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Volume: 2: Issue-1: Jan-Mar -2011 ISSN 0976-4550
CHARACTERIZATION OF PLANT GROWTH PROMOTING BACTERIA FROM SOIL OF
CENTRAL AND UPPER HIMALAYAN REGION
Agrawal Pavan. Kumar.*, Agrawal Shruti2, Verma, Satish.Kumar.3 Singh
Santosh.Kumar4 and Shukla Keshav. Prashad.5
3Department of Biotechnology, Sai institute of paramedical and allied Sciences, 26/26A, Rajpur road, Near
Meedo grand hotel, Dehradun-248001
2Department of Microbiology, Sai institute of paramedical and allied Sciences, 26/26A, Rajpur road,
Near Meedo grand hotel, Dehradun-248001
4Department of Biotechnology, SBS PG Inst. of Biomedical Science & Research, Balawala, Dehradun-
248001
5Department of Biotechnology, MNNIT, Allahabad
ABSTRACT: Natural ecosystems are directly dependent on beneficial microorganisms present in the rhizosphere
for soil health and plant productivity. Soil bacteria were isolated from soil of central and upper Himalayan region
with a view to screen/evaluate their Plant growth promoting potential. Plant growth-promoting rhizobacteria
(PGPRs) are employed as inoculants for biofertilization, phytostimulation and biocontrol. Dominant morphotypes
were picked up from King`s B, CAS, YEMA and Pikovaskaya Agar by employing dilution plating. A total of 55
isolates were evaluated for growth promotion using Paper Towel Assay on lentil and 9 isolates were selected for
detailed characterization. In dual plate assay all 9 isolates inhibited Fusarium solani; few isolates were inhibitory
towards Ganoderma lividense and Colletotrichum dematium.
Key words: Plant growth promotory rhizobacteria, Paper towel assay, lentil
INTRODUCTION
Bacterial diversity is of particular importance in human sustenance since these small creatures comprise the
majority of earth’s species diversity. Bacterial diversity is considered as one of the most useful resource with
considerable significance in the global form of bioremediation and bio-prospecting (Homer-Devin et al 2004).
Interaction between bacteria and roots of plants has been reported to be beneficial, detrimental or neutral and this
delicate balance is a consequence of both soil and plant type( Latour et al 1996) . Bacteria, beneficial to plants
may be symbiotic or free living, and are abundant near the roots. Such beneficial free-living bacteria have been
termed PGPR or plant growth promotory rhizobacteria( Glick ,1995). They benefit plants through, (a) Production
of plant hormones, such as auxins by Gutierrez et al 1996 (b) asymbiotic N2 fixation by Kennedy et al 1997 (c)
antagonism against phytopathogenic microorganisms by production of antibiotics (Sharma et al 2003) ,
siderophroes( Meyer2000), β-(1,3)-glucanase, chitinase(Renwick et al 1991) and cyanide(Flaishman et al 1996)
and (d) solubilization of mineral phosphates and other nutrients(de Freitas et al 1997) (e)ability to effectively
colonize roots are responsible for plant growth promotion [Burdetal 2000, Duffy BK, Defago G (1999),. A
number of PGPR such as Bacillus (Holl et al 1988), Pseudomonas(O’ Neill et al 1992) and Arthrobacter (Beall
and Tipping 1989) have been used for enhancement of plant performance.
Phytopathogens are major and chronic threats to food production and ecosystem stability worldwide. As
agricultural production intensified over the past few decades, producers became more and more dependent on
agrochemicals as a relatively reliable method of crop protection helping with economic stability of their
operations. Despite inconsistency in field performance, biological control is considered as an alternative or a
supplemental way of reducing the use of chemicals in agriculture ( Gerhadson 2002)
The Himalayan region represents a unique combination of plant and soil type that changes drastically with
altitude however only limited efforts have so far been made to explore the available bacterial diversity. In the
present study soil samples were collected from Chaubatia (Ranikhet) Uttaranchal Himalayas and Leh region, for
assessment of PGPR characterization.
International Journal of Applied Biology and Pharmaceutical Technology Page:363
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Agarwal pavan et al ISSN 0976-4550
MATERIAL AND METHODS
Sampling Sites:Five soil samples viz, MFA-1, MFB-1, FQP, FMP and FPA were collected from Ranikhet
(Chaubatia) at latitude 29.60C and longitude 79.50C; soil sample A-2 was from Leh (latitude, 340C and longitude,
77.50C). Characteristics of the habitat and their physico-chemical properties are presented in Table 1.
Soil Analysis: Soils were analyzed for pH and organic carbon by Walkely & Black 1934. Available and total ‘P’
were analyzed according to Olsen 1954. Dehydrogenase activity was measured according to Thalman1968.
Isolation of bacteria: Bacterial population was enumerated using 10-fold serial dilutions (Johnson and Curl1972)
and expressed as CFU g-1 dry soil; serially diluted soil samples (upto 10-5) were plated on King’s B medium for
pseudomonads by Buysen1996, yeast extract mannitol agar for rhizobia (Vincet et al 1991), Pikovaskya’s agar
for phosphate solubilizer (Pickovaskaya1948) and chromeazurol ‘S’ (CAS) agar for siderophore
producers(Schwyn and Neilands 1987). Plates were incubated in triplicates at 280C and counts (CFU) were
recorded after 72 h. A total of 55 colonies representing all the morphotypes were picked up and categorized on the
basis of soil type and media used.
Screening of bacteria for plant growth: Lentil (Lens esculentus var. PL406) seeds were surface sterilized with
0.1% mercuric chloride for 5 min, rinsed with sterilized distilled water (SDW) and soaked in bacterial suspension
(3×108 cfu ml-1) using 1% carboxymethyl cellulose (CMC). Air dried seeds were placed on a paper towel (ten
seeds per paper) and incubated at 28±20C for 21 d in a growth chamber. Percentage germination was recorded
along with root and shoot length (ISTA1993). Non-bacterized seeds served as control. Five replicates were used.
Functional attributes of the bacterial isolates: On the basis of plant growth performance in paper towel assay,
nine promising isolates were tested for their ability to produce indole acetic acid (Gordon and Weber 1951).
IAA production: Bacteria were grown overnight in five ml of M-9 minimal medium(John et al 2005)
supplemented with L-tryptophan to achieve a final concentration of 0, 50, 100, 200 and 500 µg ml-1. After
incubation for 42 h, bacterial growth was measured spectrophotometrically at 600 nm; cells were removed from
culture medium by centrifugation at 7,500 rpm for 10 min. A 1 ml aliquot of supernatant was mixed with 4 ml of
Salkowski‘s reagent (150 ml of concentrated H2SO4, 250 ml of D.W, 7.5 ml of 0.5 M FeCl3, 6H2O). Samples were
left at 28±20C for 25 min and absorbance was read at 535 nm. The concentration of IAA was determined by
referring to a standard curve.
In vitro antifungal assay: Bacteria were tested for antagonism against Colletotrichum dematium, Fusarium
solani and Ganoderma lividans using a dual culture plate assay (Sharma and Johri 2003).
RESULTS AND DISCUSSIONS
Soil analysis: Soil samples (MFA-1, MFB-1, FPA, FQP, FMP and A2) used in this investigation had slightly
acidic pH (6.25 to 6.60). Organic matter and organic carbon content was high in all samples except FPA (0.64%)
(Table 2) which represents a forest soils. Sample MFB-1 (11.84 kg ha-1), MFA-1 (27.87 kg ha-1) and FMP (50.32
kg ha-1) showed medium to high level of available ‘P’ whereas others were low (Table 1). Presence of organic
matter and availability of nutrients such as phosphorus influences microbial activity of the soil studied by Johri et
al 1999; Nautiyal et al 2000. A forest soil with mixed root fragments (FMP) depicted high dehydrogenase activity
(448.87 µg TPF ml-1 16 h-1) compared to soil without such material (FPA 52.61 µg TPF ml-1 16 h-1). FMP also had
population count (7.0×107 CFU g-1 soil) Table 1.
Dehydrogenase activity and organic matter content of a soil are correlated with soil health. There was a direct
relationship between total biological activity and organic carbon. Microbial counts in FMP were also directly
related with biological activity and organic carbon ( Lodha et al 2002).
International Journal of Applied Biology and Pharmaceutical Technology Page:364
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Agarwal pavan et al ISSN 0976-4550
Table 1: Characteristics of soils from Central and Upper Himalayan region
Code Sample site pH
±sem*
Organic
matter (%)
±sem*
Organic
‘C’ (%)
±sem*
Available
‘P’ Kg ha-1
±sem*
Total‘P’ Kg
ha-1
±sem*
Dehydrogenas
e activity µg
TPF ml-1
±sem*
MFA-1
Modern farming
upper profile
Padhandsyar,
Rampur (3100 msl)
6.55±0.12 2.98 ±0.11 1.73±0.15 27.87±0.37 124.0±o.66 352.48±0.33
MFB-1
Modern Farming
Lower profile
Padhandsyar,
Rampur (3100 msl)
6.50±0.19 2.17±0.17 1.26±0.16 11.84±0.89 43.83±0.67 131.54±0.18
FQP
Forest Soil (Oak,
Banj) Ganju (3500
msl) 6.45±0.32 3.93±0.53 2.28±0.29 9.15±0.59 258.14±0.11 437.60±0.79
FMP Forest Soil mixed
with roots (5000 msl) 6.40±0.22 4.59±0.41 2.66±0.32 50.32±0.12 196.00±0.17 448.87±0.16
FPA
Forest Soil (Planted
1987, Rampur road
(3300 msl) 6.25±0.19 1.10±0.13 0.64±0.37 5.73±0.25 159.46±0.13 52.61±0.27
A-2
Leh cold Desert at an
altitude of 12,000-
13,000 feet (Mixed
farming of wheat
Brassica and Potato
6.60±0.43 2.07±0.17 0.92±0.47 33.80±0.32 115.52±0.17 109.50±0.38
*sem: standard error of mean
Screening of bacteria for plant growth promotion: A total of 55 isolates were used to asses their influence on
seed germination and root/ shoot length of lentil in a paper towel assay. On the basis of their growth performance,
nine isolates namely MFA-1SD-2, MFA-1R-3, MFB-1R-3, FPASD-1, FQPR-2, FQPR-3, FMPPB-3, FA2K105
and FA2K1003, were used for further characterization.
Bacterized lentil seeds showed improved plant growth compared to untreated control (Table 2). A significant
difference in percentage of germination was observed compared to control; values ranged between 14.18 to
38.36%. Isolates exhibiting improved seed germination also supported improved root and shoot length (Table 3).
Seeds coated with bacterial isolates derived from soil representing modern farming practices (MFB-1R-3) showed
maximum germination (38.36%), root length (9.41 cm) and shoot length (2.83 cm). On the contrary, seeds coated
with bacterial isolates derived from Leh soil (FA2K1003) showed minimum seed germination (14.18%), root
length (9.41 cm) and shoot length (2.83 cm) compared to control (Table 3). Seed bacterization (or seed coating)
has proven to be a method of choice for studying bacterial growth promotion and biological control of plant
diseases including pre emergence and post-emergence diseases.In this study, seed treatment with the bacterial
isolates significantly improved seed emergence together with plant root and shoot length.
Functional attributes of bacteria: Except MFA-1R3, all other bacterial isolates were positive for siderophore
production, phosphate solubilization and indole acetic acid production (Table 2) but were negative for HCN.
Isolate MFB-1 R-3 produced highest level of IAA in liquid broth (33.55 µg ml-1) and FA2K1003, the least (1.75 µg
ml-1).
In vitro fungal growth inhibition assay: Under in vitro condition, five isolates restricted growth of test fungus
G. lividans in dual culture test. Inhibition level was 31.5% for MFB-1 R-3 and 39.4% for FA2K1003. Growth of
Colletotrichum dematium was inhibited by five bacterial isolates with a value of 22.5% for MFB-1 R-3 and 47.5%
for FPA SD-1. Growth of F. solani was inhibited by all nine bacterial isolates; inhibition level ranged from 7.14%
to 52.3% (Table 3).
International Journal of Applied Biology and Pharmaceutical Technology Page:365
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Agarwal pavan et al ISSN 0976-4550
Table 2: Growth promotory potential of selected bacterial isolates in a paper towel assay
on lentil (Lens esculentus var. PL 406)
Isolates IAA (
µ
g ml-1)
±sem*
Seed germination
(%) 3d
Root length (cm)
7d
Shoot length (cm)
7d
Control 53.252 1.70 3.29
MFA-1 SD-2 1.80±0.21 68.31 4.19 12.60
MFA-1R-3 6.22±0.19 73.62 2.04 10.40
MFB-R-3 33.55±0.37 73.62 4.53 12.70
FPA-SD-1 2.88±0.31 73.62 2.98 11.50
FQP R-2 2.25±0.48 68.31 2.80 10.90
FQP R-3 1.83±0.29 66.17 3.21 12.20
FMP PB-3 6.31±0.27 66.17 3.57 11.50
FA2K10513.55±0.16 68.31 2.45 12.52
FA2K1051.75±0.24 60.77 1.99 9.45
CD at 5% - 7.79 0.623 1.77
SEm - 2.80 0.224 0.640
Table 3: In vitro antagonistic behaviour of select bacterial isolates against
phytopathogenic fungi
Percent inhibition of growth (%)
Isolates G. lividens C. dematium F. solani
MFA-1 SD-2 ND ND 35.70±0.77
MFA-1R-3 34.00±0.19 ND 42.80±0.58
MFB-R-3 31.50±0.82 22.50±0.58 45.02±0.24
FPA-SD-1 36.80±0.56 47.50±0.24 52.30±0.16
FQP R-2 ND ND 19.00±0.29
FQP R-3 ND ND 23.80±0.17
FMP PB-3 36.80±0.12 25.00±0.16 7.14±0.13
FA2K105ND 32.50±0.29 40.00±0.26
FA2K100339.40±0.77 25.00±0.17 33.30±0.12
Based on dual culture
In vitro fungal growth inhibition assay showed variable antagonism against G. lividans, C. dematium and F.
solani. There is considerable evidence to suggest that there is unlimited diversity of genetically dissimilar
microorganisms in the rhizosphere, among which communities, suppressive to plant pathogens are abundant in
the soil.
Biological control of pathogenic and other deleterious microorganisms in soil-root interface is often attributed to
antibiosis where antibiotics produced by Gram -ve and Gram +ve antagonistic bacteria play direct role in disease
suppression. In this study, isolates were prominent antagonistic against F. solani. The highly effective disease-
suppressive fluorescent pseudomonad species strains owe their suppressive potential to antibiotics produced in the
rhizosphere. Phenazine derivatives were the first antibiotics implicated in biocontrol produced by fluorescent
pseudomonads, such as Pseudomonas fluorescens and P. aureofaciens. Several other antibiotics and
antimicrobials are produced in the rhizosphere by P. fluorescens, including HCN, 2, 4-diacetylphloroglucinol
(DAPG), and pyoluteorin, which directly interfere with growth of various pathogens and contribute to disease
suppression (Dwivedi and Johri 2003 ).
From a more general perspective, the diversity within populations of antagonistic microorganisms with a common
biocontrol trait is a means to improving biocontrol.
International Journal of Applied Biology and Pharmaceutical Technology Page:366
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Agarwal pavan et al ISSN 0976-4550
This approach builds on existing knowledge of mechanisms while exploiting genetic differences that have
evolved to enable microbial populations to compete successfully in diverse soil and rhizosphere environments.
Understanding the diversity within populations of biocontrol agents holds the promise of pairing specific
genotypes with their most supportive plant hosts or soil environments to maximize root colonization and disease
suppression. Finally, establishing the presence and functionality of individual populations within a particular soil
is just one first step toward fully understanding the nature of suppressiveness within that soil. Ultimately, the
parameters within which the activities of functionally important microbial populations combine to produce a
suppressive soil also must be defined. To identify those parameters, new and more detailed studies will be
required to characterize the soil structure and composition, the environmental conditions under which suppression
occurs, the molecular interactions among functionally important populations under different conditions, and the
biogeography and population dynamics of beneficial as well as pathogenic microbial populations in the field.
Because of the complexity of field soils, high through put methods will be required to adequately characterize
these populations, but the pay-off will be worth the effort. The future studies of biologically based soil
suppressiveness will present new insights in to the microbial ecology of agricultural soils and lay the foundation
for the development of creative management strategies for the suppression of soil borne diseases.
Acknowledgment:
The author wish to thank to chairmain Mr Harish Arora and Vicechairperson Mrs Rani arora of SIPAS, Dehradun.
We acknowledge the sincere thanks to Dean life science and Principal SIPAS for providing us research facilities.
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