plants

Review
The Microbial Connection to Sustainable Agriculture
Kalaivani Nadarajah * and Nur Sabrina Natasha Abdul Rahman

Department of Biological Sciences and Biotechnology, Faculty of Sciences and Technology, University Kebangsaan
Malaysia, Bangi 43600, Malaysia; [email protected]
* Correspondence: [email protected]

Abstract: Microorganisms are an important element in modeling sustainable agriculture. Their role
in soil fertility and health is crucial in maintaining plants’ growth, development, and yield. Further,
microorganisms impact agriculture negatively through disease and emerging diseases. Deciphering
the extensive functionality and structural diversity within the plant–soil microbiome is necessary
to effectively deploy these organisms in sustainable agriculture. Although both the plant and soil
microbiome have been studied over the decades, the efficiency of translating the laboratory and
greenhouse findings to the field is largely dependent on the ability of the inoculants or beneficial
microorganisms to colonize the soil and maintain stability in the ecosystem. Further, the plant
and its environment are two variables that influence the plant and soil microbiome’s diversity
and structure. Thus, in recent years, researchers have looked into microbiome engineering that
would enable them to modify the microbial communities in order to increase the efficiency and
effectiveness of the inoculants. The engineering of environments is believed to support resistance to
biotic and abiotic stressors, plant fitness, and productivity. Population characterization is crucial in
microbiome manipulation, as well as in the identification of potential biofertilizers and biocontrol
agents. Next-generation sequencing approaches that identify both culturable and non-culturable
microbes associated with the soil and plant microbiome have expanded our knowledge in this area.
Additionally, genome editing and multidisciplinary omics methods have provided scientists with
a framework to engineer dependable and sustainable microbial communities that support high
yield, disease resistance, nutrient cycling, and management of stressors. In this review, we present
an overview of the role of beneficial microbes in sustainable agriculture, microbiome engineering,
Citation: Nadarajah, K.; Abdul translation of this technology to the field, and the main approaches used by laboratories worldwide
Rahman, N.S.N. The Microbial to study the plant–soil microbiome. These initiatives are important to the advancement of green
Connection to Sustainable technologies in agriculture.
Agriculture. Plants 2023, 12, 2307.
https://doi.org/10.3390/ Keywords: microbiome; biofertilizer; biocontrols; beneficial organisms; microbiome engineering;
plants12122307
multidisciplinary omics technologies
Academic Editor: Mario De
Andrade Lira Junior

Received: 13 May 2023

1. Introduction
Revised: 1 June 2023
Accepted: 8 June 2023 One of the major issues confronting today’s modern agriculture is optimizing sus-
Published: 14 June 2023 tainable crop output in order to ensure global food security. Furthermore, climate change
has exacerbated the impact of environmental stresses such as drought, flooding, heat, and
salinity on world food productivity [1]. In addition, the current agricultural practice of
utilizing agrochemicals for optimizing yield has resulted in devastating environmental
Copyright: © 2023 by the authors. consequences to soil health and fertility [2–4]. Hence, to maximize crop output, it is clear
Licensee MDPI, Basel, Switzerland. that novel methods must be developed and investigated. One of these methods is the
This article is an open access article incorporation of beneficial microbes into agricultural practices [5,6]. While several studies
distributed under the terms and
have reported the role of microbes in plant fitness as well as in soil health and fertility,
conditions of the Creative Commons
there is still a need to elucidate the relationship between the microbiome and plant health.
Attribution (CC BY) license (https://
Among the aspects of plant–microbe interactions that should be studied are their role in
creativecommons.org/licenses/by/
the plant immune responses, signaling pathways (both in plants and microbes), positive
4.0/).

Plants 2023, 12, 2307. https://doi.org/10.3390/plants12122307 https://www.mdpi.com/journal/plants

Plants 2023, 12, 2307 2 of 25

and negative interactions between plants and microorganisms, and microbial function in
plant productivity [7,8] These findings will enlighten us regarding the entire process of
plant–microbe interaction and the discovery of microorganisms that can be exploited to
boost crop output in the near future [9,10].
By studying the plant microbiome, we are able to expound on the functional and struc-
tural diversities of the microbial communities linked to specific plants and ecosystems. The
microbial diversity observed across regions and organs in the phyllosphere, rhizosphere,
and endosphere has been well-documented by researchers [11,12]. Plants, in general, use
a variety of tactics to favor and support microbial colonization, such as the presence of
specialized structures (e.g., hairs, trichomes) or production of secondary metabolites. A
comprehensive approach towards deciphering the microbial population and its relation-
ship with plants remains a developing area of research pursued by many laboratories
worldwide [11,13]. The diversity in the microbial population is influenced by factors such
as host species, selection pressure, environment, developmental stage, and agricultural
practices [14]. Several studies have focused on specialized or niche communities to link the
population/community diversity with specific stresses or environmental pressures [15].
These observations clearly highlight the need for more thorough and in-depth stud-
ies to contribute towards the information and mechanisms that underly the microbiome
assembly [16,17]. Technologies for studying microbial diversity and the composition of a
specific plant microbiome have advanced significantly. These technologies have moved us
from culture-dependent identification, which has its limits in terms of providing a complete
picture of the microbiome, to more high-tech methods which achieve higher-resolution
images of the microbiome. The development of novel high-throughput techniques and
technologies has revealed multitrophic interactions in the black box of plant–microbe
interactions [18]. Plant-beneficial microorganisms can now be altered thanks to the ad-
vancement of these high-throughput technologies. Microbiome engineering may be an
alternate method for understanding, manipulating, and developing corresponding technol-
ogy for building microbial populations which are critical to plant health and productivity
in this scenario [19]. The new and emerging technologies will prove to be useful in de-
ciphering the depth of microbiome diversity in any given ecosystem [20]. Hence, in this
review we will address the microbiome in terms of its benefits; its shaping; its response
to the environment; plant- and soil-associated microbiomes; and the tools that have been
developed to elucidate, understand, and modify plant–microbe interactions.

2. Connected Plant Microbiome

Microorganisms naturally colonize soil and plant systems. These plant–microbe
interactions are regulated through interrelated chemical signaling. One of these micro-
communities, the rhizosphere, harbors microbial diversity that is regulated by the plant
root–soil dynamics; it involves root exudates that recruit microbial communities. The
rhizosphere also contributes to the development of endophytic communities within the
plant. Endophytes cause no negative effects to the plant and are free from environmental
control. Finally, the phyllosphere colonizes any of the above-ground plant parts [21,22].
These three micro-communities connect the above- and belowground environments with
their respective microbial diversities and communities. These micro-communities have
been studied in order to shed some light on their potential for agricultural sustainability,
growth, and development [23–25].
Beneficial microorganisms found in the plant environment have numerous benefits
to the plant ecosystem, including nutrient fixation and solubilization, stress management,
phytohormone production, plant phenology, improved yield, and many other positive
effects. Previous studies have linked these positive effects to the root exudates/semio-
chemicals, which basically function as secondary metabolites with the ability to elicit
structural and physiological changes in the rhizosphere [22,23]. The majority of these
metabolites are formed through complex pathways, such as polypropanoids, alkaloids,
and polyketides. The beneficial interactions between plants and microbes are facilitated
Plants 2023, 12, 2307 3 of 25

by chemical mediators such as terpenoids, flavonoids, and ethylene, which are induced in
response to particular triggers [22,26].
These rhizodeposits cause quorum-sensing responses in microbial communities and
release a variety of signaling substances, such as antibiotics, carbohydrates, hormones,
organic acids, and amino acids, which boost the plant’s defense against attacks and stres-
sors [27]. Jin et al. [28] revealed that root systems are able to secrete chemical compounds
that potentially induce chemotactic responses, swarming, and biofilm production. Sev-
eral reports have concluded that root exudates are versatile in nature and can mitigate
both biotic and abiotic stresses by establishing suitable rhizospheric microbiomes [29–32].
Understanding the chemically regulated process of plant–microbe and microbe–microbe
interactions in plants has provided many answers to the black box of plant–microbe inter-
action and regulation [33–37].

3. Microbiomes: Above- and Belowground Connection

Through the latest technologies, such as next generation sequencing (NGS), it has
become increasingly possible for the microbial profiles of above- and below-ground commu-
nities to be examined at the taxonomic and functional levels [38]. Extensive metagenome
profiling has been conducted for commercially viable crops. This is largely due to the ex-
ploitation of plant–soil-associated microbes for sustainable agriculture. These studies show
that plant microbiomes are predominantly populated by bacteria, viruses, fungi, oomycetes,
nematodes, and algae, and bacterial communities such as Proteobacteria, Actinobacteria,
and Bacteroidetes remain dominant communities [38]. Unlike the root microbiome, the
leaf microbiome is genetically impacted by the host plant, where many leaf microbiome
species are shared with root microbiomes, suggesting soil acquisition. All of these studies
also highlighted that there are conserved microbial taxa that exist across all plant–soil
environments. However, more studies are required in order to comprehend how plant–
microbe and microbe–microbe interactions can impact the growth and development of
plants [38–40]. The microbiome data between studies, however, cannot be compared due
to the use of different sampling methods, primers, and sequencing platforms [39,40].

Factors Affecting the Microbiome

All plant microbiome studies have shown that the structure and profile of the com-
munity is determined by the environment. These metagenome studies clearly indicate
that biotic and abiotic stresses exert an influence on the predominance of taxa within the
community. One important factor that dictates the microbiome’s composition is the geno-
type of the host. The genotype varies in several aspects, including immunity, secretions,
age, morphology, and physiology [41]. This results in the rhizosphere and phyllosphere
communities being different among different plant species. Given that the plant genotype
determines nutritional quantity, chemical signaling pathways, and other characteristics
of the plant, these factors individually or collectively control the exudates, which in turn
control the recruitment of microbes to the plant [42]. One example of a genotype differing
between varieties was seen in the microbiomes of indica and japonica root [42,43]. The
diversity and abundance seen in and around indica show that the number of nitrogen-
fixing organisms was especially high in indica as compared to japonica. This positive
plant selection pressure results in symbiotic species colonizing the rhizospheric and phyllo-
spheric niches [43]. However, in the event that there are plant–pathogenic interactions or
an anthropogenic environment in a particular ecosystem, a shift in the microbiome will
occur, resulting in negative consequences for the plant [44]. Further, the geographical factor
also influences the microbiome distribution, as there may be differences in the genera and
species found to be endemic to certain locations [43,44].
The bacterial and fungal populations in the rhizosphere are significantly influenced by
the characteristics of the soil. Microbes are a natural source of nutrients through the various
biogeocycles found in the environment. The establishment of microbial communities is
influenced by soil characteristics such as pH, moisture content, and soil structure [45,46].
Plants 2023, 12, 2307 4 of 25

Even when grown in a comparable soil environment, different microbial communities

are attracted by different plant species in the rhizosphere, rhizoplane, and endosphere.
As opposed to this, where specific microbe populations are related to plant species or
genotypes, there is also a population of core plant microbiome which is attracted to the
plants regardless of the environment or soil structure. Typically, interactions between a
plant’s genetics, related microbiome, and environmental variables will lead to changes in
the plant’s phenotype. Overall, the organization of the microbial community is shaped by
all of the elements mentioned above, and is acquired vertically through propagation or
changes in the environment [47].
Many reviews have focused on how biotic and abiotic stresses influence the plant
microbiome. Generally, disease and pests play a crucial role in changing the microbial
dynamics in and above the soil. Similarly, abiotic stressors such as drought, flooding,
salinity, heat stress, etc., have a profound effect on the plant and soil microbiome. For
instance, drought results in loss of water in the plants as well as the soil, which impacts
the plant–soil microbiome [48,49]. This results in a drop in certain plant soil microbial
taxa, while certain others that help cope with drought stress and water acquisition increase
within this stress period. Similarly, in submergence, the soil goes through an anoxic state
while the plant tissue goes through stress from submergence in water. This too results
in a change in microbial profiles, which results in an increase in microbes that survive
in anoxic environments and, at the same time, performs normal functions in the soil to
provide protection and nutrients to the plant [48,49].

4. Role of Core and Hub Microbiome

Through high-throughput sequencing, the core microbiome that exists within the host
was identified. A few bacterial communities may have had a long-standing relationship
with plants because of the high overlap of the core microbiome in multiple accessions as well
as phylogenetically distinct plants. This relationship extends to the colonization in specific
tissues and the stages of development [22,50–54]. Though persistent in the host, they make
up only a small portion of the microbiome’s richness, but remain relatively abundant. Some
of the core genera that have been reported consistently by researchers are Sphingomonas,
Burkholderia, Pseudomonas, Bradyrhizobium, Rhizobium, and Mesorhizobium. These microor-
ganisms are not only widespread and persistent, but also very numerous [51–53,55].
Generally, the core microbiome has been linked to plant development and colonization.
However, these links have not been established experimentally. While microbes are known
to improve plant function, there is no clear indication of co-evolutionary ties between a plant
and its microbes [16,56,57]. The location at the cellular/tissue level of these microbes has
been studied bioinformatically. Through these studies, a group of “hub” microorganisms
has been identified, which are presumed to hold a role in overseeing the network structure
and processes at the community level within the microbiome. Therefore, if there is any
interruption in the function of a particular community, this disruption can be attributed
to the lost or impacted hub community [51,58,59]. Niu et al. [60], in their report, stated
that the loss of the hub species Enterobacter cloacae resulted in the extinction of the other
communities in the ecosystem. Thus, it is necessary to identify hub communities and to
determine their functionality in normal and stress conditions.

5. Beneficial Microbes for Sustainable Agriculture

5.1. Beneficial Microbes and Their Mode of Action
Studies on plant microbiomes have indicated that microbes act on plant hosts either
directly or indirectly. The direct influence of the plant microbiome includes nitrogen fixation;
phosphate and iron sequestering; hormonal regulation (gibberellic acid (GA), abscisic
acid (ABA), cytokinin (CK), and auxin/indole-3-acetic acid (AUX)); and the production
of enzymes such as 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase [61]. The
reduction in host damage as a result of infection can be achieved through one or more of the
following, i.e., production of antibiotics (such as bacteriocins, lipopeptides, proteases, and
Plants 2023, 12, 2307 5 of 25

siderophores); volatile compounds; competitive exclusion; predation; or microbe-mediated

immunity, which can activate or suppress innate immune responses in the host. This is the
indirect means by which microbes control processes in the host [62].

5.2. Plant Growth Promoting Bacteria and Biotic Stressors

The overuse of agrochemicals in agriculture has resulted in negative implications
for the environment, impacted human health, and resulted in the deterioration of soil
health and fertility. In addition, harmful microbes (Ralstonia sp., Xanthomonas sp., and
Rhizoctonia sp.) that cause disease incidence become more rampant when chemicals are
overused [63]. Hence, the current trend is moving towards green agricultural practices,
where beneficial plant microbes are used to improve crop production and reduce disease
incidence. Decades of studies have shown that microbes are able to inhibit disease and also
ameliorate abiotic stresses on plants [49,64]. They have been implicated in regulating soil
health, maintaining nutrient cycles in terrestrials, and mitigating the negative influences of
climate change [4,65]. Together, these traits make beneficial microbes an amenable target
for crop yield improvement.
Some important groups of microorganisms that address the above are plant growth-
promoting bacteria (PGPB/PGPR) [66,67]. This group of organisms promotes plant health
and yield through the production of hormones and enzymes [68]. PGPB include microor-
ganisms that have the ability to control diseases, and have been labeled as biocontrol
agents [69]. PGPB inhibit pathogens by lowering iron availability to protect against dis-
eases [70]. Furthermore, advances in plant biotechnology have resulted in the development
of resistant crops through molecular breeding or transgenics, with varieties carrying genes
resistant towards disease, better nutrient content and uptake, and various other modi-
fications [71]. Transgenics is an environmentally friendly solution to the issue of biotic
stressors facing agriculture [72]. The post-genomic era offers open opportunities for the
development of superior strains or transgenic lines carrying PGPB genes for both abiotic
and biotic stress resistance [73].

5.3. Plant Growth-Promoting Bacteria and Abiotic Stressors

Abiotic stressors affect plants’ physiology and metabolism. While some plants are
able to acclimatize to stress, others are overcome by it. In the above section, we indicated
the role of PGPB in disease control through the secretion of hormones and enzymes. PGPB
secrete secondary metabolites that enable the acclimatization of plants to stress [72,74].
Several genera of Pseudomonas, Rhizobium, Bacillus, and Enterobacter have been identified
as good candidates for abiotic stress management [48]. Microbes are rapidly evolving
organisms that adapt to their environments quickly through the production of biofilms and
exopolysaccharides (EPS), through the adsorption of osmolytes, or by staying dormant [74].
Under these stresses, plants also produce hormones which help to maintain growth and
development [75]. For instance, in response to drought, the Azospirilium species develops
enhanced levels of ABA [76]. In high-salinity conditions, PGPB have the ability to absorb
and store osmolytes in the cell without any negative consequences, as EPS produced by
these isolates bind the cations, resulting in microbial cell turgidity [77,78].

5.4. The Application of PGPB in Sustainable Agriculture

The future direction of sustainable agriculture has microbes as a primary focus. PGPB
and fungi have been identified, isolated, and characterized for application in the field.
PGPB have several potential uses, especially in enhancing nutrient acquisition and se-
questering [68]. These microorganisms are known to form symbiotic interactions with
the plants’ root systems and to extend root growth and increase surface area for better
absorption of water and nutrients [79]. Some of these isolates are useful for bioremediation,
breakdown of toxic materials, improved growth, and biocontrols [72,80]. Nitrogen-fixing
organisms such as Nitrosomonas sp., Nitrobacter sp., and Rhizobium sp. have been reported
to increase nitrogen availability for plants, thus leading to increases in the plant yield and
Plants 2023, 12, 2307 6 of 25

biomass [81]. Fungi have been utilized in plant growth and yield promotion, and most
have been reported to solubilize phosphate and inhibit pathogen proliferation [82]. The
advancement in the omic approaches has flooded us with potential microbes [38] for use in
biotic and abiotic stress management [73].

6. Microbiome Engineering
The role of the plant genotype in the diversity and phylogeny of the soil microbial
population implies that the signature communities in a niche ecosystem can be tailored by
both the genotype and the environment [83,84] to provide better host performance. Hence,
engineering a rhizospheric system for plants can positively influence the plants [85]. How-
ever, in order to engineer the community, the desired impact on the host and environment
must be established. The following sections will elaborate further on the role of microbiome
engineering in sustainable agriculture.

6.1. Rhizosphere Microbiome

The ecto- and endorhizosphere are inhabited by a plethora of macro- and micro-
fauna [86]. Several studies have explored the microbial communities within these envi-
ronments and determined the association between the microorganisms and the hosts’ root
systems [86–88]. Most of these plant associations are positive to ensure plant fitness [61,89].
Many studies have reported on root endophytes such as Rhizobium spp. and Brevibacillus
spp. in nodulating and non-nodulating plants, respectively, as these plants have contributed
efficiently to nitrogen fixation. Further, through the use of metagenome data, researchers
have been able to address the taxonomic diversity in different strains that colonize different
plant tissues. The overall microbial communities associated with the host regulate various
physiological and biological functions, such as stress management, nutrient uptake, and
sequestering. Hence, identifying and mapping the function of microorganisms in each
type of plant tissue is necessary in order to understand how the plant–microbe associations
boost plant function [90,91]. However, very little is known about the trend of colonization
in agricultural crops [92,93].

6.2. Rhizosphere Engineering

In engineering the rhizospheric community, there are several questions that need to be
addressed. Essentially, we would want to engineer the environment to optimize the benefit
to the plant. Thus, how would we positively impact the plant? The obvious response
would be for us to improve nutrient cycling, nutrient sequestering, resistance/tolerance
to salinity and heavy metals, resistance to pests and diseases, and water-holding capacity.
Hence, to engineer the environment, we need to utilize all available tools to decipher the
microbiome and its functions. Although we are bombarded with a variety of data, many of
the host–microbe interactions still remains obscure. Currently, many products available in
the market are from the microbial consortium of PGPB and fungi, such as Bactophospin,
which utilizes Bacillus mucilaginosis (Russian); Flavobacterin, which uses Flavobacteria spp.
(Russian); and Mamezo, which uses Rhizobium sp. (Japan). In our local context, Malaysia
produces MYCOGold, which utilizes 4 genera of AMF [94,95]. However, there have also
been PGPB identified in the laboratory and greenhouse trials, which showed much promise,
but did not perform well when translated to the field [96,97]. This lack of performance
was largely due to the inability of these strains to colonize the environment. Therefore, a
crucial factor to bear in mind is that the bioinoculant must be able to form strong and stable
associations in the soil and with the plant to ensure lasting positioning in the community
and structure within that ecosystem [97,98].
The current omics tools has made it possible for us to make designer changes to the
environment and to plants [98,99]. In the years to come, with the advent of more sophisti-
cated tools, we will be able to engineer the environment more efficiently. The procedure,
however, will remain the same, i.e., that we will need to understand the mechanism that
shapes the environment and imitate the symbiotic relationships that exist between the soil,
Plants 2023, 12, 2307 7 of 25

microbes, and plants in nature by engineering identical environments in the field. Once
this is accomplished, the bioinoculant will flourish in the seeded ecosystem.

6.3. Shaping the Microbiome

In shaping the microbiome, effort needs to be focused on isolating and identifying
microbes and their functionality in the rhizosphere. Therefore, there needs to be a database
of microorganisms and their specific contributions to the rhizosphere so that, in developing
agricultural applications, a more astute decision may be made regarding formulating the
consortium [100]. Plant–microbe symbiotic associations should be made an important part
of this database, and organisms conducting specific functions should be grouped accord-
ingly. For instance, from previous studies, it has been reported that rhizobacteria such
as Rhizobium, Bradyrhizobium, Mesorhizobium, Azospirilium, Azotobacter, and Acetobacter are
required for nitrogen fixation [100,101]. In addition, the presence of phosphate-solubilizing
and siderophore-producing bacteria increases the nutrient availability for the plant. Further,
there is another group of organisms which produces antimicrobials that are important
in inhibiting soil pathogens. These organisms exude antibacterial compounds such as
2,4-diacetylphloroglucinol (DAPG), hydrogen cyanide (HCN), oligomycin, bacteriocine,
and antifungals [96,102]. Additionally, research has shown that inoculating plants with con-
sortia of plant growth-promoting microorganisms and arbuscular mycorrhizal (AM) fungi
aids against biotic and abiotic stresses by producing defense-related chemicals [97,103–106].
Further, the growth and development of plants is also dependent on phytohormones,
which are necessary for plant–microbe interactions [107]. Microbial communities create
a number of phytohormones, including gibberellins (GA), auxins or indole-3-acetic acid
(IAA), and cytokinins. The major role of phytohormones in plant growth and development
has been proven through transcriptome studies. The crosstalk between these chemical
and signal molecules, such as jasmonic acid and salicylic acid, are crucial in inducing
systemic acquired resistance (SAR); they induce systemic resistance (ISR) in plants. Plants
can develop resistance to a wide spectrum of pathogens both under- and aboveground by
being inoculated with non-pathogenic bacteria. This ISR primarily relies on ethylene and
jasmonate signaling pathways. Through this, plants are able to respond more rapidly to the
onslaught of pathogens. ISR has been reported in response to various microorganisms, the
microbe-associated molecular patterns (MAMPs) of which, including cell envelope compo-
nents, flagella, and siderophores, activate ISR [24,108,109]. It is interesting to note that some
PGPB cause ISR reactions and stimulate plant development by emitting volatile organic
compounds (VOCs) [110,111]. A number of Bacillus, Pseudomonas, Serratia, Azozpirilium,
and Trichoderma species are well-known ISR-inducing microorganisms (Figure 1).
Knowledge of how these organisms are regulated and what the active compounds
produced by these organisms are is essential information that is required to shape a micro-
biome. Hence, the database mentioned above is a necessary and detailed description of
functionality. The exuded chemical compounds will make the identification of key microbes
for specific functions in growth, development, and biotic and abiotic stress management
more precise and efficient [74,112].
 Plants 2023, 12, x FOR PEER REVIEW 8 of 26
Plants 2023, 12, 2307 8 of 25

Figure1.1.Interactions
Figure Interactions between
between plants
plants and
and microorganisms
microorganismsininbioticbioticand
andabiotic interactions.
abiotic interactions.ThisThis
diagram shows how abiotic and biotic stresses can be perceived by the plant, causing changes in
diagram shows how abiotic and biotic stresses can be perceived by the plant, causing changes in
cellular metabolism and affecting plant physiology, growth, and development. The stress manage-
cellular
ment inmetabolism
plants, whichandisaffecting
elicited by plant physiology,
these stresses, is growth,
managedand development.
through crosstalk The stress manage-
and interplay of
ment in plants,
hormones. which isthere
In addition, elicited by thesebetween
is crosstalk stresses, is managed
signal moleculesthrough
such ascrosstalk
jasmonic and
acid interplay
and sali- of
cylic acid In
hormones. that regulatethere
addition, SAR is and ISR, respectively.
crosstalk Both molecules
between signal of these mechanisms induceacid
such as jasmonic a defense re-
and salicylic
sponse
acid thatin plants. On
regulate SAR theand
left,ISR,
we see how abioticBoth
respectively. and of
biotic stresses
these affects plant
mechanisms physiology,
induce a defense growth,
response
inand development. These interactions result in negative impacts on susceptible varieties. Reactive
plants. On the left, we see how abiotic and biotic stresses affects plant physiology, growth, and
oxygen species (ROS) are produced in response to these stresses. However, plants do possess mech-
development. These interactions result in negative impacts on susceptible varieties. Reactive oxygen
anisms of homeostasis that keep the ROS at levels non-detrimental to the plant. On the right, we
species (ROS)
have the ISR are
andproduced
SAR, which in response to these
are produced stresses.toHowever,
in response crosstalk plants
betweendosignal
possess mechanisms
molecules and of
homeostasis
hormones inthat keep
plants. ThetheSAR
ROS andat ISR
levels
arenon-detrimental
instrumental in the to defense
the plant.
andOn the right,
response we have
of plants the ISR
against
current
and SAR,and future
which areinfiltration
producedor inassociations
response toby microorganisms,
crosstalk respectively.
between signal molecules and hormones in
plants. The SAR and ISR are instrumental in the defense and response of plants against current and
future Knowledge
infiltration orofassociations
how theseby organisms are regulated
microorganisms, and what the active compounds
respectively.
produced by these organisms are is essential information that is required to shape a mi-
6.4. Rules That
crobiome. Govern
Hence, the Microbiome Engineering
database mentioned above is a necessary and detailed description
of functionality.
Understanding The the
exuded chemical
ecological compoundsthat
mechanisms will control
make thetheidentification
emergence,ofpersistence,
key mi-
crobes for specific functions in growth, development, and biotic and abiotic stress man-
and regional adaptability of the plant-associated microbiome is essential for the successful
agement more precise and efficient [74,112].
field application of microbiome therapies. However, up to this point, the majority of the
microbiome investigations have concentrated on questions such as “what is in there?”,
6.4. Rules That Govern Microbiome Engineering
“what are their functions?”, and “how do they interact with environment and plant?”. We
have yetUnderstanding
to arrive at a the ecological
conceptual mechanisms
framework thatthat control
enables thecomprehend
us to emergence, how
persistence,
ecological
and regional
processes adaptability
control of the plant-associated
the microbiome’s assembly andmicrobiome is essential
function [113]. For thefor the successful
purpose of creating
field application
models that favor of microbiome
successful therapies.proper
colonization, However, up to this point,
understanding of thethe majorityprocesses
ecological of the
microbiome
that investigations
produce and have concentrated
sustain different on questions
plant-associated such as
microbiomes “what is in
is essential. there?”, to
According
“what
the are their
ecological functions?”,
[114] and “how do they
and meta-community interact
theories with
[115], environmentassemblages
multi-species and plant?”.are
Wethe
have yet to arrive at a conceptual framework that enables us to comprehend how
result of the interaction between four main co-evolutionary processes in microbiome–host ecolog-
ical processesThese
interactions. control
arethe microbiome’s
dispersal, assembly and
diversification, function
selection, and[113]. ForAmong
drift. the purpose
theseof four
creating models that favor successful colonization, proper understanding of the ecological
processes, dispersal enriches the process of diversification. Meanwhile, drift and selection
processes that produce and sustain different plant-associated microbiomes is essential.
contribute towards the relative abundance of the microbial species. There exists a complex
interaction between these mechanisms and the ecological traits of microbial communi-
ties (such as resistance, resilience, and functional redundancy), which are important in
preserving community stability and formation.
Plants 2023, 12, 2307 9 of 25

In the late stages of plant development, selection plays a role in community assembly,
while dispersal and drift become significant in the seed and root developmental stages [116].
At different stages of development, the processes shift in relative importance. The assembly
and performance of plant-associated microbiomes can be impacted by the arrival order,
which is referred to as priority effects [16]. By increasing the number of suppressive
bacteria or by activating the plant immune system, uncommon taxa, for instance, may have
a priority effect on the microbiome during different stages of plant development [117]. The
significance of priority effects for structuring the composition of the microbial communities
can be studied using SynCom. Findings from these studies indicate that the founding
taxa have a long-lasting impact on the formation of microbial communities, and are, thus,
resistant to invasion by newcomers.
Therefore, based on the above conclusions, we may conclude that microbiome in-
oculation can be effective when applied in the early stages of development. Local scale
community assembly can be impacted by abiotic stresses, migration from other ecosystems,
community structure, and internal ecological interactions [115]. Microbial communities
should be considered as dynamic, ever-changing systems that warrant careful study. Ad-
ditionally, studies focusing on arbuscular mycorrhizal plants have offered evidence that
genetic variations of isolates influence their effects on host–microbe interactions and, thus,
affect their potential in influencing host–plant fitness [118,119].

6.5. Shaping the Microbiome through Plant-Mediated Strategies

When addressing plant-mediated strategies, two distinct methods—plant breeding
and genetic engineering—are used to control plant characteristics. Using plant breeding
techniques to determine the microbial community is of interest for boosting crop yield and
resilience [120]. Microbiome selection has been incorporated into plant breeding programs,
focusing on specific taxa and functions that can produce good yields. Transgenic plants
with increased resistance to disease and soil physicochemical tolerance (phosphate-limited
soils) have improved disease suppression as well as ability of plants to grow on acidic
soils [121,122]. Further, transgenic tobacco and Arabidopsis plants have produced exudates
which altered the rhizospheric pH to acidic, thus improving the plants’ resistance, growth,
and ability to mineralize nutrients from the soil [123,124].
According to the research conducted by Ellouze et al. [125], certain chickpea cultivars
attract a more advantageous group of microorganisms that shape the microbiome as well
as increase the yield potential of durum wheat. Numerous studies have been conducted
to regulate plants by altering the synthesis of crucial exudates that govern the formation
of particular plant–microbiome interactions. However, the importance of breeding or
molecular breeding with emphasis on reshaping the microbiome have not received as much
attention. Plant-mediated strategies regarding the rhizosphere microbiome are currently
being addressed using molecular- and genome-based techniques that are more precise and
informative. These techniques will be elaborated on in Section 8.

7. Translation of Knowledge to Field Application

All works conducted in the discovery phase must be translated into implementation to
improve plant growth and development under all types of environmental pressure. Hence,
there needs to be technology development, translational plans, and policies developed to
ensure translation from the laboratory to the field. In this section and the following, we
will look into translational technologies to support this transition.

7.1. Engineering a Host-Mediated Microbiome for Sustainable Agriculture

The successful implementation of microbial-based solutions into the enhancement of
plant performance depends on the capacity to develop communities with beneficial traits.
Due to their superior ability to acclimatize to various environments, soil types, and/or
plant niches, native microbiota have a higher likelihood of developing and expressing
favorable traits [126]. Hence, it is possible to select for a robust, plant-optimized microbiome
Plants 2023, 12, 2307 10 of 25

that is resistant to random invasion utilizing experimental evolutionary techniques [127].

Beneficial microbiomes that are optimized for a particular environment can remain in, on,
and around plants for many generations [128].
Host-mediated microbiome engineering utilizes the knowledge of microbial commu-
nities adapted to the host to accomplish specific functionalities [18,129]. For instance, by
artificial selection, specialized microbial communities can be built up that alter plant traits
in a highly reproducible manner [130]. In order to maximize their chance of reproducing
under various stresses, plants have evolved a method known as accelerated flowering
time [131]. In this case, altering the plant microbiome artificially leads to the development
of stress-tolerant phenotypes with improved productivity in response to volatile climatic
conditions [132].

7.2. Engineering Plant Microbiomes for Sustainable Agriculture

The results of meta-analyses have revealed that domestication has caused taxonomic
alterations in root microbial communities, highlighting the importance of understanding
the function of any missing microorganisms that can give wild species a competitive ad-
vantage over their domesticated counterparts [133]. The heredity of microbiomes and their
connection to agronomic traits are both supported by recent studies. Studies conducted on
different genotypes of canola showed that there was a notable difference in the percentage
of microbial species [134,135]. As there is growing evidence that the microbiome affects
host performance, breeding programs must successfully integrate genotype, environment,
microbiome, and management interactions [136]. Therefore, to identify plant genes with
real impact on the microbiome, multi-year field and location studies must be conducted to
identify connections between the genotype, environment, and microbes. The connection
between the three is essential for the sustainable translation of beneficial microbes to the
field [43,137].
Genome-wide association studies (GWAS) have shown that plant genes play a role
in plant-associated microbiome assembly. The abundance of particular rhizosphere mi-
crobiome subgroups is strongly connected with the presence of these genes, which are
shared by diverse plant types [138,139]. By modifying these potential genes, it is possible to
produce designer plants with particular microbiomes and advantages. For instance, genes
implicated in stress responses and glucose metabolism have an impact on the phyllosphere
microbiome of rice [129]. The phyllosphere microbiome undergoes changes in the overex-
pressing line that are related to structural changes and induced immune responses [140].
The genetic basis of interactions between the microbiome and the host can be better under-
stood by GWAS, but these may also reflect false connections and are not always capable
of establishing causal relationships [141]. By affecting the expression of various genes
involved in the production of plant hormones, the resultant assemblage of soil microbiomes
affects the plant’s physiology [142]. Altered chemical signals in rhizobium–legume symbio-
sis have changed how plants are able to preferentially attract nitrogen-fixing rhizobia [143].
In addition, plant hormones such as salicylic acid [144] or secondary metabolites such as
coumarin are known to shape the root microbiota under stress, and may be utilized to
genetically engineer crops for sustainable agriculture.

7.3. Management Practices That Optimize the Microbiome

While small amounts of conducive and disease-suppressing soils can be combined
to transfer disease-suppressive properties, disease-suppressing soils can also be created
through specific management techniques, such as tillage and crop rotation [145,146]. It
is possible to steer agroecosystems in healthier directions by adopting the advantageous
microbial impacts of some management practices. For example, the intercropping of
leguminous crops with non-legumes can result in gaseous signal exchange between plant-
associated microbiomes [29]. Overall, it has been reported that intercropping enriches
soil microbiota, resulting in better fitness and yield. The impacts of organic farming
and conservation tillage on the soil microbiome result in increased soil nutrient cycling,
Plants 2023, 12, 2307 11 of 25

which leads to good growth and yield [16,147]. The use of fertilizers and machinery, site
conditions, profitability, and crop varieties are only a few variables that influence the overall
management decisions and practices that influence the optimization of the microbiome.
Further studies must be conducted to optimize the use of management practices and
microbiome engineering for the purpose of positively impacting the agricultural industry.

7.4. Optimized of Microbiomes through Genetical Engineering

Genome-based methods have uncovered a large number of genes in microbes that
mediate plant–microbe interactions [148]. The creation of superior microbial inoculants
has a great deal of promise thanks to the engineering of microbial genes that promote
advantageous features. Recently, the symbiotic Snodgrassella alvi was genetically modified
via RNA interference (RNAi) [149] to shield honeybees from viral infection and parasite
mites. Using similar techniques, it is possible to produce bacterial endophytes that cause
plants to react defensively to pathogens and pests. The creation of genome-editing tools,
such as CRISPR and CRISPR/Cas 9, has enabled the modification of genes and genomes to
achieve advantageous plant- and microbe-associated traits. This technology, according to
Farrar et al. [9], allows for the construction of improved and enhanced traits by influencing
the regulation of genes/genomes. According to Goold et al. [150], significant work is
being carried out to rebuild microbial circuitry (new gene networks created using synthetic
biology methods) and biological sensors that may be utilized in agriculture. A genetically
engineered strain of Pseudomonas putida called BananaGuard has been created to combat
the Fusarium oxysporum that causes Panama disease in banana. This altered bacteria detects
fusaric acid produced by F. oxysporum and produces inhibitors of fungal growth. The
system is inbuilt with a kill-switch that triggers self-destruction of the inhibitors when the
fungus is no longer found [151].
Recent advances in nitrogen fixation have become possible through the engineering of
naturally existing endophytic or epiphytic bacteria of cereal crops [152]. N2 -fixing strains
have been further genetically modified such that they would express N2 -fixing genes in
response to the presence of naturally existing chemical signals in plant-associated micro-
biomes. Targeted genome editing has advanced to the point that it is now conceivable to
develop designer genetic microbial circuits that respond to signals from plants to maximize
host–microbe interactions [153]. Although there are some regulatory uncertainties within
some jurisdictions, gene editing will remain a frontier science with immense potential for
plant–microbiome associations. Currently, there is some work being conducted that aims to
develop microbiomes with altered nitrogen-fixing abilities to successfully replace chemical
fertilizers in the field [150]. Inoculating natural systems with non-indigenous microbial
strains, however, requires much research to determine that there are no detrimental ef-
fects on the diversity of native plant and microbial species, and it is essential that all of
these processes are conducted in accordance with policies and jurisdictions regarding the
application of foreign organisms into the field [154,155].

8. Methods of Microbiome Studies

Methods must be developed that can provide us with data to make an informed and
educated selection of suitable candidates for the development of biofertilizers, biocontrols,
etc. The following are some of the most commonly used methods for above- and below-
ground microbial community identification, which involves organisms in and on plants, as
well as microorganisms in the soil.

8.1. CRISPR/Cas9
CRISPR/Cas9 is a new, emerging technology for addressing plant–microbe interac-
tions [156]. In addition, CRISPR/Cas9 has been used to genetically modify microorganisms to
increase their beneficial effects on crops [157]. Alternaria alternata, Colletotrichum sansevieriae,
Fusarium proliferatum, Phytophthora spp., and Sclerotinia sclerotiorum are only a few of the
phytopathogens that have been studied using the CRISPR/Cas9 system [156,158]. Fusarium
Plants 2023, 12, 2307 12 of 25

specifically causes a number of plant diseases by producing mycotoxins such as fumonisins.

The polyketide synthase gene FUM1 in Fusarium is in charge of producing fumonisins.
The FUM1 was deactivated using the CRISPR-Cas9 method, and the altered mutants failed
to make fumonisins [159]. Through the creation of CRISPR-Cas9 endogenous gene tagging,
the infection process of fungal diseases could be elucidated. It is possible to research the
subcellular localization of fungal proteins through these endogenous fluorescent tagged
genes [98]. Another example of editing is shown in Trichorderma atroviride, namely, the ace1
gene, which enhances the production of polyketide biosynthesis genes, heightening its
control over certain soil pathogen diseases [160]. As a result, the activation of gene clusters
increased the potential of these microorganisms to be used as biocontrols. Hence, this
method could uncover brand-new ways in which genes and pathways could be enhanced
in microbes to provide better control of biotic and abiotic stresses.
In addition, CRISPR-Cas9 has been utilized to modify beneficial microorganisms by
improving their functionality. It has also been applied to study processes connected to the
soil microbiome, such as nitrification and the breakdown of lignocellulose. The nitrate trans-
porter in the rice gene NRT1.1 B controls the root microbiota in indica cultivars [142,156].
The efficiency of nitrogen consumption in japonica rice was considerably improved by
targeted manipulation of this gene [161]. Two other genes (OSH15, OsAt10) modified plant
cell wall constituents in order to increase saccharification [156,162]. CRISPR elements are
naturally present in microorganisms [163] against foreign elements such as bacteriophages.
Additionally, it has been demonstrated that polar soil contains more CRISPR genes than
tropical soil, which explains the higher disease incidence observed in the tropics [156].
Further studies have also shown that genome editing using integrative plasmids reduces
horizontal gene transfers considerably [164]. Therefore, having CRISPR repetitions may
give plant-beneficial bacteria an evolutionary edge for improved adaptation (Figure 2).

8.2. Genome-Wide Association Studies (GWAS)

Numerous studies on the relationships between plants and microbes have used GWAS to
investigate the relationship between the plant genotype and microbial recruitment [165,166].
GWAS was used to identify loci associated with blast resistance in rice [167,168]. A single-
nucleotide polymorphism (SNP) chip of African rice cultivars identified genomic areas
connected to rice blast resistance [169]. Further, the effectiveness of GWAS in identifying
disease resistance was also observed in maize [166,170]. Recent studies have also shown the
ability of GWAS to identify the relationship between leguminous plants and their nodulat-
ing bacteria [171]. Horton et al. [172] employed a panel of 196 Arabidopsis thaliana genotypes
to correlate the leaf microbial population through taxonomic marker gene sequencing of
the bacterial or fungal populations. Through this study, they found that there was an
additive genetic variety association between the host and the microbial community in the
leaves and roots. With the help of these limited community datasets, Horton et al. [172]
were able to successfully correlate host SNPs to community-level characteristics of the
most common individual taxa that are connected to the leaf microbiota. To date, not many
studies have been conducted using GWAS to study the microbiome relationships above-
and belowground. However, GWAS have been used extensively in human–microbiome
interaction, proving to be a potentially useful tool for association studies of plant–microbe
interactions [173–175] (Figure 2).
Plants 2023, 12, x FOR PEER REVIEW 13 of 26
Plants 2023, 12, 2307 13 of 25

schematic diagram
Figure 2. A schematic diagram of
of several
several culture-dependent
culture-dependent and
and non-culture-dependent
non-culture-dependent methods
that have
that have been
been utilized
utilized extensively
extensively in
in the
the study
study of
of plant–soil
plant–soil microbiomes.
microbiomes. The
The methods
methods listed
listed for
for
the four omics technologies are the most commonly used techniques. The direct and indirect char-
the four omics technologies are the most commonly used techniques. The direct and indirect charac-
acterization of PGPB is also provided in this diagram. Legend: NMR—nuclear magnetic resonance;
terization of PGPB is also provided in this diagram. Legend: NMR—nuclear magnetic resonance;
LC-MS—liquid chromatography–mass spectrometry; GC-MS—gas chromatography–mass spec-
LC-MS—liquid chromatography–mass spectrometry; GC-MS—gas chromatography–mass spectrom-
trometry; RNA-Seq—RNA sequencing; DNA Seq—DNA sequencing.
etry; RNA-Seq—RNA sequencing; DNA Seq—DNA sequencing.
8.2. Genome-Wide
8.3. Association
Microbiome Sequencing Studies (GWAS)
Platforms
Numerous
The two primarystudies on the utilized
strategies relationships between plants
in metagenomic research andto microbes
target the have used
enormous
GWAS to investigate the relationship between the plant genotype and
diversity of environmental investigations are targeted and shotgun sequencing. Since these microbial recruit-
ment [165,166].
methods enableGWASresearcherswas used to identify
to expedite loci associated
the entire process atwith blastcost,
a lower resistance in rice
the Illumina
[167,168].isAthe
platform single-nucleotide
choice method for polymorphism
metagenomic (SNP) chip of African
sequencing. It providesricemany
cultivars identi-
millions of
fied genomic
brief, incredibly areas connected
accurate randomto rice
readsblast
thatresistance [169]. Further,
can be combined the effectiveness
or utilized as markers for of
GWAS in
certain identifying
metabolic disease and/or
pathways resistance was also
groups observed in maize
of microorganisms. [166,170].other
Although Recent stud-
cutting-
ies have
edge also shown
platforms, such theasability of GWAS
Ion Torrent andtoPacBio,
identifyhave
the relationship betweenthe
also been created, leguminous
Illumina
plants and
platform their
still nodulating
remains the most bacteria [171].
effective Horton platform
sequencing et al. [172] employed a panel of 196
[173,174,176].
Arabidopsis
In recentthaliana
years, genotypes
many PGPBtowith correlate
various the leaf microbial
phenotypic traits population
connected tothrough taxo-
plant growth
capacity have been
nomic marker gene characterized
sequencing ofusing whole-genome
the bacterial or fungalsequencing.
populations. For instance,
Through Brevibac-
this study,
terium frigoritolerans
they found that thereZB201705, isolated
was an additive fromvariety
genetic salt and the drought
association stressthe
between rhizosphere
host and the of
maize,
microbialis able to manufacture
community a large
in the leaves andnumber
roots. of
Withproteins
the help[177]
of from
these alimited
complete genome.
community
This shows
datasets, that B.
Horton et frigoritolerans
al. [172] were ZB201705 could be employed
able to successfully correlate hostas an inoculant
SNPs to boost
to community-
crop
level characteristics of the most common individual taxa that are connected to theused
yields, even in the presence of abiotic challenges. Pyrosequencing has also been leaf
to elucidate To
microbiota. thedate,
genome sequence
not many of Pseudomonas
studies sp. [178]. Through
have been conducted using GWAS this to
study,
studyPseu-
the
domonas was shown to produce chemicals such as siderophores, phosphate solubilizers,
Plants 2023, 12, 2307 14 of 25

ACC deaminase, and indole acetic acid, which contribute to plant growth and development
(Figure 2).
There are numerous molecular DNA fingerprinting methods which can be used
to examine the endophytic population that inhabits plant tissues, including amplified
rDNA restriction analysis (ARDRA), denaturing gradient gel electrophoresis (DGGE),
temperature gradient gel electrophoresis (TGGE), and terminal restriction fragment length
polymorphism (T-RFLP) [179]. To acquire community fingerprinting, alternative methods
such as automated ribosomal intergenic spacer analysis (ARISA), which analyzes the
extremely variable area between the 16S and 23S rDNA, can also be used [179]. However,
as of now, metagenomics techniques utilizing NGS have largely surpassed the development
of all these DNA fingerprinting techniques [180]. In an effort to better understand the
potential advantages of endophytes for the host plant, efforts have been made to sequence
entire genomes of over twenty genera of endophytes isolated from various hosts [181].

8.4. Metatranscriptomics, Metaproteomics, and Metabolomics for Understanding the Microbiomes

Through rhizosphere microbiome metatranscriptomics of wheat, oat, and pea,
kingdom-level variations have been identified between these plant systems [182]. Stress
(drought stress) also influences the microbiome associated with the root, hence causing it to
demonstrate increased transcriptional activity of genes related to amino acids and glucose
metabolism [183]. Microbial communities have been more recently developed as biosen-
sors using metagenomic and metatranscriptomic profiling [184]. The development of
proteomic and metabolomic approaches to supplement transcriptome data is a result of
the poor correlation between the transcriptional and translational processes. With the
use of metaproteomic analysis, a thorough understanding of the molecular phenotypes of
microbial communities in agricultural plants’ rhizospheres and phyllospheres has been
attained [185]. (Figure 2).
These studies have shown notable stability in the dominant microbiomes of organisms
and proteins associated with host plants and the environment [89]. Metaproteomics-based
inferences of the physiology of the microbiomes also revealed metabolic pathways that
permit specialized colonization and adaption in the rhizosphere compartments [89,130,186].
Although microbiome science is still in its infancy, the metabolome has been frequently
utilized for identifying plant diseases and causative agents [185]. According to early re-
search [185], the phyllosphere metabolome is altered by the rhizosphere microbiome, and
these alterations are related to distinct insect feeding strategies. Studies have also shown
that alterations to the root metabolome determine the recruitment of specialized microbial
communities that colonize the rhizobiome and plant, affecting the plant’s performance and
its interactions with pests and diseases [187,188]. For the detection and measurement of
small chemicals that drive plant–microbiome communication and interactions, metabolome
information is essential [130,185]. Recent advances in multiomics and integrated informat-
ics have uncovered the complex relationship between plant traits, metabolites, microbes,
and minerals in an agroecosystem. We believe that improved sample preparation; ex-
traction techniques; database compilation of proteins, metabolites, and genes; and the
development of computational tools to support bioinformatic analyses of big data will
advance this field to fully realize the potential of the omic approaches in deciphering the
genotype–phenotype relationships [189,190].

8.5. Culturomics to Support the Molecular Techniques

The use of microbiomes to produce commercial inoculants requires the use of cul-
tivated bacteria [191]. Microbial cultures are also required for us to progress into omic
studies and, at the same time, provide bacterial reference genome sequences for func-
tional studies [192,193]. Many agricultural research universities and institutions maintain
culture collections that are related to plant-associated microbiomes. Huge collections of
isolates have been subjected to comparative genomics, through which several important
genes have been identified. The validation of these genes through molecular techniques
Plants 2023, 12, 2307 15 of 25

is possible due to the availability of cultured members [194]. Despite recent advances in
culturomics, genomic diversity in cultured bacterial isolates is still far from reaching satura-
tion [195]. Future studies into the putative functions of plant-associated microorganisms
will require impartial culture sequence collections obtained from a wide range of plants
and soil types [196]. These collections must also include mechanisms to curate, share, and
standardize metadata for the strains contained within them (Figure 2).
In addition to supporting omics platforms with cultures, it is also possible to analyze
the direct and indirect mechanisms of plant growth-promoting bacteria using these cultures.
According to Glick [197], PGPB can stimulate plant growth both directly and indirectly.
Direct methods are those that make use of bacterial features that directly promote plant
development, such as nitrogen fixation, siderophores synthesis, ACC deaminase activity,
phytohormone generation, biofilm production, and phosphate solubilization. Some PGPB
can fix nitrogen from the atmosphere and transform it into a form that plants can use,
encouraging plant growth [198,199]. In a study by Sarkar [200], culturomics was utilized to
assess the capability of drought-tolerant Pseudomonas for nitrogen fixation. In addition, an
essential form of PGPB such as siderophilic bacteria can release siderophores to chelate Fe3+
in the soil for plant development [201]. By utilizing culturomics, Flores-Felix et al. [202]
revealed that the PGPB had the ability to promote blueberry development by dissolving
dicalcium phosphate to form siderophores. PGPBs also generate ACC deaminase, an
enzyme that reduces ethylene levels in plants to prevent ethylene-induced suppression of
root growth and trigger stress reactions [203–205]. Culturomics was also applied by Chicca
et al. [206], who demonstrated that PGPB (Microbacterium, Achromobacter, and Pseudomonas
spp.) produced ACC deaminase activity, which promotes plant development. Further,
phytohormones including auxins, cytokinins, and gibberellins, which control different
aspects of plant growth and development, can also be produced by PGPB [207]. A study by
Sahu et al. [208] combining metabarcoding and culturomics methods in profiling the rice
phyllosphere microbiome identified extensive and useful microbial groups for blast disease
mitigation. The results also demonstrated increased expression of genes related to defense,
including OsCERK1 and OsCEBiP, as well as genes related to phytohormones, including
OsFMO, OsPDF2.2, OsNPR1, OsPR1.1, OsEDS1, and OsPAD4. PGPB also had the ability
to create biofilms, which are microbial communities that adhere to surfaces and promote
improved colonization, nutrition substitution, and adaptation to stress [209]. PGPB also
have the capacity to solubilize phosphate and increase its availability for absorption and
use by plants. By utilizing culturomics, Flores-Duarte et al. [210] identified 13 features in
PGPB, including biofilm development and phosphate solubilization, in their study.
The indirect mechanisms of PGPB action include the production of exopolysaccha-
rides (EPS), hydrolytic enzymes, hydrogen cyanide (HCN), ISR, antimicrobial compounds,
quorum quenching, competition, and siderophores. The production of EPS by PGPB can
improve soil aggregation, provide pathogen protection, and encourage nutrient and water
retention [211]. Furthermore, the production of HCN by PGPB has antibacterial properties
and can halt the growth of plant diseases [212]. Additionally, many PGPB have secretion
systems that enable them to create antimicrobial substances, including antibiotics, organic
volatile compounds, and lytic enzymes, which can limit the development of potentially
phytopathogenic microbes [213]. PGPB also support plant resistance by inducing systemic
resistance to a variety of diseases and initiating the plant’s defense mechanisms [214,215];
they secrete hydrolytic enzymes, including chitinases, proteases, and cellulases, that break
down different parts of the pathogen’s cellular wall or extracellular structure [216]. Com-
bining a culturomics approach with molecular methods would help to characterize the
indirect and direct mechanisms of PGPB.

9. Conclusions
In the process of moving towards sustainable agriculture, agricultural practices must
reduce the extensive usage of agrochemicals. In this review, we have presented the potential
of beneficial microbes to enhance growth, development, and disease suppression in the
Plants 2023, 12, 2307 16 of 25

field. However, the effectiveness of beneficial microbes in field applications has been
less than satisfactory. This is largely due to the fact that the newly introduced microbes
must thrive in the environment and maintain a steady and stable community for the
benefits to be harnessed. This has brought forth technologies such as sequencing and
multiomics platforms, which have enabled us to visualize, to some extent, the diversity,
communities, and structures of microorganisms in any given environment. This has created
opportunities for technologies such as microbial engineering to offer designer solutions for
specific environments thus achieving greater efficiency and sustainability. This technique
promises answers to various gaps in knowledge, such as providing the right microbial
consortia based on the plant species and soil environment to ensure proper recognition
and colonization of the soil and roots by the inoculants. In this regard, the advancement
of “microbiome-driven cropping systems” may herald the next agricultural revolution
and a more sustainable method of plant production. Furthermore, the development of
modified crops or organisms may yield the desired advancement towards zero hunger
for the continuously expanding human population. This will be made possible by the
application of multiomics approaches combined with genome editing techniques such as
CRISPR for improving nutritional status, disease resistance, and crop yield.
In the years to come, however, there is much work that needs to be conducted to com-
prehend the genetics and engineering of the intricacies behind the ecological and metabolic
networks that govern plant-associated microbe interactions. For instance, research needs to
go beyond the identification of causative or beneficial organisms into “how” this informa-
tion may be used reproducibly to enhance plant growth and development and to reduce
disease incidence and spread. We also need to reduce wastage and redundancies in research
by producing standardized techniques and a center for the collation and annotation of
meta-data. These techniques must be made such that they can be utilized by any laboratory
worldwide for the translation of laboratory-raised products to the field. We also need to
focus on developing new technologies that are more efficient, accurate, cost-effective, and
quick to use both in the laboratory and in the field. The technologies that are developed
should not be restrictive to only the well-funded research groups, but should be attainable
for small laboratories. Bioinformaticians need to continuously come up with new software
or upgraded versions of the existing platforms to increase the depth, speed, and quantity
of information harnessed from the data. The upgrade does not stop with the equipment
and the techniques; the upskilling of researchers needs to evolve along with trends in the
field of plant-associated microbiome research. This field has a bountiful future, and there is
room to push the boundaries of knowledge and technologies further.

Author Contributions: Conceptualization, K.N.; methodology, K.N.; software, K.N.; validation, K.N.
and N.S.N.A.R.; writing—original draft preparation, K.N. and N.S.N.A.R.; writing—review and
editing, K.N. and N.S.N.A.R.; supervision, K.N.; project administration, K.N.; funding acquisition,
K.N. All authors have read and agreed to the published version of the manuscript.
Funding: This paper was funded by the Ministry of Education, Malaysia (FRGS/1/2019/STG03/
UKM/01/2) through a grant awarded to Kalaivani Nadarajah.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Malhi, G.S.; Kaur, M.; Kaushik, P. Impact of Climate Change on Agriculture and Its Mitigation Strategies: A Review. Sustainability
2021, 13, 1318. [CrossRef]
2. Tilman, D. Global environmental impacts of agricultural expansion: The need for sustainable and efficient practices. Proc. Natl.
Acad. Sci. USA 1999, 96, 5995–6000. [CrossRef] [PubMed]
3. Gomiero, T. Soil Degradation, Land Scarcity and Food Security: Reviewing a Complex Challenge. Sustainability 2016, 8, 281.
[CrossRef]
4. Nadarajah, K. Soil Health: The Contribution of Microflora and Microfauna. In Mycorrhizosphere Pedogenesis; Springer: Singapore,
2019; pp. 383–400. [CrossRef]
Plants 2023, 12, 2307 17 of 25

5. Koskey, G.; Mburu, S.W.; Awino, R.; Njeru, E.M.; Maingi, J.M. Potential Use of Beneficial Microorganisms for Soil Amelioration,
Phytopathogen Biocontrol, and Sustainable Crop Production in Smallholder Agroecosystems. Front. Sustain. Food Syst. 2021, 5,
130. [CrossRef]
6. Lopes, M.J.D.S.; Dias-Filho, M.B.; Gurgel, E.S.C. Successful Plant Growth-Promoting Microbes: Inoculation Methods and Abiotic
Factors. Front. Sustain. Food Syst. 2021, 5, 606454. [CrossRef]
7. Harman, G.; Khadka, R.; Doni, F.; Uphoff, N. Benefits to Plant Health and Productivity from Enhancing Plant Microbial Symbionts.
Front. Plant Sci. 2021, 11, 2001. [CrossRef]
8. Nishad, R.; Ahmed, T.; Rahman, V.J.; Kareem, A. Modulation of Plant Defense System in Response to Microbial Interactions.
Front. Microbiol. 2020, 11, 1298. [CrossRef]
9. Farrar, K.; Bryant, D.; Cope-Selby, N. Understanding and engineering beneficial plant–microbe interactions: Plant growth
promotion in energy crops. Plant Biotechnol. J. 2014, 12, 1193–1206. [CrossRef]
10. Bhattacharyya, P.N.; Jha, D.K. Plant growth-promoting rhizobacteria (PGPR): Emergence in agriculture. World J. Microbiol.
Biotechnol. 2012, 28, 1327–1350. [CrossRef]
11. Gupta, R.; Anand, G.; Gaur, R.; Yadav, D. Plant–microbiome interactions for sustainable agriculture: A review. Physiol. Mol. Biol.
Plants 2021, 27, 165–179. [CrossRef]
12. Fendrihan, S.; Pop, C.-E. Biotechnological potential of plant associated microorganisms. Rom. Biotechnol. Lett. 2021, 26, 2700–2706.
[CrossRef]
13. Sergaki, C.; Lagunas, B.; Lidbury, I.; Gifford, M.L.; Schäfer, P. Challenges and Approaches in Microbiome Research: From
Fundamental to Applied. Front. Plant Sci. 2018, 9, 1205. [CrossRef]
14. Gupta, A.; Singh, U.B.; Sahu, P.K.; Paul, S.; Kumar, A.; Malviya, D.; Singh, S.; Kuppusamy, P.; Singh, P.; Paul, D.; et al. Linking Soil
Microbial Diversity to Modern Agriculture Practices: A Review. Int. J. Environ. Res. Public Health 2022, 19, 3141. [CrossRef]
15. Anguita-Maeso, M.; Navas-Cortés, J.A.; Landa, B.B. Insights into the Methodological, Biotic and Abiotic Factors Influencing the
Characterization of Xylem-Inhabiting Microbial Communities of Olive Trees. Plants 2023, 12, 912. [CrossRef]
16. Trivedi, P.; Leach, J.E.; Tringe, S.G.; Sa, T.; Singh, B.K. Plant–microbiome interactions: From community assembly to plant health.
Nat. Rev. Microbiol. 2020, 18, 607–621. [CrossRef]
17. Fitzpatrick, C.R.; Schneider, A.C. Unique bacterial assembly, composition, and interactions in a parasitic plant and its host. J. Exp.
Bot. 2020, 71, 2198–2209. [CrossRef]
18. Kaul, S.; Choudhary, M.; Gupta, S.; Dhar, M.K. Engineering Host Microbiome for Crop Improvement and Sustainable Agriculture.
Front. Microbiol. 2021, 12, 1125. [CrossRef]
19. Afridi, M.S.; Ali, S.; Salam, A.; Terra, W.C.; Hafeez, A.; Sumaira; Ali, B.; AlTami, M.S.; Ameen, F.; Ercisli, S.; et al. Plant Microbiome
Engineering: Hopes or Hypes. Biology 2022, 11, 1782. [CrossRef]
20. Cullen, C.M.; Aneja, K.K.; Beyhan, S.; Cho, C.E.; Woloszynek, S.; Convertino, M.; McCoy, S.J.; Zhang, Y.; Anderson, M.Z.;
Alvarez-Ponce, D.; et al. Emerging Priorities for Microbiome Research. Front. Microbiol. 2020, 11, 136. [CrossRef]
21. Brunel, C.; Pouteau, R.; Dawson, W.; Pester, M.; Ramirez, K.S.; van Kleunen, M. Towards Unraveling Macroecological Patterns in
Rhizosphere Microbiomes. Trends Plant Sci. 2020, 25, 1017–1029. [CrossRef]
22. Compant, S.; Samad, A.; Faist, H.; Sessitsch, A. A review on the plant microbiome: Ecology, functions, and emerging trends in
microbial application. J. Adv. Res. 2019, 19, 29–37. [CrossRef] [PubMed]
23. Santoyo, G.; Moreno-Hagelsieb, G.; del Carmen Orozco-Mosqueda, M.; Glick, B.R. Plant growth-promoting bacterial endophytes.
Microbiol. Res. 2016, 183, 92–99. [CrossRef] [PubMed]
24. Vandenkoornhuyse, P.; Quaiser, A.; Duhamel, M.; Le Van, A.; Dufresne, A. The importance of the microbiome of the plant
holobiont. New Phytol. 2015, 206, 1196–1206. [CrossRef] [PubMed]
25. Wang, X.; Whalley, W.R.; Miller, A.J.; White, P.J.; Zhang, F.; Shen, J. Sustainable Cropping Requires Adaptation to a Heterogeneous
Rhizosphere. Trends Plant Sci. 2020, 25, 1194–1202. [CrossRef]
26. Chiaranunt, P.; White, J.F. Plant Beneficial Bacteria and Their Potential Applications in Vertical Farming Systems. Plants 2023, 12,
400. [CrossRef]
27. Li, P.; Liu, J.; Saleem, M.; Li, G.; Luan, L.; Wu, M.; Li, Z. Reduced chemodiversity suppresses rhizosphere microbiome functioning
in the mono-cropped agroecosystems. Microbiome 2022, 10, 108. [CrossRef]
28. Jin, Y.; Zhu, H.; Luo, S.; Yang, W.; Zhang, L.; Li, S.; Jin, Q.; Cao, Q.; Sun, S.; Xiao, M. Role of Maize Root Exudates in Promotion of
Colonization of Bacillus velezensis Strain S3-1 in Rhizosphere Soil and Root Tissue. Curr. Microbiol. 2019, 76, 855–862. [CrossRef]
29. Chen, Y.; Bonkowski, M.; Shen, Y.; Griffiths, B.; Jiang, Y.; Wang, X.; Sun, B. Root ethylene mediates rhizosphere microbial
community reconstruction when chemically detecting cyanide produced by neighbouring plants. Microbiome 2020, 8, 4. [CrossRef]
30. Carrión, V.J.; Perez-Jaramillo, J.; Cordovez, V.; Tracanna, V.; de Hollander, M.; Ruiz-Buck, D.; Mendes, L.W.; van Ijcken, W.F.J.;
Gomez-Exposito, R.; Elsayed, S.S.; et al. Pathogen-induced activation of disease-suppressive functions in the endophytic root
microbiome. Science 2019, 366, 606–612. [CrossRef]
31. Liang, S.; Liu, H.; Wu, S.; Xu, S.; Jin, D.; Faiola, F.; Zhuang, X.; Zhuang, G.; Qu, D.; Fan, H.; et al. Genetic diversity of diazotrophs
and total bacteria in the phyllosphere of Pyrus serotina, Prunus armeniaca, Prunus avium, and Vitis vinifera. Can. J. Microbiol. 2019,
65, 642–652. [CrossRef]
Plants 2023, 12, 2307 18 of 25

32. Stringlis, I.A.; De Jonge, R.; Pieterse, C.M.J. The Age of Coumarins in Plant–Microbe Interactions. Plant Cell Physiol. 2019, 60,
1405–1419. [CrossRef]
33. Lombardi, N.; Vitale, S.; Turrà, D.; Reverberi, M.; Fanelli, C.; Vinale, F.; Marra, R.; Ruocco, M.; Pascale, A.; D’errico, G.; et al. Root
Exudates of Stressed Plants Stimulate and Attract Trichoderma Soil Fungi. Mol. Plant-Microbe Interact. 2018, 31, 982–994. [CrossRef]
34. Shaposhnikov, A.I.; Shakhnazarova, V.Y.; Vishnevskaya, N.A.; Borodina, E.V.; Strunnikova, O.K. Aromatic Carboxylic Acids
in Barley-Root Exudates and Their Influence on the Growth of Fusarium culmorum and Pseudomonas fluorescens. Appl. Biochem.
Microbiol. 2020, 56, 344–351. [CrossRef]
35. Arif, I.; Batool, M.; Schenk, P.M. Plant Microbiome Engineering: Expected Benefits for Improved Crop Growth and Resilience.
Trends Biotechnol. 2020, 38, 1385–1396. [CrossRef]
36. Velmourougane, K.; Saxena, G.; Prasanna, R. Plant-Microbe Interactions in the Rhizosphere: Mechanisms and Their Ecological
Benefits. In Plant-Microbe Interactions in Agro-Ecological Perspectives; Springer: Singapore, 2017; Volume 2, pp. 193–219. [CrossRef]
37. Chagas, F.O.; Pessotti, R.D.C.; Caraballo-Rodríguez, A.M.; Pupo, M.T. Chemical signaling involved in plant–microbe interactions.
Chem. Soc. Rev. 2018, 47, 1652–1704. [CrossRef]
38. Nadarajah, K. Metagenomics for Improving Soil Fertility. In Soil Nitrogen Ecology. Soil Biology; Springer: Cham, Switzerland, 2021;
Volume 62, pp. 267–282. [CrossRef]
39. Navgire, G.S.; Goel, N.; Sawhney, G.; Sharma, M.; Kaushik, P.; Mohanta, Y.K.; Mohanta, T.K.; Al-Harrasi, A. Analysis and
Interpretation of metagenomics data: An approach. Biol. Proced. Online 2022, 24, 18. [CrossRef]
40. Nwachukwu, B.C.; Babalola, O.O. Metagenomics: A Tool for Exploring Key Microbiome with the Potentials for Improving
Sustainable Agriculture. Front. Sustain. Food Syst. 2022, 6, 206. [CrossRef]
41. Dastogeer, K.M.; Tumpa, F.H.; Sultana, A.; Akter, M.A.; Chakraborty, A. Plant microbiome–An account of the factors that shape
community composition and diversity. Curr. Plant Biol. 2020, 23, 100161. [CrossRef]
42. Noman, M.; Ahmed, T.; Ijaz, U.; Shahid, M.; Azizullah; Li, D.; Manzoor, I.; Song, F. Plant–Microbiome Crosstalk: Dawning from
Composition and Assembly of Microbial Community to Improvement of Disease Resilience in Plants. Int. J. Mol. Sci. 2021, 22,
6852. [CrossRef]
43. Saad, M.M.; Eida, A.A.; Hirt, H. Tailoring plant-associated microbial inoculants in agriculture: A roadmap for successful
application. J. Exp. Bot. 2020, 71, 3878–3901. [CrossRef]
44. Sivakumar, N.; Sathishkumar, R.; Selvakumar, G.; Shyamkumar, R.; Arjunekumar, K. Phyllospheric Microbiomes: Diversity,
Ecological Significance, and Biotechnological Applications. In Plant Microbiomes for Sustainable Agriculture; Springer: Cham,
Switzerland, 2020; Volume 25, pp. 113–172. [CrossRef]
45. Zheng, Q.; Hu, Y.; Zhang, S.; Noll, L.; Böckle, T.; Dietrich, M.; Herbold, C.W.; Eichorst, S.A.; Woebken, D.; Richter, A.; et al. Soil
multifunctionality is affected by the soil environment and by microbial community composition and diversity. Soil Biol. Biochem.
2019, 136, 107521. [CrossRef] [PubMed]
46. Xiong, Q.; Hu, J.; Wei, H.; Zhang, H.; Zhu, J. Relationship between Plant Roots, Rhizosphere Microorganisms, and Nitrogen and
Its Special Focus on Rice. Agriculture 2021, 11, 234. [CrossRef]
47. Yang, C.; Yue, H.; Ma, Z.; Feng, Z.; Feng, H.; Zhao, L.; Zhang, Y.; Deakin, G.; Xu, X.; Zhu, H.; et al. Influence of plant genotype
and soil on the cotton rhizosphere microbiome. Front. Microbiol. 2022, 13, 3674. [CrossRef] [PubMed]
48. Abdul Rahman, N.S.N.; Abdul Hamid, N.W.; Nadarajah, K. Effects of abiotic stress on soil microbiome. Int. J. Mol. Sci. 2021, 22,
9036. [CrossRef]
49. Koza, N.A.; Adedayo, A.A.; Babalola, O.O.; Kappo, A.P. Microorganisms in Plant Growth and Development: Roles in Abiotic
Stress Tolerance and Secondary Metabolites Secretion. Microorganisms 2022, 10, 1528. [CrossRef]
50. Blaustein, R.A.; Lorca, G.L.; Meyer, J.L.; Gonzalez, C.F.; Teplitski, M. Defining the Core Citrus Leaf- and Root-Associated
Microbiota: Factors Associated with Community Structure and Implications for Managing Huanglongbing (Citrus Greening)
Disease. Appl. Environ. Microbiol. 2017, 83, e00210-17. [CrossRef]
51. Hamonts, K.; Trivedi, P.; Garg, A.; Janitz, C.; Grinyer, J.; Holford, P.; Botha, F.C.; Anderson, I.C.; Singh, B.K. Field study reveals
core plant microbiota and relative importance of their drivers. Environ. Microbiol. 2018, 20, 124–140. [CrossRef]
52. Xu, J.; Zhang, Y.; Zhang, P.; Trivedi, P.; Riera, N.; Wang, Y.; Liu, X.; Fan, G.; Tang, J.; Coletta-Filho, H.D.; et al. The structure and
function of the global citrus rhizosphere microbiome. Nat. Commun. 2018, 9, 4894. [CrossRef]
53. Simonin, M.; Dasilva, C.; Terzi, V.; Ngonkeu, E.L.M.; Diouf, D.; Kane, A.; Béna, G.; Moulin, L. Influence of plant genotype and soil
on the wheat rhizosphere microbiome: Evidences for a core microbiome across eight African and European soils. FEMS Microbiol.
Ecol. 2020, 96, fiaa067. [CrossRef]
54. Singh, B.K.; Trivedi, P.; Egidi, E.; Macdonald, C.A.; Delgado-Baquerizo, M. Crop microbiome and sustainable agriculture. Nat.
Rev. Microbiol. 2020, 18, 601–602. [CrossRef]
55. de Souza, R.S.C.; Okura, V.K.; Armanhi, J.S.L.; Jorrín, B.; Lozano, N.; da Silva, M.J.; González-Guerrero, M.; de Araújo, L.M.;
Verza, N.C.; Bagheri, H.C.; et al. Unlocking the bacterial and fungal communities assemblages of sugarcane microbiome. Sci. Rep.
2016, 6, 28774. [CrossRef]
56. Dai, Y.; Yang, F.; Zhang, L.; Xu, Z.; Fan, X.; Tian, Y.; Wang, T. Wheat-associated microbiota and their correlation with stripe rust
reaction. J. Appl. Microbiol. 2020, 128, 544–555. [CrossRef]
Plants 2023, 12, 2307 19 of 25

57. Lemanceau, P.; Blouin, M.; Muller, D.; Moënne-Loccoz, Y. Let the Core Microbiota Be Functional. Trends Plant Sci. 2017, 22,
583–595. [CrossRef]
58. Banerjee, S.; Schlaeppi, K.; Van Der Heijden, M.G.A. Keystone taxa as drivers of microbiome structure and functioning. Nat. Rev.
Microbiol. 2018, 16, 567–576. [CrossRef]
59. Roman-Reyna, V.; Pinili, D.; Borja, F.N.; Quibod, I.L.; Groen, S.C.; Mulyaningsih, E.S.; Rachmat, A.; Slamet-Loedin, I.H.;
Alexandrov, N.; Mauleon, R.; et al. The rice leaf microbiome has a conserved community structure controlled by complex
host-microbe interactions. bioRxiv 2019, 615278. [CrossRef]
60. Niu, Y.; Bainard, L.; Bandara, M.S.; Hamel, C.; Gan, Y. Soil residual water and nutrients explain about 30% of the rotational effect
in 4-year pulse-intensified rotation systems. Can. J. Plant Sci. 2017, 97, 852–864. [CrossRef]
61. Egamberdieva, D.; Wirth, S.J.; Alqarawi, A.A.; Abd_Allah, E.F.; Hashem, A. Phytohormones and Beneficial Microbes: Essential
Components for Plants to Balance Stress and Fitness. Front. Microbiol. 2017, 8, 2104. [CrossRef]
62. Vannier, N.; Agler, M.; Hacquard, S. Microbiota-mediated disease resistance in plants. PLoS Pathog. 2019, 15, e1007740. [CrossRef]
63. Ebrahimi-Zarandi, M.; Riseh, R.S.; Tarkka, M.T. Actinobacteria as Effective Biocontrol Agents against Plant Pathogens, an
Overview on Their Role in Eliciting Plant Defense. Microorganisms 2022, 10, 1739. [CrossRef]
64. Inbaraj, M.P. Plant-Microbe Interactions in Alleviating Abiotic Stress—A Mini Review. Front. Agron. 2021, 3, 667903. [CrossRef]
65. Munir, N.; Hanif, M.; Abideen, Z.; Sohail, M.; El-Keblawy, A.; Radicetti, E.; Mancinelli, R.; Haider, G. Mechanisms and Strategies
of Plant Microbiome Interactions to Mitigate Abiotic Stresses. Agronomy 2022, 12, 2069. [CrossRef]
66. Poria, V.; D˛ebiec-Andrzejewska, K.; Fiodor, A.; Lyzohub, M.; Ajijah, N.; Singh, S.; Pranaw, K. Plant Growth-Promoting Bacteria
(PGPB) integrated phytotechnology: A sustainable approach for remediation of marginal lands. Front. Plant Sci. 2022, 13, 999866.
[CrossRef] [PubMed]
67. Ren, X.-M.; Guo, S.-J.; Tian, W.; Chen, Y.; Han, H.; Chen, E.; Li, B.-L.; Li, Y.-Y.; Chen, Z.-J. Effects of Plant Growth-Promoting
Bacteria (PGPB) Inoculation on the Growth, Antioxidant Activity, Cu Uptake, and Bacterial Community Structure of Rape
(Brassica napus L.) Grown in Cu-Contaminated Agricultural Soil. Front. Microbiol. 2019, 10, 1455. [CrossRef] [PubMed]
68. Hamid, N.W.A.; Nadarajah, K. Microbe Related Chemical Signalling and Its Application in Agriculture. Int. J. Mol. Sci. 2022, 23,
8998. [CrossRef]
69. El-Saadony, M.T.; Saad, A.M.; Soliman, S.M.; Salem, H.M.; Ahmed, A.I.; Mahmood, M.; El-Tahan, A.M.; Ebrahim, A.A.M.; El-
Mageed, T.A.A.; Negm, S.H.; et al. Plant growth-promoting microorganisms as biocontrol agents of plant diseases: Mechanisms,
challenges and future perspectives. Front. Plant Sci. 2022, 13, 923880. [CrossRef]
70. Olanrewaju, O.S.; Glick, B.R.; Babalola, O.O. Mechanisms of action of plant growth promoting bacteria. World J. Microbiol.
Biotechnol. 2017, 33, 197. [CrossRef]
71. Dong, O.X.; Ronald, P.C. Genetic Engineering for Disease Resistance in Plants: Recent Progress and Future Perspectives. Plant
Physiol. 2019, 180, 26. [CrossRef]
72. Shah, A.; Nazari, M.; Antar, M.; Msimbira, L.A.; Naamala, J.; Lyu, D.; Rabileh, M.; Zajonc, J.; Smith, D.L. PGPR in Agriculture: A
Sustainable Approach to Increasing Climate Change Resilience. Front. Sustain. Food Syst. 2021, 5, 667546. [CrossRef]
73. Singh, J.; Saini, D.K.; Kashyap, R.; Kumar, S.; Chopra, Y.; Sandhu, K.S.; Goraya, M.; Aggarwal, R. Omics technologies for agricul-
tural microbiology research. In Trends of Applied Microbiology for Sustainable Economy; Elsevier: Amsterdam, The Netherlands,
2022; pp. 343–394. [CrossRef]
74. Nadarajah, K.; Rahman, N.S.N.A. Plant–Microbe Interaction: Aboveground to Belowground, from the Good to the Bad. Int. J.
Mol. Sci. 2021, 22, 10388. [CrossRef]
75. Verma, V.; Ravindran, P.; Kumar, P.P. Plant hormone-mediated regulation of stress responses. BMC Plant Biol. 2016, 16, 86.
[CrossRef]
76. Cohen, A.C.; Bottini, R.; Pontin, M.; Berli, F.J.; Moreno, D.; Boccanlandro, H.; Travaglia, C.N.; Piccoli, P.N. Azospirillum brasilense
ameliorates the response of Arabidopsis thaliana to drought mainly via enhancement of ABA levels. Physiol. Plant. 2015, 153, 79–90.
[CrossRef]
77. Kumawat, K.C.; Sharma, B.; Nagpal, S.; Kumar, A.; Tiwari, S.; Nair, R.M. Plant growth-promoting rhizobacteria: Salt stress
alleviators to improve crop productivity for sustainable agriculture development. Front. Plant Sci. 2022, 13, 1101862. [CrossRef]
78. Morcillo, R.J.L.; Manzanera, M. The Effects of Plant-Associated Bacterial Exopolysaccharides on Plant Abiotic Stress Tolerance.
Metabolites 2021, 11, 337. [CrossRef]
79. Vacheron, J.; Desbrosses, G.; Bouffaud, M.-L.; Touraine, B.; Moënne-Loccoz, Y.; Muller, D.; Legendre, L.; Wisniewski-Dyé, F.;
Prigent-Combaret, C. Plant growth-promoting rhizobacteria and root system functioning. Front. Plant Sci. 2013, 4, 356. [CrossRef]
80. Bala, S.; Garg, D.; Thirumalesh, B.V.; Sharma, M.; Sridhar, K.; Inbaraj, B.S.; Tripathi, M. Recent Strategies for Bioremediation of
Emerging Pollutants: A Review for a Green and Sustainable Environment. Toxics 2022, 10, 484. [CrossRef]
81. Gopalakrishnan, S.; Sathya, A.; Vijayabharathi, R.; Varshney, R.K.; Gowda, C.L.; Krishnamurthy, L. Plant growth promoting
rhizobia: Challenges and opportunities. 3 Biotech 2015, 5, 355. [CrossRef]
82. Tariq, M.R.; Shaheen, F.; Mustafa, S.; Ali, S.; Fatima, A.; Shafiq, M.; Safdar, W.; Sheas, M.N.; Hameed, A.; Nasir, M.A. Phosphate
solubilizing microorganisms isolated from medicinal plants improve growth of mint. PeerJ 2022, 10, e13782. [CrossRef]
83. Nadarajah, K.; Kumar, I.S. Molecular Microbial Biodiversity Assessment in the Mycorrhizosphere. In Mycorrhizosphere and
Pedogenesis; Springer: Singapore, 2019; pp. 401–420. [CrossRef]
Plants 2023, 12, 2307 20 of 25

84. Pérez-Izquierdo, L.; Zabal-Aguirre, M.; González-Martínez, S.C.; Buée, M.; Verdú, M.; Rincón, A.; Goberna, M. Plant intraspecific
variation modulates nutrient cycling through its below ground rhizospheric microbiome. J. Ecol. 2019, 107, 1594–1605. [CrossRef]
85. Lumibao, C.Y.; Bernik, B.M.; Formel, S.K.; Kandalepas, D.; Mighell, K.L.; Pardue, J.; Van Bael, S.A.; Blum, M.J. Rhizosphere
microbial communities reflect genotypic and trait variation in a salt marsh ecosystem engineer. Am. J. Bot. 2020, 107, 941–949.
[CrossRef]
86. Dubey, A.; Malla, M.A.; Khan, F.; Chowdhary, K.; Yadav, S.; Kumar, A.; Sharma, S.; Khare, P.K.; Khan, M.L. Soil microbiome: A
key player for conservation of soil health under changing climate. Biodivers. Conserv. 2019, 28, 2405–2429. [CrossRef]
87. Dubey, A.; Kumar, A.; Abd_Allah, E.F.; Hashem, A.; Khan, M.L. Growing more with less: Breeding and developing drought
resilient soybean to improve food security. Ecol. Indic. 2019, 105, 425–437. [CrossRef]
88. Lakshmanan, V.; Selvaraj, G.; Bais, H.P. Functional Soil Microbiome: Belowground Solutions to an Aboveground Problem. Plant
Physiol. 2014, 166, 689–700. [CrossRef] [PubMed]
89. Mendes, R.; Garbeva, P.; Raaijmakers, J.M. The rhizosphere microbiome: Significance of plant beneficial, plant pathogenic, and
human pathogenic microorganisms. FEMS Microbiol. Rev. 2013, 37, 634–663. [CrossRef] [PubMed]
90. Yang, J.; Kloepper, J.W.; Ryu, C.-M. Rhizosphere bacteria help plants tolerate abiotic stress. Trends Plant Sci. 2009, 14, 1–4.
[CrossRef] [PubMed]
91. Lundberg, D.S.; Yourstone, S.; Mieczkowski, P.; Jones, C.D.; Dangl, J.L. Practical innovations for high-throughput amplicon
sequencing. Nat. Methods 2013, 10, 999–1002. [CrossRef]
92. Igiehon, N.; Babalola, O. Rhizosphere Microbiome Modulators: Contributions of Nitrogen Fixing Bacteria towards Sustainable
Agriculture. Int. J. Environ. Res. Public Health 2018, 15, 574. [CrossRef]
93. Khalifa, A.Y.Z.; Alsyeeh, A.-M.; Almalki, M.A.; Saleh, F.A. Characterization of the plant growth promoting bacterium, Enterobacter
cloacae MSR1, isolated from roots of non-nodulating Medicago sativa. Saudi J. Biol. Sci. 2016, 23, 79–86. [CrossRef]
94. Mishra, J.; Arora, N.K. Bioformulations for Plant Growth Promotion and Combating Phytopathogens: A Sustainable Approach.
In Bioformulations: For Sustainable Agriculture; Springer: New Delhi, India, 2016; pp. 3–33. [CrossRef]
95. García-Fraile, P.; Menéndez, E.; Rivas, R. Role of bacterial biofertilizers in agriculture and forestry. AIMS Bioeng. 2015, 2, 183–205.
[CrossRef]
96. Khan, N.; Bano, A.; Rahman, M.A.; Guo, J.; Kang, Z.; Babar, M.A. Comparative physiological and metabolic analysis reveals a
complex mechanism involved in drought tolerance in Chickpea (Cicer arietinum L.) induced by PGPR and PGRs. Sci. Rep. 2019, 9,
2097. [CrossRef]
97. Hashem, A.; Kumar, A.; Al-Dbass, A.M.; Alqarawi, A.A.; Al-Arjani, A.-B.F.; Singh, G.; Farooq, M.; Abd_Allah, E.F. Arbuscular
mycorrhizal fungi and biochar improves drought tolerance in chickpea. Saudi J. Biol. Sci. 2019, 26, 614–624. [CrossRef]
98. Wang, Q.; Coleman, J.J. CRISPR/Cas9-mediated endogenous gene tagging in Fusarium oxysporum. Fungal Genet. Biol. 2019, 126,
17–24. [CrossRef]
99. Moreira, Z.P.M.; Chen, M.Y.; Ortuno, D.L.Y.; Haney, C.H. Engineering plant microbiomes by integrating eco-evolutionary
principles into current strategies. Curr. Opin. Plant Biol. 2023, 71, 102316. [CrossRef]
100. Hayat, R.; Ahmed, I.; Sheirdil, R.A. An Overview of Plant Growth Promoting Rhizobacteria (PGPR) for Sustainable Agriculture.
In Crop Production for Agricultural Improvement; Springer: Dordrecht, The Netherlands, 2012; pp. 557–579. [CrossRef]
101. Kour, D.; Rana, K.L.; Yadav, N.; Yadav, A.N.; Kumar, A.; Meena, V.S.; Singh, B.; Chauhan, V.S.; Dhaliwal, H.S.; Saxena,
A.K. Rhizospheric Microbiomes: Biodiversity, Mechanisms of Plant Growth Promotion, and Biotechnological Applications for
Sustainable Agriculture. In Plant Growth Promoting Rhizobacteria for Agricultural Sustainability; Springer: Singapore, 2019; pp.
19–65. [CrossRef]
102. Caulier, S.; Nannan, C.; Gillis, A.; Licciardi, F.; Bragard, C.; Mahillon, J. Overview of the Antimicrobial Compounds Produced by
Members of the Bacillus subtilis Group. Front. Microbiol. 2019, 10, 302. [CrossRef]
103. Hashem, A.; Abd_Allah, E.F.; Alqarawi, A.A.; Radhakrishnan, R.; Kumar, A. Plant defense approach of Bacillus subtilis (BERA 71)
against Macrophomina phaseolina (Tassi) Goid in mung bean. J. Plant Interact. 2017, 12, 390–401. [CrossRef]
104. Tsurumaru, H.; Okubo, T.; Okazaki, K.; Hashimoto, M.; Kakizaki, K.; Hanzawa, E.; Takahashi, H.; Asanome, N.; Tanaka, F.;
Sekiyama, Y.; et al. Metagenomic Analysis of the Bacterial Community Associated with the Taproot of Sugar Beet. Microbes
Environ. 2015, 30, 63–69. [CrossRef]
105. Massart, S.; Martinez-Medina, M.; Jijakli, M.H. Biological control in the microbiome era: Challenges and opportunities. Biol.
Control. 2015, 89, 98–108. [CrossRef]
106. Elias, F.; Woyessa, D.; Muleta, D. Phosphate Solubilization Potential of Rhizosphere Fungi Isolated from Plants in Jimma Zone,
Southwest Ethiopia. Int. J. Microbiol. 2016, 2016, 5472601. [CrossRef]
107. Gupta, A.; Sharma, A.; Pathak, R.; Kumar, A.; Sharma, S. Solid State Fermentation of Non-Edible Oil Seed Cakes for Production
of Proteases and Cellulases and Degradation of Anti Nutritional Factors. J. Food Biotechnol. Res. 2018, 2, 1–6.
108. Zamioudis, C.; Pieterse, C.M.J. Modulation of Host Immunity by Beneficial Microbes. Mol. Plant Microbe Interact. 2012, 25,
139–150. [CrossRef]
109. De Vleesschauwer, D.; Höfte, M. Rhizobacteria-Induced Systemic Resistance. Adv. Bot. Res. 2009, 51, 223–281. [CrossRef]
110. Chowdhury, F.T.; Islam, M.R.; Islam, M.R.; Khan, H. Diversity of Plant Endophytic Volatile Organic Compound (VOC) and Their
Potential Applications. In Endophytes and Secondary Metabolites; Springer International Publishing: Cham, Switzerland, 2018; pp.
1–27.
Plants 2023, 12, 2307 21 of 25

111. Farag, M.A.; Zhang, H.; Ryu, C.-M. Dynamic Chemical Communication between Plants and Bacteria through Airborne Signals:
Induced Resistance by Bacterial Volatiles. J. Chem. Ecol. 2013, 39, 1007–1018. [CrossRef] [PubMed]
112. Rajesh, P.; Rai, V.R. Quorum quenching activity in cell-free lysate of endophytic bacteria isolated from Pterocarpus santalinus Linn.,
and its effect on quorum sensing regulated biofilm in Pseudomonas aeruginosa PAO1. Microbiol. Res. 2014, 169, 561–569. [CrossRef]
[PubMed]
113. Cordovez, V.; Dini-Andreote, F.; Carrión, V.J.; Raaijmakers, J.M. Ecology and Evolution of Plant Microbiomes. Annu. Rev.
Microbiol. 2019, 73, 69–88. [CrossRef] [PubMed]
114. Fierer, N.; Ferrenberg, S.; Flores, G.E.; González, A.; Kueneman, J.; Legg, T.; Lynch, R.C.; McDonald, D.; Mihaljevic, J.R.; O’Neill,
S.P.; et al. From Animalcules to an Ecosystem: Application of Ecological Concepts to the Human Microbiome. Annu. Rev. Ecol.
Evol. Syst. 2012, 43, 137–155. [CrossRef]
115. Miller, E.T.; Svanbäck, R.; Bohannan, B.J. Microbiomes as Metacommunities: Understanding Host-Associated Microbes through
Metacommunity Ecology. Trends Ecol. Evol. 2018, 33, 926–935. [CrossRef]
116. Yuan, H.; Li, T.; Li, H.; Wang, C.; Li, L.; Lin, X.; Lin, S. Diversity Distribution, Driving Factors and Assembly Mechanisms of
Free-Living and Particle-Associated Bacterial Communities at a Subtropical Marginal Sea. Microorganisms 2021, 9, 2445. [CrossRef]
117. Wei, Z.; Gu, Y.; Friman, V.-P.; Kowalchuk, G.A.; Xu, Y.; Shen, Q.; Jousset, A. Initial soil microbiome composition and functioning
predetermine future plant health. Sci. Adv. 2019, 5, eaaw0759. [CrossRef]
118. Van Geel, M.; Aavik, T.; Ceulemans, T.; Träger, S.; Mergeay, J.; Peeters, G.; van Acker, K.; Zobel, M.; Koorem, K.; Honnay, O. The
role of genetic diversity and arbuscular mycorrhizal fungal diversity in population recovery of the semi-natural grassland plant
species Succisa pratensis. BMC Ecol. Evol. 2021, 21, 200. [CrossRef]
119. Ramírez-Flores, M.R.; Perez-Limon, S.; Li, M.; Barrales-Gamez, B.; Albinsky, D.; Paszkowski, U.; Olalde-Portugal, V.; Sawers, R.J.
The genetic architecture of host response reveals the importance of arbuscular mycorrhizae to maize cultivation. Elife 2020, 9,
e61701. [CrossRef]
120. Trivedi, P.; Schenk, P.M.; Wallenstein, M.D.; Singh, B.K. Tiny Microbes, Big Yields: Enhancing food crop production with biological
solutions. Microb. Biotechnol. 2017, 10, 999. [CrossRef]
121. Yin, C.; Hulbert, S.H.; Schroeder, K.L.; Mavrodi, O.; Mavrodi, D.; Dhingra, A.; Schillinger, W.F.; Paulitz, T.C. Role of Bacterial
Communities in the Natural Suppression of Rhizoctonia solani Bare Patch Disease of Wheat (Triticum aestivum L.). Appl. Environ.
Microbiol. 2013, 79, 7428. [CrossRef]
122. Lebedev, V.; Lebedeva, T.; Tikhonova, E.; Shestibratov, K. Assessing Impacts of Transgenic Plants on Soil Using Functional
Indicators: Twenty Years of Research and Perspectives. Plants 2022, 11, 2439. [CrossRef]
123. Gévaudant, F.; Duby, G.; von Stedingk, E.; Zhao, R.; Morsomme, P.; Boutry, M. Expression of a Constitutively Activated Plasma
Membrane H+ -ATPase Alters Plant Development and Increases Salt Tolerance. Plant Physiol. 2007, 144, 1763–1776. [CrossRef]
124. Cameron, D.D.; Neal, A.L.; Van Wees, S.C.; Ton, J. Mycorrhiza-induced resistance: More than the sum of its parts? Trends Plant Sci.
2013, 18, 545. [CrossRef]
125. Ellouze, W.; Hamel, C.; Vujanovic, V.; Gan, Y.; Bouzid, S.; St-Arnaud, M. Chickpea genotypes shape the soil microbiome and
affect the establishment of the subsequent durum wheat crop in the semiarid North American Great Plains. Soil Biol. Biochem.
2013, 63, 129–141. [CrossRef]
126. Gu, Y.; Dong, K.; Geisen, S.; Yang, W.; Yan, Y.; Gu, D.; Liu, N.; Borisjuk, N.; Luo, Y.; Friman, V.-P. The effect of microbial inoculant
origin on the rhizosphere bacterial community composition and plant growth-promotion. Plant Soil 2020, 452, 105–117. [CrossRef]
127. Morella, N.M.; Weng, F.C.-H.; Joubert, P.M.; Metcalf, C.J.E.; Lindow, S.; Koskella, B. Successive passaging of a plant-associated
microbiome reveals robust habitat and host genotype-dependent selection. Proc. Natl. Acad. Sci. USA 2020, 117, 1148–1159.
[CrossRef]
128. Arias-Sánchez, F.I.; Vessman, B.; Mitri, S. Artificially selecting microbial communities: If we can breed dogs, why not microbiomes?
PLoS Biol. 2019, 17, e3000356. [CrossRef]
129. Thakur, N.; Nigam, M.; Mann, N.A.; Gupta, S.; Hussain, C.M.; Shukla, S.K.; Shah, A.A.; Casini, R.; Elansary, H.O.; Khan, S.A.
Host-mediated gene engineering and microbiome-based technology optimization for sustainable agriculture and environment.
Funct. Integr. Genom. 2023, 23, 57. [CrossRef]
130. Trivedi, P.; Batista, B.D.; Bazany, K.E.; Singh, B.K. Plant–microbiome interactions under a changing world: Responses, conse-
quences and perspectives. New Phytol. 2022, 234, 1951–1959. [CrossRef]
131. Kazan, K.; Lyons, R. The link between flowering time and stress tolerance. J. Exp. Bot. 2016, 67, 47–60. [CrossRef] [PubMed]
132. Müller, D.B.; Vogel, C.; Bai, Y.; Vorholt, J.A. The Plant Microbiota: Systems-Level Insights and Perspectives. Annu. Rev. Genet.
2016, 50, 211–234. [CrossRef] [PubMed]
133. Kavamura, V.N.; Robinson, R.J.; Hughes, D.; Clark, I.; Rossmann, M.; de Melo, I.S.; Hirsch, P.R.; Mendes, R.; Mauchline, T.H.
Wheat dwarfing influences selection of the rhizosphere microbiome. Sci. Rep. 2020, 10, 1452. [CrossRef] [PubMed]
134. Taye, Z.M.; Helgason, B.L.; Bell, J.K.; Norris, C.E.; Vail, S.; Robinson, S.J.; Parkin, I.A.P.; Arcand, M.; Mamet, S.; Links, M.G.; et al.
Core and Differentially Abundant Bacterial Taxa in the Rhizosphere of Field Grown Brassica napus Genotypes: Implications for
Canola Breeding. Front. Microbiol. 2020, 10, 3007. [CrossRef]
135. Wohor, O.Z.; Rispail, N.; Ojiewo, C.O.; Rubiales, D. Pea Breeding for Resistance to Rhizospheric Pathogens. Plants 2022, 11, 2664.
[CrossRef]
Plants 2023, 12, 2307 22 of 25

136. Busby, P.E.; Soman, C.; Wagner, M.; Friesen, M.; Kremer, J.; Bennett, A.; Morsy, M.; Eisen, J.A.; Leach, J.E.; Dangl, J.L. Research
priorities for harnessing plant microbiomes in sustainable agriculture. PLoS Biol. 2017, 15, e2001793. [CrossRef]
137. Wille, L.; Messmer, M.M.; Studer, B.; Hohmann, P. Insights to plant-microbe interactions provide opportunities to improve
resistance breeding against root diseases in grain legumes. Plant Cell Environ. 2019, 42, 20–40. [CrossRef]
138. Deng, S.; Caddell, D.F.; Xu, G.; Dahlen, L.; Washington, L.; Yang, J.; Coleman-Derr, D. Genome wide association study reveals
plant loci controlling heritability of the rhizosphere microbiome. ISME J. 2021, 15, 3181–3194. [CrossRef]
139. Wang, Y.; Wang, X.; Sun, S.; Jin, C.; Su, J.; Wei, J.; Luo, X.; Wen, J.; Wei, T.; Sahu, S.K.; et al. GWAS, MWAS and mGWAS provide
insights into precision agriculture based on genotype-dependent microbial effects in foxtail millet. Nat. Commun. 2022, 13, 5913.
[CrossRef]
140. Jochum, M.D.; McWilliams, K.L.; Pierson, E.A.; Jo, Y.-K. Host-mediated microbiome engineering (HMME) of drought tolerance in
the wheat rhizosphere. PLoS ONE 2019, 14, e0225933. [CrossRef]
141. Awany, D.; Allali, I.; Dalvie, S.; Hemmings, S.; Mwaikono, K.S.; Thomford, N.E.; Gomez, A.; Mulder, N.; Chimusa, E.R. Host and
Microbiome Genome-Wide Association Studies: Current State and Challenges. Front. Genet. 2019, 9, 637. [CrossRef]
142. Li, Y.; Liang, J.; Deng, B.; Jiang, Y.; Zhu, J.; Chen, L.; Li, M.; Li, J. Applications and Prospects of CRISPR/Cas9-Mediated Base
Editing in Plant Breeding. Curr. Issues Mol. Biol. 2023, 45, 918–935. [CrossRef]
143. Geddes, B.A.; Paramasivan, P.; Joffrin, A.; Thompson, A.L.; Christensen, K.; Jorrin, B.; Brett, P.; Conway, S.J.; Oldroyd, G.E.D.;
Poole, P.S. Engineering transkingdom signalling in plants to control gene expression in rhizosphere bacteria. Nat. Commun. 2019,
10, 3430. [CrossRef]
144. Lebeis, S.L.; Paredes, S.H.; Lundberg, D.S.; Breakfield, N.; Gehring, J.; McDonald, M.; Malfatti, S.; Del Rio, T.G.; Jones, C.D.;
Tringe, S.G.; et al. Plant Microbiome. Salicylic acid modulates colonization of the root microbiome by specific bacterial taxa.
Science 2015, 349, 860–864. [CrossRef]
145. Palojärvi, A.; Kellock, M.; Parikka, P.; Jauhiainen, L.; Alakukku, L. Tillage System and Crop Sequence Affect Soil Disease
Suppressiveness and Carbon Status in Boreal Climate. Front. Microbiol. 2020, 11, 534786. [CrossRef]
146. De Corato, U. Soil microbiota manipulation and its role in suppressing soil-borne plant pathogens in organic farming systems
under the light of microbiome-assisted strategies. Chem. Biol. Technol. Agric. 2020, 7, 17. [CrossRef]
147. Hartman, K.; Van Der Heijden, M.G.A.; Wittwer, R.A.; Banerjee, S.; Walser, J.-C.; Schlaeppi, K. Correction to: Cropping practices
manipulate abundance patterns of root and soil microbiome members paving the way to smart farming. Microbiome 2020, 8, 14.
[CrossRef]
148. de Souza, R.S.C.; Armanhi, J.S.L.; Damasceno, N.D.B.; Imperial, J.; Arruda, P. Genome Sequences of a Plant Beneficial Synthetic
Bacterial Community Reveal Genetic Features for Successful Plant Colonization. Front. Microbiol. 2019, 10, 1779. [CrossRef]
149. Leonard, S.P.; Powell, J.E.; Perutka, J.; Geng, P.; Heckmann, L.C.; Horak, R.D.; Davies, B.W.; Ellington, A.D.; Barrick, J.E.; Moran,
N.A. Engineered symbionts activate honey bee immunity and limit pathogens. Science 2020, 367, 573. [CrossRef]
150. Goold, H.D.; Wright, P.; Hailstones, D. Emerging Opportunities for Synthetic Biology in Agriculture. Genes 2018, 9, 341. [CrossRef]
151. Kemal, R.; Islamiah, P.W.N.; Lusiany, T. Synthetic Biology for BioControl: A Mini-Review. In Proceedings of the 6th International
Conference on Global Resource Conservation, Malang, Indonesia, 1–2 December 2015; Volume 6, pp. 92–94.
152. Ryu, M.-H.; Zhang, J.; Toth, T.; Khokhani, D.; Geddes, B.A.; Mus, F.; Garcia-Costas, A.; Peters, J.W.; Poole, P.S.; Ané, J.-M.; et al.
Control of nitrogen fixation in bacteria that associate with cereals. Nat. Microbiol. 2019, 5, 314–330. [CrossRef] [PubMed]
153. Goyal, R.K.; Schmidt, M.A.; Hynes, M.F. Molecular Biology in the Improvement of Biological Nitrogen Fixation by Rhizobia and
Extending the Scope to Cereals. Microorganisms 2021, 9, 125. [CrossRef] [PubMed]
154. Kumar, S.; Diksha; Sindhu, S.S.; Kumar, R. Biofertilizers: An ecofriendly technology for nutrient recycling and environmental
sustainability. Curr. Res. Microb. Sci. 2021, 3, 100094. [CrossRef] [PubMed]
155. Mahmud, K.; Makaju, S.; Ibrahim, R.; Missaoui, A. Current Progress in Nitrogen Fixing Plants and Microbiome Research. Plants
2020, 9, 97. [CrossRef] [PubMed]
156. Prabhukarthikeyan, S.R.; Parameswaran, C.; Keerthana, U.; Teli, B.; Jagannadham, P.T.K.; Cayalvizhi, B.; Panneerselvam, P.;
Senapati, A.; Nagendran, K.; Kumari, S.; et al. Understanding the Plant-microbe Interactions in CRISPR/Cas9 Era: Indeed a
Sprinting Start in Marathon. Curr. Genom. 2020, 21, 429–443. [CrossRef] [PubMed]
157. Glandorf, D.C.M. Re-evaluation of biosafety questions on genetically modified biocontrol bacteria. Eur. J. Plant Pathol. 2019, 154,
43–51. [CrossRef]
158. Nakamura, M.; Okamura, Y.; Iwai, H. Plasmid-based and -free methods using CRISPR/Cas9 system for replacement of targeted
genes in Colletotrichum sansevieriae. Sci. Rep. 2019, 9, 18947. [CrossRef]
159. Ferrara, M.; Haidukowski, M.; Logrieco, A.F.; Leslie, J.F.; Mulè, G. A CRISPR-Cas9 System for Genome Editing of Fusarium
proliferatum. Sci. Rep. 2019, 9, 19836. [CrossRef]
160. Fang, C.; Chen, X. Potential biocontrol efficacy of Trichoderma atroviride with cellulase expression regulator ace1 gene knock-out. 3
Biotech 2018, 8, 302. [CrossRef]
161. Xin, W.; Wang, J.; Li, J.; Zhao, H.; Liu, H.; Zheng, H.; Yang, L.; Wang, C.; Yang, F.; Chen, J.; et al. Candidate Gene Analysis for
Nitrogen Absorption and Utilization in Japonica Rice at the Seedling Stage Based on a Genome-Wide Association Study. Front.
Plant Sci. 2021, 12, 670861. [CrossRef]
162. Mohapatra, S.; Mishra, S.S.; Bhalla, P.; Thatoi, H. Engineering grass biomass for sustainable and enhanced bioethanol production.
Planta 2019, 250, 395–412. [CrossRef]
Plants 2023, 12, 2307 23 of 25

163. Luo, M.L.; Leenay, R.T.; Beisel, C.L. Current and future prospects for CRISPR-based tools in bacteria. Biotechnol. Bioeng. 2016, 113,
930–943. [CrossRef]
164. Wheatley, R.M.; MacLean, R.C. CRISPR-Cas systems restrict horizontal gene transfer in Pseudomonas aeruginosa. ISME J. 2021, 15,
1420–1433. [CrossRef]
165. Kim, S.-M.; Reinke, R.F. A novel resistance gene for bacterial blight in rice, Xa43(t) identified by GWAS, confirmed by QTL
mapping using a bi-parental population. PLoS ONE 2019, 14, e0211775. [CrossRef]
166. Xiao, Y.; Liu, H.; Wu, L.; Warburton, M.; Yan, J. Genome-wide Association Studies in Maize: Praise and Stargaze. Mol. Plant 2017,
10, 359–374. [CrossRef]
167. Raboin, L.-M.; Ballini, E.; Tharreau, D.; Ramanantsoanirina, A.; Frouin, J.; Courtois, B.; Ahmadi, N. Association mapping of
resistance to rice blast in upland field conditions. Rice 2016, 9, 59. [CrossRef]
168. Zhu, D.; Kang, H.; Li, Z.; Liu, M.; Zhu, X.; Wang, Y.; Wang, D.; Wang, Z.; Liu, W.; Wang, G.-L. A Genome-Wide Association Study
of Field Resistance to Magnaporthe oryzae in Rice. Rice 2016, 9, 44. [CrossRef]
169. Mgonja, E.M.; Balimponya, E.G.; Kang, H.; Bellizzi, M.; Park, C.H.; Li, Y.; Mabagala, R.; Sneller, C.; Correll, J.; Opiyo, S.; et al.
Genome-Wide Association Mapping of Rice Resistance Genes against Magnaporthe oryzae Isolates from Four African Countries.
Phytopathology 2016, 106, 1359–1365. [CrossRef]
170. Bartoli, C.; Roux, F. Genome-Wide Association Studies in Plant Pathosystems: Toward an Ecological Genomics Approach. Front.
Plant Sci. 2017, 8, 763. [CrossRef]
171. Oladzad, A.; González, A.; Macchiavelli, R.; De Jensen, C.E.; Beaver, J.; Porch, T.; McClean, P. Genetic Factors Associated with
Nodulation and Nitrogen Derived from Atmosphere in a Middle American Common Bean Panel. Front. Plant Sci. 2020, 11,
576078. [CrossRef]
172. Horton, M.W.; Bodenhausen, N.; Beilsmith, K.; Meng, D.; Muegge, B.D.; Subramanian, S.; Vetter, M.M.; Vilhjálmsson, B.J.;
Nordborg, M.; Gordon, J.I.; et al. Genome-wide association study of Arabidopsis thaliana leaf microbial community. Nat. Commun.
2014, 5, 6320. [CrossRef] [PubMed]
173. Bartram, A.K.; Lynch, M.D.J.; Stearns, J.C.; Moreno-Hagelsieb, G.; Neufeld, J.D. Generation of Multimillion-Sequence 16S rRNA
Gene Libraries from Complex Microbial Communities by Assembling Paired-End Illumina Reads. Appl. Environ. Microbiol. 2011,
77, 3846–3852. [CrossRef] [PubMed]
174. Caporaso, J.G.; Lauber, C.L.; Walters, W.A.; Berg-Lyons, D.; Huntley, J.; Fierer, N.; Owens, S.M.; Betley, J.; Fraser, L.; Bauer,
M.; et al. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. ISME J. 2012, 6,
1621–1624. [CrossRef] [PubMed]
175. Huttenhower, C.; Gevers, D.; Knight, R.; Abubucker, S.; Badger, J.H.; Chinwalla, A.T.; Creasy, H.H.; Earl, A.M.; FitzGerald, M.G.;
Fulton, R.S.; et al. Structure, function and diversity of the healthy human microbiome. Nature 2012, 486, 207–214. [CrossRef]
176. Krishna, B.M.; Khan, M.A.; Khan, S.T. Next-Generation Sequencing (NGS) Platforms: An Exciting Era of Genome Sequence
Analysis. In Microbial Genomics in Sustainable Agroecosystems; Springer: Singapore, 2019; pp. 89–109. [CrossRef]
177. Zhang, C.; Li, X.; Yin, L.; Liu, C.; Zou, H.; Wu, Z.; Zhang, Z. Analysis of the complete genome sequence of Brevibacterium
frigoritolerans ZB201705 isolated from drought- and salt-stressed rhizosphere soil of maize. Ann. Microbiol. 2019, 69, 1489–1496.
[CrossRef]
178. Duan, J.; Jiang, W.; Cheng, Z.; Heikkila, J.J.; Glick, B.R. The Complete Genome Sequence of the Plant Growth-Promoting Bacterium
pseudomonas sp. UW4. PLoS ONE 2013, 8, e58640. [CrossRef]
179. Johnston-Monje, D.; Mejia, J.L. Botanical microbiomes on the cheap: Inexpensive molecular fingerprinting methods to study
plant-associated communities of bacteria and fungi. Appl. Plant Sci. 2020, 8, e11334. [CrossRef]
180. Afzal, I.; Shinwari, Z.K.; Sikandar, S.; Shahzad, S. Plant beneficial endophytic bacteria: Mechanisms, diversity, host range and
genetic determinants. Microbiol. Res. 2019, 221, 36–49. [CrossRef]
181. Orozco-Mosqueda, M.D.C.; Santoyo, G. Plant-microbial endophytes interactions: Scrutinizing their beneficial mechanisms from
genomic explorations. Curr. Plant Biol. 2021, 25, 100189. [CrossRef]
182. Turner, T.R.; Ramakrishnan, K.; Walshaw, J.; Heavens, D.; Alston, M.; Swarbreck, D.; Osbourn, A.; Grant, A.; Poole, P.S.
Comparative metatranscriptomics reveals kingdom level changes in the rhizosphere microbiome of plants. ISME J. 2013, 7,
2248–2258. [CrossRef]
183. Chen, Y.; Yao, Z.; Sun, Y.; Wang, E.; Tian, C.; Sun, Y.; Liu, J.; Sun, C.; Tian, L. Current Studies of the Effects of Drought Stress on
Root Exudates and Rhizosphere Microbiomes of Crop Plant Species. Int. J. Mol. Sci. 2022, 23, 2374. [CrossRef]
184. Xie, J.; Dawwam, G.E.; Sehim, A.E.; Li, X.; Wu, J.; Chen, S.; Zhang, D. Drought Stress Triggers Shifts in the Root Microbial
Community and Alters Functional Categories in the Microbial Gene Pool. Front. Microbiol. 2021, 12, 744897. [CrossRef]
185. Srivastava, A.K.; Das, A.K.; Jagannadham, P.T.K.; Bora, P.; Ansari, F.A.; Bhate, R. Bioprospecting Microbiome for Soil and Plant
Health Management Amidst Huanglongbing Threat in Citrus: A Review. Front. Plant Sci. 2022, 13, 858842. [CrossRef]
186. Knief, C.; Delmotte, N.; Chaffron, S.; Stark, M.; Innerebner, G.; Wassmann, R.; von Mering, C.; Vorholt, J.A. Metaproteogenomic
analysis of microbial communities in the phyllosphere and rhizosphere of rice. ISME J. 2011, 6, 1378–1390. [CrossRef]
187. Huang, A.C.; Jiang, T.; Liu, Y.-X.; Bai, Y.-C.; Reed, J.; Qu, B.; Goossens, A.; Nützmann, H.-W.; Bai, Y.; Osbourn, A. A specialized
metabolic network selectively modulates Arabidopsis root microbiota. Science 2019, 364, aau6389. [CrossRef]
188. Hong, Y.; Zhou, Q.; Hao, Y.; Huang, A.C. Crafting the plant root metabolome for improved microbe-assisted stress resilience.
New Phytol. 2022, 234, 1945–1950. [CrossRef]
Plants 2023, 12, 2307 24 of 25

189. Muthuramalingam, P.; Jeyasri, R.; Rakkammal, K.; Satish, L.; Shamili, S.; Karthikeyan, A.; Valliammai, A.; Priya, A.; Selvaraj, A.;
Gowri, P.; et al. Multi-Omics and Integrative Approach towards Understanding Salinity Tolerance in Rice: A Review. Biology
2022, 11, 1022. [CrossRef]
190. Sirangelo, T.M.; Rogers, H.J.; Spadafora, N.D. Multi-Omic Approaches to Investigate Molecular Mechanisms in Peach Post-
Harvest Ripening. Agriculture 2022, 12, 553. [CrossRef]
191. Martínez-Hidalgo, P.; Maymon, M.; Pule-Meulenberg, F.; Hirsch, A.M. Engineering root microbiomes for healthier crops and soils
using beneficial, environmentally safe bacteria. Can. J. Microbiol. 2019, 65, 91–104. [CrossRef]
192. Land, M.; Hauser, L.; Jun, S.-R.; Nookaew, I.; Leuze, M.R.; Ahn, T.-H.; Karpinets, T.; Lund, O.; Kora, G.; Wassenaar, T.; et al.
Insights from 20 years of bacterial genome sequencing. Funct. Integr. Genom. 2015, 15, 141. [CrossRef]
193. Sarhan, M.S.; Hamza, M.A.; Youssef, H.H.; Patz, S.; Becker, M.; ElSawey, H.; Nemr, R.; Daanaa, H.-S.A.; Mourad, E.F.; Morsi, A.T.;
et al. Culturomics of the plant prokaryotic microbiome and the dawn of plant-based culture media—A review. J. Adv. Res. 2019,
19, 15–27. [CrossRef] [PubMed]
194. Fagen, J.R.; Leonard, M.T.; McCullough, C.M.; Edirisinghe, J.N.; Henry, C.S.; Davis, M.J.; Triplett, E.W. Comparative Genomics of
Cultured and Uncultured Strains Suggests Genes Essential for Free-Living Growth of Liberibacter. PLoS ONE 2014, 9, e84469.
[CrossRef] [PubMed]
195. Bai, Y.; Müller, D.B.; Srinivas, G.; Garrido-Oter, R.; Potthoff, E.; Rott, M.; Dombrowski, N.; Münch, P.C.; Spaepen, S.; Remus-
Emsermann, M.; et al. Functional overlap of the Arabidopsis leaf and root microbiota. Nature 2015, 528, 364–369. [CrossRef]
[PubMed]
196. Tosi, M.; Mitter, E.K.; Gaiero, J.; Dunfield, K.E. It takes three to tango: The importance of microbes, host plant, and soil
management to elucidate manipulation strategies for the plant microbiome. Can. J. Microbiol. 2020, 66, 413–433. [CrossRef]
[PubMed]
197. Glick, B.R. The enhancement of plant growth by free-living bacteria. Can. J. Microbiol. 1995, 41, 109–117. [CrossRef]
198. Kuan, K.B.; Othman, R.; Rahim, K.A.; Shamsuddin, Z.H. Plant Growth-Promoting Rhizobacteria Inoculation to Enhance
Vegetative Growth, Nitrogen Fixation and Nitrogen Remobilisation of Maize under Greenhouse Conditions. PLoS ONE 2016, 11,
e0152478. [CrossRef]
199. Vejan, P.; Abdullah, R.; Khadiran, T.; Ismail, S.; Nasrulhaq Boyce, A. Role of Plant Growth Promoting Rhizobacteria in Agricultural
Sustainability—A Review. Molecules 2016, 21, 573. [CrossRef]
200. Sarkar, S.; Kamke, A.; Ward, K.; Hartung, E.; Ran, Q.; Feehan, B.; Galliart, M.; Jumpponen, A.; Johnson, L.; Lee, S.T.M. Culturomics
of Andropogon gerardii rhizobiome revealed nitrogen transforming capabilities of stress-tolerant Pseudomonas under drought
conditions. bioRxiv 2022, 500515. [CrossRef]
201. Wang, Y.; Zhang, G.; Huang, Y.; Guo, M.; Song, J.; Zhang, T.; Long, Y.; Wang, B.; Liu, H. A Potential Biofertilizer—Siderophilic
Bacteria Isolated from the Rhizosphere of Paris polyphylla var. yunnanensis. Front. Microbiol. 2022, 13, 870413. [CrossRef]
202. Félix, J.D.F.; Sanchez-Juanes, F.; Silva, L.R.; Alves, G. Metagenomic and Culturomic Approaches for Blueberry Biofertilizer Design.
Biol. Life Sci. Forum 2021, 3, 49. [CrossRef]
203. Singh, R.P.; Shelke, G.M.; Kumar, A.; Jha, P.N. Biochemistry and genetics of ACC deaminase: A weapon to “stress ethylene”
produced in plants. Front. Microbiol. 2015, 6, 937. [CrossRef]
204. del Carmen Orozco-Mosqueda, M.; Glick, B.R.; Santoyo, G. ACC deaminase in plant growth-promoting bacteria (PGPB): An
efficient mechanism to counter salt stress in crops. Microbiol. Res. 2020, 235, 126439. [CrossRef]
205. Glick, B.R.; Penrose, D.M.; Li, J. A Model For the Lowering of Plant Ethylene Concentrations by Plant Growth-promoting Bacteria.
J. Theor. Biol. 1998, 190, 63–68. [CrossRef]
206. Chicca, I.; Becarelli, S.; Bernabei, G.; Siracusa, G.; Di Gregorio, S. Innovative Culturomic Approaches and Predictive Functional
Metagenomic Analysis: The Isolation of Hydrocarbonoclastic Bacteria with Plant Growth Promoting Capacity. Water 2022, 14,
142. [CrossRef]
207. Tsukanova, K.A.; Chebotar, V.K.; Meyer, J.J.M.; Bibikova, T.N. Effect of plant growth-promoting Rhizobacteria on plant hormone
homeostasis. S. Afr. J. Bot. 2017, 113, 91–102. [CrossRef]
208. Sahu, K.P.; Patel, A.; Kumar, M.; Sheoran, N.; Mehta, S.; Reddy, B.; Eke, P.; Prabhakaran, N.; Kumar, A. Integrated Metabarcoding
and Culturomic-Based Microbiome Profiling of Rice Phyllosphere Reveal Diverse and Functional Bacterial Communities for Blast
Disease Suppression. Front. Microbiol. 2021, 12, 780458. [CrossRef]
209. Haque, M.; Mosharaf, K.; Khatun, M.; Haque, A.; Biswas, S.; Islam, S.; Islam, M.; Shozib, H.B.; Miah, M.U.; Molla, A.H.; et al.
Biofilm Producing Rhizobacteria with Multiple Plant Growth-Promoting Traits Promote Growth of Tomato Under Water-Deficit
Stress. Front. Microbiol. 2020, 11, 542053. [CrossRef]
210. Flores-Duarte, N.J.; Pajuelo, E.; Mateos-Naranjo, E.; Navarro-Torre, S.; Rodríguez-Llorente, I.D.; Redondo-Gómez, S.; López,
J.A.C. A Culturomics-Based Bacterial Synthetic Community for Improving Resilience towards Arsenic and Heavy Metals in the
Nutraceutical Plant Mesembryanthemum crystallinum. Int. J. Mol. Sci. 2023, 24, 7003. [CrossRef]
211. Bouremani, N.; Cherif-Silini, H.; Silini, A.; Bouket, A.C.; Luptakova, L.; Alenezi, F.N.; Baranov, O.; Belbahri, L. Plant Growth-
Promoting Rhizobacteria (PGPR): A Rampart against the Adverse Effects of Drought Stress. Water 2023, 15, 418. [CrossRef]
212. Rijavec, T.; Lapanje, A. Hydrogen Cyanide in the Rhizosphere: Not Suppressing Plant Pathogens, but Rather Regulating
Availability of Phosphate. Front. Microbiol. 2016, 7, 1785. [CrossRef]
Plants 2023, 12, 2307 25 of 25

213. Santoyo, G.; Urtis-Flores, C.A.; Loeza-Lara, P.D.; Orozco-Mosqueda, M.D.C.; Glick, B.R. Rhizosphere Colonization Determinants
by Plant Growth-Promoting Rhizobacteria (PGPR). Biology 2021, 10, 475. [CrossRef] [PubMed]
214. Glick, B.R. Plant Growth-Promoting Bacteria: Mechanisms and Applications. Scientifica 2012, 2012, 963401. [CrossRef] [PubMed]
215. Saeed, Q.; Xiukang, W.; Haider, F.U.; Kučerik, J.; Mumtaz, M.Z.; Holatko, J.; Naseem, M.; Kintl, A.; Ejaz, M.; Naveed, M.; et al.
Rhizosphere Bacteria in Plant Growth Promotion, Biocontrol, and Bioremediation of Contaminated Sites: A Comprehensive
Review of Effects and Mechanisms. Int. J. Mol. Sci. 2021, 22, 10529. [CrossRef] [PubMed]
216. Ajijah, N.; Fiodor, A.; Pandey, A.K.; Rana, A.; Pranaw, K. Plant Growth-Promoting Bacteria (PGPB) with Biofilm-Forming Ability:
A Multifaceted Agent for Sustainable Agriculture. Diversity 2023, 15, 112. [CrossRef]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.