**2. Effects and mechanisms of plant growth promotion by microorganisms**

As mentioned above, some bacteria are capable of promoting plant growth (PGPB and PGPR) through direct (biofertilization) and indirect (biocontrol) mechanisms. The direct mechanisms are based on the direct promotion of plant development, among which are nitrogen fixation, phosphate solubilization, production of phytohormones (auxins, cytokinins, and gibberellins), the enzymatic activity of the aminocyclopropane carboxylic acid (ACC) deaminase, and iron complexation by bacterial siderophores. On the other hand, indirect methods are responsible for inhibiting pathogenic organisms for plants. Among these methods are the synthesis of antibiotics, enzymes that degrade the cell wall, or the induced systemic resistance (ISR) process [42]. The mechanisms carried out by PGPB and PGPR will depend on the host plant and will be influenced in turn by biotic (such as plant defense mechanisms and genotype) and abiotic (such as weather conditions and soil composition) factors [43].

However, bacteria are not the only microorganisms that are able to promote plant growth. Mycorrhizal fungi also can carry out this function, there being a symbiotic association between them and most terrestrial plants [44] favoring micronutrient absorption, resistance to diseases caused by pathogens, or reduction of plant stress caused by environmental factors [45].

#### **2.1 Direct mechanisms**

*Legume Crops - Prospects, Production and Uses*

NH4 +

fixation [25, 26].

of the plants being able to do it by direct or indirect mechanisms. Regarding legumes, they also interact with rhizobacteria, and the main interaction is the symbiosis between legumes and nitrogen-fixing rhizobia [4]. Rhizobia are bacteria that belong to the family Rhizobiaceae, and the most important genera are *Bradyrhizobium*, *Ensifer*, *Mesorhizobium*, *Rhizobium*, and *Sinorhizobium* [6]. During this interaction, rhizobia invade the root and group into a specialized organ called nodule. Inside the nodules, rhizobia become bacteroids, which transform N2 to

, molecule that can be assimilated by plants [7]. Inoculation of legumes with

Climate change is one of the most important problems in the planet nowadays. Because of that, temperature and drought are increasing, involving an increase in arid and semi-arid zones and generating a loss of arable soils [12–14]. Drought is an abiotic stress that causes the highest losses in agriculture, so it is a very important factor in crop productivity [15]. Drought and heat involve the appearance of saline soils [16], although some human activities, like the increase of irrigation with bad water quality, are also responsible [17]. Salinity affects around 800 million of hectares in the world, and it is considered a global problem [18] being another stress that limits plant growth, productivity, nitrogen fixation in legumes, and the seed germination [19, 20] due to the uptake excess of NaCl by the plants [21]. Furthermore, the salt excess decreases the organic matter in soils and modifies the microbial population in the rhizosphere [22], so salinity also affects the nodulation negatively [23]. Finally, an additional abiotic stress is heavy metals. The increase of heavy metal concentrations becomes a pollution problem, being humans the main responsible of it [24]. Heavy metals affect plants and soils as the rest of abiotic stress does, and in legumes, they dramatically reduce nodule number and nitrogen

To try to recover these affected soils, phytoremediation is being used, and several studies confirm that it is a very efficient tool, particularly in combination with bacterial inoculation since PGPB improve the potential of plants to phytoremediate soils [27–31]. Legumes belong to the plants used in phytoremediation because this family is one of the most diverse among other plant families in the world, and some of them are able to grow in degraded soils and can be used as pioneer plants in order to repopulate these degraded areas [25, 32]. In fact, legumes are usually used in intercropping with other crops to decrease the amount of pesticides and improve the quality of soils making legumes one of the most promising components of the Climate Smart Agriculture concept [33]. As described above, the symbiosis of legumes with rhizobia improves the growth of legumes and allows them to grow better in the degraded soils, but all the named abiotic stresses interfere with this interaction. For that, authors look for rhizobia resistant to these stresses that able to grow and form nodules even in degraded soils [34–37]. Furthermore, several studies demonstrate that coinoculation of legumes with rhizobia and another PGPR increases nodulation, plant growth, and the potential to phytoremediate soils of plants in the presence of abiotic stresses [38–41]. This improvement in legumes occurs for the interaction between plants and bacteria through different direct and

indirect mechanisms that help the plant to grow in the presence of stress.

In this chapter, the different bacterial mechanisms used to improve the plant growth in the presence of the most important abiotic stresses nowadays are been reviewed, in order to help legumes to grow under stress situations and recover the degraded soils using the interaction between legumes and bacteria. Furthermore,

rhizobacteria produces a bioaugmentation of the microbial population in soils, thus contributing to plants growing bigger and faster [8–10], and this can solve the problem of the rapid growth of the world population that causes a great pressure in the area of land destined for food [11]. Moreover, the arable lands are decreasing

because of the climate change and some human activities.

**116**

#### *2.1.1 Fixation of nitrogen*

Nitrogen is one of the essential elements for life that is present in biochemical structures as important as nucleotides and proteins, but atmospheric nitrogen is mostly nonassimilable for plants since about 78% is in a gaseous state, so it must become ammonia, thanks to the nitrogenase enzyme to be assimilable. This reaction is carried out by rhizobia under symbiosis with legumes, thanks to which the rhizobia obtain carbon provided by the legumes from photosynthesis, and they provide the plant with nitrogen [46].

Atmospheric nitrogen fixation occurs mainly in leguminous plants where rhizobia/plant interactions are highly specific [42]. However, certain free-living bacteria (such as *Frankia* spp. or Actinobacteria) are also able to fix atmospheric nitrogen to a much wider range of plants than rhizobia [47]. For example, coinoculation of *Bradyrhizobium* sp. UFLA 03-84 with *Actinomadura* sp. 183-EL, *Bacillus* sp. IPACC11, or *Streptomyces* sp. 212 in cowpea plants improves the nitrogen fixation even in the presence of salinity conditions [48]. Another example of the improvement of nitrogen fixation in the presence of a different abiotic stress is reported by Saia et al. [49] in which plants of *Trifolium alexandrinum* were inoculated with arbuscular mycorrhizal (AM) fungi leading to an improvement of nitrogen fixation and the plant growth in water stress.

#### *2.1.2 Solubilization of phosphate*

Phosphorus is also an essential element for life that is involved in such important processes as energy transfer, respiration, or photosynthesis [50]. After

nitrogen, phosphorus is the second most limiting element for plant growth [51]. The content of this element in the soil is 0.05% (w/w) of which only 0.1% can be used by plants, a problem to which the use of phosphorus-based chemical fertilizers that are fixed within the soil must be added and limited the bioavailability of the element [52].

There are a wide variety of microorganisms that can solubilize phosphate to make it assimilable to plants by releasing phosphorus from soil insoluble phosphates. An example of this is the endophytic bacterium *Gluconacetobacter diazotrophicus* that is capable of solubilizing phosphate by acidification [53]. Bacteria also can solubilize phosphates in the presence of different stresses such as *Serratia* sp. J260, *Pantoea* sp. J49, *Acinetobacter* sp., L176, and *Planomicrobium* sp. MSSA-10 that kept this property in the presence of salinity conditions or *Bacillus* sp. L55 in the presence of high temperatures [54, 55]. Besides, other bacteria are able to solubilize organic phosphorous because they produce phytase and phosphatases enzymes that act by dephosphorylating phytates and organic phosphorus [51]. This kind of bacteria can help to legume crops to improve their growth and productivity in these abiotic stress conditions.

#### *2.1.3 Phytohormones production*

There are certain microorganisms capable of producing phytohormones, which can promote or modify the development of plants at low concentrations [43]. Among the most common phytohormones are gibberellins, cytokinins, auxins, ethylene, and abscisic acid [53], and their effects can be stimulants or inhibitors of plant growth. The most widely studied are auxins, particularly the indole-3-acetic acid (IAA), being the one that is produced in a greater proportion by plants and PGPR where up to 80% of rhizobacteria synthesize it as a secondary metabolite [56]. This auxin is present in different cellular responses such as cell division, gene expression, or root development and lengthening and affects photosynthesis, pigment formation, and resistance to stress conditions [53].

When the concentration of IAA in plants is adequate, the application of bacterial IAA can have positive, negative, or neutral effects [46], so that this bacterial synthesis will determine the stimulation or inhibition of plant growth and may change the hormone level from optimal to supraoptimal. In this way, the PGPR will stimulate growth when the IAA concentration is below the optimum levels for the plant [42]. It is important that IAA producing bacteria must keep the property even in the presence of any abiotic stress to help plants to grow in these conditions. **Table 1** shows some examples of IAA producing microorganisms that cause improvements in legume crops under stress conditions.

#### *2.1.4 ACC deaminase activity*

Ethylene, also known as the stress hormone, is a phytohormone present in all higher plants, making it a key element in a wide range of biological activities, intervening in processes that affect the growth and development of plants where almost all plant tissues and their stages of development are affected by it. It is produced from 1-aminocyclopropane-1-carboxylic acid (ACC) and is catalyzed by the enzyme ACC oxidase [59, 60]. Most abiotic stresses cause a large increase in ethylene concentration causing wilting of the flowers or initiating senescence in the leaves among other consequences, so its increase translates into harmful effects on the growth and health of plants and therefore to a reduction in crop production. However, when its concentration is adequate, it decreases the wilting, stimulates the germination of seeds, and influences the fruit ripening [61]. More recently,

**119**

*Helping Legumes under Stress Situations: Inoculation with Beneficial Microorganisms*

**Microorganism Plant host Plant improvement Abiotic** 

*Medicago sativa* Larger root size Heavy

Improved the growth and increased the nodulation

and Zn and increased the

osmoprotectants, greater Rubisco availability

Higher salt tolerance and osmoprotectants

and other growth features

nodulation

*Glycine max* Improved the plant biomass

**stress**

metals

Heavy metals

Salinity [57]

Drought [8]

Salinity [9]

Heat [10]

**References**

[37]

[58]

evidence is being presented showing that ACC deaminase producing microorganisms can facilitate nodulation of legumes under stress conditions, called as nodulat-

*Beneficial interactions between IAA producing bacteria and legumes under stress conditions.*

The great importance of this hormone is the reason for it having being extensively studied, so a wide range of microorganisms capable of secreting the enzyme ACC deaminase is known. ACC deaminase hydrolyses ACC in ammonia and 2-oxobutanoate, thus causing a reduction in the concentration of ethylene in plants [64]. Among ACC deaminase producing microorganisms are the following genera: *Bacillus*, *Pseudomonas*, *Klebsiella*, *Serratia*, *Arthrobacter*, *Azospirillum*, *Streptomyces*,

*Alcaligenes*, *Enterobacter*, *Agrobacterium*, and *Rhizobium* [59, 60]. The interaction of these microorganisms with legumes enhances plant growth and crop productivity

Iron is another essential micronutrient for plants that, in the oxygen-rich conditions of the rhizosphere, is in the form of Fe+3 that is insoluble for plants and microorganisms [53]. Siderophores, involved in both direct and indirect mechanisms of plant growth promotion, are small molecules of a peptide nature formed by side chains and functional groups that act as ligands with high affinity for the Fe+3 ions [65]. A wide range of bacteria and fungi are capable of secreting siderophores that occur in rhizospheric soils in neutral-alkaline pH conditions, where there is a deficiency of this element due to its low solubility in these conditions [66]. These microorganisms can subsequently absorb the siderophore-Fe+3 complex by means of a specific receptor and release the Fe in its bioavailable form (Fe+2) to support bacterial growth [61]. The creation of this complex also assumes an important role in the adsorption of Fe by plants, in the presence of other metals such as nickel or cadmium [43]. Another very important function of siderophores is to prevent the proliferation of pathogens by competing for the available iron. In this way, rhizobacteria help plant growth by releasing these biocontrol agents against

phytopathogens (antagonism of PGPR against pathogens) [61].

*Microbacterium*, *Achromobacter*, *Acinetobacter*, *Acidovorax*, *Agrobacterium*,

*DOI: http://dx.doi.org/10.5772/intechopen.91857*

*Galega officinalis*

*Ensifer meliloti* RD64 *M. sativa* Accumulation of

*Stylosanthes guianensis*

*Bradyrhizobium* sp. RM8 *Vigna radiata* Reduced the uptake of Ni

*Ochrobactrum cytisi*

*Bradyrhizobium* sp.

*Aspergillus japonicus*

RJS9–2

EuR-26

**Table 1.**

*extremorientalis* TSAU20 and *P. trivialis* 3Re27

Azn6.2

*Pseudomonas* 

ing helper bacteria [62, 63].

under stress conditions (**Table 2**).

*2.1.5 Production of siderophores*

*Helping Legumes under Stress Situations: Inoculation with Beneficial Microorganisms DOI: http://dx.doi.org/10.5772/intechopen.91857*


#### **Table 1.**

*Legume Crops - Prospects, Production and Uses*

the element [52].

abiotic stress conditions.

*2.1.3 Phytohormones production*

nitrogen, phosphorus is the second most limiting element for plant growth [51]. The content of this element in the soil is 0.05% (w/w) of which only 0.1% can be used by plants, a problem to which the use of phosphorus-based chemical fertilizers that are fixed within the soil must be added and limited the bioavailability of

There are a wide variety of microorganisms that can solubilize phosphate to make it assimilable to plants by releasing phosphorus from soil insoluble phosphates. An example of this is the endophytic bacterium *Gluconacetobacter diazotrophicus* that is capable of solubilizing phosphate by acidification [53]. Bacteria also can solubilize phosphates in the presence of different stresses such as *Serratia* sp. J260, *Pantoea* sp. J49, *Acinetobacter* sp., L176, and *Planomicrobium* sp. MSSA-10 that kept this property in the presence of salinity conditions or *Bacillus* sp. L55 in the presence of high temperatures [54, 55]. Besides, other bacteria are able to solubilize organic phosphorous because they produce phytase and phosphatases enzymes that act by dephosphorylating phytates and organic phosphorus [51]. This kind of bacteria can help to legume crops to improve their growth and productivity in these

There are certain microorganisms capable of producing phytohormones, which

When the concentration of IAA in plants is adequate, the application of bacterial IAA can have positive, negative, or neutral effects [46], so that this bacterial synthesis will determine the stimulation or inhibition of plant growth and may change the hormone level from optimal to supraoptimal. In this way, the PGPR will stimulate growth when the IAA concentration is below the optimum levels for the plant [42]. It is important that IAA producing bacteria must keep the property even in the presence of any abiotic stress to help plants to grow in these conditions. **Table 1** shows some examples of IAA producing microorganisms that cause improvements

Ethylene, also known as the stress hormone, is a phytohormone present in all higher plants, making it a key element in a wide range of biological activities, intervening in processes that affect the growth and development of plants where almost all plant tissues and their stages of development are affected by it. It is produced from 1-aminocyclopropane-1-carboxylic acid (ACC) and is catalyzed by the enzyme ACC oxidase [59, 60]. Most abiotic stresses cause a large increase in ethylene concentration causing wilting of the flowers or initiating senescence in the leaves among other consequences, so its increase translates into harmful effects on the growth and health of plants and therefore to a reduction in crop production. However, when its concentration is adequate, it decreases the wilting, stimulates the germination of seeds, and influences the fruit ripening [61]. More recently,

can promote or modify the development of plants at low concentrations [43]. Among the most common phytohormones are gibberellins, cytokinins, auxins, ethylene, and abscisic acid [53], and their effects can be stimulants or inhibitors of plant growth. The most widely studied are auxins, particularly the indole-3-acetic acid (IAA), being the one that is produced in a greater proportion by plants and PGPR where up to 80% of rhizobacteria synthesize it as a secondary metabolite [56]. This auxin is present in different cellular responses such as cell division, gene expression, or root development and lengthening and affects photosynthesis, pig-

ment formation, and resistance to stress conditions [53].

in legume crops under stress conditions.

*2.1.4 ACC deaminase activity*

**118**

*Beneficial interactions between IAA producing bacteria and legumes under stress conditions.*

evidence is being presented showing that ACC deaminase producing microorganisms can facilitate nodulation of legumes under stress conditions, called as nodulating helper bacteria [62, 63].

The great importance of this hormone is the reason for it having being extensively studied, so a wide range of microorganisms capable of secreting the enzyme ACC deaminase is known. ACC deaminase hydrolyses ACC in ammonia and 2-oxobutanoate, thus causing a reduction in the concentration of ethylene in plants [64]. Among ACC deaminase producing microorganisms are the following genera: *Bacillus*, *Pseudomonas*, *Klebsiella*, *Serratia*, *Arthrobacter*, *Azospirillum*, *Streptomyces*, *Microbacterium*, *Achromobacter*, *Acinetobacter*, *Acidovorax*, *Agrobacterium*, *Alcaligenes*, *Enterobacter*, *Agrobacterium*, and *Rhizobium* [59, 60]. The interaction of these microorganisms with legumes enhances plant growth and crop productivity under stress conditions (**Table 2**).

### *2.1.5 Production of siderophores*

Iron is another essential micronutrient for plants that, in the oxygen-rich conditions of the rhizosphere, is in the form of Fe+3 that is insoluble for plants and microorganisms [53]. Siderophores, involved in both direct and indirect mechanisms of plant growth promotion, are small molecules of a peptide nature formed by side chains and functional groups that act as ligands with high affinity for the Fe+3 ions [65]. A wide range of bacteria and fungi are capable of secreting siderophores that occur in rhizospheric soils in neutral-alkaline pH conditions, where there is a deficiency of this element due to its low solubility in these conditions [66]. These microorganisms can subsequently absorb the siderophore-Fe+3 complex by means of a specific receptor and release the Fe in its bioavailable form (Fe+2) to support bacterial growth [61]. The creation of this complex also assumes an important role in the adsorption of Fe by plants, in the presence of other metals such as nickel or cadmium [43]. Another very important function of siderophores is to prevent the proliferation of pathogens by competing for the available iron. In this way, rhizobacteria help plant growth by releasing these biocontrol agents against phytopathogens (antagonism of PGPR against pathogens) [61].


#### **Table 2.**

*Beneficial interactions between ACC deaminase producing bacteria and legumes under stress conditions.*


#### **Table 3.**

*Beneficial interactions between bacteria that produce siderophores and legumes and plant improvements under stress conditions.*

In general, plants can benefit in many ways, thanks to the production of siderophores since they are also involved in the improvement of nitrogen fixation or in the prevention of toxicity by heavy metals [75]. Thus, PGPR able to produce siderophores have a certain competitive advantage over other microorganisms in the rhizosphere [64]. Some of these microorganisms are shown in **Table 3** showing the repercussions caused by the synthesis of iron chelating compounds depending on the crop and the conditions of the plant-microbe interaction.

#### **2.2 Indirect mechanisms**

Indirect mechanisms are those processes where PGPR prevent or neutralize the harmful action of phytopathogens by the production of substances that confer

**121**

**Table 4.**

*Helping Legumes under Stress Situations: Inoculation with Beneficial Microorganisms*

that produce antibiotics against phytopathogens reflected in **Table 4**.

the soybean against *S. sclerotiorum* under drought stress [78].

*Rhizobium* sp. RS12 *C. arietinum Fusarium solani* and

*Pseudomonas* sp. YL23 *G. max Erwinia amylovora* and *Dickeya* 

*Pantoea ananatis* RM2 *P. sativum Trichoderma longibrachiatum* and

*Phaseolus vulgaris*

*PGPR that produce antibiotics and their effects as biocontrol agents.*

greater natural resistance to the host plant, protecting it against infections (biotic stress), although they also help plants to grow actively under conditions of environ-

Antibiotic production is the main mechanism by which a large and heterogeneous group of bacteria fight the harmful effects of plant pathogens (usually fungi). The antibiotics produced by PGPR are low molecular weight compounds that negatively interfere with the metabolic processes of other microorganisms, thus delaying their growth [64] or inhibiting it [56]. There are some examples of PGPR

The effectiveness with which these molecules interfere with pathogen suppression will depend on the metabolite secreted by the PGPR and environmental conditions (mineral content of the soil, osmotic conditions, carbon sources, etc.) [76]. Moreover, some phytopathogens may develop resistance to specific antibiotics by repeated use of the same strain that produces a particular antibiotic, so it is preferable to inoculate plants with PGPR that produce several antibiotics [59]. There are some PGPR that have antagonistic activities against some phytopathogens in addition to improve plant growth in the presence of some stress as it is the case of *Cellulosimicrobium funkei* AR6 that improves the root elongation in crops of *P. vulgare*, *V. radiata,* and *V. mungo* in the presence of Cr(VI) and also has a strong antagonistic activity against *Aspergillus niger* [77]. Another example is *B. thuringiensis* UFGS2 that improves plant growth, physiologic parameters, and the resistance of

Some PGPR produce enzymes that are involved in the lysis of cell walls and neutralization of pathogens by interrupting a particular stage of development or the cell cycle [79], playing an important role in promoting plant growth by protecting them of biotic and abiotic stresses due to the suppression of these pathogens. Among the produced enzymes for this purpose are chitinases, dehydrogenases, β-glucanases, lipases, phosphatases, or proteases [59]. The cell wall of most fungi is formed by residues of β-1,4-N-acetyl-glucoseamine and chitin, so that the bacteria that produce β-1,3-glucanase and chitinase can control the growth of phytopathogen [43]. Furthermore, some PGPR are able to produce this kind of enzymes and protect the crops under abiotic stress like *Bacillus licheniformis* A2 that produces

**Microorganism Plant host Phytopathogen Reference**

*Macrophomina phaseolina*

*chrysanthemi*

*Fusarium oxysporum*

*Sclerotinia sclerotiorum* [83]

[80]

[81]

[82]

*DOI: http://dx.doi.org/10.5772/intechopen.91857*

mental stress (abiotic stress) [43].

*2.2.2 Lytic enzymes of the cell wall*

*Bacillus* sp. B19, *Bacillus* sp. P12, and *B. amyloliquefaciens* B14

*2.2.1 Production of antibiotics*

greater natural resistance to the host plant, protecting it against infections (biotic stress), although they also help plants to grow actively under conditions of environmental stress (abiotic stress) [43].
