. Later, O’Connell et al.
(1996) proposed the idea of “rhizosphere engineering,” which also recommended
modifying plant and its root exudates production, i.e., nutrients like sugars, proteins,
flavonoids, organic acids, mucilage, and amino acids, to attract a favorable or advan-
tageous micro-organism that impacts plant phenotype itself. Rhizosphere engineer-
ing is the process of modifying a plant’s root system and immediate environment to
produce a “biased” environment that will significantly boost plant survival and crop
yield (Ryan et al., 2009). There is great emphasis on identifying ways to enhance the
establishment of advantageous native or introduced microbial populations, such as
rhizobia and mycorrhiza, which aid in nutrient uptake, directly stimulate plant
growth, or inhibit plant diseases. The core elements of the rhizosphere engineering
include the identification and selection of suitable crop species, soil amendment, the
introduction of adventitious microbes, and genetic modification of activity of micro-
organisms and crop plants (Ryan et al., 2009). However, there are many techniques
of rhizosphere engineering. Additionally, it is feasible to apply the particular nutri-
ents, fertilizers, and bioinoculants to bacteria and fungi. Another aspect of rhizo-
sphere engineering is bioremediation, which makes use of naturally occurring or
genetically modified organisms or plants to break down toxins in soil and regain
the ecological and environmental balance (Bisht et al., 2015;Haldar &Sengupta,
2017). Rhizosphere engineering has become a crucial tool for offering a financially
feasible and environmentally sound solution to address the several global issues
brought on by population growth.
236 CHAPTER 11 Bioengineering of rhizobiome toward sustainable

The purpose of plant-microbiome engineering is to promote a variety of phyto-
microbiome interactions that will ultimately lead to a more favorable overall
outcome for crop plant (Quiza et al., 2015). The introduction of organic fertilizers
like composts, biosolids, and animal manure is a well-known technique in organic
agriculture around world (Haldar &Sengupta, 2017;Lim et al., 2015). Furthermore,
it is impossible to ignore the biohazards resulting from heavy metals and hazardous
compounds found in biowaste (Quiza et al., 2015). The rhizosphere is made up of
three essential components: soil, plants, and microbes described in detail in
Fig. 11.1. To increase crop production, all can be engineered (Hakim et al., 2021).
3.1 Plant-based rhizosphere engineering
Since invention of agriculture, natural ecosystems have been modified or engineered
to sustain human needs. Since the 1980s, it has been possible to alter the genetics of
plants to produce desired phenotypes, i.e., resistance against pathogens, storage
longevity, resistance to stressful environmental conditions, insect resistance, in-
crease growth or simply having a particular color or appealing shape of flowers or
FIGURE 11.1
Plants, microorganisms, and soil are the three main components of rhizosphere
engineering for sustainable agriculture. These are amenable to engineering in order to
increase crop yield sustainably. The rhizosphere can be affected by the soil amendment
to encourage plant growth and development. Plant characteristics like root architecture
and root exudates, which can alter the rhizosphere’s nature and activity, can be modified
through plant breeding.
3. Why rhizosphere engineering for sustainable agriculture? 237

fruits it produces (CarmenOrozco-Mosqueda, 2022). Plant breeding is one method
of achieving plant-based rhizosphere engineering. Development and selection of
crops/cultivar lines with potential for improved systemic resistance to disease and
environmental stressors, root exudation, and accelerated mutual symbiosis are cor-
nerstones of plant breeding (Haldar &Sengupta, 2017). Plant genotype is carefully
regulated in agroecosystems through breeding and varietal selection (Dries et al.,
2021). Despite importance of rhizosphere microbiome for sustainability of plant
ecosystem, plant microbiome is not taken into account in conventional breeding
techniques (Mendes et al., 2018). Creation and maintenance of the wholesome
and advantageous microbial communities in rhizosphere could be facilitated by
the addition of an understanding of plant-microbial interactions to plant breeding
strategies. Upcoming agricultural practices and plant breeding will eventually pro-
duce sustainable ways to decrease harm caused by soil-borne diseases by incorpo-
rating knowledge of interactions between plants and microorganisms (Wille et al.,
2019). Both conventional plant breeding techniques and plant genome editinge
based techniques show promise for accumulating favorable alleles linked to stress
tolerance in genome of crop plants (Dries et al., 2021). Genetically engineered plants
are endowed with ability to produce greater amounts of exudates that are extremely
specialized for adventitious microorganisms, change efflux of organic anions from
soil and their transportation through roots, alters the soil properties such as pH,
and salinity, and encourage disease suppression in soils (Haldar &Sengupta,
2017;Mazzola, 2007;Savka et al., 2002).
3.2 Microbiome-based rhizosphere engineering
Rhizosphere engineering can be done by changing local microbes, which would in-
fluence the metabolism of crop plants (Arif et al., 2020;Orozco-Mosqueda et al.,
2018). To increase agricultural production, microbiome-based strategies either
include the direct inoculation of specific microorganisms or co-inoculation of mixed
cultures of PGPR, ectomycorrhizal fungi (EMF), and endophytes (Haldar &
Sengupta, 2017). For growth of sustainable agriculture, crops must be biotic and
abiotic stress tolerant and have higher nutritional content. As a result, soil-
dwelling microorganisms like bacteria, algae, and fungi, can be used to achieve
the desired crop traits (Rai et al., 2020). Plant growth-promoting rhizobacteria are
among the most effective soil microorganisms that have been shown to boost plant
growth and productivity, while also accelerating plant growth rate without polluting
the environment (Calvo et al., 2014). Numerous plant PGPRs strains are identified,
some of which are commercial, have been documented for many years. These strains
include Enterobacter,Bacillus,Azotobacter,Serratia,Variovorax,Pseudomonas,
Azospirillum, and Klebsiella (Glick, 2012). Crop production is increased by PGPRs
as they improve the fertility of soil for nutrient availability (Rai et al., 2020). Under-
standing interactions between microorganisms and plants can aid in the creation of
new ecofriendly, economically viable, and sustainable agricultural production sys-
tem (Rakshit et al., 2015). To engineer the micro-organisms that give plants their
238 CHAPTER 11 Bioengineering of rhizobiome toward sustainable

disease resistance, it is necessary to comprehend how they interact with the environ-
ment and plant in establishing sustainable agriculture. The PGPR inoculation pro-
motes efficient and sustainable crop production to fulfill the demands of an
increasing global population at a time when agriculture is subject to a variety of
environmental stresses. By making nutrients more readily available in root zone,
PGPR improves nutrient uptake (Rai et al., 2020). Fixation of atmospheric nitrogen
by prokaryotes (like Azospirillum) is a crucial phenomenon as nitrogen becomes a
limiting factor for plant growth (Lloret &Martı
´nez-Romero, 2005;Raymond
et al., 2004). Rhizobacteria that encourage the plant growth, including Kocuria tur-
fanensis strain 2M4, solubilize phosphate and increase number of phosphate ions
that are available to plants in soil (Goswami et al., 2014;Rai et al., 2020;Wani
et al., 2007b). Lavakush et al. (2014) found that Pseudomonas putida and Pseudo-
monas fluorescents improve the ability of rice crops to absorb nutrients. Addition-
ally, it has been noted that Pseudomonas putida also improves the uptake of
nitrogen, potassium, and phosphorus in chickpeas (Israr et al., 2016;Rai et al.,
2020). Bacillus pumilus and Bacillus amyloliquefaciens have been shown to
improve uptake of nitrogen and phosphorus in tomato crops (Fan et al., 2017).
The uptake of nitrogen and phosphorus in wheat was said to be enhanced by Serratia
marcescens (Sood et al., 2019). Inquiry into function of genes involving interaction
between microbes and plants will eventually help create new methods for enhancing
health of plants and soil. Biofertilizer is made up of living microbes and encourages
plant growth by making more primary nutrients available to them when applied to
seeds, plant surfaces, or soil. Biofertilizer is a microorganism-based fertilizer that
boosts plant growth and has a significant impact on enhancing worldwide agricul-
tural production (Yadav &Nath Yadav, 2018). Biofertilizers are microorganism in-
oculants that improve plant growth and productivity (Khalid et al., 2009).
Accordance to Mishra et al. (2013), a biofertilizer increases nitrogen fixation, phos-
phate solubilization, or cellulolytic microorganisms, which boosts plant production
and yield by permitting easier nutrient uptake and enhances soil quality in the rhizo-
sphere zone (Malusa
´&Vassilev, 2014). PGPM can be grouped into three classes:
plant growth promoting rhizobacteria (PGPR) (Podile &Kishore, 2006), arbuscular
mycorrhizal fungi (AMF) (Jeffries et al., 2003), and free-living nitrogen-fixing bac-
teria (Franche et al., 2009). The growth of plants depends on phosphate and nitrogen.
The ability of crop plants to withstand stress, maturity, quality, and fix nitrogen are
all significantly influenced by phosphate. A fungus called Penicillium bilaii aids in
phosphate availability from the soil. It has the potential to produce an organic acid
that breaks down soil phosphate so that the roots may use it (Mosttafiz et al., 2012).
For a very long period, biofertilizers have included Rhizobium and Azotobacter. The
most often exploited bacteria in the production of biofertilizers is Rhizobium. Nod-
ules in plant roots are home to this bacterium. Nodules serve as biological factories
for nitrogen fixation. Rhizobacterium can get nitrogen from the atmosphere and
transform it into an organic form that can be taken by plants and utilized. Many bac-
teria, including cyanobacteria, can convert atmospheric nitrogen into ammonia,
which plants use as part of their nutrition cycle. Many PGPR viz. Pseudomonas
3. Why rhizosphere engineering for sustainable agriculture? 239

spp., Bacillus amyloliquefaciens,Bacillus spp., Bacillus cereus, and Bacillus subtilis
are considered to be very effective and prospective biocontrol agents (Francis et al.,
2010). Because these bacterial strains produce endospores and can tolerate chal-
lenging biotic stress, they are significant components of biofertilizers (Pe
´rez-
Garcı
´a et al., 2011). In addition to promoting plant growth, biofertilizers with
high PGPR concentrations also increase the amount of N, P, and K in soil, protect
plants from many diseases, and transform major and minor nutrient molecules
that are difficult for plants to absorb (Yang et al., 2011). Biopesticide is an ecolog-
ically sound substitute for chemical pesticides for controlling pests including weeds,
insects, and fungi that reduce crop productivity (Rai et al., 2020). Numerous PGPR
can be utilized as biopesticides that encourage plant growth and development. Many
PGPRs have been documented and can be exploited as biopesticides, including Pae-
nibacillus,Enterobacter,Serratia,G. mossae,Pseudomonas,Azotobacter,Klebsi-
ella,Glomus fasciculatum,Bacillus,Burkholderia,Streptomyces,Arthrobacter,
Azospirillum,Gigaspora margarita, and Alcaligenes (Bhattacharyya &Jha, 2012).
With aid of biofertilizers, plants can utilize all of the nutrients present in soil and
air, which decreases the expense of agriculture production for farmers.
3.3 Soil-based rhizosphere engineering
The ability of rhizosphere to support plant growth can be influenced by the soil
amendment, like plant residues, silicon, coal fly ash, biochar, sewage sludge, zeo-
lites, and cattle manure (Hakim et al., 2021;Jakkula &Wani, 2018). Despite recent
advancements in plant genetics, soil analytical technologies, and microbial ecology,
soil amendments continue to be an empirical technique that provides descriptive in-
formation. Microbial communities have an impact on availability of nutrients in soil,
and biochar can have an impact on these communities as well (Rasse et al., 2022).
This indicates that interaction between BCF and soil microbes may have an impact
on both availability of native soil nutrients and uptake of nutrient supplied by
biochar-based fertilizer. Ye et al. (2016) observed that total soil NO
3
was boosted
by biochar-treated compost more than by compost alone and attributed this impact
to stimulation of soil nitrifier populations. As a result, top soil retains nutrients,
serving as a nutrient pool for plants and fostering the plant growth and biochar
also enhances cations exchange capacity of soil (Rasse et al., 2022). According to
Zhang et al. (2014), biochar dramatically boosted the soil microbial activity and
increased soil moisture, which in turn affected decomposition of SOM. Due to
porous nature of biochar, it has been able to serve as an appropriate habitat and sub-
strate for microbial breakdown of bioavailable minerals in soil. As a consequence,
incorporation of biochar improved the soil porosity, soil microbial habitat, soil car-
bon sequestration, and soil carbon source for energy. Instead of being extracted and
refined, organic fertilizers are made from resources that have undergone the mini-
mum processing, i.e., organic matter and plant and animal waste. Organic matter,
such as animal dung, bird droppings, sewage sludge, and food waste, is broken
down by microorganisms in soil, which release vital nutrients. Improve soil texture,
240 CHAPTER 11 Bioengineering of rhizobiome toward sustainable

increases water retention, and increases activity of soil bacteria and fungi make this
natural fertilizer more environmentally friendly (Prapagdee &Tawinteung, 2017).
The main nutrients released from this material are N, P, and K, which safeguard
plants from pests and diseases (Prapagdee &Tawinteung, 2017). Recent research
has indicated that adding biochar to soil may enhance a variety of soil microorgan-
isms; these phenomena may be related to release of nonlabile biochar-associated
organic compounds or changes in soil characteristics brought on by adding biochar
to soil (Zhang et al., 2017). According to Kolton et al. (2017), increased plant per-
formance from biochar was associated with both better rhizosphere metabolic poten-
tial and more microbial diversity. Effects of root exudates on rhizosphere microbiota
may change with the addition of biochar. According to Gu et al. (2017), biochar can
change rhizosphere’s microbial populations by promoting propagating of bacteria
through absorption of root exudates or by improving physical characteristics due
to its porous nature.
4.Rhizosphere engineering for abiotic
It has been found that different types of microorganisms that live in the phyllo-
sphere, rhizosphere, and/or endorhizosphere aid plants in adapting to an abiotic
stress environment (Morales-Cedeno et al., 2021;Phour &Sindhu, 2022). These
bacteria that live in soil and rhizospheric microorganisms have an impact on the
health of plants and pollutants removal (De Vries et al., 2020). The use of microbial
population and microbiological systems can reduce abiotic stresses and increase
agriculture productivity in a sustainable and environment-friendly manner in gen-
eral. PGPR may improve the growth and development of plants in response to
abiotic stress. For example, some PGPR enhance plant growth directly by synthesis
of different types of phytohormones such as abscisic acid, auxins, cytokinin, gibber-
ellins, and aminolevulinic acid, by enhancing nutrient availability and absorption,
for example, BNF and phosphate solubilization by increasing siderophores synthesis
(Kumar et al., 2022;Phour &Sindhu, 2022). Rhizobacteria that boost plant growth
have drawn interest from all around the world in the last 10 years as a means of
improving crops under salt stress, as they directly aid plant growth by increasing
the uptake of nutrients from the soil and indirectly inhibit diseases development
in the plants (Sultana et al., 2021). Table 11.1 Illustrates the various types of bene-
ficial microorganisms used in sustainable agriculture through promoting plant
growth and reducing abiotic stresses in various crops. As a result of their capacity
to synthesize the exopolysaccharides and create hydrophilic biofilms, some strains
of plant growth promoting bacteria (PGPB), including the Rhizobium,Pseudo-
monas, and Bacillus have been found to promote the growth of soybean and wheat
(Rossi &De Philippis, 2015). Osmoprotectants like trehalose, proline, and glycine
betaine are synthesized by PGPR strains and have multiple functions for supporting
plants in stressful situations (Enebe &Babalola, 2018). Osmolytes that produce
PGPR are usually involved in ROS scavenging, protein and membrane integrity
4. Rhizosphere engineering for abiotic 241

maintenance, salt stresserelated protein maintenance, cytosolic acidity regulation,
and lipid peroxidation reduction (Phour &Sindhu, 2022). According to Linic
et al. (2019), there is a positive link between levels of phenolic acid and the ability
to withstand salt in three different Brassica crops, for example, Chinese cabbage,
kale, and white cabbage. Cucumber plants were inoculated with the Rhodopseudo-
monas palustris strain, which can synthesize IAA and ALA, fix nitrogen, solubilize
phosphate/potassium, and enhance plant development as well as bestow cucumber
with salt tolerance (Ge &Zhang, 2019). The ability of some strains of PGPB,
including Rhizobium, Bacillus, and Pseudomonas, to synthesize exopolysaccharides
and form hydrophilic biofilms, has been reported to promote the development of
soybean and wheat (Rossi &De Philippis, 2015). Osmoprotectants like proline,
trehalose, and glycine betaine are generated by some PGPR strains and have various
functions for protecting plants in stressful situations (Ahmad et al., 2013;Phour &
Sindhu, 2022). Salt-tolerant strains of Azospirillum improved wheat plant develop-
ment more robustly and produced heavier grains under salt-stressful environments
(Hakim et al., 2021;Nia et al., 2012). Through the production of auxin and abscisic
acid as well as the regulation of the expression of many salt-stressed genes,
B. amyloliquefaciens inoculation in rice increases salinity tolerance and boosts
growth (Shahzad et al., 2017). On plant cultivation under water-stressed, drought-
tolerant conditions, the effects of inoculation with various PGPR strains that are
drought-tolerant were assessed. For example, some rhizobacteria, including Rhizo-
bium, Azotobacter, Bacillus, Serratia, Xanthomonas, and Azospirillum, have been
proven to be effective in reducing the effects of drought stress. These bacteria can
produce several traits that aid in plant growth (Bazany et al., 2022;De Vries
et al., 2020). Exopolysaccharide-producing bacteria, that is, Pseudomonas,Rhizo-
bium, and Bacillus, are highly effective mechanisms for enhancing soil structure
by binding soil particles together, which in turn mitigated the effects of drought
stress (Belimov et al., 2009;Sandhya et al., 2009). Inoculation of wheat (Triticum
aestivum L.) with several indole-3-acetic acid-producing rhizobacteria, including
B. simplex,B. amyloliquefaciens,Moraxella pluranimalium,B. thuringiensis,Bacil-
lus muralis, and B. muralis, led to an improvement in a number of spikelets and til-
lers in drought conditions (Raheem et al., 2018). The coinoculation of three plant
growth promoting strains of bacteria known to promote plant growth, namely Raoul-
tella planticola,Pseudomonas fluorescence, and Klebsiella variicola, resulted in
increased drought resistance and glycine betaine and choline accumulation in maize
(Gou et al., 2015;Phour &Sindhu, 2022). Drought-tolerant rhizobacterial strains
including Alcaligenes faecalis,Proteus penneri, and P. aeruginosa produce protein,
proline, and sugar content in maize plants, which helps to improve water potential,
growth, and water loss for fostering resistance against drought conditions (Naseem
&Bano, 2014). Nutritional value, proline content, shoot and root weight, antioxidant
enzymes, sprouting in sugarcane and sprouting were all improved by treatment with
Bacillus megaterium BMSE7 strains and Bacillus subtilis BSSC11 (Chandra et al.,
2018;Phour &Sindhu, 2022). Soybean plants exposed to drought stress had
decreased levels of chlorophyll and photosynthetic activity; however, P. putida
242 CHAPTER 11 Bioengineering of rhizobiome toward sustainable

treatment reduces the effects of water-deficit conditions by increasing biomass and
the photosynthetic apparatus (Etesami, 2018;Tiwari et al., 2016). Coinoculation
with B. thuringiensis and P. putida in Trifolium increases proline concentration,
which lowers electrolyte leakage and stromal conductivity (Hakim et al., 2021;Ortiz
et al., 2015). Higher temperature-induced cellular damage also led to protein dena-
turation and aggregation. Lahsini et al. (2022) isolated 160 rhizobacterial isolates,
from the rhizosphere of olive tree orchards, of which 80% were halotolerant (4%
NaCl) and 65% were thermotolerant (45C). The three bacterial strains namely Ba-
cillus megaterium (MSRC23), Arthrobacter globiformis (MSRC52), and Bacillus
licheniformis (MSRC76) were found to possess the most notable multitrait PGP ac-
tivities, such as the production of phosphate solubilization, indole-3-acetic acid, and
siderophores, as well as a high tolerance for salinity and temperature. The inocula-
tion of P. putida in the chickpea plant exhibited thermotolerance through the devel-
opment of a thick biofilm and the production of transcription factors linked to stress.
Another efficient defense mechanism of PGPR (Pseudomonas AKMP6) against
abiotic stress is protein accumulation in heat-stressed sorghum plants (Ali et al.,
2009). When potatoes are subjected to temperature stress, a rhizobacterium like Bur-
kholderia phytofrmans which produces ACC deaminase has been reported to
enhance stem height, shoot biomass, and root biomass (Bensalim et al., 1998). Ba-
cillus safeness and Ochrobactrum pseudogrignonense were injected into wheat
plants, and it was reported that this activated antioxidant signaling under high-
temperature stress, increasing redox enzyme activity and causing the formation of
proline and glycine betaine (Sarkar et al., 2018). Another prevalent challenge that
typically comes in temperate regions is frost injury brought on by freezing temper-
atures. Additionally, it was discovered that freezing temperatures had an impact on
rhizosphere microbiota and chemical synthesis in root exudates (Cloutier et al.,
2021). It was reported that Pseudomonas syringae and Erwinia herbicola defend
plants from supercooling at subzero temperatures. These epiphytic bacteria, which
have ice-nucleating ability, for example, ice þbacteria, were isolated from the phyl-
losphere (Lindow &Leveau, 2002). In related research, novel cold-tolerant PGPR
species, such as Serratia marcescens, Exiguobacterium acetylicum, Pseudomonas
lurida, Pantoea dispersa, and Pseudomonas fragi, enhanced plant growth in freezing
temperatures (Selvakumar et al., 2008). Additionally, it was observed that Pseudo-
monas putida strain UW4 promoted the growth of the canola plants in salinity stress
situations and low temperatures (Cheng et al., 2007). Alkaline and acidic soils can
cause serious mineral nutritional problems, which weaken and weaken the plant.
Soil microorganisms like mycorrhiza and phosphate-solubilizing bacteria or fungi
can be exploited in acidic or alkaline environments to overcome nutrient and mineral
toxicity problems (Paul &Clark, 1989;Phour &Sindhu, 2022). These advantageous
bacteria were capable of producing siderophores, N
2
fixation, and phosphate solubi-
lization, among other PGP traits. A variety of rhizobacteria with PGP activity,
including Azospirillum,Rhizobium,Bacillus,Pseudomonas, and Azotobacter were
found in degraded hill slopes, alkaline soil, and acidic soil (Selvakumar et al.,
2009). From an acidic soil with a pH of 4.5, root-nodulating strains of Rhizobium
4. Rhizosphere engineering for abiotic 243

loti and R. tropici were found (Cunningham &Munns, 1984;Zahran, 1999). The
development of legume symbiotic factors in arid conditions was facilitated by the
use of acid-tolerant rhizobacterial strains, which boosted growth, improved plant
health, and increased crop production (Zahran, 1999;Phour &Sindhu, 2022).
Some PGPR strains can be bioremediating soil that has been contaminated with
heavy metals and lessen the negative impact that heavy metals have on plants (Barra
Caracciolo &Terenzi, 2021;Chakraborty et al., 2018). Heavy metal stress may be
mitigated by using soil microbes like PGPR and arbuscular mycorrhizal fungus
(AMF), which can absorb heavy metals in their tissues (Phour &Sindhu, 2022;Tse-
gaye et al., 2017). Pantea agglomerans and Bacillus megaterium were shown to
effectively tolerate an aluminum concentration of 8 mM, and the growth of Vigna
radiata was improved (Silambarasan et al., 2019a,2019b).
5.Rhizosphere engineering for biotic stress
World crop productivity is being threatened by the biotic stresses. The intensive use
of the pesticides to control the pathogenesis and proliferation of pathogenic micro-
organisms has degraded and polluted the soil quality abruptly (Igiehon &Babalola,
2018). Biocontrol of phytopathogenic microorganisms through other microbes has
become more popular in recent years for a sustainable agricultural production sys-
tem (Karthika et al., 2020). Rhizobacteria that produce siderophores colonize plant
roots and remove all other microorganisms from this milieu. Pseudomonas sp.
released siderophores that enhanced the plant development and slowed spread of dis-
ease in green gram (Hakim et al., 2021). Pyochelin and pyoverdine are forms of the
siderophores produced by Pseudomonas fluorescens and Pseudomonas aeruginosa.
These microorganism-produced siderophores increase the intake of Fe and inhibit
the growth and development of pathogenic microbes by competing with hazardous
microorganisms for Fe scavenging (Shen et al., 2013). The Table 11.1 illustrates
various types of beneficial microorganism used in sustainable agriculture by promot-
ing the plant growth and reducing biotic stresses in various crops. Rhizobacteria that
produce siderophores include Rhizobium, Bacillus, Streptomyces, Serratia, Brady-
rhizobium, and Klebsiella (Mustafa et al., 2019). The capacity of soil bacteria to pro-
duce ACC deaminase enzyme is the main factor in lowering ethylene produced by
pathogens and harmful effects. Bacteria, including Ralstonia solanacearum,Azo-
spirillum lipoferum,Rhizobium, and Bacillus, Pseudomonas, are known to produce
ACC-deaminase and aid plants in coping with stress (Ali et al., 2020;Hakim et al.,
2021). The PGPR can manufacture ACC deaminase to act as a soldier for the detri-
mental impact of ethylene; in addition to this, it also acts as a biocontrol agent
against various pathogens like those caused by Sclerotium rolfsii,Botrytis cinerea,
Xanthomonas oryzae, Pythium ultimum,Rhizoctonia solani,Phytophthora sp., and
Fusarium oxysporum. Additionally, pseudomonads synthesize phenazine, an anti-
biotic with redox activity that can neutralize the phytopathogens including Fusarium
oxysporum and Gaeumannomyces graminis. Numerous antibiotics produced by
244 CHAPTER 11 Bioengineering of rhizobiome toward sustainable

Table 11.1 An outlook of various soil microorganism effects on plant growth and their
role in the mitigation of stress.
Crops Microorganisms
Contributions toward
sustainable agriculture References
Rice Glutamicibacter sp.1-aminocyclopropane-1-
carboxylic acid (ACC)
deaminase activity, indole-3-
acetic acid production (helps
in reducing salt stress)
Hakim et al. (2021)
Bacillus amyloliquefaciens Proline content. Chauhan et al.
(2019).Phour &
Sindhu (2022)
Acinetobacter soli, Bacillus
sp.,Pseudomonas putida,
Pseudomonas mosselii,
Arthrobacter woluwensis
Phosphorus solubilization, 1-
aminocyclopropane-1-
carboxylic acid (ACC)
deaminase activity,
siderophores production.
Xiao et al. (2020),
Rasul et al. (2019)
Pseudomonas sp.Resistance toward:
Xanthomonas oryzae
(peroxidase activity,
polyphenol-oxidase &
phenylalanine-ammonia
lyase).
Yasmin et al.
(2016),Hakim
et al. (2021)
Micrococcus sp.Indole-3-acetic acid and
siderophore production.
Sukweenadhi
et al. (2015)
Wheat Agrobacterium fabrum,
Bacillus amyloliquefaciens,
Leclercia adecarboxylata,
Pseudomonas aeruginosa
Significant improvement in
nitrogen, phosphorus, and
potassium of shoots and
seeds. 1-aminocyclopropane-
1-carboxylic acid deaminase
activity.
Danish &Zafar-ul-
Hye (2019)
Aeromonas sp.Exopolysaccharides (EPS)
production
Phour &Sindhu
(2022),Ashraf
et al. (2004)
Pseudomonas libanensis Siderophore, ammonia, IAA,
P-solubilization, 1-
aminocyclopropane-1-
carboxylic acid (ACC)
deaminase. Helps in reducing
the drought stress.
Kour et al. (2020)
B. safensis Antioxidant enzymes,
osmolyte accumulation.
Reduces heat stress.
Sarkar et al. (2018)
Planococcus rifietoensis 1-aminocyclopropane-1-
carboxylic acid (ACC)-
deaminase activity,
phosphorus solubilization,
indole-3-acetic acid
production. Reduced salt
stress.
Rajput et al. (2018)
Continued
5. Rhizosphere engineering for biotic stress 245

Table 11.1 An outlook of various soil microorganism effects on plant growth and their role in the
mitigation of stress.dcont’d
Crops Microorganisms
Contributions toward
sustainable agriculture References
Aeromonas spp. 1-aminocyclopropane-1-
carboxylic acid (ACC)-
deaminase activity, zinc and
phosphorus solubilization,
indole-3-acetic acid
production. Reduced salt
stress.
Rajput et al. (2018)
Enterobacter sp., A.
chlorophenolicus, S.
marcescens, B. megaterium
Phosphorus-solubilization,
indole-3-acetic acid
production, HCN, nitrogen-
fixation, gibberellin,
siderophores.
Hakim et al. (2021)
Azospirillum brasilense,
Bacillus amyloliquefaciens
Secondary metabolites,
antioxidant enzymes.
Reduces heat stress.
Abd El-Daim et al.
(2014)
P. putida Antioxidant activity, proline,
total proteins, sugars, amino
acids. Reduces heat stress.
Ali et al. (2020),
Hakim et al. (2021)
Pseudomonas sp. (54RB) Crop yield increase. Barman et al.
(2019)
Rhizobium leguminosarum
(Thal-8)
Improves phosphorus uptake
by plant roots.
Afzal &Bano
(2008)
Azospirillum brasilense Boosts plant growth. Barman et al.
(2019),
Dobbelaere et al.
(2002)
Barley Curtobacterium sp.Produces proline, indole-3-
acetic acid.
Cardinale et al.
(2015)
Maize (Zea
mays L.)
Geobacillus sp.Proline content increases. Phour &Sindhu
(2022)
Aeromonas veronii, Bacillus
safensis, B. pumilus,
Enterobacter aerogens
Nitrogen fixation, phosphate
solubilization, indole-3-acetic
acid production, siderophore
and HCN, 1-amino
cyclopropane-1-carboxylic
acid (ACC) deaminase activity.
Mukhtar et al.
(2020),Phour &
Sindhu (2022)
Acinetobacter johnsonii Nutrient uptake and
antioxidant defense,
enzymatic activities increase.
Shabaan et al.
(2022)
Pseudomonas entomophila IAA, phosphorus,
siderophore.
Sandhya et al.
(2010)
Kocuria rhizophila strainY1 Tolerance to salt stress, better
redox potential, ion
homeostasis, increased
growth, nutrient acquisition
induction of stress-responsive
genes, production of indole-3-
acetic acid.
Li et al. (2020)
246 CHAPTER 11 Bioengineering of rhizobiome toward sustainable

Table 11.1 An outlook of various soil microorganism effects on plant growth and their role in the
mitigation of stress.dcont’d
Crops Microorganisms
Contributions toward
sustainable agriculture References
Cupriavidus necator 1C2 and
Pseudomonas fluorescens
S3X
Boost uptake of phosphorus
and nitrogen, amelioration of
drought stress, indole-3-
acetic acid, siderophore
production, and ACC
deaminase activity.
Pereira et al.
(2020),
CarmenOrozco-
Mosqueda (2022)
Azospirillum irakens Helps in germination and
growth of seed.
Barman et al.
(2019)
Pseudomonas putida (R-168,
DSM-291)
It increases crop vigor and
production.
Nezarat &
Gholami (2009)
Pseudomonas fluorescens
(DSM-50090; R-98)
Promotes growth of seedling. Nezarat &
Gholami (2009),
Barman et al.
(2019)
Azospirillum lipoferum (DSM-
1690)
Promotes crop growth. Nezarat &
Gholami (2009)
Azospirillum brasilense (DSM-
1691)
Promotes growth of seedling. Nezarat &
Gholami (2009),
Barman et al.
(2019)
Achromobacter xylosoxidans,
Azospirillum brasilense,
Bacillus subtilis, Bacillus
megaterium, Pseudomonas
stutzeri, Rhodococcus
rhodochrous
IAA production, phosphorus
solubilization, zinc
solubilization.
Goteti et al. (2013),
Qaisrani et al.
(2014),Zahid et al.
(2015)
Azospirillum sp.Siderophore, nitrogen fixation,
IAA, phosphorus
solubilization. Amelioration of
drought stress.
Garcı
´a et al.
(2017),Hakim
et al. (2021)
Mung bean
(Vigna radiata
L.)
Rhizobium sp., LSMR-32, and
Enterococcus mundtii
LSMRS-3
Promote phosphatase and
dehydrogenase activity, boost
proline and antioxidative
enzymes.
Kumawat et al.
(2022)
Pantoea sp.1-aminocyclopropane-1-
carboxylic acid (ACC)
deaminase activity.
Panwar et al.
(2016),Phour &
Sindhu (2022)
Bacillus cereus, B. drentensis,
B. pumilus, B. subtilis,
Enterobacter cloacae,
Pseudomonas putida,
Rhizobium, Bradyrhizobium,
Ochrobactrum
Nitrogen fixation, solubilization
of phosphorus, indole-3-
acetic acid production,
siderophore production, 1-
aminocyclopropane-1-
carboxylic acid (ACC)-
deaminase activity.
Mahmood et al.
(2016),Tariq et al.
(2012)
Bradyrhizobium sp.(RM-8) Remediates Ni and zinc
stress.
Wani et al. (2007a)
Continued
5. Rhizosphere engineering for biotic stress 247

Table 11.1 An outlook of various soil microorganism effects on plant growth and their role in the
mitigation of stress.dcont’d
Crops Microorganisms
Contributions toward
sustainable agriculture References
Cicer arietinum
L.
Bacillus sp.IAA, HCN, EPS, ammonia
production. Ameliorates
drought stress.
Khan et al.
(2019a),Hakim
et al. (2021)
Pseudomonas sp Promotes crop growth. Rokhzadi et al.
(2008),Barman
et al. (2019)
Azospirillum sp.Root development. Rokhzadi et al.
(2008)
Azotobacter sp.Nutrients acquisition and
growth.
Rokhzadi et al.
(2008)
Mesorhizobium ciceri,
Ochrobactrum ciceri, Serratia
marcescens
Phosphorus solubilization, IAA
production, nitrogen fixation.
Zaheer et al.
(2016),Imran et al.
(2015)
Medicago
ciliaris
Sinorhizobium Production of high proline
content.
Ben Salah et al.
(2013)
Medicago
sativa L (alfalfa)
Bacillus megaterium
NRCB001, Bacillus subtilis
subsp. subtilis NRCB002, and
Bacillus subtilis NRCB003
Promote production of auxins
and siderophores,
solubilization of phosphate
and potassium, excretion of
NH
3
and 1-
aminocyclopropane-1-
carboxylic acid (ACC)
deaminase activity.
Zhu et al. (2020)
Soybean
(Glycine max)
Pseudomonas fluorescens
LBUM677
Promotes more fatty acids
content, high seed oil
production, and plant growth
promotion.
Jime
´nez et al.
(2020),
CarmenOrozco-
Mosqueda (2022)
Pseudomonas sp.Promotes production of
indole-3-acetic acid, EPS, 1-
aminocyclopropane-1-
carboxylic acid (ACC)
deaminase activity.
Del Carmen
Orozco-
Mosqueda et al.
(2019),Kumari
et al. (2015)
Arthrobacter woluwensis AK1 Promote production of indole-
3-acetic acid, abscisic acid
(ABA).
Khan et al. (2019b,
2019c)
Bacillus firmus SW5 Glycine, betaine, proline,
antioxidant activity.
El-Esawi et al.
(2018)
B. aryabhattai IAA, antioxidant enzymes
(heat stress).
Park et al. (2017)
Bacillus cereus Siderophore, indole-3-acetic
acid, phosphorus
solubilization, EPS.
Arif et al. (2017)
Groundnut
(Arachis
hypogea L.)
Ochrobactrum sp. Production of IAA and 1-
aminocyclopropane-1-
carboxylic acid (ACC)
deaminase activity.
Phour &Sindhu
(2022),Paulucci
et al. (2015)
248 CHAPTER 11 Bioengineering of rhizobiome toward sustainable

Table 11.1 An outlook of various soil microorganism effects on plant growth and their role in the
mitigation of stress.dcont’d
Crops Microorganisms
Contributions toward
sustainable agriculture References
Brassica
napus L.
Azospirillum sp.Promote better growth. Yasari &
Patwardhan
(2007)
Azotobacter sp.Increased yield. Yasari &
Patwardhan
(2007)
Pseudomonas putida (G 12-2) Seedling establishment. Barman et al.
(2019)
Rhizobium leguminosarum Boosts better growth of shoot. Noel et al. (1996)
Brassica
juncea L.
Bacillus sp.(32) Reduces stress caused by Cr. Rajkumar et al.
(2006)
Pseudomonas (A4) Reduces stress caused by Ni. Zaidi et al. (2006),
Barman et al.
(2019)
Tomato Bacillus (6 isolates),
Brevibacillus (1 isolate),
Pseudomonas (3 isolates),
and Trichoderma (8 isolates)
Protect from Ralstonia
solanacearum-induced
bacterial wilt of tomatoes.
Konappa et al.
(2020),
CarmenOrozco-
Mosqueda (2022)
Bacillus cereus 1-aminocyclopropane-1-
carboxylic acid (ACC)
deaminase activity, EPS.
Ameliorate heat stress.
Mukhtar et al.
(2020)
Bacillus pumilus, Bacillus
amyloliquefaciens
HCN, siderophore, nitrogen
fixation, indole-3-acetic acid,
phosphorus solubilization.
Fan et al. (2017),
Hakim et al. (2021)
Achromobacter sp.Reduce ethylene level by 1-
aminocyclopropane-1-
carboxylic acid (ACC)
deaminase activity.
Mayak et al.
(2004a,2004b)
Okra
(Abelmoschus
esculentus)
Bacillus baekryungensis
DPM17
Phosphate solubilization,
nitrogen fixation, production
of ammonia, IAA, and
gibberellins.
AlAli et al. (2022),
Phour &Sindhu
(2022)
Enterobacter sp.1-aminocyclopropane-1-
carboxylic acid (ACC)
deaminase, antioxidant
activity. Ameliorate salt stress.
Habib et al. (2016)
Mentha
arvensis
Exiguobacterium sp.Production of EPS. Bharti et al. (2014)
Pepper
(Capsicum
annum L.)
Bacillus fortis SSB21 Reduces level of lipid
peroxidation, ROS, ethylene.
Yasin et al. (2018),
Phour &Sindhu
(2022)
Watermelon Trichoderma asperellum
M45a
Uses as biocontrolling agent
against Fusarium wilt
Zhang et al.
(2020),
CarmenOrozco-
Mosqueda (2022)
5. Rhizosphere engineering for biotic stress 249

Bacillus sp., like colistin, circulin, and polymyxin are efficient in combating phyto-
pathogens (Liu et al., 2018;Maksimov et al., 2011). The main traits of biological
controlling agents that stop growth of pathogenic bacteria are the secretion and syn-
thesis of lytic enzymes (Hakim et al., 2021). The main way that PGPR contributes
through action of these lytic enzymes play a pivotal role to plant growth is by
defending plants from a variety of pathogenic fungi, including Fusarium oxysporum,
Botrytis cinerea, Phytophthora sp.,Rhizoctonia solani, Sclerotium rolfsii, and
Pythium ultimum (Chen et al., 2020). Using PGPR to bio-prime plants results in sys-
temic resistance to a variety of plant diseases (Naznin et al., 2013). The development
of induced systemic resistance in plants is influenced by rhizosphere bacteria and
fungi that support plant growth (Pieterse et al., 2014). The ISR-producing beneficial
bacteria can locally inhibit immune response in roots. For instance, in tomato,
different groups of microorganisms living in endosphere and hemisphere have a
role in modulating phenylpropanoid metabolism, which strengthens cell wall and
protects against Fusarium oxysporum f. sp. Lycopersici (Hakim et al., 2021). As a
result, it is possible to engineer the immune system of plants to draw in microorgan-
isms and give e resilience for thousands of generations.
6.Conclusions and future outlook
Food and environmental security are facing severe threats from rapid land degrada-
tion, loss of nutrient-rich top soils due to erosion threats, contamination with toxic
substances, and depletion of the soil organic matter. This is concluded that devel-
oping field of rhizosphere engineering is more than a potential path toward a
more sustainable agricultural production system. Agricultural sustainability is a
key issue today as ecosystem is being drained by pollution and other factors, which
affect the human population, animals, and crop plants. Rhizosphere engineering is
becoming more and more prominent as its potential importance to agricultural sus-
tainability. Timing is right to combine the technological advancements with an un-
derstanding of rhizosphere biology to produce intriguing findings that will benefit
humanity. Rhizo-microbiome interactions with plant roots play a critical part in
reducing anomalies in crop plants brought on by various biotic and abiotic stress
conditions, which have been considered important to development of sustainable
agricultural production systems. Rhizo-microbiome is very fascinating, particularly,
when it comes to engineering the rhizosphere by changing resident plants and/or mi-
crobial communities in response to environmental stress and climate change to see
how rhizosphere might react to specific engineering interventions to boost its capa-
bilities. Biofertilizers based on the microbial inoculants are appealing as they act to
fix N, SO
4
, K, and Zn and solubilize nutrients and enhance plant growth by hormonal
action or antibiosis. They are the main drivers to hasten the restoration of land qual-
ity and ensure food security for the next generation. Plant growth promoting rhizo-
bacteria (PGPR) are important for growth and crop yield in agriculture as well as for
maintaining soil fertility status. They can also be used as biocontrol agents to assist
250 CHAPTER 11 Bioengineering of rhizobiome toward sustainable

in the control of plant pests and pathogens, promote the plant growth, and make nu-
trients available in biotic and abiotic stress conditions in an environmentally friendly
way. Thus, it is necessary to characterize rhizo-microbiome in more detail and depth
to achieve sustainable agriculture. Future studies should concentrate on how plant-
microbe interactions function in harsh conditions for welfare of plants through
rhizosphere engineering. The stabilization of these bacteria in the soil ecosystems
can be used as biofertilizers and biopesticides and can reduce the usage of
chemical-based products in agricultural production systems. Scientists also need
to address other concerns, such as increased effectiveness of biofertilizers. Through,
use of genetic engineering novel or improved PGPR strains can be developed by
changing their specific features. This altered PGPR is a low-input, ecologically
sound, and sustainable technology that can assist in controlling plant stresses.
Biotechnological methods might become an increasingly adept at utilizing rhizo-
biome to fullest extent possible. To support the rhizosphere engineering for sustain-
able agriculture, it might be viable to maintain and restore this soil microbial
diversity. Rhizosphere engineering makes use of microorganism inoculants as one
of recently emerging opportunities for addressing agricultural constraints, and
obtaining high crop production with improving the soil quality is the main challenge
for sustainable agriculture.
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266 CHAPTER 11 Bioengineering of rhizobiome toward sustainable

Bioinformatics study to
unravel the role of
rhizobiome to biologically
control the pathogens in
vegetables
12
Vanya Bawa, Meghna Upadhyay, Sheetal Verma
Department of Botany, Central University of Jammu, Jammu, Jammu &Kashmir, India
1.Introduction
The rhizosphere interface is a nexus of plant-microbe interactions and is a key that
assists plant uptake of moisture and mineral nutrients (Subhalakshmi &Indira,
2017) to maximize the microbiome functions. Millions of microorganisms reside
in the rhizosphere, a confined space connected to plant roots. The term “rhizo-
biome” or “root microbiome” refers to the group of bacteria that are linked with
the rhizosphere. Through control over plant growth and development, these micro-
biomes play a crucial part in plant health (Zhou et al., 2021a). The research is still
focused on how to relate organisms and is still evolving using genome-based and
high-throughput methods. Even though the importance of the microbial commu-
nity in the rhizosphere is widely understood, the characterization of the many mi-
croorganisms that have colonized the rhizosphere has not been done. Research on
microbiomes continues to focus on how to connect organisms utilizing genome-
based and high-throughput methods. This natural culture system is a key compo-
nent of the agricultural microbiome engineering system, as it has been used to
maximize microbiome functions in agroecosystems to increase plant nutrient up-
take (Pang et al., 2021;Vurukonda et al., 2018) and resistance to biotic and abiotic
stresses (Dagdas et al., 2012;Jia et al., 2021). The understanding of microbial
functions and interaction can be of great potential in agriculture (Azizoglu,
2019;Moon et al., 2021). The principal area here in particular is the main focus
on the identification of rhizospheric microbiome taxa that further can be utilized
to improve agriculture sustainability; however, the number of potential targets in
the entire rhizobiome, and the variation between crops is immense and need sepa-
rate efforts (Pan et al., 2015;Zhang &Sun, 2018). The multiplication rate of soil
microbes is always on exponential rate and determination along with the charac-
terization of the “core” rhizobiome regarding multiple members of microbial
assemblage associated with habitat is a tedious task. Much potential can be
CHAPTER
267
Rhizobiome. https://doi.org/10.1016/B978-0-443-16030-1.00015-8
Copyright ©2023 Elsevier Inc. All rights reserved.

harnessed by narrowing down the search to target only core microbiota that has a
greater influence on soil nutrient cycling and influencing other edaphic factors
ratedtoplantgrowth(Luo et al., 2019;Pang et al., 2021;Spence et al., 2014).
Yet multiple definitions of the term core rhizobiome have been used across studies
to try and identify key microbes based on their presence within a host population,
spatial distribution, temporal stability, or contribution to host function and fitness.
To try and identify important bacteria based on their presence within a host popu-
lation, spatial distribution, temporal stability, or contribution to host function and
fitness, several definitions of the term “core rhizobiome” have been employed
throughout research (Jin et al., 2020;Subhalakshmi &Indira, 2017)Itisstudied
that the relevance to dissect the rhizosphere has been increased so far as salt-
sensitive (SS) and salt-resistant (SR) plants directly affected root-derived bacteria
and play relevant functions when obstructed by the stresses, salt-induced recruit-
ment of certain RDB led to a constant growth boost in plants regardless of their
salinity tolerance capacities, although SSs and SRs recruited different RDB and
pertinent functions when challenged by salinity. The second genome of a plant
is thought to be its related microbiome (Pan et al., 2015) because it is not only
important for regulating plant metabolism (Luo et al., 2019) but also critical for
the plant immune system (Jin et al., 2020;Weietal.,2020). Nevertheless, only
a small number of researchers have attempted to investigate whether and how
plants develop particular RDB when subjected to salt stress, or whether the ability
of RDB to alleviate salt stress in salt-tolerant plants differs from that in SS ones.
Specific RDB strains have been shown to boost plant performance under salinity
conditions through several advantageous mechanisms, including regulating ion ho-
meostasis, generating phytohormones, encouraging osmolyte accumulation,
improving antioxidant activity, and improving nutrient absorption (Olanrewaju
et al., 2021). The recent development of high-throughput sequencing in conjunc-
tion with a variety of “omics” approaches enables researchers to uncover host in-
teractions and microbiome structure and dynamics at a previously unheard-of
level. Modern sequencing methods offer in-depth information on the identity
and relative abundance of the microbial partners of plants. Because sequences
are generated directly from the environmental sample, the cultivation of microbial
isolates is not necessary (Epstein, 2013;Hug et al., 2016). However, the freedom
gained through sequencing technology can result in a deluge of data which must be
countered by selecting an experimental design and sequencing methodology
appropriate to the scientific question being asked. A thorough understanding of
the types of expected biases and errors should be considered carefully when
choosing a particular sequencing method. All of the methodologies thus far
described call for particular bioinformatics techniques and apparatus for data
reduction, processing, and interpretation. Here, we provide instructions for
researchers on how to plan and carry out computational investigations on plant-
microbe interactions. We discuss the quality of public genome data, software
pipelines to analyze amplicon and metagenomic sequencing data, and present
workflows of data analysis for both approaches. Data integration of additional
268 CHAPTER 12 Bioinformatics study to unravel the role of rhizobiome

“omics” techniques will be addressed to promote much-needed multidisciplinary
research that could shed light on the interlinked complexity of plante-microbiome
interactions and their dynamics (Zhang &Sun, 2018). The present chapter will re-
view the utilization and flow of information via the pipeline method in bioinfor-
matics (Fig. 12.1).
1.1 Potential spatial effect
The spatial scanning of the rhizosphere is one of the stringent means to analyze its
biological components. Soil sampling with adequate investment in replication is
required to eliminate biases and inherent variability (Prosser, 2010;Lennon &
Jones, 2011). The rhizosphere dynamics are always changing and given that the
abundance and community structure of the microbial populations associated
with leaves and roots differ according to the stage of plant development (Copeland
et al., 2015;Edwards et al., 2018;Wagner et al., 2016), to maintain the precision of
statistical interpretation of molecular data a sufficient number of replicates is
necessary. Additionally, stochastic factors, such as the timing of species’ arrival,
may affect the distribution of species on roots and possibly even on leaves (Ken-
nedy et al., 2009). To advocate the intrinsic variability before sequencing, the
FIGURE 12.1
The flow of information and data processing under the bioinformatics pipeline.
1. Introduction 269

collection of adequate samples minimum of five replicates per plant organ, and
bulk soil samples within a 2 cm zone of roots. Furthermore, when immature tissues
are the subject of comparative community analysis, it is important to take into ac-
count the considerable diversity in microbial colonization density and community
structure among plant organs/tissues (such as roots and ectomycorrhizal root tips)
of individual plants (Richter-Heitmann et al., 2016). To avoid microbial commu-
nity composition associated with plant organs changing due to changing climatic
circumstances and affected by previous sowing or adjacent crop stand of cereal
crops and vegetable or fruits (Tang et al., 2020). Wheat and watermelon show
this type of mixed interaction in terms of the rhizosphere to the rhizosphere and
disease development (Kro
¨ber et al., 2014), and that affects the sampling; hence,
it should be done of plant material at the location of plant growth (such as a field
or greenhouse) (e.g., phylloplane-colonizing bacteria). The soil amendments are
also one of the important factors to be considered before sampling (Tang e t al.,
2020). The importance of the sample in terms of the spatial arrangement of root
architecture defines the efficient results in sequencing and data generation. The
locally applied tools include the availability of liquid nitrogen or dry ice in the field
so that the samples should be immediately snap-frozen or preserved in commercial
stabilization solutions (such as RNAlater, and LifeGuard). Washing with solutes is
a common process in sample preparation. To learn about potential contaminations
that can impair microbiome investigations and hinder the discovery of low-
abundance community members is crucial, these should be sanitized and
sequenced independently (Laurence et al., 2014;Quinn &Keough, 2002;Salter
et al., 2014). The potential of soil microfauna should be under great care by har-
vesting and treating plant material and associated microbiomes when using
sequencing techniques to answer research questions (Bulgarelli et al., 2012;Lund-
berg et al., 2012).
1.2 Microbiome on the molecular level
DNA extraction and further processing for sequence data generation work coher-
ently with the methods minimizing the loss of microbiome DNA. A grouping of
setting-up methods for minimizing nonmicrobiome DNA during experiments and
in-silico is important to get transcriptomics and genomics data sets of plant-
associated microorganisms, such DNA can come from a variety of sources. To main-
tain stringency, when extracting microbial nucleic acids from plant material,
researchers should be aware that chloroplast and mitochondrial DNA will also be
extracted through milling and physico-chemical lysis (Lutz et al., 2011). Although
it is difficult to remove the rhizosphere, samples from plant roots might be extremely
polluted, as was previously described (Kryukov &Imanishi, 2016). While working
with a microbiome, the contamination source could be human DNA which can be
added during the processing of the samples’ DNA (Kryukov &Imanishi, 2016).
Additionally, estimations of soil microbial diversity may be questionable due to relic
DNA (Carini et al., 2016). Researchers are now able to learn about host relationships
270 CHAPTER 12 Bioinformatics study to unravel the role of rhizobiome

as well as the structure and dynamics of the microbiome at a level that was previ-
ously unheard of because of the recent advancements in high-throughput sequencing
and several “omics” methods. Modern sequencing techniques and processing
through the use of multiple platforms make it possible to gain in-depth knowledge
about the identification and relative abundance of the microbial partners of plants.
Since sequences are acquired directly from the ambient material, the culture of
microbiological isolates is not necessary (Epstein, 2013;Hug et al., 2016). Even
though sequencing technology’s freedom can result in a deluge of data, this must
be avoided by selecting an experimental strategy and sequencing methodology
that are appropriate for the specific scientific topic at hand. The types of predicted
biases and errors should be carefully analyzed before choosing a specific sequencing
method.
High-throughput sequencing of marker gene amplicons is being employed in
plant microbiome investigations to better understand the makeup, organization,
and location of microbial populations in the environment (Knief, 2014). Amplicon
sequencing has the advantage of allowing for the emphasis on particular microbial
communities (such as bacteria or archaea) or even functional genes (Herbold et al.,
2015). Even rare species can be positively identified with amplified sequencing
thanks to its high specificity, but because of its sensitivity, it is also susceptible to
contamination (Glassing et al., 2016). Any study that significantly relies on ampli-
con sequencing must include both positive (known mock communities) and negative
controls. Shotgun metagenomics can “bin” the data into draught genome sequences;
however, it is less effective than amplicon sequencing at identifying the existence of
uncommon organisms (Poretsky et al., 2014). Data can be “binned” via amplified
sequencing to create a more precise abundance value. Through these, it is feasible
to determine whether symbionts may be able to “communicate” with plants via
secretion systems or effectors or link taxonomic identity to important plant pro-
cesses like nitrogen fixation (Eichinger et al., 2016). Other high-throughput molec-
ular techniques, including transcriptomics, proteomics, and metabolomics, can be
used to supplement metagenomic approaches. The study of the human microbiome
has made extensive use of metatranscriptomics (Bashiardes et al., 2016), which can
serve as a paradigm for the research of the plant microbiome. Various methods have
been developed for high through put RNA-sequencing and data analysis (RNA-Seq)
as a high-throughput gene expression quantification technology and still there is no
accurate pipeline to study variety of analysis at a single click of analysis (Conesa
et al., 2016). Whereas, Meta-proteomic data can be used to enhance gene models,
uncover posttranslational modifications and frameshifts, and provide insights into
the complete microbial communities in plants in addition to providing evidence
for protein expression and quantification (Butterfield et al., 2016;Nesvizhskii,
2014). Researchers can learn about surface exudates secreted in phylloplane and
rhizoplane on the exterior as well as secondary and main plant metabolites that could
affect the host’s microbiome can be analysed using genomics approaching and
computational predictions. To itigate the technical problems, efforts have been
made to create artificial plant microbiomes include computational modeling
1. Introduction 271

(Scheuring &Yu, 2012) and synthetic community experiments combined with
multi-omics (Vorholt et al., 2017). This provides a better way to compare phenotypic
and genotypic data.
1.3 Databases and methods for sequence classification
To better understand the makeup, structure, and spatial distribution of microbial
communities in the environment, high-throughput sequencing of marker gene ampli-
cons is increasingly used in plant microbiome studies (Knief, 2014). Amplicon
sequencing has the advantage of being able to target single groups of microbes
(such as bacteria or archaea) or even functional genes (such as DsrA, AmoA, etc.)
(Herbold et al., 2015). Amplicon sequencing’s high specificity makes it possible
to utilize it to positively identify even rare organisms, but because of its sensitivity,
it is also vulnerable to contamination (Glassing et al., 2016). For any experiment that
largely relies on amplicon sequencing, including both positive (recognized mock
communities) and negative controls is vital.
Shotgun metagenomics can be used to “bind” data into draft genome sequences,
although it is less accurate than amplicon sequencing at detecting the existence of
uncommon organisms (Poretsky et al., 2014). These allow for the connection of
taxonomic identity to crucial plant activities, such as nitrogen fixation, or the assess-
ment of whether symbionts may be able to “communicate” with plants via secretion
systems or effectors (Eichinger et al., 2016).
Various high-throughput molecular techniques, including metabolomics, prote-
omics, and transcriptomics can be used in conjunction with metagenomic ap-
proaches to enhance their effectiveness. The application of meta-transcriptomics
in human microbiome research is well established and it can be used as a model
for plant microbiome research. Recent reviews of RNA-seq data analysis best prac-
tices have been published (Bashiardes et al., 2016;Conesa et al., 2016), whereas,
meta-proteomic data can be used to improve gene models, uncover posttranslational
modifications and frame-shifts, and provide insights into entire microbial commu-
nities in plants in addition to providing evidence for protein expression and quanti-
fication (Butterfield et al., 2016;Nesvizhskii, 2014). Studying plant metabolome
reveals details on both the interior surfaces of the host (plant solute transport) and
the secondary and primary plant metabolites that could affect the microbiome (phyl-
loplane, rhizoplane).
Our understanding of the functions of microbes and how they interact with plants
has greatly benefited through bioinformatic analysis (Cha et al., 2016;Koberl et al.,
2013;Spence et al., 2014). For instance, metagenomic data analysis was initially
used to determine that Pseudomonas spp. was to blame for sugar beet affliction in
soil that was resistant to Rhizoctonia solani (Mendes et al., 2011). Testing compu-
tational predictions in the lab or on the job, though, is frequently not an easy task.
Computational modeling (Sokal, 1963) and synthetic community experiments
paired with multi-omics are recent efforts toward artificial plant microbiomes
(Vorholt et al., 2017). To give more emphasis on sequencing, NGS data must be
272 CHAPTER 12 Bioinformatics study to unravel the role of rhizobiome

properly processed to de-noise readings and categorize them into reliable OTUs or
amplicon sequence variations (ASVs) to produce biologically significant results.
OTUs are a common component of contemporary microbial ecology and were first
presented as a practical substitute for classification at the species level to help with
quantitative ecological comparisons (Sokal, 1963) (The fundamentals of numerical
taxonomy, San Francisco: W.H. Freeman, 1963). A few tools, such as MOTHUR
(Schloss et al., 2009), QIIME (Schymanski et al., 2014), and UPARSE, are available
for creating OTUs (Caporaso et al., 2010). Similar processing processes, such as
filtering sequencing reads for quality and length, as well as generating and catego-
rizing OTUs from microbial 16S rRNA amplicons are present in all three pipelines.
PIPITS (Edgar, 2013) represents a method for processing ITS amplicons. The num-
ber of databases that categorize microbial taxa into guilds or lifestyles is increasing.
For instance, the FUNGENE (Nguyen et al., 2016) database divides fungal taxa into
lifestyle categories such as symbiotroph, phototroph, and saprotroph, while the
FUNGENE database serves as a repository for the most prevalent functional marker
genes utilized in microbial ecology. The PHYTOPATH (Benli et al., 2013) database
stores information on plant pathogen species, while the NIFH database (encoding
the dinitrogenase reductase portion of nitrogenase) helps analysis of the evolution
and ecology of nitrogen-fixing organisms. This presents chances to evaluate
macro-ecological queries regarding the functional variety of rhizosphere micro-
biomes as mentioned in Table 12.1.
1.4 Sequencing protocols and data processing
After choosing the right primer pair, compatibility with the appropriate sequencing
platform (such as IlluminaMiseq) must be guaranteed. Short barcodes in primer se-
quences and the addition of Illumina adaptors, for instance, allows for the parallel
sequencing of several samples. A single PCR process using already integrated
primers can do this.Amplicon sequence variants (ASVs) and/or group them into
trustworthy OTUs to produce biologically significant results. Similar processing
Table 12.1 Database used for processing microbiome-rhizosphere.
Database Utilization References
FUN GUILD Division of fungal taxa Gweon et al. (2015)
FUNGENE Used in microbial ecology Nguyen et al. (2016)
PHYTOPATH Used for plant pathogen studies Benli et al. (2013)
NIFH Evolution and ecology of the nitrogen-fixing
organism
Pedro et al. (2016)
ORTHOMCL Identification of ortholog groups for eukaryotic
genomes
Gaby &Buckley
(2014)
KEGG Molecular-level functions Li et al. (2003)
1. Introduction 273

processes, such as quality and length filtering of sequencing reads and OTU creation
and classification of microbial 16S rRNA amplicons, are present in all three pipe-
lines. PIPITS (Edgar, 2013) is a collection of instructions for processing ITS ampli-
cons that need software, such as VSEARCH (Kanehisa et al., 2017), an open-source
software equivalent to USEARCH (Rognes et al., 2016) of which many are already
available in the public domain. We recommend the reader to visit for a more thor-
ough review of ITS amplicon sequencing (Edgar &Flyvbjerg, 2015).
Beta diversity analyses between pyrosequencing datasets (2010 vs. 2011), and
operational taxonomic units (OTUs) were identified using the open reference
OTU picking pipeline implemented in QIIME V1.8.0 (Edgar &Flyvbjerg, 2015).
For the OTU picking, the algorithm with a 97% clustering identity and the GREEN
GENES database release 13.8 was used. Pyrosequencing revealed the presence of 16
phyla or candidate divisions, 39 bacterial classes, 44 bacterial orders, 96 families, or
250 different genera in the rhizosphere soil sample (Nilsson et al., 2019). Currently,
three main techniques use microbial sequence data: whole genome sequencing, mi-
crobial meta-omics, and taxonomy classification utilizing rRNA-gene and internal
transcribed spacer (ITS) sequences. When performing 16S- and 18S-rRNA gene
or ITS analyses, microbial diversity may be underestimated despite improved meth-
odologies and data availability if the assignment depends on unfinished reference
gene databases (Pascual et al., 2016).
Sequencing of complete microbial genome initiatives is also being done in to-
matoes using the WGS approach following the bioinformatics tool (Kro
¨ber et al.,
2014). For instance, the related packages OrthoMCL, KEGG, and others enable
the prediction of orthologs among various species and increase the likelihood of
identifying a microbial lifestyle through a genomic signature. Sequence characteris-
tics like base composition, GC-skew, purine-pyrimidine ratio, dinucleotide abun-
dance, codon bias, and oligonucleotide composition, the presence of particular
gene families, horizontal acquisition of genome islands, and the processes of
genome shrinkage/expansion are just a few examples of niche-specific genome sig-
natures. There are reasons to believe that this is only the tip of the iceberg for the
remarkable diversity in the niche-specific signature features that have been charac-
terized thus far, underscoring the evolutionary plasticity of the microbial genomes.
Most of the signature features have yet to be explored and revealed. One can antic-
ipate the discovery of many more novel niche-specific genomic signatures in micro-
organisms that have evolved to distinct specialized lifestyles or extreme ecological
niches given the growing quantity of microbial genome sequences that are available
to the public. If appropriately evaluated, these fingerprints may not only provide in-
formation on the molecular mechanisms behind microbes’ niche specialization, but
they may also have significant ramifications (Brunel et al., 2020).
The first metaanalysis of this methodology was just recently published in the
context of pooling 606 microbiomes that had been exposed to varied environmental
conditions and sampled in various habitats (Dutta &Paul, 2012). Using publicly
accessible raw 16S rRNA gene sequencing data from the IlluminaMiSeq V4 hyper-
variable area, the authors chose nine independent research on 16S rRNA gene
274 CHAPTER 12 Bioinformatics study to unravel the role of rhizobiome

amplicon sequencing (reprocessed into ASVs). Their findings contrast with those of
Hendershot (Rocca et al., 2019), whose findings were previously mentioned but
which, when employing different analytical techniques, were unable to detect global
patterns associated with temperature. Consequently, the study (Dutta &Paul, 2012)
demonstrated the effectiveness of merging sequence data using ASVs and produced
a more in-depth understanding of how particular environmental parameters influ-
ence microbiome responses utilizing ASVs in conjunction with meta-analyses
from numerous large-scale studies in the context of the rhizosphere (Dutta &
Paul, 2012).
1.5 Omics to metagenomic approaches
Being the newer approach to understanding the microbial world, metagenomics of-
fers a powerful lens for assessing and exploiting the taxonomic and metabolic diver-
sity of microbial communities on an environmental level. Metagenomics has been
defined as functional-based or sequence-based cultivation-independent analysis of
the collective microbial genomes present in a given habitat (Hendershott &Vale,
2014). The extraction and analysis of metagenome from the diverse environment
are done to pursue objectives viz., Discovery of novel biomolecules and biocatalysts,
understanding the dynamic composition of microbial communities, and most impor-
tantly harnessing functional attributes of the community genome (Hassan et al.,
2021;Riesenfeld et al., 2004;Robinson et al., 2010;Xia et al., 2019). Metagenomics
is subdivided into two major approaches that target different aspects of the local mi-
crobial community; structural metagenomics which deals with the study of the struc-
ture of uncultured microbial population and functional metagenomics which is used
to identify genes that code for the function of interest. It is crucial to have better
knowledge regarding the microbial species, the presence in the human microbiome
fuels attempts to adjust diets to achieve the beneficial balance of bacterial species
(Kukkar et al., 2022). Further research is required to understand the interactions be-
tween phytopathogenic bacteria, bacteria that promote plant growth, and bacteria
that can cause disease in people and animals (Mendes et al., 2011).
Whole genome sequencing (WGS) makes use of the sequence data from the
entire genome, which illustrates various degrees of conservation. This offers better
phylogenetic resolution than amplicon sequencing and allows function prediction.
Advanced high-throughput metagenomic approaches resulted in millions of environ-
ment gene sequences that paved the way to the so-far hidden phylogenetic compo-
sition of the complex environment microbial communities (Umu et al., 2013). For
the majority of large-scale microbiome research efforts, high throughput sequencing
technologies like the IlluminaNextseq and HiSeq can quickly generate billions of
highly accurate 100e300 bp reads at a low cost and that shotgun sequence-
derived dataset’s evaluation provide a reliable estimate about the microbial diversity
stored in metagenomic libraries (Sjo
¨ling &Cowan, 2008). The major advantage of
this cloning-independent approach is the avoidance of phylogenetic marker genes
and cloning. However, it is crucial to first take into account how much sequencing
1. Introduction 275

data is required to fully utilize the richness of metagenomic datasets to address spe-
cific scientific problems. Unfortunately, this is not an easy task. Because of the sig-
nificant strain diversity found in plant-associated communities, there may be less
coverage of some genomes and poorer assembly (Mayorga et al., 2022). Existing
16S rDNA amplicons data (Sczyrba et al., 2017;Sha et al., 2020;Tamames et al.,
2012) and/or early metagenomic data are needed for methods to estimate the amount
of sequencing required to retrieve data for a target genome.
To estimate the appropriate sequencing depth for the sort of analysis that will be
performed, The rationale for producing a metagenomic dataset must be carefully
considered. For the computational study of shotgun metagenomes, four strategies
are typically used: taxonomic profiling, target-gene reassembly, and genome binning
are examples of taxonomic binning. Moreover, computational speed is an important
factor. Computational speed can be increased by the use of multiple compute nodes.
Additionally, several internet sites offer software tools, tutorials, protocols, and
instructions for processing, statistical analysis, and visualizing metabolomic data.
Platforms that specialize in biomarker development and categorization, such as
the Metabolic Workbench (Ni et al., 2013), XCMS for MS-based data (Sud et al.,
2016), or MetaboAnalyst (Gowda et al., 2014), offer a wealth of resources for this
goal. These software tools are available in the public domain.
Plant-specific databases, such as Gramene/PlantReactome, a free and open-
source, curated plant pathway database site, or PLANT SEED, which offers
annotation and model data for 10 plant genomes (Xia et al., 2015), can be used to
annotate plant genomes and build metabolic models (Naithani et al., 2017;Seaver
et al., 2014). The PLANTCYC database, which as of version 12.0 has 1200 path-
ways in more than 350 plant species, is part of the Plant Metabolic Network, which
is another enormous resource for plant metabolic networks (Sha et al., 2020). Over-
all, comprehensive annotation of plant metabolomes has not yet been accomplished,
despite ongoing advancements in nontargeted metabolomics (Tello-Ruiz et al.,
2018).
With the advancement of high-throughput sequencing technology, it is now
possible to use biological control agents to investigate dynamic changes in micro-
bial communities. In this work, techniques were utilized to make it easier to eval-
uate soil Tri choderma biological control and its effects on the P. b r a s s i c a e
population. Hence, was estimated its effect on plant-microbe interaction under
the effect of Tr ichoderma inoculation in the control of P. b r a s s i c a e clubroot dis-
ease on rhizosphere microbial communities (Li et al., 2020;Mazzola et al.,
2015). In contrast, the Plant Growth Promoting Rhizobacteria (PGPR) can stimu-
late plant growth in vegetables through biological activity. Their application is seen
as a potential, long-term strategy for crop growth. Additionally, a large number of
biosynthetic gene clusters (BGCs) for the formation of secondary metabolites are
being discovered in PGPR, which aids in the discovery of possible antimicrobial
properties for the control of tomato disease (Zhou et al., 2021b). The effects of
helpful bacteria on the rhizosphere microbiota and the growing circumstances of
vegetables during plug seedling, however, are not well understood. In this study,
276 CHAPTER 12 Bioinformatics study to unravel the role of rhizobiome

we used a culture-independent next-generation sequencing-based methodology to
examine the effects of Bacillus amyloliquefaciens L-S60, a plant-beneficial bacte-
rium, on the diversity and dynamics of the rhizosphere microbiota and the growth
circumstances of cucumber seedlings. The presence of beneficial rhizosphere spe-
cies like Bacillus,Rhodanobacter,Paenibacillus, Pseudomonas, Nonomuraea,and
Agrobacterium was higher upon L-S60 treatment than in the control group. L-S60
application also significantly altered the structure of the bacterial community asso-
ciated with the cucumber seedling in the rhizosphere. The bacterial community
composition and the growing conditions of the vegetables during plug seedling,
however, were positively impacted by plant growth promoting rhizosphere bacteria
(Qin et al., 2017). The rhizosphere microbial communities can be easily changed in
the growth of leafy vegetables, and vice versa, by external manipulation. So far,
lettuce was treated with B. amyloliquefaciens FZB42 and treated and nontreated
plants were profiled by high-throughput metagenome sequencing of whole com-
munity DNA. However, under experimental conditions, metagenomics and com-
parison of taxonomic community profiles only revealed minor changes, whereas,
taxonomic profiling and functional analysis of annotated sequences revealed no
major differences between samples regarding the application of the inoculants
strain (Kro
¨ber et al., 2014). In contrast, antagonistic activity evidenced so far
from some studies after using biocontrol agents and the genome analysis revealed
advantageous results in prospects for microbial and plant growth (Phazna et al.,
2022). After a thorough examination of the rhizosphere’s microbial communities,
the dynamics of the soil to microbial communities exhibit a wide range of out-
comes. Significant variations between crops cultivated on the same soil type and
between crops that were planted in other soil types as well. The necessity to deploy
soil microbe investigations more beneficially is necessitated by stringent equip-
ment and methodologies to obtain the desired findings. The rhizosphere microbial
community structure is unquestionably influenced by elements particular to the
soil and the kind of plants (Liu et al., 2020).
2.Conclusion
The availability of tools and techniques can revolutionize the approach to geno-
mics through phenomics, which is impossible without the branch assistance of bio-
informatics. As far as the plant sciences are concerned, it demands intelligent
searching along with efficient processing and filtering of numerous, complex
data, uncovering and addressing specific tasks. To evaluate the current state of
the art and provide useful recommendations for experimental and computational
considerations in molecular plant-microbiome, the investigations in terms of
sequencing and “omics” techniques with an emphasis on the conditions create
importance to customize various methods for certain research trajectories of data
mining. The relevant requirements concerning genomic databases are taken into
consideration and can offer a potent tool to detect and quantify small molecules
2. Conclusion 277

and molecular changes at the plant-bacterial interface. And cutting edge technol-
ogies can efficiently combine data generation and its mobilization through the bio-
informatics pipeline to provide scientific information to the public domain for the
plant holobiont in the future.
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284 CHAPTER 12 Bioinformatics study to unravel the role of rhizobiome

Azospirillum—a free-living
nitrogen-fixing bacterium 13
M.D. Jehani
1
, Shivam Singh
2
, Archana T. S.
1
, Devendra Kumar
1
, Gagan Kumar
3
1
Department of Plant Pathology, School of Agriculture, Lovely Professional University, Phagwara,
Punjab, India;
2
Krishi Vigyan Kendra-Baghpat, S.V.P University of Agriculture and Technology,
Meerut, Uttar Pradesh, India;
3
Krishi Vigyan Kendra, Narkatiaganj, Dr. Rajendra Prasad Central
Agricultural University, Samastipur, Bihar, India
The most frequently lacking nutrient, nitrogen, is a vital component of plants and
contributes to poor agricultural production globally. Although it makes up a signif-
icant portion of dietary proteins, plants have low absorption efficiency, and signifi-
cant amounts can be lost through denitrification and leaching. The Haber-Bosch
process generates about 83 million tonnes of N fertilizers annually worldwide. How-
ever, this method consumes a lot of fossil fuel and has high energy and capital ex-
penditures. Unfortunately, plants only utilize less than 50% of provided N fertilizer.
Concern over environmental pollution, rising consumer demand for organic agricul-
tural and horticultural products, and rising costs of chemical fertilizers have reig-
nited interest in improving biological nitrogen fixation (BNF) as a biofertilizer to
meet crop N-requirements.
The linkage between BNF and sustainable agriculture is a recurring theme in the
majority of studies. BNF is almost always associated with sustainability. Systems
that are able to increase their own N use their own environment less, and many
even add to their own N. There is a range of 100e290 million tonnes of potential
BNF (symbiotic and nonsymbiotic) in natural ecosystems per year. The estimations
are larger for the tropics than for more temperate places because symbiotic N
2
-fix-
ation is the predominant method. However, there are significant knowledge gaps
about BNF, especially nonsymbiotic N
2
-fixation.
A tonne of wheat grain, including straw, requires about 26 kg of nitrogen (N), yet
at the height of crop growth, N demand exceeds N mineralization supply. Nitrogen
fertilizers currently make up the majority of this deficiency. It should be able to
decrease the need for increasingly expensive industrially fixed N fertilizers if biolog-
ical systems can be modified to boost N inputs from nonsymbiotic N
2
-fixation.
Nonsymbiotic N
2
-fixation measurements were frequently done more than 30
years ago. Since then, our farming practices all across the world have seen tremen-
dous modifications. A shift toward intensive cropping methods, the use of no-tillage
techniques, the retention of stubble, and the cultivation of genetically modified crops
are some of the major developments in farming practices. Through the provision of
more carbon (C) resources for biological activity and the development and
CHAPTER
285
Rhizobiome. https://doi.org/10.1016/B978-0-443-16030-1.00001-8
Copyright ©2023 Elsevier Inc. All rights reserved.

maintenance of optimum conditions for nonsymbiotic N
2
-fixation, all of these fac-
tors are anticipated to have a considerable impact on nonsymbiotic N
2
-fixation. As a
result, it is crucial to reconsider biological systems in the context of these elements.
1.Diazotrophic (nitrogen-fixing) population
It has been well over a century since an experimental demonstration of the presence
of microorganisms that can biologically fix atmospheric nitrogen (N) took place.
The ability of the legume-rhizobial symbiosis to fix N
2
was solidly established
only 100 years ago. Since then this symbiotic system has been thoroughly studied
and used as a reliable method of improving the soil’s N condition and supplying
N for crops and pastures. The discovery of many N
2
-fixing organisms, or diazo-
trophs, in the 1950s sparked optimism that nonleguminous crops could gain similar
advantages in the 1970 and 1980s. These diazotrophs appeared to create a limited
number of relationships with nonlegume plants.
Endophytes, both obligatory and facultative, have been found to include certain
nonsymbiotic N
2
-fixing bacteria. The only endophytic N
2
-fixing bacteria known
before their discovery were those in the Casuarinas-Frankia or legume-Rhizobium
relationship. Bereiner was the first to bring the term endophyte to the study of N
2
-
fixation in graminaceous plants (1992a). The word, in general, refers to all bacteria
that are able to colonize the interior tissues of plants during part of their life cycle
without obviously harming the host. Because they have ready access to nutrients
and water from the plant and are less susceptible to competition from other micro-
organisms in the rhizosphere or soil, endophytic bacteria have an advantage over
free-living or rhizosphere bacteria. The presence of low oxygen (O
2
) and compara-
tively high carbon in the plant interior may also contribute to the environment being
favorable for N
2
-fixation. With a plant host, the bacteria can fix N
2
more effectively.
It is more likely that endophytic organisms will be effective as inoculants. Noncul-
turable N
2
-fixing microorganisms appear to predominate, similar to nonendophytic
diazotrophs. In order to identify and measure nitrogenase activity by a N
2
-fixing bac-
terial endophyte, Azoarcus spp., which was active in an uncultivable state in kallar
grass, Hurek et al. (2002) employed polymerase chain reaction-amplified nifH tran-
scripts (Leptochloa fusca). They anticipated that uncultured grass endophytes are
ecologically prominent and may be crucial in N
2
-fixation in natural grass environ-
ments based on their phylogenetic study of nitrogenase sequences.
1.1 Systems of associative diazotrophs
There have long been efforts to identify beneficial interactions between plants and
N
2
-fixing microorganisms. Simply because this is where growth substrates are found
in the greatest number, it follows that a significant amount of the overall microbial
286 CHAPTER 13 Azospirillum—a free-living

activity in soil is likely to be connected to the growth of plants, or their subsequent
breakdown. It should be kept in mind that there is very little chance that such diaz-
otrophs’ N
2
-fixation will have a substantial impact on the plant as long as it is
restricted to the bacterial growth. However, some bacterial species, particularly
those belonging to the genus Azospirillum, are thought to have a close relationship
with plants that verges on symbiosis.
1.1.1 Azospirillum
Azospirillum-genus bacteria are free-living microbes that aid in plant growth
(PGPB). Numerous plant species, many of which are important in terms of
agronomy and ecology, are affected in terms of growth and productivity (Pii
et al., 2015). The growth promotion of Azospirillum, which involves nitrogen fixa-
tion (Santos et al., 2017) as well as the generation of phytohormones, polyamines,
and trehalose, is the most widely recognized idea about the mechanism of action
of this organism (Bashan &Bashan, 2010). Azospirillum has several methods of ac-
tion, and the significance of each one can change based on the soil and climate con-
ditions, as well as the solubilization of minerals that the plant needs, including iron
and phosphorus (Bashan &Bashan, 2010). These processes eventually result in
larger, and frequently more productive, plants (Garcı
´a et al., 2017;Machado
et al., 1991). Wheat, rice, sugarcane, and maize, crop yields have all increased
because of Azospirillum. Cacti, fruit trees, and chilli peppers have all benefited
from its use. Twenty-five species isolated from various habitats have been described
for the genus Azospirillum. Majority of these species were cultured from watery set-
tings and polluted places after being separated from the roots of wild plants (Ben
et al., 1997;Xie &Yokota, 2005).
1.1.1.1 Azospirillum species genetics
Some of these species have been sequenced thanks to the development of molecular
biology, adding to our understanding of the variety of genes involved in this bacte-
rium’s various features, including those that promote plant growth. The size of each
Azospirillum species’ genome varies; in the case of A it is 4800 kb in size. 9600 kb
in A.lipoferum, roughly 7000 kb in A. brasilense. Megaplasmids have distinctive ge-
nomes, some of which are linear (Martin-Didonet et al., 2000;Wisniewski-Dye
´et al,
2012). These megaplasmids are frequently found. They are among the earliest ge-
netic traits for the genus Azospirillum to be described. Depending on the species,
they might have as few as 7 or as many as 10 replicons (Martin-Didonet et al.,
2000). One copy of each plasmid, ranging in size from 100 kb to 1.7 Mb, is found
in Azospirillum strains, and each strain has a distinct profile. Minichromosomes
have also been observed in Azospirillum strains in addition to plasmids. It’s inter-
esting to note that the genome of A. brasilense consists of many chromosomes
with replicons that are 600, 1000, and 1700 kb in size. A. brasilense was found to
have several chromosomes; nonetheless, a 2500 kb additional chromosome was
observed (Garcı
´a et al., 2020;Kwak &Shin, 2016).
1. Diazotrophic (nitrogen-fixing) population 287

2.Modes of action
Symbiosis, parasitism, commensalism, amensalism, and neutralism are only a few of
the diverse and complex ways that microbes interact with plants (Glick &Gamalaro,
2021). These microorganisms’ growth is reliant on plant photosynthesis, and they
influence plant growth in turn. As a result, they are collectively referred to as the
plant microbiome (Klaus &Bulgarelli, 2015;Lebeis et al., 2012;Muller et al.,
2016;Wang et al., 2008;Zhang et al., 2021) In recent years, crop productivity
has increased thanks to the use of advantageous plant-microbiome relationships.
Plant-beneficial bacteria produce plant growth stimulating hormones in addition
to enhancing pathogen resistance, soil characteristics, nutrient availability, and plant
development (Chaparro et al., 2012;Wasai &Minamisawa, 2018). Even though the
soil microbiome is made up of bacteria, fungi, algae, protozoa, viruses, and other
organisms, beneficial bacterial populations play a significant role in increasing
crop yield for sustainable agriculture.
Microorganisms that live in the rhizosphere support plant growth through direct
or indirect methods. Increased nutrient availability and the production of phytohor-
mones are directly related to the promotion of plant growth (Malik and Sindhu,
2011;Santoyo et al., 2021a), whereas the indirect mechanisms that contribute to
improved plant health and crop productivity include the suppression of diseases
by biocontrol agents, alleviation of abiotic stresses, and bioremediation of pollutants
and contaminants (Glick &Gamalaro, 2021;Zhang et al., 2021).
2.1 Mechanisms for promoting plant growth
Without adding any chemicals to the soil, beneficial bacterial inoculants supply ni-
trogen, phosphorous, potassium, and other plant nutrients to the crop, improving
plant development and increasing crop output (Basu et al., 2021;Singh &Gupta,
2018;Tiwari et al., 2018;Vimal et al., 2018). Additionally, IAA, gibberellins
(GA), and cytokinin synthesis and excretion have been shown to enhance root sur-
face area enabling greater uptake of soil nutrients by plants (Duca et al., 2014;Jangu
&Sindhu, 2011;Khan et al., 2016,2020).
2.1.1 Increase in the availability of nutrients
The growth of plants essentially depends on 16 different micro- and macronutrients,
and if any of these nutrients are deficient, the growth of the plant may be dysfunc-
tional or unbalanced. Different soil, climate, and agricultural plant characteristics
have an impact on nutrient availability. Better plant development and agricultural
output are produced as a result of soil bacteria maintaining the optimal concentration
of soil nutrients (Hirel et al., 2011;Kumar et al., 2021;Richardson et al., 2009). Uti-
lizing beneficial bacteria in the rhizosphere improves soil nutrient availability for
greater plant growth through the solubilization of zinc, potassium, and phosphate;
nitrogen fixation; and phytohormone synthesis (Sehrawat &Sindhu, 2019;Sharma
et al., 2019).
288 CHAPTER 13 Azospirillum—a free-living

The use of beneficial microorganisms as biofertilizers contributes to higher
nutrient levels in three ways: (i) by altering plant metabolism and the composition
of root exudates; (ii) by affecting the solubility and availability of nutrients; and
(iii) by enhancing interactions with other soil microbes. By secreting substances
including oxalate, gluconate, citrate, catechol, lactate, and pseudobactin, microbes
can mineralize nutrients through acidolysis, oxidoreduction, chelation, or other pro-
cesses. Arbuscular mycorrhizal fungi usually work in symbiosis with terrestrial
plants to boost the availability and uptake of water and minerals in exchange for
eating the carbon from the plant. According to studies (Etesami et al., 2021;Jiao
et al., 2021;Patel et al., 2021;Santoyo et al., 2021), the use of nutrient-
mobilizing microbial inoculants stimulates root and shoot growth, improves nutrient
uptake, and increases seed yield of various crops grown in pot houses and in fields
under various agro-environmental conditions.
Azospirillum brasiliense and Rhizobium tropici seed inoculation had a synergis-
tic effect and increased plant biomass, accumulated nitrogen, thousand-grain weight,
and grain yield of common bean (Phaseolus vulgaris L.) in a study conducted to
establish the role of PGPR in increasing nutrient availability and plant growth pro-
motion by Filipini et al. (2021).
2.1.2 Production of phytohormones
Some phytohormones, also known as plant growth regulators, are synthesized by
bacteria and plants in extremely low concentrations. These hormones affect physi-
ological processes such as cell division, development, gene expression, and stress
responses as well as root and shoot growth, shape, flowering, senescence, and
seed growth. Because phytohormones increase root surface area and root hair length,
plant roots are better able to absorb nutrients and water (Tsegaye et al., 2017).
Increased metabolic activity brought on by phytohormone production aids in de-
fense, normal cellular function, and abiotic stress management (Khan, Enshasy,
et al., 2020). During biotic and abiotic stressors, hormone-secreting microorganisms
that promote plant growth either produce hormones or change the concentration of
hormones within the plant.
According to Cassian and Diaz-Zorita (2016), phytohormones can be divided
into five classes: auxins, cytokinins, gibberellins, ethylene, and abscisic acid. Other
classes have also been found, such as strigolactones, jasmonates, and brassinoste-
roids, which serve as targets for metabolic engineering to create crop plants that
can survive abiotic stress.
On diverse crop plants, pathogenic bacteria, fungi, and viruses called plant path-
ogens cause a variety of diseases. These plant pathogen-caused infections reduce
crop output globally and are responsible for annual yield losses of 20%e40% in a
variety of cereal and legume crops (Oerke, 2006). For this reason, efforts are being
made to characterize antagonistic microorganisms for use as biopesticides for boost-
ing agricultural crop production (Santoyo et al., 2012;Anand et al., 2020;Jiao et al.,
2021). Inappropriate application of pesticides for disease control causes environ-
mental pollution leading to public health hazards. The creation and secretion of
2. Modes of action 289

siderophores, hydrolytic enzymes, antibiotics, volatile organic chemicals, hydrogen
cyanide, and establishment of systemic resistance are some of the strategies used to
control the diseases.
2.1.3 Production of siderophores
A lack of iron can result in improper respiration and photosynthesis because it is one
of the essential components involved in plant metabolism (Zuo &Zhang, 2011).
Aerobic settings contain iron as Fe
3þ
, which is a significant resource in soil.
When consumed by microorganisms and plants as Fe
þ2
form, Fe
3þ
rapidly produces
hydroxides and oxyhydroxides, rendering it inaccessible to them (Ghazy &El-
Nahrawy, 2021;Pahari &Mishra, 2017). Through the secretion of siderophores,
which are chelating substances with low molecular weight, ferrous is acquired. After
the siderophore and Fe
3þ
form a complex, the Fe
3þ
form is reduced to Fe
2þ
and
released into the cell (Kashyap et al., 2017). This readily absorbed Fe
2þ
is either
taken up directly as an iron-siderophore complex or is exchanged for another metal
via a ligand (Novo et al., 2018;Rasouli-Sadaghiani et al., 2014). Oxygen and nitro-
gen are electron-rich atoms that make up siderophores, which bind to cations (Chu
et al., 2010;Ghavami et al., 2017).
2.1.4 Synthesis of enzymes
Any organism’s metabolic activity is controlled by the activity of numerous en-
zymes. Structure-rich biomolecules in soil are depolymerized and mineralized by
extracellular enzymes released by bacteria, archaea, and fungi. These enzymes’ syn-
thesis and activity could be controlled to promote plant growth, carbon sequestra-
tion, and bioremediation (Burns et al., 2013). Ascorbate peroxidase (APX),
catalase (CAT), glutathione/thioredoxin peroxidase (GPX), and glutathione S-
transferase all contribute to the reduction of stress under abiotic stress circumstances
(Mittler et al., 2004;Nivetha et al., 2021;Wagner et al., 2002;Willekens et al.,
1995). It is also known that the hydrogen peroxidase enzyme serves as a signaling
molecule during biotic and abiotic stress, photosynthesis, and the cell cycle (Sofo
et al., 2015).
2.1.5 Producing antibiotics
The growth and maintenance of a variety of species, including commensals, pathogens,
and symbionts, depend on soil as a microbial pool (Mendes et al., 2013). Competition
for food and space grows as the number of organisms rises, which forces various mi-
crobial species to alter their survival and establishment tactics (Song et al., 2005;
Deman’eche et al., 2008;Philippot et al., 2010;Arora et al., 2013a). Antibiotic syn-
thesis is the most often used survival tactic in microbial competition (Jiao et al., 2021;
Sehrawat &Sindhu, 2019). Low molecular weight heterogenous chemicals known as
antibiotics are harmful to competing microbial strains (Duffy, 2003). Aldehydes,
ketones, alcohols, and sulfides are examples of volatile antibiotics. Nonvolatile
antibiotics include phenylpyrrole, cyclic lipopeptide amino polyols, and
heterocyclic nitrogenous compounds (Fernando et al., 2018;Gouda et al., 2017).
290 CHAPTER 13 Azospirillum—a free-living

Antibiotics may have antibacterial, antiviral, antioxidant, anticancer, antihelminthic,
phytotoxic, and/or cytotoxic properties. At low concentrations, they may also operate
as chemicals that encourage plant development (Kim, 2012). Microbes respond by
developing IAR (intrinsic antibiotic resistance) against the drugs; as a result, the
competing IAR-positive strain and the antibiotic-producing strain offer survival stra-
tegies. According to Glick et al. (2007) and van Loon (2007), antibiotics produced by
PGPR are also antagonistic compounds created against phytopathogens. Due to cell
membrane deformation, translational inhibition, arrest at the stage of ribosomal
RNA production, and suppression of cell wall synthesis, antibiotics prevent the growth
of harmful organisms (Maksimov et al., 2011).
2.1.6 Induced systemic resistance
Plants have defense mechanisms that include systemic acquired resistance (SAR)
and induced systemic resistance (ISR) in response to pathogenic attacks (SAR).
Two powerful responses, the ethylene and jasmonate pathways, called for the
signaling molecules involved, are used to defend against pathogen attacks on plants
(Pangesti et al., 2016). ISR signaling molecules include flagellar proteins, the
O-antigen side chain, chitin, pyoverdine, lipopeptide surfactants, and salicylic
acid (Doornbos et al., 2012). The secretion of phytohormones, PAMPs (pathogen-
associated molecular patterns), MAMPs (microbes-associated molecular patterns),
and the production of elicitor molecules, such as volatile organic compounds, side-
rophores, phytases, and miRNAs, are some of the different strategies used by
biocontrol agents to ensure ISR in plants.
3.Measurement/quantification
Depending on the N
2
-fixing system in question, the amounts of fixed nitrogen vary,
but put simply, free-living and associative N
2
-fixers would fix a lot less nitrogen than
symbiotic systems where host plants directly provide the microsymbiont with en-
ergy and shield the nitrogenase enzyme from being deactivated by oxygen.
Nonsymbiotic N
2
-fixation measuring techniques now in use are far from perfect,
and measurement may be inaccurate if improper methodologies or insufficient con-
trols are applied. Methodological issues are the most obvious weaknesses. The lim-
itations of this type of research include the low activities offered by free living
bacteria, the application uncertainties of the C
2
H
2
reduction assay of soils, the insen-
sitivity of the
15
N
2
-fixation method, the high variability of nitrogenase in soil cores,
and the complexity of such systems. However, when properly used, these techniques
might still offer some insightful information about the function and significance of
nonsymbiotic N
2
-fixation.
3.1 Assay for C
2
H
2
reduction
The C
2
H
2
reduction assay is an extremely sensitive assay that relies on nitrogenase,
which is generally responsible for biological N
2
-fixation, to reduce C
2
H
2
to C
2
H
4
.
3. Measurement/quantification 291

The only enzyme known to carry out this reduction is nitrogenase (Witty, 1979).
Hardy et al. (1968) conducted a thorough evaluation of the assay and discovered
a quantitative association between C
2
H
2
reduced to N
2
fixed of between three and
four and a direct correlation between N
2
-fixation (N
2
2NH
3
) and C
2
H
2
C
2
H
4
in
pure cultures of diazotrophs and in legumes. Witty (1979) also discovered that the
assay correctly predicted N
2
-fixation in pure cultures, but that in soils that were
either left alone or that contained pasture grasses, the amount of N
2
-fixation was
overestimated because C
2
H
2
hindered the oxidation of the natural C
2
H
4
created in
the soil. Nohrstedt (1983) proposed straightforward controls with low quantities
of C
2
H
2
, adequate to suppress natural C
2
H
4
production but insufficient for C
2
H
2
reduction.
The C
2
H
2
reduction test is a quick, easy, and inexpensive technique that, when
combined with the right controls and calibrations, can be used to assess nitrogenase
activity both in time and space. If the absolute values of N
2
-fixation are not crucial, it
can be quite helpful for comparative reasons under controlled circumstances.
3.2 Measurement of N
2
-fixation with
15
N
2
gas
Burris et al. (1943) published the first report on the use of
15
N
2
gas to confirm N
2
-
fixation. Its general applicability has historically been severely constrained by the
lack of materials rich in
15
N and mass spectrometers to examine the samples. But
this measurement technique has been around for a while. This technique can demon-
strate N
2
-fixation with certainty and has been used to show N
2
-fixation in soils
(Witty &Day, 1978;Roper, 1983;Azam et al., 1988), cereals, and grasses (Giller
et al., 1988;Witty &Day, 1978).
If the
15
N
2
gas is devoid of impurities like
15
NH
3
, the procedure can be sensitive
and precise. A procedure for creating “clean””
15
N
2
gas for use in such studies was
disclosed by Ergersen in 1980. The cost of
15
N
2
prevents its widespread use,
although it is a very helpful technique for calibrating other N
2
-fixation measure-
ments. Using the technique to gauge plant-related N
2
-fixation can be challenging
due to the inability to regulate ambient factors. Witty and Day (1978) created a com-
plex system for managing the environment around a developing plant while it was
exposed to
15
N
2
.
Using a chamber for the incubation of whole root systems and growth medium in
15
N
2
enriched gas mixtures, an experiment by Giller et al. (1984) was able to clearly
demonstrate that detectable levels of N were fixed by bacteria in growth medium.
They proved that within 3 days of the plant’s initial exposure to the gas, fixed N
had been integrated into its roots and shoots. After an additional week of growth,
the roots had higher
15
N enrichment, and the enrichment in the shoots had nearly
doubled.
It is significantly trickier to show that
15
N
2
is incorporated into free-living micro-
bial communities in the soil. Although the incorporation of
15
N
2
validated N
2
-fixa-
tion in soils in both the laboratory and the field, precise measurements of N
2
-fixation
in the field using
15
N
2
gas were not feasible. Numerous diazotrophic bacteria that are
292 CHAPTER 13 Azospirillum—a free-living

free-living and require low oxygen concentrations to fix nitrogen can be found in low
oxygen environments called microsites. Sites that limit O
2
availability may also
restrict access by
15
N
2
, which could lead to a reduction in the amount of N
2
-fixation.
3.3 Isotope
15
N dilution
A different method of employing
15
N
2
is to mark soil with
15
N in the form of salts,
such as urea or (15NH
4
)2SO
4
, and then watch as this
15
N is diluted with air
14
N from
BNF. Boddey et al. (1983),Chaulk (1985), and Boddey (1987) provide specifics on
the isotope dilution technique. In order to make the soil noticeably different from the
naturally occurring enrichment of the atmospheric N
2
, the isotope dilution technique
entails feeding the soil with a
15
N enriched or deficient, source of N. The ratios of N
derived from mineral sources in the soil and from the air (through fixation) can then
be computed.
The main precautions while using the procedure have to do with selecting the
nonfixing control, whose roots should investigate the same amount of soil as the
fixing plant and be closely related to it (Boddey et al., 1983). Chaulk (1985) notes
that if the isotope’s geographical and temporal availability is not consistent, the
fixing and reference plants might not acquire the same percentage of labeled and un-
labeled N from the soil.
The measurement of total N, which is necessary to compute the quantity of N
2
-
fixed, will be the biggest source of error, and measurements of other N cycle fluxes,
like N deposition, leaching, and denitrification, may be of the same order of magni-
tude as free-living N
2
-fixation. However, yield-independent measurement of the per-
centage of soil total N that has originated via N
2
-fixation is now possible thanks to
the capacity to monitor tiny changes in the
15
N isotope composition of soils (Unko-
vich &Baldock, 2008).
Estimating N
2
-fixation in soil-grown sugarcane, forage grasses, cereals, and acti-
norhizal plants has been done using the
15
N isotope dilution method, primarily in
tropical environments.
3.4 N-fixed using a different approach
Giller and Merckx (2003) stated that the best way to determine the role of nonsym-
biotic N
2
-fixation is to quantify net N inputs over extended periods of time in the
field (i.e., an N budget). Controlling and monitoring all the activities that contribute
to N in the soil can, however, provide major challenges. The amount of dirt utilized
might be a significant cause of mistake (Dart, 1986). The incorporation of fixed ni-
trogen into plant tissue or the soil is not directly measured by the total N balance
approach. The total of gains from diverse sources, including fixed and unmeasured
losses, constitutes any net growth. However, investigations have shown significant N
increases that can only be explained by N
2
-fixation inputs.
There aren’t many published N-balance studies for field-grown cereals or
grasses. Monitoring N inputs and outputs over an extended period of time is
3. Measurement/quantification 293

important to account for variability and inaccuracies caused by slight variations in
soil N relative to the total (Dart, 1986;Vallis, 1973). The reproducibility and accu-
racy of N readings must also be very good, which requires following stringent sam-
pling procedures and taking a huge number of samples.
From a scientific perspective, unless all inputs and outputs can be precisely
recorded throughout time, N balancing studies may not provide a direct estimate
of N gains due to nonsymbiotic N
2
-fixation. However, it does offer a net balance
of N accumulation, which is helpful information for growers when deciding how
much N fertilizer to apply to a crop.
The majority of data on estimations of N
2
-fixation using methods now in use
relate to a single instant in place and time. However, if environmental circumstances
in those locations are known, knowledge of the factors that encourage N
2
-fixation
and the rates at which fixation responds to changes in environmental conditions
can be utilized to produce estimates for a wider region (such as meteorological re-
cords, cropping statistics and soil maps). This theory was applied by Gupta et al.
(2006) to produce estimates for a portion of Australia’s southern agroecological
zones. A GIS spatial analysis tool was used to calculate the potential N
2
-fixation
in various zones utilizing data from earlier studies on the impacts of various soil
moistures, temperatures, and carbon sources. By applying this theory to a variety
of measurement techniques, it may be possible to learn more about the areas that
will benefit the most from nonsymbiotic N
2
-fixation and where new discoveries
can be made.
4.The nonsymbiotic N
2
-fixation-related factors
4.1 Oxygen
Iron (Fe) protein is quickly and irreversibly inactivated when exposed to air because
nitrogenase proteins are very sensitive to oxygen (O
2
)(Eady, 1996). Therefore,
before N
2
-fixation (nitrogenase activity) can take place, nonsymbiotic N
2
-fixing
bacteria require mechanisms to keep O
2
out of the cell. Azotobacter, Beijerinckia,
Derxia, and Azomonas are only a handful of the culturable bacteria that can fix N
aerobically (Havelka et al., 1982;Stewart, 1980). Large cells in the first two genera
have a quick growth rate, exclude O
2
through rapid respiration, and produce some
extracellular polysaccharides (Dalton, 1980;Postgate, 1971). The latter two genera
have enormous internal lipid bodies and create an abundance of slime (Dalton,
1980). Even while other growth processes take place under aerobic circumstances,
the remaining majority of culturable diazotrophic bacteria are much less skilled at
excluding O
2
and will only fix N in these conditions (Dalton, 1980).
The necessity to keep O
2
out might significantly limit the times and locations
where nonsymbiotic N
2
-fixation can take place. Soil saturation can produce anaer-
obic conditions. Under these circumstances, significant levels of N
2
-fixation have
been measured (Rice &Paul, 1972). But plant growth is not favored by anaerobic
conditions to promote O
2
transfer. Dryer soils require far more subtle systems.
294 CHAPTER 13 Azospirillum—a free-living

4.2 The presence of C
As an energy source, carbon is necessary. Free-living N
2
-fixing bacteria typically
feed on above- and below-ground plant matter from pastures and crops that is
degrading. Root exudates are used by associative N
2
-fixing bacteria in a rhizosphere
community with plants and other species. Other microbial communities are in fierce
competition with one another for scarce energy supplies in both settings. Contrarily,
endophytes can reach the plant’s internal nutrients (Wilson, 2006).
Crop residues’ main structural constituents are cellulose and hemicellulose. The
majority of N
2
-fixing bacteria require breakdown into smaller components first,
although certain species (Azospirillum spp.) can utilize straw directly for fixation
(Halsall et al., 1985). A wide variety of bacteria, fungi, protozoa, and microfauna
are among the organisms that participate in the breakdown of cellulolytic materials.
The breakdown products of cellulose, such as carbohydrates and certain organic
acids and alcohols, can be utilized by almost all diazotrophic heterotrophic bacteria
(Roper &Halsall, 1986). Rates of N
2
-fixation are proportional to available agricul-
tural residue and decomposition rates (Roper, 1983).
4.3 Aggregates of soil
The soils are not uniform. In the soil, interactions between plant-derived particulate
organic matter and enhanced labile organic fractions from soil microbes result in the
formation of different kinds of aggregates (Six et al., 2001). Undisturbed soils tend
to have more macro aggregates, and macro aggregates tend to have higher C con-
tents than microaggregates (Six et al., 2000). The dynamics of soil organic matter
are primarily governed by aggregate dynamics and their interactions with microbial
communities (Denef et al., 2001). Microaerobic and anaerobic conditions can
coexist alongside aerobic conditions in aerated soils leading to aggregate develop-
ment in the soil. Low O
2
concentrations in zones of microbial consumption are
caused by limitations of gas transport in soil aggregates and root tissues, resulting
in steep O
2
gradients (Angert et al., 2001). Gas diffusion restrictions notwith-
standing, substrates like dissolved organic C, can be divided into aerobic and anaer-
obic fractions and processes (Li et al., 2000). Therefore, it is conceivable for soluble
byproducts of aerobic organic matter decomposition to provide C energy to
aggregate-dwelling microaerobic and anaerobic N
2
-fixing bacteria.
4.4 Temperature
Nitrogen fixation has been demonstrated to occur in situations with extremely low
temperatures, such as deserts and Antarctica’s ice caps. Most N
2
-fixation in deserts
happens in the cooler morning hours using dew or after summer rains (Rychert et al.,
1978), but N
2
-fixing bacteria can survive in the hot, dry circumstances in between
times up to 60C(Jensen, 1981).
Temperature has a substantial impact on nitrogen fixation (Roper, 1983).
Although there appeared to be a shift in the temperature ranges for activity
4. The nonsymbiotic N
2
-fixation-related factors 295

consistent with climatic temperature ranges at each of the sites from which samples
were collected, laboratory studies revealed that the most favorable temperatures for
N
2
-fixation were between 30C and 35C, with a range from 4Cto45
C(Roper,
1985). The optimal temperature range for activity is between 25C and 37C, ac-
cording to several research conducted around the world (Jensen, 1981).
4.5 Moisture
Moisture is necessary for the growth and functioning of all microorganisms.
Reduced O
2
at the sites of fixation has been used to encourage N
2
-fixation in soils
under high soil moisture conditions (Rice &Paul, 1972). However, studies evalu-
ating the moisture needs for N
2
-fixation in soils from the same site, both in field
and in laboratory trials, underlined the significance of preserving aggregate structure
and O
2
gradients in unsaturated soils. While nitrogenase activity in undisturbed soils
in the field occurred at moistures below 30% field capacity, nitrogenase activity in
disturbed soils in the laboratory needed a minimum of 50% field capacity moisture
(Roper, 1985).
In some situations, cyanobacteria or other free-living bacteria in conjunction
with a fungus can contribute considerable amounts of biologically fixed N through
the growth of lichens, which are severe semi-arid habitats (Russow et al., 2005;
Rychert et al., 1978). The presence of rhizosheaths around the roots of several spe-
cies of perennial grasses is another important adaptation to dry settings. Sand grains
are used to make rhizosheaths, which are bonded to the root by substances like muci-
lage and other root exudates. Rhizosheaths sustain enhanced organic materials,
higher moisture contents, and a larger density of microorganisms, including associa-
tive diazotrophs, compared to the nearby sand (Othman et al., 2004). Although the
proportions of N
2
-fixed were minor, the grass plants are likely to be the main ben-
eficiaries because this activity was isolated within the rhizosheath and was not
affecting the bulk soil.
4.6 Supplemental nutrition and minerals
It is well known that combining N in soil can prevent nonsymbiotic microbes from
fixing N
2
(Knowles, 1980). However, C:N ratios have a significant impact on the dy-
namics of N
2
-fixing microbial communities. According to research by Kavadia et al.
(2007), when C was in sufficient supply, the microbial population assimilated extra
ammonium N for growth purposes, and N
2
-fixing bacteria were able to live and even
fix N
2
. In contrast, increased ammonium N concentrations hindered the N
2
-fixing
population under the same circumstances but with low C.
Crop residue degradation products are frequently utilized as a source of energy
for non-symbiotic N
2
-fixation. However, because to high C:N ratios, rates of break-
down can be slow (Roper &Ladha, 1995). When mineral N is added to soils altered
with agricultural residues that have a high C:N ratio, the rate of decomposition ac-
celerates, releasing C for utilization by N
2
-fixing bacteria (Barder &Crawford,
296 CHAPTER 13 Azospirillum—a free-living

1981;Sain &Broadbent, 1977). As a result, there must be a careful balance between
providing enough mineral N for efficient crop residue breakdown and avoiding hav-
ing too much extra mineral N that prevents N
2
-fixation.
4.7 Managing techniques
Macroaggregate stability is supported by no-tillage, but any increase in soil distur-
bance diminishes aggregation, lowers soil C, and disturbs the pore network that al-
lows soil organisms to communicate (Six et al., 2000,2001;Young &Ritz, 2000).
When compared to cultivated soil, no-till soil conserves more of its water content.
All of these contribute to the characteristically higher biological activity under
no-till than in cultivated soils (Young &Ritz, 2000). However, biological adapta-
tions to modified tillage techniques can be gradual, often taking years to materialize
(Cookson et al., 2008).
Using crop residues as an energy source, Roper (1985) evaluated N
2
-fixation by
free-living bacteria in field trials using various tillage techniques, including no-
tillage, scarification (lightly mixing soil and straw at the surface), burnt
and cultivated stubble, and stubble incorporated (disc ploughing). The incorporated
treatment, where there was good soil-straw contact and thus good microbe-straw
interaction, had the highest nitrogenase activity 1 year after the tillage treatments
were first imposed. But 5 years later, the less disruptive scarified technique was
preferred for N
2
-fixation. Activity in the scarified treatment was somewhat lower
than that under no-tillage, whereas activity in the “integrated” treatment was sub-
stantially lower. This implies that over time, each tillage treatment’s soil structure
and microbial function underwent changes. If the experiment had gone on, it is
possible that the no-tillage method would have worked best in the long run. This
was unquestionably the case in a field experiment carried out by Lamb et al. in
1987 at a site where tillage (no-till, stubble mulch, and plough) had been installed
12 years earlier. When compared to disturbed soils, the amount of N
2
-fixed was
considerably higher in no-till areas.
5.Plants and other creatures actualized nitrogen from
diazotrophs
The site at which N
2
-fixation takes place is expected to have a significant impact on
the transfer of N
2
-fixed nonsymbiotically to plants. Endophytic diazotrophs are
probably able to provide physiologically fixed N to the host directly (Sturz et al.,
2000). James et al. (2000) noted that there isn’t any proof that endophytic diazo-
trophs, as those in a rhizobium-legume symbiosis, directly transmit nitrogen to
plants. Endophytic diazotrophs have not been found in living host cells, only in inter-
cellular gaps, vascular tissue, aerenchyma, and dead cells (McCully, 2001). As a
result, it is likely that the death of these organisms and the release of fixed N to
the plant will be necessary for N transfer from them (James et al, 2000).
5. Plants and other creatures actualized nitrogen from diazotrophs 297

The main exception to this rule is ammonium-emitting diazotroph mutants, such as
endophytic Azospirillum in paranodulated cereals (Christiansen-Weniger, 1998;
Sriskandarojah et al., 1993), however it is improbable that these organisms could
survive and fix N in the field if used as inoculants. To evaluate the contributions
of N from endophytic diazotrophs, additional integrated interdisciplinary research
is essential (James et al, 2000). In order for N
2
-fixed by diazotrophs or N present
in nonfixing microbial biomass in the soil or rhizosphere to be transferred to plants,
these bacteria must typically die and release ammonium or amino acids. In this
manner, large amounts of fixed N can be liberated (Lethbridge &Davidson,
1983a;1983b). However, some diazotrophic bacteria, like Beijerinckia derx (Miya-
saka et al., 2003) and cyanobacteria, are able to excrete nitrogenous chemicals while
they are growing (Balachandar et al., 2004;Jones &Wilson, 1978).
The death of these bacteria and the subsequent release of ammonium or amino
acids are typically required for the transfer to plants of N
2
-fixed by diazotrophs or
N contained in nonfixing microbial biomass in the soil or rhizosphere. This allows
for the discharge of large amounts of fixed N (Lethbridge &Davidson, 1983a;
1983b). Beijerinckia derx (Miyasaka et al., 2003) and cyanobacteria are two exam-
ples of diazotrophic bacteria that can excrete nitrogenous compounds while growing
(Balachandar et al., 2004;Jones &Wilson, 1978).
6.Extending the utility of nonsymbiotic N
2
-fixation
6.1 Inoculation
An easy-to-use and cost-effective carrier material, such as an organic substance or a
synthesis of specific molecules, is used to hold one or more bacterial strains or spe-
cies in a diazotroph inoculant formulation. The inoculant is the vehicle used to
deliver bacteria from the manufacturing facility or laboratory to the living plant.
The formulation of the inoculant has a significant impact on the immunization pro-
cedure since it impacts the inoculant’s likelihood of effectiveness (Bashan, 1998).
The effective inoculation of plants by bacteria is always regarded as being
largely dependent on the colonization of the roots by helpful microbes (Suslow,
1982). The injection site for Azospirillum will probably influence whether it survives
and colonizes the roots or not. The bacteria are subjected to the physical forces and
interactions that naturally exist between soil bacteria and soil particles during this
time of establishment, including adsorption, encapsulation by clay minerals, and
wet and dry soil regimes.
6.2 Conserving consistent N (reducing N losses)
Even at low rates, the benefit of nonsymbiotic N
2
-fixation will be significantly
increased if soil N losses are decreased or eliminated. Numerous papers detail sig-
nificant losses of nitrogen (N) caused by leaching of mobile NO-3 ions below the
root zone, denitrification, and loss to the atmosphere as NO and N
2
O. Others need
to be developed; however, several strategies to decrease N losses from the soil are
already recognised.
298 CHAPTER 13 Azospirillum—a free-living

In addition to supporting nonsymbiotic N
2
-fixation, crop residues with high C:N
ratios can immobilize soil nitrogen for subsequent release and utilization by new
crops. Early studies by Allison and Klein (1962) showed that N was immobilized
by expanding populations of decomposer bacteria within the first 7 days after wheat
straw was added to soil. About 25 days later, nitrogen started to be released, but it did
so gradually over the course of 6e8 weeks. This method is more likely to help a new
crop and prevent N losses than a single massive N input.
7.Conclusion
Nonsymbiotic N
2
-fixation is a topic covered in a lot of literature, and a variety of
inputs are attributed to this source. Studies on nonsymbiotic N
2
-fixation in Gramina-
ceous plants have advanced significantly in a number of areas, according to a histor-
ical overview of those studies. Although the endophytic diazotrophic bacteria/plant
association exhibits several traits that are similar to the legume symbiosis, the
assumption that nitrogen fixation efficiency may be identical to rhizobia/legume
symbiosis was not realized.
In order to improve the accessibility of nutrients like N, P, K, Zn, and S, as well
as to modulate phytohormones, suppress plant diseases, and reduce abiotic stresses,
a variety of plant growth promoting microorganisms have been identified. Under
greenhouse and field circumstances, inoculations of a single beneficial microbe or
a group of them have been found to increase plant biomass and crop output. The
inoculation of multifunctional PGPR strains does not enhance plant growth, crop
production, or agri-produce quality in some circumstances, and a number of restric-
tions have been observed to limit crop development under field settings in a variety
of agricultural environments. Because bacteria develop in usually different ways in a
lab and a greenhouse, this has an impact on how well inoculated microorganisms
survive and function in the field.
Microbial function is greatly influenced by environmental and managerial con-
ditions, but some of this variability is probably due to methodological variations,
particularly the use of particular techniques. This review has made an effort to
emphasize the benefits of current approaches and how they should be used. Combi-
nations of techniques have proven particularly helpful for calculating absolute levels
of N
2
-fixed and confirming the accuracy of other measurements like the C
2
H
2
reduc-
tase assay and N balances. The use of expensive
15
N techniques in conjunction with
less expensive and more sensitive assays, such as the C
2
H
2
reduction test, is essential
to advancing our understanding of nonsymbiotic N
2
-fixation. Although there are still
some challenges in achieving this for free-living N
2
-fixing bacteria in the soil, veri-
fication of the C
2
H
2
reduction test using
15
N
2
should be done routinely to verify con-
version ratios of C
2
H
4
:N
2
. However, research indicates that, except in anaerobic
settings when conversion factors are significantly bigger, C
2
H
4
:N
2
ratios are often
1 to 2 times the theoretical range of 3e4, which can lead to significant overestima-
tions of N
2
-fixation. Our understanding of nonsymbiotic N
2
-fixation will grow as a
7. Conclusion 299

result of new molecular technologies, especially when paired with existing tech-
niques. In particular, microarray technologies give researchers the chance to look
at the diversity and operation of diazotrophic microbial communities at the same
time.
Designing farming systems that encourage N inputs from fixation can benefit
from a general grasp of the environmental parameters governing nonsymbiotic
N
2
-fixation. Over the past 20 years, farming techniques have shifted toward intense
cropping, no-tillage, and stubble retention. A crucial factor in nonsymbiotic N
2
-fix-
ation is higher C inputs, which are provided by these modified behaviors. Additional
factors that contribute to N
2
-fixation include decreased N mineralization over the
summer due to extensive nonlegume rotations and increased P availability. A
well-developed soil structure with big aggregates is encouraged by no-tillage tech-
niques’ lack of soil disturbance, which makes it possible for anaerobic/microaerobic
N
2
-fixation and aerobic cellulitic processes to coexist in close proximity. These pro-
cedures also preserve soil moisture and keep microbial interactions and activity
going. Under these new agricultural practices, it is crucial to reevaluate nonsymbi-
otic N
2
-fixation and consider alternate systems, including pasture cropping, which
could only need small amounts of N fertilizer because of gains from N
2
-fixation
and decreased N losses.
In order to maximize the benefits of nonsymbiotic N
2
-fixation, any techniques to
boost it should be combined with processes that decrease N losses from the soil. Un-
derstanding these relationships, especially the endophytic ones, could be aided by
defining the activity of genes found in nitrogen-fixing bacteria and knowledge
gained by the genome sequences of various plants of interest to growers.
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308 CHAPTER 13 Azospirillum—a free-living

Plant-microbe
interactions: different
perspectives in promoting
plant growth and health 14
Belur Satyan Kumudini, Sunita Mahadik, Amrisha Srivastava,
Savita Veeranagouda Patil
Department of Biotechnology, School of Sciences, JAIN (Deemed-to-be University), Bengaluru,
Karnataka, India
1.Introduction
Various anthropogenic and natural activities have led to the changes perceived in the
ecosystem. This has driven the disturbance in the natural likelihood of host-microbe
interactions (Sharma et al., 2022). With these climatic manifestations, the gross
yield obtained by the major crops has been reduced which is thereby unable to
meet the demands of the food. Hence, food security is the major concern associated
with climate change, increased population, and scarcity of cultivable land. Not only
do the plants circumvent climatic changes, but even the life underneath the ground is
also prone to these changes and poses a major threat to the growth of the plants, indi-
rectly influencing food security.
Plants recruit various bacterial microbiomes in the rhizosphere, phyllosphere,
endosphere, and surrounding soils that play a vital role in plant growth and health
(Afzal et al., 2019;Azeem et al., 2022;Dong et al., 2019;Kusale et al., 2021). A
symbiotic association between plants and microbes is important and advantageous
for proper microbial colonization (Nemeske
´ri et al., 2022;Vishwakarma et al.,
2020). This rhizosphere hotspot is embedded with multifarious, mutualistic, and
symbiotic microbes such as plant growth promoting rhizobacteria (PGPR), plant
growth promoting fungi (PGPF), arbuscular mycorrhizal fungi (AMF) influencing
the growth and enhancement of plants under stressed and nonstressed conditions.
The dynamic features of these rhizospheric microorganisms and their interaction
with the host plants relate to growth enhancement. Further in-depth information
regarding their properties is emphasized.
CHAPTER
309
Rhizobiome. https://doi.org/10.1016/B978-0-443-16030-1.00014-6
Copyright ©2023 Elsevier Inc. All rights reserved.

2.Plant-microbe interactions: a dynamic association
The rhizospheric area of soil surrounding the roots of plants is the most significant
center with utmost microbial activity. Most plant growth-promoting microbes rely
on root exudates for their continued existence as root exudates incorporate a range
of compounds including polysaccharides and proteins (Whipps, 1990). Several
fungal and bacterial species reside in the rhizosphere interacting with each other
and with plants which can be congenial or antagonistic (Saharan &Nehra, 2011;
Nadeem et al., 2013;Ding et al., 2019;Kumudini &Patil, 2021). These interactions
occur above as well as below the ground level and help in enhancing cultivation and
environment constancy (Bais et al., 2004). This plant-microbe consociation helps in
enhancing plant growth under normal as well as under stress conditions. Rhizobac-
teria and mycorrhizal fungi play an important role in supplying the essential mineral
nutrients required for the growth of plants in return for carbon necessary for their
survival.
Few studies (Belimov et al., 2005;Trivedi &Sa, 2008;Numan et al., 2018;SkZ
et al., 2018;Mahadik &Kumudini, 2020;Kumudini &Patil, 2021) have been re-
ported that show how microbes help enhance the growth and development of plants
under stress conditions like salinity, drought, pathogen, temperature, and heavy
metals. Bacillus subtilis helped in enhancing the rate of photosynthesis and less-
ening the level of reactive oxygen species (ROS) in chickpeas under salinity stress
conditions (Allah et al., 2018). Likewise, arbuscular mycorrhiza also play a signif-
icant role in enhancing the growth of plants under different stress conditions by
increasing the production of antioxidant system and osmoprotectants to provide
essential nutrients to the host plant (Habibzadeh, 2015;Quiroga et al., 2019;Kumu-
dini &Patil, 2021).
Microbes with pathogenic nature hamper the plant growth and development by
causing diseases. Interaction type may be congenial or antagonistic, which
completely depends on microbial species and their mode of action (Xiong &Fuhr-
mann, 1996;Pamp &Tolker-Nielsen, 2007;Vacheron et al., 2013). For example,
cyanide-producing bacterial strains obstruct the growth of plants, whereas,
phytohormone-producing bacterial strains enhance growth. In totality, plant growth
promoting microbes help in increasing the nutrient uptake efficiency of plants, help-
ing their plant growth and promotion (Table 14.1).
2.1 Plant-beneficial fungi
The mutual association between plants and fungi helps in the survival of both, as
fungi decay organic matter to provide nutrients for plant growth. On the other
hand, plants release amino acids, sugars, and organic acids, stimulating fungal colo-
nization in plant roots. Arbuscular mycorrhiza is the most prominent one and grows
in most of the crops residing in or operating on the roots of the plants (Bonfante &
Genre, 2010;Delavaux et al., 2019). This mycorrhiza not only protects crops from
harmful pathogens but also helps in the absorption of nutrients like nitrogen and
310 CHAPTER 14 Plant-microbe interactions: different perspectives

Table 14.1 Plant-microbe interactions on abiotic stress with different host systems.
Organism Host plant
Abiotic
stress Effect References
Bacillus cereus Black
mustard
Heavy metal
(chromium)
Improved plant growth and reduced chromium toxicity Akhtar et al.
(2021)
Bacillus cereus Rice Heavy metal
(cadmium)
Increased antioxidant enzyme activities, enhanced plant
growth, biomass production, photosynthetic pigments,
micronutrients, and lowered electrolytes leakage
Jan et al.
(2019)
Pseudomonas
fluorescence
Ragi Heavy metal
(chromium)
Enhanced enzymatic and nonenzymatic antioxidants Varsha &
Kumudini
(2016)
Bacillus sp.(12D6),
Enterobacter sp.(16i)
Wheat,
maize
Drought Significantly increased root branching, root length, root
surface area, and number
Jochum et al.
(2019)
Bacillus paramycoides
(DT-85), Bacillus
paranthracis (DT-97)
Wheat Drought Enhanced production of drought-combating molecules like
superoxide dismutase, peroxidase, catalase, and proline
Yadav et al.
(2022)
Trichoderma asperellum Wheat Drought Decreased in the proline, H
2
O
2
, and MDA contents. Increased
crop yield
Illescas et al.
(2022)
Trichoderma harzianum Rice Drought Significantly enhanced antioxidative defense, secondary
metabolites, and hormonal upregulation. photosynthetic and
antioxidative such as plastocyanin, a small chain of Rubisco,
PSI subunit Q, PSII subunit PSBY, osmoproteins, proline-rich
protein, aquaporins, stress-enhanced proteins, and
chaperonins
Bashyal et al.
(2021)
Trichoderma asperellum Sugarcane Drought Increased photosynthesis rate, stomatal conductance, and
water-use efficiency. Enhanced superoxide dismutase and
peroxidase enzyme activities, as well as the proline
concentration, root and stalk development, and sugar
portioning
Scudeletti
et al. (2021)
Continued
2. Plant-microbe interactions: a dynamic association 311

Table 14.1 Plant-microbe interactions on abiotic stress with different host systems.dcont’d
Organism Host plant
Abiotic
stress Effect References
Trichoderma parareesei,
T. harzianum
Rapeseed Salinity and
Drought
Increased the expression of genes related to the hormonal
pathways of abscisic acid (ABA) under drought stress, and
ethylene (ET) under salt stress.
Poveda (2020)
Pseudomonas
pseudoalcaligenes,
Bacillus subtilis
Soybean Salinity Significantly enhanced plant biomass, relative water content,
and osmolytes. Increased activities of defense-related systems
such as ion transport, antioxidant enzymes, proline, and MDA
content in shoots and roots
Yasmin et al.
(2020)
Trichoderma harzianum Common
bean
Salinity Soluble proteins, soluble carbohydrates, and amino acids
were increased in shoots and decreased in roots. Significantly
increased proline in both shoots and roots
Abd El-Baki
et al. (2022)
Brevibacterium linens
RS16
Rice Heat stress Decreased ethylene emission levels. Enhanced the expression
profiles of glutathione S-transferase. Decreased ROS
concentrations and thereby increased heat stress tolerance
Choi et al.
(2022)
Bacillus cereus SA1 Soybean Heat stress Increased the ascorbic acid, peroxidase, superoxide
dismutase, and glutathione contents. Decreased reactive
oxygen species generation, altered auxin, and ABA stimuli,
and enhanced potassium gradients
Khan et al.
(2020)
Trichoderma harzianum
(BHU P4)
Okra Heat stress Increased fresh weight, dry weight, chlorophyll content, and
nutrient content, as well as total phenolic content and SOD
Singh et al.
(2021)
Trichoderma koningii Tomato Heat stress Increase in the starch, protein, and total phenol content,
reduction in H
2
O
2
generation, and lignin deposition
Tripathi et al.
(2021)
Trichoderma asperellum Tomato Chilling and
drought
stress
Reduced most of the negative impacts of drought and cold
stress, with no effect on plant height or chlorophyll (a, b)
content
Cornejo-Rı
´os
et al. (2021)
Trichoderma harzianum Tomato Low
temperature
stress
Enhanced photosynthetic and growth rates, reduced lipid
peroxidation rate, and electrolyte leakage, also improved
expression of TAS14 and P5CS
Ghorbanpour
et al. (2018)
312 CHAPTER 14 Plant-microbe interactions: different perspectives

phosphorus and increases the water uptake capability. In return, they get carbon syn-
thesized by plants during the process of photosynthesis (Ziedan et al., 2011;van der
Heijden et al., 2017). Protease is produced by ectomycorrhizae which are respon-
sible for protein degradation, whereas endo-mycorrhizae help in the absorption of
nutrients from the soil (Howard et al., 2022).
Mycorrhiza in association with plants helps in the protection of ozone layer deple-
tion by reducing the emission of nitrous oxide from soil. The synergistic effect of plant
and fungi function positively for plants and negatively for pests. Previous studies by
Estrada et al. (2013) showed that the synergistic effect of Cucumis sativus and Col-
letotrichum tropicale lowered the foliage destruction caused by Atta colombica.
2.2 Plant-beneficial oomycetes
Oomycetes like Pythium oligandrum help in reducing the inflammation caused by
harmful microbes via mycoparasitism and antagonism or by incorporating a defense
mechanism (Yacoub et al., 2018). The formation of pathogen-related molecular pat-
terns P. oligandrum helps in the activation of jasmonic acid a growth-regulating sub-
stance, thus decreasing plant systemic resistance against harmful microbes
(Benhamou et al., 2012;Lazar et al., 2022;Wilson &McDowell 2022).
P. oligandrum during the first step of mycoparasitism transcripts the productivity of
encoding proteases, glucanases, cellulases, protease inhibitors, putative effectors, and
elicitors, after this attachment of antagonist to the host, occurs (Horner et al., 2012).
Hydrolytic enzyme deteriorates the host cell wall, therefore enabling entry to the
cell and helping in the growth and development by providing carbon. After the multi-
plication of antagonistic cells, P. oligandrum develops many papilla-like structures at
the entry point. As a result of this interaction, the cells of P. oligandrum canbeseenin
host hyphae. P. oligandrum helps in protecting plants from harmful pathogensby insti-
gating resistance against several microbial and biotic responses. Its presence in the rhi-
zospheric area of soil does not affect the native microflora but reduces the effect of
harmful microbes on plants. The mode of action of P. oligandrum protecting plant
roots in the rhizospheric area of soil from harmful microbes is the same as that of Tri-
choderma species. The synthesis of phytohormones and secondary metabolites by
favorable microbes is a well-known fact for enhancing plant growth. The beneficial
microbe P. oligandrum produces an ample amount of auxin and TNH
2
in the rhizo-
spheric area that enhances plant growth. Therefore, P. oligandrum can be considered
a plant growth promoting oomycete (Le Floch et al., 2003).
2.3 Plant-bacteria interactions
Many bacteria in the rhizospheric area of soil rely on root exudates for their exis-
tence. The interaction between plants and bacteria can be beneficial and fatalistic
and affects the growth and development of plants. The positive and negative effect
of these interactions depends on complex molecular signaling (Whipps, 1990;
Kumudini et al., 2018;Kumudini &Patil, 2021).
2. Plant-microbe interactions: a dynamic association 313

2.4 Plant beneficial bacteria
The growth and development of plants completely rely on the beneficial interaction
between plants and microbes. Among all plant growthpromoting bacteria, rhizobac-
teria are the most prominent ones that possess plant growth promoting characteris-
tics, like hormone production; solubilization of nutrients; nitrogen fixation;
production of siderophore, and enzymes like chitinase and ACC-deaminase. Pseu-
domonas,Bacillus,Serratia,Enterobacter,Erwinia,Beijerinckia,Klebsiella,Flavo-
bacterium,Burkholderia, and Gluconobacter are some significant genera to which
plant growth promoting rhizobacteria (PGPR) belong (Podile &Kishore 2006;Patil
et al., 2016;Varsha &Kumudini, 2016;Jayamohan et al., 2020;Mahadik &Kumu-
dini, 2020). The PGPR help in enhancing the growth and development of plants un-
der normal as well as under-stress conditions. With the help of their enzyme activity,
these bacteria help in increasing the nutrient uptake ability of plants. For example,
under salinity and drought stress situation, the level of ethylene rises, which nega-
tively affects the growth and development of plants, which is surpassed by the
ACC-deaminase activity of plant growth promoting rhizobacteria (Glick et al.,
2007;Jayamohan et al., 2020). Likewise, PGPR with the capability of producing
exopolysaccharides help plants living under water-restricted conditions (Gall
et al., 2021;Naseem &Bano, 2014;Sandhya et al., 2009). PGPR with phosphate
and potassium solubilizing activity play an important role in proliferating the acces-
sibility of these nutrients to plants (Archana et al., 2012; Boubekri et al., 2021; Pan-
hwar et al., 2014; Patil et al., 2016), along with the uptake of macronutrients like
calcium for the plant growth.
PGPR also help in protecting plants from harmful pathogens by either deterio-
rating their cell wall or limiting the need for some essential nutrients. Siderophore
production is helpful in binding iron, making it inaccessible to pathogens (Bhatta-
charyya &Jha, 2012;Kumudini et al., 2017;Patil et al., 2016). Enzymes like chiti-
nases, cellulases, glucanases, proteases, and lipases produced by bacteria play an
important role in lysing the cell wall of some detrimental fungi (Beneduzi et al.,
2012;Jayamohan et al., 2020;Patil et al., 2016). During the interaction between
plant and bacteria, the bacteria commence a reaction in the plant roots helping in
transfer of signals throughout the plant. This results in the activation of plant defense
mechanisms in case of pathogen attack and the production of antimicrobial phyto-
alexins (Jayamohan et al., 2020;Patil et al., 2020;Singh et al., 2022;Van loon,
2007).
3.Plant-microbe interactions in enhancing plant growth
and health
Various climatic and pedological challenges affect plant health and productivity. In
the soil, plant-microbe interactions play a pivotal role in several vital ecosystem pro-
cesses such as carbon sequestration and nutrient cycling. However, its composition
314 CHAPTER 14 Plant-microbe interactions: different perspectives

and diversity vary across soil horizons (Luo et al., 2021). Beneficial microbes asso-
ciated with plants are plant growth promoting microorganisms (PGPM) that can help
plants to maintain plant growth under different environmental stress conditions (Ete-
sami &Beattie, 2017). Rhizosphere microorganisms have the closest relationship
with crops, which can selectively harbor specific beneficial microorganisms
(PGPM) that promote the transformation of plant nutrients and facilitate better plant
health, growth, and development (Lopes et al., 2021). Previous literature showed
that rhizosphere microorganisms offer more beneficial services through their inter-
actions with the host plants resulting in overall plant yield enhancement.
Beneficial plant-microbe interactions can influence soil fertility and plant growth
by providing essential nutrients and stimulating the growth and development of host
plants under biotic and abiotic stress (Kumar &Verma, 2018). Plant-associated mi-
croorganisms facilitate plant growth and health through direct and indirect mecha-
nisms. Direct plant growth promotion can be achieved by the interaction between
the host plant and beneficial microbes. Various microbial genera have been reported
to enhance plant survival and growth under different environmental stresses
(Fig. 14.1). The direct mechanisms include the biosynthesis of phytohormones.
For example, indole-3 acetic acid (IAA) (Duca &Glick, 2020), cytokinins, and gib-
berellins (Pavlu et al., 2018), 1-aminocyclopropane-1-carboxylate (ACC) deaminase
enzyme (Gupta &Pandey, 2019;Sagar et al., 2020;Singh et al., 2022), phosphate
and potassium solubilization as well as atmospheric nitrogen fixation (Batista
et al., 2018; Khatoon et al., 2020), siderophore production (Nithyapriya et al.,
2021;Saraf et al., 2017), minerals uptake improvement (Sarita et al., 2021), biofilm
formation for protection from external stresses (Nasab et al., 2022), production of
HCN (Patil et al., 2016), and exopolysaccharide quorum sensing (Mushtaq et al.,
2021). On the other hand, the indirect mechanism is the potential of microbes to sup-
press the deleterious effect of plant pathogens and mitigate biotic and abiotic
FIGURE 14.1
Plant growth enhancement under stress conditions by rhizomicrobiome.
3. Plant-microbe interactions in enhancing plant growth and health 315

stresses (Khatoon et al., 2020). It includes abiotic and biotic stress management,
induced systemic resistance (Patil &Kumudini, 2019), production of hydrolytic en-
zymes (b-1,3-glucanase and chitinase), competition for nutrients in the rhizosphere,
and the production of secondary metabolites (Zin &Badaluddin, 2020).
These beneficial microorganisms can enhance plant growth under stress conditions
by the production of low molecular weight nonenzymatic antioxidants (a-tocopherol,
ascorbic acid, phenols, flavonoids); enzymatic antioxidants such as superoxide
dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase
(APX); osmolytes (proline, glycine betaine, and organic acids), as well as
pathogenesis-related proteins (Hasanuzzaman et al., 2020;Nanda et al., 2019).
3.1 Phytohormone production
Phytohormones are plant growth regulators produced by plants for proper growth
and productivity. They regulate various biochemical and physiological processes
in plants. The important phytohormones including auxins (IAA, IBA), gibberellins,
abscisic acid, ethylene, and cytokinins function during cell division and cell enlarge-
ment, seed germination, root formation, and stem elongation, resulting in improved
plant health and growth under abiotic stress conditions (Fahad et al., 2015;Sharma
&Kaur, 2017). Shahzad et al. (2016) reported that inoculation with ABA-producing
Bacillus amyloliquefaciens RWL-1 under normal and saline conditions significantly
increased root and shoot growth and the concentration of salicylic acid in rice under
salinity stress conditions. Studies by Mahadik and Kumudini (2020) revealed signif-
icant beneficial effects on finger millet plants treated with fluorescent pseudomo-
nads. The PGP attributes of fluorescent pseudomonads such as IAA and
siderophore production along with ACC deaminase activity enhanced enzymatic
and nonenzymatic antioxidants that helped in the enhancement of salinity stress
tolerance in these plants. The research findings by Khan et al. (2020) suggested
that the production of plant hormones such as IAA and GA in Bacillus cereus
(SA1) increased the ascorbic acid peroxidase, superoxide dismutase, glutathione
as well as amino acid contents in soybean plants to ameliorate the effect of high tem-
peratures, so it could be used as a thermotolerant bacterium for the mitigation of heat
stress damage in soybean plants. Uzma et al. (2022) reported that IAA-producing
Pseudomonas aeruginosa induced drought tolerance and growth promotion in
mung bean plants. It improved photosynthetic activity, membrane stability, water
content, and antioxidant efficacy by inoculation with P. aeruginosa. Therefore, these
IAA-producing strains have the potential for plant growth promotion in areas facing
water deficiency.
3.2 ACC deaminase activity
1-Aminocyclopropane-1-carboxylate (ACC) is a precursor of the phytohormone
ethylene, helpful in improving crop productivity under stress, by regulating the
growth, development, and stress response of plants (Orozco-Mosqueda et al., 2020).
316 CHAPTER 14 Plant-microbe interactions: different perspectives

Several researchers (Jamil et al., 2018;Nadeem et al., 2017) have evaluated the effec-
tiveness of using Pseudomonas fluorescens strain under water scarcity possessing
ACC-deaminase activity in combination with L-tryptophan in wheat crops and combi-
nation with compost and biochar in cucumber, respectively, to reduce the effect of
drought stress. The studies by Gupta et al. (2021) revealed that Bacillus marisflavi
and Bacillus cereus having ACC deaminase activity increased plant-biomass, carbohy-
drates, reducing sugars, protein, chlorophylls, and phenolic content in rice to protect
the plant health under salinity stress, A recent study by Singh et al. (2022) demon-
strated that inoculation of wheat plants with Enterobacter cloacae strain ZNP-4 signif-
icantly improved the growth parameters such as shoot and root length, fresh and dry
weight, chlorophyll a and chlorophyll b. It also improved the proline content and
decreased malondialdehyde (MDA) levels under salinity (NaCl) and heavy metal
(Zn) stress.
3.3 Phosphate (P) solubilization
Phosphorus (P) is the most essential nutrient that plays a vital role in the nutrition of
plants next to nitrogen (N). It plays an important role in various metabolic processes
such as photosynthesis and respiration, flowering and seed setting, nitrogen fixation
in legumes, energy transfer, and signal transduction (Dakshayini et al., 2020;Kumar
et al., 2018). Studies by Tandon et al. (2019) reported that Trichoderma koningiopsis
(NBRI-PR5) produced organic acids for solubilizing insoluble tri-calcium phos-
phate (TCP) at high pH stress, whereas, under drought conditions, it accumulated
polyphosphate in its mycelia and produced an alkaline phosphatase enzyme. The
microscopic observations revealed an accumulation of P in the form of polyphos-
phate granules, which could be the source for soluble P released as orthophosphate
through enzymatic activity, and therefore, it can be used in the management of
stressed soils. Studies by Kour et al. (2020a) suggested that Acinetobacter calcoace-
ticus EU-LRNA-72 and Penicillium sp. EU-FTF-6 efficiently mitigated the adverse
effects of drought in foxtail millet by enhancing the accumulation of glycine betaine,
proline, and sugars, and decreasing lipid peroxidation. Another report by Kour et al.
(2020b) showed that P-solubilizing isolates Streptomyces laurentii EU-LWT3-69
and Penicillium sp. EU-DSF-10 were found to be efficient in terms of enhancing
the accumulation of different osmolytes such as glycine betaine, proline, sugars,
increased chlorophyll content, and decreasing lipid peroxidation in great millet (Sor-
ghum bicolor) in vitro under greenhouse.
3.4 Siderophore production
Siderophores or biochelators are low molecular weight compounds that have a
strong attraction for iron, an essential nutrient for plant growth. It chelates ferric
ions secreted by many fungal and bacterial species that grow under normal or
iron stress. Iron is involved in biological processes such as photosynthesis, respira-
tion, and chlorophyll production; therefore, it is an important element involved in the
3. Plant-microbe interactions in enhancing plant growth and health 317

life of plants. Siderophore-producing microbes reduce iron deficiency and enhance
all physiological and biochemical processes such as photosynthesis, respiration, and
chlorophyll production of the plant under drought conditions (Kumar et al., 2016),
saline soil (Sultana et al., 2021), and heavy metal-stressed soil (Hofmann et al.,
2021). Siderophore-producing microorganisms have the potential to produce PGP
attributes such as plant hormones (IAA, GA, ABA), phosphate solubilization ability,
and secondary metabolites that enhance plant growth under stressed conditions
(Breitkreuz et al., 2021).
The plant inoculation study by Sultana et al. (2020) revealed significant benefi-
cial effects on the inoculation of siderophore-producing bacterial strains Bacillus
tequilensis and Bacillus aryabhattai strains that showed enhanced photosynthesis,
transpiration, and stomatal conductance of the coastal rice plants that resulted in a
higher yield. The Bacillus tequilensis and Bacillus aryabhattai strains showed
good potential as PGPR for salinity mitigation practice for coastal rice cultivation.
Lastochkina et al. (2020) reported that siderophore-producing B. subtilis enhanced
the nutrient level in soil resulting in the growth of wheat plants under drought con-
ditions. Similarly, Pseudomonas strains having siderophore-producing ability
enhanced the soil nutrients as well as phosphate and potassium solubilization under
drought conditions (Breitkreuz et al., 2021). Studies by Vishnupradeep et al. (2022)
denoted that inoculation with two siderophore-producing bacterial strains Providen-
cia sp. (TCR05) and Proteus mirabilis (TCR20) enhanced the plant growth, pig-
ments, proteins, phenolics, and relative water content and decreased the lipid
peroxidation, proline, and superoxide dismutase activity in maize plants under chro-
mium (heavy metal- contaminated and drought conditions. They reported that these
strains could be bioinoculants for improving plant growth and phytostabilization
practices in chromium-contaminated sites even under drought conditions. Oubohs-
saine et al. (2022) reported that inoculation with siderophore-producing Rhodococ-
cus qingshengii strain LMR340 in Sulla spinosissima enhanced plant biomass,
chlorophyll, and carotenoid content and antioxidant enzyme activities. This bacterial
inoculant restored plant growth under harsh conditions of different heavy metal (Zn,
Pb, and As) stress.
4.Perspectives on plant productivity in a different scenario
Today’s agriculture has been adversely affected all around the globe by different
abiotic stresses such as salinity, drought, water logging, heavy metal deposition,
nutrient depletion, and temperature, which are major hindrances affecting plant
growth and agricultural productivity (Kannepalli et al., 2020;Kapadia et al., 2021).
To overcome the detrimental effects of climate change and abiotic stresses, on plants,
there is growing interest in the application of ecofriendly and effective plant growthe
promoting microorganisms for strengthening plant defenses against abiotic stresses.
Several studies reported that rhizosphere microorganisms play a substantial role in
mitigating abiotic stress such as salinity (Mahadik &Kumudini, 2020), extreme
318 CHAPTER 14 Plant-microbe interactions: different perspectives

temperatures (Khan et al., 2020), drought (Nemeske
´ri et al., 2022), and heavy metal
remediation (Oubohssaine et al., 2022; Vishnupradeep et al., 2022).
To date, a large number of PGPR taxa including Pseudomonas (Mahadik &
Kumudini, 2020), Bacillus (Azeem et al., 2022), Serratia (Khan &Singh, 2021)
Enterobacter (Singh et al., 2022), Klebsiella (Kusale et al., 2021), Azotobacter,Bur-
kholderia (Yang et al., 2020), and Rhizobium (Irshad et al., 2021) that possess multi-
farious traits have been isolated and characterized to ameliorate abiotic stresses in
different plants (Ullah et al., 2021). Similarly, treatment with PGPF particularly
of the genus Aspergillus,Trichoderma,Penicillium,Alternaria,Fusarium, and
Phoma has been reported to enhance plant growth under abiotic stress conditions
in different crops such as maize (Kumar et al., 2016), wheat (Zhang et al., 2019),
barley (Gupta et al., 2021), rice (Anshu et al., 2022).
5.Future prospects
With the current prospective of the plant microbe interactions in maintaining plant
health and growth, it is also important to understand the underneath mechanism
involved. This knowledge will envisage more insights for the sustainable use of
these microbes in agriculture. The host and nonhost rhizospheric microorganism in-
teractions also have to be harnessed for further gain of the evidence on usage of these
microbes at large scale as an application. It is also important to draw an uniformity
on screening prior to the formulations.
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328 CHAPTER 14 Plant-microbe interactions: different perspectives

Recent advances in
discovery of new drugs
from plants-associated
microbes 15
Sharav Desai, Vipul Patel, Neha Hajare
Sanjivani College of Pharmaceutical Education and Research, Kopargaon, Maharashtra, India
1.Introduction
One of mankind’s greatest achievements is the development of medications for the
treatment of infectious diseases. Every higher organism, including plants, contains
microbes on its surface or within. Most of viruses, bacteria, fungi, nematodes, and
oomycetes found in plants have a significant impact on plant health. While some
microorganisms cause disease, others protect it or help plants to develop. More
or less severe illness is the outcome of interactions between microbes on or in
plants. The variety of organisms that interact with plants within each group of mi-
crobes is enormous. For instance, it is estimated that more than 10,000 pathogenic
species of fungus and viruses infect plants and many of these are crucial for the
economy (Smulson &Suhadolnik, 1967). Since long back, plants have been uti-
lized as medicines for various types of illnesses all over the world to the bio-
prospecting of secondary metabolites produced by those plants (Egamberdieva
et al., 2017).
Around 80% of people, especially in developing nations, rely on herbal medi-
cines for their main healthcare (Chen et al., 2016). These herbal medications are
more affordable and significantly safer than contemporary synthetic drugs. Today’s
medications, which make up more than half of the market, are made from these
natural materials. Attention has now shifted to bacterial endophytes, which are
located intracellularly and/or intercellularly in medicinal plants, proving to be a
promising source of new drug discovery. However, due to the widespread use of
medicinal plants for the development of new drugs, many medicinal plants are
endangered.
Endophytes and rhizosphere bacteria, fungi, and other plant-associated microor-
ganisms are important and largely untapped natural products with chemical struc-
tures that have been chosen by evolution for their biological and ecological
utility. These bacteria have been found to produce a diverse range of bioactive small
molecules. With details on their hosts, biological activity and, culture conditions,
many clinically significant compounds that were isolated and described from
CHAPTER
329
Rhizobiome. https://doi.org/10.1016/B978-0-443-16030-1.00009-2
Copyright ©2023 Elsevier Inc. All rights reserved.

more than 70 plant-associated microbial strains during the route of the previous
5 years are provided. According to research reported, plant tissues are a rich source
of natural molecules for medicinal and biotechnological interests. Most of these sub-
stances are formed by microorganisms, so-called endophytes, that coexist in the im-
mediate vicinity of the host plant without harming it (Joseph &Priya, 2011).
The microbiome of the rhizosphere can also have significant impact on the
health, nutrition, and growth of plants in natural environment. Microbial commu-
nities such as bacteria, viruses, fungi, oomycetes, archaea, and arbuscular mycor-
rhizae are attracted to and nourished by nutrients, peripheral border cell, exudates,
and mucilage released by plant root (Philippot et al., 2013). Actinomycetes and
fungi have historically been discovered to be the most active makers of secondary
metabolites among all examined microorganisms.
According to various research, fungi are crucial for every terrestrial ecosystem’s
sustainability and biodiversity as well as its health and prosperity. The deep and
pervasive relationships between the fungi and plant life cycles have long been recog-
nized by evolutionary biologists. There is a lot of circumstantial evidence to support
the idea that symbiosis between fungi and plants helped plants invade early hard
terrestrial ecosystem that were devoid of nutrients, constantly desiccating, and inad-
equately sheltered, exposing these habitats to the sun’s intense light. The funda-
mental idea that symbiotic relationships between arbuscular mycorrhizal (AM)
fungi and the roots of higher plants were crucial in the migration of plants to land
is still widely accepted, even though recent developments in fungal systematic
have rendered some of the evolutionary hypotheses that led to the aforementioned
assertion obsolete (Blackwell, 2000).
Numerous pharmaceutical companies have discovered several new bioactive
compounds as a result of the research. The bioactive substances are naturally pro-
duced by several fungi and bacterial species, but Actinobacteria, and in particular,
Actinomycetes, are the most interesting group of microorganisms capable of pro-
ducing these secondary metabolites. Due to their capacity to manufacture various
kinds of medications in terms of chemical structure and methods of action, this order
is significant. According to recent study, Actinomycetes are also a valuable source
for discovering new natural products because of their special enzyme sets, which
enable the production of molecules that may be helpful for a variety of applications
(Lo Grasso et al., 2016). As a result of the diversity in their biological and metabolic
evolution, microbes have the capacity to utilize a variety of solid substrates,
including live plants. Many plants have been reported to create cooperative relation-
ships with both bacteria and fungi.
This chapter’s goal is to analyze the distribution and biological properties of
substances produced naturally by microbes that coexist closely with plants. Mi-
croorganisms from the rhizosphere and endophytes are among them. This chapter
includes brief descriptions of the distribution, biological activities, relevance,
and implications of the presence of natural products of plant-associated mi-
crobes. It covers the literature covered by Chemical Abstracts up to November
2021.
330 CHAPTER 15 Recent advances in discovery of new drugs

1.1 Actinobacteria
The actinobacteria is one of the largest groups within the bacteria domain (Ludwig
et al., 2012). The actinobacterial genomes are important for biotechnology, ecology,
and for both human and veterinary medicine (Ventura et al., 2007). Most actinobac-
teria are independent, free-living organisms that can be found in both aquatic and
terrestrial environments (Macagnan et al., 2006). Actinobacteria have high
guanine-plus-cytosine in their genomes. They grow by extension and branching of
the hyphae. The Greek words for ray and muks are what gave them their name.
The actinomycetes were considered transitional between the two. A significant num-
ber of actinomycetes produce mycelium in order to reproduce by sporulation. The
cells of actinomycetes are thin and susceptible to antibacterial agents, but the com-
parison is superficial. They can be either heterotrophic or chemoheterotrophic, but
most are able to use a wide variety of nutrition sources. Actinobacteria can be inhab-
itants of soil or aquatic environments (Bugg et al., 2011;Lechevalier &Lechevalier,
1965). Most of actinobacteria’s life cycles are lived as semidormant spores, and
they’re soil-dwelling saprophytes (Khan &Williams, 1975). Actinomycetes can
be found in soils, fresh and salt water, and the air. In soils rich in organic matter
and alkaline soils, they are more abundant than other media. Actinobacteria can
be found on the soil surface and at depths of more than 2 m below ground (Barka
et al., 2016). Ninety percent of the commercialized antibiotics used for clinical or
research purposes were produced using more than 5000 substances reported from
actinobacteria (Table 15.1)(Jose &Jha, 2016). Members of the Actinobacteriaceae
family create polyketides and nonribosomal polyketide peptides, which are the main
routes for the production of secondary metabolites in the family (Passari et al.,
2015). Streptomyces is the largest group of actinobacteria with over 800 species.
The species of this genus are the most significant sources of natural compounds
with a past record of producing novel bioactive molecules, including with several
commercially significant pharmaceuticals including ivermectin, tetracycline, strep-
tomycin, and nystatin (Miao &Davies, 2010). Several compounds were character-
ized from Streptomyces spp. over the past decade (Das et al., 2018). Many
research groups are trying to find novel actinobacterial strains that can be used to
find new drugs against diseases (Das et al., 2018).
In the article titled “Actinobacteria in natural product research: progress and
prospects” authored by Polpass Arul Jose et al. has explored more than 500 naturally
produced bioactive substances which come from actinobacterial origin (Jose et al.,
2021).
1.2 Algae
Algae are generally polyphyletic in nature, and consist of one maybe more cells linked
together in colonies that have very little in common with each other (Egamberdieva
et al., 2017;Montasser et al., 2016;Torres et al., 2014). Natural products from algae
have been used as food and medical treatments for a long time. Because of their
1. Introduction 331

abundant supply of fiber, minerals, antioxidants, vitamins, steroids, even lectins, algae
are a useful source of many compounds and products that are utilized extensively and
commercially (‘Feeding Common Carp Fish (Cyprinus Carpio) on Natural Foods
(Algae, Phytoplankton, Zooplankton and Others) on Tigris River in Mosul Dam/
Duhok, Kurdistan Region of Iraq’, 2016; Jose &Kurup, 2016;P&J, 2016;Perez,
2016). Algae included the diverse range of chemical elements and biologically active
nutraceuticals. These items have such a high market value currently. Currently
commercially marketed products include carotenoids, phycobilins, fatty acids, poly-
saccharides, vitamins, and physiologically active compounds for use in both human
and animal health (Merida &Zepka, 2015;El-Sharony et al., 2015). The international
market for pharmaceuticals is growing fast. Within India, pharmaceutical companies
currently account for 70%e80% in the market and their sector share is improving
each year. Since the start of time, algae have the ability to be beneficial to people,
and there is presently a great deal of interest in using cyanobacteria (blueegreen
algae) to make antibiotics and other pharmacologically active chemicals. Algal items
comprise antimicrobials, antivirals, therapeutic proteins, drugs, antifungals, and many
other items (Ariyanti, 2012;Budiyono &Kusworo, 2012;El-Sheekh et al., 2012;
Sulaymon, 2013;Parab &Tomar, 2012;Silva AMS, 2013). Antioxidants help to
Table 15.1 Biologically active compounds from Actinobacteria.
Targets Class Compounds
Ribosomes 30s Antimicrobial compounds Erythromycin
Chloramphenicol
Ribosome 50s Antimicrobial compounds Gentamycin
Kanamycin
Neomycin
Streptomycin
Tetracycline
Cell membrane and cell wall Antimicrobial compounds Vancomycin
Daptomycin
Platensimycin
RNA synthesis Antimicrobial compounds Rifamycin
DNA Anticancer substances Bleomycin
Actinomycin D
Anthracyclines
Mitomycin
Immunoglobulins Immuno suppressive compounds Rapamycin
Tacrolimus
Neurons and
neurotransmitters
Antiparasitic/Insecticidal
compounds
Avermectins
Milbemycins
Spinosyns
Amino acid synthesis Herbicidal compounds Bialaphos
Glufosinate
332 CHAPTER 15 Recent advances in discovery of new drugs

prevent premalignant lesions start emerging, which delays the cancer’s progression.
Numerous algae species, per a study, have helped to reduce oxidative injury by scav-
enging free radicals and active oxygen, which limits the growth of cancer (Ariyanti,
2012;Bisen, 2016;El-Sheekh et al., 2012;Ichihara et al., 2016;Pastorino &Bregni,
2016). Oral cancer can be cured with algae that have antioxidant properties such as
carotenes. Algal floral compounds are also used in cancer treatments (Mahmoud
et al., 2016;Bisen, 2016;El-Sheekh et al., 2012;Ichihara et al., 2016;Pastorino &
Bregni, 2016). Prior to now, freshwater and seawater were thought to be the biological
habitats of algae. Because algae could not be removed from the rhizosphere, phyllo-
sphere, or endosphere, it has been debatable whether algae should be included in the
plant microbiome (phytobiome) (GANTAR et al., 1991;Gantar &Elhai, 1999;Treves
et al., 2016;Zhu et al., 2018). Additionally, biochemical and molecular investigations
have not been used extensively to assess the role of algae in plant fitness. Algae have
just recently been shown to belong to the phytobiome, according to research. Chlor-
ella species, for example, has been discovered in the soil and on plant leaf surfaces,
and Nostoc and Anabaena spp. have been detected on the root surfaces of plants (Gan-
tar &Elhai, 1999;Liu &Chen, 2016;Spiller et al., 1993;Treves et al., 2016;Zhu
et al., 2018).
1.3 Endophytes
Endophytes belong to a group of organisms that spend at least some of their lives in
plant tissues without damaging their hosts (Wilson, 1995). De Bary coined the word
“endophyte” to describe these kinds of microbes (Bary, 2011). Plants, weeds, and
ornamental and fruit trees from wild and domesticated setting have been reported
to have endophytes (Ting et al., 2009). As per Germida et al. the population of en-
dophytes is considered to be a subset of the rhizospheric microbial population (Ger-
mida et al., 1998). They enter plants primarily through the roots and the aerial parts
of the plant, such as leaves, flowers, stems, and cotyledons. Natural products from
endophytic microbes can be used in a wide range of industries. They can spread
in the whole plant body from the point of entry. They reside within cells or intracel-
lular spaces after entering the host. After residing in plant tissues, endophytes are
reported to create a variety of natural compounds that could serve as efficient and
fruitful sources of medications (Fig. 15.1)(Christina et al., 2013;Germida et al.,
1998;Jacobs et al., 1985;Patriquin &Do
¨bereiner, 1978). The pharmaceutical sector,
as well as other industries, has a huge potential regarding natural products made
from microorganisms.
Endophyte-derived natural products have been found to have antibacterial, anti-
viral, anticancer, and antidiabetic effects. An unexplored and underused endophytic
bacterium is responsible for the production of antibiotics. The potential source of
novel antibiotics is believed to be the endophytic bacteria. The source of most of
the antibiotics has been the soil bacteria. The endophytic bacteria seem to be a prom-
ising alternative source of antibiotics (Angela, Meireles et al., 2008;Apgar et al.,
2021;Colombo &Ammirati, 2011;Guo et al., 2000;Zhang et al., 1999).
1. Introduction 333

Obligate and facultative are the two main types of endophytic bacteria (Baldani
et al., 1997). Obligate endophytes are able to exist inside plant tissues for the whole
of entire lives whereas facultative endophytes can remain in the soil, on the top of
plants, within plants, and on synthetic nutrients. Facultative endophytic bacteria
are extensively found throughout the plant world and can be isolated from other
plant species to evaluate their potential for producing naturally occurring goods
with substantial economic value (James, 2000). From a wide range of plants,
many species of cultivable endophytic bacteria, both in Gram-positive and Gram-
negative strains, have already been isolated, characterized, and reported (Bhore &
Sathisha, 2010;Sturz &Christie, 1996). The host plant offers no advantages to
the Endophytic bacteria. It’s crucial to remember that Endophytic bacteria have
an extremely low population density. In a variety of ecological and environmental
situations, endophytes are known to promote the growth and development of host
plants (Dudeja et al., 2012;Long et al., 2008). According to Bhore et al. the endo-
phytic bacteria are also known to boost host plants’ resistance to diseases and to
encourage biological nitrogen fixation (Waryono, 2019). However, in the pharma-
ceutical business, the capacity of endophytic bacteria to produce a range of natural
compounds is crucial (Strobel and Daisy, 2003). Many research teams around the
world are working in screening endophytes for different natural products and to
learn more about endophytic bacteria (bioactive compounds). It has been demon-
strated that some endophytic bacteria create organic substances like phytohormones,
low molecular weight chemicals, enzymes, siderophores, and antibiotics (Table 15.2)
(Frommel et al., 1991;Glick et al., 1998).
FIGURE 15.1
Biologically active products form endophytes.
334 CHAPTER 15 Recent advances in discovery of new drugs

Table 15.2 Compounds with biological activity from endophytes.
Compounds Activity References
Ecomycin In fungal infection Miller et al. (2004)
Bacilysocin In fungal infection Tamehiro et al. (2002)
Nystatin In fungal infection Fjærvik &Zotchev (2005)
KB425796-A In fungal infection Kai et al. (2013)
Bacillomycin In fungal infection, in lysis Aranda et al. (2005)
Munubicin In bacterial infection Castillo et al. (2002)
Hemomycin In bacterial infection Bae et al. (2015)
Subtilin In bacterial infection Sidorova et al. (2018)
Bacteriocins In bacterial infection Sansinenea &Ortiz (2011)
Amicoumacin Active in malarial infections Pinchuk et al. (2002)
Artemisinin Active in malarial infections Li et al. (2012)
Coronamycin Active in malarial infections Ezra et al. (2004)
Spectinomycin Infection associated with
Mycobacterium tuberculosis
Barry (2014)
Treponemycin Infection associated with
Mycobacterium tuberculosis
Yassien et al. (2015)
Androprostamines Found to be active in prostate
cancer
Yamazaki et al. (2015)
Camptothecin In cancer Shweta et al. (2013)
Indolocarbazoles In cancer Xu et al. (2014)
Doxorubicin Mammary carcinoma and cancer
treatment
Wisher (2012)
Anthracyclin Against tumor Li et al. (2015)
Daptomycin Infectious illnesses of the mucosa
and epidermis caused by bacteria
Miao et al. (2005)
Monensin Effective in parasitic infection like
coccidiosis
qowicki &Huczy
nski (2013)
Mitomycin C Against cancer Danshiitsoodol et al. (2006)
Saadamycin Effective in fungal skin diseases El-Gendy &El-Bondkly
(2010)
Streptav idin Associated with building
immunotherapy to fight with cancer
Pesic et al. (2014)
Thaxtomin A Cellulose synthesizing blocker Francis et al. (2015)
Xiamycin Effective in AIDS Ding et al. (2010)
Beta exotoxin Effective in destroying or controlling
insects
Espinasse et al. (2002)
Albaflavonol B As sesquiterpene Raju et al. (2014)
1. Introduction 335

Metabolites produced by microorganisms, plants, and animals are known as nat-
ural products (Baker et al., 2007). These natural products are significant because
they have historically served as sources of medicines. Natural products have
frequently been used as sources for the lead compounds that give rise to numerous
synthesized medications. Paclitaxel (Taxol), the first compound effective in cancer
treatment to reach the $1000 million place, is a key instance of a natural chemical
derived from the Taxus wallichiana, also known as the Yew tree (Wani et al.,
1971). The traditional immunosuppressive drug cyclosporine, which was shown to
be derived from the Tolypocladium inflatum, further increased the value and signif-
icance of endophytes (Fouda et al., 2015). Endophytic bacteria have the capacity to
create brand-new organic products. Researchers are examining novel and distinctive
natural compounds with significant commercial potential. Endophytes are well-
known to produce natural compounds effective in bacterial, fungal infections.
They are also known to have significant role in the management of diabetes and
also involved in the management of immunity as immunosuppressive agents. Endo-
phytes are seen as a great source of natural products. The endophytic bacteria, which
make a range of antibiotics, are among the potential sources of novel antibiotics. The
antibiotics formed by endophytic bacteria represent some of the most unusual anti-
biotics; examples comprise ecomycins, pseudomycins, munumbicins, and kakadu-
mycins. Endophytic fungi could be a better source for the creation of novel
medications to treat cancer and multidrug-resistant bacteria. They are easy to culture
in a laboratory and ferment. It will contribute to environmental protection.
1.4 Future outlook
It is now attractive and full of promise for the fields of agriculture, pharmacology,
and medicine to study the microbiome of plants. Scientists are paying more attention
to the effectiveness of medicinal plants and the associated microbes as they evaluate
their pharmacological potential in the creation of the bioactive substances that are
naturally present in them. Numerous phytochemical components found in medicinal
plants have the potential to be investigated as treatments for human ailments. The
current search for novel biologically active metabolites from plant endophytes has
varied the therapeutic chemicals and increasing demand for benign medications.
On the bioactivities of its related fungi endophytes, there is a limited amount of
knowledge.
Endophytes and actinomycetes produced chemicals are important for treating
human diseases such as cancer, diabetes, bacterial infections, oxidative stress, and
inflammation. Fungal endophytes are an alternative source for the creation of natural
substances in the current period of the emergence of new diseases. TALEN (Tran-
scription Activator-Like Effector Nucleases), CRISPR (Clustered Regulatory Inter-
spaced Short Palindromic Repeats)-Cas9, and ZFN (Zinc-Finger Nucleases)-Cas9
are a few biotechnological tools that are used to create the substances in big quan-
tities. Other approaches, such as in vitro regeneration, electroporation technology for
the creation of transgenic therapeutic plants, combinatorial biosynthesis, and genetic
336 CHAPTER 15 Recent advances in discovery of new drugs

transformations, could potentially be used to manipulate genes. Future research
should concentrate on the mechanisms underlying the interactions between plants
and endophytes, actinomycetes, their biogeographical distribution, elucidating the
fundamental mechanisms of bioactive compound synthesis, as well as the methods
to manipulate the identified pathways for the synthesis of natural bioactive com-
pounds from plant-associated endophytes and actinomycetes.
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Further reading 343

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Plant health: feedback
effect of root exudates and
rhizobiome interactions 16
Shrikrishna Bhagat, Pranali Shete, Ashish Jain
Dept. of Microbiology, Smt. C.H.M. College Ulhasnagar, University of Mumbai, Mumbai,
Maharashtra, India
1.Introduction
Substantial advancements provide innovative technologies in crop protection, even
though plant diseases and changing environmental conditions affect crop yields
worldwide. Particularly, to feed the expanding population, the world’s food con-
sumption is anticipated to rise above 70% by 2050 (Singh et al., 2020). Conse-
quently, extensive research has been done on how diseases cause pathogens and
their interaction with plants, thus finding solutions to food-related problems has
proven difficult (Gao et al., 2021;Pischetsrieder, 2018).
Several microorganisms are constantly present in plants, which include fungi,
bacteria, viruses, nematodes, and organisms feeding on roots collectively mentioned
as root microbiomes. These microorganisms populate the soil nearby the roots,
which is a significant and active area designated the rhizosphere (Vacheron et al.,
2013). The size, width, and extent of the rhizosphere can vary, majorly depending
upon the root structure of individual plants and the composition and characteristics
of the soil. A complex system rhizosphere may host a variety of biological and
nonbiological communications (Fitzpatrick et al., 2018;Reinhold-Hurek et al.,
2015). Plant health and performance depend on the microbiome found in the rhizo-
sphere (Berendsen et al., 2012;Marasco et al., 2012). During difficult and adverse
growing conditions, rhizobiome plays a crucial role in services fundamental to agri-
culture and, more broadly, to host biology including nutrient absorption, disease
resistance, and stress tolerance, nutrient recycling, maintaining soil structure (Evelin
et al., 2009).
In reply to external environmental stimulations, the plant root releases active
compounds known as root exudates (rhizodeposits) which perform a vital part as
the messenger between the plant system and rhizobiome (Kawasaki et al., 2016).
There are various compounds released as rhizodeposits comprising primary metab-
olites like sugars, organic acids, and amino acids, as well as a large number of
distinctive secondary metabolites flavonoids, glucosinolates, lignins, auxins, and
many chemoattractants (Mavrodi et al., 2021;Richardson et al., 2020). Root exu-
dates are essential for enhancing the diversity and number of microorganisms in
CHAPTER
345
Rhizobiome. https://doi.org/10.1016/B978-0-443-16030-1.00007-9
Copyright ©2023 Elsevier Inc. All rights reserved.

the soil around a plant. Positive and negative interactions in the rhizosphere are co-
ordinated by root exudates. Root exudates tempt healthy microorganisms such as
mycorrhizae, plant growth promoting rhizobacteria (PGPR), and rhizobium in addi-
tion to forming a symbiotic association with them and preventing pathogenic mi-
crobes to attack the plant. Associations with parasitic plants, dangerous
microorganisms, and invertebrate herbivores are examples of harmful interactions
(Lundberg &Teixeira, 2018).
Inorganic ions, water, electrons, protons, and carbon compounds are formed in
process of photosynthesis, which is released as root exudates into the rhizosphere.
The rhizospheric deposits mostly consist of phytosiderophores and polysaccharides
in addition to photosynthates. The former facilitates nutrient uptake, but the latter,
when combined with rhizosphere bacteria, generates a mucigel that protects symbi-
otic microbes and allows for their free movement within the rhizosphere. Exudates,
which operate as signal molecules, can also facilitate these connections by openly
interceding the number of interactions around the rhizosphere including plant-
microbe, plant-plant, and microbe-microbe interactions (Olanrewaju et al., 2018).
Comprehensive research has been done on the interactions between plants and
rhizospheric bacteria for biocontrol, stimulating plant development, and studying
the effect on biogeochemical cycles (nitrogen and carbon cycles) each of which is
essential for maintaining the plant’s health. This has made it necessary to improve
and recognize the assortment of bacteria in the rhizosphere to make the most use
of helpful microbes for promoting plant development. The primary route for exuda-
tion into the rhizosphere is through a plant’s root system. Additionally, certain soil
physicochemical characteristics that are partially altered and impacted by roots con-
trol the development of plant root systems. The interactions between the root
exudate and rhizobiome must consider various parameters, including the impact
on the rhizospheric environment and soil characteristics. However, this chapter
will solely include the impacts of root exudates-rhizobiome interaction on plant
health.
2.Rhizosphere and rhizobiome: a dynamic system
Soil is a fundamental system on the Earth that is dynamic and very complex and
comprises a variety of physiochemical and biological components (Lehmann
et al., 2020). Soil is necessary for holding down the roots which are a source of nu-
trients, oxygen, and water that support plant development. The rhizosphere, a region
of the soil that is significantly impacted by and connected to the roots of the plants, is
formed as a result of the plants growing their roots (Bakker et al., 2020). Also, a
segment of the soil that is influenced by the root has been described as the rhizo-
sphere. Because some types of soil tend to make it difficult for roots to attach,
some rhizospheric soil has been observed to hold roots firmly in place even after
shaking. It also depends on the appearance, moisture content, and nature of the
soil. Therefore, a definition of the rhizosphere that includes “the amount of the
346 CHAPTER 16 Feedback effect of root exudates

soil and also its residing biodiversity, together with their relationships, which are
impacted by the plant roots”” seems to be the most accurate (Philippot et al.,
2013;York et al., 2016).
Each plant’s root system determines the rhizosphere’s size, width, and extent
rather than the soil’s characteristics. Since the majority of root-free soils are oligo-
trophic, that is, contain low amounts of nutrients, thus microorganisms present there
typically survive on a low-calorie diet, with the availability of organic nutrients,
particularly those that provide energy, acting as the main productivity barrier.
Organic nutrients that are easy to use can be found in much greater quantities in
rhizosphere soil. This nutritional enrichment in the rhizosphere causes changes in
respiration, gas exchange, nutrient and moisture availability, and consumption, as
well as more intensive microbial metabolism and production than what is sustained
in bulk soil. The rhizosphere is a complex system with a wide range of biological and
abiotic interactions that greatly affect it (Fitzpatrick et al., 2018;Reinhold-Hurek
et al., 2015). Rhizospheres primarily contain rhizoplane made up of root epidermis,
soil attached to root, and mucigel, as well as rhizosheath, consisting of soil glued to
root hairs and mucigel (York et al., 2016).
The root system of the plants is the key element to enriching the rhizosphere with
microorganisms (either beneficial or harmful) from the surrounding which affects
the plant health called rhizospheric effects. These microbes can help plants by pro-
tecting against pathogens, providing nutrition, and protecting against stress condi-
tions. Thus, the rhizosphere comprises these chemical, physical, and biological
complex activities and is considered the most dynamic habitat for microbial interac-
tions (Olanrewaju et al., 2017).
2.1 Rhizobiome: microbial collection in the rhizosphere
The biological components in the rhizosphere’s vicinity are essential for its pro-
cesses. A collection of microorganism communities that lives in the rhizosphere
is referred to as a rhizomicrobiome or rhizobium. Also, some other scientists
denoted genomes that interact with the plant root called rhizobiome (Olanrewaju
et al., 2018;Pollak &Cordero, 2020;Santoyo et al., 2016). The rhizosphere is
home to a wide range of microorganisms, ranging from prokaryotes, viruses, and eu-
karyotes (bacteria, fungi, nematodes, protists, and phage) which are supportive or
damaging (Venturi &Keel, 2016). The majority of research on the rhizobiome
has focused on the bacterial strains and fungi that dominate the microbiome there.
Bacteriophages influence the crescendos of the rhizosphere microbiome including
Microviridae, Siphoviridae, and Podoviridae families are the most prevalent exist
in the soil (Pratama et al., 2020). In the experiment “rhizobox” it was found that Bac-
teroidetes, Methylobacterium, Rhizobium, Arthrobacter, Pseudomonas, Stenotro-
phomonas, and Enterobacter are the most prevalent bacterial communities found
in Lolium perenne rhizobiome (Chen et al., 2016). Like the gut, the rhizospheric
microbiota’s complexity provides information about the individual plants and the
entire plant community. It is significant to note that the rhizobiome may coexist,
2. Rhizosphere and rhizobiome: a dynamic system 347

flourish, and engage in a variety of interactions with the plant, with the root tissue
serving as the primary site of contact. Therefore, selecting efficient bioinoculation
agents in the future necessitates identifying beneficial rhizobiome activities with
favorable interactions and a profound effect on engaging with the plant as well as
other microbes (Sasse et al., 2018;Vasseur-Coronado et al., 2021).
2.2 Communication in the rhizome
To communicate with one another, the rhizospheric microbes deliver indicating fac-
tors that change their transcriptional activity, affecting the rhizospheric environment.
Rhizomicrobiome coordinates with one another in the same or different species by
the process of quorum sensing/signaling, which produces molecules that serve as
signals. Quorum signaling involves the synthesis, and release of signaling molecules
in a satisfactory quantity to detect and get an answer after the recipient microorgan-
isms. This signaling may take place between harmful microbes, helpful-harmful mi-
crobes, and helpful microorganisms (Helman &Chernin, 2014;Hong et al., 2012).
These autoinducers (chemical signals) regulate the gene expression of some tasks of
the receiver by, biofilm-forming ability, motility, proliferation (Hassan et al., 2016;
Weihua et al., 2011), virulence factors, metabolic activity (Atkinson &Williams,
2009), and their adhesion properties.
Due to the activation of multiple developmental pathways during signal trans-
mission between the plant body and the microorganisms, such messages might be
essential for the rhizomes’ future growth. This highly evolved and intricate network
of interspecies communication is crucial for controlling and modifying the rhizo-
biome. There are different types of quorum-sensing signals in bacteria and fungi
which are detailed in Table 16.1.
3.Role of rhizobiome in plant health
It is significant to note that the rhizobiome flourishes plant health by engaging in
numerous interactions with the root epithelium acting as the main point of contact
and promoting plant growth (Berendsen et al., 2012;Kwak et al., 2018). Microor-
ganisms present in the rhizosphere help the plant by controlling pathogenesis,
manufacturing plant growth factors, acquiring nutrients, and dealing with stress
related to nutrient competition, which are all necessary for the continued existence
of the plant (Ahemad &Kibret, 2014).
4.Rhizobiome contributes to limiting nutrient acquisition
By cooperating with plants in mutually supportive as well as nonmutualistic ways,
the rhizobiome makes nutrients available to plants. Macro- and micronutrients have
an impact on many critical biochemical and physiological processes, including
348 CHAPTER 16 Feedback effect of root exudates

cellular division, cell expansion, respiration, fruit and grain quality, the activity of
enzymes, and biomolecule biosynthesis (Hakim et al., 2021). Nitrogen, zinc, phos-
phorous, and iron are some of the fundamental elements needed for these activities.
These elements are also crucial components of biomolecules and energy carrier
groups (Fredeen et al., 1990).
Only nitrate and ammonia forms of nitrogen are absorbed by plants. Nitrogen-
fixing bacteria are free-living and symbiotically construct ammonia for plant expen-
diture, by transforming nitrogen present in the atmosphere to ammonia (Prasad et al.,
2014). Rhizobium species, a group of bacteria that converts atmospheric nitrogen
(N) to ammonia, form nodules on the roots or stems of legume plants. It includes
Mesorhizobium sp., Bradyrhizobium sp., Sinorhizobium sp., Azorhizobium sp.,
and Allorhizobium sp., which follow the same path to fix atmospheric nitrogen (Mfi-
linge et al., 2014;Ravikumar, 2012). Nitrogen-fixing microorganisms that freely live
in the rhizosphere are mainly Burkholderia sp., Acetobacter sp., Herbaspirillum sp.,
Table 16.1 Quorum signaling molecule released by rhizomicrobiome for
communication.
Quorum signaling
molecules Description References
Acyl-homoserine
lactone (AHL) and
autoinducer
peptides (AIPs)
•2-alkyl-quinolones, fatty
acid methyl esters, g-butyr-
olactones, and furanone
•Produced by Gram-positive
and Gram-negative
bacteriadBacillus subtilis,
Bacillus velezensis, Pseudo-
monas syringae, Pseudo-
monas putida, and Erwinia
Ferluga et al. (2008),Sindhu
et al. (2017),Zhang &Li
(2016)
Antibiotics •Produced by
bacteriadcyclic
lipopeptides, cyclic and
polycyclic tetrameter
macrolactams
•Produced by fungi-g-
heptalactone, tyrosol,
g-butyrolactone, dodecanol,
and farnesol
Brescia et al. (2020),
Hartmann &Schikora (2012)
Volatile organic
compounds (VOCs)
•Low molecular weight lipid
soluble combinations (100
e500 Da)
•alkanes, ketones, alkene,
terpenoids, and sulfates
•P.aeruginosa,B.
licheniformis, and S.
marcescens
Barraud et al. (2009),Kai
et al. (2016),Kanchiswamy
et al. (2015),Tyc et al. (2015)
4. Rhizobiome contributes to limiting nutrient acquisition 349

Azospirillum sp., Azoarcus sp., Azotobacter sp., Bacillus sp., and empower plant ni-
trogen nourishment (Igiehon &Babalola, 2018;Mahmud et al., 2020;Vessey, 2003).
Co-inoculation of Rhizobium spp. with some other bacteria Bacillus,Pseudomonas,
Serratia, and Azotobacter increases nitrogen absorption in leguminous plants
(Remans et al., 2008;Tchebotar et al., 1998).
Phosphate solubilizing microorganisms make inorganic phosphate available for
plants by secreting phosphatase. There are several phosphate-solubilizing bacteria
are reported including Bacillus, Klebsiella, Enterobacter, Xanthomonas, and Pseu-
domonas (Billah et al., 2019;Zaidi et al., 2009). In addition to phosphorus and ni-
trogen, plants may also get iron through symbiotic relationships with soil
rhizobacteria. Even though soil contains more ferric ions (Fe3þ), plants often
choose to consume Fe mostly in the state of reduced form (Fe2þ). Microorganisms
release a variety of siderophores chelators that attach to Fe3þand transferred to
roots transforming to Fe2þand absorbing immediately (NicolausKhodr &Hider,
2000). According to some explanations, mycorrhizal fungi help plants acquire
more than 80% of the phosphorus and nitrogen they need (Nouri et al., 2015).
4.1 Rhizobiome produce plant growth hormones
Plant growth hormones essential for cellular and organ development (fruit, leaves,
stem, and root) includes auxins, gibberellins, ethylene, cytokinins, abscisic acids,
jasmonic acids, brassinosteroids, and salicylic acid. Several rhizospheric microor-
ganisms yield plant growth hormones that encourage plant development (Backer
et al., 2018;Olanrewaju &Babalola, 2019). Indole acetic acid is one of the utmost
essential auxin, developed by more than 80% of rhizospheric bacteria which plays an
essential role in cell division, apical dominance, and progress in root growth (Morffy
&Strader, 2020). One study reported that Bacillus amyloliquefaciens SQR9 in-
creases the tryptophan level in the cucumber plant which is a precursor for the indole
acetic acid (Liu et al., 2016). Rhizobacteria Azospirillum sp., Erwinia herbicola,
Agrobacterium sp., Rhodococcus fascians, and Pseudomonas sp., were found to
be promising IAA producers (Brandi et al., 1996;Costacurta &Vanderleyden,
1995). Ethylene is a multipurpose phytohormone that controls senescence as well
as growth. Under organic phenomenon and abiotic pressure circumstances, ethylene
synthesis starts in plants. Some rhizobacteria produce 1-aminocyclopropane-1-
carboxylase (ACC) deaminase which stunts plant growth by suppressing ethylene
manufacturing capacity (Li et al., 2000). Salt strain was minimized in pea plants
by ACC deaminase (Wang et al., 2016). Many bacterial species including Arthro-
bacter,Azotobacter,Pseudomonas,Bacillus,Acinetobacter,Azospirillum,Flavo-
bacterium,Rhizobium,Burkholderia,Bradyrhizobium, and Xanthomonas are
found to be gibberellin producer in the rhizosphere (Bottini et al., 2004;Gutie
´r-
rez-Man
˜ero et al., 2001;MacMillan, 2001). Cytokines contributed to seed germina-
tion and vascular expansion in plants produced by rhizobiome Azotobacter,
Paenibacillus, Rhizobium, Bacillus, and Azospirillum genera (Garcı
´a de Salamone
et al., 2001;Taller &Wong, 1989;Timmusk et al., 1999).
350 CHAPTER 16 Feedback effect of root exudates

4.2 Rhizobiome as biocontrol agent
The threat posed by pathogenic microbes to crop production globally has resulted in
the extensive use of fungicides and pesticides to combat them. The use of these
chemicals has contaminated the rhizospheric soil and it affecting the environment
as well as human beings. Plant growth-promoting rhizobacteria (PGPR) fights path-
ogenic microorganisms in various ways, producing lytic enzymes, siderophores, and
antimicrobial compounds, and also induces resistance in plants (Costacurta &Van-
derleyden, 1995).
PGPR produces lipase, chitinases, proteases, and other lytic enzymes which
inhibit the bacterial as well as fungal phytopathogens Sclerotium rolfsii, Fusarium
oxysporum, and Rhizoctonia solani (Aris et al., 2011). The study conducted by Bar-
nett and his co-workers found that the combined effect of Pantoea agglomerans,
Exiguobacterium acetylicum, and Microbacteria sp. effectively suppressed the
Rhizoctonia patch disease (Barnett et al., 2006). Another study proved the antimi-
crobial potential of innate tomato rhizobiome when inoculated in different tomato
cultivation fields against Ralstonia solanacearum (Prasad et al., 2014). Many mi-
crobes majorly Pseudomonas sp. produce 2, 4-Diacetylphloroglucinol (DAPG) hav-
ing potential antimicrobial activity against zoospore forming Pythium sp. (Pal &
Gardener, 2006). Several siderophores-producing bacteria transported the bulk of
the iron and other nutrients to the plants, which limits these nutrients for pathogens
and reduces their action or death (Ferreira et al., 2019). Some genera of Bacillus and
Pseudomonas produce lipopeptides which are helpful in the control of various path-
ogenic bacteria and fungi (Mejri et al., 2017). Rhizospheric PGPR activates some
defense signaling in plants which boosted plants’ tolerance against pathogens, as
well as abiotic factors, termed induced systemic resistance (ISR). One study con-
ducted by Jung et al. (2011) observed that the Pseudomonas aureofaciens 63-28
strain brought a security mechanism in soybean seedlings against Rhizoctonia solani
AG-4 pathogenic strain (Jung et al., 2011).
It has been widely demonstrated that using the plant rhizobiome skillfully could
indeed start reducing the need for pesticides and chemical mixed fertilizers which
have detrimental consequences on both human beings and animals’ well-being as
well as pollute the environment. The plant roots and the rhizobiome can engage
in a variety of physical and chemical exchanges. In the rhizospheric region, mi-
crobes are free but the majority of the time they start forming distinct bacterial spots
where they form their colony by secreting some molecules named biofilms (Wei &
Zhang, 2006). Numerous biological and physical dynamics influence the composi-
tion of the rhizobiome including the soil properties, root architecture, seed micro-
biome, plant species, plant age and genotype, environmental conditions, bulk soil
biodiversity, and plant defense response (Bulgarelli et al., 2012;Chaparro et al.,
2013a;O’Brien et al., 2018;Santoyo et al., 2017).
So how do the plants recruit the specific microorganisms in the rhizosphere re-
gion? How are harmful microbes not cultivated in the phyllosphere and rhizosphere?
How does rhizobium know which nutrient is limited and required by the plant?
4. Rhizobiome contributes to limiting nutrient acquisition 351

Numerous rhizobium practices require external force to act upon them to take place.
A signal that establishes the start or stop of a process might exist, or there could be a
mediator operating as a coupler to connect the procedure mediators. The mediators
in most interactions must, however, be linked together, that’s where the root exu-
dates come into the picture. Although at least a few of the exudates were previously
thought to be plant garbage, there are those that could inhibit or attract organisms to
the rhizosphere, connect complex interrelationships performing in the rhizosphere,
and have a noteworthy impact on the plant’s overall fitness (Badri &Vivanco, 2008).
5.Root exudates: the spray of chemical signals
Plants constantly release different ingredients into their environment through leaves,
stems, or roots, different forms (solid, liquid, gas) are acknowledged as exudation.
This study concentrated on root exudates, which haveparticipated in a range of different
rhizosphere conversations and aid in the movement of both carbon and nitrogen from
the soil through the root. A fundamental component of the rhizosphere, in addition to
the abiotic components, is the microbes, whose activities are essential to multiple
nutrient cycling and the proper operation of crop fields. Aside from performing com-
mon functions, a vast array of highly lucrative small molecular weight molecules exist
that are released by plant roots into the rhizosphere termed root exudates (Preece &
Pen
˜uelas, 2020). Root exudates set off a chain reaction of feedback loops involving
the soil particles, the associated microbiome, and the roots. Root exudates in soil
set off a chain reaction of feedback loops involving the soil particles, the associated
microbiome, and the roots. This active interaction comprises, promoting soil mass
accumulation, mobilizing nutrients, removing highly harmful metals and toxic mate-
rials, signal exchange between and within plants, releasing plant defense compounds,
and signal exchange between plants and microbes (Bais et al., 2006;Philippot et al.,
2008). Exudate-driven rhizosphere processes take place on a very small scale, but
their overall effects have great influence.
Root exudates are the mixture of both primary and secondary metabolites produced
by plants including molecular weight
˂
1000 Da as well as
˃
1000Daltons; contain muci-
lage and enzymes (Badri et al., 2009;Oburger &Jones, 2018). Primary metabolites
mainly include sugars (glucose, fructose, galactose, and oligosaccharides), amino
acids (cysteine, proline, glycine, and phenylalanine), organic acids (malic and acetic
acid), growth factors, and vitamins whereas secondary metabolite contains alkaloids,
phenolic compounds (flavonoids), terpenes, glycosides, and tannins (Das &Gezici,
2018;Mavrodi et al., 2021;Richardson et al., 2020;Rolfe et al., 2019).
Table 16.2 represents all the details about primary and secondary root exudates
produced by different plants and their effect on plant health.
6.Root exudation transport mechanism
The tip of the root is a widely accepted part where the root exudate secretion occurs.
The root tip plays a crucial role in the root’s reactions to environmental stimuli and is
352 CHAPTER 16 Feedback effect of root exudates

Table 16.2 Different primary and secondary metabolites produced by plants and their effect on plant health.
Plant name
Metabolites
produced Microbial response Microorganisms
Plant
promotion References
1. Brachypodium
distachyon
Malic acid, fumaric
acid, citric acid,
and succinic acid
Chemotaxis, biofilm
formation
Bacillus sp.,
Microbacterium sp.
Stress
tolerance,
nutrient
exchange,
overall growth
promotion
Ramey et al.
(2004),Saleh
et al. (2020)
2. Arabidopsis
thaliana
Malic acid, citric
acid, succinic
andand oxalic acid
Chemotaxis, biofilm
formation
Bacillus velezensis Strain
S3-1
Biofertilizer and
biocontrol agent
Jin et al. (2019)
3. Tomato Malic acid, citric
acid, succinic acid,
and oxalic acid
Chemotaxis Bacillus
amyloliquefaciens SQY
162
Competes with
tomato bacterial
wilt (TBW)
caused by
pathogen
Ralstonia
solanacearum
Wu et al.
(2017)
4. Tomato Lactic acid and
hexanoic acid
Chemotaxis, biofilm
formation
Bacillus cereus AR156 Elicits plant
immune
response,
assists in
biocontrol of
tomato wilt
disease
Wang et al.
(2019)
5. Water-melon Malic acid and
citric acid
Induces motility and root
colonization
Paenibacillus polymyxa
SQR-21
Biocontrol of
pathogens
Ling et al.
(2011)
6. Brachypodium
distachyon
Several organic
acids
Activate regulatory and
stress response genes
Pseudomonas
fluorescens group
Competitive
colonization and
disease
prevention
Mavrodi et al.
(2021)
Continued
6. Root exudation transport mechanism 353

Table 16.2 Different primary and secondary metabolites produced by plants and their effect on plant health.dcont’d
Plant name
Metabolites
produced Microbial response Microorganisms
Plant
promotion References
7. Rice Organic acids,
especially malic
acid
Chemotaxis, root
colonization
Pseudomonas putida
RRF3
Re-organizes
plant root
transcriptome to
elicit defense
response
Kandaswamy
et al. (2019)
8. Cucumber Citric acid Chemotaxis, biofilm
formation
Bacillus
amyloliquefaciens SQR9
Defense against
by Fusarium
oxysporum
Fusarium sp.
cucumerinum J.
H. Owen (FOC)
Xu et al. (2013),
Zhang et al.
(2013)
9. Banana Fumaric acid Chemotaxis, biofilm
formation
Bacillus subtilis N11 Reduce the wilt
disease affected
by Fusarium
oxysporum f. sp.
cubenserace 4
(FOC4)
Zhang et al.
(2011) (2013)
10. Arabidopsis
thaliana
Salicylic acid
organic acids
Growth signal Diverse bacteria Promotes
growth of PGPR
Lebeis et al.
(2015)
11. Oryza sativa L Salicylic and malic
acids
Growth promotion Bacillus subtilis RR4 Stress
alleviators, plant
growth
promotion
Rekha et al.
(2020)
12. Arabidopsis
thaliana
Amino acids Increase in microbial
community
Pseudomonas,
sphingomonas
Foliar pathogen
resistance
Wen et al.
(2020)
13. Arabidopsis
thaliana
Amino acids Chemotaxis and selective
growth promotion
Proteobacteria Prevented
dissolved
oxygen carbon
release
Wen et al.
(2022)
354 CHAPTER 16 Feedback effect of root exudates

14. Tea plants Amino acids Diversify rhizobiome Diverse bacteria Promote plant
growth
Ghatak et al.
(2022)
15. Cucumber Amino acids Chemoattractant Bacillus
amyloliquefaciens SQR9
Promotes
growth of PGPR
Feng et al.
(2018)
16. Arabidopsis
thaliana
Sugars Increase in PGPR PGPR Plant growth
promotion
Lopes et al.
(2022)
17. Arabidopsis
thaliana
Sugars Increase in alkaline
phosphatase
Saccharimonadales Phosphorus
cycling
Wang et al.
(2022)
18. Durum wheat All primary
metabolites
Increase in microbes Diverse bacteria Plant growth
promotion
Iannucci et al.
(2021)
19. Leguminous
plants
Flavonoids Chemoattractant Arbuscular mycorrhizal
fungi
Promotes
nodulation
Steinkellner
et al. (2007)
20. Lotus japonicus Phenolic acids Induces
lipochitooligosaccharides
(LCOs) called NOD factors
Mesorhizobium Root
deformation and
increase in
nodule numbers
Shimamura
et al. (2022)
21. Abelmoschus
esculentus
Phenolic acids Chemotaxis, biofilm
formation
Alcaligenes faecalis Activates
defensive
pathway, plant
growth
promotion
Ray et al.
(2018)
22. Arabidopsis
thaliana
Benzoxazinoid Attraction PGPR growth
promotion, e.g.,
Pseudomonas
plant growth
promotion
Hu et al.
(2018),
Kudjordjie et al.
(2019)
23. Arabidopsis
thaliana
Phenylpropanoids Attraction of PGPR Pseudomonas putida Plant growth
promotion;
efficiently
degrades soil
pollutants
Wang et al.
(2021)
Continued
6. Root exudation transport mechanism 355

Table 16.2 Different primary and secondary metabolites produced by plants and their effect on plant health.dcont’d
Plant name
Metabolites
produced Microbial response Microorganisms
Plant
promotion References
24. Soybean plant Isoflavones Attracting nitrogen-fixing
bacteria like rhizobium
Rhizobium Plant growth
promotion
Sugiyama
(2019)
25. Medicago sativa 7,40-
dihydroxyflavone
Attracting beneficial
bacteria and symbiosis
Gaiellales,
Nocardioidaceae, and
Thermomonosporaceae
Nod gene
induction and
plant growth
promotion
Szoboszlay
et al. (2016)
26. Acer
saccharum
Phenolic
compounds
Increase in selective
bacteria
Paraburkholderia and
Caballeronia
Enhance soil
organic matter
decomposition
and increase its
availability to
plants
Zwetsloot et al.
(2020)
356 CHAPTER 16 Feedback effect of root exudates

the first plant component to explore new soil environments (Helman &Chernin,
2014). The exudation in the rhizosphere involves a full range of mechanisms, which
are either passively or actively released dependent on the nature of the exudate. The
exudation has traditionally remained thought of as a passive practice, facilitated
through various ways, including the transportation by vesicles, ionic channels,
and diffusion through the root membrane, in contrast, side proteins found in the
root plasmatic membrane facilitate the root exudation of chemical compounds via
active transport (Baetz &Martinoia, 2014).
The vast electrochemical potential gradient persists all over the plasma mem-
brane because of the low concentration of organic compounds in the soil which
allow the exudates having a minimum molecular weight (amino, acids sugars,
and organic acids) to diffuse out in the soil by passive diffusion. This is
gradient-dependent process and is regulated by factors like the permeability of
the root membrane, root cells, and the exudate’s chemical nature (Jones et al.,
2009;Rolfe et al., 2019). The plasma membrane is impermeable to the large
and polar molecules; therefore, these molecules passed out through it with the
help of specific membrane proteins and ion channels. These channels allowed
the polar molecules to diffuse without affecting their integrity while passing the
lipid membrane (Yang &Hinner, 2014). Efflux of these primary metabolites is
up- and downregulated by their specific gene expression levels. Channels found
in the plasma membrane are slow anion channels (SLACs) which took some
time to come into action whereas some rapidly activated channels present were
called quick anion channels (Dreyer et al., 2012). Some malate transferring trans-
porter is activated by AL3
þ
ions called aluminum-activated-malate transporters
(ALMT), majorly found in Arabidopsis thaliana, Vitis vinifera, Oryza sativa plants
(Sharma et al., 2016). Also, there are some metabolite-specific transporters present
in the plasma membrane such as SWEET transporter for sugar (Chen et al., 2015),
for amino acid transportation there are GDU transporters (Pratelli et al., 2009),
CAT transporters (Yang et al., 2010), UMAMIT transporters (Dinkeloo et al.,
2018;Moe, 2013), and MATE/citrate transporters present for organic acids
(Mora-Macı
´as et al., 2016). The mechanism of exocytosis transported some large
metabolites packed in vesicles to protect from pathogenic attack (Wes ton e t al. ,
2012). Transpiration of high molecular weight compounds required energy,
some transporters use energy from ATP hydrolysis and transport these compounds.
There are ATP-dependent pumps, with H
þ
antiports present such as ATP-
dependent ABC(ATP Binding Cassette), citrate, and MATE (Multidrug and toxic
compound extrusion) transporters that have Hþ-coupled antiport mechanisms
(Meyer et al., 2010;Wu et al., 2017). The family of ABC transporters is the
most prevalent principal transporters in living things, including mammals. In addi-
tion, analysis of the rhizobiome of the Arabidopsis thaliana abcg30 mutant showed
that this transporter can modify the rhizobiome composition, this genetically
altered transporter passage extra phenolic complexes and very few sugar content
than the natural unmodified genotype which suggests that root exudates interact
with soil microorganisms in a significant way (Wu et al., 2017).
6. Root exudation transport mechanism 357

One of the major concerns in rhizosphere research is whether the root, that is,
origin or the nearby associated microorganisms regulate the quality and frequency
of exudate secretion from the root. To simply remove root exudates from the soil,
the microbial community maintains the greater concentration gradient that motivates
efflux. However, there is also confirmation that microbes can release substances that
make root membranes more permeable, allowing for higher exudate release rates.
Exudation is thus a result of the way process microbial lift and root press (Kudoyar-
ova et al., 2014;Phillips et al., 2004).
7.Factors affecting root exudate profile
It has been discovered that root exudates composition and pattern of exudation could
be altered qualitatively as well as quantitatively by dynamics of abiotic and biotic
influences. The biological factors include pathogenic attack, herbivory, plant devel-
opment stage and its genotype, and plant species. Abiotic factors contain some phys-
ical as well as chemical influences such as drought conditions, salinity, soil nutrient
composition, extreme temperature, and atmospheric CO
2
level. Ultimately all these
events directly affect the plant’s health. Abiotic and biotic stresses are highly corre-
lated, and biotic stress can increase resistance to physical and chemical stress con-
ditions Fig. 16.1 (AbuQamar et al., 2009).
FIGURE 16.1
Illustration of biological, chemical, and physical factors affecting the root exudation.
358 CHAPTER 16 Feedback effect of root exudates

8.Root exudates and rhizobiome: synergistic influence on
plant health
Root exudates produced by plants participated in various biological processes such
as root-rhizobiome interactions, root-root interactions, and root-insect interactions,
which positively and negatively affect plant health (Haldar &Sengupta, 2015;Sub-
rahmaniam et al., 2018). Root exudates show themselves to be more than just a sup-
porting character in the rhizospheric system where several biological and chemical
reactions are carried out. Even though exudates are generated in small quantities,
they are engaged in a variety of rhizosphere operations (Jin et al., 2019).
8.1 Relation with nitrogen-fixing bacteria
Several microorganisms can convert free nitrogen (N
2
) into ammonia that can absorb
plants. Different plants take advantage, including rice, wheat, and maize create
mutualistic interactions with nitrogen-fixing bacteria that allow free-living
nitrogen-fixing microorganisms to go into the roots by releasing secondary metab-
olites (Chaparro et al., 2013b). Plants released specific flavonoids to attract or repel
desirable bacteria species to maintain this relationship. During nodule formation,
rhizobacteria require specific flavonoids, one is naringenins to express Nod factors
(Hu, 2000). Zhang and his lab members (2009) silenced the gene of
chalcone synthase pathways which is crucial for the synthesis of two flavonoids, first
was isoflavonoids (formononetin and biochanin A) and second flavones (7,40-
dihydroxyflavone) in plant M. truncatula, as result, no nodulation formed even in
the presence of rhizobacteria S. meliloti (Zhang et al., 2009). When Nod factors
are created chemically, they are altered using, in addition to other substances,
various kinds of sugars, acetate, or by changing the acyl tail’s saturation level. A
recent study offers fresh insight into the beneficial interactions between rare fungi
residing in plants, i.e., endophytic, nodule formation in roots, and fixation of atmo-
spheric nitrogen. Researchers discovered that Phomopsis liquidambaris an endo-
phytic fungus improved the activity of genetic factors required in the synthesis of
phenolic and flavonoid compounds in the Arachis hypogaea L (peanut plant), and
root-derived phenolic mixtures and flavonoids might successfully upturn the activity
of the gene responsible for nodule formation in Bradyrhizobium to escalate crop pro-
duces (Xie et al., 2019). Pyrosequencing studies of the microbial populations indi-
cate that they are highly responsive to fluctuations in the phenolic compound
combinations present in exudates obtained from maize plants, which can either
excite or hinder the development of various communal fellows such as some legume
species that can issue precise groupings of flavonoids to entice bacteria that fix ni-
trogen (Hassan &Mathesius, 2012). These results demonstrate the potential of mi-
crobes and root exudates to enhance nitrogen uptake.
8.2 Relation with PGPR
Rhizobacteria and root exudates relation is not only limited to nitrogen fixation,
but by spending these signal molecules the plant can affect the expression of
8. Root exudates and rhizobiome: synergistic influence on plant health 359

certain microbial genes, especially those that code for beneficial traits for the plant.
About 8.2% of gene expression level altered in the presence of root exudates in a
Gram-positive rhizobacterium Bacillus amyloliquefaciens FZB42, which indicates
the important number of the induced genes from these were linked to chemotaxis
and motility (Fan et al., 2012). The presence of strigolactones a root exudate in
Fabaceae plants’, attracts endomycorrhiza which grows in branching hyphae
necessary for root establishment (Akiyama et al., 2005). The roots of tomato plants
release fumaric and citric acids, which bring plant development supporting Pseu-
domonas fluorescens into the rhizospheric region (de Weert et al., 2002).
B. subtilis, a plant growth-promoting bacterium, formed biofilms when secreted
polysaccharides arabinogalactan, pectin, and xylan from Thalle cress roots (Beau-
regard et al., 2013). Pseudomonas putida is drawn into the rhizosphere from the
loose soil by the benzoxazinoids, exudate produced by maize, which is advanta-
geous for plant growth (Neal et al., 2012). As already mentioned, root exudates
affect microbial attraction among other things and there are numerous options
once the microorganism enters the rhizosphere.
8.3 Root exudates: role in limiting nutrient and mineral acquisition
Through the root system plants obtain a significant amount of inorganic nutrients
from the rhizosphere. Plants secrete a variety of root metabolites into the rhizo-
spheric surrounding which alters the acidity or alkalinity (pH) and constructs com-
plexes of metals present in soil and metabolites to control nutrient bioavailability
and combat environmental metal stresses (Dakora &Phillips, 2002). Early in the
1960s, it was discovered that the roots of tobacco plants secreted riboflavin
(Rbfl), and later it was discovered that some dicotyledonous plants, including sugar
beet (Beta vulgaris L.) and Hyoscyamus albus, secreted riboflavin when grown in
Fe-deficient environments (Higa et al., 2012). The phenolic compound coumarin
is secreted in Brassica napus and maize to acquire iron from alkaline substrates
(Clemens &Weber, 2015). The chelation-grounded approach for iron [ Fe(II) ]
acquirement employed by graminaceous plants entails the secretion of Fe(II)
chelating agents known as phytosiderophores rhizosphere surrounding (Kobayashi
&Nishizawa, 2012). Root-secreted exudates play crucial roles in enhancing plant
nutrient uptake as well as being crucial for plants’ ability to tolerate heavy metals.
For their significant roles in the detoxification of cadmium (Cd), aluminum (Al), gal-
lium (Ga), and copper (Cu), low molecular weight organic acids have been studied
(Meier et al., 2012;Zhu et al., 2011). The plants Zea mays (maize), Triticum aesti-
vum (wheat), and Glycine max (soybean) secrete different organic acids whereas
Arabidopsis thaliana primarily secretes citrate found to be the most effective
Al
þ3
chelating agent to remove from the rhizosphere (Agnello et al., 2014;Chang
et al., 2017). Additionally, root exudates can make the micronutrient phosphorus
(P) more readily available. When phosphorus in the soil reacts with cations in the
soil, phosphates are produced, which are only sporadically miscible. It has been
shown that when the soil lacks phosphate, the presence of citric acid and phytase,
360 CHAPTER 16 Feedback effect of root exudates

exudates of tobacco plants enhances the effectiveness of phosphorus procurement
(Giles et al., 2018). Additionally, a range of root exudates is produced in reply to
changing phosphorus concentrations (Pantigoso et al., 2020).
8.4 Root exudates: mystical ingredient of plant defense
The features and relative richness of the root exudates significantly influence path-
ogen levels in the soil. The enormous metabolic diversity of root exudates is being
gradually revealed through the identification, classification, and studying of their
propertiesdinnumerable unique bioactive substances as well as previously undis-
closed groups of specific defensive system substances. Phenolic and terpenoids
secreted by plants have potent antibacterial and antifungal properties on rhizo-
spheric pathogens. Notably, phenolic metabolites can positively affect the native
soil microbial community and are effective at luring some soilborne microorganisms
(Bais et al., 2002a). Canavanine was discovered to have the ability to stimulate some
beneficial bacteria while repressing a significant portion of other soil microorgan-
isms (Cai et al., 2009). The root system of the barley plant shows great antifungal
potential against Fusarium graminearum attack by secretion of phenylpropanoids,
a derivative of cinnamic acid (Lanoue et al., 2009). Arabidopsis thaliana produced
root exudates, which contain derivatives of tryptophan bioactive molecules like
some glucosinolates or the indole derivative camalexin, which are potent antifungal
as well as antibacterial agents (Consonni et al., 2009). One study discovered that
noninfected A. thaliana roots constitutively produce and release the diterpene rhiza-
thalene (Vaughan et al., 2013). In plant-plant interactions, root exudates can also be
beneficial, particularly because some root exudates boost the resistance of nearby
plants to herbivory. One of the phytotoxic (poisonous to the plants) compounds pro-
duced by the couch grass Elytrigia repens is carboline which inhibits the nearby
plants (Glinwood et al., 2003). Besides this, the plant secretes some root exudates
to defend against herbivores indirectly by luring predators and parasites that attack
it. When Phaseolus lunatus (lima bean) is attacked, its root induces volatile com-
pound production to attract the mites which attack spider mites and undamaged
P. lunatus plants (Guerrieri et al., 2002). Defense root exudates produce a diverse
and flexible protective coating of organic molecules in the rhizosphere.
9.Future viewpoints
Through confirmation of substantial dissimilarities in the microbial societies among
species and cultivators, exudates’ composition and consequently their ability to
modulate the rhizobium depend on some biotic and abiotic factors. Currently,
very little information is known about how specific plant root exudates affect the
microbiome to enhance plant health and well-being, and growth. When investigating
rhizosphere metabolites, it is challenging to find a sample’s composition that pre-
cisely corresponds to the root exudates in situ because root exudates are influenced
9. Future viewpoints 361

by a variety of elements, particularly root microbes. Studying plant-plant and plant-
microbe communications controlled by root exudates is most difficult because roots
are hidden underground. Studying root exudation requires an understanding of both
the structure and function of a root system, in addition to an extensive analysis of the
rhizospheric communal. It is important to consider the variety and richness of plant
species as well as the functional mixture and duplication that exist in microbial
groups (Bais et al., 2002b). One of the main goals of comprehending the mechanism
controlling plant-microorganism interference is to identify and regulate the plant
rhizobiome, enabling the full potential development of the plant (Hirsch et al.,
2013). As the field of functional genomics expands and is used, among other things,
to examine the arrangement and purpose of the genomes of the rhizobiome, a Func-
tional Genomics Database has been created to characterize genetic markers that help
organisms acclimate to plant ecosystems (Levy et al., 2018). The metabolomics and
genome sequencing instruments advancement have the potential to help gain a better
understanding of the genetic underpinnings of exudates and microbiological absorp-
tion priorities and, consequently, how and when to regulate them (Dreyer et al.,
2012).
Utilizing the new techniques will therefore enable the delivery of compounds to
areas where root exudates are scarce and the modification of both plant and micro-
bial traits, leading to the development of new crop management techniques
(Fig. 16.2).
FIGURE 16.2
Schematic overview of the root exudates and their effects on plant health.
362 CHAPTER 16 Feedback effect of root exudates

10.Summary
Through the production of different signaling molecules that serve a variety of pur-
poses to maintain the rhizospheric integrity, plants contribute to the stability of the
rhizobiome. Constructive microorganisms, in detail, assist and support plant growth,
by, among many other factors, adapting root systems, acquiring nutrients and min-
erals, and defending against pathogenic microorganisms. The rhizobiome conse-
quently makes a significant contribution to the nutrition of the plant. Its complete
evidence that plants require more or fewer fundamentals for optimal growing and
enlargement is provided by the rhizobiome. To find out how to make these rhizo-
spheric microbes more effective for ongoing crop production, more research should
be done on how they communicate with each other and include their host species.
Due to the biological system’s inherent complexity, little is known about how root
exudates affect the rhizosphere microbiome and how this affects plant fitness and
health. Even though the fact that this aspect of plant-microbe exchanges is still rela-
tively new, investigations concentrating on methodologies of plant secretions and
operational verification of these secretions from plants must be a priority. Addition-
ally, methodologies for studies into plant exudates and secretions should be devel-
oped. The findings obtained from these experimental studies can assist in the
identification and characterization of responsive genes that are responsible for the
overexpression of these metabolic byproducts. Targeting these genes will enable bet-
ter plant breeding. Due to the intricate nature of multicellular operations in response
to environmental changes, uncovering these exudations mechanisms and data vali-
dation can be a frustrating challenge. However, merging leading and topmost inves-
tigations to understand plant-microbe interconnections and bacterial community
structure has tremendous potential for multiomics, modeling techniques, imaging
practices, and recombinant methods. To address the issues of food security, next-
generation agriculture can be developed sustainably using these fundamental princi-
ples, and ideas and these advanced approaches soon will make the investment
worthwhile.
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Ecotypic adaptation of
plants and the role of
microbiota in ameliorating
the environmental
extremes using
contemporary approaches
17
Mohan Singh Rana
1
, Jyoti Ranjan Rath
2
, Chejarla Venkatesh Reddy
3
,
Sangay Pelzang
4
, Rahul G. Shelke
5
, Smit Patel
6
1
Department of Environment and Life Sciences, Sherubtse College, Royal University of Bhutan,
Kanglung, Bhutan;
2
Department of Plant Sciences, School of Life Sciences, University of
Hyderabad, Gachibowli, Hyderabad, Telangana, India;
3
Department of Civil Engineering,
National Institute of Technology, Farmagudi, Ponda, Goa, India;
4
Department of Life Sciences and
Biotechnology, South Asian University, New Delhi, India;
5
Pepthera Laboratories Pvt Ltd., DL-3,
Atal Incubation Center, Gujarat Technological University, Chandkheda, Ahmedabad, Gujarat,
India;
6
Department of Biotechnology, Amity University, Kant Kalwar, Kant, Rajasthan, India
1.Introduction
An ecotype of a plant is a group of plants that has evolved to thrive in a certain
habitat. Native plant adaptation to environmental factors is gaining more attention,
especially in light of climate variability and the microbiota that accompany it. In
plant evolutionary ecology, differentiating genetic from environmental variation
has necessitated the establishment of rigorous experimental methodologies. Tures-
son first used ecotype in 1922 to describe plant populations uniquely adapted to spe-
cific environments. A healthy plant has a good amount of taxonomically diverse
microbiota associated with them. Plant scientists today are still expanding upon
his legacy of using garden experiments to learn how natural selection creates variety
within plant species. These ecotypes were distinguished from one another by a set of
observable characteristics that had a genetic basis and were present in common gar-
den experiments (VanWallendael et al., 2022). Understanding the mechanisms un-
derlying plant-microorganism coadaptation is critical for sustainable agriculture.
However, the impact of the local adaptability challenge faced by ecotype on rhizo-
sphere microbiota association remains to be understood. To achieve the objectives of
climate-smart agriculture, particularly sustainable crop production, addressing this
knowledge gap is a crucial prerequisite. There is a growing understanding that plant
CHAPTER
377
Rhizobiome. https://doi.org/10.1016/B978-0-443-16030-1.00005-5
Copyright ©2023 Elsevier Inc. All rights reserved.

microbiota plays a role in plant growth and can protect plants from invading patho-
gens. Plant microbiota has received a lot of attention in recent years, and plant phe-
notypes are increasingly being recognized as the result of multipartite interactions.
Microbial interactions can help plants learn and adapt to edaphic stress (Santoyo
et al., 2021). Plants also aid in the organization of the soil’s microbial community.
Their interactions with the plant can be beneficial or harmful. Furthermore, these mi-
crobes can cooperate or compete with one another. The phenotypic difference may
emerge along ecological gradients due to natural selection on trait diversity together
with environment-driven malleable alterations. Plants must adapt to geographical
environmental stresses. Individual plants’ capacity to adapt differently to changes
in the environment often results in adaptation mechanisms. For example, two eco-
types of a species that are locally suited to different settings interact between plant
roots and soil microorganisms associated with them differently by colonizing new
habitats and recruiting rhizosphere communities that are phylogenetically related.
Regardless of the increasing elevation, low-elevation ecotypes typically produced
higher chemical defenses (Bakhtiari et al., 2019). Important microbes with Fe-
and Zn-solubilizing properties can be used in agriculture to biofortify micronutrients
in various cereal crops. Understanding population differentiation and the emergence
of new species is critical for identifying natural selection patterns and mechanisms
in plant ecotypes. Environmental extremes like fast land degradation, deficiency in
soil water, climate change, pests, diseases, heavy metals, and salt stress have signif-
icantly affected crop production worldwide. A microbiome is the collection of mi-
croorganisms found in the soil environment. They engage in a variety of plant
growth promoting activities, such as fixing, mineralizing, solubilizing, and mobi-
lizing nutrients, producing siderophores, antagonistic substances, antibiotics, and
releasing substances that stimulate plant growth, like auxin and gibberellin hor-
mones, through interactions with the roots of the host plant (Egamberdieva et al.,
2017). Many plant species create symbiotic associations with microorganisms and
profit from mineral nutrient supplies while expending minimal energy. The abun-
dance of root exudates, crop species, and cultivars determine the distribution of these
plants (Suman et al., 2016). By producing antioxidant enzymes and accumulating
osmolytes like proteins, proline, glycine betaine, phenolic compounds, and soluble
sugars, plants adjust their cellular osmotic and water potential in response to envi-
ronmental factors and activate their natural defense mechanisms. Phytohormones
regulate developmental processes and signaling networks, enabling plants to
respond to biotic and abiotic stresses, thereby ensuring their survival. Understanding
the synthesis of ethylene, salicylic acid, and jasmonates has advanced our under-
standing of their functions in plant responses to abiotic stress. Other plant hormones,
including auxin, gibberellic acid, brassinosteroids, abscisic acid, and peptide hor-
mones, have been associated in a variety of ways with the signaling pathways
used by plants to defend themselves (Suman et al., 2022). Over time, interactions
between plant-associated microbes and their host may have favoured the synthesis
of biologically active secondary metabolites. The analysis and identification of
the microbial community that coexists with plants are now possible as a result of
378 CHAPTER 17 Ecotypic adaptation of plants

advancements in molecular, analytical, and next-generation sequencing technolo-
gies. With the advent of cutting-edge and novel high-throughput sequencing tech-
nologies like genomics, metabolomics, metagenomics, and proteomics datasets
are becoming increasingly available for accessions cultivated in geographically
dispersed ecotypes (Pais et al., 2021). Datasets from barley (Bedada et al., 2014)
and avocado (Ge et al., 2019) genomes provide valuable insights into understanding
the effects pronounced by ecotypes. Efforts to improve plant tolerance to such
extremes through breeding and genetic engineering techniques have yielded limited
results. It is, therefore, necessary to find a sustainable alternative means for agricul-
ture production, especially under extreme conditions.
In this chapter, we summarize the major recent findings and cutting-edge
methods in plant ecotype and related microbiota research in the context of traits
that are advantageous for nutrient uptake and plant health.
2.Plant ecotype and the associated microbiota
An ecotype, also known as ecospecies, is a geographical variation, population, or
race that belongs to a species and is genotypically suited to a particular environment.
Majority of the time, despite the phenotypic variations that ecotypes show due to
environmental heterogeneity, they may interbreed with other ecotypes that are
located nearby without losing their fertility or vigor (Molles, 2015;Turesson,
1981). Ecotypic diversity within a species is frequently the outcome of natural selec-
tion. This has turned out to be a rather common occurrence in both annual and peren-
nial grasses, trees, shrubs, and plants (McMillan, 1960). The microbiota, a varied
group of microbes, coexists with plants in living systems. These microorganisms
either reside inside plant tissues (the endosphere) or outside (the episphere). All
of the microorganisms present in an environment, such as bacteria, viruses, and
fungi, are considered as the microbiota as a whole. Microbes significantly influence
the ecology and physiology of plants. The community structure and diversity of the
plant microbiota are influenced by a number of variables relating to the host, micro-
organisms, and environment (Dastogeer et al., 2020). All multicellular species have
a variety of microbial associations commensal, symbiotic or parasitic (De Sordi
et al., 2019). By modifying themselves with the aid of internal or external factors
by creating ecotypes plants can adapt to biotic difficulties, and edaphic stresses
such as nutrient deficiency, toxicity, etc (Young et al., 2018). For plant health and
the ability to respond to abiotic stress, plants maintain a strong relationship with
the soil microbes in their immediate surroundings (Cavicchioli et al., 2019). Several
elements, including the soil’s pH, moisture level, temperature, humidity, root
exudate, nutritional values, and human activities affect the organization of the soil
microbial population (Daniel, 2005). Primarily, two types of microorganisms live
on plants endophytes and epiphytes (Whipps et al., 2008). In the form of leaf litter
and root exudates, plants appear to be the major source of carbon and, consequently,
energy for soil microbes. In response, plant adaptability is promoted by soil
2. Plant ecotype and the associated microbiota 379

microbes by the release of growth regulators or by enhancing the production of
phytohormone (Gouda et al., 2018). The plant often exchanges hexose sugars
with the fungal symbiont for inorganic phosphate in mutualistic circumstances
(Chibucos &Tyler, 2009).
In plants growth promoting bacteria (GPB) provide critical functions to plants
such as nitrogen fixation, mineral solubilization, manufacture of plant hormones,
direct improvement of mineral absorption, and pathogen defense. GPBs may
compete with the pathogen for an ecological status or a substrate, and produce antag-
onistic compounds, or develop immunity in host plants to the pathogen in order to
protect plants (Bloemberg &Lugtenberg, 2001). Root-associated microorganisms
can improve the capacity of plants’ drought tolerance systems by influencing their
physiology and biochemistry. It has been demonstrated that interactions between
plants and microbes in the rhizosphere increase host resistance to stress and plant
growth (Fig. 17.1). Plant-associated microbial activities include the production of
biofilms, control of hormone levels, osmotic regulation, enhancement of antioxida-
tive enzymes, facilitating water and food ingestion, and management of gaseous ex-
change (Sarkar Soumyadev et al., 2022). It has been discovered that when drought
stress occurs, the fungal community evolves more than the bacterial population (He
et al., 2020).
Two superior ecotypes of coconut, Bedakam, and Annur from Kerala, India,
were identified for drought and salinity resistance (Rajesh et al., 2014). Four Crocus
FIGURE 17.1
Symbiotic interaction between plants and microbes is present in the environment or soil.
There is a continuous cycle in nature where the plants provide carbohydrates and
micronutrients that is taken up by the microbes, while they fix the nitrogen and other
minerals in the soil benefitting the plant.
Created with BioRender.com.
380 CHAPTER 17 Ecotypic adaptation of plants

sativus ecotypes from Mediterranean regions were studied for enhanced adaptation
(Cardone et al., 2021). The ecotypic adaptations for pH tolerance, notably lignin
production in limestone ecotypes in conjunction with nonarbuscular mycorrhiza,
are also verified in the wild grass Holcus lanatus by transcriptome analysis (Young
et al., 2018). The development of plant ecotypes is substantially influenced by the
surroundings. Locally adapted Mimulus guttatus ecotypes differ genetically in vari-
ables influencing rhizosphere ecosystems (Bowsher et al., 2020). In Arabidopsis
thaliana leaves, 19 significant microbiota were found that are vital to the composi-
tion of microbial communities (Almario Juliana et al., 2022). In the rhizospheres of
three ecotypes of the major prairie grass (Andropogon gerardii), bacterial and fungal
communities were examined. It was discovered that the composition of bacterial
populations varied across the A. gerardii ecotypes, but not microbial communities
(Sarkar Soumyadev et al., 2022). Domestication and breeding selection have grad-
ually distinguished current crop microbiota from those of their wild counterparts. A
rhizosphere assessment of four domesticated and 20 wild barley (Hordeum vulgare)
genotypes indicated unique eco-geographical restrictions imposed by their host
plants (Alegria Terrazas et al., 2020). Bacterial communities colonize several plant
species’ roots, which are unique even when hosts share the same environment. The
study of Lotus japonicus and A. thaliana in a multispecies culture condition revealed
signs of host preference among commensal bacteria in a community context (Wippel
et al., 2021).
Research on the microbial population architecture of Arabidopsis ecotypes has
shown that microbes have also been shown to be helpful to plants during cold stress
(Etemadi et al., 2018). Acinetobacter and Citrobacter were considerably enriched in
rhizosheath soil following drought stress, indicating that drought stress promotes
rhizosheath microbial aggregation. Furthermore, ecotype-specific rhizosheath
microbiome recruitment revealed heterogeneity in drought stress responses in
Switchgrass (Liu, Ye, et al., 2021;Singer et al., 2019). The bacterial microbiome
of Donggang pasque-flower (DPF) was more varied in the wild than in farmed plants
in the karst habitat. Despite some significant variations, both wild and domesticated
DPF plants typically have the same fundamental bacterial microbiome that served as
endophytes. Plants profited from the detected bacterial strains via nitrification,
nutrient uptake, or phytohormone synthesis, resulting in noticeable growth differ-
ences in A. thaliana (Dutta et al., 2021). Gupta et al. (2020) looked into using
two rhizosphere-growing strains of Pseudomonas fluorescens to lessen Alternaria
brassicae caused mustard blight. In Switchgrass (Panicum virgatum), plant-
associated microbiota is enriched in Alphaproteobacteria and Actinobacteria
(Singer et al., 2019). In the case of Cd hyperaccumulators, members of the Burkhol-
deriaceae and Sphingomonas families shared microbiota cores (Luo et al., 2022).
Among root microbes, the family Enterobacteriaceae was observed to be predomi-
nant in overall response to highland climates in case of rice ecotypes (Pang et al.,
2020).
2. Plant ecotype and the associated microbiota 381

3.Secondary metabolites associated with microbiota
Plant secondary metabolites (PSMs) are a diverse group of structurally different
compounds based on their biosynthetic pathways. They are estimated to be around
100,000 compounds, and more compounds are being discovered rapidly. Secondary
metabolites are classified into various classes: Flavonoids, alkaloids, steroids, ter-
penes, and phenolics (Kessler &Kalske, 2018). Secondary metabolites are produced
in plants in response to different environmental stimuli, the interaction of various
microbes, and also against pathogenic microorganisms. Microbial colonization
sometimes might lead to alterations in the biosynthetic pathways of a particular
metabolite, which might lead to the synthesis of new metabolites (Huang et al.,
2014). Microbes that colonize in the internal plant tissues known as endophytes
are rapidly increasing and creating a plethora of enormous plant metabolic pathways
which might result in various metabolic syntheses of secondary metabolites. It has
been reported that poplar plants inoculated with Paenibacillus sp. showed increased
levels of asparagine, urea, and threitol.
Enterobacter ludwigii has been reported to show a significant increase in vanillic
acid and low levels of catechin, esculin, arbutin, astringin, pallidol, ampellopsin, D-
quadrangularin, and isohopeaphenol. Secondary metabolites (Fig. 17.2)suchas
calexin and glucosinolates have been reported to show an increased level of production
against encounters with pathogenic microbes Pseudomonas syringae py tomato (Pst)
FIGURE 17.2
Plants produce secondary metabolites as a defense mechanism against stress and
pathogens. Some beneficial microbes induce the plant to produce secondary metabolites,
which increase the value of the plant. PSMs exhibit antimicrobial, antiviral, and
antioxidant properties.
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382 CHAPTER 17 Ecotypic adaptation of plants

(Brotman et al., 2012). Plant secondary metabolites (Table 1 7. 1) can act as abiotic
stress response mediators; their interaction with microbe can help in better intake of
nutrients and water and lower the generation of reactive oxygen species (ROS) by
increasing the production of root peroxidases (Naylor &Coleman-Derr, 2018).
4.Mechanism of action (nutritional absorption and plant
health)
Plant ecotypes are unique species that evolve from the interaction with their local
environment. They are the outcome of genetic adaptations to certain ecosystems.
Table 17.1 Plant-microbe interaction.
Plant
Secondary
metabolite Microbe References
Artemisia annua Artemisinin Pseudonocardia sp. Li et al. (2012)
Catharanthus
roseus
Vindoline,
serpentine, and
ajmalicine
Staphylococcus sciuri
Micrococcus sp.
Tiwari et al.
(2013)
Vindoline Curvularia sp. and
choanephora infundibulifera
Pandey et al.
(2016)
Bookoo kush,
Burmese, maui
wowie, white widow,
and sour diesel
Cannabidiol Pseudomonas,Cellvibrio,
Oxalobacteraceae,
Xanthomonadaceae,
Actinomycetales, and
Sphingobacteriales
Winston et al.
(2014)
Rice plant p-Coumaric and
ferulic acid
Azospirillum sp.
B510
Yasuda et al.
(2009)
Maize cultivars
(PR37Y15 and
DK315)
Zoxazinoids A. brasilense
CFN-535, Az. lipoferum
CRT1 and A. brasilense
UAP-154
Walker et al.
(2011)
Rice cultivars
(cigaron and
nipponbare)
Cigaron Azospirillum lipoferum Chamam
et al. (2013)
Taxus globosa and
Taxus media
Taxane Pseudomonas syringae Ramirez-
Estrada et al.
(2015)
Ambrosia
artemisiifolia
Thiarubrine A Protomyces gravidus Bhagwath &
Hjortsø
(2000)
Hyoscyamus
muticus
Sesquiterpenes Rhizoctonia solani Singh et al.
(2014)
Cichorium intybus Coumarin Phytophthora parasitica Bais et al.
(2000)
4. Mechanism of action (nutritional absorption and plant health) 383

Studies have demonstrated that the root-associated microbiota is dynamic and re-
sponds to various abiotic and biotic stimuli in various settings (Liu, Wang, et al.,
2021). Mutualistic microbes help with nutrient uptake, may increase stress tolerance
in leaves, and are partitioned differently across different parts of roots, according to
studies on ecotypic plant (Switchgrass, Panicum virgatum) pathogens (VanWallen-
dael et al., 2022). The microbial communities in soil are incredibly diverse and
include a range of microorganisms, including bacteria, viruses, fungi, algae, and
archaea. Most of these microbes primarily use nutrients found in plant roots, like
root exudates and secondary metabolites (Subrahmanyam et al., 2020). Flavonoids,
strigolactones, and terpenoids are substances in plant root exudates used in subter-
ranean chemical communication techniques. Strong differences were found in the
microbiomes of various plant species and accessions, which led to the theory that
exudates play a key role in determining plant-microbe interactions. However, it
has been demonstrated that plants in particular draw advantageous interaction part-
ners via signals originating from their roots (Jacoby et al., 2017).For Switchgrass
plants, microorganisms in the soil are essential for sustaining plant health and pro-
ductivity, controlling soil fertility, and cycling nutrients (Wagg et al., 2014). In an
experiment involving reciprocal transplants, two ecotypes of Mimulus guttatus
that are locally suited to different settings, it was discovered that interactions be-
tween plant roots and soil microorganisms significantly affect plant health and pro-
ductivity, demonstrating that diverse M. guttatus ecotypes can colonize new habitats
and recruit rhizosphere communities that are phylogenetically related. Nevertheless,
the rhizosphere communities of the two ecotypes differed at both sites, at least partly
because of variations in the presence or absence of microbial taxa, particularly un-
common (low abundance and low occupancy taxa (Bowsher et al., 2020). In ecotypic
plant production systems, proper assessment of mutualistic plant-microbe interac-
tions could boost yields, promote nutrient uptake and stress tolerance, and provide
ecological integrity including carbon sequestration and improved soil biodiversity
(Hestrin et al., 2021). Abiotic stress tolerance, food uptake, and defense against plant
pathogen infection are major purposes mediated by the rhizosphere microbiome. Re-
searchers consequently need a fundamental understanding of the molecular mecha-
nisms and the nature of the rhizosphere microbiome. Direct and indirect
mechanisms can categorize the underlying mechanisms (Subrahmanyam et al.,
2020).The most frequent direct method produces phytohormones, ACC deaminase,
nitrite synthesis, sulfide oxidation, organic phosphate mineralization, and inorganic
phosphorus solubilization. In contrast, indirect mechanisms include quorum sensing
signaling interference; induced systemic resistance; biofilm formation; competition
for nutrients and space, synthesis of antibiotics, antimicrobial compounds, or lytic
enzymes; and inhibition of quorum sensing signaling (Dı
´az-Rodrı
´guez et al.,
2021).Increased nutrient absorption may be associated with growth-promoting ac-
tivities. It was proposed that better nutrient availability and uptake could partially
account for the improved plant development seen in all stages of plant growth pro-
moting rhizobacteria (PGPR)-inoculated plants (Calvo et al., 2019).
384 CHAPTER 17 Ecotypic adaptation of plants

5.Nutritional absorptions by bacteria
The absorption of vital nutrients and micronutrients from the soil is facilitated by
bacterial chemical synthesis. Additionally, these bacteria contribute to the produc-
tion of plant growth regulators such as IAA (indole-3-acetic acid), deaminase, and
ACC, which aid in enhancing plant growth. These growth enhancing substances
accelerate growth and stop stress-induced ethylene from impairing plant growth
excessively. Additionally, the bacteria aid in the adsorption of iron and zinc, the sol-
ubilization of phosphorus and potassium, atmospheric nitrogen fixation, and the
production of plant hormones (Fig. 17.3)(Munir et al., 2022). Plant growth promot-
ing rhizobacteria (PGPR) and other helpful bacteria (such as actinomycetes) are
combined in bacterial-based plant biostimulants (BSs) such as Acetobacter,Agro-
bacterium,Azospirillum,Azotobacter,Bacillus,Burkholderia,Enterobacter,
Frankia,Pseudomonas,Rhizobia,Serratia,andStreptomyces, which support root
formation, growth, and stress tolerance. To sustain plant nutrition, diazotrophs
(i.e., N
2
-fixing bacteria) are crucial, especially when resources are scarce. Using
nitrogenase, a protein found in rhizobial bacteroids, to convert atmospheric
elemental nitrogen into ammonia, symbiotic rhizobia can meet up to 90% of the ni-
trogen needs of the host legumes. It has also been demonstrated that a number of
nonsymbiotic diazotrophic and N-fixing bacterial species increase plant uptake of
FIGURE 17.3
Bacteria induce plant growth regulators such as IAA (indole-3-acetic acid), deaminase,
and ACC and inhibit ethylene. N
2
-fixing bacteria convert N
2
from the air and produce
NH
3
. Arbuscular mycorrhiza increases tolerance to stress such as drought and salinity. It
also regulates Na
þ
and K
þ
pump. Fungi increase the soil organic matter regulating plant
hormones.
Created with BioRender.com.
5. Nutritional absorptions by bacteria 385

nitrogen (Bardi &Malusa
`, 2012). Nitrogen can alter the concentration of cellular
nitrates (NO
3
-
), which alters the hydrodynamic characteristics of root cell mem-
branes. This causes water potential gradients, which boost water transport from
the soil into root cells. Since aquaporins are variably expressed in plant roots in
response to nitrogen availability; this might also be connected to aquaporins. The
expression of the roots-specific aquaporin genes OsPIPs and OsTIPs, which are
linked to root hydraulic conductivity, can be increased by nitrogen input. The
NRT2.1 nitrate transporter might control a process like this because it was discov-
ered to influence the transcript abundance of PIP aquaporins in Arabidopsis,which
is also associated with root hydraulic conductivity. Additionally, it has been
claimed that ammonium (NH4
þ
), as opposed to nitrate, can enhance the drought
resilience of ecotypic plants (Kang et al., 2022). Some Gram-positive microorgan-
isms, like Thermoleophilia and Actinobacteria, can enhance the transition of the
lowland rice ecotype from paddy field to upland (Xiong et al., 2021).
6.Nutritional absorptions by fungi
Following inoculation of seedlings with rhizosphere microflora from mature Switch-
grass stands, seedlings experienced significant increases in shoot and root weights as
well as in N and P uptake (Brejda et al., 1998). For example, Trichoderma species
increased root-to-shoot signaling by promoting the biosynthesis of several hor-
mones. These hormones improve nutrient solubility, uptake, and assimilation. It
has been shown that Arbuscular Mycorrhizal (AM) can increase salt tolerance
through a number of mechanisms, including altering the ratio of K
þ
/Na
þ
in plant
cells, ion salt transfer to the vacuole, production of growth hormones, and improve-
ment of soil and rhizospheric conditions, as well as improvement of photosynthetic
efficiency or water-use efficiency. Additionally, AM acts as an osmoregulator by
boosting sugar and electrolyte concentrations to reduce the negative consequences
of salt stress. In a series of studies, AM increased antioxidant capacity by turning
on the plant glutathione-ascorbate cycle, which induced resistance to salt stress
(Koza et al., 2022). Under drought stress, the increased influence of AMF on root
morphology was noticeably more pronounced, demonstrating the greater signifi-
cance of mycorrhizas in root adaptation. The mycorrhizal contributions to the ab-
sorption of P were greater than the contributions to the uptake of N and K among
the three nutrient uptakes examined. The primary nutrients that affect plant cell
structure and function for growth patterns are phosphorus and nitrogen, whereas
K
þ
levels influence the upregulation of plant defense, osmolyte accumulation, anti-
oxidant profiles, and enzymatic regulation of plant metabolism. Transcriptome re-
sponses in the plant Holcus lanatus (L.) suggested that AM and non-AM fungi of
some Ascomycota genera may contribute to P and Fe nutrition in limestone soil,
whereas other non-AM Ascomycota, particularly those related to Phialocephala,
may improve plant N and K nutrition, increase tolerance to metal(loid) ions in
acid bog soils, and facilitate ecotype adaptation (Young et al., 2018). The three
386 CHAPTER 17 Ecotypic adaptation of plants

sorghum (Sorghum bicolor) ecotypes showed that increased GS (glutamine synthe-
tase) activity in the roots and leaves and GDH (glutamate dehydrogenase) activity in
the roots might be evidence that the NO
3
-
ingested by AMF can be transported
directly to root cells for utilization and integration into organic structures. It has
been claimed that these enzymatic changes can also improve plant development
and health (Anass et al., 2021).
7.Role of the microbiota in amelioration of environmental
extremes
Microbiota associated with plants plays a crucial role in ameliorating environmental
extremes such as disease pest and other environmental stresses.
7.1 Disease and pathogens
The world’s fragile agroecosystems are under a great deal of stress due to the need to
boost agricultural production on a base of land resources that is constantly diminish-
ing and degrading. It is thought that soil microbial diversity is crucial for preserving
the sustainability of agricultural production systems. Microbial diversity and
ecosystem processes are intricately linked. A microorganism aids a plant’s develop-
ment, productivity, and adaptability (Yadav et al., 2018). Crop-related microbes can
be divided into three types, such as rhizospheric, phyllospheric, and endophytic mi-
crobes. The area of soil that is influenced by roots through the release of substrates
that have an impact on microbial activity is known as the rhizosphere. Numerous
microbial species from the genera Azospirillum, Alcaligenes, Arthrobacter, Acineto-
bacter, Bacillus, Paenibacillus, Burkholderia, Enterobacter, Erwinia, Flavobacte-
rium, Methylobacterium, Pseudomonas, Rhizobium, and Serratia (Lavania et al.,
2006;Shah et al., 2017) have been linked to the plant rhizosphere. Endophytic bac-
teria inhabit the tissues of plants without actually harming the host. Endophytes can
persist at the site of entry or spread widely throughout the plant. These microorgan-
isms may exist in the circulatory system, within cells (Jacobs et al., 1985), or in
intercellular gaps (Patriquin &Do
¨bereiner, 1978). Numerous genera of endophytic
bacteria, including Achromobacter, Azoarcus, Burkholderia, Enterobacter, Glucona-
cetobacter, Herbaspirillum, Klebsiella, Microbiospora, Micromonospora, Nocar-
dioides, Pantoea, Planomonospora, Pseudomonas, Serratia, Streptomyces, and
Thermomonospora are present. They have been categorized into different host plants
such as wheat, chickpea, pea, maize, rice, common bean, and soybean (Yadav et al.,
2018). The phyllosphere is a typical environment for bacterial and plant synergism.
The most adapted bacteria live on leaf surfaces because they can withstand high tem-
peratures (40Ce55C) and UV rays. Numerous bacteria have been identified in the
phyllosphere, including Agrobacterium, Methylobacterium, Pantoea, and Pseudo-
monas. Crop-associated microbes can stimulate plant development. Epiphytic, endo-
phytic, and rhizospheric plant microbiomes have been shown to directly support
7. Role of the microbiota in amelioration of environmental extremes 387

plant growth through nitrogen fixation, the solubilization of minerals like phos-
phorus, potassium, and zinc, and the production of siderophores and plant growth
hormones like cytokinin, auxin, and gibberellins.
The microbiome plays a significant role in plant disease emergence and spread
(Erlacher et al., 2014). The two biggest classes of plant pathogens, fungi and bacte-
ria, are discussed as the root causes of plant ailments. It is believed that host fitness is
inversely connected with the severity of a disease. Additionally, relationships be-
tween plant-fungal pathogens could be both race- and race nonspecific. Race-
specific interaction is documented for several leaf infections, such as rust or powdery
mildew fungus (Sacrista
´n&Garcı
´a-arenal, 2008), whereas, race-non-specific inter-
action is recorded for a variety of soil-borne pathogens. Numerous infections prolif-
erate on plant surfaces through tissues, while others live inside plant tissues. As plant
infections are often investigated primarily on damaged plants, our current under-
standing of the factors influencing the ecology of plant pathogens is still quite
restricted. Numerous plant pathogens are either cultivable or have diagnostic
methods for those that are known to be obligate pathogens but cannot be grown.
Despite the significant advancements in cultivation-dependent methods, not all mi-
croorganisms found in natural habitats can be grown (Bai et al., 2015). A micro-
biome imbalance (dysbiosis) of the soil microbiota is also expected to play a role
in certain diseases, like replant diseases, for which no pathogen has yet been discov-
ered. Regardless of a plant’s mode of life, pathogens can interact with a wide range
of plants (biotroph, necrotroph, obligate, or facultative).
Fungal pathogens can adopt a different nonpathogenic lifestyle when growing on
many other nonhost plant species in contrast to their endophytic pathogenic lifestyle
in a host plant. Verticillium dahliae, a fungus plant pathogen, was discovered to
frequently reside in the endosphere in many plants (Go
¨tz et al., 2006), and it is
employed for biological control. Another intriguing example is Rhizoctonia, which
can have major effects on various crops and is partially host-specific. The prevalence
of potential pathogens on plants begs the question of whether microbial diversity is
always necessary for the emergence of certain “microbiome ailments.” Shifts in the
microbiome can result from microbial invasions, and endophytes may play a role in
disease control in a similar way. Mendes Rodrigo et al. (2007) noted the endophytic
isolates of the Burkholderia cepacia suppressing Fusarium moniliforme on sugar-
cane. By interfering with quorum sensing, microorganisms can also prevent the in-
duction of harmful genes; otherwise, pathogens would have to compete with
microbes for plant resources. In addition, by creating antagonistic chemicals in plant
tissue or on the surfaces of the plants, microorganisms can directly affect the defense
response of the plants (Fravel, 1988). The microbes which support diseases
frequently come from the Enterobacteriaceae family (Erlacher et al., 2014), which
is well-known for its ability to degrade plant tissues. Biblical scriptures mention
crop rotation as one of history’s earliest plant protection techniques. Crop rotation
is a very effective way to prevent many ailments. This is due to the plant micro-
biome’s high level of specificity, which increases the overall microbial diversity
in the soil.
388 CHAPTER 17 Ecotypic adaptation of plants

Monitoring the relationship between the plant microbiome and diseases has sig-
nificant potential in the future. Microbiome-wide association studies in human med-
icine discovered complex connections between the microbiota and the environment
as the initial keys for creating microbiome-based precision diagnostics and treat-
ments (Gilbert et al., 2016). This can be advocated as a worthwhile goal for the
development of soil health and the closely related promotion of plant health.
7.1.1 Water
Abiotic stress is anticipated to rise due to changing precipitation patterns caused by
climate changes in the availability of nutrients essential for plant growth due to
intensive land use and increased fertilizer use (Trenberth, 2011). Understanding
how the root microbiome reacts to such environmental changes could be a crucial
step in the development of microbial methods to assist plants to raise their stress
tolerance. These tactics could involve the identification and selective introduction
of new PGP microbes into farming areas or the active management of soil commu-
nities through agricultural practices that support plant abiotic stress tolerance
(Schlaeppi &Bulgarelli, 2015). Scientists still don’t fully comprehend the relative
contributions and intricate interactions between soil and plant variables that result
in the root microbial communities which further complicate the matter (Naylor &
Coleman-Derr, 2018). Here, we discuss how water stress, such as flooding and
drought, affects plants that influence the root microbiome. Land plants, especially
crops, suffer when submerged for an extended period, and it is anticipated that floods
will become more frequent and severe. One of the most severe stresses plants expe-
rience is when nonphotosynthetic plant tissues, such as roots, are submerged. This
causes oxygen levels to drop, which prevents cellular respiration (Voesenek et al.,
2006). Understanding how microbial populations respond to flooding is crucial
for the potential acquisition of PGP bacteria or fungi that may help confer stress
tolerance to plants subjected to hypoxic or anoxic growth conditions. For instance,
it has been shown that a variety of bacteria produce the enzyme 1-
aminocyclopropane-1-carboxylate (ACC) deaminase, which is used to control
ethylene levels in plants. ACC is ethylene’s immediate precursor that is produced
by plants. Plant damage from stress is decreased by bacteria that produce ACC-
deaminase because they cleave ACC and limit ethylene. This was demonstrated in
basil (Ocimum sanctum), where plants inoculated with ACC-deaminase-generating
bacteria benefited from enhanced growth and decreased ethylene levels when grown
in damp soil compared to uninoculated plants (Barnawal et al., 2012). Similar to this,
Pseudomonas putida UW4 which produces ACC-deaminase was inoculated into cu-
cumber (Cucumis sativus) plants, and the plants were then grown in hypoxic condi-
tions. Proteomic profiling of the cucumber plants revealed a transition in the protein
profile toward the proteins involved in nutrient metabolism, defense stress, and anti-
oxidant activity, potentially explaining the growth-promoting mechanisms of the
bacteria (Li et al., 2013). Despite the demonstrated ability of plant-beneficial mi-
crobes to flourish in flooded environments, very few studies solely concerned with
bacterial communities have examined root-associated microbial community
7. Role of the microbiota in amelioration of environmental extremes 389

responses in nonwetland plant species exposed to flooding. For instance, rootless
bulk soil, rhizosphere, and whole root (rhizoplane and root endosphere) samples
from poplar (Populus sp.) seedlings subjected to an experimental flood revealed a
significant shift in bacterial community composition in the rhizosphere and whole
root compartments, compared to bulk soil (Graff &Conrad, 2005). Similar results
were found in a study of wheat (Triticum aestivum), where flooding and N restriction
reduced the abundance of some denitrifying bacteria and shifted the makeup of their
community in the rhizosphere in comparison to bulk soil samples taken away from
the physical influence of roots (Hamonts et al., 2013). The transition from aerobic to
facultative to stringent anaerobes is a result of changes in oxygen availability. It was
speculated that this shift toward anaerobic bacteria could be one reason for the rise in
the relative abundance of Aquaspirillum in flooded poplar rhizosphere and root sam-
ples (Whitman et al., 2015). Many plants develop unique, gas-filled tissues called
aerenchyma in response to floods, which help move oxygen from the still -oxic
shoots to the anoxic roots. Such tissue has been observed in significant crop species
including rice (Oryza sativa), barley (Hordeum vulgare), maize (Zea mays), wheat,
soybean (Glycine max), and sugarcane (Saccharum spp.). Such tissue is well devel-
oped in aquatic and wetland plants, but in nonwetland plant species it typically only
forms as a stress response to environmental conditions, like flooding (Hartman &
Tringe, 2019). To avoid cell damage brought on by floods, plants have also been
shown to release phytotoxic substances including ethanol, lactic acid, and alanine,
which accumulate in root tissue of tomato, pea, and maize in response to aerobic
respiration under low oxygen conditions (Badri &Vivanco, 2009). In light of the
likelihood that agricultural environments will experience more flooding in the
future, more focus should be placed on how agricultural plant species respond to
flooding. Furthermore, additional studies are needed to fill in knowledge gaps about
how root exudates change when various crop species are subjected to flooding
growth conditions, how these exudates affect the diversity and composition of bac-
terial and fungal communities in roots, and the implications for plant growth and
function.
In drought conditions, AMF can promote plant drought fitness through improved
nutrition or more direct effects on stomatal conductance and enhanced water use ef-
ficiency. However, the outcomes are frequently AMF species-specific (Auge
´,2001).
Studies describing drought-induced changes in nonmycorrhizal, root-associated
fungi are scarce. Those that do exist have found that these fungi are either unrespon-
sive to drought or show negligible changes possibly because bulk soil fungi have
also been reported to be generally unresponsive to drought conditions. Reports of
exceptions to this rule undoubtedly demonstrate that specific fungal communities
may be drought-sensitive and that factors other than soil moisture content affect
these communities. Similarly, recent research by Santos-Medelln Christian et al.
(2017) found that rice exposed to drought experienced significant root-associated
fungal community rearrangement. However, inadequate taxonomic resolution pre-
vented the distinction of specific drought-responsive taxa. Therefore, more investi-
gation is required to determine how particular fungal taxa react to dryness in bulk
390 CHAPTER 17 Ecotypic adaptation of plants

soil and plant roots. Many recent studies describing the root bacteria microbiomes of
rice, sorghum, and diverse lineages of plant species under drought stress have re-
ported enrichment of bacteria from the phylum Actinobacteria. The relative abun-
dance of Actinobacteria, particularly the genus Streptomyces, in root endosphere
communities increased roughly sixfold under drought conditions, according to a
study of 30 genetically diverse plant species that were subjected to drought or wa-
tering treatments, although the results varied by plant species (Fitzpatrick et al.,
2018). As soils dry out, diffusion channels that carry water-soluble substances be-
tween soil particles and bacteria are diminished or eliminated, and water potentials
drop, putting microbes under osmotic stress (Schimel, 2018). To reduce their inter-
nal solute potential and prevent losing water to their surroundings, bacteria are
assumed to store osmolytes inside of their cells. Gram-positive bacteria are expected
to accumulate osmolytes both naturally and in response to drought, which increases
their tolerance to osmotic stress (Harris, 1981), and their cell wall layer is thought to
increase their resistance to desiccation, which further increases their tolerance to
drought (Schimel et al., 2007). Actinobacteria are therefore physiologically suited
to drought environments, and this competitive advantage also enables them to
flourish in the droughted root microbiome. However, current data emphasize plants’
functions in modulating drought responses in the root-associated microbial popula-
tion. It is interesting to note that drought only significantly changed the makeup of
the bacterial community in soils where grassland plant communities were present.
These alterations were caused by greater relative abundances of Actinobacteria in
droughted soils. The observed changes in the root microbiome may be explained
by changes in root metabolites brought on by drought. Changes in the root metab-
olomic profile were connected with a shift in the relative abundance toward
Actinobacteria-dominated communities in sorghum grown under drought circum-
stances. Drought-affected roots had a considerable increase in numerous carbohy-
drates and amino acids, with the molecule glycerol-3-phosphate (G3P), a crucial
precursor to peptidoglycan production, exhibiting the greatest enrichment. Further,
it has been demonstrated that the transcriptional activity of root microbiome constit-
uents, including recognized PGP bacteria, actively reacts to plant stress situations.
According to Sheibani-Tezerji Raheleh et al. (2015), the root endophytic bacteria
Burkholderia phytofirmans colonizing potato (Solanum tuberosum) plants that
have been exposed to drought has up-regulated genes that are probably involved
in the detoxification of reactive oxygen species (ROS). However, during periods
of drought, photosynthetic pathways can change, and ROS generation can rise to
levels that cause oxidative damage and cell death (Cruz de Carvalho, 2008). Thus,
the intriguing thought is that activation of these bacterial genes may be a common
trait in the bacterium intended to reduce oxidative stress in its plant host
(Sheibani-Tezerji Raheleh et al., 2015).
Therefore, it is essential to continue characterizing crop species genomically to
understand the interactions between hosts and microbes that regulate the PGP mech-
anisms of bacteria and fungi and the genetic factors that control root microbiome
assembly and function under droughted growth conditions. The development of
7. Role of the microbiota in amelioration of environmental extremes 391

new crop cultivars designed specifically for improved responsiveness to the admin-
istration of particular microbial inoculants could result from advances in this
understanding.
7.1.2 Temperature
Global temperatures are rising, and many countries are experiencing more frequent
and severe heat and chilling temperatures as a result of climate change. These
anthropogenic pressures seriously jeopardize the health of plants and their ability
to produce crops. The effects of biotic and abiotic challenges on plant survival are
moderated by the microbiome associated with plants (Fig. 17.4). However, changes
in the content and activities of plant microbiota brought on by climate change may
impact host functioning. Additionally, there is a greater need to understand how to
exploit the microbiome to increase crop yields and reduce losses brought on by envi-
ronmental challenges due to the high demand for food and the ever-growing
population.
FIGURE 17.4
Bacteria such as Enterobacter and Burkholderia reduce temperature stress.
Aquaspirillum protects plants from water logging and salinity by regulating the osmotic
pressure. Mycorrhiza and some PGPR bacteria break down heavy metals. They also
behave as a protective barrier or immune boosters protecting the plant from pathogens.
Created with BioRender.com.
392 CHAPTER 17 Ecotypic adaptation of plants

The temperature in the environment has an impact on the soil’s moisture levels,
aggregation, pH, and nutrient diffusion, and these modifications also affect plants
and microorganisms (Onwuka &Mang, 2018). Numerous plant metabolic processes
are altered by heat stress, including the formation of ROS, modification of phytohor-
mone signaling, reduction of photosynthetic rate and respiration, inactivation of pro-
teins, and alteration of fluidity and permeability of the cellular membrane. Cold
stress also impacts plants (Ding et al., 2019). A change in temperature affects the
composition and structure of nucleic acids, proteins, and membranes in the micro-
biota. These changes affect the microbes’ physiological functions (Zhang &Gross,
2021). The combined effects of heat and drought stress on the bacterial population in
the roots of sorghum were studied by Wipf et al. (2021). Different Actinobacteria
were enriched for drought and heat, suggesting that sorghum plants recruit different
microbiota members under drought and heat stress. Even though single heat and
drought stresses increased the comparative abundance of Actinobacteria and
reduced the frequency of Proteobacteria in roots and soil. The processes by which
various bacteria are recruited by plants and the impact of these microbial assem-
blages on plant heat stress tolerance require more investigation. According to
some studies, microbiota help plants tolerate heat better. The mechanisms for this
improvement include enhanced plant growth and nutrient uptake as well as reactive
oxygen species detoxification, which reduce cellular damage (Shekhawat et al.,
2021). For example, in moderate and hot temperatures, B. cereus boosted soybean
growth and chlorophyll content. Heat-induced increases in ABA were mitigated
by B. cereus injection. Improved heat tolerance following inoculation with the
AM fungus Glomus fasciculatum was linked to increased antioxidative activity
(Maya &Matsubara, 2013). These results suggest that bacteria may increase plant
heat tolerance, although the precise mechanism by which microbes do this is still
largely unknown. The laboratory- and open-field-grown wheat plants were more
tolerant of heat after receiving an injection of the root endophyte Enterobacter sp.
SA187. Furthermore, via modifying the trimethylation of lysine 4 on histone H3
(H3K4me3), which is a constitutive change, in the promoters of the heat-stress genes
APX2 and HSP18.2, SA187-induced thermotolerance in A. thaliana was shown to
be mediated by ethylene signaling. These epigenetic changes would prime, but
not necessarily activate, a heat stress response harmful to plant growth. These
show how useful root endophytes are for improving the ability of agricultural crops
to withstand heat stress. Ethylene signaling contributes to the ability of tomatoes and
rice to withstand heat stress (Pan et al., 2019). These findings add credibility to
ethylene signaling involvement in SA187-induced thermotolerance.
Microbes can also improve plants’ resistance to cold. Some plant physiological
changes linked to microbial inoculation have been described, despite processes be-
ing poorly understood. As an illustration, Burkholderia phytofirmans inoculation
increased carbon fixation and the accumulation of starch, proline, carbohydrates,
and phenolics, which improved their ability to withstand cold temperatures (Ait
Barka Essaid et al., 2006). Additionally, AM fungus strengthened plants’ resistance
to cold stress (Caradonia et al., 2019). For instance, under normal and cold-stress
7. Role of the microbiota in amelioration of environmental extremes 393

circumstances, the inoculation of tomato with G. mosseae improved the plant’s
growth, chlorophyll content, and antioxidative enzyme activities. Malondialdehyde
buildup brought on by cold was reduced by G. mosseae inoculation, indicating that
G. mosseae lessens oxidative damage to lipids brought on by cold stress. AMF are
members of the monophyletic phylum Glomeromycota. AMF are considered oblig-
atory symbionts because, in order to complete their life cycle, they require to ingest
carbon from their host plants (Abdel Latef &Chaoxing, 2011). Employing plant-
environment-symbiont connections as a basis for agricultural management would
enable farmers to harvest crops with higher yields and better performance. So, sym-
biosis and agricultural management are essential for enhancing crop tolerance and
performance.
8.Conclusion and future prospect
Burkholderia phytofirmans,Citrobacter, Enterobacter, and Acinetobacter are some
of the most common microbes associated with plant ecotypes. Plant ecotypes asso-
ciated with microbiota are species-specific. These microbes have been linked to
plant ecotypes that are favored by key factors such as soil and climatic conditions.
The benefits of their harmonious relationships have been extensively studied, partic-
ularly the secondary metabolic productions and health benefits brought to the asso-
ciated plants. However, exploiting their relationships to address environmental
issues in the face of climate change remains unexplored. Plant microbiota could
be a viable option for improving plant nutrition and health. Inoculation of such mi-
crobes in crops could boost efforts toward sustainable agriculture while also playing
a role in microbial remediation.
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402 CHAPTER 17 Ecotypic adaptation of plants

Ecological and structural
attributes of soil
rhizobiome improving
plant growth under
environmental stress
18
Ali Reza Mirzaei
1
, Bahman Fazeli-Nasab
2
, Moharram Valizadeh
3
1
Department of Agronomy and Plant Breeding, Faculty of Agriculture and Natural Resources,
University of Mohaghegh Ardabili, Ardabil, Iran;
2
Department of Agronomy and Plant Breeding,
Agriculture Institute, Research Institute of Zabol, Zabol, Sistan and Baluchestan, Iran;
3
Research
Center of Medicinal Plants, University of Sistan and Baluchestan, Zahedan, Sistan and
Baluchestan, Iran
1.Introduction
Stress is an unpredictable limitation or fluctuation that is imposed on the natural
metabolic behavior of the plant and causes injury or disease. This phenomenon is
caused by factors that disturb the balance. The flexibility of natural metabolisms
causes the response to environmental changes (Gaspar et al., 2002). There are
many environmental factors that have negative effects on the growth and develop-
ment of plants. Lack and excess of food, high and low temperature, drought, and
salinity are the most important environmental limitations in the production of plant
products around the world (Shaddad, 2010). The term tension is derived from the
Latin word Stringere, which means imposed force or forced force. From the plant’s
point of view, the external pressures that limit the dry matter production ratio of the
whole or part of plant growth are called stress (Amozadeh &Fazeli-Nasab, 2012;
Fazeli-Nasab &Amozadeh, 2012;Khan et al., 2001;Shirazi et al., 2016). In other
words, stress is an unpredictable limitation or fluctuation that imposes on the regular
metabolic patterns of the plant and leads to disease, injury, or aberrant physiological
conditions (Jaleel et al., 2009). Environmental stress and air pollution have signifi-
cant harmful effects on humans, animals, and plants, but their negative effects on
plants are more significant, because plants, unlike other organisms, live in one place
and cannot change their place of living in adverse environmental conditions (Zhu,
2002). Stress is divided into two categories of living and nonliving stresses that
negatively affect plant growth and production. Living stresses include the attack
of pests and diseases on plants, and nonliving stresses include stresses such as
lack of water, salinity, and temperature, which are widespread worldwide and cause
CHAPTER
403
Rhizobiome. https://doi.org/10.1016/B978-0-443-16030-1.00003-1
Copyright ©2023 Elsevier Inc. All rights reserved.

great damage to plants. Stress causes changes in plant morphology, physiology, and
genetics (Jaleel et al., 2009).
2.Drought stress
Drought stress is one of the most important abiotic stresses and one of the biggest
global environmental restrictions for agriculture in arid and semi-arid regions, which
has caused a significant decrease in the yield of plant products and has affected food
security all over the world. Severe water stress may lead to the stop of photosyn-
thesis, metabolic disorder, and ultimately the death of the plant (Fujita et al.,
2005;Jaleel et al., 2009). Drought stress refers to a period without significant rainfall
and generally occurs when the available water in the soil decreases and atmospheric
conditions cause continued water scarcity through evaporation and transpiration.
Drought tolerance has been seen in almost all plants, but with different levels
from one species to another and even within a species (Gaspar et al., 2002). Drought
is the most important nonliving stress in crops. Drought is a global problem that
threatens agricultural production both quantitatively and qualitatively, and with
the increase in world population and climate change, it has created very critical con-
ditions in the world. Such tension affects the performance of products and often
causes a drop in it (Andersson et al., 2004;Damghan, 2009). Due to the unpredict-
ability of the occurrence, intensity, time, and duration of drought, as well as its inter-
action with other nonliving stresses, especially salinity and cold stress, drought
stress is one of the most damaging factors in agriculture (Jaleel et al., 2009). In order
to survive environmental stress, plants respond to stress and adapt. Drought stress
induces a range of physiological and biochemical responses in plants. These re-
sponses include the closing of the stomata, stopping the growth of cells and photo-
synthesis, activating respiration, and inducing the expression of genes related to
stress; many of these factors are caused by the signals that are induced as a result
of soil dryness (Seki et al., 2007;Zhu, 2002). In the conditions of salt and drought
stress, the plant hormone abscisic acid is produced quickly and plays an essential
role in the response and tolerance of these stresses, which is why it is known as
the stress hormone (Bray, 2004). The expression of many inducible genes by abiotic
stresses is regulated by the hormone abscisic acid. Genes that respond to drought,
salt, and cold stress are also induced by applying abscisic acid hormone treatment
(Azad et al., 2017;Fazeli-Nasab et al., 2012a,2012b;Shinozaki et al., 2003).
Drought resistance engineering in plants is of great economic importance. In order
to develop new strategies, one of the most important goals of research related to
plant drought tolerance is to understand the mechanisms involved in drought resis-
tance. Drought triggers various responses in plants. These responses include changes
in gene expression, accumulation of metabolites such as plant hormones including
abscisic acid, and an increase in the amount of osmotically active compounds
such as highly hydrophilic proteins, proteins that absorb oxygen radicals, and chap-
erone proteins (Shinozaki et al., 2003).
404 CHAPTER 18 Ecological and structural attributes

3.Salinity stress
The increasing spread of soil salinity, which is one of the most important obstacles to
the optimal and quality production of crops, has resulted in efforts of producing
plants that tolerate salinity stress (Katiyar-Agarwal et al., 2005). New developments
in the rhizosphere along with increased knowledge about ion transporters and stress
signaling proteins have led to a greater understanding of the mechanism of ion bal-
ance and its regulation in plants. Also, a lot of research has been done to identify ion
transporters and regulatory mechanisms that play a role in Na
þ
ion balance and
maintain the cytoplasmic K
þ
/Na
þ
ratio (Katiyar-Agarwal et al., 2005). More than
half of the inducible genes by drought stress are also induced by salinity stress or
abscisic acid treatment, which is the reason for the significant interaction between
plant responses to drought stress, salinity, and abscisic acid (Shinozaki et al.,
2003). Transgenic plants tolerant to salinity show tolerance to other stresses such
as cold, frost, heat, and drought (Katiyar-Agarwal et al., 2005).
4.Abscisic acid hormone (ABA)
Abscisic acid (ABA) was first recognized as a plant growth inhibitor. Additional
studies have attributed different physiological roles in plants, such as the stimulation
of bud and seed dormancy, separation of leaves and fruits from the plant, senescence,
and resistance in plants under stress, to this hormone. The researchers stated that the
reason for the adaptation of tomato, potato, winter wheat, spinach, and Arabidopsis
plants to drought or cold is the increase in abscisic acid level. Abscisic acid appears
to regulate plant responses under various conditions. Abscisic acid is weakly
involved in germination and accumulates during drought stress and changes the tran-
scription level of a number of genes. The level of this hormone increases during wa-
ter stress and therefore it has been stated that ABA is a mediator of water-plant
communication (Go
´mez et al., 1988).
According to the role of the ABA hormone, the genetic pathways of response to
abiotic stresses in plants are divided into ABA-dependent and ABA-independent
pathways (Fig. 18.1). There is an overlap between abscisic acid-dependent and
abscisic acid-independent regulatory pathways in drought stress tolerance. In these
regulatory pathways, there are several transcription factors that control gene clusters
(Buitink et al., 2006). A transcription factor can control the expression of several
target genes by binding to specific sequences in their promoters. This type of tran-
scriptional regulatory system is called regulon. Examining the expression of induc-
ible genes by stress has shown that there are at least four different regulatory systems
to respond to stress, which are (Nakashima &Yamaguchi-Shinozaki, 2005):
1. DREB regulator
2. NAC and ZF-HD regulators
3. AREB regulator (ABF)
4. MYC/MYB regulator
4. Abscisic acid hormone (ABA) 405

DREB regulator, NAC regulator, and ZF-HD are independent of abscisic acid,
and do not respond to abscisic acid and cold treatment. MYC/MYB and AREB
(ABF) regulators are dependent on abscisic acid. By controlling the expression of
the regulatory system, it is hoped to improve the tolerance of plants to environmental
stresses (Nakashima &Yamaguchi-Shinozaki, 2005).
Although soil is a valuable and nonrenewable ecological system, it has always
been subject to widespread degradation due to human activities such as transporta-
tion, urban sprawl, and illegal dumping or landfilling (Pietrelli et al., 2020;Vo c -
ciante &Meshalkin, 2020). In some cases, the entry of heavy metals, mineral
oils, polycyclic aromatic hydrocarbons, etc., into the soil is associated with
FIGURE 18.1
Inducible regulatory networks in environmental stress (Yamaguchi-Shinozaki &
Shinozaki, 2005).
406 CHAPTER 18 Ecological and structural attributes

dangerous effects to the environment and natural resources and endangers human
health. Therefore, remediation of contaminated soils and places is an important
step in protecting the environment and living organisms. Today, various methods
have been developed to deal with polluted water (Puri et al., 2021;Reverberi
et al., 2018). The use of modified plant species that remove soil and water contam-
inants can be very effective, but this method requires the consumption of raw mate-
rials and sometimes the use of chemicals that inevitably affect the plant. But another
method is phytoremediation. Phytoremediation is a cost-effective and sustainable
technology used to clean up pollutants and enable plant species to make good use
of soil and water resources. Plants are able to use the right bacteria and excrete con-
taminants that reduce growth using root secretions. Meanwhile, plant growth pro-
moting rhizobacteria (PGPR) have a great effect on facilitating plant growth even
when the plant is in critical condition. In addition, PGPR in agriculture can be an
excellent support to counteract the destructive effects of abiotic stress, such as exces-
sive salinity, and drought, and replace expensive mineral fertilizers that are harmful
to the environment (Conte et al., 2021;Pedron et al., 2017,2021). In addition, phy-
toremediation biomass can be converted into bioenergy as well as support, leading to
biodiversity and soil stabilization (Pedron et al., 2017,2021). In studying the phy-
toremediation system, understanding how the host plant is organized is useful and
necessary. Also, root secretions play an important role in the composition of the mi-
crobial community (Vives-Peris et al., 2020). Studies in Zea mays and Solanum lyco-
persicum have shown that these two plants absorb more beneficial and active
bacteria due to their root secretions. These studies show that the rhizosphere has
different chemical and physical properties that change with different microbial com-
munities, plant status, and plant species, making the rhizosphere one of the most
complex ecosystems (Phour et al., 2020). In fact, the host plant can choose the
most suitable one through complex mechanisms. Some studies consider this choice
the result of joint evolution between the plant and microbiomes. Another recent
study shows that the intervention of hydrocarbon-contaminated sediments improves
the activity of microorganisms and increases the activity of rhizosphere microbial
communities (Truyens et al., 2015). In this chapter, we examine the effects of Plant
Growth Promoting Rhizobacteria (PGPR) and their role in improving stress condi-
tions and increasing plant adaptation to stressful conditions.
5.Properties and potential of plant growth promoting
rhizobacteria (PGPR)
Plants react to changes in ethylene hormone levels in the face of various environ-
mental and biological stresses such as salinity, cold, heat, and heavy metals.
Ethylene is produced in the soil by biological and nonbiological mechanisms
(Saghafi et al., 2013). Rhizosphere bacteria can prevent the increase of ethylene con-
centration in plants during stress and reduce the negative effects of this hormone on
5. Properties and potential of PGPR 407

the growth and development of plant organs, especially roots. PGPR regulate
ethylene production by producing the enzyme deaminase-ACC. This enzyme indi-
rectly and mainly by reducing the level of stress ethylene in the plant causes plant
growth to continue (Grichko &Glick, 2001). Researchers also investigated the effect
of different strains of PGPR on modulating the effects of salinity stress on a variety
of plants (Shukla et al., 2012), Arachis hypogea (Zhang et al., 2007), soybeans
(Cheng et al., 2007) rapeseed (Mayak et al., 2004), tomato (Nadeem et al., 2007),
corn (Han &Lee, 2005), lettuce (Marcelis &Van Hooijdonk, 1999), radish (Zahra
et al., 2011), peanuts (Saravanakumar &Samiyappan, 2007). The researchers attrib-
uted the ability of different strains to increase stress resistance to the decrease in
ethylene activity by the deaminase-ACC enzyme in these bacteria. When the plant
is exposed to drought and salinity stress, it responds to stress through rhizosphere
microorganisms that create protective mechanisms. In fact, plants are subjected to
a complex network of functional interactions created by PGPR to avoid the effects
of stress. The beneficial molecules and activities described in Fig. 18.2 communicate
with the roots and form a support network for plant survival.
PGPR are soil bacteria that live in the rhizosphere and are in direct contact with
the roots and are involved in enhancing plant growth and secretion by secreting
various regulatory molecules. These bacteria interact with the host plant within plant
tissues and exert their beneficial effects much more effectively (Fig. 18.3)(Giauque
et al., 2019).
PGPRs are able to, directly and indirectly, affect plant growth and cope with
stress. Direct effects include nitrogen fixation, facilitating the uptake of nutrients
FIGURE 18.2
Complex and efficient network of functional interactions created by PGPR (Vocciante
et al., 2022).
408 CHAPTER 18 Ecological and structural attributes

from the plant growth medium, production or release of secondary metabolites, side-
rophores, ACC deaminase, and effects of growth regulators or hormones such as
auxins, cytokinins, gibberellins, and the like. However, the indirect effect of
PGPR) on plant growth stimulation occurs when the harmful effects of one or
more pathogens are reduced or prevented altogether. Some of the most common in-
direct mechanisms are the production of hydrogen cyanide, antibiotics, and enzymes
that are able to do this and destroy the cell wall of pathogens (Olanrewaju et al.,
2017).
PGPRs, in addition to coexisting with rhizobium bacteria, contain other benefi-
cial rhizosphere bacteria such as the genus Bacillus,Pseudomonas aeruginosa,Ace-
tobacter,Enterobacter,Herbaspirillum,Azospirillum, and many other unknown
bacteria.
FIGURE 18.3
Simplified scheme of the main activities of plant growth promoting bacteria (PGPR) and
their interaction with the root system (Vocciante et al., 2022).
5. Properties and potential of PGPR 409

It was observed (Dobbelaere et al., 2003) that inoculation of plants with a variety
of bacteria capable of producing auxin resulted in longer roots, longer lethal fibers,
and more rootlet branches than controls. Growth retardation can be due to changes in
the balance of plant growth regulators (phytohormones) due to stress. Some strains
of PGPRs are able to increase plant growth through the concentration of known phy-
tohormones. These phytohormones affect the root growth pattern of the plant and
cause it to produce larger roots, with more branching and more effective surface
area. Therefore, the treatment of plants with growth-promoting bacteria (as a gener-
ator in regulating plant growth) as a reciprocal factor on stress-affected plants is used
to improve the effects of nonbiological environmental stresses.
6.Siderophores
There are four types of siderophores chemically: catecholates, phenolates, hydrox-
amates, and carboxylates. Because iron is often present in the soil as a trivalent insol-
uble hydroxide, plants cannot absorb it easily. Small siderophore molecules are
produced and secreted by PGPR and have a very high affinity for iron and facilitate
the absorption of iron in plant cells (Saha et al., 2016).
7.Phosphate solubilization
Phosphorus is an essential element for plant growth, but is rarely available to plants.
Phosphorus is present in the soil as organic (50%) and inorganic (50%), both
of which are insoluble and therefore cannot be absorbed by the roots. What soil
bacteria can do is essential for the soil. In fact, the bacteria convert phosphorus
into H
2
PO-4 and HPO-24 b y dissolution, which can be used by plants. This action
is mainly associated with a decrease in pH due to the production of low molecular
weight organic acids. The availability of sufficient amounts of phosphorus following
phosphate dissolution activity can lead to a significant reduction in the use of
chemical fertilizers (Alori et al., 2017).
8.Nitrogen fixation
Nitrogen is also an essential element for plant growth. Nitrogen fixation is the pro-
cess by which nitrogen in the atmosphere (N
2
) is converted to ammonium (NH
4
).
Seventy-eight percent of nitrogen is not absorbed by the soil. This element is essen-
tial for the production of amino acids and proteins. Biological nitrogen fixation
(BNF) is an enzyme reaction that is catalyzed by the enzyme nitrogenase and con-
verts nitrogen into useable ammonia for the soil. Because the basic structure of
almost all biomolecules, including the amino acids of proteins, DNA, RNA, and
410 CHAPTER 18 Ecological and structural attributes

nucleic acids, contains nitrogen, nitrogen fixation is necessary for the survival of all
living organisms. Some organisms in the nitrogen cycle stabilize it. Autotrophs play
this role in nature. In addition to carbon stabilization, green plants also stabilize ni-
trogen (Aasfar et al., 2021;Sickerman et al., 2019).
9.Auxins, cytokinins, gibberellins
Phytohormone IAA is the most common auxin among plant-related bacteria and
plays a major role in plant-bacterial interactions (Luo et al., 2018). Strengthening
the roots, especially capillary roots and secondary roots, is one of the main functions
of this hormone, which ultimately leads to increased root secretion. Cytokinins play
a major role in vascular development and embryogenesis. It also plays an important
role in responding to environmental changes and stresses (Hamza &AL-Taey,
2020). Gibberellins are involved in the transport of metabolites and in the formation
of chloroplasts and leaf aging. They also cause cell division and stem morphogenesis
(Rizza &Jones, 2019).
10.ACC Deaminase
ACC Deaminase is one of the most important direct mechanisms in PGPRs. Inacti-
vating the precursor ethylene ACC, and the production of ammonia and alpha keto-
butyrate do the action of this enzyme. High levels of ethylene can inhibit plant
growth and even kill them. Environmental stress increases the activity of ACC syn-
thase and ACC oxidase and ultimately increases ethylene. PGPR help plants reduce
the negative effects of stress by increasing the enzyme ACC deaminase, and by
increasing adaptation, help the plant survive under stress (Glick, 2014).
11.Effectiveness of PGPR in hydrocarbons and heavy
metals contaminated soils
PGPR through nitrogen fixation, nutrient enhancement, inhibition of ethylene pro-
duction, direct production of plant hormones, and increase in oxygen concentratio-
n,in general, can help break down organic pollutants and eliminate the harmful
effects of pollutants. PGPR include two types of root-related bacteria that remain
near or on the root surface and are known as rhizosphere bacteria. Those that suc-
ceed in entering the root tissue are called endophytes. Because endophytes are pro-
tected by roots, they can protect the host from the harmful effects of contaminants
(Lumactud et al., 2016).
Organic compounds are not the only toxic pollutants for plants. Heavy metals
can also cause huge changes in the chemical-physical properties of the soil. Heavy
11. PGPR in hydrocarbons and heavy metals contaminated soils 411

metals are often present as insoluble salts. Plants need to create a hemostatic
network to control oxidative stress caused by the presence of heavy metals
(DalCorso et al., 2019). To do this, plants stimulate physiological and molecular
mechanisms, such as the active transport of ions, and direct these substances from
the roots to cellular vacuoles (Ma et al., 2016). Chemicals such as ethylenediamine-
tetraacetic acid (EDTA), ethylenediamine-N, N-disuccinic acid (EDDS) and plant
growth-promoting bacteria (PGPR) increase the transfer of these substances to the
vacuole and reduce the toxic effects of heavy materials (Mishra et al., 2017).
The reduction of heavy metals by this method has been observed in the following
plants: Bacillus pumilus (Ma et al., 2016), Rhodococcus erythropolis (Liu et al.,
2015), Bradyrhizobium sp. (Guo &Chi, 2014), Ralstonia eutropha,Chryseobacte-
rium humi (Moreira et al., 2014).
Plants inoculated with PGPR had a significantly lower accumulation of reactive
oxygen species (ROS) and showed higher levels of antioxidant enzymes, especially
peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and ascorbate
peroxidase (APX). Other studies have shown that in inoculated plants, photosyn-
thetic pigments, the number of leaf shoots, and root length increased (Raklami
et al., 2019).
12.PGPR to face salinization and drought facing the abiotic
stresses
The effect of PGPR in conditions of abiotic stress caused by drought, salinity, and
soil alkalinity is critical for plant growth (Enebe &Babalola, 2018). Prolonged
drought conditions cause plant growth promoting bacteria to secrete phytohor-
mones, osmolytes, and antioxidant molecules that can induce structural and morpho-
logical changes in the root that ultimately increase the plant’s stress tolerance. The
development and branching of the root system in drought conditions causes the root
tip to reach the deeper layers of the soil and absorb the necessary materials. In this
way, the plant adapts to adverse conditions and provides its water and minerals
(Misra et al., 2020).
12.1 Drought and salinity pressure
Utilizing plant-microbial interactions can have a positive effect on salinity and
drought response. Plants have a convincing strategy against drought and in general
against all abiotic stresses, and those are the roots that show high structural and
morphological flexibility. Drought leads to significant changes in the phospholipid
composition of the root membrane. Under conditions of water stress, many PGPRs
produce the hormone ABA, which is able to regulate the plant. Increased ABA hor-
mone leads to control of stomatal closure, reduction of leaf transpiration, and conse-
quently water retention in the plant in drought conditions (Vurukonda et al., 2016).
However, the main function of PGPR is to increase the elasticity of the roots, which
412 CHAPTER 18 Ecological and structural attributes

allows new roots to penetrate into the soil to reach deeper water basins (Koevoets
et al., 2016). Another activity of PGPR in drought is the storage of salts in plant cells
through osmotic regulation, which leads to an increase in the accumulation of sub-
stances such as the amino acids proline, glycine, phenylalanine, organic acids,
sugars, and mineral ions which can protect plants against possible oxidative damage
(Gagne
´-Bourque et al., 2016).
Another nonliving factor is soil salinization, which is a growing global threat.
Salinity stress can drastically reduce the productivity of various products. High con-
centrations of salt often damage the soil, especially organic matter (Schirawski
Perlin, 2018). Salinity stress can drastically reduce the productivity of various
products. The phenomenon of salinization is related to all salts. These salts include
sodium chloride (NaCl), sodium sulfate (Na
2
SO
4
), magnesium sulfate (MgSO
4
), and
magnesium chloride (MgCl
2
). However, NaCl is certainly the most vital,
causing excessive salinity and biochemical and physiological damage to the plant
(Flowers &Colmer, 2015). Numerous microorganisms in the soil are able to tolerate
the presence of salt even at high concentrations, often belonging to the bacteria
Bacillus,Pseudomonas agrobacterium,Enterobacter,Klebsiella,Streptomyces,
and Ochromobactrum. In the presence of high salinity, plants are subjected to
significant stress, which leads to several adverse effects. These include reduced
photosynthetic activity, limited nutrient uptake, and destruction of cell membranes,
and plant dehydration, which can lead to plant death (Zhang et al., 2018).
Abiotic stresses such as drought and salinity have a strong impact. The selection
of stress-resistant species and the understanding of the biochemical mechanisms un-
derlying it are certainly very effective. However, without a doubt, PGPR can have
the greatest impact on reducing plant damage due to salinity and drought (Ma
et al., 2020). In general, PGPR can increase plant salinity tolerance through the accu-
mulation of osmolytes; increasing the absorption of nutrients; nitrogen fixation; the
solubilization of phosphorus and other essential elements; ACC deaminase activity;
production of auxins, siderophores, and exopolysaccharides (Saghafi et al., 2019).
13.Water phytodepurationdconstructed wetlands (CW)
Our planet has huge reserves of water, but only 3% of fresh water is available in the
world. This means that about three billion people have limited access to water re-
sources for daily use. The increase in global temperature has caused the drying of
rivers, springs, and sources of fresh water in the world. Many rivers, lakes, and un-
derground water tables can no longer support different ecosystems, and feeding the
population has unfortunately faced a serious problem (Carvalho et al., 2017, p. 397).
Meanwhile, new strategies are needed to reuse and improve water. Constructed wet-
lands (CW) systems are systems that operate on the basis of water reproduction and
treatment (Fig. 18.4). Plants can significantly improve water quality by removing
pollutants from water. Rhizobacteria that stimulate plant growth in the conditions
of drought, salinity, and the presence of pollutants minimize plant metabolism
13. Water phytodepurationdconstructed wetlands (CW) 413

and reduce pollutants, especially heavy substances in the plant (Alavi et al., 2022;
Backer et al., 2018;Dhuldhaj &Malik, 2022;Rajkumar et al., 2012).
The most critical PGPR activities beneficial to support and to alleviate environ-
mental stresses (salinity, heavy metals, hydrocarbons) are the production of phyto-
hormones, 3-indole acetic acid (IAA), abscisic acid (ABA), cytokines (CKs),
gibberellins (GAs) modulating plant physiology; ACC deaminase (ACCD) activity
lowering ethylene level; activation of antioxidant enzymes (superoxide dismutase,
SOD; catalase, CAT; peroxidase, POD); Naþ(Kþ)/Hþpumps regulating ions ho-
meostasis; production of exopolysaccharides (EPS)); production of osmolytes (i.e.,
proline, glycine, betaine) to stabilize protein conformation (Vocciante et al., 2022).
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420 CHAPTER 18 Ecological and structural attributes

Modulation of rhizosphere
microbial populations
using Trichoderma-based
biostimulants for
management of plant
diseases
19
Efath Shahnaz
1
, Saba Banday
2
, Ali Anwar
2
, M.N. Mughal
2
, G.H. Mir
2
, Qadrul Nisa
2
,
Gazala Gulzar
2
, Atufa Ashraf
2
, Diksha Banal
2
1
Dryland Agriculture Research Station, SKUAST-Kashmir, Rangreth, Jammu and Kashmir, India;
2
Division of Plant Pathology, Sher-e-Kashmir University of Agricultural Sciences &Technology of
Kashmir, Shalimar, Jammu &Kashmir, India
1.Introduction
Trichoderma is one of the most well-studied fungal biocontrol agents with diverse
modes of action and multiple benefits to the plant, soil, and environment. It can
be isolated from soil, air, water; as endophytes of plants; as opportunistic pathogens
of humans and from animals. Besides being used as a biocontrol agent, it finds in-
dustrial applications due to the production of large array of enzymes, for the biore-
mediation of problem soils and as a plant growth biostimulant. It belongs to the class
Hypocreaceae of the order Hypocreales. Although clearly distinguishable on the ba-
sis of microscopic studies, species differentiation is often fraught with errors due to
overlapping characters (Thokala et al., 2021). Dou et al. (2020) suggested MIST as a
multi-locus identification system for Trichoderma. Currently many new species are
being reported on a daily basis with the result that the database is continuously
increasing.
The most common and frequent use of Trichoderma is as a biocontrol agent for
the management of diseases. Almost all types of diseases, be it soil borne or seed or
air borne; or root or foliar diseases are managed by different species of Trichoderma
(Ghazanfar et al., 2018;Al-Ani &Mohammed, 2020;Shahnaz et al., 2022;Ali &
Shahnaz, 2022). Some of the diseases managed by Trichoderma are black spot of
rose, tomato root rot, black foot disease of grapes, rice blast, bacterial wilt of tomato,
charcoal rot of mungbean, sugarcane smut and basal rot of onion (Akhtar &Javaid,
2018;Chou et al., 2020;Faheem et al., 2018;Javaid et al., 2018;Kariuki et al., 2020;
Kashyap et al., 2020;Tegene et al., 2021;van Jaarsveld et al., 2021), besides
CHAPTER
421
Rhizobiome. https://doi.org/10.1016/B978-0-443-16030-1.00013-4
Copyright ©2023 Elsevier Inc. All rights reserved.

innumerable diseases caused by viruses and nematodes. Various species of Tricho-
derma find use in bioremediation of diesel oil spill (Nazifa et al., 2018); copper
contamination (Cuevas &Banaay, 2022); heavy metals in waste water (Hussain &
Mutlag, 2021); and even soils contaminated with explosives or other toxic hazards
like TNT (Alothman et al., 2020), though mostly for bioremediation of saline and
alkaline soils.
Trichoderma has been found to play a major role in the modulation of soil rhi-
zobiome. It can modulate the beneficial microorganisms for enhancing the nutrient
uptake by plants; increase their antagonistic potential; or help them in various
growth promoting properties (Table 19.1). It can also directly modulate the plants
genes for alleviation of biotic or abiotic stresses or production of secondary
Table 19.1 Effect of different Trichoderma species on nutrient uptake by
some plants.
S.
No.
Trichoderma
spp. Plant/Crop Nutrient
Change
direction Reference
1. T. virens (GV41)
or T. harzianum
(T22)
Iceberg lettuce
(Lactuca sativa
L.) and rocket
(Eruca sativa
Mill.)
Ascorbic acid,
N-use
efficiency
Increases Fiorentino
et al. (2018)
2. Trichoderma
atroviride
Arabidopsis Sucrose
transport and
metabolism
Increase Esparza-
Reynoso
et al. (2021)
3. Selected
Trichoderma
strains (T22,
TH1, and
GV41)
Strawberry Anthocyanins
and other
antioxidants
Increased Lombardi
et al. (2020)
4. Trichoderma
asperellum T34
Wheat Phosphorus Increase Garcia-
Lopez et al.
(2018)
5. T. reesei Rice Fe, Co, Cu,
and Mo
Increased Singh et al.
(2019)
6. T. atroviride Vegetables P, Mg, Fe, Zn,
and b
Increased Colla et al.
(2015)
7. T. asperellum
isolate CHF 78
Tomato P, K, Mg, and
Zn
Increased Li et al.
(2018)
8. T. harzianum Sugarcane N, P, K Increased Singh et al.
(2010)
9. Trichoderma
asperellum and
Trichoderma
harzianum
Arabidopsis,
tomato
Fe Increases Martı
´nez-
Medina
et al. (2017)
422 CHAPTER 19 Modulation of rhizosphere microbial populations

metabolites (Table 19.2). In grape, Trichoderma harzianum T39 had a dual effect of
directly modulating the genes related to microbial recognition machinery and
enhancement in the expression of defense-related processes after pathogen inocula-
tion (Perazzolli et al., 2012). T. harzianum isolate ALL-42 also modulated the com-
mon beans plant’s metabolism and triggered its defense response (Pereira et al.,
2014). Plasma membrane (PM) H þ-ATPase activity may be enhanced and MAP
kinase 6 activated (Guo et al., 2020). In some cases, there may not be any significant
effect on the metabolic diversity of the community but there may be change in the
utilization of carbohydrates, complex carbon compounds, and organic phosphorus
compounds (Senkovs et al., 2021).
Table 19.2 Modulation of gene expression by different Trichoderma species.
S.
No. Trichoderma spp. Plant/Crop Genes involved Reference
1. T. hamatum Tomato 45 genes associated with
abiotic or biotic stress and
metabolism
Alfano et al.
(2007)
2. T. atroviride Arabidopsis AtSPS1F, AtSPS2F, and
AtSPS3F
Esparza-
Reynoso
et al. (2021)
3T. longibrachiatum Tomato Genes involved in
photosynthesis,
antioxidant defenses, and
maintenance of a juvenile
state
De Palma
et al. (2021)
4. T. asperelloides Arabidopsis
thaliana,
Cucumis
sativus
WRKY18 and WRKY40 Brotman
et al. (2013)
5. T. asperellum Rice J10sPR10 and LOX-RLL De Sousa
et al. (2020)
6. T. atroviride Arabidopsis HAD-2 Estrada-
Rivera et al.
(2019)
7. T. atroviride Arabidopsis abi1-1 and abi2-1 Contreras-
Cornejo
et al.
(2015b)
8. T. asperelloides Arabidopsis Receptor genes Gupta et al.
(2014)
9. T. erinaceum Tomato WRKY Aamir et al.
(2019)
1. Introduction 423

2.Improvement of soil nutrient uptake
The potential of Trichoderma species to improve the nutrient uptake efficiency has
resulted in a renewed interest in this fungus. Inoculations with Trichoderma are a
viable strategy to increase yields and nutrient uptake efficiency, particularly in leafy
vegetables (Fiorentino et al., 2018). In rice, the application of Trichoderma-based
biofertilizer under nutrient-deficient conditions, increased the uptake of micronu-
trients like Fe, Co, Cu, and Mo and resulted in higher levels of indole acetic acid
and gibberellic acid (Singh et al., 2019). The volatile compounds from Trichoderma
were found to locally trigger a readjustment of iron homeostasis in roots, which was
further linked to systemic elicitation of ISR by priming of jasmonic acidedependent
defenses (Martı
´nez-Medina et al., 2017). Under System of Rice Intensification
(SRI), it was found that inoculation with T. asperellum leads to significant increase
in rice seedling growth, germination rate, vigor index, chlorophyll content, nutrient
uptake, and subsequently yield of plants (Doni et al., 2017). In case of Pinus sylvest-
ris var. mongolica, seedling biomass, root structure index, soil nutrients, and soil
enzyme activity were significantly increased as compared to control after inocula-
tion with T. harzianum and T. virens (Halifu et al., 2019).
3.Adaptation under different climatic conditions
Trichoderma has been found to activate mechanisms and processes in plants that
help them to withstand various types of abiotic and biotic stresses with the result
that they are able to show a better survival rate under adverse climatic conditions.
Arabidopsis wild-type seedlings when inoculated with T. virens and T. atroviride,
showed decreased stomatal aperture and reduced water loss when compared with un-
inoculated seedlings, which can help the plants to adapt better to climatic changes,
particularly water stress (Contreras-Cornejo et al., 2015a). Pretreatment of tomato
plants with T. longibrachiatum had a significant effect on genes involved in mitiga-
tion of stress damage (De Palma et al., 2021). T. reesei NBRI 0716 (NBRI 0716)
modulated the arsenic speciation and improved grain yield and quality of chickpea
as well as prevented diversity loss caused by high arsenic (Tripathi et al., 2015). Un-
der water logging conditions also, Trichoderma can be applied for reaping its bene-
ficial properties. T. harzianum was found to improve plant physiology and
metabolism, maintain plant nutrient status, and improve photosynthetic pigments
and water logging in tomato (Elkelish et al., 2020). Treatment of Arabidopsis and
cucumber with T. asperelloides prior to salt stress imposition significantly improved
seed germination and affected the expression of several genes related to osmoprotec-
tion and general oxidative stress in the roots (Brotman et al., 2013).
424 CHAPTER 19 Modulation of rhizosphere microbial populations

4.Response to plant pathogens
There is continuing and accumulating evidence that points to the fact that plants
respond actively to various beneficial microbes, particularly Trichoderma. However,
the nature of response is highly complex and intricate. In tomato, there was upregu-
lation of genes involved in the ethylene and jasmonate (ET/JA) and salicylic acid
(SA)emediated signaling pathways as well as stimulation of response to Rhizoc-
tonia solani by expression of several defense-related genes (Manganiello et al.,
2018). Tomato seedlings inoculated with T. asperellum reduced disease severity of
wilt caused by Fusarium oxysporum f. sp. lycopersici, and there was a significantly
negative correlation between disease severity and nutrient uptake (Li et al., 2018).
Some species of Trichoderma are more efficient than others under different envi-
ronmental conditions. T. virens was found to be more effective than T. atroviride in
promotion of biomass gain, whereas, both were effective in induction of systemic
resistance against Alternaria solani, Botrytis cinerea and Pseudomonas syringae
pv. tomato through the secretion of proteins (Salas-Marina et al., 2015). Similarly,
T. atroviride was demonstrated to be more effective in growth promotion of tomato
and reduction in the severity of disease caused by Phytophthora nicotianae than
T. asperellum (La Spada et al., 2020).
There is usually a negative correlation between biocontrol agents and severity of
diseases caused by plant pathogens. It has also been found that generally biocontrol
agents do not induce a negative effect on the nontarget microbial communities (Cucu
et al., 2020). Li et al. (2020) found that T. harzianum inoculation reduced the inci-
dence and severity of club root of Chinese cabbage, caused by Plasmodiophora
brassicae, and this reduction coincided with reduction of other pathogenic fungi
like Alternaria and Fusarium, whereas, there was relative abundance of dominant
bacterial genera Delftia and Pseudomonas, and increase in population of other bac-
teria including Bacillus. A consortium of a strain of Bacillus subtilis and a strain of
Trichoderma harzianum were able to cause suppression of common scab disease
caused by Streptomyces spp. in potato and increased tuber yield by increasing the
beneficial bacteria in the rhizosphere (Wang et al., 2019).
Singh et al. (2016) found that the optimum spore load for enhancement in seed
germination and radicle length was different for different vegetable crops, viz. 10
3
spores mL
1
in tomato and ridge gourd, 10
4
spores mL
1
in brinjal and okra, while
10
6
spores mL
1
in chilli and guar. At higher doses there was reduced seed germi-
nation percentage and radicle growth. This implies that spore load, concentration,
viability, and other factors play a significant role in the efficacy and final results ob-
tained from different Trichoderma species.
4. Response to plant pathogens 425

5.Trichoderma-based biostimulants
Trichoderma exerts its beneficial properties through the production of various bio-
stimulants, in addition to its direct effect through mycoparasitism, competition, anti-
biosis, and induction of systemic resistance (Shahnaz et al., 2022). A biostimulant
from T. virens strain GV41 increased marketable yield and biomass production of
lettuce under optimal and suboptimal fertilization, whereas, rocket responded to
T. virens treatment only in absence of fertilization (Visconti et al., 2020).
T. harzianum and other biostimulants were found to positively influence growth,
development, and health of carrot plants along with the promotion of growth of
antagonistic fungi (Patkowska et al., 2020). Similarly, T. saturnisporum increased
the commercial production of melon without any negative effects on fruit quality
(Fernando et al., 2018). However, Silletti et al. (2021) concluded from their studies
that though biostimulants many increase growth and stress tolerance, these re-
sponses are heavily influenced by nutrient availability in soil and environmental con-
ditions. Recently, different Trichoderma species have been used for the production
of biostimulants based on different nanoparticles like selenium (Barbieru et al.,
2019;Joshi et al., 2021), silver (Qu et al., 2021), and zinc (Imran et al., 2022).
6.Conclusion
Trichoderma is a versatile fungi that has ubiquitous presence in various soil systems.
It is easily isolated and cultured on various media and hence finds immense uses in
agricultural systems. Its numerous properties make it a fungi of choice in agricul-
ture, industry, pharmaceuticals, and various other enterprises. It can be used for
the management of various soil and foliar diseases through its biocontrol properties.
Its enzymatic properties make it an excellent fungi for use in food and fabric indus-
tries. It finds use for the amelioration of problematic soils, under changing climatic
conditions as well as for the modulation of soil rhizobiome and genetic modulation.
Inspite of all these benefits, lots of research needs to be done to harness the full ge-
netic potential of this fungi and find its true worth in the changing scenario of today
and tomorrow.
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Multiomics analysis of
rhizosphere and plant
health 20
Tulasi Korra
1
, Thiru Narayanan Perumal
1
, Uday Kumar Thera
2
1
Mycology and Plant Pathology, Banaras Hindu University, Institute of Agricultural Sciences,
Varanasi, Uttar Pradesh, India;
2
Department of Plant Science and Landscape Architecture,
University of Maryland, College Park, MD, United States
1.Introduction
Plants can’t reach most soil minerals. Ninety percent of soil nitrogen in a forest
ecosystem is bound in proteins and humus, limiting plant use. Soil bacteria play
key roles in both the conversion of restricted nitrogen into inorganic form (minerals)
and the solubility of nitrogen in other nutrients including potassium, phosphate, and
iron (Michalzik &Matzner, 1999). Plants secrete organic compounds and solubilize
soil minerals to communicate with microorganisms. Microorganisms and metabo-
lites help plants to communicate with soil. The rhizosphere, rhizoplane, and endo-
sphere are root-system environments. Roots directly influence the microclimate.
It’s where the roots and soil meet each together. Many studies have defined its mi-
crobes, metabolites, and minerals (Gaskins et al., 1990). According to a study, plants
emit around 10% of photosynthates through their roots. The rhizosphere’s high car-
bon content stimulates soil microorganisms, making it a microbiological hotspot and
one of the world’s most active environments. Roots create secondary metabolites,
including antioxidants, for rhizobacteria. Flavonoids interact with nitrogen-fixing
rhizobia and act as antibacterial phytoalexins to prevent root infections. Ca
2þ
,
Mg
2þ
and Fe
2þ
at the root zone tip if their intake exceeds mass flow (Liu &Murray,
2016). Microbial activity affects rhizosphere mineral availability. Chemical stimuli
and positive interactions can affect crop development and reflect physiological sta-
tus. Full knowledge of these parameters is critical for optimal yield and long-term
crop production, especially with environmental changes. The host plant’s activity af-
fects each ingredient. Due to the many links between each component, environ-
mental and molecular mechanisms of development must be examined holistically
using omics data (Timmusk et al., 2011). Recent research shows that multiomics
analysis can boost crop output in agroecosystems. Environmental disruptions may
affect plant growth and agricultural productivity via rhizosphere changes. The rhi-
zosphere’s bacterial population affects mineral availability. A greater understanding
of the root-associated environment and proper management will increase crop yield
in a changing environment (Marschner et al., 2001). This chapter provides an
CHAPTER
433
Rhizobiome. https://doi.org/10.1016/B978-0-443-16030-1.00022-5
Copyright ©2023 Elsevier Inc. All rights reserved.

overview of multiomics in the rhizosphere with data for the rhizosphere soil commu-
nity (Fig. 20.1).
2.Rhizospheric microbe metabolomics
Metabolomics studies organisms of metabolomes at a certain time and condition. A
metabolome contains thousands of biological substrates of primary and secondary
metabolites. These metabolites perform signaling and specialized response roles
(Gachon et al., 2005). The metabolome is far more flexible to environmental
changes than the proteome or transcriptome. Metabolomics studies the metabolic
processes involved in phenotypic responses. Targeted and untargeted metabolomics
exist (i.e., global and unspecific). Targeted metabolomics detects and quantifies pre-
selected compounds (Srivastava, 2019). Internal standards are used to identify and
measure metabolites. Untargeted global metabolomics examines as many metabolic
pathways as possible in a single assay, revealing hundreds of compounds. A more
specialized technique can be used for a subset of biologically important metabolites.
FIGURE 20.1
Outline of multiomics in the rhizosphere.
434 CHAPTER 20 Multiomics analysis of rhizosphere and plant health

3.Metabolomics uses
Metabolomics monitors chemical variation in rhizosphere samples from the ecology,
microbiology, and physiology of plants. Metabolomics, a relatively young field, can
help understand the rhizosphere and other complex systems (Paraskevaidi et al.,
2020). Liquid and Gas Chromatography-Mass spectrometry (LC-MS, GC-MS)
and Nuclear Magnetic Resonance (NMR) spectroscopy are used to investigate
metabolomes in biological samples because they can identify several metabolites
in a single cycle run (Tikunov et al., 2010). NMR finds a bulk amount of metabolites
without extensive sample preparation or fractionation. NMR has a 1-micromolar-
detection limit. NMR intensity corresponds linearly with relative mixture concentra-
tions; a standard (as) molecule with a known concentration (C) provides absolute
concentration. NMR shows the sample’s most abundant metabolites NMR can quan-
tify SMWVOCs such as alcohols and SCFAs like acetate, propionate, and butyrate.
GC-MS is used to study biogenic volatile organic molecules. GC-MS can detect pri-
mary metabolic products such as carbohydrates, fatty acids, and organic acids after
extract derivatization (White et al., 2017). LC-MS is used for resolving nonvolatile
compounds.
4.Metabolomics challenges
Complex microbial community metabolome characterization is difficult. Exudate
extraction is one of the rhizospheric metabolomics’ biggest hurdles (Mashabela
et al., 2022). Total data from microorganisms and plant components and their ulti-
mate products are needed to completely understand plant and bacterial metabolism.
Using cutting-edge mass spectrometers on germ-free plants could advance rhizo-
sphere studies (Agrawal &Verma, 2021).
4.1 Metagenome
A “metagenome” is a list of microbes produced by sequencing bacteria, archaea, and
protists. The metagenome encompasses all environmental genes and taxa. The meta-
transcriptome contains transcriptionally active genes and taxa. The metaproteome
shows whether genes are functionally active at the moment of samples to control
gene expression levels either at transcription or translation. Meta-epigenome and
meta-epiproteome are nonsequence DNA/RNA and protein changes (White et al.,
2017). NGS monitors the metaepigenome, or methylation of nucleic acids, while
MS detects the metaepiproteome, as changes in proteins (e.g., protein-
phosphorylation). Despite limited varied-omics findings in the rhizosphere, a hand-
ful of excellent studies have employed omics techniques to assess the structure and
function of rhizosphere soil properties. The study of microbial community and its
structure, such as NGS 16S rRNA PCR amplicons (i.e., ribosomal RNA polymerase
chain reaction amplification-based sequencing) in the rhizosphere of Arabidopsis
4. Metabolomics challenges 435

thaliana (Haichar et al., 2012), common annual grass (Avena fatua)(Miya &
Firestone, 2000). Metagenomics investigation in soybean (Aris et al., 2011) and
rice (Breidenbach et al., 2016) has shown the rhizosphere’s metabolic capacity. A
metatranscriptome study in cereal grains (maize and soybeans) demonstrated that
glyphosate affects bacterial cell nutrition, carbohydrates, and amino acid meta-
bolism. Another metatranscriptomic study comparing cereal grains (wheat, oat)
and legumes (peas) found that peas have a greater rhizosphere microbiome due to
the “rhizosphere effect” and more rhizosphere-specific microbiomes (Lee et al.,
2021). Pyrosequencing (16S rRNA) of bacteria from plant compartments (Rhizo-
sphere, Endosphere, Rhizoplane) (Table 20.1).
5.Multiomics investigation on an agroecosystem
demonstrates organic nitrogen’s function in increasing
crop output
Liebig (1840) formulated the notion of minerals useful for plant growth and devel-
opment. Knowing the agroecosystem, which includes living entities like plants and
microorganism is vital in agriculture due to the misuse of artificial fertilizer. Organic
nitrogen (N) is an important mineral for crop output during soil solarization, even in
the presence of inorganic nitrogen. Using organic nitrogen from soil solarization to
make crop production more sustainable. Globally, inorganic fertilizer inputs, crop
yields, and nitrogen leaching from soils have grown (Ichihashi et al., 2020). Multio-
mics on a field planted with mustard (Brassica rapa) was to explore agro-ecosystem
network structure, including ecologically friendly soil solarization. Solarization
increased shoot biomass regardless of fertilizer. Omics technology links with agro-
ecosystem, including plant properties heterogeneously linked to soil metabolites,
minerals, and microorganisms. Surprisingly, solarization’s organic nitrogen boosts
crop yield (Ichihashi et al., 2020). In in vitro and a laboratory test could use agricul-
tural soils to confirm that alanine and choline would increase the biomass of plants
by functioning as a nitrogen (N) source and physiologically active chemicals.
Organic nitrogen is crucial for agroecosystem plant growth (Deng et al., 2022).
5.1 General rhizosphere-omics challenges
Rhizosphere and soil science are rapidly adopting multidisciplinary methodologies
that require collaboration across geographical science and plant biology. Future
rhizosphere investigations will require advances in technique, technologies, data an-
alytics, and software. Advanced omics methodologies and technology are applied to
the rhizosphere (Lombi &Susini, 2009). A recent study using Arabidopsis thaliana
16S rRNA PCR amplicon sequencing on 613 samples showed the diversity of rhizo-
sphere microorganisms. It found almost 2000 species (in the form of OTUs) per
gram of rhizosphere soil. Since the mean of the microbial genome has 3000
436 CHAPTER 20 Multiomics analysis of rhizosphere and plant health

Table 20.1 Pyrosequencing (16S rRNA) of bacteria from plant compartments.
Plant/crop Rhizosphere Endosphere
Sequencing
technique Dominant species References
Para grass
(Urochloa
mutica)
þþþ Bacillus, Chloroflexi, Microcoleus Clostridium,
Caldilinea
Mukhtar et al.
(2016)
Wheat plants
(Triticum
aestivum)
þþþ Achromobacter, Clostridia, Cellulomonas,
Bacillus, Gallionella, Herbaspirillum,
Pseudomonas, Rhizobium, Xanthomonas,
Sinorhizobium, Burkholderia, Pantoea,
Enterobacter, Geobacter, Stenotrophomonas,
Nocardia, Mycobacterium, Microbacterium
Valverde et al.
(2016)
Taxus cuspidate
var. Nana
þþþ 16S rRNA Actinobacteria, Chloroflexi Hao et al.
(2016)
Aloe vera (Aloe
barbadensis)
þþþ 16S rRNA
variable gene
(V3eV4)
Proteobacteria, Firmicutes, Actinobacteria,
Bacteriodetes
Akinsanya et al.
(2015)
Rice (Oryza
sativa)
þþþ 16S rRNA Geodermatophilus, Actinokineospora,
Actinoplanes, Streptomyces, Kocuria
Mahyarudin
et al. (2015)
Triticum
aestivum
(Wheat)
þþþ þþþ 16S rRNA Bacillus, Acetobacter, Stenotrophomonas Abbasi et al.
(2015)
(Wheat) Triticum
aestivum
þþþ Azoarcus, Balneimonas, Bradyrhizobium,
Gemmatimonas, Lysobacter,
Methylobacterium, Mesorhizobium, Microvirga,
Rubellimicrobium, Rhodoplanes, Skermanella
(Wheat)
Naz et al.
(2014)
Soybean
(Glycine max)
þþþ 16S rRNA Bacillus, Bradyrhizobium rhizobium,
Stenotrophomonas, Streptomyces
Sugiyama et al.
(2014)
Continued
5. Multiomics investigation on an agroecosystem demonstrates 437

Table 20.1 Pyrosequencing (16S rRNA) of bacteria from plant compartments.dcont’d
Plant/crop Rhizosphere Endosphere
Sequencing
technique Dominant species References
Lettuce
(Lactuca sativa)
þþþ 16S rRNA sequencing Alkanindiges, Sphingomonas,
Burkholderia, Novosphingobium, Sphingobium
Gan et al.
(2014)
Arabidopsis
thaliana (Thale
cress)
þþþ Arthrobacter, Kineosporiaceae,
Flavobacterium, Massilia
Bodenhausen
et al. (2013)
Arabidopsis
thaliana (Thale
cress)
þþþ þþþ 16S rRNA.
Variable gene
(V5eV6)
Acidobacteria, Planctomycetes,
Proteobacteria, Actinobacteria, Bacteroidetes
Bulgarelli et al.
(2012)
Pennisetum
spp.
þþþ BOX-
PCR,16S
rRNA, and
nifH
sequences
Azospirillum brasilense, Gluconacetobacterdi
azotrophicus, Gluconacetobacter liquefaciens,
Gluconacetobacter sacchari, Burkholderia
silvatlantica, Klebsiella sp., Enterobacter
cloacae and Enterobacter oryzae
Videira et al.
(2012)
C. microphylla,
H. mongolicum,
and
H. scoparium
þþþ 16S rRNA Proteobacteria Zhou et al.
(2020)
Tricholoma
matsutake
þþþ 16S rRNA Tricholoma, Umbelopsis, Oidiodendron,
Sagenomella, Cladophialophora, and
Phialocephala
Jeong et al.
(2021)
438 CHAPTER 20 Multiomics analysis of rhizosphere and plant health

protein-coding genes, we get 6 10
6
bacterial proteins. 9 10
7
proteins equal
30,000 species per gram. Rhizosphere soil contains interfering humic acids, plant
polyphenols, and other degraded macromolecules, making it difficult to extract
DNA, RNA, proteins, and metabolites (Lundberg et al., 2012). Coextraction inter-
feres with PCR and protein/metabolite ionization. High-resolution omics data differ-
entiate biological molecules from interfering substances. Many bacteria are resistant
to lysis, resulting in extraction bias, which distorts microbial populations. Genomes
are widely extracted from the rhizosphere zone using a single commercial kit or
technique. Metabolomic studies provide robust extraction and lysis for complete
downstream analysis with minimal bias (Riemann &Middelboe, 2002).
6.NGS of rhizospheric microbes
NGS platforms measure individual genomes, transcriptomes, metagenomes, and
metatranscriptomes. NGS is less expensive and time-consuming than MS-based pro-
teomics and metabolomics. MinION2012, Oxford (1000e90,000) 90.0% 1 Gb Illu-
mina is the most experienced NGS platform manufacturer, providing high-utility
sequencing (White et al., 2017). Illumina’s technologies include (1) oligonucleotide
flow cells (2) reversible chain terminators and (3) bridge PCR amplification to
sequence DNA. Fragmenting nucleic acids to the desired insert size and inserting
oligonucleotide barcodes results in sequencing-read libraries. The library barcode
sequences complement array flow cells, which are then annealed as well as amplified
using bridge PCR. Illumina offers overlapping insert libraries with paired-end reads,
various read lengths, and short reads for high throughput. Illumina offers 8e15 kbp
read lengths. TruSeq synthetic long-read replaces Moleculo. NGS has been used to
predict core microbial community and plant-microbe interactions.
6.1 NGS difficulties
Data processing, data storage, and nucleic acid extraction are barriers to NGS in the
rhizosphere. Rhizosphere science’s biggest challenge is data processing. NGS mi-
crobial community data analysis and storage receive high marks. Genome extraction
bias is the biggest obstacle to using NGS in rhizosphere research. Gram-positive mi-
crobes have thick cell walls, making lysis difficult. Dormant microorganisms in soil
and rhizosphere samples make nucleic acid extraction harder (Vidal et al., 2018).
Multiple extraction methods and lysis processes must be used to cover a microbial
community thoroughly. If enough material is available, we could recommend
extracting nucleic acids using multiple methods. Dead microbial cell nucleic acids
are another concern. DNA from these cells can misunderstand quantity and meta-
bolic potential. Metatranscriptomes can confirm that DNA-based and 16S amplicon
PCR investigations are trustworthy in predicting transcriptionally active functions
and microbial populations (Marcelino et al., 2019).
6. NGS of rhizospheric microbes 439

7.Mass spectrometry (MS)
Metaproteomics studies proteins in a sample. Proteins and metabolites from samples
have been analyzed. MS is presently the most used technique for both metaproteo-
mics and metametabolomics. Combining chromatography and mass analysis helps
to characterize complicated proteins and metabolites. MS-based proteomics in-
volves protein extraction using solvents, detergents, or physical techniques such
as chloroform and methanol by method sonication. After being extracted, proteins
are broken into peptides (usually trypsin) for further study (Bierla et al., 2013). Elec-
trospray ionization (ESI) is utilized for LC-MS, proteomics, and metabolomics. ESI
allows LC-MS to detect intact species by gently ionizing substances. ESI uses a high
voltage (V) electric field to create millions of nanometric-charged droplets from a
liquid sample passing via a capillary tube (Pacholarz &Barran, 2016). GC-MS
uses electron impact (EI) alone in metabolomics, causing significant fragmentation.
In a conventional EI, an electron beam in a vacuum ionizes the analytes. A mass
analyzer determines the mass-to-charge ratio (m/z) after ionizing the peptides or me-
tabolites, and a detector counts ions at each m/z value (Buchberger et al., 2018).
8.Rhizospheric metaproteomics
A study of the metaepigenome in the rhizosphere or any other environment would
help comprehend gene regulation in a complex microbial community. Ijaz et al.
(2022) examined the rhizosphere’s metaepigenome using epigenomics. Metaproteo-
mics is applied to identify specific diversified microbes (e.g., acid mine waste)
(Baker &Banfield, 2003). The rhizosphere’s metaepiproteome could be used to
study protein organisms’ downstream control and function. Studies on rhizospheres
include leaf litter decomposition, and methanotrophs in rice rhizosphere/root tissues
(Uroz et al., 2019).
9.Conclusions
Omics is an analytical technique used to assist researchers to comprehend rhizo-
sphere organisms’ involvement in plant growth development and ecosystem health.
We have much to learn about the diverse, plant-specific rhizospheric communities
(374,000 are expected to exist on our planet). Multiomic approaches in rhizosphere
science have great potential, despite substantial challenges. They can help use the
rhizosphere for increased plant growth and quality, sustainable crop output, and
higher soil carbon storage in arable soils.
440 CHAPTER 20 Multiomics analysis of rhizosphere and plant health

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444 CHAPTER 20 Multiomics analysis of rhizosphere and plant health

Chemical profiling of
metabolites of Bacillus
species: A case study 21
Aurelio Ortiz, Estibaliz Sansinenea
Facultad de Ciencias Quı
´
micas, Beneme
´rita Universidad Auto
´noma de Puebla, Puebla, Pue,
Me
´xico
1.Introduction
The chemical study of an organism and the molecular structure elucidation of the
compounds isolated from this organism can lead to several applications in different
research areas. One of these areas is focused on the industrial production of some
chemical compounds with great value oriented to pharmaceutical, cosmetic or
food industry, or agriculture. The discovery of each new category of medicinal
agents has potential economic significance to the national economy because it can
yield a more productive population. If successful, it benefits the discoverers and their
licensed manufacturers. The antibiotics are obvious examples of economically sig-
nificant natural products (Woodruff, 1980). Besides, chemical compounds produced
by the microorganisms can be antifungals against some phytopathogens which cause
crop diseases or compounds that promote the growth of a plant. Therefore, many mi-
croorganisms such as fungi or bacteria are useful in agriculture since they are attrac-
tive ecofriendly alternatives to further applications of mineral fertilizers and
chemical pesticides (Ortiz &Sansinenea, 2021).
The Bacillus is one such genus that has received the most attention from scien-
tific community due to its capacity to produce a vast array of chemical compounds
with wide biological activities including antifungal, antibacterial, antiviral, or plant
growth promoter (Be
´rdy, 2005). The bacteria belonging to Bacillus genus have a
very long and distinguished history in biotechnology field; due to this there are
some bacteria such as B. thuringiensis defined as biologic control agents, which
are very effective in treating different insect pests. Besides, several Bacillus species
are capable of producing a wide range of natural products with different chemical
structures and different biological activities, which give to Bacillus the capacity
to survive in the environment surrounding it (Salazar et al., 2022;Sansinenea &
Ortiz, 2011).
These natural compounds produced by Bacillus can act as natural weapons
against other pathogens such as bacteria, fungi, or insects, can function as metal
transporting agents, or can promote the growth of plants through secreting hormones
(Ortiz &Sansinenea, 2019). Among the chemical compounds secreted by Bacillus
CHAPTER
445
Rhizobiome. https://doi.org/10.1016/B978-0-443-16030-1.00004-3
Copyright ©2023 Elsevier Inc. All rights reserved.

the most important can be classified as bacteriocins, antibiotics, lipopeptides and
polyketides. However, there are some other compounds such as indoles, diketopiper-
azines, siderophores, and hormones among others which can have a relevant role in
agriculture (Keswani et al., 2022;Ortiz &Sansinenea, 2021).
It is important to know the chemical profiles of several Bacillus species. Also, it
is common in Bacillus families to group some Bacillus based on their genetic anal-
ysis similarity. In this chapter we will analyze the general process to isolate the
chemical compounds of Bacillus and chemical profiles of the most important com-
pounds isolated from Bacillus species.
2.Chemical extraction and isolation of natural products
The quantity of natural products which are isolated from microorganisms is always
very small. The extraction and isolation processes are time consuming and require
the work of many laboratories and for this reason the extraction of these compounds
has been the bottleneck for the application of natural products in drug development.
There is an urgent need to develop effective and selective methods for the extraction
and isolation of bioactive natural products.
The extraction is the first step to achieve natural products from biological sources
and the solvent extraction is the most used method. The extraction walks through
four steps: (1) the solvent penetrates into the solid matrix; (2) the solute dissolves
in the solvents; (3) the solute diffuses out of the solid matrix; (4) the extracted sol-
utes are collected. Any factor that improves diffusivity and solubility in the above
steps will facilitate extraction. The properties of the extraction solvent, the particle
size of the raw materials, the extraction temperature, and the duration of extraction
will affect the extraction efficiency (Katz &Baltz, 2016).
The extraction can be done using adsorbent resins, which are polymeric adsor-
bents with macroporous structures and without ionic exchange groups. These resins
have been widely used as isolation method or as purification method to eliminate
impurities due to their advantages such as the high adsorption capacity, low cost,
easy regeneration and easy scaling. The resins mechanisms involved in adsorption
of natural products include electrostatic forces, hydrogen bonds, complex formation
and size sieving actions between resins and natural products in solution (Zhang
et al., 2018).
The compounds of the crude extract are a complex mix of several natural com-
pounds that need to be purified to achieve an active and pure fraction. This purifica-
tion depends of the physical and chemical characteristics of the compound.
Chromatography is the essential technique to achieve a pure compound from a com-
plex mix and specially column chromatography (Zhang et al., 2018).
Silica gel is the main adsorbent used as stationary phase. It is a polar adsorbent
containing silanol groups. The molecules are retained by silica gel through hydrogen
bonds and dipole-dipole interactions. Therefore, polar compounds are retained more
time in the silica gel column than on polar compounds. The desorption of polar
446 CHAPTER 21 Chemical profiling of metabolites

compounds will be enough to use a polar mobile phase, even adding water (Zhang
et al., 2018). Alumina (aluminum oxide) is a strong polar adsorbent used in the sep-
aration of natural products especially in the separation of alkaloids. However, its use
has decreased due to dehydration, decomposition, or isomerization that can occur
during separation.
The compounds’ separation is based on the adsorption affinity differences of the
natural compounds. This is the reason to select adequate stationary phase and sol-
vents as mobile phase. This selection can guarantee the maximum recovery of the
target compounds. In the early stage, one liquid phase was coated to a solid matrix
(silica gel, carbon, cellulose, etc.) as the stationary phase and another liquid phase
was employed as the mobile phase.
The separation of natural products can be done based on their molecular size us-
ing membrane filtration (MF) or gel filtration chromatography (GFC). However, the
separation can be also done based on ionic strength using ion-exchange chromatog-
raphy, which separates molecules based on the differences in their net surface charge
(Zhang et al., 2018).
Finally, with the aim of elucidating structures, chromatographic and spectro-
scopic or spectrometric techniques are used.
3.Antifungals
Several fungi act as parasites for different types of plants destroying important crops.
This has great consequences due to great economic losses. The genus of Bacillus sp.
has an extraordinary machinery to secrete several secondary metabolites, lytic en-
zymes and toxins against the phytopathogens, which cause plant diseases, promoting
plant growth (Ortiz &Sansinenea, 2019). Besides, these bacteria can induce the sys-
temic resistance of the plant (Borriss et al., 2019). There are some mechanisms that
can control pathogens causing plant diseases. Some of these mechanisms include:
(a) Competition where the biocontrol agent is more effective than the pathogen at
gathering critical nutrients or space and, therefore, must be in place before disease
onset, (b) Antibiosis where the biocontrol agent produces a chemical compound of
some type (antibiotic or toxin) that acts against the pathogen, (c) Predation or para-
sitism where the biocontrol agent directly attacks the pathogen, (d) Induction of host
plant resistance where the biocontrol agent triggers a defensive response in the host
plant that limits the ability of the pathogen to invade the plant (De Cal et al., 2012).
Biofungicides have several advantages that make them the perfect option to use
against the pest-infested crops. Biofungicides have strong selectivity, since they act
only against pests, being safe for humans and animals including beneficial insects,
such as pollinators. Since biofungicides are present and derived from natural ecosys-
tems, their impact on the environment is insignificant. Besides, they are easily
decomposed by sunlight, plants, or various soil microorganisms completing a natu-
ral life cycle. Most natural products degrade rapidly in sunlight, air, or moisture, or
when exposed to insect detoxification enzymes if ingested. Rapid breakdown
3. Antifungals 447

implies that these products do not persist long in the environment, which reduces the
risk to nontarget organisms (Salazar et al., 2022).
The control of fungal diseases by Bacillus-based biopesticides represents an
interesting opportunity for agricultural biotechnology, since these microorganisms
improve soil quality, soil health, and the growth, yield, as well as quality of crops
(Ortiz &Sansinenea, 2021). Antagonistic metabolites that are secreted by Bacillus
include lipopeptide surfactants. Lipopeptides are potent biofungicides which have
been applied in several crops. There are three main lipopeptides families named
as iturin, surfactin, and fengycin families (Fig. 21.1). These compounds are hydro-
philic cyclic peptides, consisting of 7e10 a-amino acids, attached only to a single b-
amino (iturin) or b-hydroxy (surfactin and fengycin) fatty acid. The high capacity of
these compounds to act against phytopathogens has drawn attention to the agricul-
tural sector. Many biosynthetic gene clusters are responsible for the synthesis of a
specific chemical and among Bacillus spp. surfactin, fengycin, and plipastatin or
iturin are the most frequent and prevalent biosynthetic gene clusters (Salazar
et al., 2022). It has been reported that direct action of surfactins against phytopath-
ogenic fungi (antibiosis) is low in contrast with other lipopeptides; however, their
indirect effect in suppressing fungal plant disease can be important (Shahid et al.,
2021). In contrast, iturins have a high antifungal activity. Basically, the antifungal
FIGURE 21.1
Lipopeptides of Bacillus sp.
448 CHAPTER 21 Chemical profiling of metabolites

activity of iturins depends on their lipid chain and peptide ring (Ali et al., 2022).
Fengycin acts as a fungicide specifically active against filamentous fungi and can
be used to treat various diseases in plants (Zhang et al., 2018). Fengycins increase
cell permeability and induce ROS production, which can oxidize lipids, proteins,
DNA, and carbohydrates within biological systems, leading to the breakdown of
the cellular membrane or cell death (Sur et al., 2018). Bacillus subtilis has been
one of the most studied species of Bacillus genus and has been demonstrated as a
producer of lipopeptides (Cherepanova et al., 2021;Cruz Mendoza et al., 2022).
There are other compounds produced by Bacillus with antifungal activity such as
Zwittermicin A (Sansinenea &Ortiz, 2012). Indole alkaloids have important biolog-
ical activities which is the reason for their extensive use in medicine (Fig. 21.2).
Indole is the precursor for a wide variety of tryptamine-indole, and 2,3-
dihydroindole-containing secondary metabolites. Recently some of these interesting
compounds have been studied for their competency to have antifungal activity
against F. oxysporum, Alternaria sp., and M. roreri (Vaca et al., 2020). Bacillus
spp. also secrete several lytic enzymes, such as chitinases, b-1,3-glucanases, b-
glucosidase, lipases, and proteases, which have the ability to degrade the compo-
nents of fungal cell wall such as chitin, b-glucans, and proteins (Berini et al.,
2018). Therefore, this enzymatic machinery is perfect acting against different phy-
topathogens (Huang et al., 2017;Shrestha et al., 2015).
FIGURE 21.2
Zwittermicin A and indole alkaloids structures.
3. Antifungals 449

4.Antibacterials
There are several compounds with antibacterial activity secreted by Bacillus sp.
(Fig. 21.3). Bacillaene-type compounds are highly unstable molecules due to their
molecular structure. In NMR studies, it has been demonstrated that the bacillaene
compounds appeared as open chains of polyene system. Difficidin and oxydifficidin
were identified as products suppressing plant pathogenic bacterium E. amylovora,
which causes fire blight disease at orchard trees (Keswani et al., 2020). Macrolactins
are macrolides containing three separate diene structure elements in a 24-membered
lactone ring, which possess potent antibacterial activity (Ortiz &Sansinenea, 2020).
However, it was also demonstrated that these compounds can function as antifungals
against phytopathogenic fungi such as Fusarium oxysporum and Moniliophthora
roreri (Salazar et al., 2020). Cyclic dipeptides, called diketopiperazines, are very
simple peptide derivatives, with the ability to bind to a wide range of receptors
and have several biological activities. The most studied activity that has been re-
ported is the antimicrobial activity (Ortiz &Sansinenea, 2017).
Isocoumarins or the saturated analogs 3,4-dihydroisocoumarins are a small
group of interesting isocoumarins that show some types of substitution with different
FIGURE 21.3
Chemical structures of some antibacterials.
450 CHAPTER 21 Chemical profiling of metabolites

biological activities and that are produced by Bacillus sp. (Fig. 21.4). The biological
activity of many of them is antibacterial activity against several Gram-positive and
Gram-negative bacteria. Values of antibiotic activity obtained by different authors
depend on the method of determination and, above all, the used test strains. Most
of dihydroisocoumarins found in bacteria including the amicoumacins, bacilosar-
cins, were found in Bacillus sp. (Ortiz et al., 2019).
However, the most powerful antibacterial secreted by Bacillus sp. are the com-
pounds called bacteriocins. These compounds, called bacteriocins, are small pep-
tides showing stability in a wide range of pH and temperatures which have a
potent antibacterial activity. Most of bacteriocins are generally recognized as safe
because they are degraded by proteases in the human intestine (Vaca et al., 2022).
Bacillus is considered as a producer of bacteriocins, and it can be seen through
the large number of species which produce a wide variability of bacteriocins with
antibacterial activity against pathogen enemies (Salazar et al., 2017). Bacteriocins
have a variable antibacterial activity, especially against different Gram-positive bac-
teria, through destabilizing the anionic polymers of the cell wall. Due to their anti-
bacterial activity, they can be employed in food industry as natural preservatives of
food since they extend the product shelf life. Besides, they can be a good option to
treat antibiotic multiresistant pathogen strains, which affect human health and
currently is being a public health problem worldwide (Fischbach &Walsh, 2009).
They can prevent infections since they have narrow spectrum of activity (Sumi
et al., 2015) being quite specific (Fuochi et al., 2021). There are many different iden-
tified bacteriocins isolated from several Bacillus sp. strains which have been revised
in recent reviews (Basi-Chipalu et al., 2022;Vaca et al., 2022). Bacteriocins from
Bacillus species are classified based on lactic acid bacteria bacteriocins (Abriouel
FIGURE 21.4
Chemical structures of dihydroisocoumarins.
4. Antibacterials 451

et al., 2011). Based on this classification, there are three classes of bacteriocins: class
I that includes antimicrobial peptides that undergo different kinds of posttransla-
tional modifications, class II that includes small bacteriocins, ribosomally synthe-
sized, nonmodified and linear peptides, and class III that includes large proteins
with phospholipase activity. Bacteriocins form pores in the cell membrane of bacte-
ria. First, bacteriocins, which are positively charged, are attracted to the target bac-
teria through electrostatic forces interacting with the membrane phospholipids.
Then, hydrophobic region of the bacteriocin penetrates the nonpolar interior of
the cell membrane forming pores. This generates losing of ions and other molecules
leading to cell death (Kumariya et al., 2019).
5.Plant growth promoting compounds
The various direct and indirect mechanisms of plant growth promotion by Bacillus
spp. are nitrogen fixation, solubilization, and mineralization of phosphorus and other
nutrients, phytohormone production, production of siderophores, antimicrobial
compounds, and hydrolytic enzymes, induced systemic resistance (ISR), and toler-
ance to abiotic stresses (Saxena et al., 2020).
Biological nitrogen fixation involves highly specialized and intricately evolved
interactions between soil microorganisms and higher plants for harnessing the atmo-
spheric elemental nitrogen. There are several Bacillus species that can fix atmo-
spheric nitrogen such as B. cereus, B. circulans, B. firmus, B. pumilus, B.
licheniformis, B. megaterium, B. subterraneous, B. aquimaris, B. vietnamensis,
and B. aerophilus (Ding et al., 2015;Yousuf et al., 2017). Many species of Bacillus
have been reported as phosphorus solubilizers (Saeid et al., 2018;Saxena et al.,
2020). It has been reported that the mechanism of mineral phosphate solubilization
by these bacteria has been associated with the release of low molecular weight
organic acids, such as succinic acid, that help solubilize the fixed phosphorus into
an exchangeable form. Some microorganisms can solubilize micronutrients such
as iron (Fe) and zinc (Zn) by excreting small molecules (siderophores) or organic
acids. Different species of Bacillus are siderophore producers helping plants in
acquisition of iron and zinc besides acting as biocontrol agents (Ortiz &Sansinenea,
2019). Some siderophores secreted by Bacillus sp. are bacillibactin, a 2,3-
dihydroxybenzoyl-Gly-Thr trilactone siderophore, petrobactin and 3,4-
dihydroxybenzoic acid (3,4-DHB) which were considered virulence factors for
B. anthracis and B. cereus (Zawadzka et al., 2009).
Bacillus spp. are known as plant growth promoters, both due to the suppression
of pathogens, through before mentioned mechanisms, and secreting some com-
pounds and hormones, which directly affect the plant growth. Auxins, gibberellins,
cytokinins, ethylene (ET), and abscisic acid are the well-known phytohormones pro-
duced Bacillus spp. in soil, which play different roles affecting plant cell enlarge-
ment, division, and enlargement of roots (Ambreetha et al., 2018;Mukhtar et al.,
2017). Some auxins producing Bacillus strains are capable of producing IAA,
452 CHAPTER 21 Chemical profiling of metabolites

indole-3-carboxylic acid and indole-3-lactic acid alleviating the drought stress in
wheat (Raheem et al., 2018). Different Bacillus species produce gibberellins (Kes-
wani et al., 2022;Radhakrishnan &Lee, 2016). Bacillus subtilis,B. megaterium, B.
licheniformis, and B. velezensis have been demonstrated for cytokinin production
stimulating the growth of some plants such as Arabidopsis thaliana through lateral
root elongation and root hair formation (Asari et al., 2017). Enhanced photosyn-
thetic activity is also reported as shown in leaves of inoculated plants (Vinci
et al., 2018). In almost all cases several mechanisms and compounds are involved
in the health of the plant since Bacillus can solubilize some nutrients like phosphorus
and Zn, and at the same time secrete antifungals against pathogens and phytohor-
mones to stimulate the plant growth. All mechanisms improve the plant health
and growth.
6.Conclusions
Bacillus spp. is a producer of a wide variety of chemical compounds with different
biological activities. The wide variability of these secondary metabolites with broad
biological activities has great potential applications in the agriculture, food and
pharmaceutical industries to prevent or control spoilage and pathogenic microorgan-
isms. It is important to know that some compounds are very common of different
Bacillus species; however, other compounds are very specific of some species.
Therefore, it is important to have a compound library for each species. This can
be affected by nutrients contained in the culture medium which can change the meta-
bolism forming another product. For this reason although the compounds produced
by Bacillus have been the topic of researchers during past years, it is important to
follow the developments of new compounds produced by the species or even by
new strains that are found in nature.
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456 CHAPTER 21 Chemical profiling of metabolites

Achievements of Professor
Hiltner vis a vis the
contributions toward
rhizosphere science 22
Aparna Baban Gunjal
Department of Microbiology, Dr. D.Y. Patil, Arts, Commerce &Science College, Pimpri, Pune,
Maharashtra, India
1.Introduction
Rhizosphere is the region around the plants where the beneficial microorganisms
survive. These beneficial microorganisms are called plant growthepromoting rhizo-
bacteria (PGPR). These PGPR mainly include bacteria, fungi, and actinobacteria.
The PGPR improve the growth of various plants and crops by direct and indirect
mechanisms. These plant growth promotion mechanisms by the PGPR include sol-
ubilization of phosphorus and potassium, production of iron chelating compounds
called as siderophores, plant growth hormones such as indole acetic acid, gibberel-
lins and cytokinins, nitrogen fixation, production of enzymes, i.e., cellulase, chiti-
nase, amylase, protease, lipase, etc. The rhizosphere science includes the aspects
related to study of PGPR, plant growth promotion by PGPR by various mechanisms,
molecular level study of plant growth promotion, etc. The research in the field of
rhizosphere science is gaining wide interest by many scientists (Figs. 22.1e22.3).
Many scientists have carried out immense research related to rhizosphere sci-
ence. Professor Lorenz Hiltner is the first scientist who has studied and contributed
immensely in the field of rhizosphere science. This chapter describes mainly the
achievements and contribution of Professor Lorenz Hiltner in the field of rhizo-
sphere science.
2.Rhizosphere science
Rhizosphere science deals with the study of beneficial bacteria near the roots of
plants. The mechanisms of PGPR behind growth of plants or crops are also included
under this aspect. The study of rhizosphere science is now gaining immense
importance.
CHAPTER
457
Rhizobiome. https://doi.org/10.1016/B978-0-443-16030-1.00020-1
Copyright ©2023 Elsevier Inc. All rights reserved.

FIGURE 22.1
Professor Lorenz Hiltner.
FIGURE 22.2
Bacteria near the rhizosphere region.
458 CHAPTER 22 Achievements of Professor Hiltner

3.Lorenz Hiltner
Lorenz Hiltner (Fig. 22.1) was born on November 30, 1862, in Neumarkt, Upper
Palatinate (Oberpfalz), Bavaria. He was the first to coin the word ‘rhizosphere’ in
1904. Hiltner studied the beneficial microorganisms, nitrogen-fixing bacteria, as
well as symbiotic bacteria found in the nodules of legumes. Hiltner further studied
the interactions among the beneficial microorganisms as well interactions among the
symbiotic bacteria in the nodules of the legumes. The first nitrogen-fixing bacteria
reported by Hiltner was Rhizobium beijerinckii. The interactions of symbiotic bac-
teria with the nodules of legumes and the exact mechanism of nitrogen fixation were
studied by Hiltner. He realized that nitrogen in the soil has influence on the legumi-
nous plants. This is due to the beneficial bacteria present near the rhizosphere region.
This is how Hiltner termed the word ‘rhizosphere’ and carried out research related to
rhizosphere science aspects. There is specificity between the Rhizobium sp. and the
leguminous plants. Hilter and Nobbe together developed Rhizobium inoculum called
as ‘Nitragin.’ The Rhizobium inocula were given to the farmers for their use in agri-
culture. This led to the discovery of ‘rhizotechnology.’ Hiltner further studied that
the plants release some chemicals (rhizodeposits) near their rhizosphere region
which attracts beneficial microorganisms. The microorganisms around the rhizo-
sphere region include PGPR, nitrogen-fixing bacteria, mycorrhizal fungi, biocontrol
microorganisms, mycoparasitic fungi, and protozoa. During his study of rhizosphere
science, Hiltner described the phenomenon of “soil sickness.” The work of Hiltner is
described in Handbook on Agricultural Bacteriology by Lohniss (1910). Lorenz
Hiltner is recognized as the founder of soil microbiology and organic farming.
The rhizosphere is a very dense region around the roots.
FIGURE 22.3
Microorganisms near the rhizosphere region.
3. Lorenz Hiltner 459

4.Rhizosphere soil
The rhizosphere is of immense importance in various plantemicrobe interactions
(Pinton et al., 2007). The plants and the characteristics of the soil influence the rhizo-
sphere. The rhizosphere microorganisms can be used to get biomass (Pinton et al.,
2007;Yang &Crowley, 2000). The studies related to rhizosphere aspects have
attracted many researchers. The metabolites such as enzymes, toxins, and antibiotics
are also essential for the study of plantemicrobe interactions. Molecular cross-talks
are also important in plantemicrobe interactions, for e.g., between Rhizobium sp.
and leguminous and nonleguminous plants. The composition of the rhizosphere so-
lution is also suggested to enhance the plant growth.
5.Beneficial microorganisms near the rhizosphere region
The beneficial microorganisms near the rhizosphere region include Pseudomonas,
Bacillus, Serratia, Streptomyces, Burkholderia, Herbaspirillum, Acinetobacter
sp., etc. (Fig. 22.2). They are known to promote the growth of various plants and
crops by direct and indirect mechanisms which are helpful to the farmers in their
agriculture. These microorganisms also are important as biocontrol agents which
will control the growth of pathogens. There is diversity of these microorganisms
near the rhizosphere region.
6.Group of microorganisms near the rhizosphere region
The group of microorganisms near the rhizosphere region is represented in Fig. 22.3.
6.1 Bacteria
Bacteria are common in the rhizosphere region with their population about 1 106
and 1 109 organisms g
1
of the soil (Oyewole &Asiotu, 2012).
6.2 Actinobacteria
Actinobacteria population is wide in the rhizosphere soil after the bacteria (Thanga-
pandian et al., 2007). Several species of actinomycetes such as Streptomyces,Nocar-
dia,Actinomadura, and Microbispora are commonly present in the rhizosphere soil
(Nithya &Ponmurugan, 2012;Zhang et al., 2012).
6.3 Protozoa
Some species of protozoa are also found in the rhizosphere soil.
460 CHAPTER 22 Achievements of Professor Hiltner

7.Factors affecting microbial population in the rhizosphere
region
7.1 Soil type and its moisture
The microorganisms near the rhizosphere region are more in sandy soils with less
amount of moisture.
7.2 Soil amendments and fertilizers
The soil amendments and fertilizers do not have much effect on the rhizosphere re-
gion (Oyewole &Asiotu, 2012).
7.3 Rhizosphere pH
The rhizosphere microbial population with more activity, then the pH is acidic.
7.4 Proximity of root with soil
The close interactions of the plant roots and soil are necessary for the rhizosphere
effect (Oyewole &Asiotu, 2012).
7.5 Plant species
The rhizosphere microbial population will vary with the plant species.
7.6 Root exudates
The rhizosphere effect also depends on the root exudates.
8.Conclusion
The rhizosphere science aspect is very important. The microorganisms in the rhizo-
sphere region play important role in the plant growth promotion. Further study is
required to understand the molecular mechanisms involved by these microorganisms
in plant growth promotion. The novel microorganisms near the rhizosphere region
must be isolated, characterized, and identified as well as their applications mainly
in the agriculture field.
8. Conclusion 461

References
Lohnis, F. (1910). Handbuch der landwirtschaftlichen Bakteriologie. Gebru
¨der Borntraeger.
Nithya, B., & Ponmurugan, P. (2012). Studies on actinomycetes diversity in Eastern Ghats
(Yercaud hills) of southern India for secondary metabolite production. International Jour-
nal of Agricultural Research, 7(3), 152e159. https://doi.org/10.3923/ijar.2012.152.159
Oyewole, O., & Asiotu, N. (2012). Rhizosphere microbial communities: A review. Research
and Reviews in BioSciences, 6, 271e279.
Pinton, R., Veranini, Z., & Nannipieri, P. (2007). The rhizosphere. Biochemistry and organic
substances at the soil-plant interface. LLC. Review of Microbiology, 19, 241e266.
Thangapandian, V., Ponmurugan, P., & Ponmurugan, K. (2007). Actinomycetes diversity in
the rhizosphere soils of different medicinal plants in Kolly Hills-Tamilnadu, India, for sec-
ondary metabolite production. Asian Journal of Plant Sciences, 6(1), 66e70. https://
doi.org/10.3923/ajps.2007.66.70
Yang, C. H., & Crowley, D. E. (2000). Rhizosphere microbial community structure in relation
to root location and plant iron nutritional status. Applied and Environmental Microbiology,
66(1), 345e351. https://doi.org/10.1128/AEM.66.1.345-351.2000
Zhang, J., Ma, Y., & Yu, H. (2012). Arthrobacter cupressi sp. nov., an actinomycete isolated
from the rhizosphere soil of Cupressus sempervirens.International Journal of Systematic
and Evolutionary Microbiology, 62(11), 2731e2736. https://doi.org/10.1099/
ijs.0.036889-0
462 CHAPTER 22 Achievements of Professor Hiltner

Index
‘Note: Page numbers followed by “f ” indicate figures and “t” indicate tables.’
A
Abiotic stress, 233e235, 290, 315e316, 318e319,
389e390
alter root exudation, 149e150
VOCs against, 71e72
increased salt tolerance, 71e72
Abiotic stressors, 289
Above-ground processes, 145e146
Abscisic acid (ABA), 73, 241e244, 289, 404e407,
414, 452e453
abscisic acid-dependent regulatory pathways, 405
abscisic acid-independent regulatory pathways,
405
Acetic acid, 73e74
Acetobacter, 106, 385e386, 409
Acetoin, 61e62
Achromobacter,22e23, 82, 387e388
Acidithiobacillus ferrooxidans, 171
Acidovorax,22e23
Acinetobacter, 82, 173e174, 212, 381, 387e388
A. calcoaceticus EU-LRNA-72, 317
A. lactucae, 147e148
Actinobacteria, 331, 385e386, 390e391, 393,
460
Actinobacterial genomes, 331
Actinomycete isolation agar (AIA), 220e221
Actinomycetes, 330, 336e337
Adenosine monophosphate (AMP), 22e23
Adoption of biofertilizers, lack of, 100
Advanced high-throughput metagenomic
approaches, 275e276
Advances in analytical techniques and
microbiome genome sequencing
technology, 151e152
Advances in discovery of new drugs from
plants-associated microbes
actinobacteria, 331
algae, 331e333
endophytes, 333e336
Aerenchyma, 389e390
Aerobic bacteria, 82
Aeromonas, 173e174
Aerosols, 48e49
Aggregates of soil, 295
Agricultural policy, 233e235
Agricultural production, 147, 167e168
Agricultural products, 19
Agricultural systems, 168
Agriculture, 237e238
importance of, 98
Agrobacterium, 108, 173e174, 212, 233e235,
276e277, 350, 385e386
A. rhizogenes,26
A. tumefaciens,64
Agrochemicals, 103
Agrostis tenuis,84e85
Air pollution, 403e404
Albeit amino acids, 8
Alcaligenes,22e23, 82, 209, 387e388
Alcohol compounds, 68e69
Aldehyde compounds, 69
Algae, 331e333
Alkanes compounds, 70
Alkenes compounds, 70
Allorhizobium, 173e174, 349e350
Alpha-HCH, 113
Alternaria spp., 67
A. tenius, 172e173
Alumina, 446e447
Aluminum (Al), 214e215, 360e361
Amensalism, 288
Amicoumacins, 450e451
Amino acids, 7
Amino corrosive biosynthesis, 8
1-aminocyclopropane-1-carboxylate deaminase
enzyme (ACC deaminase enzyme), 22e23,
27e28, 315e317, 350, 389e390, 411, 414
Aminolevulinic acid, 241e244
Ammonia, 349e350
Ammonium (NH4
+
), 385e386, 410e411
Amplicon sequence variations (ASVs),
272e274
Amplicon sequencing, 271e272
Amplified ribosomal DNA restriction analysis
(ARDRA), 195e196
Anabaena, 169e170
Anaerobic bacteria, 83
Ancient context, 145e146
Antagonistic metabolites, 448e449
Anthrobacter,63
Antibacterials, 450e452
chemical structures, 450f
Antibiosis, 447
of VOCs, 68
463

Antibiotics, 290e291, 445e446
producing, 290e291
synthesis, 290e291
Antifungals, 447e449
Antimicrobial compounds, biosynthesis of,
153e154
Arabidopsis, 385e386
A. thaliana, 130, 147e148, 357, 361
16S rRNA PCR amplicon sequencing,
436e439
plants, 72
Arbuscular mycorrhiza (AM), 310e313, 330,
386e387
plant supplement carriers for arbuscular mycor-
rhizal beneficial interaction, 5e7
Arbuscular mycorrhizal fungi (AMF), 26, 85,
171e172, 238e244, 289, 309
Archaea, 330
AREB regulator (ABF), 406
Arthrobacter, 82, 107, 170e171, 173e174, 209,
387e388
A. globiformis, 241e244
A. sulfonivorans,87e89
Artificial fertilizers, 108
Ascorbate peroxidase (APX), 290, 316, 412
Aspergillus, 110, 170e173, 221
A. niger, 171e172
A. terreus, 171
Associative diazotrophs, systems of, 286e287
ATP Binding Cassette (ATP-dependent ABC), 357
Aulosira, 169e170
Aureobasidium pullulans, 172e173
Autoregulation of nodulation (AON), 6
Auxins, 241e244, 289, 411, 452e453
Azoarcus, 173e174, 286, 387e388
Azolla, 169e170
Azomonas, 106, 294
Azorhizobium, 169e170, 173e174, 349e350
Azospirillum sp., 21e22, 24e25, 106, 168e174,
209, 212, 215e216, 238e244, 286e287,
298, 350, 385e388, 409
A. brasilense,23e24, 192, 289
A. lipoferum, 244e250
diazotrophic population, 286e287
extending utility of nonsymbiotic N
2
-fixation,
298e299
genetics, 287
measurement/quantification, 291e294
assay for C
2
H
2
reduction, 291e292
isotope
15
N dilution, 293
measurement of N
2
-fixation with
15
N
2
gas,
292e293
N-fixed using different approach, 293e294
modes of action, 288e291
mechanisms for promoting plant growth,
288e291
nonsymbiotic N
2
-fixation-related factors,
294e297
plants and creatures actualized nitrogen from
diazotrophs, 297e298
strains, 24e25
Azotobacter, 106e107, 168e171, 173e174, 209,
212, 216e217, 238e244, 294, 319,
385e386
A. chroococcum,21e22
B
BacA protein, 7
Bacillaene-type compounds, 450
Bacillibactin, 452
Bacillus,46e47, 61, 74, 81e82, 84e85, 95,
106e108, 110, 150e152, 156, 170e174,
209, 212, 217, 233e235, 238e250,
276e277, 314, 319, 350, 385e388, 409,
445
antibacterials, 450e452
antifungals, 447e449
B. amyloliquefaciens,30e32, 61e63, 71, 134,
238e240
L-S60, 276e277
RWL-1, 316
SQR9, 350
B. aryabhattai, 318
B. cereus, 63, 238e240, 316
B. circulans, 171, 214e215
B. edaphicus, 171
B. lentus, 192
B. licheniformis, 30, 241e244
B. marisflavi, 316
B. megaterium, 241e244
B. mucilaginosus, 171, 214e215
B. pumilus, 81, 238e240
B. subtilis,24e26, 30e32, 61e63, 71, 81, 97e98,
108, 148, 153e154, 188e192, 238e240,
310
B. tequilensis, 318
B. thuringiensis, 171e172, 445
B. velezensis, 132e133
chemical extraction and isolation of natural
products, 446e447
plant growth promoting compounds, 452e453
Bacilosarcins, 450e451
Bacteria, 212, 330, 445, 460
nutritional absorptions by, 385e386
464 Index

Bacterial consortia, 174e176
Bacterial elements in RN beneficial interaction,
6e7
Bacteriocins, 445e446, 451e452
Barley (Hordeum vulgare), 389e390
Basic alignment search tool (BLAST), 129
Basic Local Alignment Search Tool (BLAST),
130
Basil plant (Ocimum basilicum), 26
Baumannia, 8
Bean (Phaseolus vulgaris L.), 289
Beijerinckia, 170e171, 294, 314
B. derx, 297e298
Beneficial microorganisms, 289, 460
Beneficial plant-microbe interactions, 315e316
Beneficial rhizobacteria, 61e62
Benzothiazole, 68, 71
Benzoxazinoid (BX), 148
Beta diversity analyses, 274
b-1,3-glucanases, 188e192, 449
b-glucans, 449
b-glucosidase, 449
b-hydroxybutyrate granules, 215e216
Bin data, 135
Bio-based alcohols, 52
Bioactive substances, 330
Biochelators, 317e318
Biocontrol agents, 110, 144e145
plant defense with, 30e32
rhizobiome as, 351e352
Bioengineering of rhizobiome, 235e236
rhizosphere engineering for abiotic, 241e244
rhizosphere engineering for biotic stress,
244e250
rhizosphere engineering for sustainable agricul-
ture, 236e241
Biofertilization, 112
Biofertilizers, 102e113, 167e169, 193e194,
238e240. See also Chemical fertilizers
biological nitrogen fixation, 108e109
carrier constraints, 194
challenges and constraints with microbial
consortiaebased, 193e195
HCH degradation, 113
importance and applications of PGPR, 107e108
increase in growth, 109
lack of adoption of, 100
mechanisms of PGPR, 108
microbial antagonism, 110e112
microorganisms with PGPR, 107
pesticide-specific biosurfactants, 112e113
PGPR, 104e106
phosphate solubilization, 109e110
Biofungicides, 447e448
Biogenic VOCs, 63
Biogeochemical setting, 145e146
Biogeographical setting, 145e146
Bioinformatics, 128e129
impact on genomics, 129e130
resources and platforms for plant microbes inter-
action study, 131e133
tools, 130e131
Biologic stresses alter root exudation, 149e150
Biological nitrogen fixation (BNF), 108e109,
151e152, 285, 410e411, 452
Biomass, 1, 46
Biome
marine biome, 47e49
terrestrial biome, 49e50
Biopesticides, 235e236, 238e240
Biopesticides and Pollution Prevention Division
(BPPD), 180e182
Bioremediation techniques, 82e83
microorganisms used for, 82e83
techniques employed in, 83e85
rhizoremediation, 83e84
rhizoremediation of heavy metals, 84e85
Biostimulants (BSs), 385e386
Biosurfactants (BS), 113
Biosynthetic gene clusters (BGCs), 276e277,
448e449
Biotic elements, 21e22
Biotic stress, 290, 315e316. See also Abiotic
stress
rhizosphere engineering for, 244e250
by using rhizobacteria for sustainable crop
produce
biotic stresses effects on plants, 20e21
PGPR impact on root architecture, 27e28
PGPRs as growth enhancer, 21e23
PGPRs systemic effects on functioning and
physiology of plant, 23e25
plant and rhizobacteria interaction effect on
secondary metabolites, 25e27
plant defense with biocontrol agents, 30e32
stimulating defense reaction of rhizobacteria in
plants, 28e30
Biotic stressors, 289
Blueegreen algae (BGA), 168e170
Botrytis spp., 67
B. cinerea, 244e250
Bradyrhizobium, 106, 156, 169e170, 173e174,
209, 349e350
B. japonicum,96e97
Index 465

Branched-chain amino acids (BCAAs), 132e133
Brassica napus,87e89, 360e361
Brassinosteroids, 289
Brevibacterium halotolerans,81
Brevundimonas sp., 85
Brucella abortus,7
Buchnera,10e11
Bug microorganism amino corrosive digestion,
7e10
dynamic capability of coordinated bug microor-
ganism amino corrosive digestion, 10e11
Burkholderia,21e22, 61e62, 106, 108, 170e171,
173e174, 314, 319, 385e388
B. ambifaria,63
B. azospirillum, 107
B. cenocepacia, 171e172
B. cepacia, 388
B. phytofirmans,24e25, 390e391, 393e394
Burkholderiaceae, 152e153
2,3-butanediol, 68e69, 73
C
C
2
H
2
reduction, assay for, 291e292
Cadmium (Cd), 360e361
Caenorhabditis elegans, 70, 130
Calexin, 382e383
Camalexin, 23e24
Candida steatolytica, 212e213
Capillary roots, 411
Carbenicillin, 218e219
Carbon (C), 6, 285e286
compounds, 346
fluxes, 96
presence of, 295
sequestration, 314e315
microbes for, 51
ocean MCP, 51
Carbon dioxide (CO
2
), 50, 52
Carboxylates, 410
Carex arenaria, 150
Carotenes, 331e333
Carotenoids, 8
Carriers, 178e180
Carsonella ruddii,9
Catalase (CAT), 290, 316, 412, 414
Catecholates, 410
Caulobacter, 173e174
Cell biology mass carbohydrates, 154e155
Cellulose, 1
Cellulose agar (CAM), 222
Cellvibrio, 131
Cenchrus ciliaris,84e85
Cereal grains, 435e436
Chaetomium, 170e171
Chemical compounds, 445
Chemical fertilizers, 100e102, 167e168,
213e214. See also Biofertilizers
nitrogen, 101
nitrogen, phosphorus, and potassium in Indian
agriculture, 101
phosphate, 102
potassium, 102
Chemical signals, spray of, 352
Chemoeffectors, 85
Chitin, 449
Chitinases, 188e192, 449
Chloramphenicol, 221
Chlorella species, 331e333
Chromatography, 446
Chromobacterium, 173e174
Chryseobacterium, 170e171
C. balustinum,24e25
Citrobacter, 381
Climate change, 182e188
affecting ecosystem, 44e45
extreme events causing, 45
interaction between species, 45
role of primary producers in, 44e45
different class of microbes impact on, 47e50
effect on marine biome, 48
effect on terrestrial biome, 49
human and interactions impact on, 44e45
microorganisms effect on, 48e49
terrestrial microbes effect on, 49e50
Cloning-independent approach, 275e276
Clostridium, 107
Clustered Regulatory Interspaced Short Palin-
dromic Repeats-Cas9 (CRISPR-Cas9),
336e337
Colletotrichum spp., 67
C. tropicale, 313
Commensalism, 288
Commercial stabilization solutions, 269e270
Community of microorganisms, 46e47
Competition, 447
Constructed wetlands (CW), 413e414, 414f
Contamination, 82
Copper (Cu), 360e361
Core microbiome, 156e158
Core rhizobiome, 267e269
Core rhizosphere microorganisms
Aspergillus, 221
Azospirillum, 215e216
Azotobacter, 216e217
466 Index

Bacillus, 217
bacteria, 212
cultivation strategies for beneficial rhizospheric
microbes, 215
Enterobacter, 217e218
Frankia, 218
fungi, 212e213
Klebsiella, 218e219
members of rhizomicrobiome, 211
Metarhizium, 221
Methylobacterium, 219
nutritional strategies for beneficial rhizospheric
microbes, 215
Penicillium, 222
Pseudomonas, 219
Rhizobium, 220
rhizospheric microbiome, 213e215
Streptomyces, 220e221
Trichoderma, 222
Corynebacterium,82
Crocus sativus, 380e381
Crops, 29
crop-associated microbes, 387e388
crop-related microbes, 387e388
management, 155
multifunctional plant growth-promoting attri-
butes of microbial consortia on, 182e192
production, 213e214
productivity, 156, 167e168
residues, 295, 297
degradation products, 296e297
Cucumber (Cucumis sativus), 313, 389e390
Cultivation strategies for beneficial rhizospheric
microbes, 215
Cupriavidus,84e85
Cyanobacteria, 296
Cyclic dipeptides, 450
Cyclohexanol, 71
Cylindrocladium parasiticum, 177e178
Cysteine, 9e10
Cytokines (CKs), 414
Cytokinins (CK), 169e170, 241e244, 289, 411,
452e453
D
Data processing, 273e275
Databases
and methods for sequence classification,
272e273
VOCs, 63
Deaminase, 385e386
Decaying root systems (DR systems), 132
Defined propionate media (DPM), 218
Dehydration, 72
Delftia sp., 170e171, 173e174
Derxia, 294
2,4-Diacetylphloroglucinol (DAPG), 351
Diatoms, 48
Diazotrophic bacteria, 297e298
Diazotrophic population, 286e287
systems of associative diazotrophs, 286e287
Diazotrophs, plants and creatures actualized
nitrogen from, 297e298
Dicholoran, 221
Different analytical techniques, 274e275
Difficidin, 450
2,3-dihydroindole-containing secondary
metabolites, 449
Dihydroisocoumarins, 450e451
chemical structures, 451f
3,4-dihydroisocoumarins, 450e451
3,4-dihydroxybenzoic acid (3,4-DHB), 452
Diketopiperazines, 445e446, 450
Dimethyl disulfide (DMDS), 67, 74
Dimethyl trisulfide, 71
Dimethylhexadecylamine (DMHDA), 70
Dipole-dipole interactions, 446e447
Disease, 329, 387e394
control, 114
disease-suppressive soils, 152e153
pest, 387
Dissolved Organic Matter (DOM), 51
Diverse soils, 46
DNA extraction, 270e271
Donggang pasque-flower (DPF), 381
DREB regulator, 406
Drosophila,70
D. melanogaster, 130
Drought, 389e390, 404, 412e413
stress, 404
tolerance, 404
Drugs, 331e333
E
Ecospecies. See Ecotype
Ecosystem, 43
approaches, 50
microbes as tools for achieving ecosystem
approaches, 50e52
need for, 50
human and interactions impact on, 44e45
Ecotype, 379e380
of plant, 377e379
Ecotypic plant production systems, 383e384
Index 467

Ectomycorrhizal fungi (EMF), 238e240
Electron impact (EI), 440
Electrons, 346
Electroporation technology, 336e337
Electrospray ionization (ESI), 440
Elytrigia repens, 361
Endophyte-derived natural products, 333
Endophytes, 286, 329e330, 333e337, 411
Endophytic Azospirillum, 297e298
Endophytic bacteria, 336
Endophytic diazotrophs, 297e298
Endophytic organisms, 286
Endosphere, 433e434
Enterobacter,22e23, 62, 107, 170e174, 209,
217e218, 238e240, 314, 319, 350,
385e388, 409
E. asburiae,21e22
E. cloacae, 214e215
E. ludwigii, 382e383
Entomopathogenic fungi, 221
Entomopathogenic nematodes (EPN), 213
Environment type, 83
Environmental disruptions, 433e434
Environmental extremes, microbiota role in
amelioration of, 387e394
Environmental Protection Agency (EPA),
180e182
Environmental stress, 403e404
ABA, 405e407
ACC deaminase, 411
auxins, cytokinins, gibberellins, 411
drought stress, 404
effectiveness of PGPR in hydrocarbons and heavy
metals contaminated soils, 411e412
inducible regulatory networks in, 406f
nitrogen fixation, 410e411
PGPR to face salinization and drought facing
abiotic stresses, 412e413
phosphate solubilization, 410
properties and potential of plant growth promot-
ing rhizobacteria, 407e410
salinity stress, 405
siderophores, 410
water phytodepuration, 413e414
Enzymatic antioxidants, 316
Enzymes, synthesis of, 290
Epicoccum nigrum, 172e173
Erwinia, 170e171, 233e235, 314, 387e388
E. herbicola, 241e244, 350
Escherichia coli, 143e144
2-ethyl 1-hexanol, 71
Ethylene (ET), 289, 350, 407e408, 452e453
synthesis, 29
Ethylene and jasmonate (ET/JA), 425
Ethylene diamine tetra acetic acid (EDTA), 220
Ethylenediamine-N,N-disuccinic acid (EDDS),
411e412
Ethylenediaminetetraacetic acid (EDTA),
411e412
European Food Safety Authority (EFSA),
180e182, 195
Exiguobacterium acetylicum, 241e244, 351
Exopolysaccharides (EPS), 414
Extracellular PGPR, 96e97
Extracellular plant growth promoting rhizobacte-
ria (ePGPR), 212
Exudates, 346
extraction, 435
F
Facultative endophytic bacteria, 334
Fengycin, 448e449
Ferric ions (Fe3+), 350
Fertilizers, 23
chemical fertilizers, 100e102
improper use of, 99e100
Field-level constraints, 194e195
Flavobacterium, 82, 170e171, 173e174, 209,
314, 387e388
Flavonoids, 6e7, 433e434
Flooding, 389e390
Food grains, 100
Food security, 309
Fossil fuels, 52, 233e235
Frankia, 173e174, 218, 385e386
Frateuria aurantia, 171
Free-living N
2
-fixing bacteria, 295
Functional Genomics Database, 361e362
Fungal cell, 28e29
Fungal consortia, 176e177
Fungal diversity, 212
Fungal endophytes, 336e337
Fungal pathogens, 388
Fungal-bacterial consortia, 177e178
FUNGENE database, 272e273
Fungi, 212e213, 329e330, 445
nutritional absorptions by, 386e387
Fungicides, 114
Fusarium, 170e171
F. culmorum, 150
F. moniliforme, 388
F. oxysporum,67e68, 98, 151e154, 188e192,
212e213, 244e250, 351, 450
F. verticillioides, 153e154
468 Index

G
Gallium (Ga), 360e361
Gas chromatography-mass spectrometry
(GC-MS), 435
Gel filtration chromatography (GFC), 447
Gelatin, 1
Gene expression by roots, regulation of, 85
Gene regulatory networks, 131
Genetically modified organisms (GMOs), 235
Genomes of heritable symbionts, 5
Genomics, 6, 32, 270e271
bioinformatics impact on, 129e130
Gibberellic acid, 21e22
Gibberellins (GA), 169e170, 241e244, 288e289,
316, 411, 414, 452e453
Glomus fasciculatum, 393
Gluconacetobacter, 173e174, 387e388
G. diazotrophicus, 171e172
Gluconate, 217e218
Glucose, 217e218
Glucosinolates, 23e24, 382e383
Glutamate dehydrogenase (GDH), 386e387
Glutamine synthetase (GS), 386e387
Glutathione S-transferase, 290
Glutathione/thioredoxin peroxidase (GPX), 290
Glycerol, 217e218
Glycerol-3-phosphate (G3P), 390e391
Glycine betaine, 317
Good agricultural practices (GAP), 182
Gordonia, 170e171
Gram positive microorganisms, 385e386
Gram-positive rhizobacterium Bacillus amyloli-
quefaciens FZB42, 359e360
Gramene/PlantReactome, 276
Greenhouse gas (GHG), 48, 52, 233e235
Growth, 19
increase in, 109
retardation, 410
Growth promoting bacteria (GPB), 380
H
Haber-Bosch process, 285
Haemophilus influenza, 129e130
Harmful microbes, 153e154
Health, plant-microbe interactions in enhancing
plant growth and, 314e318
Healthy production systems, 144e145
Heavy metals
contaminated soils, 411e412
rhizoremediation of, 84e85
Helminthsporium sativum,71
Hemicellulose, 1
Herbal metabolites, 25
Herbaspirillum, 387e388, 409
H. seropedicae,23e25
Herbivorous bugs, 1
Heritable symbionts, 5
Heteropsylla cubana,10
Hexa-chlorocyclohexane (HCH), 113
degradation, 113
High throughput sequencing technologies,
275e276
High-throughput molecular techniques, 271e272
High-throughput sequencing
in conjunction, 267e269
of marker gene amplicons, 271e272
technology, 276e277
Holcus lanatus, 380e381
Holobiont-based control of rhizospheric biota, 156
Homalodisca vitripennis,8
Hormone-secreting microorganisms, 289
Hormones, 445e446
Host plants, 387e388
resistance induction, 447
Human diseases, 336e337
Human-pathogenic microbes, impact of, 156e158
Hydrocarbons, 411e412
Hydrogen bonds, 446e447
Hydrogen cyanide, 81
Hydrogenophaga, 107
Hydrophilic cyclic peptides, 448e449
Hydroxamates, 410
Hyoscyamus albus, 360e361
I
Illegal dumping, 406e407
In vitro regeneration, 336e337
Indian agriculture, nitrogen, phosphorus, and po-
tassium in, 101
Indian Institute of Horticultural Research (IIHR),
182
Indole acetic acid (IAA), 169e170, 214, 220,
315e316, 350, 385e386
Indole alkaloids, 449
Induced Systemic Resistance (ISR), 21, 23e24,
95, 104, 176e177, 291, 351, 452
signaling molecules, 291
Inoculation, 298
Inorganic fertilizers, 100e102
Inorganic ions, 346
Inositol, 218e219
Intergovernmental Panel on Climate Change
(IPCC), 50
Internal standards, 434
Index 469

Internal transcribed spacer (ITS), 274
Intracellular PGPR, 96e97
Intracellular plant growth promoting rhizobacteria
(iPGPR), 212, 233e235
Intrinsic antibiotic resistance (IAR), 290e291
Ion-exchange chromatography, 447
Iron (Fe), 214e215, 452
iron-deficiency response, 151e152
optimization of iron homeostasis, 74e76
protein, 294
Isocoumarins, 450e451
Isotope
15
N dilution, 293
Isotope dilution technique, 293
Iturin, 448e449
J
Jasmonates, 289
K
Kenknight and Munaier agar (KMA),
220e221
Ketone compounds, 69
Klebsiella,22e23, 106, 170e171, 173e174, 209,
218e219, 238e240, 314, 319, 350,
387e388
K. oxytoca,21e22
K. variicola, 214e215, 241e244
Kluyvera ascorbata,85
Knob capability, 6
Knockout mutants, 149
L
Lactose, 217e218
Laccaria laccata,87e89
Landfilling, 406e407
Legumes, 435e436
Leguminous plants, 6
Lignin, 1
Lima bean (Phaseolus lunatus), 361
Lipases, 449
Lipopeptides, 445e446, 448e449
Liquid chromatography-mass spectrometry
(LC-MS), 435
Living stresses, 403e404
Lolium perenne, 110e111
Lotus japonicus,6
Low molecular weight heterogenous chemicals,
290e291
Low-molecular-weight chemicals, 61e62
Lysinibacillus, 150
Lysobacter spp., 61e62
Lytic enzymes, 29
M
Macro-nutrients, 348e349
Macroaggregate stability, 297
Macrolactins, 450
Macrophomina phaseolina, 188e192
Magnesium chloride (MgCl
2
), 413
Magnesium sulfate (MgSO
4
), 413
Maize (Zea mays), 83e84, 319, 360e361,
389e390, 406e407
Malondialdehyde (MDA), 316
Malt extract agar (MEA), 222
Marine biome, 47e49
climate change effect on, 48
microorganisms effect on climate change, 48e49
Marine phytoplankton, 48
Mass spectrometry (MS), 133, 440
Medicago truncatula,6
Megaplasmids, 287
Meloidogyne incognita,67
Membrane filtration (MF), 447
Mentha piperita,30
Mesorhizobium, 106, 169e170, 173e174, 188,
209, 349e350
M. chacoense,96e97
M. ciceri,21e22
M. pluriforium,96e97
Meta-epigenome, 435e436
Meta-epiproteome, 435e436
Metabolic Workbench, 276
Metabolites, 336, 433e434
Metabolome, 434
Metabolomics, 272
challenges, 435e436
uses, 435
Metagenome, 435e436
Metagenomic approaches, omics to, 275e277
Metagenomics, 275
Metallothioneins (MTs), 83e84
Metaproteome, 435e436
Metaproteomic data, 135, 271e272
Metaproteomics, 133
in plant microbialeassociated studies, 134
Metarhizium, 221
Metatranscriptome, 435e436
Metatranscriptomics, 135
Methane (CH
4
), 50
Methanethiol, 70
Methanol, 219
Methionine, 9e10
Methyl dehydrogenase (MDH), 219
Methyl rhamnolipids, 113
Methylobacterium,22e23, 219, 387e388
470 Index

Microbacteria sp., 351
Microbacterium,21e22, 170e171
Microbes, 82, 136e137, 143e144, 310, 329e330,
393e394
as active ingredients of microbial consortia,
168e174
nitrogen fixation microorganisms, 169e170
PGPR, 173e174
phosphate solubilizing microorganisms,
170e171
potassium solubilizing microorganisms, 171
sulfur oxidizing microorganisms, 172e173
zinc solubilizing microorganisms, 171e172
different class of microbes impact on climate
change, 47e50
marine biome, 47e49
terrestrial biome, 49e50
as tools for achieving ecosystem approaches,
50e52
microbes as sustainable fuel, 52
microbes for carbon sequestration, 51
microbes to reduce methane emissions, 51e52
nitrous oxide mitigation, 52
Microbes-associated molecular patterns
(MAMPs), 291
Microbial biological control agents (MBCA),
176e177
Microbial carbon pump (MCP), 51
Microbial communities, 330
rhizosphere differentials affect microbial com-
munity assembly, 147e148
Microbial consortia, 174e178
bacterial consortia, 174e176
beneficial microbes as active ingredients of,
168e174
carrier materials for, 178e180
challenges and constraints with microbial
consortiaebased biofertilizers, 193e195
biofertilizer carrier constraints, 194
biological constraints, 193
field-level constraints, 194e195
quality control constraints, 194
regulatory constraints, 195
technical constraints, 193e194
fungal consortia, 176e177
fungal-bacterial consortia, 177e178
multifunctional plant growth-promoting
attributes of microbial consortia on crops,
182e192
regulatory framework for commercialization of
microbial consortium biofertilizers,
180e182
Microbial consortium, 144e145, 174, 195
regulatory framework for commercialization of
microbial consortium biofertilizers,
180e182
Microbial control, 156
Microbial genomes, 144e145
Microbial inoculants, 168e169
Microbial population in rhizosphere region,
factors affecting, 461
Microbial symbionts, 4e5
Microbial VOCs, 63
Microbiology, 459
Microbiome, 144e145, 388
assembly, 148e150
biodiversity
biochemical mediators in plant growth
promoting microorganisms, 151e152
ecology, 145e146
holobiont-based control of rhizospheric biota,
156
management of rhizosphere microbiota,
154e156
PDRM, 152e154
plant growth variations, 148e150
impact of plant-friendly, plant-pathogenic, and
human-pathogenic microbes, 156e158
rhizosphere differentials that affect microbial
community assembly, 147e148
rhizosphere microbiome assembly and impact
on plant growth, 146e147
rhizosphere microbiota, 144e145
microbiome-based rhizosphere engineering,
238e240
microbiome-mediated protection, 144e145
on molecular level, 270e272
of rhizosphere, 330
rhizospheric microbiome, 213e215
Microbiomics, 131
Microbiospora, 387e388
Microbiota
disease and pathogens, 387e394
temperature, 389e392
water, 389e392
role in amelioration of environmental extremes,
387e394
secondary metabolites associated with,
382e383
Micrococcus, 173e174, 233e235
Micromonospora sp., 98, 387e388
Micronutrients, 348e349
Microorganisms, 2, 4, 19e20, 23, 30, 47, 61, 82,
97e98, 109e110, 127, 143e146,
Index 471

233e235, 241e244, 288, 345, 348,
433e434, 445, 460
different class of microbes impact on climate
change, 47e50
ecosystem approaches, 50
effect on climate change, 48e49
human and interactions impact on ecosystem and
climate change, 44e45
microbes as tools for achieving ecosystem
approaches, 50e52
with PGPR, 107
soil microbes, 45e47
used for bioremediation, 82e83
Microsites, 292e293
Millet (Sorghum bicolor), 317
Mimulus guttatus, 380e381, 383e384
Mineral acquisition, role in limiting,
360e361
Mineral phosphate solubilization,
452
Minichromosomes, 287
Minimal microbiome, 156e158
miRNAs, 291
Mode of action, 70
Modern sequencing methods, 267e271
Molecular techniques, 46
Moniliophthora roreri, 450
Moths (Catharanthus roseus), 26
MOTHUR, 272e273
Mugineic acid, 151e152
Multiomics, 433e434
investigation on agroecosystem, 436e439
general rhizosphere-omics challenges,
436e439
NGS of rhizospheric microbes, 439
metabolomics challenges, 435e436
metabolomics uses, 435
MS, 440
rhizospheric metaproteomics, 440
rhizospheric microbe metabolomics,
434
Mutualistic microbes, 383e384
MYC/MYB regulator, 406
Mycobacterium,21e22, 81e82
M. tuberculosis, 129e130
Mycoplasma genitalium, 129e130
Mycorrhiza, 127, 313
Mycorrhiza-helper bacteria (MHB),
177e178
Mycorrhizae, 212, 345e346
Mycorrhizal fungi, 176e177, 310
Myrothecium cinctum, 172e173
N
N-Acyl Homoserine Lactones (AHLs), 87
N-decanal, 71
15
N
2
gas, measurement of N
2
-fixation with,
292e293
NAC regulator, 406
Nanotechnology, 153e154
Natural culture system, 267e269
Natural ecosystem, 43
Natural products, 333, 336
Nematodes, 213
Neutralism, 288
New Fabian broth (NFb), 216
Next-generation sequencing (NGS), 143e144
difficulties, 439
of rhizospheric microbes, 439
Nicotiana attenuate,74
Nitrate, 349e350
Nitrogen (N), 1, 49, 100e101, 103e104,
108e109, 285e286, 298, 317, 349e350,
359
conserving consistent, 298e299
fertilizer, 100e101
fixation, 61, 272, 295, 410e411
microorganisms, 169e170
fixing population, 286e287
in Indian agriculture, 101
measurement of N
2
-fixation with
15
N
2
gas,
292e293
N-fixed using different approach, 293e294
N
2
-fixing system, 291
plants and creatures actualized nitrogen from
diazotrophs, 297e298
Nitrogen-fixing bacteria, 144e145, 349e350
relation with, 359
Nitrogen-fixing microorganisms, 133, 349e350
Nitrogen-fixing microscopic organisms, 3
Nitrogen-fixing organ, 6
Nitrogenous fertilizers, 101
Nitrosomonas,82
Nitrous oxide (NO), 50
mitigation, 52
No objection certificate (NOC), 180
Nocardia,82
Nocardioides, 387e388
Nonanal, 71
Nonculturable N
2
-fixing microorganisms,
286
Nongovernmental organizations (NGOs),
103e104
Nonliving stresses, 403e404
Nonomuraea, 276e277
472 Index

Nonsymbiotic N
2
-fixation
extending utility of, 298e299
conserving consistent N, 298e299
inoculation, 298
measurements, 285e286
measuring techniques, 291
nonsymbiotic N
2
-fixation-related factors,
294e297
aggregates of soil, 295
managing techniques, 297
moisture, 296
oxygen, 294
presence of C, 295
supplemental nutrition and minerals, 296e297
temperature, 295e296
Nonvolatile antibiotics, 290e291
Nostoc, 169e170
Nucleic acid, 21
Nutrients, 30, 215
acquisition, role in limiting, 360e361
availability, 105e106
cycling, 314e315
increase in availability of, 288e289
Nutritional absorption
by bacteria, 385e386
by fungi, 386e387
and plant health, 383e384
Nutritional strategies for beneficial rhizospheric
microbes, 215
Nutritive quality, 1
O
Oatmeal agar (OMA), 222
Obligate endophytes, 334
Ocean acidification, 48
Ocean MCP, 51
Ochrobactrum pseudogrignonense, 73, 241e244
1-octen-3-ol, 68
Omics to metagenomic approaches, 275e277
“Omics” techniques, 135, 267e269
Oomycetes, 28e29, 213, 313, 330
Operational taxonomic units (OTUs), 147e148, 274
Oral cancer, 331e333
Organic agricultural practices, 167e168
Organic food products, 167e168
Organic matter, 240e241
Organic nitrogen, 436
Oryza sativa plants, 357
Osmolytes, 316e317
Osmoprotectants, 241e244
Oxalobacteriaceae, 152e153
Oxydifficidin, 450
Oxygen (O
2
), 286, 294
P
Pachypsylla,10
Paclitaxel, 336
Paenibacillus, 106, 171, 173e174, 276e277, 382,
387e388
P. polymyxa,21e22
Pandemic COVID-19 incidence, 167e168
Pantoea, 173e174, 387e388
P. agglomerans, 214e215, 351
P. dispersa, 241e244
Paracoccus denitrificans, 172e173
Parasitism, 288, 447
Particles of Matter (POM), 51
Pathogen-associated molecular patterns (PAMPs),
291
Pathogenesis-related proteins (PR proteins), 95
Pathogens, 387e394
Pea (Pisum sativum), 9e10
Pea aphids (Acyrthosiphon pisum), 8
Pectobacterium carotovorum,64
Penicillium, 110, 170e173, 222
EU-DSF-10, 317
EU-FTF-6, 317
P. bilaii, 238e240
P. luteum, 171e172
Peroxidase (POD), 188e192, 316, 412, 414
Pesticide-specific biosurfactants, 112e113
Pesticides, 114
Petrobactin, 452
Pharmaceutical companies, 330
Phenol, 215
Phenolates, 410
Phenolic metabolites, 361
Phenylalanine ammonia-lyase (PAL), 188e192
Phomopsis liquidambaris, 359
Phosphate fertilizers, 102
Phosphate solubilization (P solubilization),
109e110, 317, 410
Phosphate solubilizing bacteria (PSB), 170e171
Phosphate solubilizing microorganisms (PSM),
110, 170e171, 350
Phosphate starvation responses (PSRs), 149
Phosphorus (P), 49, 100e101, 109e110, 149,
170e171, 317, 360e361, 410
fertilizer, 100e101
in Indian agriculture, 101
Phosphorus-solubilizing bacteria (PSB), 168e169
Phototroph, 272e273
Phylae, 215
Phyllobacterium, 170e171
Phytases, 291
Phytochelatins (PCs), 83e84
Index 473

Phytohormones, 27e28, 81, 173e174, 377e379,
410, 414
IAA, 411
production of, 289e290, 316
PHYTOPATH database, 272e273
Phytopathogenic fungi, 450
Phytopathogens targeted by PGPR VOCs, 67e68
Phytophthora sp., 213, 244e250
P. capsici,30e32
P. infestans,67
Phytoplankton, 48e49
Phytoremediation, 406e407
Pinus sylvestris var. mongolica, 424
PIPITS, 273e274
Planomonospora, 387e388
Plant bacteria interactions in rhizoremediation,
85e87
colonization of root, 85
regulation of gene expression by roots, 85
signal exchange/communication in rhizosphere,
87
Plant beneficial bacteria, 314
Plant biostimulant, 195
Plant boom promoting, 106
Plant breeding, 237e238
Plant defense
with biocontrol agents, 30e32
mystical ingredient of, 361
Plant development and disease resistance (PGBR),
147
Plant disease-resisting microorganisms (PDRM),
152e154
biosynthesis of antimicrobial compounds,
153e154
disease-suppressive soils, 152e153
Plant diseases, 152e153
Plant diversity, 49
Plant ecosystems, 156e158
Plant ecotypes, 383e384
and associated microbiota, 379e381
Plant engineering studies, 235
Plant growth
management of plant growth under drought stress
and rhizosphere microbiota, 154
mechanisms for promoting, 288e291
increase in availability of nutrients, 288e289
ISR, 291
producing antibiotics, 290e291
production of phytohormones, 289e290
production of siderophores, 290
synthesis of enzymes, 290
regulators, 385e386
rhizobiome produce plant growth hormones, 350
rhizosphere microbiome assembly and impact on,
146e147
variations, 148e150
abiotic and biologic stresses alter root exuda-
tion, 149e150
plant growth and disease resistance, 150
Plant growth and development regulator, 19e21,
30
as growth enhancer, 21e23
impact on root architecture, 27e28
phytopathogens targeted by PGPR VOCs, 67e68
relation with, 359e360
systemic effects on functioning and physiology of
plant, 23e25
PGPRs effect on plant metabolome, 24e25
PGPRs effect on plant nutrition, 23
PGPRs effect on plant transcriptome, 23e24
Plant growth promoting bacteria (PGPB),
241e244
Plant growth promoting fungi (PGPF), 309
Plant growth promoting microbes (PGPMs),
168e169, 214
Plant growth promoting microorganisms (PGPM),
193, 314e315
biochemical mediators in, 151e152
Plant growth promoting rhizobacteria (PGPR), 95,
104e106, 111e112, 173e174, 233e235,
238e240, 276e277, 309, 314, 345e346,
351, 385e386, 406e407, 411e412, 457
activities, 409f
applications, 99e100
biofertilizers, 102e113
chemical fertilizers, 100e102
complex and efficient network of functional in-
teractions created by, 408f
effectiveness of PGPR in hydrocarbons and heavy
metals contaminated soils, 411e412
to face salinization and drought facing abiotic
stresses, 412e413
importance and applications of, 107e108
mechanisms of, 108
microorganisms with, 107
properties and potential of, 407e410
rhizosphere, 95e100
Plant growth promotion (PGP), 154, 213e214,
220
Plant growth-beneficial rhizosphere bacteria, 147
Plant growth-promoting attributes of microbial
consortia on crops, multifunctional,
182e192
Plant health, 144e145, 156e158
474 Index

factors affecting root exudate profile, 358
nutritional absorption and, 383e384
rhizobiome contributes to limiting nutrient
acquisition, 348e352
rhizosphere and rhizobiome, 346e348
communication in rhizome, 348
role of rhizobiome in plant health, 348
root exudates, 352
root exudates and rhizobiome, 359e361
mystical ingredient of plant defense, 361
relation with nitrogen-fixing bacteria, 359
relation with PGPR, 359e360
role in limiting nutrient and mineral acquisi-
tion, 360e361
root exudation transport mechanism, 352e358
synergistic influence on, 359e361
Plant hormones, 404
Plant interaction effect on secondary metabolites,
25e27
Plant Metabolic Network, 131, 276
Plant metabolome, PGPRs effect on, 24e25
Plant microbes interaction study, bioinformatic
resources and platforms for, 131e133
Plant microbialeassociated studies, metaproteo-
mics in, 134
Plant microbiomes, 233e235, 288
Plant microbiota, 377e379
Plant nutrition, PGPRs effect on, 23
Plant pathogens, 289e290
Plant production through rhizosphere manage-
ment, 155e156
Plant productivity in different scenario, perspec-
tives on, 318e319
Plant rhizosphere, 96e97
Plant roots, 96, 145e146
exudates, 233e235
Plant secondary metabolites (PSMs), 382e383
Plant soil, 26
Plant species, 20e21, 461
Plant supplement carriers for arbuscular
mycorrhizal beneficial interaction, 5e7
bacterial elements in RN beneficial interaction,
6e7
plant factors in rhizo nodule beneficial
interaction, 6
Plant tissues, 1
Plant transcriptome, PGPRs effect on, 23e24
Plant-associated microbiota, 381
Plant-associated microorganisms, 315e316
Plant-bacteria interactions, 313
Plant-based rhizosphere engineering, 237e238
Plant-beneficial bacteria, 288
Plant-beneficial fungi, 310e313
Plant-beneficial oomycetes, 313
Plant-friendly, impact of, 156e158
Plant-microbial interactions, 96e98
Plant-microbial relationship, 3
Plant-microbiome engineering, 237
Plant-pathogenic, impact of, 156e158
Plant-rhizobacteria interactions, 19e20
Plant-specific databases, 276
PLANTCYC database, 276
Plantemicrobe interactions, 310e314
in enhancing plant growth and health, 314e318
ACC deaminase activity, 316e317
phosphate solubilization, 317
phytohormone production, 316
siderophore production, 317e318
perspectives on plant productivity in different
scenario, 318e319
plant beneficial bacteria, 314
plant-bacteria interactions, 313
plant-beneficial fungi, 310e313
plant-beneficial oomycetes, 313
recent and new approaches to study, 135e137
Plants, 19, 21, 111e112, 291
Plants-associated microbes, advances in discovery
of new drugs from
actinobacteria, 331
algae, 331e333
endophytes, 333e336
Plasma membrane (PM), 357, 422e423
Plectonema, 169e170
Polar compounds, 446e447
Pollinators, 447e448
Polyethylene terephthalate (PET), 87e89
rhizoremediation of PET plastics, 87e89
Polyketides, 445e446
Polylactide (PLA), 87e89
rhizoremediation of PLA plastics, 87e89
Polyphenol oxidase (PPO), 188e192
Poplar (Populus sp.), 389e390
Populus deltoids, 192
Potassium (K), 100e101, 171
fertilizers, 102
in Indian agriculture, 101
Potassium solubilizing bacteria (KSB), 171
Potassium solubilizing microorganisms, 171
Potato (Solanum tuberosum), 390e391
Potato dextrose agar, and yeast extract (PDAY),
221
Potential spatial effect, 269e270
Poyhydroxyl alkanoates (PHA), 81
Prairie grass (Andropogon gerardii), 380e381
Index 475

Predation, 447
Primary metabolites, 352
Production
of phytohormones, 289e290
of siderophores, 290
Proline, 317
Proteases, 449
Proteins, 21, 449
phosphorylation, 32
sequence database, 134
Proteomics, 32, 133e134, 272
computational proteomics, 133e134
experimental methodologies, 133
metaproteomics in plant microbialeassociated
studies, 134
Proteus mirabilis, 318
Protons, 346
Protozoa, 460
Providencia sp., 318
Proximity of root with soil, 461
Pseudomonas spp., 22e23, 46e47, 61e63, 68,
70e71, 81e82, 84e85, 97e98, 106, 108,
110, 136e137, 156, 170e174, 192, 209,
212, 214e215, 219, 233e235, 238e250,
272e273, 276e277, 314, 319, 350,
385e388
P. aeruginosa,21e22, 73e74, 113, 188, 316,
409
P. aureofaciens 63e28, 351
P. chlororaphis,73
P. fluorescence, 171e172, 241e244
P. fluorescens,23e24, 30, 71, 85, 316e317,
359e360, 381
P. fragi, 241e244
P. lurida, 241e244
P. luteola, 151e152
P. phaseolicola,64
P. protegens, 151e152
P. pseudoalcaligenes,24e25, 81
P. putida,21e24, 83e84, 108, 238e240,
359e360
CRN-09, 188e192
P. simiae, 151e152
P. stutzeri,30e32, 132e133, 151e152
P. syringae, 64, 150, 241e244,
382e383
strains, 24e25, 318
Pyrosequencing of bacteria from plant
compartments, 437te438t
Pythium, 213
P. oligandrum, 213, 313
P. ultimum, 108, 244e250
Q
QIIME, 272e273
Quality control constraints, 194
Quorum sensing (QS), 87, 147e148
quorum sensing/signaling, 348
R
2R,3R-butanediol, 61e62
Ralstonia solanacearum, 244e250, 351
Raoultella planticola, 241e244
Raw data, 129
Reactive oxygen species (ROS), 310, 382e383,
390e391, 412
Reduce methane emissions, microbes to, 51e52
Regulon, 405
Rhizo nodule beneficial interaction, plant factors
in, 6
Rhizobacteria, 61e62, 107, 110e111, 310,
413e414
interaction effect on secondary metabolites,
25e27
stimulating defense reaction of rhizobacteria in
plants, 28e30
direct mechanisms, 28
indirect mechanisms, 28e30
Rhizobacterial volatile compounds
alcohol compounds, 68e69
alkanes and alkenes compounds, 70
antibiosis of VOCs, 68
defense against water loss, 72e74
different type of VOCs, 64e67
enhancement of sulfur acquisition, 74
ketone and aldehyde compounds, 69
optimization of iron homeostasis, 74e76
phytopathogens targeted by PGPR VOCs, 67e68
sulfur compounds, 70e71
VOCs, 62e63
against abiotic stress, 71e72
producing rhizobacteria, 63e64
Rhizobia, 127, 385e386
Rhizobiome, 267e269, 347e348
contributes to limiting nutrient acquisition,
348e352
as biocontrol agent, 351e352
produce plant growth hormones, 350
role in plant health, 348
root exudates and, 359e361
Rhizobium,22e23, 106, 110, 156, 168e171,
173e174, 212, 220, 241e250, 319,
347e348, 387e388
R. beijerinckii, 459
R. cicero,96e97
476 Index

R. leguminosarum,21e22
R. leguminosarum bv. trifolii,85
R. tropici, 289
Rhizobox, 347e348
Rhizoctonia, 388
R. bataticola, 188e192
R. solani, 67, 136e137, 153e154, 212e213,
244e250, 272e273, 351
AG-4, 351
Rhizodeposits, 209
Rhizome, communication in, 348
Rhizomicrobiome, 132, 209e210, 233e235,
347e348
members of, 211
Rhizophagus, 24e25
Rhizophagus irregularis,25e26
Rhizoplane, 96, 209, 433e434
Rhizoremediation, 81, 83e84
of heavy metals, 84e85
of PET and PLA plastics, 87e89
plant bacteria interactions in, 85e87
Rhizosphere, 19e20, 83, 95e100, 145e146,
210e211, 233e235, 309, 315e316,
346e348, 387e388, 405e407, 433e434,
436e439, 457
bacteria, 329e330
differentials affect microbial community assem-
bly, 147e148
dynamics, 269e270
effect, 147e148, 435e436
importance of agriculture, 98
improper use of fertilizers, 99e100
institutional constraints, 100
interface, 267e269
lack of adoption of biofertilizers, 100
management of rhizosphere microbiota, 154e156
improved soil and plant production through
rhizosphere management, 155e156
management of plant growth under drought
stress and, 154
role of plants in rhizosphere development,
154e155
microbes, 127, 215
bioremediation, 82e83
plant bacteria interactions in rhizoremediation,
85e87
rhizoremediation of PET and PLA plastics,
87e89
techniques employed in bioremediation,
83e85
microbial collection in, 347e348
microbial communities, 214, 276e277
microbiomes, 132
assembly and impact on plant growth,
146e147
microorganisms, 314e315, 318e319
pH, 461
plant-microbial interactions, 96e98
rhizosphere-focused syntheses, 145e146
science, 457
Hiltner, Lorenz, 459
signal exchange/communication in, 87
socioeconomic constraints, 100
soil, 433e434, 460
Rhizosphere engineering, 235
for abiotic, 241e244
for biotic stress, 244e250
for sustainable agriculture, 236e241
microbiome-based rhizosphere engineering,
238e240
plant-based rhizosphere engineering, 237e238
soil-based rhizosphere engineering,
240e241
Rhizospheric biota, holobiont-based control of,
156
Rhizospheric ecology, 132
Rhizospheric effects, 347
Rhizospheric metaproteomics, 440
Rhizospheric microbes, 209, 215
cultivation strategies for, 215
metabolomics, 434
nutritional strategies for, 215
Rhizospheric microbiome, 213e215
bioinformatic resources and platforms for plant
microbes interaction study, 131e133
bioinformatic tools, 130e131
bioinformatics, 128e129
bioinformatics impact on genomics,
129e130
proteomics, 133e134
recent and new approaches to study plant-microbe
interactions, 135e137
Rhizospheric shape, 154e155
Rhizospheric soil, 212e213
Rhizotechnology, 459
Rhodanobacter, 276e277
Rhodococcus,46e47, 81e82, 170e171,
209
R. fascians, 350
Rhodopseudomonas palustris, 241e244
Rice (Oryza sativa), 319, 389e390
Root-associated microorganisms, 380
Root-insect interactions, 359
Root-root interactions, 359
Index 477

Roots, 209e210
colonization, 96, 105
colonization of, 85
exudates, 96, 144e145, 210e211, 345e346, 352,
360e361, 461
factors affecting root exudate profile, 358
and rhizobiome, 359e361
exudation transport mechanism, 352e358
exudes, 85
metabolism, 148e150
microbiome, 267e269
plant growth and development regulators impact
on root architecture, 27e28
regulation of gene expression by, 85
rhizobiome interactions, 359
secretions, 406e407
structure index, 424
system of plants, 347
Roseiflexus genus, 150
Rosellinia necatrix, 153e154
S
Saccharomyces
S. cerevisiae, 130
S. unispora, 212e213
Salicylic acid (SA), 21e22, 150, 425
Salinity pressure, 412e413
Salinity stress, 405
Salix viminali,87e89
Salt excessively sensitive (SOS), 177e178
Salt stress, 404
Salt-resistant (SR), 267e269
Salt-sensitive (SS), 267e269
Saprophytic, 221
Saprotroph, 272e273
Sclerotinia rolfsii,67
Sclerotium rolfsii, 244e250, 351
Scolecobasidium constrictum, 172e173
Scytonema, 169e170
Secondary metabolites, 209e210, 447
associated with microbiota, 382e383
plant and rhizobacteria interaction effect on,
25e27
Secondary roots, 411
Seedling biomass, 424
Sequence classification, databases and methods
for, 272e273
Sequencing protocols, 273e275
Serratia,61e63, 71, 107, 170e174, 233e235,
238e244, 314, 319, 385e388
S. liquefaciens, 171e172
S. marcescens, 241e244
S. plymuthica,71,87e89
Shot gun technique, 129e130
Shotgun metagenomics, 271e272
Siderophores, 81, 291, 317e318, 410, 445e446,
452
production of, 290, 317e318
siderophore-producing microbes, 317e318
siderophore-producing microorganisms,
317e318
Silica gel, 446e447
Silicon (Si), 214e215
Single nucleotide polymorphisms, 128e129
Single-cell genomics (SCG), 195e196
Sinorhizobium, 169e170, 349e350
S. meliloti,7
Slow anion channels (SLACs), 357
Sodium chloride (NaCl), 413
Sodium sulfate (Na
2
SO
4
), 413
Software tools, 131
Softwares, 128
Soil, 346e347
aggregates of, 295
amendments and fertilizers, 461
bacteria, 433e434
control, 155e156
ecosystem, 132
enzyme activity, 424
fertility, 109e110
microbes, 45e47
importance of, 46
nature and composition, 46e47
microbial networks, 233e235
microbial variety, 3
microorganisms, 168
nutrients, 424
organisms, 4, 145e146
production through rhizosphere management,
155e156
salinity, 405
salinization, 413
sampling, 269e270
science, 436e439
soil-based rhizosphere engineering, 240e241
soil-borne plant diseases, 144e145
solarization, 436
systems, 233e235
texture, 127
type, 144e145
and moisture, 461
Solanum lycopersicum, 406e407
Solarization, 436
Sorghum (Sorghum bicolor), 386e387
478 Index

Soybean (Glycine max), 241e244, 360e361,
389e390
Spatial scanning of rhizosphere, 269e270
Special nutrient agar (SNA), 222
Sphingobacteriaceae, 152e153
Sphingomonas,81e82
Standard operating procedures (SOPs), 195e196
Stenotrophomonas maltophilia,21e22
Streptomyces,61e63, 87e89, 98, 108, 131, 150,
173e174, 209, 220e221, 233e235, 331,
385e388, 390e391
S. griseorubiginosus,98
S. laurentii EU-LWT3e69, 317
Streptosporangium sp., 98, 233e235
Stress, 20e21, 403e404
hormone, 404
resilience, 20e21
Strigolactones, 289
Sugar beet (Beta vulgaris L.), 360e361
Sugarcane (Saccharum spp), 389e390
Sugars, 317
Sulfur (S), 74
absorption, 9e10
compounds, 70e71
enhancement of sulfur acquisition, 74
sulfur oxidizing microorganisms, 172e173
sulfur-containing amino acids, 9e10
Sulfur oxidizing bacteria (SOB), 172e173
Sulla spinosissima, 318
Superoxide dismutase (SOD), 21, 316, 412, 414
Supplemental nutrition and minerals, 296e297
Surfactin, 448e449
Sustainability, 233e235, 285
Sustainable agricultural practices, 233e235
Sustainable agriculture, 105e106, 285
rhizosphere engineering for, 236e241
Sustainable fuel, microbes as, 52
Switchgrass (Panicum virgatum), 381
Symbionts, 2
categories of, 4e5
Symbiosis, 174, 288
Symbiotic associations between microbes and host
plants
categories of symbionts, 4e5
dynamic capability of coordinated bug microor-
ganism amino corrosive digestion, 10e11
inescapable herbivore symbiont ventures into sap-
taking care of specialties, 7e10
plant supplement carriers for arbuscular mycor-
rhizal beneficial interaction, 5e7
Symbiotic microbes, 309
Symbiotroph, 272e273
System of Rice Intensification (SRI), 424
Systemic acquired resistance (SAR), 104,
176e177, 291
Systems of associative diazotrophs, 286e287
Azospirillum, 287
T
Targeted metabolomics, 434
Taxus wallichiana, 336
Tellurian plants, 146e147
Temperature, 295e296, 389e392
Terminal restriction fragment-length
polymorphism (T-RFLP), 195e196
Terrestrial biome, 49e50
climate change effect on, 49
effect on climate change, 49e50
Terrestrial ecosystems, 132
Thermobifida sp., 98, 233e235
Thermoleophilia, 385e386
Thermomonospora, 387e388
Thiobacillus sp., 172e174
T. acidophilus, 172e173
T. aquaesulis, 172e173
T. denitrificans, 172e173
T. intermedius, 172e173
T. neapolitanus, 172e173
T. novellus, 172e173
T. thiooxidans, 172e173
T. thioparus, 172e173
Thiomicrospira thyasirae, 172e173
Thiosphaera pantotropha, 172e173
Tolypocladium inflatum, 336
Tolypothrix, 169e170
Transcription Activator-Like Effector Nucleases
(TALEN), 336e337
Transcription factor, 405
Transcriptomics, 272
Transportation, 406e407
Tri-calcium phosphate (TCP), 317
Tricarboxylic acid (TCA), 220
Trichoderma, 170e171, 212e213, 222, 276e277,
313, 421
adaptation under different climatic conditions,
424
effect of different Trichoderma species on
nutrient uptake by plants, 422t
improvement of soil nutrient uptake, 424
modulation of gene expression by different Tri-
choderma species, 423t
response to plant pathogens, 425
T. atroviride, 425
T. harzianum, 188, 425
Index 479

Trichoderma (Continued)
T39, 422e423
T. koningiopsis, 317
T. virens, 425
Trichoderma-based biostimulants, 426
Trichoderma medium E (TME), 222
Trichoderma selective medium (TSM), 222
Tridecane, 70
Tryptamine-indole, 449
U
1-undecene, 68
Untargeted global metabolomics, 434
Untargeted metabolomics, 434
UPARSE, 272e273
Urban sprawl, 406e407
Urea, 293
V
Variovorax,22e23, 238e240
Vegetables, bioinformatics study of role of
rhizobiome to biologically control
pathogens in
databases and methods for sequence
classification, 272e273
microbiome on molecular level, 270e272
omics to metagenomic approaches, 275e277
potential spatial effect, 269e270
sequencing protocols and data processing,
273e275
Vegetative organs, 19
Vegetative storage protein (VSP), 72
Verticillium spp., 67
V. biguttatum, 212e213
V. dahliae, 388
Vesicular-arbuscular mycorrhiza (VAM),
168e169, 212
Viruses, 330
Vitamins, 169e170
Vitis vinifera, 357
Volatile organic compounds (VOCs), 61e63
against abiotic stress, 71e72
antibiosis of, 68
different type of, 64e67
phytopathogens targeted by PGPR VOCs, 67e68
producing rhizobacteria, 63e64
Volatile organic compounds, 291
VSEARCH, 273e274
W
Water, 346, 389e392
defense against water loss, 72e74
molds, 213
phytodepuration, 413e414
stress, 389e390
Wheat (Triticum aestivum L.), 241e244, 319,
360e361, 389e390
Whole genome sequencing (WGS), 275e276
Wild-type plants (WT plants), 71e72
X
Xanthobacter,82
X. agilis, 170e171
X. tagetidis, 172e173
Xanthomonas, 170e171, 241e244, 350
X. campestris,64
X. oryzae, 244e250
Xenobiotics, 83, 105e106
Xylem, 9e10
Y
Yersinia pestis, 129e130
Z
ZF-HD regulator, 406
Zinc (Zn), 452
solubilizing microorganisms, 171e172
Zinc-Finger Nucleases-Cas9 (ZFN-Cas9),
336e337
Zwittermicin A, 449
and indole alkaloids structures, 449f
480 Index