Perspectives for sustainable agriculture from the microbiome in plant rhizosphere

Abstract

The ever-growing human population globally has resulted in the quest for solutions to the problem of hunger by providing food security. The importance of plant-root-associated microorganisms cannot be overlooked, plants rely on them. These root colonizers dominate the rhizosphere due to the abundance of available nutrients, relying on their host plant for nutrients and other essential requirements. The relationships between microbial communities and plants are controlled by the type of plant and microorganism involved. Advances in modern molecular techniques have led to the evolution of omic technology using nucleic acid molecules to study plant-microorganism associations capable of stimulating plant growth, improve yield, and induce disease suppression. This review elucidates the activities of microbial communities, especially nitrogen-fixing rhizobacteria associated with plant roots, nitrogen fixation as a mechanism of promoting plant growth, their importance, and the challenges employing bioinoculants. Prospecting plant growth promoters using omic technology will advance sustainable agriculture globally.

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Plant Biotechnology Reports
https://doi.org/10.1007/s11816-021-00676-3
REVIEW
Perspectives forsustainable agriculture fromthemicrobiome inplant
rhizosphere
BlessingChidinmaNwachukwu1· OlubukolaOlurantiBabalola1
Received: 19 March 2021 / Revised: 20 April 2021 / Accepted: 27 April 2021
© Korean Society for Plant Biotechnology 2021
Abstract
The ever-growing human population globally has resulted in the quest for solutions to the problem of hunger by providing
food security. The importance of plant-root-associated microorganisms cannot be overlooked, plants rely on them. These
root colonizers dominate the rhizosphere due to the abundance of available nutrients, relying on their host plant for nutrients
and other essential requirements. The relationships between microbial communities and plants are controlled by the type of
plant and microorganism involved. Advances in modern molecular techniques have led to the evolution of omic technology
using nucleic acid molecules to study plant-microorganism associations capable of stimulating plant growth, improve yield,
and induce disease suppression. This review elucidates the activities of microbial communities, especially nitrogen-fixing
rhizobacteria associated with plant roots, nitrogen fixation as a mechanism of promoting plant growth, their importance, and
the challenges employing bioinoculants. Prospecting plant growth promoters using omic technology will advance sustain-
able agriculture globally.
Keywords Nitrogen-fixing genes· Omic technology· Plant growth-promoting rhizobacteria· Root nodulation process·
Soil fertility
Introduction
Life dwelling on earth depends on the celestial environment
and is controlled by the soil beneath our feet. Soil is a critical
part of the ecosystem with valuable resources important to
us, as the topmost layer of the earth; it holds plants at the
root and support animal life. Above all, the soil is composed
of numerous microorganisms interacting with each other.
Soil ecosystem is a source of minerals, organic matters, liq-
uids, and gases. Moreover, soil acts as a water filter and a
medium that encourages plant growth (Beckers etal. 2017).
All soil houses millions of organisms that contribute to the
biodiversity and distribution of most of the antibiotics for
fighting plant borne diseases (Zhou etal. 2020).
In the context of this review, the soil region around the
plant root that connects the network of plant root-associated
microorganisms, especially bacteria are found in the plant
root system called the rhizosphere. Microbial communities
in the rhizosphere perform dynamic beneficial interrelated
functions in the plant root zone (Olanrewaju etal. 2019). The
entire set of microbial populations inhabiting the plant root-
associated region is referred to as the rhizosphere microbi-
ome or rhizobiome. Some of these microorganisms may be
pathogenic (harmful to plant growth) or non-pathogenic hav-
ing symbiotic relationships with other rhizospheric microor-
ganisms (Ali etal. 2017).
Lorenz Hiltner in 1904 introduced the term ‘rhizosphere’
due to Beijerinck’s discovery of bacterial community inhab-
iting the soil with the ability to fix nitrogen. According to
the author, rhizosphere was described as root-associated soil
compartment influenced by root activity. Furthermore, rhizo-
sphere is the ecological niche where microorganisms dwell,
serving as a connection to other plant root zones. Micro-
organisms in the compartment perform significant activi-
ties that contribute to plant health, mostly nitrogen fixation
(Olanrewaju etal. 2019). The rhizosphere inhabits myri-
ads of microorganisms and is considered a well-advanced
external functional plant habitat, which is the plant’s sec-
ond genome because plants are referred to as metaorgan-
isms. Therefore, understanding the exact contributions of
Online ISSN 1863-5474
Print ISSN 1863-5466
* Olubukola Oluranti Babalola
olubukola.babalola@nwu.ac.za
1 Food Security andSafety Niche, Faculty ofNatural
andAgricultural Sciences, North-West University, Private
Mail Bag X2046, Mmabatho2735, SouthAfrica

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rhizobiome mainly nitrogen (N) fixing bacteria concerning
plant health and productivity are important (Ali etal. 2017).
Bacterial species usually do not work in isolation but in
association with other microbial species that have diverse
beneficial impacts on plants, hence promoting plant growth
or non-beneficial with damaging effects on the host plants
(Bagyaraj and Ashwin 2017; Murphy etal. 2015). In the
rhizosphere, the association between plant and the microbial
communities occurs within the soil matrix (Pii etal. 2015;
Sabale etal. 2019). Rhizosphere is a diverse habitat with a
high abundance of microbial community called rhizobiome.
The bacterial species are the most dominant microorganisms
found in the rhizosphere, colonizing the microenvironment
with mutual benefits (Bagyaraj and Ashwin 2017).
The beneficial rhizobiome in the regions have close
interaction with host plants primarily contributing to the
soil health for various agricultural purposes (Alawiye and
Babalola 2019). Among other benefits of rhizobiome that are
of attention is their interaction with other microorganisms
and plants. Understanding the interactions in the rhizosphere
is not solely for comprehending their involvement in plant
growth and development. Likewise, to understand the differ-
ent metabolic activities that occurs in the rhizosphere (Liu
etal. 2015; Preece and Penuelas 2016). Also, for exploiting
their association in phytoremediation approaches that can
lead to the sustainable production of metabolites and crops
(Schlemper etal. 2017).
More so, the microbial community control plant rhizos-
phere has led to competitors cohabiting in the environment
in a mutual relationship (Liu etal. 2015). The positive rela-
tionship is a symbiotic relationship with the host plants or
negative with damaging impacts that are from predators
or pathogens (Igiehon etal. 2019). Either of the microbial
community associations in the rhizosphere influence plant
growth and tolerance to stressors, however, its significance
is creating awareness (Rasmann and Turlings 2016).
Studies have shown that the rhizosphere is the hotspot for
diverse abundance of microbial populations conducting sev-
eral taxonomical operations. The root region (rhizosphere)
is a complex soil environment that attracts much microbial
mainly bacterial genera from the bulk soil. The bacterial
community attracted to the plant root region interacts with
the plant and other microbial communities, as a result,
release metabolites for promoting plant growth. The plant
roots and soil environment control the associations between
microorganisms and plants as well as plants and microorgan-
isms (Alawiye and Babalola 2019).
The rhizobiome play an essential role in sustaining and
stabilizing the ecosystem. These roles influence the plant
through gaseous and nutrient exchanges, and also, plant
liter, water filter, and rhizodeposits. The plant root region is
a path for nutrient and mineral absorption, which releases
a several of organic and inorganic compounds known as
rhizodeposits. The presence of rhizodeposits forms an exclu-
sive environment in the rhizosphere, which serve as a pointer
that diverse rhizobiomes are bound to be in abundance in the
rhizosphere with high nutrients (Preece and Penuelas 2016).
Moreover, the rhizosphere contains a pool of microbial
diversity conducting different interactions and functions,
such as growth promotion and pathogen resistance. The
rhizosphere is controlled by root exudates (low molecular
weight organic compounds secreted by plant roots, which
are released into the rhizosphere, capable of influencing
the rhizosphere, inhibit harmful microorganisms, and pro-
mote plant growth), mucilages (polysaccharide substances
extracted as gelatinous or viscous solution produced by most
plants and some microorganisms), and border cells (those
cells that separate from plant root tips and disperse into soil
environment) released into the rhizospheric soil by plant
roots.
Plants secrete border cells, exudates, rhizodeposit nutri-
ents, and mucilages that attract microorganisms to the
region, which serve as food for microorganisms inhabiting
in the rhizosphere. The rhizosphere is structured by groups
of microorganisms mostly bacterial species that are mesmer-
ized by the rhizodeposits disseminating from the plant roots
(Orlikowska etal. 2017). Ultimately, contributing to making
the soil the most complex and diverse habitat in the ecosys-
tem to a point that many microbial species colonizing the
rhizosphere release regulatory substances that can influence
plant ability to survive in challenging soil environmental
conditions, including in waterlogged areas, extreme cold or
desert on the planet that is a threat to sustainable agriculture
(Alawiye and Babalola 2019; Sabale etal. 2019).
Sustainable agriculture is an integrated farming system
having a site-specific approach that would over time satisfy
the human need for food, improve environmental quality,
and natural resources. Generally, improve the economy,
result in enhancing the farmers and society in the quality of
life. Sustainable agriculture relies on fertile soil, but rapid
desertification and land degradation have damaged the soil
caused an approximate loss of about 24 billion tons of fertile
arid land world-wide (Pii etal. 2015; Zhou etal. 2016).
Plant microbial communities participate in transforma-
tions that impact on the composition of litter, which could
lead to some changes in the turnover of nutrients, soil struc-
ture, physiology, and characteristics. These might cause
changes in the structure and composition of plant microbial
communities (Xu etal. 2019). In addition, biotic and abiotic
factors control the microbial activities in the rhizosphere
region. The rhizobiome coexisting in the rhizosphere plays
an essential role in improving soil quality, plant health, and
crop production (Zhang etal. 2017).
The inoculation of diverse bacterial species into the soil
helps in improving plant-microorganism interactions, bio-
geochemical cycles, and biological engineering. Therefore,

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could encourage agriculturists from the application of
agrochemicals, substituting the functions of these toxic
chemical-based products for suitable beneficial bacteria (Li
etal. 2019). The overall understanding of the biodiversity
and structure of functional and active root and free-living
microorganisms in the rhizosphere, such as nitrogen-fixing
bacteria, capable of fixing nitrogen for plant use, which is
paramount in enhancing crop performance and growth, in
turn, increase agricultural crop production (Lagos etal.
2015).
To gain a complete understanding of the microbial net-
work in the rhizosphere, it is necessary to integrate the
interactions between the most abundant soil, and plant root
microorganisms (Zhou etal. 2020). A detailed review of the
interactions of the diverse nitrogen-fixing microbial com-
munity in the rhizosphere was discussed. Also, a compre-
hensive review of the mechanism of nitrogen fixation, con-
tributions, and challenges of applying PGPR in sustainable
agriculture was discussed. Conclusively, omics techniques
for identifying and characterizing microbial communities
were highlighted.
Rhizobiome ofagricultural plants: plant growth
promoters
Rhizobiome referred to as rhizobacteria are a class of bac-
terial species found in the rhizosphere with the ability to
enhance host plant growth. Plant growth is stimulated by
diverse microbial communities present in the rhizosphere.
Several plant growth-promoting microorganisms are cohab-
iting in the rhizosphere. Plant root assembles many plant
growth-promoting rhizobacteria communities such as Azos-
pirillium, Rhizobium, Arthrobacter, and Pseudomonas into
the internal, surface of plant roots, and surrounding the plant
environment (Lagos etal. 2015).
The rhizobiome play critical beneficial roles in the host
plant, including plant nutrient cycling, phytohormone pro-
duction, and immunity against phyto-pathogens., couple
with, the specific capacity to further improves soil fertil-
ity (Lee etal. 2019). In this instance, the plant microbial
community has been described to accouter plant hosts with
new pools of genes attributed as extended genome or plant
genome (Igiehon etal. 2019; Vandenkoornhuyse etal. 2015).
Meanwhile, some scientists have reported the richness of
rhizobacteria in plant root regions and rhizospheric soils.
Hence, any shift in the balance or changes in the edaphic
conditions will certainly have impact on the soil microbiota
and crop yield (Lee etal. 2019). Various agricultural benefi-
cial microorganisms and their functions in the rhizosphere
of different plants are outlined in Table1.
Recently, the attentions of some scientists have been
drawn to the critical effects of plant growth-promoting rhizo-
bacteria (PGPR) on plants. The impacts have unveiled the
different mechanisms of interactions between plants and bac-
terial communities in the rhizosphere. PGPR has been iden-
tify as a means to enhance sustainable food production in the
future (Babalola 2010; Finzi etal. 2015; Igiehon etal. 2019).
Various mechanisms are employed by PGPR to either pro-
mote plants growth or have general impact on the plants, for
example, the production of inhibitory substances (antifungal
and antibiotics metabolites) against plant pathogens (Igiehon
etal. 2019; Vandenkoornhuyse etal. 2015). In particular,
Firmicutes, Proteobacteria and Actinobacteria are involved
in suppression of diseases caused by the pathogenic fungus
Rhizoctonia solani. Another study showed that certain bac-
teria were able to eliminate the impact of R. solani on let-
tuce, further suggesting that some bacteria have impacts on
rhizosphere-assocaited bacteria and plant tissue-associated
fungi (Scherwinski etal. 2008), especially the pathoegenic
ones. Another example is Pseudomonas sp. AF-54 isolated
from sunflower rhizosphere with antagonistic effects on
Fusarium sp. The bacterium can be a promising biocontrol
agent against sunflower pathogens (Majeed etal. 2018a). It
was however shown that beneficial bacterial species, such
as Pseudomonas fluorescens F113, produce antifungal sub-
stance 2, 4-diacetylphloroglucinol that are not detrimental to
the mycorrhizal fungus Glomus mossea, but rather helps the
fungus to colonize host plants’ roots more effectively (Barea
etal. 1998). On the other hand, like bacteria, certain fungi
can contribute to mycorrhizal root colonization while they
hamper the growth of other fungi in the rhizosphere (Igiehon
and Babalola 2018b), meaning that, there are some fungal
species that inhibit the growth and/or survival of other fungi
in the soil ecosystem.
Other mechanisms of PGPR are mainly through bio-
geochemical cycles, significantly in the nitrogen fixation
process by nitrogen-fixing bacterial species, which spe-
cifically possess nitrogen-fixing genes (nif) that provide
nitrogen for usage (Chen etal. 2019a; Igiehon etal. 2019)
by plants, especially nodule-forming plants. The process
involved in the formation of nodules has been reviewed in
detailed in previous studies (Igiehon etal. 2018a; Oldroyd
etal. 2011; Udvard and Poole 2013). However, some bac-
teria cannot initiate nodulation process since the process
entails complex plant-microbial interactions. Some nitro-
gen fixing bacteria contribute to nitrogen fixation process
for host plant development by penetrating root cracks and/
or wounds possibly created by root movement within the
soil (Gaiero etal. 2013; Santi etal. 2013). Azoarcus sp.
BH72, which is a kallar grass root tissue-associated bac-
terial species improved the dry weight of the grass when
grown in nitrogen-deficient soil compared to the ‘nifk
mutant strain of BH72’. Nevertheless, the bacterial spe-
cies can mutate from free-living to endophytic, as well as
from endophytic to free-living forms. Also, soybean plants
inoculated with Rhizobium spp. alone and co-inoculated

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with Rhizobium spp. and mycorrhizal fungal consortium
were observed to nodulate the plants in the field at North-
West Province of South Africa under semi-arid condition
(Igiehon etal. 2020). The Rhizobium spp. were shown to
possess nif and nod genes upon analyses of their whole
genomes through Kbase pipeline (Igiehon etal. 2019,
2020).
Chen etal. (2019b) studied PGPR communities of
wheat plants and their beneficial effects on plant devel-
opment. The results provided insights that proved a shift
Table 1 Some plant root-associated microorganisms and their influence on plants
Microorganisms Effects
(Positive and negative)
References
Burkholderia sp., Achromobacter sp.,
Azospirillum sp., Chryseobacterium sp.
Stimulatory effects on plant growth (Ambrosini etal. 2015)
Rhizobium spp. YAS34 Promote plant growth under stressed conditions, and resistance to
water
Make use of fertilizer more effectively by increasing nitrogen uptake
(Ali etal. 2017)
Proteobacteria, Acidobacteria Indicates soil nutrient (Beckers etal. 2017)
Azotobacter vinelandii; Azotobacter
Chroococcum Promote plant growth (Ali etal. 2017)
Frankia Nitrogen-fixing, and
P-solubilization
(Beckers etal. 2017)
Candidatus
Saccharibacteria High indicator:
To biotic and abiotic stresses;
strict nutrient requirements;
plant innate immune responses;
Interact with host plant genotype and microorganism-to-microorgan-
ism interaction
(Starr etal. 2018)
Azospirillum lipoferum Inhabits plant root niche and crude oil contaminated soil (Singh etal. 2018)
Enterobacter Produces siderophore, Indole Acetic Acid (IAA), and HCN; Fixes
nitrogen, and
P-solubilization
(Starr etal. 2018)
Pyrococcus furiosus, Flavobacterium Biocontrol activity;
P and K solubilization
(Singh etal. 2018)
Bacillus ceres Causes rootlet rot (Singh etal. 2018)
Curtobacterium, Phosphorus solubilization, and IAA production (Ali etal. 2017)
Pseudomonas putida GR12-2 Ethylene, and
ACC-deaminase inhibitor
(Alawiye and Babalola 2019)
Pseudomonas fluorescens angstrom 313 Causes shunted growth in plant (Mosimann etal. 2017)
Bacillus substilis Causes sour skin, and soft-rotting disease of onion (Murphy etal. 2015)
Pseudomonas fluorescens;
Pantoea agglomerans Antifungal activity (Alawiye and Babalola 2019)
Azospirillum brasilense Promotes the uptake of NO3,
K + , and H2PO4
(Ali etal. 2017)
Burkholderia
ambifaria MCI7
Antifungal activity,
siderophore production,
Increase shipped weight, and
plant performance
(Mosimann etal. 2017)
Burkholderia pseudomallei Causes melioidosis (Manivanh etal. 2017; Mora-
Ruiz etal. 2018)
Halobacillus sp Biocontrol activity, IAA production, and P-solubilization (Mosimann etal. 2017)
Pseudorhodoplanes, Paenibacillus, ocuria Indole Acetic Acid production,
P-solubilization, and nitrogen-fixing
(Şeker etal. 2017)
Arthrobacter P-solubilization, production of IAA, and biocontrol
Agent
(Singh etal. 2018)
Pseudomonas fluorescens, and
P. fluorescens biotype F
ACC deaminase
activity, Chitinase
activity; increased
grain yield, and weight
(Şeker etal. 2017)

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in root rhizobiome can affect plants and the microbiome.
There are shreds of evidence that has of course implicated
the activities in the rhizosphere that trigger the release
of rhizodeposits that are of benefits to plant. So to tradi-
tionally observe the effects of various cultivated plants on
the composition of PGPR found in the rhizosphere, there
is a need to understand the abundance and activities of
the PGPR in rhizospheric soil (Babalola 2010; Chen etal.
2019a; Hu etal. 2018).
The activities of the PGPR determine the number of
organic compounds excreted by the roots. Thus, these com-
pounds are collectively known as exudates (Babalola 2010;
Hu etal. 2018). The exudates and other rhizodeposits influ-
ence the physical and chemical structures of the rhizobac-
teria (Cregger etal. 2018). PGPR assimilate and released
exudates deposits, the exudates are then discharged into the
rhizospheric soil after modification (Crawford and Knight
2017). In return, it is important for the soil quality that
increases when these deposits are absorbed by the PGPR
that can finally boost plant growth (Orlikowska etal. 2017).
For example, root polysaccharide enhanced the production
of biofilm matrix by Bacillus subtilis, this rhizobacteria is of
benefit to plants generally (Olanrewaju etal. 2019). Figure1
outline the different mechanisms of action PGPR uses in
improving plant growth.
PGPR perceives different signals secreted from plant
roots. Ultimately, it releases different signaling molecules
that control various stress responses that are caused by
edaphic conditions. This usually equips the plant with the
ability to resist or tolerate the different stresses that would
lead to root development, plant growth, and increase in crop
productivity (Crawford and Knight 2017; Lyu etal. 2019).
PGPR coexisting in the rhizosphere respond differently to
root metabolic activities by stimulating biomass and activi-
ties (Lyu etal. 2019).
The growing pieces of evidence in the study of plant
growth promotion have proved that PGPR improves soil
health and quality, for this reason, promotes plant develop-
ment and growth. For example are PGPR that speeds up
nutrients cycling, influences, increases available nutrients,
and nitrogen-fixation (Finzi etal. 2015; Lyu etal. 2019).
Adding further, the PGPR does not perform activities in iso-
lation, there is an association between the plant and micro-
biota in the micro-environment. This coexistence influence
seedling survival that results in the advancement of plant
growth performance and productivity (Crawford and Knight
2017).
There are shreds of evidence that backs up the compe-
tence of PGPR in sustainable agriculture. Nevertheless,
PGPR was first explored only for improving crop production
Fig. 1 Mechanisms of plant growth promotion. PGPR promotes
plant growth by direct and/or indirect mechanisms. The direct PGPR
mechanism includes nitrogen fixation, siderophore production, phos-
phate solubilization, potassium solubilization and phytohormone
production, while the indirect PGPR mechanism includes antibiotic
production, hydrogen cyanide production, hydrolytic enzyme produc-
tion, polysaccharide production and induced systemic resistance. The
PGPR also helps the plant to fight against phyto-pathogens. PGPR
stands for plant growth promoting rhizobacteria, while ISR stands for
induced systemic resistance

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(Lyu etal. 2019). Recently, it has been proposed that it plays
a critical role in the suitable functioning of the agroeco-
system (Oak 2019). Scientists have shown that PGPR is
beneficial for the improvement of soil quality, combating
pathogens, and the effects of climate change. Identically, it
can help in the restoration of degraded land and decrease the
effects of environmental soil pollutants (Lyu etal. 2019).
Evidently, Majeed etal. (2018b) recorded different strains
of Pseudomonas fluorescence and Pseudomonas sp. strains,
for example, Pseudomonas sp. AF-54 from sunflower rhizo-
sphere as promising microorganisms with plant growth-pro-
moting traits.
Other agmricultural beneficial microorganisms present
in the rhizosphere are mycorrhizal fungi (MF), which have
pesticidal genes that could destroy the negative effects of
chemical-based pesticides applied on soil and indirectly
enhance plant biomass (Bulgarelli etal. 2015). Few of the
agricultural important fungal strains that provide mineral
nutrients for plants are genera frequently isolated from the
root region in high abundances, such as Fusarium, Xylaria,
Cladosporium, Dactylonectria, and Trichoderma (Muller
etal. 2018). Although, there are reports on some fungal
genera in low abundance, for example, Aspergillus, Alter-
naria, Mucor, Leptoxyphium, and Chloridium (Crawford and
Knight 2017; Lee etal. 2019; Pelagio-Flores etal. 2017).
However, not only the rhizobacteria and AM are the
members of the rhizobiome present in the rhizosphere.
Other microorganisms cohabiting in the rhizosphere have
been documented. Lee etal. (2019) identified and reported
an abundance of archaea groups commonly known as ammo-
nium oxidizers in the rhizosphere than the bulk soil. Some
phyla of archaeal from the rhizosphere of the tomatoes plant
recorded are Aenigmarchaeota, Crenarchaeota, Diaphero-
trites, Euryarchaeota, Pacearchaeota, Woesearchaeota, and
Thaumarchaeota.
Plant root‑bacterial interactions intherhizosphere
Plant root plays different essential roles, including anchor-
ing and supporting of the plant to the ground, absorption
of water, inorganic nutrients, and food. Additionally, nutri-
ent storage, translocation of minerals and water to the stem;
secretion and accumulation of various potential compounds,
such as the secondary and primary metabolites peptides,
sugars, carboxylic acids, and proteins (Kuzyakov and Bla-
godatskaya 2015). The underground world of plants is a net-
work of interactions facilitated by the microbial community
and plant roots. The plant root regions and soil are colonized
by arrays of bacteria that promote the symbiotic relationship,
resulting in the enhancement of nutrients bioavailability and
the release of organic substances into the habitat (Beckers
etal. 2017; Nwachukwu etal. 2021).
In our previous study Nwachukwu etal. (2021), the
effects of plant roots on rhizosphere bacteria was reported to
have been caused by root length, volume, biomass and sur-
face area. These root-associated parameters encourage the
bacterial species to interact in the rhizosphere (Phour etal.
2020). These interactions can be in different forms including
antagonism, parasitism, amensalism and symbiosis, which
play a critical role in providing the host plants with different
benefits (Igiehon and Babalola 2018b), thereby contributing
to the sustenance of the plants. Also, these microbial interac-
tions in the rhizosphere is relevant to agriculatural practices
that depend less on chemical fertilizer applications.
The relationship between plant root and bacteria was first
recorded by Foster and Rovira in 1976, they considered dif-
ferent ultrathin sections of the wheat bacterial community
with the aid of a transmission electron microscope (Kuzya-
kov and Blagodatskaya 2015). The result showed that bac-
teria sparsely colonized immature roots in the root regions.
Conversely, in the cortical cells and cell walls, rhizobiome
were more abundant. Correspondingly, in an experiment, it
was reported that bacterial species in the rhizosphere had a
significant difference compared to the bacteria in the bulk
soil in different capacities, including numbers, types, and
sizes (Muller etal. 2018). Contrary to what was reported,
abundant bacterial species in the tomato plant endosphere
compared to other niches studied (Zhou etal. 2016).
Recently, in a study, bacterial genera of more beneficial
functional impact were abundant in the rhizosphere. The
abundant bacterial genera were Alphaproteobacteria, Fir-
micutes, and Actinobacteria. Outside the rhizosphere, Ver-
rucomicrobia were higher in the bulk soil compared to other
niches (Lyu etal. 2019). However, there was a disparity in
the results of a related study on soybean plant rhizosphere. A
group of bacterial communities with a particular nutritional
function was abundant in the root surface or rhizoplane
while the least was in the surrounding bulk soil (Lee etal.
2019; Zhou etal. 2020).
Besides, the metabolism of nitrogen, potassium, and
phosphorus, the bacterial community is involved in iron
metabolism and uptake, also transportation of membranes
(Fonseca-García etal. 2016). The diversity of bacterial com-
munities that colonize the different below ground zones has
been comprehensively studied to interpret the mechanisms
that allow various interactions between the internal tissues,
root surface, rhizosphere, and bulk soil. Varieties of sub-
strates are released through organic nutrients and growth-
promoting materials that bacterial species act on, resulting
in the promotion of plant growth (Tassi etal. 2017). Plants
dispense peculiar organic substrates that are utilized by
bacteria. Hence, it provides inorganic nutrients and growth-
promoting substances that stimulate plant growth.
Accordingly, Singh etal. (2019) isolated different species
of rhizobacteria possessing multiple plant growth-promoting

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traits from sunflower rhizosphere soil. The interaction
between PGPR and other resident microorganisms produced
secondary metabolites with plant growth-promoting proper-
ties. Some of the rhizobacteria was member Bacillus subtilis
strain Rhizo SF48, Bacillus thuringiesis strain Rhizo SF 23,
Acinetobacter sp. strain N15254. Also, various strains of
Pseudomonas and Enterbacteria spp were reported. Some
of the identified PGPR inhibited the growth of Fusarium
oxysporum.
Several factors, such as soil properties and type, plant
genotype, different plant developmental stages, and
rhizodeposits influence the level of interactions between
bacterial communities and plant root systems (Beckers etal.
2017). The different interactions that occur in the rhizos-
phere produce various available rhizodeposits, including
those that depend on different abiotic and biotic factors;
plant variants and developmental stages (Beckers etal. 2017;
Lagos etal. 2015). Although the upper part of the plant is
held by soil at the root, plants need a variety of macro and
micronutrients that play beneficial roles for their growth and
development. Some nutrients are required in major quantity
and are called macronutrient, namely, nitrogen, carbon, sul-
fur, calcium, and phosphorus. However, some nutrients are
needed in smaller portions which are referred to as micronu-
trients, for example, zinc, iron, magnesium, and nickel (Finzi
etal. 2015; Fonseca-García etal. 2016).
Moreover, toxic elements, such as heavy metals in the
soil have been reported to get accumulated in plant tissues
over time, when present in extreme conditions and above the
permissible limits, for example, barium (Finzi etal. 2015).
The negative impact of the presence of toxic elements in the
soil is that it becomes toxic to plants as a result limit plant
growth. Ordinarily, a high amount of toxic elements within
plant tissues have a direct or indirect impact on the plant
(Beckers etal. 2017). The direct impacts generate oxidative
stress that increases the inhibition of cytoplasmic enzymes
and damages the cell structures (Vandenkoornhuyse etal.
2015). The indirect impact substitute’s nutrients that is
essential to plant cation exchange sites. In that case, the ions
control the roles of different enzymes and proteins. Thereby,
stops metabolism and reveals the extent of toxicity in the
plant, for example, ions (boron) which have been reported
to be toxic to plant even at low concentrations (Tassi etal.
2017).
Soil fertility: role ofPGPR
The soil is a habitat for various macro-organisms, such as
ants, earthworms, insects, termites, and with a substan-
tial number of microorganisms that participate in nutrient
cycling, mineralization of organic compounds, synthesis of
phytohormones, and other important processes that advance
plants productivity. Furthermore, they help in improving and
maintenance of soil fertility (Susilowati etal. 2015).
PGPR colonize plant roots and have been implicated in
all important activities that occur in the soil such as the pro-
motion of plant health by the aiding plant take-up in many
nutrients. In addition, control the pathogens that could affect
the host plant negatively. The moisture content in the soil
determines the colonization of PGPR in the rhizosphere and
long-term soil fertility. PGPR is responsible for synthesizing
many biomolecules that enhance soil health (Vandenkoorn-
huyse etal. 2015).
Several organic compounds that operate as plant residues
undergo decomposition and mineralization. These minerals
increase soil health and fertility (Fig.2). The high amount
of phosphate available to plants is as a result of P solubili-
zation, an activity performed by PGPR. Additionally, other
chemicals in charge of plant growth are formed and they
influence the plant root morphology and enhance soil health
and fertility. The soil environments are improved mainly
by bacteria species thus readily makes nutrients available
(Suyal etal. 2015). The presence of nitrogen (N) in plants
is critical for protein and amino acid synthesis. Nitrogen is
acquired from the atmosphere, rhizosphere, and soil through
the fixing of nitrogen. However, volatile compounds and
metabolites are released by PGPR to improve plant and soil
health (Igiehon etal. 2019).
Likewise, different enzymes are released to control the
increase and effect of pathogens, hence contribute to the
biocontrol of phytopathogens and improvement of plant
growth. These compounds boost crop yield and enhance
soil health. The overall mechanisms are essential to achieve
a more fertile soil (Jiang etal. 2016). Apart from promot-
ing plant growth, PGPR inoculant as a biofertilizer could
enhance resident soil microbial diversity without causing
any harm in the soil or to humans, unlike the traditional
chemical-based fertilizers that have been implicated in the
contamination of farmland after application. For example,
nitrate from chemical fertilizer capable of contaminating
below groundwater, which can run-off from the soil after
rainfall and contaminate the surface water.
Interestingly, some PGPR acts as both biopesticides and
biofertilizers, for example, Burkholderia cepacia. A study on
maize showed that these microorganisms stopped the nega-
tive impact of Fusarium species and promoted maize growth
in iron-deficient soil by producing siderophore. Some PGPR
species produce siderophore and antibiotics with antago-
nizing properties that could stimulate systemic resistance,
which may suppress the impacts of many pathogens which
can be used for many agricultural purposes, including
improving soil fertility (Manivanh etal. 2017).
Additionally, arbuscular mycorrhizal fungi having plant
growth-promoting and pesticidal traits have been recorded
as a beneficial rhizospheric microorganism with the potential

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of destroying the damaging effects of chemical fertilizers,
resulting in increased plant biomass and improve soil fertil-
ity significantly proves that AMF is a promising microor-
ganism for sustainable agriculture. Jadhav and Anil (2020)
reported the influence of AMF on soil fertility and sunflower
plant growth. Therefore, it is important to harness agricul-
tural beneficial rhizobiome with the potential of enriching
agricultural soil for sustainable agricultural development.
Biological nitrogen fixation: PGPR asnitrogen fixers
The rhizosphere is made up of a unique and enormous vari-
ety of bacterial species. Some are cultivable and known
(1%), while 99% are yet uncultured, which makes it chal-
lenging for researchers to assess them. The variations of
bacterial communities mainly PGPR, shape the integral
structure of the rhizosphere (Alawiye and Babalola 2019;
Igiehon and Babalola 2018a). According to Olanrewaju etal.
(2019), a large proportion of bacteria colonize different land
plant roots and niches. These bacteria are assembled to make
up the rhizobiome.
Among the plant root microenvironment the rhizosphere
is a thin soil layer that surrounds plant roots, hence acts as
crossroads for microbial interactions, which makes this zone
an exceptionally special, active, and important zone with the
potential for diverse root activities and unique metabolic
processes (Zhou etal. 2020). Rhizobacteria communities co-
existing in the rhizosphere are plant growth promoters that
play several roles, including nitrogen fixation, increasing
available nutrients, phytopathogens suppressions, and dis-
ease inhibitions. Again, improve plant persistence and toler-
ance to environmental stress, such as high salinity and water
shortage (Babalola 2010; Olanrewaju etal. 2019).
Rhizosphere as a haven for diverse bacterial species with
a unique structure and composition that is impacted by plant
regulated mechanisms (Mukhtar etal. 2019). Variations in
the rhizosphere are modified by most bacterial populations
broadly advantageous to plants (Bulgarelli etal. 2015; Lee
etal. 2019). Recently, there were detailed reports docu-
mented on rhizosphere, bacterial components, and their
influence on plants (Kuan etal. 2016; Mukhtar etal. 2019).
It has been recorded that in the rhizosphere, nitrogen-fixing
is the fundamental mechanism that enhances plant growth
(Lee etal. 2019; Susilowati etal. 2015).
Nitrogen (N) is a major macronutrient important for the
growth and productivity of all living organisms, bacteria,
and plants inclusive. Nitrogen is found in membrane lipids
and nucleotide (Mukhtar etal. 2019; Zhang etal. 2017). It
is among the major essential component of plant dry mass
because it is the main constituent of chlorophyll (compound
plants use from sunlight to produce sugars from water and
carbon dioxide) and amino acids. When plants do not get
enough nitrogen, they are unable to produce amino acids,
without amino acids plants cannot make the unique protein
that plant cells require for growth (Babalola 2010).
The biological fixing of atmospheric nitrogen is an essen-
tial microbial activity that supports life on earth. This pro-
cess takes place when atmospheric nitrogen is transformed
Fig. 2 Contributions of plant growth-promoting rhizobacteria to
enhance soil fertility. Plant growth promoting rhizobacteria contrib-
ute to soil fertility by fixing atmospheric nitrogen (nitrogen fixation),
enhancing iron availability through siderophores production, enhanc-
ing nutrient availability through phytohormone production and phos-
phate solubility, as well as priming the defense mechanisms of host
plants to improve resistance against wide range of phyto-pathogens

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to ammonia (NH3) by a highly complex oxygen unstable
enzyme system found in free-living symbiotic diazotrophs
called nitrogenase (Igiehon etal. 2019). Nitrogen fixation is
an energy-demanding process that requires the hydrolysis of
a minimum of 16mol of ATP per mole of reduced nitrogen
(Souza etal. 2015). Taking into consideration the two kinds
of nitrogen fixation (symbiotic and non-symbiotic) base on
the category of related organisms and plant involved.
Gram-negative rhizobacteria species possessing plant
growth-promoting traits capable of fixing nitrogen domi-
nated the rhizosphere, these categories of rhizobacteria can
be referred to as nitrogen fixers (Alawiye and Babalola 2019;
Igiehon and Babalola 2018a). The common range of nitro-
gen-fixing rhizobacteria genera with the potential of improv-
ing plant growth and development, which consequently lead
to increase in crop productivity, include Azospirillum, Azo-
tobacter, Acetobacter, Arthrobacter, Bacillus, Burkholde-
ria, Serratia, Pseudomonas, and Mitsuaria (Babalola 2010;
Bagyaraj and Ashwin 2017; Manivanh etal. 2017). Other
important rhizobacteria strains such as Pseudomonas fluo-
rescens Pf153 and Pseudomonas sp. DSMZ 13,134 from a
particular locality having different plant growth-promoting
traits with influence on maize crop yield was reported by
Mosimann and teammates (2017). Correspondingly, Vibrio
fluvialis NWU37, Pseudomonas fluorescens NWU65, and
Ewingella Americana NWU59 were identified in our labora-
tory, likewise Streptomyces species and lots of Proteobacte-
ria (Alawiye and Babalola 2019).
The common broad range of nitrogen-fixing bacteria
present in the rhizosphere, which symbiotically fix nitrogen
with a particular plant is Pseudomonas, Rhizobium, Frankia,
Azotobacter Paenibacillus, Klebsiella, Salinibacter, Festuca
juncifolia, Agrostis capillaris, Holcus lanatus, Poa pratensis,
Bradyrhizobium, Sinorhizobium, Mesorhizobiu, and Des-
champsia cespitosa in synergistic association with legumi-
nous and non-leguminous plants and trees that employ their
functions (Kuan etal. 2016; Mukhtar etal. 2019). Distinct
features are used to differentiate members of nitrogen-fixing
bacterial communities in various plant rhizospheres. Some
plants inhabit different nitrogen-fixing bacterial communi-
ties, such as sunflower (Majeed etal. 2015a), rice (Miller
2018), poplar (Mukhtar etal. 2019), halophytes (Mora-Ruiz
etal. 2018), Arabidopsis (Ali etal. 2017), and Tomato (Lee
etal. 2019).
A study of different plant varieties reported that the genus
Pleomorphomonas known for their nitrogen-fixing capac-
ity was abundant in the rhizosphere (Kang etal. 2019).
Similarly, Ambrosini etal. (2018) recorded nitrogen-fixing
Bacilli belonging to the genus Paenibacillus spp from the
sunflower rhizosphere. Many processes trigger bacterial-
plant interactions, which have led to the development of
beneficial relationships and have enhanced adjustments to
different environmental challenges (Pii etal. 2015). Another,
distinct nitrogen-fixing bacterial species from the rhizos-
phere plant root system of mature Populus deltoides trees
have been reported (Salas etal. 2017).
Notably, non-symbiotic bacteria fix the fewer amount of
nitrogen, sufficient to the associated host plants compared
to bacteria found in the root nodule (rhizobia) (Mosimann
etal. 2017). Despite the slower fixing capability of non-
symbiotic bacteria, few PGPR has proved to be very efficient
in increasing this process by making the nitrogen nutrient
that is short in supply accessible to plants. During a non-
symbiotic nitrogen-fixing activity, free-living diazotrophs
excite the growth of non-leguminous plants.
The common genera of this group are Anabaena, Diazo-
trophicus, Azotobacter, Azocarus, Cyanobacteria, and Glu-
conoacetobacter diazotrophicus (Zarraonaindia etal. 2015;
Zhang etal. 2017). The application of cultures with non-
symbiotic nitrogen-fixing PGPR, mainly Azospirillum and
Azotobacter enhanced the productivity of crops (Pii etal.
2015; Souza etal. 2015). Just as Rhizobium nitrogen fixation
is important in the cultivation of various plants (Ambrosini
etal. 2012; Jadhav and Anil 2020). Ambrosini etal. (2018)
reported that novel nitrogen-fixing Paenibacillus helianthi
sp. nov. encouraged high growth and yield of sunflower
plants.
The majority of rhizospheric nitrogen-fixing bacterial
species populate the host plant root, some may emanate from
other tissues, such as internal tissues, leaf, flowers, and bulk
soil (Salas etal. 2017). A high abundance of members from
the phyla Proteobacteria and Spirochaetes were identified
from the rhizosphere and bulk soils of rice farm, whereas
Gemmatimonadetes, Acidobacteria, and Planctomycetes
were in depletion (Miller 2018).
Among the nitrogen-fixing bacterial communities in the
rhizosphere are bacteria that colonize the host plant root by
establishing themselves in the root surface, while a frac-
tion finds their way into the plant’s internal tissues and is
controlled by the innate immunity of the plant (Jiang etal.
2016). Some phyla recorded within the root surface and
internal tissues are Alpha, Beta, and Deltaproteobacteria.
Few indicated classes are Chloroflexi and Bacteroidetes (Li
etal. 2019; Miller 2018).
Bacterial communities in the rhizosphere use of a wide
variety of substances that increase the higher coloniza-
tion and persistence rates serving as sources of the nutri-
ent (Crawford and Knight 2017; Cregger etal. 2018). The
rhizoplane, which is the plant root surface is one of the plant
root systems where bacteria inhabit and attach themselves
to the root surface structures with the aid of either the cell
surface polysaccharides or fimbriae or flagella (Miller 2018;
Olanrewaju etal. 2019).
Similarly, some reports stated that there is a fragile
boundary between the rhizoplane and rhizosphere (Beck-
ers etal. 2017; Mukhtar etal. 2019; Olanrewaju etal.

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2019). Before now, researchers have recorded that some
nitrogen-fixing bacterial species, such as Caulobacter sp.
and Zoogloea ramigera identified from rhizoplane of some
plants (Beckers etal. 2017; Edwards etal. 2015). These
bacterial species are capable of controlling the nutrient
status of the surrounding soils and other factors, such as
pH (Elmagzob etal. 2019).
Most scientists have overlooked the activities occur-
ring in the internal tissues (endosphere), which makes this
region the least studied. Endosphere is a plant microen-
vironment that harbours a lesser concentration of bacte-
rial communities called endophytic bacteria (Gomes etal.
2018; Zhou etal. 2016). The interior root region being an
exclusive niche has a fewer number of bacterial density
compared to the root surface (rhizoplane) while rhizos-
phere has more abundant bacterial diversity (Elmagzob
etal. 2019). Table2 describes nitrogen-fixing genes and
the functions.
The beginning of molecular communication between soil
bacterial and host plants occurs through the discharge of
signals as communication chemicals, for example, flavonoid
capable of improving plants-microorganism relationship
(Alawiye and Babalola 2019; Ali etal. 2017; Carrell and
Frank 2015). Majeed etal. (2018b) recorded that this chemi-
cal helped in the choice of the greater befitting partners for
their development and the destruction of the pathogenic
once. The communication signal is recognized by NodD
(bacteria receptor) which acts as a transcriptional activa-
tor of nodulation genes (NodA, NodB, NodC, and NodFE).
Evidently, Nod factors stimulate agent of root nodules inhab-
iting in the rhizobia.
Contributions ofplant growth‑promoting
rhizobacteria tosustainable agriculture andfood
security
Generally, farmers worldwide need new innovative ideas for
farming to increase farm production that can take care of the
human population, which is estimated to be approximately
7 billion globally. For food security to be actualized, it
requires a process of producing adequate food and improving
its quality. This process must maintain the ever-increasing
human population without sabotaging environmental protec-
tion and impacting the environment negatively, this process
is referred to as the global green revolution (Igiehon and
Babalola 2018a). However, sustainable agricultural develop-
ment is needed to ease these issues. According to National
Research Council, the advancement of recent eco-friendly
agricultural practices, energy conservation strategies, and
natural resources that are promising to food security are the
crucial goals of sustainable food production (Grafton etal.
2015; Igiehon etal. 2019).
Researchers have indicated that the most promising
approaches to accomplish these objectives are to substitute
harmful pesticides and inorganic fertilizers with environ-
mental friendly symbiotic microbial formulations, for exam-
ple, Bradyrhizobium species with the possible attribute of
improving plant health and crop yields, while protecting
Table 2 Some rhizobial nitrogen-fixing genes and their predicted functions
Nitrogen-fixing
genes
Functions References
nifA Particular nitrogen-fixing regulatory protein and transcriptional stimulator; regulates the
expression of genes encoding accessory functions by the aid of their RpoN-dependent -24/-12
promoters and the nitrogenase structural genes
(Brink etal. 2017)
nifB Protein that controls biosynthesis iron-molybdenum cofactor (Lemaire etal. 2018)
nifD Nitrogenase molybdenum-iron protein and forms alpha chain (Backer etal. 2018)
nif H Nitrogen-fixation enzyme protein (Chauhan etal. 2015)
nifK Nitrogen-fixating enzyme (nitrogenase) molybdenum to iron protein; forms beta chain (Kuan etal. 2016)
nifQ Protein that aid in nitrogen fixation and binding of molybdenum to iron (Lemaire and Gastal 2016)
nifW Stabilizes nitrogen-fixing enzyme, nitrogenase (Srivastavia 2017)
nif X Molybdenum-iron cluster binding protein
nif Z Nitrogen-fixing protein (Srivastavia 2017)
fixA Nitrogen-fixing gene, electron transfer flavor protein, and beta chain
fixB Nitrogen-fixing gene, proteins aid electron transfer flavor, and alpha chain (Brink etal. 2017)
fixC Nitrogen-fixing protein and oxidoredutase enzyme (Kumari etal. 2019)
fixH Nitrogen-fixing and cation transport gene (Gastal etal. 2015)
fixJ Double component nitrogen-fixing regulatory protein and a direct positive regulator of nifA (Lemaire and Gastal 2016a)
fixK Expression of nifA, fixGHIS, and fix NOQP Ferrodoxin and FNR/CRP transcriptional activator
and regulatory gene; Ferrodoxin, such as protein, control the gene for nitrate respiration and
one of two stigma 54 proteins
(Backer etal. 2018)

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plants from biotic stressors, for example, pests, phytopatho-
gens, and abiotic stressors, such as drought, climate change,
and different environmental pollutants (Lang etal. 2019;
Lugtenberg 2015). There are reports on the identification
and application of PGPR as a substitute to chemical-based
fertilizers. The usage of PGPR as an alternative approach
has enhanced the abundance and richness of soil microor-
ganisms, have influenced plants health and yield positively
(Babalola etal. 2007, 2021).
Bacterial (microbial) cultures are combined with chemi-
cally advanced carriers with the help of liquid or solid fer-
mentation approaches. The bacterial isolates are alterna-
tively integrated into plants in mixed or pure culture through
seed or soil application, biological priming, and seedling
dip. Again, it is the application of individual bacterial and
identification of functionally appropriate rhizobiome. Their
importance in promoting crop productivity is another basic
and critical task since the rhizobiome is essential, thus it is a
description of the plant host second genome (Babalola 2010;
Beckers etal. 2017).
Therefore, to attain food security, there is a need to
depend on the advancement of plant diversity, growth, and
seed yield. The major way this can be accomplished is by
inoculation of crops with rhizobacteria, such as Rhizobium
species (Igiehon and Babalola 2018a; Igiehon etal. 2019),
as discussed below.
The effects ofrhizobial inoculant asaplant growth
promoter
Rhizobium species are bacteria capable of reducing atmos-
pheric nitrogen to ammonia through nodule formulation in
the stems or roots of host plants. Due to the agricultural
and environmental significance of Rhizobium species, many
researches have been conducted on the bacteria (Ambrosini
etal. 2018). The inoculation of seeds with Rhizobium spe-
cies was recorded to improve the formation of the nodule,
nitrogen absorption, and seed protein. Some seeds, such as
sunflower (Helianthus annuus L), soybean (Glycine max L.),
lentil, and pea inoculated with Rhizobium species enhanced
root nodulation, root and shoot microbial diversity, seed-
ling height, and shoot biomass, as a result, increased plant
growth and crop yield significantly (Ambrosini etal. 2018).
Other yield constituents included the number of seeds per
pod, total number of pods per crop, and sum of branches
bearing pod per plant was as well improved (Brader etal.
2017; Enebe and Babalola 2018).
In a field and greenhouse study the researchers reported
that Vicia sativa L. plants were modified with Azospirillium,
the result showed that there was a notable increase in nitro-
gen-fixing activities, N content, and percentage (Igiehon
and Babalola 2018a). A similar experiment on peanut plant
showed that there was an improvement in the number of root
nodules, root dry weight, and plant growth. Chicken pea
treated with Rhizobium species both in greenhouse and field
experiments compared to the controls improved plant growth
(Lawson etal. 2017). Rhizobium species were more effective
on the plant growth when co-inoculated with other plant
growth-promoting microorganisms. Rhizobium species co-
inoculated with few bacterial species will not only improve
the effectiveness of Rhizobium species but will advance crop
production (Kuan etal. 2016; Lang etal. 2019).
Some beneficial bacterial species that have favorably
improved crop productivity by co-inoculation with Rhizo-
bium species are Azotobacter, Bacillus, Enterobacter, Ser-
ratia, and Pseudomonas (Ambrosini etal. 2016). The co-
inoculation of Rhizobium leguminosarum bv. trifoli and
Azospirillium lipoferum enhanced white clover root nodu-
lation (Igiehon and Babalola 2018a). Furthermore, there are
reports on Rhizobium and Azospirillium co-inoculation on
the bean with increased nitrogen-fixing activity in the crop
(Igiehon and Babalola 2018a; Lang etal. 2019; Lugtenberg
2015).
The co-inoculation of Rhizobium and Bacillus species on
cowpea resulted in the bacterial species stimulating plant
growth by increasing the nutrient uptake and root weight
(Igiehon and Babalola 2018a; Lang etal. 2019; Lugtenberg
2015). The combination of Bacillus, Rhizobium, and Azos-
pirillium species advanced nitrogen fixation and root nodula-
tion in pigeon pea. The co-inoculation of Streptomyces lylius
WYEC108 and Rhizobium sp. promoted the growth of pea
by increasing the number of nodules. Again, enhanced the
colonization of Streptomyces at the root and root nodules (Li
etal. 2019). The combinations of various inoculants have
been explored in different crop experiments but there is still
a need for further research.
Another, the incorporation of Rhizobium sp. in crops
stimulates crop growth and serves as a substitute to expen-
sive chemical-based fertilizers too. Using relevant species
as an inoculant in environments with nitrogen gas depletion
can be a better avenue to improve the growth and develop-
ment of crops (Igiehon and Babalola 2018a; Lyu etal. 2019).
Taking in mind the inexpensive rate of inoculation and many
agricultural benefits, farmers are encouraged to take advan-
tage of the numerous benefits of these bioinoculants and
biofertilizers on crops.
The effects ofrhizobial inoculant onplants
photosynthesis andchlorophyll concentration
Nitrogen is an essential plant regulating element for growth
because it is limited in the environment. Plants require an
abundance of nitrogen than other mineral nutrients for their
growth. Nitrogen is a major component of many organic
compounds, including proteins, chlorophylls, and plant
growth regulators (Lyu etal. 2019). The visible appearance

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of the crop with nitrogen deficiency is stunted growth,
reduced branching, and yellow colouration of the leaves.
Nitrogen as a monomer of protein is essentially required
for the total plant cell enzymatic processes. It is present in
proteins and vitamins. Also, act as a key player in photosyn-
thetic processes in photosynthetic plants (Zhou etal. 2016).
Plants inoculated with rhizobial strains in field and green-
house experiments indicated a considerable increase in the
chlorophyll levels of the leaves. As well, there was 80 and
140% notable increase in photosynthetic activities in plants
modified with rhizobial strains compared to the controls
(Xiao etal. 2017). Again, soybean inoculated with B. japoni-
cum resulted in the buildup of chlorophyll content. There
was an increase in diverse growth indices, such as height,
number of leaves, the width of the stem, number of flowers,
pod development, and root extension, which distinguished
the treated plant from the control (Bulgarelli etal. 2015;
Igiehon and Babalola 2018a).
Another, the impact of P and B. japonicum was studied in
a field experiment on the growth of cowpea. Consequently,
there was a drastic increase in the chlorophyll level of the
leaves. However, there is still a study gap on the application
of K, P, and Rhizobium as well as the formation of chlo-
rophyll in Phaseolus vulgaris, which needs to be studied
(Deng etal. 2019).
The effects ofrhizobial inoculants onthemineral
nutrient assimilation byplants
The availability and assimilation of mineral nutrients are
important for plant growth. Mineral nutrients, such as nitro-
gen (N), magnesium (Mg), calcium (Ca), and potassium
(K) are required by various plant varieties for their growth
and increase crop productivity. The mutualistic association
between Rhizobium species and plants can be the main N
source in majority of the cropping systems, where approxi-
mately 80% of the N originating is from biological fixing
of N. In a study, amendment of crop seeds with Rhizobial
cultures improved N uptake and root nodulation (Deng etal.
2019; Finzi etal. 2015).
Similarly, seeds modified with Rhizobium spp. showed
an increase in plant N from 19 to 42mg per plant (Lange
etal. 2015; Lugtenberg 2015). In a study in Brazil Pha-
seolus vulgaris amended with Rhizobium sp. cultures sig-
nificantly increased N from about 20–60kg per of N per
hectare (Garrido-Oter etal. 2018; Igiehon etal. 2019).
Potassium is another essential mineral nutrient conserved
in the soil, mostly in a non-soluble form which cannot be
easily absorbed by plants as a result limits plant growth.
Assimilation of potassium is essential for plant function and
development. For the non-soluble P form to be solubilized
by PGPR, it should pass through acidification, chelation, and
different enzymatic activities (Resende etal. 2014).
Mineral nutrient uptake depends on different factors, such
as the interactions at the rhizosphere, the concentration of
the minerals, and the ability to replace them in the soil. This
challenge can be resolved by the introduction of Rhizobium
spp or other phosphates solubilizing bacterial species, such
as Burkholderia, Bacillus, Erwinia, and other important
mineral nutrients into the rhizosphere soil (Manivanh etal.
2017; Souza etal. 2015). Apart from the inadequacies of the
main mineral nutrients required by plants, micronutrients
can also become limited in the rhizosphere, causing low crop
production. Some of the essential micronutrients needed
by plants are iron (Fe), molybdenum (Mo), zinc (Zn), and
boron (B). It was recorded in a study that the amendment
of Pigeon pea (Cajanus canjan L. Millsp) with Rhizobium
strains inoculant relatively increase the number of available
mineral nutrients in the rhizosphere as a result boosted the
capacity of plant nutrient uptake (Lyu etal. 2019).
The impact of Rhizobium strains inoculants on the
increase of minerals nutrients uptake in the rhizosphere and
plant growth have been reported by authors (Igiehon and
Babalola 2018a; Majeed etal. 2015b; Pii etal. 2015). A
study showed the influence of Rhizobium strains on min-
eral nutrients assimilation by Phaseolus vulgaris, which
resulted in a notable increase in Ca, P, K, S, and Mg uptake
in all plant parts. The introduction of Rhizobium strains has
improved the significant uptake of different micronutrients,
including Cu, Fe, Mn, Zn, Fe, B, and Mo in various plant
parts as recorded in some studies (Igiehon etal. 2019; Kuan
etal. 2016).
The relationship betweenofPGPR, iron acquisition,
crop productivity, andphytopathogens
extermination forsustainable agriculture
Important mineral nutrient, such as inorganic N, Fe, and P
required by plants can be acquired through symbiotic asso-
ciations with rhizobacteria. Plants often absorb Fe from
reduced ferrous ion (Fe2+), although the ferric ion (Fe3+) is
readily available in the soil. Most plants have been recorded
to release chelators and siderophore that attach to Fe3+ and
aid in retaining it in the soil solution. Chelators significantly
release Fe3+ to the root surface, where it is transformed to
Fe2+ that is then absorbed rapidly. Grasses secrete the sidero-
phores that are absorbed alongside Fe3+ across the plasma
membrane (Igiehon and Babalola 2018a; Kuan etal. 2016;
Lange etal. 2015; Lugtenberg 2015).
Nevertheless, few PGPR can separate insoluble Fe from
the soil with the help of bacterial siderophores, therefore
makes it accessible for the host plant, which is evidence
that some plants can take in bacterial Fe3+ siderophore com-
plexes require for their growth (Lange etal. 2015; Lugten-
berg 2015). There is controversy on the benefits of bacterial
Fe3+ siderophore complex absorption to plants iron nutrition.

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Some scientists recorded that the contributions of Fe from
the aforementioned sources to plants and stated that the
total iron requirement is little. Although, few accepted their
essential benefit contributions in calcareous soils (Igiehon
etal. 2019; Lugtenberg 2015). A study showed that bacterial
siderophore, such as pseudobactin and ferrioxamine B did
not provide a sufficient amount of iron for plants (Igiehon
and Babalola 2018a; Igiehon etal. 2019).
The bioavailability of Fe in the rhizosphere soil is reduced
by obtaining Fe through PGPR siderophores, as a result,
impact on the proliferation of fungi, which may be harmful
to plants in soils deficient of Fe. Plants are relatively pro-
ductive in soil rich in microorganisms than the soil with a
low amount of microorganisms. This acknowledges the fact
that PGPR helps host plants by obtaining mineral nutrients
(Beckers etal. 2017; Deng etal. 2019).
Challenges applying PGPR inthefield
forsustainable agriculture
The technology of bioinoculation holds the potential for the
future. However, several challenges are encountered in the
field application of PGPR in sustainable agriculture. Few
main bottlenecks need to be addressed to elevate the efficacy
of PGPR inoculation in field applications. The development
of recent inocula is depends on laboratory screening assays
that mainly depend on PGPR mechanisms, such as nitrogen
fixation, calcium phosphate solubilization, the activity of
ACC deaminase, and auxin synthesis (Hristeva and Denev
2017; Sessitsch and Mitter 2015). Screening of pure cul-
ture in the laboratory with plant growth promotion potential
does not result from microorganisms that can promote plant
growth in the field (Mukhtar etal. 2019; Murphy etal. 2015;
Prasad etal. 2019).
Unfortunately, some of these organisms with the potential
of improving plant growth in the field are discarded during
invitro screening in the laboratory because their mecha-
nisms are not fully understood. The most critical challenge
is the development of PGPR-based products that can be
applied commercially, coupled with the process of screen-
ing, and efficient selection of the most potential organisms
(Sessitsch and Mitter 2015). Some factors are being consid-
ered when isolating, screening, and selecting PGPR. Some
criteria that are considered include the competence of the
host plant to adapt to a specific environment, pathogens,
and climate change (Itelima etal. 2018; Salas etal. 2017).
Moreover, other means of selection and isolation are
the spermosphere model. This approach is an enrichment
method that employs different seed exudates that are used
as a source of nutrients, for instance, the one recorded as
potential nitrogen-fixing bacteria from the rice rhizosphere.
Isolating promising microorganisms with the capacity to
inhibit soil-borne pathogens is another essential method of
selection. Therefore, the potential microorganisms should
be collected particularly from soils that have suppressive
effects on the specific pathogen (Purwati and Herwati 2016).
Other methods include selection based on characteristics
associated with PGPR, such as antibiotics and siderophore
production; and activity of ACC deaminase. To solve the
problem of selecting the excellent and promising strains of
PGPR, there is a need for upgrading to advanced assessment
systems and efficient bioassays (Sessitsch and Mitter 2015).
Another is the application of PGPR as a biological control
agent against phytopathogens, such as fungal pathogens. In
an experiment performed in greenhouse systems, the result
showed that PGPR has the potential in controlling differ-
ent phytopathogens (Hristeva and Denev 2017). This was
achieved because of the controlled and consistent environ-
mental conditions in the greenhouse throughout the planting
season. Nevertheless, it is impossible to achieve a constant
environmental condition in the field, where instability in
the biotic and abiotic factors are very high and the rivalry
between indigenous micro and macrofauna is a challenge too
(Itelima etal. 2018; Schlemper etal. 2017).
However, the understanding of the previously mentioned
factors can be useful in deciding the appropriate type and
concentration of PGPR strains, and time of inoculation.
Likewise, soil types, cropping strategies, and farm manage-
ment practices that could encourage PGPR survival and
increase (Itelima etal. 2018). Hristeva and Denev (2017)
reported that sunflower rhizosphere soil inoculated with
PGPR inocula showed that there was an impact of soil type
on sunflower rhizobiome population density and structure.
For those reasons, the idea of engineering the rhizosphere
to improve the function of PGPR is becoming more popular
(Chaudhary etal. 2020; Parnell etal. 2016; Schlaeppi and
Bulgarelli 2015).
Other areas of concern are to identify the appropriate car-
rier and to establish more excellent composite formulations
that can guarantee the survival, proliferation, and actions of
PGPR in field applications. Also, concentrate on compat-
ibility with chemical-based seed treatments. To overcome
the challenges of the field application of PGPR, approaches,
such as optimizing the conditions for growth before the for-
mulation, the improvement of carriers, and application tech-
nology can be helpful (Chaudhary etal. 2020; Şeker etal.
2017).
Some farms cultivated with high-value crops are fre-
quently fumigated with broad-spectrum biocidal fumigants
that can change the structure of the biological community
in the soil (Macouzet 2016; Majeed etal. 2015b). However,
long-term exposure to these fumigants affects the micro-
bial community interactions, which can be helpful in nutri-
ents acquisition and mobilization. The biocidal fumigants
are damaging to soil health and pose a challenge to PGPR
inocula colonization in the rhizosphere. Plant breeding has

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1 3
helped achieve the green revolution, still few of the frame-
works of bioinoculants have been made to combine the
microbiome-based form of plant breeding to accomplish a
heritable PGPR population that can improve crop productiv-
ity and food security (Hristeva and Denev 2017; Macouzet
2016).
The green revolution has revealed the negative impact
of inorganic fertilizers, herbicides, and pesticides applica-
tion in the soil environments. The applications of chemical-
based products have led to soil damage, especially as soil
contaminants. The combination of plant growth promotion
and bioremediation would be an advantageous approach in
solving agricultural problems globally. Besides, plotting
microbiota consortia to mitigate different forms of plant
growth promotion and bioremediation is an important form
of this approach (Barquero etal. 2019). The production of
bioinoculant for a particular soil condition, defeating envi-
ronmental restrictions, training of farmers, and farmworkers
on how to efficiently incorporate the bioinoculants to crops
are essential in the development and deployment of many
important bioinocula (Prasad etal. 2019).
The different challenges discussed must be surmounted
before registering commercialized PGPR products for pub-
lic use. Such as, the scaling up of fermentation conditions,
large scale production of PGPR, maintaining product qual-
ity, efficacy, and stability (Bashan etal. 2016). Factors that
should be taken into consideration before commercialization
are cost, duration of shelf-life, ease of application, rate of
colonization, and compatibility. Before deciding the com-
mercialization of PGPR products, cost capitalization, and
possible markets must be considered and studied (Itelima
etal. 2018). Also, the knowledge of the levels of pathogenic-
ity, toxigenicity, and allergenicity is of utmost importance.
Studies on potential gene transfer and persistence in the
environment are needed to devise means to enhance safety
measures of commercial products. Categorizing the prod-
ucts under the proper category, either as a biocontrol agent
or biofertilizer is important as well (Barquero etal. 2019;
Orlikowska etal. 2017; Parnell etal. 2016).
Meta‑omics techniques foridentifying rhizobiomes
ofagricultural sustainable importance
Before the emergence of the meta-omics techniques, micro-
organisms were isolated and identified using the traditional
or culture-based method for isolating and classifying micro-
organisms. This conventional method for culturing could not
identify the entire microorganisms in a sample, which made
approximately 99% of the microorganisms unknown. Meta-
omic method captures the non-culturable microorganisms
and their functions (Fadiji and Babalola 2020). The meta-
omics technique as an emerging approach is used for char-
acterizing the functional and structural genomes in a given
environmental sample. It has unlocked the understanding
of diverse structures and functions exhibited by microbial
communities in the plant rhizosphere (Alawiye and Babalola
2019). Table3 highlights the different methods of identify-
ing agricultural beneficial microbial species.
The meta-omic techniques have aid in studying microbial
populations at different levels and functions. Meta-omics
technique include metatranscriptomics, used to differenti-
ate the transcriptome profile of the microbial community
and their interactions using mRNA sequencing and micro-
array applications (Vargas-Albores etal. 2019). Another is
metaproteomics an approach used to classify the totality of
complementary protein at a particular time in an environ-
mental sample, results in understanding the interactions
among microorganisms and microorganisms, and between
plants and microorganisms (Yu etal. 2019).
Metaproteomics assists in understanding microbial pro-
tein profiles, complex metabolic pathway, and identifies the
multi-functional genes of microorganisms (Adegboye and
Babalola 2013; Vargas-Albores etal. 2019). Furthermore,
metagenomics with the application of shotgun metagenomic
analysis (Illumina MiSeq, NextSeq, and HiSeq) predicts at
the gene level the entire taxonomic and molecular functions
of the microbial community in an environment. Metabo-
lomics specifically evaluates small molecules of low-molec-
ular weight in biological systems. It is a quantitative and
qualitative analytical approach used for metabolite profiling
of a huge portion of metabolomes (Jansson and Hofmockel
2018; Oulas etal. 2015).
Meta-omic applications are classified as next-generation
sequencing (NGS) analyzing tools. The NGS approaches
build on the strength of omics tools to study plant-associ-
ated microorganisms in an agricultural environment. These
meta-omics techniques have facilitated the understanding of
the variations, structures, abundance, richness, and distribu-
tions of plant rhizosphere microbial communities (Adeg-
boye and Babalola 2013; Fadiji and Babalola 2020). Aside
from comprehending the properties mentioned earlier, it
is as well used in identifying microbial community mem-
bers with exceptional qualities and functions in the rhizo-
sphere. Microbial communities of different plants studied
using the omics approaches are sunflower, wheat, maize,
sorghum, oat, cowpea, and soybean (Donovan etal. 2018;
Maropola etal. 2015). Our recent study on sunflower rhizo-
sphere and bulk soils using NGS approach (specifically, 16S
amplicon sequencing technique) revealed a mean read of
86.59–99.30% for bacteria and 7.0–13.4% for archaea. In
the study, the most abundant bacterial phyla were Proteo-
bacteria (18–36%), Firmicutes (17–51%) and Actinobac-
teria (7–38%), while Thaumarchaeota (1–13%) and Cre-
narchaeota (1%) were observed to be the most abundant in
the archaea domain (Babalola etal. 2021). Using the same
sequencing technique, a study on the microbial diversity of

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Table 3 Methods of identifying agriculturally beneficial microorganisms
Methods Applications used Functions Limitations Reference
Culture-based Planting on general or selective
culture media view with the aid of
a different microscope, biosensor
imaging
Isolate and identify agricultural
important microorganisms, identi-
fied mechanisms of a cell to cell
interactions, the pattern of coloniza-
tion and detection phyto-stimulatory
genes
Limited information; loss of uncultur-
able microorganisms
(Fadiji and Babalola 2020)
Amplicon sequencing/targeted
metagenomics
16S and 18S rRNA amplification
using PCR machine, cloning or
DGGE, high throughput sequencing,
RFLP, FISH, confocal laser scanning
microscopy (CLSM), secondary
ion mass spectrometry (SIMS),
biosensor or stable isotope labelling,
oligonucleotide probes, bioinformat-
ics tools and database soft wares
Extraction of specific genomes of
interest from environmental and
legumes samples, identifying
microbial population and functional
genes performing specific metabolic
activity; analyze microbial structure,
identification of microorganisms
involves in phyto-biocontrol and
macergens genes, characterizing
nutrient availability, analyze AMF
genome
Limited information about the
genome; damaging sampling
(Jünemann etal. 2017; Oulas etal.
2015)
Whole-genome metagenomics Shotgun metagenomics- Illumina
Hiseq or Miseq, bioinformatics tools
and database soft wares
Extraction of entire genomes in an
environmental sample, Identify
both known and novel genes and
microorganisms responsible for
plant-microorganisms interactions,
microorganism-microorganism
interactions, Identify metagenomes
with potential functional and struc-
tural genes
Number of replicates is limited;
require computational power
(Donovan etal. 2018; Martellacci etal.
2019)
Metaproteomics LC/FT-ICR, 2- dimensional electro-
phoresis
Profiling of proteins using protein
expression profile analysis
Needs computational power; compari-
son to transcriptomic data
(Alawiye and Babalola 2019; Hu etal.
2018)
Metabolomics LC–MS, GC–MS, NMR, FTIR, ESI-
Micro-T of MS
For identifying and characterization
of both novel and known; targeted
and untargeted metabolites and
their specific functions, Profiling of
metabolites with biological control
ability against phytopathogens;
assesses metagenomic pathways
Requires a few numbers of replicate
and computational power; large
amount of metabolites
(Lagos etal. 2015; Sabale etal. 2019)
Metatranscriptomics mRNA sequencing and microarray
applications
Screening of untargeted genome;
assessment of metabolic pathways
and gene expression
Few numbers of replicates; requires
computational power and compari-
son to metagenomics data
(Alawiye and Babalola 2019; Delmont
etal. 2015)

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soybean rhizosphere co-inoculated with alien Rhizobium sp.
strain R1, Rhizobium cellulosilyticum strain R3 and mycor-
rhizal consortium unveiled different classes of bacteria such
as Actinobacteria, Verrumicrobia, Cyanobacteria, Acido-
bacteria, Nitrospirae, Firmicutes, Planctomycetes, Chloro-
flexi, Proteobacteria, Bacteroidetes, and Gemmatimonadetes
(Igiehon etal. 2020).Recently, the attention of scientists
is shifting from the previous Illumina system or shotgun
metagenomics to the latest third-generation sequencing
techniques. The most recent third-generation techniques,
for example, MinION (Nanopore) and PacBio completes
its reads within 4–6h and can take up ultra-long reads of
hundreds of thousand bases, unlike the Illumina system that
takes days, which is the major advantage over the Illumina
system. Other advantages of the third-generation techniques
are that they are relatively inexpensive, affordable, portable,
and user-friendly. Furthermore, no special training or equip-
ment is required for data analysis. It can be connected to lap-
top or computer system, and can easily generate a microbial
profile from full-length 16S or 18S rRNA gene sequence
at a significant high taxonomic resolution (Pii etal. 2015;
Brader etal. 2017).
However, some limitations are encountered when using
the MinION (Nanopore) and PacBio, for instance, error rate
and low accuracy level, which may be due to the restric-
tion to a specific application and continuous long reads.
However, these limitations can be corrected with further
improvements (Fadiji and Babalola 2020). For better knowl-
edge of the microbial variations and structures in the rhizo-
biome, the combination of more than one of the omics tools
is to be employed; this significantly makes the meta-omics
technique a fascinating technique in system biology.
Conclusion andfuture prospects
This review has provided detailed insights into various
microbial communities and significant impact of PGPR
with a more focus on the nitrogen-fixing rhizobacteria with
specific roles in sustainable agriculture. The nitrogen-fixing
genes of rhizobacteria can promote plant growth even when
host plants are exposed to biotic and abiotic stressors, such
as a prolonged shortage of water and high salinity content.
The PGPR can as well act as biofertilizers that can improve
soil fertility and increase crop yields, which would reduce
the world-wide dependence on chemical-based fertilizers
and pesticides known to have a damaging impact on the
environment. Given that these biological fertilizers are inex-
pensive and ultimately eco-friendly.
The understanding of the PGPR mostly nitrogen-fixing
rhizobacteria is required because they are significant micro-
bial groups that propel the activities in the plant microen-
vironment. The knowledge of the microbial community is
advancing into evolution involving integrating different dis-
ciplines and applications that are needed. Coupled with, the
interrelationship of multiple disciplines in science, in-depth
knowledge of the rhizosphere, and for harnessing the meta-
bolic products. Advancements in studies are required when
more advanced technologies and data analysis software is
employed.
At this present time, studying the rhizosphere is an
emerging field of microbiome research that is significantly
progressing in the use of novel techniques, such as the fin-
gerprinting and third next-generation sequencing MinION
(Nanopore) and PacBio applications in understanding the
activities of the microbial communities in the rhizosphere
regions. Scientists have the challenge of quantifying the
16S rRNA or 18S rRNA or internal transcribed spacer
(ITS) sequences, a Polymerase Chain Reaction (PCR)
based method and the sequencing technique targets a single
genome, either 16S rRNA or 18S rRNA or ITS genes that do
not provide comprehensive information on the microorgan-
ism’s metagenome diversity and functions.
Omic techniques have bridged the shortfalls of the con-
ventional techniques and the phylogenetic surveys in iden-
tifying and characterizing the entire microbial genomes and
the genetic features and traits that enable microorganisms to
survive in the soil environments. The data obtained using the
meta-omic approaches will give a broader view, the descrip-
tion of the microbial components, and assists in improv-
ing the methods of combating plant diseases and improving
plants growth. For the most part, promote functions worth-
while in agriculture and have a favourable impact on food
production globally, and increase food security.
Acknowledgements B.C.N. appreciates the National Research Foun-
dation South Africa/ The World Academy of Science (NRF-TWAS)
for PhD stipend (UID121772). O.O.B., acknowledges the National
Research Foundation, South Africa for grants (UID123634; 132595)
that supported research in her laboratory.
Author’s contributions All authors equally contributed.
Funding National Research Foundation, South Africa, funded the work
in our laboratory (UID123634; 132595).
Declarations
Conflict of interest The authors declare no conflicts of interest.
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