In recent decades, several concepts have emerged to categorize microbial biological inputs according to the functional activity associated with the mechanism of action of PGPM. PGPM are classified into bio-fertilizers (enhance nutrient availability in plants), phyto-stimulants (produce phytohormones), rhizo-remediators (degrade pollutants), and bio-pesticides (produce antimicrobial compounds) [
10]. Therefore, within the bio-fertilizers category, there are grouped biological inputs that contain microorganisms mainly involved in biological nitrogen fixation, phosphorus, zinc, sulfur, potassium, and iron solubilization-mobilization. Phyto-stimulants stimulate root growth through phytohormone production, with emphasis on auxins, cytokinins, and gibberellins producers. Bio-pesticides include microorganisms capable of controlling phytopathogens from the soil or seeds through antibiosis, enzyme production, or induction of systemic resistance.
1.1. Direct Mechanisms for Promoting Plant Growth
Nitrogen constitutes approximately 80% of the gases in the atmosphere, being the fourth most abundant element in living organisms. However, this abundance is not utilized by plants, which can only make use of combined forms found in the soil in small quantities. Biological nitrogen fixation is undoubtedly the most extensively studied direct mechanism, contributing significantly to the total nitrogen supply required by plants. This process can be carried out by free-living bacteria or in symbiosis with plants, involving the transformation of atmospheric nitrogen into a plant-assimilable form through the action of the nitrogenase enzyme complex. Almost 70% of the nitrogen in the form of NH
4+ is derived from legume-rhizobium symbiosis, which can provide up to 90% of the nitrogen required by leguminous crops [
11,
12]. Among the bacterial genera that perform free-living fixation, notable ones include
Azospirillum,
Azotobacter,
Derxia,
Beijerinckia, and
Bacillus. Within the groups forming well-known symbioses, we have Nostoc, Frankia, and Rhizobium, with the latter being the most widely used in the composition of biological inputs around the world.
Bacteria capable of nitrogen fixation that form nodules in legumes are known as rhizobia. This group can be found in seven families of α and β-proteobacteria, divided into 15 genera. Among the α-proteobacteria, notable genera include
Rhizobium [
13],
Mesorhizobium [
14],
Bradyrhizobium [
15],
Sinorhizobium/
Ensifer [
16],
Azorhizobium [
17],
Devosia [
18],
Phyllobacterium [
19] and
Ochrobactrum [
20]. In the β-proteobacteria, species from the genera
Burkholderia [
21] and
Cupriavidus [
22] can be found.
Due to the notable importance of Biological Nitrogen Fixation (BNF), research has been encouraged and various commercial formulations based on bacteria belonging to these genera have been developed. As a result of this process, for example in Brazil, there is an extensive list of bacterial strains with nitrogen-fixing capability that have been evaluated and recommended, mainly for legumes due to their symbiotic efficiency. Worldwide, biological inputs based on nitrogen-fixing bacteria constitute more than 70% of commercialized fertilizers. Another important group comprises solubilizers, which represent approximately 15% of these products [
23].
Phosphorus is the second limiting element for plant growth, after nitrogen. It is abundant in soils in both organic and inorganic forms but is typically found in low availability for plants. The dynamics of phosphorus in soils are complex due to the phenomenon of phosphorus fixation, which involves the transformation of labile phosphorus into non-labile forms. This mechanism is explained by the strong affinity that phosphorus has for Ca
3+, Fe
3+, and Al
3+ ions. As a result, in most soils phosphorus exists in insoluble forms, while plants can only absorb the two soluble forms: monobasic (H
2PO
4-) and dibasic (HPO
42-) forms [
24].
In agriculture, to address this issue, phosphatic fertilizers are used. However, these fertilizers are often lost because they are quickly converted into their insoluble forms. Phosphate-solubilizing microorganisms have been studied for their ability to enhance phosphorus availability through solubilization and/or mineralization. Solubilization occurs through the production of low molecular weight organic acids, such as gluconic acid and citric acid. Several studies demonstrate that inorganic sources of phosphorus, potassium, and other soil nutrients are solubilized by bacterial species like
Achromobacter, Agrobacterium, Azotobacter, Beijerinckia, Bacillus, Burkholderia, Erwinia, Flavobacterium, Microbacterium, Rhizobium, Pseudomonas, and Serratia,, as well as fungal species like
Aspergillus,
Penicillium,
Fusarium,
Chaetomium, and
Cephalosporium [
25,
26].
On the other hand, mineralization is associated with the production of phosphatase enzymes, with bacteria involved in the mineralization of organic phosphorus considered the primary source of this enzyme's activity in soils. Organic phosphorus is present in forms such as inositol phosphate, phospholipids, and nucleic acids, with inositol phosphate being the most abundant and dominant form. Organic phosphorus comprises 30 to 70% of the total phosphorus in agricultural soils and is as important as the pool of inorganic phosphorus in contributing to the available phosphorus for plants [
27].
The availability of phosphorus in the soil can also be strongly influenced by the establishment of symbiosis between roots and Arbuscular Mycorrhizal Fungi (AMF) present in the soil. These microorganisms expand the root absorption area by producing extraradical mycelium, thus contributing to the uptake of immobile elements in the soil, such as phosphorus. They also enhance water absorption under water-deficit conditions. Many factors can affect the benefits of AMF for agriculturally important species, including plant genotypes, cultivars within the same crop, fungal species/isolates, and soil and climatic conditions. This highlights the need to select fungal isolates for symbiotic efficiency specific to the plant species of interest, as well as to evaluate this efficiency under varying soil fertility levels, particularly in assessing phosphorus availability. This is crucial since phosphorus influences the degree of colonization and the benefits for the host plant [
5].
Plant hormones are among the most important regulators of plant growth. They have a direct impact on plant metabolism and interfere with stimulating the plant's defense response against stress. The production of phytohormones by microorganisms is well-established in the literature. Recent studies have shown that microbial phytohormones can be used to induce systemic plant tolerance to stressful environmental conditions. It is known that about 80% of the bacteria inhabiting the rhizosphere produce indole-3-acetic acid (IAA), the most widely studied type of auxin.
Azospirillum spp.,
Azotobacter spp.,
Aeromonas spp.,
Burkholderia spp.,
Enterobacter spp.,
Pseudomonas spp., and
Rhizobium spp. are the main bacterial genera in the rhizosphere capable of synthesizing this phytohormone [
28,
29,
30,
31]. IAA acts on the division and elongation of plant cells, particularly in stimulating the emergence of lateral roots and root hairs. This aspect is of high importance for nutrient absorption and water acquisition, thereby mitigating the effects of abiotic stresses. Furthermore, the production of this phytohormone by microorganisms plays an important ecological role, as it helps establish a communication signal with plants, facilitating the mutualistic benefits for microorganisms through the association. Microorganisms capable of producing IAA are already present in commercial inoculants, contributing to increased productivity of economically important grasses in Brazil [
32].
Cytokinins are also an important group of plant hormones that play a crucial role in maintaining cell proliferation and differentiation and are also associated with the inhibition of premature leaf senescence. Zeatin is the most studied cytokinin. The production of these substances occurs at the root tips and is subsequently transported through the xylem. Cytokinins produced by plant growth-promoting bacteria at low hormonal levels have a stimulating effect on plants. This phytohormone can also participate in root nodule organogenesis, promoting their development. It was observed that cytokinin production in bacterial species of the genera
Arthrobacter,
Bacillus,
Azospirillum, and
Pseudomonas, which stimulated the root development of associated plants [
33].
Gibberellins are another significant group of plant growth regulators. They are known as regulators of reproductive organs and fruit formation. There are more than 89 types of gibberellins, with gibberellic acid being the most common and well-studied. They play a crucial role in cell division and elongation, seed dormancy, and germination, as well as in controlling photosynthesis rate and chlorophyll content in plants.
Acinetobacter,
Alcaligenes Azospirillum, Azotobacter, Bacillus, Bradyrhizobium, Burkholderia, Pseudomonas, Pantoea, and
Rhizobium are some of the bacterial genera where the production of this phytohormone has been observed [
34,
35]. This production has also been noted in endophytic fungi such as
Aspergillus fumigatus [
36]. Several studies have shown that gibberellic acid stimulates plant growth and development under various abiotic stress conditions [
37].
Ethylene is a hydrocarbon gas widely used in agriculture. It acts to inhibit growth, seed germination, onset of flowering, and fruit ripening. Under stress conditions, plants produce ethylene in elevated concentrations, which can lead to plant senescence [
38]. Although ethylene is essential for the growth and development of plant species, it is necessary to regulate its harmful levels [
39]. Among other adverse effects of ethylene, it has been noted that it can act as an inhibitor of nodulation in legumes. There are PGPM that possess mechanisms enabling control through the production of enzymes like rhizobitoxin and 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which participate in the biosynthetic pathway of ethylene [
40].
Rizobitoxin is an enol-ether amino acid produced by bacteria of the genus
Bradyrhizobium. For a long time, it was considered a phytotoxin as it could cause chlorosis in soybean leaves. Later, it was discovered that this substance also strongly inhibits ACC synthase, an enzyme that is a key factor in the biosynthesis of ethylene. Rizobitoxin plays a positive role in the establishment of symbiosis between
Bradyrhizobium elkanii and symbiotic legumes, acting to reduce the endogenous levels of ethylene in the roots. Currently, it is known that
rhizobitoxine production is restricted
to B. elkanii strains [
41,
42]. For this reason, several studies have questioned what mechanisms other microbial species might use to counteract the deleterious effects of ethylene on plants. As a result, the enzyme ACC deaminase (E.C. 3.5.99.7) was discovered, responsible for the irreversible conversion of ACC, the immediate precursor of ethylene, into ammonia and α-ketobutyrate [
43]. This enzyme has been detected in various microbial genera, but its distribution is not uniform as it can be absent in organisms of the same genus and species. According to Glick [
44], there are at least two direct consequences resulting from the decrease in ACC levels within the plant: a reduction in ethylene levels and the subsequent decrease in growth inhibition and cell proliferation. Consequently, plants that establish symbiotic relationships with growth-promoting bacteria possessing ACC deaminase enzyme activity may develop longer roots and potentially exhibit higher aboveground growth.
1.2. Indirect Mechanisms for Promoting Plant Growth
Some microorganisms also can promote plant growth indirectly by preventing or reducing damage caused by phytopathogens. Among the most studied indirect mechanisms are: competition for nutrients and space, synthesis of siderophores, antibiotics, hydrogen cyanide (HCN), toxins, bacteriocins, and hydrolytic enzymes. Additionally, the synthesis of volatile organic compounds and plant hormones such as salicylic acid and jasmonic acid, and the modulation of ethylene levels contribute to systemic resistance in many plant species.
Competition for nutrients and space is an important biocontrol mechanism. This occurs because both beneficial microorganisms and phytopathogens can colonize the same niches and utilize the same nutrients [
45]. Biological control through nutrient competition occurs by locally and temporally increasing highly competitive strains during critical stages of the pathogen's life cycle. The control takes place without any direct interaction between the two organisms, as the antagonist acts by producing enzymes that more rapidly degrade complex organic matter, utilizing simple carbohydrates and amino acids more quickly, or producing siderophores in case of iron competition [
46]. Thus, these strains modulate the growth environment in the target niche, making the conditions less favorable for the pathogen's development.
Spadaro and Droby [
45] described how the use of fast-colonizing yeasts is an effective mechanism against pathogen invasion. In their study, they analyzed the competition processes between
Pichia guilliermondii and the pathogens
Penicillium digitatum,
P. expansum,
Botritis cinerea, or
Colletotrichum spp. on wounds of different fruits. Yeasts, as unicellular organisms, are capable of rapidly multiplying under favorable conditions in nutrient-rich fruit wounds, making it challenging for the pathogen to establish itself.
Siderophores are chelating secondary metabolites that function by binding to Fe
3+ and transporting it across the bacterial cell membrane under conditions of limited iron availability [
47]. Siderophores produced by plant growth-promoting bacteria act as biocontrol agents, as their high affinity for Fe can prevent phytopathogens from acquiring the necessary amount of this element, thus limiting the pathogen's infection capacity [
48,
49]. Various species within the bacterial genus
Pseudomonas have frequently been described as siderophore producers, reducing the population of pathogens in the rhizosphere [
50,
51]. Another example was described by Segarra et al. [
52] who found that the fungus
Trichoderma asperellum can produce siderophores, thereby controlling diseases caused by
Fusarium sp.
Antibiosis is the ability to produce antimicrobial compounds that suppress or reduce the growth and/or proliferation of phytopathogens. These are mostly products of secondary metabolism belonging to heterogeneous groups of low molecular weight organic compounds, including antibiotics, volatile compounds, and cell wall-degrading enzymes, among others [
53,
46]. Characterization studies and elucidation of the mode of action of these compounds have formed the basis for the selection and commercialization of some strains for biological control. The production of these metabolites has been described in bacterial genera such as
Agrobacterium,
Bacillus,
Pantoea,
Pseudomonas,
Serratia,
Stenotrophomonas, and
Streptomyces; and fungal genera such as
Trichoderma,
Purpureocillium,
Boletus,
Suillus,
Chroogomphus,
Xerocomus,
Pisolithus,
Russula, and
Scleroderma [
54]. Some of the most studied examples include iturin, surfactin, fengycin, 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin, and phenazine from bacteria [
55,
56], and trichodermin, trichodermol, gliovirin, gliotoxin, viridin, and leucinostatins from fungi [
57,
58]. Among the prominent lytic enzymes are chitinases, cellulases, xylanases, pectinases, glucanases, lipases, amylases, arabinases, and proteases (Ghorbanpour et al., 2018), which can lyse the cell wall of phytopathogens such as
Botrytis,
Fusarium,
Phytophthora,
Pythium,
Plectosporium,
Rhizoctonia,
Sclerotium, and
Verticillium, among others [
59,
60].
PGPM can also trigger a phenomenon in plants known as induced systemic resistance (ISR), similar to systemic acquired resistance (SAR), where plants activate their defense mechanisms, playing a significant role in suppressing pathogens. This defense mechanism doesn't target specific pathogens, making it particularly interesting in controlling diseases caused by various agents. Bacterial molecules like the O-antigenic side chain of bacterial LPS, flagellar proteins, pyoverdine, chitin, β-glucans, cyclic lipopeptide surfactants, and salicylic acid have been described as signals for inducing this type of systemic resistance [
61]. It is relevant to highlight that categorizing PGPM into groups based on their mechanisms of action is a methodological exercise to meet regulatory requirements for registration in Brazil. In reality, in nature, interactions occur dynamically, and many times a microorganism possesses different mechanisms of action that work simultaneously, resulting in biocontrol.