Atmospheric nitrogen is reduced to ammonia (NH
3) 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 N
2 [36][26]. After photosynthesis, BNF is the second most important process on Earth, due to its significant role in agroecosystem sustainability
[34,35][27][28].
The nitrogenase activity of nitrogen-fixing microorganisms is responsible for BNF, whereas atmospheric N
2 is reduced to ammonia. Bacteria with BNF capacity are categorized into three groups: free-living, associated, and symbiotic bacteria. Free-living N
2 fixing bacteria belong to different genera such as
Gluconacetobacter,
Azospirillum, and
Azotobacter 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 N
2 in non-leguminous plants (including cereals). The nitrogenase complex is a two-component metalloenzyme.
Rhizobium bacteria are symbiotic bacteria linked to leguminous plants. The non-symbiotic or free-living type N
2-fixing bacteria are cyanobacteria (blue-green algae,
Anabaena,
Nostoc) and other species belonging to different genera, such as
Azotobacter,
Beijerinckia, 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.
Phytohormone Production
Phytohormones are organic compounds that influence physiological processes in plants even at very low concentrations. The ability of soil microbiota to produce phytohormones 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.
Phytohormones play a significant role in plant growth during cell division and enlargement, 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.
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][29].
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][30]. 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][29].
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 desiccation
[39][30]. Several plant-associated bacteria can produce ABA, thereby increasing phytohormone 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][29]. ABA-producing endophytic bacteria include
Achromobacter xylosoxidans,
Brevibacterium halotolerans,
Bacillus licheniformis,
Bacillus pumilus, and
Lysinibacillus fusiformis [42][31].
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][30]. The first described bacterium with GA production ability was
Rhizobium phaseoli, which produces GA1 and GA4.
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][14][28].
Pseudomonas fluorescens and
Rhizobium spp. are cytokinin-producing bacteria
[43][32].
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 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.
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][33][34].
Solubilization of Major Nutrients
Soil microorganisms play a major role in nutrient cycling. The crop residues incorporated 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][33].
Among the macronutrients, phosphorus (P) is essential for plant growth and development. 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][29][35].
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 orthophosphate. 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][29]. The organic acids most frequently produced by Gram-negative PGPRs are gluconic acid and 2-ketogluconic acid.
PGPRs can release from soil organic and inorganic phosphorus by producing several enzymes, such as phytases, phosphatases, phosphanatases and lyases
[38][29]. 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][14].
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 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.
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][29][36]. 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][33].
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][29].
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][36]. 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.
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][37][38]. 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][39].
Microbial control agents can exert their plant-protecting characteristics based on their mode of interaction with pathogens through direct and indirect mechanisms (
Figure 3).
Figure 3. Indirect and direct mechanisms of biocontrol agents. Indirect methods for biocontrol agents include induced systemic resistance and plant growth-promoting mechanisms. Biocontrol agents directly protect plants through antimicrobial metabolites and bacterial interactions. Arrows indicate the direction of the relationship.
Antibiotics
The production of antibiotics by various microorganisms is a biological control mechanism. 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][40][41].
Interference of Quorum Sensing with Virulence
Quorum sensing is a cell–cell communication process in bacteria that involves extracellular signaling molecules (autoinducers) and serves to share information about cell density. Because many processes are advantageous only in this group, when the bacterial population 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 colonization and expression of virulence factors in plant pathogenic strains, such as
Pseudomonas syringae,
Pantoea stewartii,
Erwinia chrysanthemi, and
Burkholderia glumae [66,67,68,69][42][43][44][45].
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][33][36].
Induced Systemic Resistance (ISR)
Disease control by various beneficial bacterial strains involves the induction of systemic resistance. Different microbial metabolites and biocontrol agents can generate an immune response in the host plant, resulting in systemic disease resistance
[46][33]. 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
Bacillus amyloliquefaciens,
B. atrophaeus,
B. cereus,
B. megaterium,
B. subtilis,
Paenibacillus alvei,
Pseudomonas fluorescens,
Pseudomonas aeruginosa, and
Streptomyces pactum [82][46].
2.4. Plant-Beneficial Function Encoding Gene Clusters and Mobile Genetic Elements
Horizontal gene transfer (HGT) is an event in which bacteria incorporate advantageous 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][47]. Up to 20% of the bacterial genome is disseminated during horizontal gene transfer events
[84][48]. 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][49][50].
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][50][51]. 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][49].
HGT is a common strategy for changing adaptation-related genes, such as those related to antibiotic resistance and heavy metal resistance among soil bacteria.
Plant growth-promoting rhizobacteria possess more than one beneficial function as a result of gene accumulation in the rhizosphere and soil environment governed by selection mechanisms. The major function genes related to plant-beneficial function are as follows: (i). nitrogen fixation-contributing
nifHDK genes (encoding nitrogenase), (ii). Mineral 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 decarboxylase/phenylpyruvate decarboxylase), (v). antimicrobial compound synthesis
hcnABC/phlACBD (hydrogen cyanide/2,4-diacetylphloroglucinol) genes, and (vi). induced systemic resistance conferring
budAB/budC (acetoine/2,3-butanediol) genes.
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. Synergistic processes between ACC deaminase and IAA production and N
2 fixation
[89[52][53][54][55][56][57][58][59],
90,91,92,93,94,95,96], ACC deaminase and IAA production and stress tolerance
[93[56][60],
97], and phosphate solubilization and ACC deaminase
[98][61] were reported.
The role of ACC deaminase and IAA in BNF fixation process is complex; they can enhance nodule formation, improve the competitiveness of rhizobia for nodulation, suppress nodule senescence, and upregulate genes associated with legume–rhizobia symbiosis
[96][59].
The role of ACC deaminase in nodule formation was studied using either knockout or overproducing strains for the ACC deaminase-producing gene
[96][59]. 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][52]. 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 N
2 fixation regulator
NifA2 [99][62]. In senescent nodules, increased gene expression of
PsACS2 encoding ACC synthase (an enzyme involved in ethylene synthesis) and increased transcription have been observed
[90][53]. Therefore, ACC deaminase-producing PGP bacteria can enhance N
2 fixation by extending the lifespan of functional nodules.
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][63][64]. 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 bioformulations
[101,102][63][64].
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 significant advantages over the above-mentioned formulations
[101,102][63][64]. Microbial cells are immobilized by adhesion/biofilm formation on solid supports or entrapment, thereby conferring a protective environment for bacterial cells
[103][65].
The use of environmentally friendly biopolymer matrices is well suited to sustainable agriculture. Microbial cells are encapsulated using various techniques such as ionic gelation, emulsification, and spray drying
[102][64]. 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][66][67].
Alginates are the most widely studied microbial carriers, mainly for
Azospirillum sp. and
Pseudomonas sp.
[102][64]. Alginate bead-entrapped
A. brasilense showed better viability during prolonged storage
[104][68]. Calcium alginate microspheres have been used for
Trichoderma viride spore encapsulation and provided a supportive environment for growth
[109][69].
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][70]. Rhizosphere microbiome engineering has gained much attention in advanced agricultural research
[113,114][71][72].
Microbiome engineering uses a microbe-focused approach that is based on constructing 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][71].