agriculture

Review
Beneficial Soil Microbiomes and Their Potential Role in Plant
Growth and Soil Fertility
Éva-Boglárka Vincze 1,2 , Annamária Becze 1,3 , Éva Laslo 2 and Gyöngyvér Mara 2, *

1 Faculty of Science, Doctoral School of Chemistry, University of Pécs, Vasvári Pál Street 4, 7622 Pécs, Hungary;
vinczeboglarka@uni.sapientia.ro (É.-B.V.); beczeannamaria@uni.sapientia.ro (A.B.)
2 Faculty of Economics, Socio-Human Sciences and Engineering, Sapientia Hungarian University of Transylvania,
Libertăţii Sq. 1, 530104 Miercurea Ciuc, Romania; lasloeva@uni.sapientia.ro
3 Department of Analytical Chemistry and Environmental Engineering, Faculty of Applied Chemistry and
Material Sciences, Politehnica University of Bucharest, 1-7 Polizu Street, 011061 Bucharest, Romania
* Correspondence: maragyongyver@uni.sapientia.ro

Abstract: The soil microbiome plays an important role in maintaining soil health, plant productivity,
and soil ecosystem services. Current molecular-based studies have shed light on the fact that
the soil microbiome has been quantitatively underestimated. In addition to metagenomic studies,
metaproteomics and metatranscriptomic studies that target the functional part of the microbiome are
becoming more common. These are important for a better understanding of the functional role of
the microbiome and for deciphering plant-microbe interactions. Free-living beneficial bacteria that
promote plant growth by colonizing plant roots are called plant growth-promoting rhizobacteria
(PGPRs). They exert their beneficial effects in different ways, either by facilitating the uptake
of nutrients and synthesizing particular compounds for plants or by preventing and protecting
plants from diseases. A better understanding of plant-microbe interactions in both natural and
agroecosystems will offer us a biotechnological tool for managing soil fertility and obtaining a
high-yield food production system.

Keywords: rhizosphere; microbiome; PGPR; ecological function; sustainable agriculture

Citation: Vincze, É.-B.; Becze, A.;

Laslo, É.; Mara, G. Beneficial Soil
Microbiomes and Their Potential Role 1. Introduction
in Plant Growth and Soil Fertility. The increased demand for agricultural products worldwide has been a consequence
Agriculture 2024, 14, 152. https:// of population growth. Increased production leads to topsoil depletion, reduced organic
doi.org/10.3390/agriculture14010152 matter content, and compromised soil ecological function. Soil ecological function is
Academic Editor: Ramakrishna
maintained by soil microbes, which confer stability to the soil environment and stability
Wusirika during disturbance.
Other issues related to inadequate land use include groundwater contamination,
Received: 22 December 2023 outbreaks of plant diseases, and air pollution. The need for sustainable and healthy food
Revised: 13 January 2024
is also reflected in a population that is sensitive to environmental problems. Therefore,
Accepted: 18 January 2024
eco-friendly strategies for sustainable agriculture are becoming increasingly popular.
Published: 20 January 2024
According to De Corato, sustainable agroecosystems are highly resilient, adaptive,
and diverse [1]. These issues are all related, with diversity conferring more adaptability,
and adaptability being a key component of resilience in agroecosystems. The diversity of
Copyright: © 2024 by the authors.
the soil microbiota, which plays a crucial role in nutrient recycling and soil formation, is
Licensee MDPI, Basel, Switzerland. therefore a key issue in sustainable agriculture. The main issue that has to be studied is
This article is an open access article not taxonomical but the functional diversity of soil bacteria. A better understanding of the
distributed under the terms and role of microbes in agroecosystem functioning in the framework of plant growth and soil
conditions of the Creative Commons fertility is key to sustainable agricultural production.
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).

Agriculture 2024, 14, 152. https://doi.org/10.3390/agriculture14010152 https://www.mdpi.com/journal/agriculture

Agriculture 2024, 14, 152 2 of 23

2. Soil Microbiome
2.1. Spatial Distribution of Soil Microbiome
Microorganisms are one of the most abundant living organisms on Earth, constituting
approximately 17% of the global biomass [2]. Soil is the most complex habitat that contains
a huge abundance of microbial life, which comprises approximately 4–5 × 1030 microbial
cells [3]. The soil microbiome is mainly comprised of soil bacteria, archaea, fungi and
viruses. Mendes et al. estimates that 108 –109 bacteria, 107 –108 viruses and 105 –106 fungal
cells are in one gram of soil [4]. Soil microbial communities provide ecosystem services
as nutrient recycling, carbon sequestration, water retention, plant growth promotion and
defense [5–7]. The focus on soil microbiota research has become noticeable due to its role in
the global carbon cycle and climate change, as well as in sustainable agriculture.
The diversity and abundance of microbes are affected by land-use patterns and soil
compartments. Agricultural ecosystems are more homogeneous than natural environments
due to lower plant diversity and frequent human disturbance. Two soil compartments can
be distinguished based on the strength of their relationship with the plant roots. Bulk soil
is defined as the part of the soil that is not or is loosely attached to the root, whereas the
attached part of the soil is considered rhizosphere soil. Bacterial community composition
also differs considerably between bulk and rhizosphere soils, decreasing diversity from the
bulk soil to the roots [8]. In tobacco and Arabidopsis plants, the number of microbes present
in the rhizosphere is approximately 10- to 100-fold higher than that in bulk soil [9].
The rhizosphere is considered a biological hotspot where plant–microbe, microbe–
microbe, and microbe–plant interactions shape microbial community composition. Plant
roots secrete organic compounds that support microbial activity [8]. Rhizosphere soil con-
tains 108 –1011 cultivable cells in one gram of soil, which corresponds to approximately 104
microbial species [10]. In addition to plant growth-promoting rhizobacteria, soil also pro-
vides habitats for plant pathogenic microorganisms and opportunistic human pathogenic
bacteria [4].
The spatial heterogeneity of the soil microbiome is determined on the one hand by
environmental factors and on the other hand by populational processes [11]. Environmental
factors can be both biotic and abiotic, and soil microbial colonization is influenced by plant
root exudates in the rhizosphere and environmental parameters. The bulk soil microbial
community is an important factor that shapes the rhizosphere microbiome, being the main
reservoir from which soil microorganisms are attracted by chemotaxis to root exudates [9].
The same taxa are therefore present in bulk and rhizosphere soils but differ in their relative
abundance [12].
Differences in the microbial community between bulk and rhizosphere soils were
studied in maize fields by Li et al. [13]. They observed that the rhizosphere soil microbiota
was enriched in Proteobacteria, Bacteroidetes, and Actinobacteria, accounting for 73–80%
of total reads versus 46–56% in bulk soil. A decreased abundance was observed for
Acidobacteria, Gemmatimonadetes, Chloroflexi, Firmicutes, and Nitrospira in the rhizosphere
relative to that in the bulk soil [13]. Fan et al. studied the microbial community of wheat
fields with an emphasis on three soil compartments, namely tightly and loosely bound soil
and bulk soil [8]. They found that Proteobacteria, Actinobacteria, and Acidobacteria dominated
all soils, whereas the abundance of Actinobacteria, Bacteroidetes, Alphaproteobacteria, and
Verrucomicrobia was higher and the abundance of Gammaproteobacteria, Chloroflexi, and
Deltaproteobacteria was lower in tightly bound soil than in the other soils. The greater
relative abundance of Actinobacteria in tightly bound soil was explained by their antibiotic-
producing potential, whereas the presence of Alphaproteobacteria was attributed to their fast
growth characteristics.
Agriculture 2024, 14, 152 3 of 23

Owing to the complexity of the soil system, in addition to the variation in space
(microhabitats), stratification also shapes the microbiome. Rchiad et al. observed that, in
a semiarid agroecosystem, although the diversity index of the soil microbiota does not
decrease with increasing soil depth, there were differences in the microbial profiles [14].
They detected 43 microbial phyla, of which the distribution of 12 was affected by soil
depth. At the phylum/family level, a transition in the microbial community from the top
(0–15 cm) to the deepest level (30–60 cm) was observed. The abundances of Verrucomicrobia
and Bacteroidetes decreased with soil depth. The abundance of soil functional genes was
also affected by depth, and most functional categories were observed either in the top layer
or in the deepest level [14].

2.2. Soil Microbiome Taxonomic Diversity: Structure and Function

2.2.1. Study Methods
Microbiological studies, as well as those for the soil microbiome, have traditionally
been based on isolation and culturing methods, using different specific growth media to
maximize culturable isolates. Owing to the development of molecular genetic methods,
we found that culture-based methods missed most of the microbial diversity. Data on
the ratio of unculturable bacterial fraction that remains to be explored reflects different
opinions: Mendes et al. and Dubey et al. reported it as approximately 90–95%, whereas
Yadav et al. reported that it might be from 100 to 1000 fold larger [4,5,15]. Each opinion
reflects that the soil and rhizosphere microbiomes are highly underestimated. Most of
the bacterial phyla have unculturable representatives whereas Archaea were reported only
using culture-independent methods [5,15].
In the late 20th century, culture-independent molecular techniques allowed us to
study bacterial genomic material. The first developed methods were based on targeted
gene sequencing (16S ribosomal RNA), whereas today we are able to decipher the meta-
genome (whole genome sequencing) and even the metatransciptome (mRNA) of a whole
environment. Metaproteomics and metatranscriptome analyses target the functional part
of the microbiome based on protein extraction and analysis. The last two methods are
important for understanding not only the taxonomic profile but also the functional role of
the microbiome [16].

2.2.2. Taxonomic Diversity

The rhizosphere microbiome includes different groups of bacteria. They belong to
the following phyla: Acidobacteria, Actinobacteria, Ascomycota, Bacteroidetes, Basidiomycota,
Deinococcus-Thermus, Euryarchaeota, Firmicutes, and Proteobacteria [15]. The bacterial commu-
nity diversity and composition differ between different crop plants, with higher differences
reported between different plant types, such as legumes, forbs, and grasses. A review of
the microbiome distribution of different crops was conducted for six important crop plant
species: maize, wheat, rape, cotton, rice, and soybean (Figure 1). Bacterial strains belonging
to phyla Proteobacteria, Actinobacteria, Acidobacteria, Gemmatimonadetes and Chloroflexi were
observed in all six crop plants [15,17].
Maize rhizosphere was preferentially colonized by Proteobacteria (class Betaproteobacte-
ria and Gammaproteobacteria), Bacteroidetes (class Sphingobacteria) and Actinobacteria, with
Massilia, Burkholderia, Ralstonia, Dyella, Chitinophaga and Sphingobium as dominant genera
accounting for from 63% to 77% of total bacteria [13]. The core rhizosphere of wheat
comprises Proteobacteria, Bacteroidetes, Actinobacteria, Acidobacteria, Gemmatimonadetes, Ar-
matimonadetes, Planctomycetes, Saccharibacteria, Verrucomicrobia, Firmicutes, Nitrospirae, and
Chloroflexi. Sphingobacteriaceae (Bacteroidetes) and Gemmatimonadaceae were the most abun-
dant families [18].
Agriculture 2024,
Agriculture 2024, 14,
14, 152
x FOR PEER REVIEW 4 of 24
4 of 23

Figure 1.1. Relative

Relativeabundance
abundance of of
different
differentbacterial communities
bacterial communities in different crops
in different (based
crops on Lion
(based et al.
Li
[13],
et al. Mahoney et al. [18],
[13], Mahoney et al. Rathore et al. et
[18], Rathore [19],
al. Ullah et al. et
[19], Ullah [20],
al. Edwards et al. et
[20], Edwards [21],
al. and
[21],Sugiyama et al.
and Sugiyama
[22]).
et al. [22]).

Maize rhizosphere
The highest waswas
similarity preferentially colonized
found between by Proteobacteria
the maize and wheat(class Betaproteobac-
rhizomicrobiomes.
teria community
The and Gammaproteobacteria), Bacteroidetes
structures of rape, (class
cotton, rice, andSphingobacteria)
soybeans wereand moreActinobacteria, with
specific. Approxi-
Massilia,
mately 99% Burkholderia,
of the rapeRalstonia,
microbiota Dyella, Chitinophagabyand
was represented theSphingobium as dominant
phyla Proteobacteria, genera
Bacteroidetes,
accounting forAcidobacteria,
Actinobacteria, from 63% to 77% of total bacteria
Verrucomicrobia, and [13]. The core
Chloroflexi. rhizosphere
Bacteria of wheat
belonging com-
to families
prises Sphingomonadaceae,
such asProteobacteria, Sphingobacteriaceae,
Bacteroidetes, Actinobacteria,Micrococcaceae
Acidobacteria,and Chthoniobacteracaea
Gemmatimonadetes, were
Armati-
among the Planctomycetes,
monadetes, most abundant Saccharibacteria,
groups [19]. TheVerrucomicrobia,
cotton microbiome, studiedNitrospirae,
Firmicutes, by Ullah etand al.,
consists mainly
Chloroflexi. of Proteobacteria,
Sphingobacteriaceae Actinobacteria,
(Bacteroidetes) andGemmatimonadetes,
Gemmatimonadaceae Chloroflexi,
were the Cyanobacteria,
most abun-
and Acidobacteria
dant families [18].at the phylum level [20]. Bacterial strains belonging to the Burkholderia,
Streptomyces, Rhizobium,
The highest Massilia,
similarity Pseudonocardia,
was found Shinella,
between the maizeSphingobium,
and wheat Agaricicola, Mesorhi-
rhizomicrobiomes.
zobium,
The Streptomyces,
community Altererythrobacter,
structures Opitutus,
of rape, cotton, rice, and and Sphingopyxis
soybeans genera
were more were the
specific. most
Approx-
abundant in the cotton rhizosphere. The rice microbiome was dominated by
imately 99% of the rape microbiota was represented by the phyla Proteobacteria, Bacteroide-Proteobacteria,
Chloroflexi, and Acidobacteria
tes, Actinobacteria, phyla,
Acidobacteria, whereas in theand
Verrucomicrobia, caseChloroflexi.
of the soybean microbiome,
Bacteria belongingbacterial
to fam-
strains
ilies belonging
such to ProteobacteriaSphingobacteriaceae,
as Sphingomonadaceae, were the dominantMicrococcaceae
phyla, followed and Actinobacteria and
byChthoniobacteracaea
Chloroflexi
were among [22].
the most abundant groups [19]. The cotton microbiome, studied by Ullah et
al., consists mainly of Proteobacteria, Actinobacteria, Gemmatimonadetes, Chloroflexi, Cyano-
2.2.3. Factors Affecting Diversity
bacteria, and Acidobacteria at the phylum level [20]. Bacterial strains belonging to the
The mainStreptomyces,
Burkholderia, environmentalRhizobium,
factors affecting the community
Massilia, structure
Pseudonocardia, of theSphingobium,
Shinella, rhizosphere
microbiome are pH, salinity, moisture, temperature, and nutrient content (C-N
Agaricicola, Mesorhizobium, Streptomyces, Altererythrobacter, Opitutus, and Sphingopyxis contentgen-
and
other nutrients); however, vegetation also has an important role [15,23].
era were the most abundant in the cotton rhizosphere. The rice microbiome was domi- Environmental
factorsby
nated create unique ecological
Proteobacteria, niches
Chloroflexi, andthat frame specific
Acidobacteria phyla,microbiomes. Numerous
whereas in the studies
case of the soy-
have shown that environmental stress can alter microbial diversity and
bean microbiome, bacterial strains belonging to Proteobacteria were the dominant phyla,shift ecosystem
function. by
followed Niche-based
Actinobacteriaresearch has shown
and Chloroflexi [22]. that the important processes of microbial
communities are influenced by microbial fitness and habitat conditions [5].
2.2.3.The spatial
Factors heterogeneity
Affecting Diversityof the soil microbiome was studied in the case of switch-
grass vegetation, where the bacterial community structure proved to be patchy, and the
The main environmental factors affecting the community structure of the rhizosphere
abundance of the dominant phyla (Verrucomicrobia) changed 2.5 fold in a 10 cm3 grid [5].
microbiome are pH, salinity,
In a study conducted moisture,
in an alpine temperature,
environment, and nutrient
environmental contentexplained
parameters (C-N content
41%
and
of theother
totalnutrients);
variation however, vegetation also
in soil communities, with has
pHanand
important role [15,23].
temperature being Environmen-
the strongest
tal factors create
influencing factorsunique ecological
[23]. Praeg niches thatthe
et al. compared frame
bulkspecific microbiomes.
and rhizosphere soils Numerous
and found
studies have shown that environmental stress can alter microbial
that Ranunculus glacialis roots explained approximately one-third of the variationdiversity and shift
[23].
Agriculture 2024, 14, 152 5 of 23

Differences among different plant type (legume, grass, or forb) microbiomes were observed
by Hannula et al. [24]. They attributed a variation of 4% in bacteria and 11% in fungi to the
difference between plant groups and 30% to species-level variations.
Mahoney et al. studied the microbiome of wheat cultivars [18]. They found higher
differences in Planctomycetes, Acidobacteria, Actinobacteria, and lower differences in Chlo-
roflexi, Fibrobacteres, and Verrucomicrobia in the nine studied wheat cultivars. The bacterial
community structures at lower taxonomic ranks (family, genus, and OTU levels) were
influenced by different growth stages in Zea mays. At early growth stages bacterial species
belonging to Massilia, Flavobacterium, Arenimonas and Ohtaekwangia genera were more abun-
dant, whereas bacterial species belonging to Burkholderia, Ralstonia, Dyella, Chitinophaga,
Sphingobium, Bradyrhizobium and Variovorax genera were dominant at later stages [13].
Anthropogenic effects that cause biotic and abiotic stresses and changing climatic
conditions modify soil microbial and plant diversity [10,25]. Ullah et al. identified drought-
tolerant bacteria in drought-treated cotton plants [20]. Thermophilic bacteria belonging to
the phyla Chloroflexi and Gemmatimonadetes were found to be dominant in drought-affected
environments [20]. The use of fertilizers, farming, and tillage methods in agroecosystems
can induce changes in soil microbiomes. In the case of the switchgrass rhizosphere mi-
crobiome, a fertilizer-induced decrease in the relative abundance of the most abundant
phylum (Verrucomicrobia) was observed [5].
The use of different soil amendments also reduced bulk soil microbiome diversity and
influenced the rhizosphere community in Zea mays. Changes in community structure were
caused by a lower abundance of Actinobacteria and Firmicutes and a higher abundance of
Proteobacteria, Bacteroidetes, Verrucomicrobia, and Acidobacteria [12]. Fertilizer type and dose
also contributed to changes in rhizobial community structure in Z. mays, and differences in
the abundance of microbial groups were attributed to their different nutrient contents [26].
Tillage practices modified the root- and shoot-associated bacterial communities in rape
plants, whereas farming practices affected the microbial community structure of rice [21].

2.2.4. Ecological Function

In microbial communities, it is important to study species composition and existing
ecological functional groups. Different microbes have different roles in the community
structure, which might support soil health and plant productivity. Microbial communi-
ties are complex dynamic networks with various interactions between microbes, such as
resource competition, metabolic dependencies (cross-feeding), spatial organization no-
tably production of biofilms, signaling, horizontal gene transfer, coevolution, and viral
looting [27]. Usually, a higher diversity of microbes increases the quantity of metabolites,
secondary metabolites, phytohormones, biocontrol substances, and other beneficial sub-
stances, thereby contributing to soil structure and fertility, root system architecture and
nutrient foraging, plant nutrition and hormonal balance, plant stress tolerance, agricultural
productivity, and resilience to climate, land use, and agronomic practices [10]. Wei et al.
studied the role of the initial microbiome in the occurrence and phytopathogeny of Rhizoc-
tonia solanum [28]. They observed higher abundances of Alphaproteobacteria, Firmicutes, and
Cyanobacteria in soils associated with healthy tomato plants, whereas diseased plants were
found in soils rich in Acidobacteria, Actinobacteria, and Verrucomicrobia [28].
In a study realized in a semiarid region regarding the functional metagenome analysis
of different soil layers, twenty-eight functional categories of subsystem level 1 were detected.
Differences between the soil layers were observed in 20 functional categories. In the top
layer (0–15 cm), which is rich in organic matter, functional genes related to DNA and RNA
metabolism, and nutrient metabolism (N, S, carbohydrate) were more abundant than in
the deeper layers. The 15–30 cm layer proved to be overlapping, showing a transition
in the microbial community and function. In the deeper soil layer (30–60 cm), where the
organic matter content is lower and mineral content is higher, functional genes related to P
metabolism, nucleosides and nucleotides, amino acids and derivatives were observed in
abundance. Understanding the changes in the microbial community and function between
Agriculture 2024, 14, 152 6 of 23

soil layers could be important for the usefulness of intercropping and further development
of sustainable agricultural practices [14].
The microbial diversity of soil is important for maintaining the function of soil ecosys-
tem services. Microbes with plant growth promotion and protection potentials are im-
portant for sustainable agricultural practices. A better understanding of plant–microbe
interactions in both natural and agroecosystems will offer us a biotechnological tool for
managing soil fertility and obtaining a high-yield food production system [29].

2.3. Beneficial Plant–Microbe Interactions

2.3.1. Biostimulant Microbes
Microorganisms and plants live in nature in association, but the microorganisms
can be free-living, attached, or enter symbiosis with the host plants. There are different
interactions such as commensalism, mutualism or parasitism. During evolution, plants
interacted with a broad range of plant growth-promoting rhizobacteria (PGPR). Owing
to recent advances in metagenomics, massive genome-sequencing strategies, and new
identification techniques, bacterial rhizobiome mapping is rapidly progressing. These
findings revealed novel bacterial species and their mechanisms involved in biocontrol and
plant growth promotion [30].
Microorganisms living in soil can indirectly promote plant growth (Figure 2), espe-
cially by fixation of atmospheric N2 , production of siderophores, plant growth hormones
(cytokinins, auxins, and gibberellins), volatile compounds, and solubilization of nutrients
and minerals (phosphorus, potassium, zinc, etc.). Additionally, some PGPR species play a
role in plant stress resistance. They can help the host plant overcome drought and7 saline
Agriculture 2024, 14, x FOR PEER REVIEW of 24
stresses and can increase the plant’s capacity to sequester heavy metals or other toxic
elements [13,26,31].

Plantgrowth-promoting
Figure2.2.Plant
Figure growth-promotingmechanisms
mechanismsby bysoil
soilmicrobes.
microbes.PGPRs
PGPRsplay
playan
animportant
importantrole
roleinin
plantgrowth
plant growthpromotion,
promotion,stress
stressresistance,
resistance,plant
planthealth
healthand
andprotection,
protection, phytoremediation,
phytoremediation, and
and ISR.
ISR.

PGPRS
Biological are agriculturally
Nitrogen Fixation important bacteria because they contribute to plant growth
by increasing germination rate, biomass, chlorophyll, and nitrogen content. These can
Nitrogen
improve from
the leaf soilshoot
area, is available to plants
and root length,inhydraulic
inorganicactivity,
forms (nitrate, ammonium)
and crop yield. Theyand
can
organic ones (urea, amino acids, small peptides). Organic forms can be used only in spe-
cial environments [33]. The Earth’s atmosphere is rich in elemental dinitrogen (N2) but
this is biologically unavailable for plants. Fowler et al. calculated the amount of biologi-
cally available nitrogen convertible from the total amount of dinitrogen gas 4 × 109 Tg N
to be 473 Tg N [34,35].
Atmospheric nitrogen is reduced to ammonia (NH3) gas, and this reduction can be
Agriculture 2024, 14, 152 7 of 23

also confer plant tolerance to biotic (pathogen bacteria, fungi, yeast, pests, insects etc.) and
abiotic stresses (drought, flood, heavy metals, salinity, temperature etc.) [15]. Fungi that
promote plant growth belong to the following genera: Aspergillus, Penicillium, Rhizoctonia,
Talaromyces, and Trichoderma, among which the most promising plant growth-promoting
fungi are Trichoderma sp. (T. viride and T. harzianum) and Penicillium chrysogenum [32].

Biological Nitrogen Fixation

Nitrogen from soil is available to plants in inorganic forms (nitrate, ammonium) and
organic ones (urea, amino acids, small peptides). Organic forms can be used only in special
environments [33]. The Earth’s atmosphere is rich in elemental dinitrogen (N2 ) but this
is biologically unavailable for plants. Fowler et al. calculated the amount of biologically
available nitrogen convertible from the total amount of dinitrogen gas 4 × 109 Tg N to be
473 Tg N [34,35].
Atmospheric nitrogen is reduced to ammonia (NH3 ) gas, and this reduction can be
made artificially by the Haber–Bosch procedure or occurs naturally as thunderstorms and
biological nitrogen fixation (BNF), which accounts for 66% of the total fixed N2 [36]. After
photosynthesis, BNF is the second most important process on Earth, due to its significant
role in agroecosystem sustainability [34,35].
The nitrogenase activity of nitrogen-fixing microorganisms is responsible for BNF,
whereas atmospheric N2 is reduced to ammonia. Bacteria with BNF capacity are catego-
rized into three groups: free-living, associated, and symbiotic bacteria. Free-living N2
fixing bacteria belong to different genera such as Gluconacetobacter, Azospirillum, and Azoto-
bacter spp., but their contribution is negligible compared with the total BNF. The highest
proportion of BNF is due to symbiotic nitrogen-fixing bacteria called rhizobia. In addition,
other PGPRS with nitrogenase complexes, called diazotrophs, fix N2 in non-leguminous
plants (including cereals). The nitrogenase complex is a two-component metalloenzyme.
The first component is dinitrogenase reductase, a homodimeric iron protein that acts as a
reductase. They have a high reducing capacity and are responsible for providing electrons.
The second is dinitrogenase, a heterotetrameric Mo-Fe protein that utilizes the electrons
provided to reduce N2 to NH3 [35].
Rhizobium bacteria are symbiotic bacteria linked to leguminous plants. The non-
symbiotic or free-living type N2 -fixing bacteria are cyanobacteria (blue-green algae, An-
abaena, Nostoc) and other species belonging to different genera, such as Azotobacter, Bei-
jerinckia, and Clostridium. Associative nitrogen-fixing bacteria, such as Azospirillum sp.
(maize, rice, and wheat), Klebsiella sp., Azotobacter sp. and Alcaligenes sp. live around
roots in the rhizosphere and they have the role to stream the fixed nitrogen to the plant.
Endophytic nitrogen-fixing microorganisms are linked to cereals, grasses, sugarcane, and
Azoarcus sp. (Kallar grass, sorghum, rice), Herbaspirillum sp. (rice, sorghum, sugarcane),
Gluconacetobacter diazotrophicus (sorghum, sugarcane) and Burkholderia sp. (rice) [33,36].

Phytohormone Production
Phytohormones are organic compounds that influence physiological processes in
plants even at very low concentrations. The ability of soil microbiota to produce phy-
tohormones is a potential source of phytohormones. Plant growth hormones, such as
auxins (indole-3-acetic acid), gibberellins, abscisic acids, ethylene, and cytokinins, are
biosynthesized by microorganisms. For synthesis, a considerable amount of metabolic
energy and nutrients are required [35,37]. Ethylene, jasmonic acid, and salicylic acid are
stress-related regulators of plant immunity that are involved in the creation of a central
signaling backbone that coordinates defense responses against phytopathogens [38].
Phytohormones play a significant role in plant growth during cell division and enlarge-
ment, seed germination, root formation, and stem elongation. Phytohormones produced
by bacteria are released into the plant body and have a positive effect on plant growth
and development. Several reports have shown that bacteria can produce 60 times more
plant growth regulator substances than plants can. Bacteria belonging to the Rhizobium,
Agriculture 2024, 14, 152 8 of 23

Sinorhizobium, Bradyrhizobium, Azospirillum, Bacillus, Paenibacillus, and Pseudomonas genera

have the capacity to produce phytohormones (auxins, ABA, cytokinins, and gibberellins),
thereby improving plant growth and productivity under natural conditions [27,35].
All plant-associated microbes produce auxins, but not all PGP microbes have the ability
to produce gibberellin. This capacity is related to root-associated microbes. Auxins, mostly
indole-3-acetic acid (IAA), are synthesized by 80% of rhizosphere bacteria. Tryptophan is
the main precursor for IAA biosynthesis in bacteria. Bacteria that promote IAA synthesis
can take up tryptophan present in root exudates. There are five different tryptophan-
dependent and tryptophan-independent pathways, as in Azospirillum brasilense, in which
the biosynthetic intermediates are unknown [38].
Among bacterial phytohormones, IAA, which promotes root elongation and lateral
root development, is the most studied. These plant hormones are highly effective under
stressful conditions. Some plants are unable to produce enough auxins to cope with stress
effects; therefore, bacterial auxins can alleviate stress conditions in plants [39]. Bacterial
strains with IAA production capacity include Pseudomonas fluorescens, Pseudomonas syringae,
Agrobacterium tumefaciens, Pantoea agglomerans, Azospirillum brasilense, Bacillus cereus, Bacillus
amyloliquefaciens, Rhizobium sp., and Bradyrhizobium sp. [38]. According to Shahzad et al.,
inoculation with Micrococcus yunnanensis RWL-2, Pantoea dispersa RWL-3, Micrococcus luteus
RWL-3, and Staphylococcus epidermidis RWL-7 in rice plants resulted in a significant increase
in dry biomass, shoot and root length, chlorophyll, and protein content [40,41]. IAA-
producing fungi include Aspergillus, Mortierella, Talaromyces, Fusarium, Penicillium, and
Trichoderma spp. Penicillium janczewskii, which produces IAA, and inhibits Rhizoctonia solani,
a phytopathogen that causes stem rot [32].
Abscisic acid (ABA) is a stress-related hormone that plays a key role in photoperiodic
induction of flowering, contributing to plant growth and development. It is involved
in plant responses to different environmental stresses such as cold, salinity, and desicca-
tion [39]. Several plant-associated bacteria can produce ABA, thereby increasing phyto-
hormone levels in plants. ABA is an important factor in modulating plant defenses, so
plant mutants with altered ABA biosynthesis or that are ABA-insensitive are more resistant
to pathogens than wild-type plants [38]. ABA-producing endophytic bacteria include
Achromobacter xylosoxidans, Brevibacterium halotolerans, Bacillus licheniformis, Bacillus pumilus,
and Lysinibacillus fusiformis [42].
The gibberellin (GA) phytohormone plays a major role in leaf expansion and stem
elongation in plants. When GA is applied exogenously, it can promote parthenocarpy in
fruits, bolting plants, breaking tuber dormancy, and increasing fruit size and the number of
buds. Several soil microorganisms have been reported to produce gibberellin, with positive
or negative effects on nodulation and plant growth. These microorganisms can induce
nodule organogenesis and inhibit nodulation during the infection stage [39]. The first
described bacterium with GA production ability was Rhizobium phaseoli, which produces
GA1 and GA4. Biologically active GA1 and GA3 are produced by Azospirillum lipoferum,
Acetobacter diazotrophicus, and Herbaspirillum seropedicae [42]. GA-producing fungi such as
Cladosporium sp. were reported by Adedayo and Babalola, and were found to improve
tomato plant growth due to GA production as well as playing an important role in pea
plant colonization [32].
Cytokinins (CK) play a role in many stages of plant development by stimulating plant
cell division, root development, and root hair formation, activating dormant buds, and
inducing the germination of plant seeds. These plant hormones affect apical dominance
and regulate nodulation and nitrogen fixation. Some pathogenic and beneficial microbes
produce cytokinin phytohormones. It has been reported that PGPRs from Pseudomonas and
Bacillus genera produce cytokinin, especially zeatin [15,35]. Pseudomonas fluorescens and
Rhizobium spp. are cytokinin-producing bacteria [43].
Ethylene (ET) is a plant stress hormone. Under stress conditions, higher amounts
of ethylene can negatively affect plant growth. Ethylene production can be modulated
by bacterial strains possessing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase
Agriculture 2024, 14, 152 9 of 23

activity. PGPRS acts as a sink for the ET precursor, 1-aminocyclopropane-1-carboxylic

acid (ACC), consequently reducing ET levels in roots and simultaneously increasing root
length and plant growth. ACC exuded by roots and seeds can be taken up by rhizobacteria,
and due to activity of ACC deaminase (ACCd) is split into ammonia and α-ketobutyrate.
ACCd activity has been detected in many PGPRs, and the genes encoding ACCd are
widespread within these bacteria. PGPRs with ACCd activity enhance plant tolerance to
biotic and abiotic stresses by reducing ET production in colonized roots and promoting
plant growth [27,35]. It has been reported that Actinobacteria alleviate stress in plants by
reducing ethylene levels in roots by secreting ACCd enzymes [44]. Patil et al. revealed that
bacterial strain Bradyrhizobium elkanii is an inhibitor in ethylene synthesis and alleviated
the negative effect of stress-induced ethylene production on nodulation [45].
The phytohormones produced and secreted by microbes are used as growth regu-
lators in crop production. These provide benefits to the host plant by facilitating root
system expansion, enhancing the absorption of water and nutrients, and improving plant
survival [15].

Enzymatic Activity
1-aminocyclopropane-1-carboxylate deaminase (ACC-deaminase, ACCd) enzyme
plays an important role in plant hormone and ethylene regulation. ACC deaminase is
found in numerous microbial species, including Gram-negative and Gram-positive bacteria
and fungi [46,47].

Solubilization of Major Nutrients

Soil microorganisms play a major role in nutrient cycling. The crop residues incor-
porated in the soil represent the carbon, energy, and nutrient sources of microorganisms.
Rhizobia can solubilize nutrients such as phosphorus, iron, potassium, and zinc, thus
increasing their availability to plants [46].
Among the macronutrients, phosphorus (P) is essential for plant growth and devel-
opment. P is abundantly available in the soil in organic (phytin) and inorganic forms (P
minerals such as apatite and secondary P minerals such as Al, Fe, and Ca phosphates). P is
a major growth-limiting nutrient despite being present in soil in abundancy in insoluble
form. The soluble level of P in soil determines the P accessibility to plants [38,44].
PGP microbes are a biological rescue system because they are capable of solubilizing
insoluble inorganic P in soil, increasing its availability to plants in the form of orthophos-
phate. The major mechanism of P solubilization involves the production of organic acids.
As a result, insoluble P is transformed into its soluble form. The produced organic acids
decrease the soil pH or chelate mineral ions, resulting in phosphate solubilization [38].
The organic acids most frequently produced by Gram-negative PGPRs are gluconic acid
and 2-ketogluconic acid. Other organic acids produced by phosphate-solubilizing PGPRs
are lactic, isobutyric, isovaleric, acetic, glycolic, oxalic, succinic, and malonic acids [38].
PGP microbes with phosphate solubilization capacity belong to different genera such as
Arthrobacter, Azotobacter, Bacillus, Beijerinckia, Burkholderia, Citrobacter, Enterobacter, Erwinia,
Flavobacterium, Halolamina, Microbacterium, Pantoea, Paenibacillus, Pseudomonas, Rhizobium,
Rhodococcus, Serratia and Thiobacillus [15,38,40].
PGPRs can release from soil organic and inorganic phosphorus by producing several
enzymes, such as phytases, phosphatases, phosphanatases and lyases [38]. In this process,
the microbes also produce organic acids (gluconate, acetate, ketogluconate, oxalate, lactate,
tartarate, succinate, citrate, and glycolate), but this depends on the type of carbon source
used as substrate. The highest amount of solubilized P was observed in in vitro conditions
when glucose, sucrose or galactose was used as the sole carbon source [15].
Potassium (K) is considered the third major macronutrient for plant growth and crop
yields. More than 90% of the potassium that exists in soil is in the form of insoluble rocks
and silicate minerals. The soluble form of K is present in soil in low concentration. One of
the major constraints in crop production is the potassium deficiency due to imbalanced
Agriculture 2024, 14, 152 10 of 23

fertilizer application. Lack of potassium causes poorly developed roots, small seeds, lower
crop yields and slow growth. An alternative indigenous source of potassium for plants is
the potassium solubilized by soil microorganisms. The potassium-solubilizing microbes
(KSMs) have an important role in transforming insoluble K into available form for uptake
by plants through production and secretion of different organic acids. Many bacteria such
as Acidithiobacillus ferrooxidans, Bacillus mucilaginosus, Bacillus edaphicus, Bacillus circulans,
Burkholderia sp., Paenibacillus sp. and Pseudomonas sp. possess the capacity to solubilize
K into available form. These bacteria contribute to the enhancement of K+ availability in
agricultural soils [48].
KSMs excrete low molecular organic acids (citric, tartaric, oxalic, succininc, syringic,
malic, coumaric, and ferulic acids), thus modifying potassium uptake. The organic acids
dissolve K+ from the minerals, thereby lowering the pH and forming metal–organic com-
plexes with Si4+ , thus bringing the potassium into solution. Through weathering, biofilms,
polymers, capsular polysaccharides, and low molecular weight ligands produced by soil
microbiota can mobilize K [33]. The application of KSMs as biofertilizers in agriculture can
reduce the use of agrochemicals and support environmental sustainability [48].
Adedayo and Babalola reported that Ceriporia lacerata, Aspergillus awamori, and Penicil-
lium digitatum were able to solubilize phosphate owing to the activity of organic acids and
phytase [32]. Trichoderma sp. improved the absorption of several minerals and nutrients (K,
N, and P), whereas T. viride fungi increased the nitrogen content of the soil [32].

Solubilization of Iron with Siderophore Production

The bioavailability of iron as an essential micronutrient is limited by the soil. Siderophores
produced by soil bacteria play a key role in plant iron nutrition. These compounds are low-
molecular-weight chelators with a high affinity for iron (III), the most common form of iron
in nature. The iron solubilization mechanism relies on the formation of a stable siderophore-
Fe+3 complex that can be absorbed by plants [38,48]. To date, more than 500 siderophores
have been identified. Plant growth-promoting Pseudomonas fluorescens produces pyoverdine
among other siderophores. Microorganisms can produce other siderophoric compounds
such as catechol, hydroxamate, carboxylate, and phenolate, which contribute to plant
protection against pathogens. Bacterial strains with iron chelation properties belong to
Azotobacter, Bacillus, Enterobacter, Arthrobacter, Nocardia, and Streptomyces [46].
The direct beneficial effect of siderophores is the improvement in the iron nutritional
status of the plant, contributing to plant growth promotion. It has been hypothesized that
bacterial siderophores chelate Fe+3 from the soil, making it accessible to phytosiderophores,
but the exact mechanism is unknown. It has been shown that plants can incorporate
Fe+3 -pyoverdine complexes resulting in an increase in the iron content of plant tissues. The
indirect beneficial role of bacterial siderophores in plant growth promotion is their capacity
to reduce the availability of iron to phytopathogens [38].
Siderophore synthesis is influenced by several environmental factors such as pH,
the level of iron, the presence of other trace elements, and an adequate supply of carbon,
phosphorus, and nitrogen sources [48]. Siderophores transport iron into bacterial cells by
means of a system involving ferric-specific ligands (siderophores) and their corresponding
membrane receptors, which are chelating agents in bacteria. Studies have shown that
siderophores suppress pathogens, and this mechanism consists of the fact that siderophores
sequester a limited supply of iron (III) in the rhizosphere, thereby limiting the availability
of iron for pathogens and suppressing their growth [46].

2.3.2. Biocontrol Activity of Microbes

The overuse of chemicals in agriculture, such as pesticides, insecticides, herbicides,
and fertilizers, negatively affects consumer health, biomagnification of chemicals, and
economic loss [49,50]. Biological control organisms are defined as living organisms other
than disease-resistant host plants that suppress the activity of plant pathogens in the soil
environment [51].
Agriculture 2024, 14, 152 11 of 23

Microbial control agents can exert their plant-protecting characteristics based on their
mode of interaction with pathogens through direct and indirect mechanisms (Figure 3).
Indirect mechanisms are those that do not require interaction with pathogens, such as
microbial-induced systemic resistance (ISR), nutrients, and space competition. The pro-
duction of antibiotics and lytic enzymes to inhibit pathogen taxa is considered a direct
mechanism [52]. Bacterial isolates with biocontrol potential produce a broad spectrum of
bioactive metabolites such as antibiotics, siderophores, volatiles, and growth-promoting
substances. These bacteria can compete aggressively with other microorganisms and can
Agriculture 2024, 14, x FOR PEER REVIEW 12 of 24
easily adapt to environmental stress [51]. Trichoderma spp. produce biological antifungal
volatile organic compounds (VOCs), such as 6-pentyl-2H-pyran-2-one (6-PP) [32].

Figure3.3.Indirect
Figure Indirectand
and direct
direct mechanisms
mechanisms of biocontrol
of biocontrol agents.
agents. Indirect
Indirect methods
methods for biocontrol
for biocontrol agents
agents include induced systemic resistance and plant growth-promoting mechanisms.
include induced systemic resistance and plant growth-promoting mechanisms. Biocontrol agents Biocontrol
agents directly protect plants through antimicrobial metabolites and bacterial interactions. Arrows
directly protect plants through antimicrobial metabolites and bacterial interactions. Arrows indicate
indicate the direction of the relationship.
the direction of the relationship.

Accordingly,the
Accordingly, themechanisms
mechanismsofofbiocontrol
biocontrol byby PGPRS
PGPRS include
include competition,
competition, antibio-
antibiosis,
sis, hydrolytic enzymes, siderophore production, and induced systemic
hydrolytic enzymes, siderophore production, and induced systemic resistance. Species resistance. Spe-
cies belonging to different genera such as Bacillus, Serratia, Enterobacter, Pseudomonas,
belonging to different genera such as Bacillus, Serratia, Enterobacter, Pseudomonas, Burkholde-
Burkholderia,
ria, Herbaspirillum,
Herbaspirillum, Staphylococcus,
Staphylococcus, Ochrobactrum,
Ochrobactrum, Streptomyces,
Streptomyces, and Stenotropho-
and Stenotrophomonas are
known as antagonistic species against plant pathogens and are also used are
monas are known as antagonistic species against plant pathogens and also used as
as biopesticides
biopesticides
for forbacterial
the control of the control
and of bacterial
fungal and whereas
diseases, fungal diseases, whereas species
species belonging to generabelonging
Bacillus,
to genera Bacillus,
Pseudomonas, Pseudomonas,
and Serratia have beenandreported
Serratia to
have beenplant
induce reported to induce
systemic plant
resistance systemic
[46,53–57].
resistance [46,53–57].
Biocontrol agents produce and release many metabolites (lipopeptides, bacteriocins,
Biocontrol agents produce
antibiotics, biosurfactants, and release many
cell wall-degrading metabolites
enzymes, (lipopeptides,
and microbial bacteriocins,
volatile compounds),
antibiotics, biosurfactants, cell wall-degrading enzymes, and microbial
which reduce pathogen growth and metabolic activity. In contrast, BCAs affect pathogen volatile com-
pounds), and
virulence which reducefor
compete pathogen
nutrientsgrowth andby
and space metabolic
producing activity. In contrast,
antimicrobial BCAs affect
compounds [58].
pathogen virulence and compete for nutrients and space by producing antimicrobial com-
pounds [58].

Antibiotics
The production of antibiotics by various microorganisms is a biological control mech-
Agriculture 2024, 14, 152 12 of 23

Antibiotics
The production of antibiotics by various microorganisms is a biological control mecha-
nism. A microbially synthetized antibiotic can inhibit the metabolic activities of pathogenic
agents. The mechanisms involved in pathogen inhibition include inhibition of cell wall and
protein synthesis, and deformation of cellular membranes. The biochemical nature of these
metabolites largely determines their modes of action. Antibiotics also play a pivotal role in
the induced systemic resistance (ISR) mechanism in plants [59,60].
Several microorganisms belonging to different genera have been characterized for their
antibiotic production capacity, including Agrobacterium sp., Pseudomonas sp., Bacillus sp.,
Pantoea sp., Serratia sp., Stenotrophomonas sp., Streptomyces sp., and Trichoderma sp. [46,52].
Ram et al. listed various bacterial species and strains that are authorized for producing
agroinoculants such as Agrobacterium tumefaciens, Agrobacterium radiobacter strain 84, Bacillus
subtilis, Streptomyces lydicus, Burkholderia cepacia, Erwinia amylovora, Alcaligenes sp., Serratia
marcescens GPS 5, and Streptomyces griseoviridis [60].
Antibiotics synthetized by biocontrol strains include: polyketides, heterocyclic ni-
trogenous compounds, phenazine, phenylpyrroles, cyclic lipopeptides, aminopolyols, and
volatile antibiotics [51,59,61]. Several reports have been published on the production of
antibiotics by various biocontrol bacterial strains to target pathogens [60–63]. Ongena
and Jacques investigated the production of iturin, fengycin, and surfactin by Bacillus
sp. [64]. Raaijmakers and Mazzola investigated the production of antibiotic metabolites (2,
4-diacetylphloroglucinol, pyrrolnitrin, phenazine) in Pseudomonas sp. [65].
Adedayo and Babalola reported that T. harzianum had an inhibitory effect on the
growth of phytopathogen fungi: F. oxysporum, Phythium sp. and R. solani [32]. Gliocladium
virens produces an antibiotic (gliovirin) that inhibits the growth of Pythium ultimum and F.
oxysporum [32].

Interference of Quorum Sensing with Virulence

Quorum sensing is a cell–cell communication process in bacteria that involves extracel-
lular signaling molecules (autoinducers) and serves to share information about cell density.
Because many processes are advantageous only in this group, when the bacterial popula-
tion increases, gene expression is altered. Processes such as biofilm formation, antibiotic
production, and virulence factor secretion are controlled by QS. Quorum sensing (QS) is
important for expressing bacterial pathogenicity in plants. QS is required for the coloniza-
tion and expression of virulence factors in plant pathogenic strains, such as Pseudomonas
syringae, Pantoea stewartii, Erwinia chrysanthemi, and Burkholderia glumae [66–69].
QS inhibitory activity in the raw extracts of plants or microbes has been used in
the food packaging industry and in the treatment of multi-drug-resistant bacteria [70].
Interference with pathogen QS, termed quorum quenching, can be used as a sustainable
biocontrol strategy [70–72]. QS interference can be accomplished in three ways, namely
preventing the signal molecule biosynthesis, degrading the signal molecules, and blocking
the signal corpuscle receptors [72]. BCAs can also interfere with QS against pathogens
due to their enzymatic degradation capacity, by inhibiting the production of molecules
(lactonases, pectinases, chitinases) which initiate infections [54,73].
Acinetobacter sp., Bacillus sp., Burkholderia sp., Lysinibacillus sp., Serratia sp., Pseudomonas
sp., Rhodococcus sp., Strepsporangium sp., Streptomyces sp., Enterobacter sp., and Myroides
sp. were reported as AHL-degrading bacteria [71,74,75]. AHL-degrading Lysinibacillus has
been shown to reduce potato soft rot disease caused by Pectobacterium carotovorum subsp.
carotovorum [76,77].

Lytic Enzymes
A potential mechanism of action against pathogens is the production of lytic enzymes.
PGPRs inhibit the growth of fungal pathogens (Fusarium oxysporum, Sclerotinia sclerotiorum,
and Botrytis cinerea) and other soil-borne pathogens through the excretion of enzymes such
as chitinases, hydrolases, proteases, and glucanases [46,48].
Agriculture 2024, 14, 152 13 of 23

The production and secretion of cell wall lytic enzymes (CWLEs) are the most im-
portant effects of BCAs in restraining the growth of soil-borne pathogens [78]. The mode
of action of CWLEs is to disrupt the structural integrity of the target pathogen cell wall.
β-1,4-N-acetyl glucoseamine and chitin are the principal components of fungal pathogenic
cell walls. Numerous CWLEs, such as β-1,3-glucanase and chitinase, have been shown
to play a role in most BCAs. CWLEs are generally involved in the lysis of the cell wall
and thus neutralize the inhibitory action of pathogens [79]. PGPRS inhibit widely known
phytopathogens such as Phytophthora capsici and Rhizoctonia solani [80,81].

Induced Systemic Resistance (ISR)

Disease control by various beneficial bacterial strains involves the induction of sys-
temic resistance. Different microbial metabolites and biocontrol agents can generate an
immune response in the host plant, resulting in systemic disease resistance [46]. Plants
recognize microbial compounds (flagellin, lipopolysaccharide, exopolysaccharide, and
chitin oligosaccharides) produced by beneficial microorganisms. Different bacterial species
are effective against fungal, bacterial, and viral infections through ISR, including Bacil-
lus amyloliquefaciens, B. atrophaeus, B. cereus, B. megaterium, B. subtilis, Paenibacillus alvei,
Pseudomonas fluorescens, Pseudomonas aeruginosa, and Streptomyces pactum [82].
In ISR, some plant hormones such as salicylic acid, jasmonic acid, and ethylene
are involved. The host plant defense response against phytopathogens occurs through
the jasmonic acid and ethylene signaling pathways. Manifestation of ISR is related to
cell wall integrity and biochemical and physiological changes. Several bacterial strains
with ISR-eliciting traits have been reported to be effective against a broad spectrum of
fungal pathogens. These bacteria include Pseudomonas aeruginosa, Pseudomonas fluorescens,
Bacillus amyloliquefaciens, B. subtilis, B. pasteurii, B. cereus, B. pumilus, B. mycoides, and B.
sphaericus [73,80].
Treatment of cotton plants with P. chrysogenum induced ISR against Verticillium dahlia.
P. simplicissimum, T. harzianum, T. viride and Acremonium sclerotigenum were effective in
inducing ISR in crop plants such as cucumber and tomato [32].

2.4. Plant-Beneficial Function Encoding Gene Clusters and Mobile Genetic Elements
Horizontal gene transfer (HGT) is an event in which bacteria incorporate advanta-
geous genes into their genomes. Horizontally transmitted genes are crucial for bacterial
adaptation to changing environments or to plant-microbe interactions. They are often
grouped into genomic islands and gene clusters [83]. Up to 20% of the bacterial genome is
disseminated during horizontal gene transfer events [84]. The rhizosphere is considered to
be one of the hotspots of microbial gene transfer whereas the microbiome is a rich reservoir
of genetic functionality [85,86].
Many function genes in soil bacteria are encoded by plasmids that act as mobile
genetic elements (MGEs). Plasmids are most commonly considered antibiotic resistance
gene carriers; however, they are also important carriers of heavy metal detoxification genes,
N fixation genes, and other plant growth stimulation genes. The pSym plasmid found
in Rhizobium sp., in addition to nodulation and atmospheric nitrogen fixation genes, is
involved in phytohormone synthesis and transport of root exudate compounds. This
conjugative plasmid is commonly transferred to the soil or rhizosphere community, mainly
after sensing certain plant compounds such as flavonoids [86,87]. An approximately
150 kb plasmid was observed in an endophytic plant growth-promoting Enterobacter sp.
(pENT638-1), which has a role in host colonization [85]. In the case of Enterobacteriaceae
and Pseudomonadaceae, the most abundant and highly efficiently transferred is the IncP
plasmid [85]. IncP plasmids have clustered restriction sites that represent an integration
site for mobile genetic elements (MGEs) carrying antibiotic and heavy metal resistance
determinant genes [87].
HGT is a common strategy for changing adaptation-related genes, such as those
related to antibiotic resistance and heavy metal resistance among soil bacteria. Lekired et al.
Agriculture 2024, 14, 152 14 of 23

performed an extensive analysis of mobile genetic elements (MGEs) in Pantoea eucrina OB49
and found a plethora of MGEs (transposons, insertion sequences, a putative integrative
and conjugative element) related to antibiotics (beta-lactamase class) and a broad range of
heavy metal tolerance [83].
In the rhizosphere, microbes sense surrounding environmental stimuli and modulate
the process of gene exchange; therefore, appropriate environmental conditions can promote
HGT events. Higher temperature values (up to 35 ◦ C), loamy soil, use of organic fertilizer,
and higher soil toxicity have been found to increase the frequency of plasmid transfer
between bacteria [86].
Plant growth-promoting rhizobacteria possess more than one beneficial function as
a result of gene accumulation in the rhizosphere and soil environment governed by se-
lection mechanisms. The major function genes related to plant-beneficial function are as
follows: (i). nitrogen fixation-contributing nifHDK genes (encoding nitrogenase), (ii). Min-
eral phosphate solubilization pqqBCDEFG genes (encoding pyrroloquinoline quinone),
(iii). inhibition of ethylene biosynthesis acdS gene (encoding 1-aminocyclopropane-1-
carboxylate), (iv). IAA-producing ipdC/ppdC genes (encoding indole-3-pyruvate decar-
boxylase/phenylpyruvate decarboxylase), (v). antimicrobial compound synthesis hcn-
ABC/phlACBD (hydrogen cyanide/2,4-diacetylphloroglucinol) genes, and (vi). induced
systemic resistance conferring budAB/budC (acetoine/2,3-butanediol) genes, which was
reported previously and studied in Proteobacteria by Bruto et al. [88].
Co-occurrence of budAB with ipdC, the nifHDK operon, clustered pqqBCDE genes, and
hcnABC with phlABCD gene clusters was observed in 25 PGPR genomes [88]. Bruto et al.
also studied the genome plasticity of the Proteobacteria and identified acquisition and loss
events of the plant-beneficial function encoding genes [88]. They observed clade specificity
in phloroglucinol-producing (phlACBD) and IAA-producing (ipdC/ppdC) genes. Among
the genes conferring induced systemic resistance, budAB was clade-specific, whereas budC
was observed several times in other taxa. Although the genes responsible for mineral
phosphate solubilization were detected in the LCA of Pseudomonas, they were acquired
15 times by other taxa. The highest number of acquisition events were observed in nitrogen
fixation-contributing genes (nifHDK) and the ACC deaminase (acdS) gene [88]. Occurrence
of function genes related to plant-beneficial function in other root-adapted, non-PGPR
bacteria was also observed [88].
Cross-kingdom MGE transfers also promote environmental adaptation. Due to the
co-existence of bacteria, fungi, and plants in the soil environment and rhizosphere, MGE
transfers occur among all parties. The network structure of fungal mycelia and root
exudates enable the transfer of MGEs among bacteria, and MGE transfers occur between
fungi and bacteria [86].

2.5. Synergistic Microbial Processes

In many cases, plant inoculation with bacterial consortia proved to be more efficient
than inoculation with a single bacterial strain. Current research is focused on deciphering
how synergistic microbial processes work and how different plant beneficial traits influence
each other. Synergistic processes between ACC deaminase and IAA production and N2
fixation [89–96], ACC deaminase and IAA production and stress tolerance [93,97], and
phosphate solubilization and ACC deaminase [98] were reported.
The role of ACC deaminase and IAA in BNF fixation process is complex; they can en-
hance nodule formation, improve the competitiveness of rhizobia for nodulation, suppress
nodule senescence, and upregulate genes associated with legume–rhizobia symbiosis [96].
The role of ACC deaminase in nodule formation was studied using either knockout or
overproducing strains for the ACC deaminase-producing gene [96]. When Mesorhizobium
loti ACC deaminase-overproducing mutant strain was tested for the efficiency of plant
colonization and nodulation, it was found to be more efficient than the wild type [89].
This relationship is relatively complex, whereas in Mesorhizobium loti, the acdS gene was
found in the symbiotic island and its expression was regulated by the N2 fixation regulator
Agriculture 2024, 14, 152 15 of 23

NifA2 [99]. In senescent nodules, increased gene expression of PsACS2 encoding ACC
synthase (an enzyme involved in ethylene synthesis) and increased transcription have
been observed [90]. Therefore, ACC deaminase-producing PGP bacteria can enhance N2
fixation by extending the lifespan of functional nodules. Nascimento et al. investigated the
physiological function of ACC deaminase in a transformed Mesorhizobium sp. expressing
ACC deaminase and its parental type strain without this activity, and they observed
nitrogenase activity in 31-day-old nodules [94]. Nascimento et al. also observed that
ACC deaminase-producing Pseudomonas fluorescens YsS6 strain increased the nodulation
ability of both alpha- and beta-rhizobia compared to its ACC deaminase mutant strain [95].
Thus, free-living ACC deaminase-producing bacteria play an important role in facilitating
rhizobia nodulation.
IAA is a biomolecule that can affect legume–rhizobia interactions because nodule
initiation and development require high levels of IAA to regulate cell division and nodule
primordium formation. IAA also alters the expression of genes associated with rhizobia
and nodule initiation in plant cells [96]. Camerini et al. demonstrated a close relationship
between IAA and nodulation when a lower nodule formation capacity was observed in
IAA-negative Rhizobium leguminosarum bv. viciae compared to the parent strain [91]. More-
over, short-chain fatty acid production (caproic acid) of IAA-producing nodule-enhancing
rhizobacteria facilitated rhizobia colonization when used as a co-inoculum [96]. The
IAA-overproducing Ensifer meliloti RD64-induced 45-day-old nodules had an extended
nitrogen-fixing zone and a reduction in the senescent zone compared with plants nodulated
with the wild-type strain [92]. In the same IAA-overproducing bacterial strain, Defez et al.
observed an increase that reached maximum induction of the fixNOQP1,2 operon gene in
the nodules after 40 days of inoculation [93].
Crosstalk between ACC deaminase and IAA-producing bacteria exists; ACC deam-
inase acts as a sink for ACC, whereas IAA may facilitate plant growth or activate the
transcription of the enzyme ACC synthase. If ethylene synthesis occurs, IAA signal trans-
duction is limited [97].
Orozco-Mosqueda et al. and Defez et al. studied the role of ACC deaminase and
IAA in the stress response [93,100]. In Pseudomonas sp. UW4 ACC deaminase (acdS) and
trehalose (treS) mutant strains (mutated at one or two traits), synergism between the two
traits was observed when used as a bioinoculant on salt-stressed tomato plants, since single
gene mutants have a better effect than double ones [100]. Stress tolerance-associated gene
expression upregulation was observed in IAA overproducing E. meliloti RD64 compared to
its wild derivative [93].
Alemneh et al. reported the presence of diverse ACC deaminase producing bacteria
that showed phosphate solubilizing capacity and proved that ACC deaminase mediated P
solubilization [98]. They also observed improved nodulation by increasing the P nutrition
of chickpeas when inoculated with ACC expressing Burkholderia sp. 12F.
Synergism occurs not only between bacteria, but also between bacterial ACC deami-
nase and arbuscular mycorrhizal fungi [97]; therefore, metabolomic studies of the whole
soil microbiome are very useful in understanding plant–soil–microbe interactions.

2.6. Innovations in Carrier Materials for Bioinoculants

Carrier materials for bioinoculants must be chemically stable, nontoxic, low-cost,
and able to provide a protective niche for microorganisms to ensure the viability of cells
during storage and controlled release [101,102]. Many types of bioinoculant carriers have
been studied in the recent decades. They can be classified as solid, liquid, organic, or
inorganic. Additives that nutritionally support microorganisms are used in these bioformu-
lations [101,102].
Peat, biochar, bagasse, cork compost, attapulgite, sepiolite, perlite, and amorphous
silica were used as media for the solid bioformulations. They provide support for beneficial
microbes, in contrast with liquid bioformulations that are more sensitive to prolonged
storage. Immobilized formulations or encapsulation is an emerging technology with
Agriculture 2024, 14, 152 16 of 23

significant advantages over the above-mentioned formulations [101,102]. Microbial cells

are immobilized by adhesion/biofilm formation on solid supports or entrapment, thereby
conferring a protective environment for bacterial cells [103]. Various polymer matrices
were tested to study their effect on microbial cell viability during encapsulation and
storage [104–107].
The use of environmentally friendly biopolymer matrices is well suited to sustainable
agriculture. Microbial cells are encapsulated using various techniques such as ionic gela-
tion, emulsification, and spray drying [102]. Additives are used to improve the stability,
encapsulation efficiency, and mechanical properties of the carrier polymer, as well as fillers
to improve microbial survival [106,108].
Alginates are the most widely studied microbial carriers, mainly for Azospirillum sp.
and Pseudomonas sp. [102]. Alginate bead-entrapped A. brasilense showed better viability
during prolonged storage [104]. Calcium alginate microspheres have been used for Tricho-
derma viride spore encapsulation and provided a supportive environment for growth [109].
Panichikkal et al. used alginate beads supplemented with salicylic acid and zinc oxide
nanoparticles to immobilize Pseudomonas sp. DN18 which proved to be a promising biofor-
mulation for Oryza sativa [108]. The drawbacks of alginate beads such as low mechanical
strength, poor appearance, and high porosity have also been reported [105].
Starch presents important characteristics as a potential biopolymeric carrier, as it is
nontoxic, shows high solubility, and confers the controlled release of PGPB [102]. Marcelino
et al. used an innovative biodegradable foam containing as major component starch
combined with low-cost industrial by-products such as sugarcane bagasse, glycerol, rock
phosphate, crystal sugar, powdered skim milk and yeast extract for Azospirillum brasilense
Ab-V5 immobilization [106]. Microbial cell viability was maintained for up to 120 d at
room temperature [106]. Starch beads combined with chitosan were tested for release of A.
brasilense and P. fluorescens. After one year of storage, the recovery of bacteria was of the
order of 108 –109 CFU/g for both bacterial strains [107]. Alginate–chitosan nanoparticles
supplemented with starch were used for encapsulation of B. licheniformis. The bioformula-
tion was tested on chili plants, and plant beneficial traits were observed [110].
Nanofibers have also been used for the immobilization of bacterial co-cultures based
on Pantoea agglomerans and Burkholderia caribensis, and on testing, showed beneficial effects
on soybean [111].
The above-mentioned studies using immobilized formulations are based mainly on
laboratory-scale experiments and rarely on field experiments. Therefore, future research
should address how the production of bioformulations can be scaled up, how efficient they
are in the field, and what their long-term effects on soil and microbiome are.

2.7. Engineering Microbiome

Many plant growth-promoting microbes and microbial consortia have been studied
and proposed as potential bioinoculants. Various carriers have been tested to maximize
their colonization and persistence in harsh soil environments. Nevertheless, limitations of
natural bioinoculant use have been reported due to the complexity of soil–microbe–plant
interconnectedness.
A better understanding of the rhizosphere biochemical and molecular specificity that
governs plant–microbe interactions is required to be used in rhizosphere microbiome
engineering [112]. Rhizosphere microbiome engineering has gained much attention in
advanced agricultural research [113,114].
Microbiome engineering uses a microbe-focused approach that is based on construct-
ing synthetic communities called SynComs. These communities can be constructed using
bottom-up strategies. The bottom-up approach involves the identification of keystone
microbial taxa (e.g., Agrobacterium, Pseudomonas, Enterobacter) and the use of a combination
of microbial isolates. SynComs complexity is important in terms of their effectiveness and
stability in a changing environment, and functional species can be substituted because of
their stable metabolic network [113].
Agriculture 2024, 14, 152 17 of 23

Other possibilities rely on the genetic engineering of PGPRs, when PGP gene clusters
isolated from rhizobacteria are introduced on broad-host-range plasmids or phage transduc-
tion. Integrative and conjugative plasmid systems and chassis-independent recombinase-
assisted genome engineering are more advantageous than conjugative plasmids, which
are rapidly lost from bacterial populations [114]. A new strategy for PGP gene cluster
transmission is based on the introduction of foreign genes in situ through a conjugal donor
strain, which has led to the emergence of microbiome engineering [114].
Plant-secreted secondary metabolites such as carbohydrates, amino acids, and flavonoids
are key drivers for the colonization of the rhizosphere [112] and play a role in microbial
gene expression [114]. Therefore, plant-derived signals can be used as controllers of the
expression of the introduced genes. Ryu et al. identified such legume-derived flavonoid-
inducible expression systems in rhizobia and used them to N-fixation in Pseudomonas
protogens Pf-5 [115].
The plant-microbe relationship specificity and their species-specific signal make it
possible to use them in a very targeted way [114]. Rhizopine was recognized as a specific
signal to select rhizobia (Rhizobium and Sinorhizobium) in legumes and was used to obtain
transgenic Medicago and barley plants. Rhizopine signaling allows the establishment of a
control-engineered PGPR [116].
Owing to recent advances in this field, at least two directions for improving plant
growth promoting biopreparates can be defined. Both synthetic microbiome/PGPR ge-
netic engineering and plant genome engineering to recruit the desired microbiome can
revolutionize the field of sustainable agriculture.

2.8. Farm-Derived Products in Sustainable Agricultural Practices

Reduced use or lack of use of external inputs such as agrochemicals and the use instead
of farm-derived organic inputs are common practices in sustainable agriculture. The main
aim of these practices is to increase the soil organic carbon level, which is important from
both environmental and agricultural points of view.
Farm-derived organic amendments, such as compost, compost tea, and manure, are
used more frequently for the substitution of agrochemicals. Organic wastes can be suc-
cessfully converted through anaerobic decomposition to form organic fertilizers [117].
The reduced use of chemical fertilizers and higher use of farm amendments mitigates
greenhouse gas (GHG) emissions [118]. In a meta-analysis by Wei et al., an amount of
0.203 MgCO2 eq/ha was calculated in the case of full substitution with organic fertilizer
(manure, compost, or commercial organic fertilizer) [119]. A considerable carbon sink
resulted when partial substitution of mineral fermentation with organic amendments was
used [118,119]. The impact of the use of fermented liquid amendments was evaluated
in comparison with mineral fertilization on lettuce growth and soil quality at the micro-
cosm and field scales [117]. Urra et al. reported that the commercial and farm-made
fermented liquid organic amendments used in experiments beside sustaining crop yield
had ameliorative effect on soil quality [117].
Sujatha et al. proposed the conversion of weed biomass to obtain an environment-
friendly and climate-friendly amendment [120]. Substances such as urea, cow dung, a
microbial consortium (Bacillus subtilis), and a farm-derived liquid organic formulation
(jeevamrutham) were used as activators. The most efficient activator was the farm-derived
organic formulation, which converted the weed biomass to a carbon and humic substance-
rich amendment [120].
Sustainable agricultural practices, such as recycling farm-derived products, are in
accordance with global strategies to reduce greenhouse gas emissions by diminishing
agricultural waste and agrochemical-related emissions.

3. Concluding Remarks and Future Perspectives

Owing to stratification and various microhabitats, the complexity of the soil system
supports the formation of a diverse microbial community. Analyzing microbial community
Agriculture 2024, 14, 152 18 of 23

data is currently the biggest challenge. Beyond taxonomy, understanding the functional
groups of bacterial taxa and the dynamics of the bacterial community structure are impor-
tant issues for better understanding soil ecosystem functioning. Because the rhizosphere is
considered one of the hotspots of microbial gene transfer and the microbiome is a rich reser-
voir of genetic functionality, PGPR genetic engineering can be an important tool in the field
of sustainable agriculture. How is the soil ecological function affected by environmental
changes and how can this function be maintained under sustainable agricultural practices?
How these are shaped by plant and microorganism interactions remains to be elucidated.
Another tool for sustainable agriculture is based on the specificity of plant-microbe com-
munication and relies on plant genome engineering. Therefore, the key to sustainable
agriculture relies on an enhanced comprehension of the complexity of soil–microbe–plant
systems and their impact on short- or long-term sustainability. In light of these facts, future
efforts and research topics should focus on the following: (i). exploitation of the potential of
genetically engineered microbes and plants for sustainable agriculture; and (ii). long-term
effects on the soil and microbiome of the immobilized bioformulations; and (iii). clean and
sustainable practices based on farm-derived product conversion for soil amendments.

Author Contributions: Conceptualization, G.M. and É.L.; investigation, É.-B.V., A.B., G.M. and É.L.;
writing—original draft preparation, É.-B.V., A.B., G.M. and É.L.; writing—review and editing, G.M.,
and É.L.; visualization, É.-B.V. and G.M. All authors have read and agreed to the published version
of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: The authors acknowledge the University of Pécs, Faculty of Sciences, Institute of
Chemistry, Chemical Doctoral School and Collegium Talentum Programme of Hungary for the PhD
scholarship accorded to É.-B.V and A.B.
Conflicts of Interest: The authors declare no conflict of interest.

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