*4.1.1 Nutrient enrichment by Nitrogen fixation*

Nitrogen is a macronutrient required by the plants for synthesizing proteins, nucleic acids and enzymes. Plants synthesize their food with the help of chlorophyll and nitrogen forms an essential component of chlorophyll. Despite the fact that the atmospheric air comprises of about 78% of nitrogen N, this gas is not available for use by the plants directly. Nitrogen application to crops has led to an enormous increase in food production which has eventually resulted in increased human population. Haber-Bosch process being the source of industrial nitrogen fertilizers, has been regarded as the primary cause of explosive growth in human population [79]. Currently, large amounts of synthetic chemical fertilizers are being used in agriculture and these fertilizers have been used beyond their limits, moreover they are expensive and polluting. Application of chemical fertilizers liberates reactive nitrogen into the atmosphere which leads to emission of green-house gases and at the same time eutrophication of water bodies. The detrimental effects of fertilizer use become much more pronounced when these are applied injudiciously. The economic and most importantly environmental concerns make the use safer and relatively cheaper alternatives necessary. Biological nitrogen fixation, whether symbiotic or non-symbiotic is a potential alternative promoting plant growth and hence increasing production [80]. Plant growth promoting-rhizobia are able to perform biological nitrogen fixation (BNF) and thus help plants in nitrogen assimilation. They live in soil and after producing specialized structures (nodules) in legumes by infecting their roots, they fix the atmospheric nitrogen (N2) and convert the same into a more readily useable form i.e., ammonia (NH3) so that the plants can utilize it for their growth. These rhizobia in turn get organic acids which serves as a source of carbon and energy. Two classes of genes: 1. Nodulation (nod) genes and 2. nitrogen fixation (nif) genes are needed

*DOI: http://dx.doi.org/10.5772/intechopen.102657 Potential Applications of Rhizobacteria as Eco-Friendly Biological Control, Plant Growth…*


#### **Table 2.**

*Genes involved in nitrogen fixation.*

for the establishment of a good association between rhizobia and plants. Bacterial genes present in plasmids, code for Nod and Nif proteins [81]. Mainly three nod genes namely nodC, nodB and nodA are involved in nitrogen fixation. In addition to this, other nod genes viz., nod, nol or noe have been found in some rhizobial species [82]. Nodulation genes code for the enzymes involved in production of nodulation factors (nod) [77]. The roots of leguminous plants produce flavonoids in the rootzone, these compounds stimulate the expression of nod genes in the bacteria. Their expression in turn produces the Nod factor, which is a lipochito-ologosachharidic nodulation signal. This signal triggers mitosis and nodule formation [83]. Nitrogen fixation genes include genes for nitrogenase. Nitrogenase forms the most important part of BNF. The enzyme has 2 components: a. dinitrogenase reductase and b. dinitrogenase. The former gives electrons to the later which reduces N2 to NH3. BNF involves different clusters of genes for nitrogen fixation and nodule formation in leguminous plants (**Table 2**) [77].

#### *4.1.2 Phosphate solubilization*

Phosphorus is another macronutrient essential for proper development of plants. Its deficiency can adversely affect plant growth. After nitrogen phosphorous is the most limiting nutrient for plant growth [84]. Phosphorus forms an integral part

of DNA and RNA, enzymes and phospholipids. Besides this, important processes like photosynthesis, formation of roots, flowers, ability of plants to cope up with diseases depend on the optimal levels of phosphorus [85, 86]. Although the soils are naturally rich in phosphorous reserves but the amount that is available to plants for their use is only a small fraction of the original amount present. This is because phosphorus is predominantly present in insoluble forms in soil and plants can only make use of phosphorus in soluble form i.e., the monobasic (H2PO4 -) and dibasic forms (H2PO4 2−). Phosphorus availability is governed by various factors such as pH of soil, soil temperature, amount of organic matter present in the soil, root system and most importantly soil microorganisms. The latter has a critical role in increasing P availability to plants. Soil P concentration ranges between 0.01-3 mg P L− 1 which is very small compared to the amount that plants need for normal growth. Therefore, to make sure that the plants are not devoid of P, remaining amount is compensated by soil rhizobia using their phosphate solubilizing property. These rhizobia are referred to as phosphate solubilizing microbes (PSMs), having the ability to hydrolyze insoluble phosphorus in soil into readily soluble form. They develop a network in the rhizosphere around the plant roots, allowing them to absorb P from a broader area. The use of PSMs is an environmentally safe and cheap method to reduce the insufficiency of phosphorous and promote its absorption and assimilation by plants. PSMs are able to convert the insoluble phosphorus into soluble form by lowering the pH, chelating cations and mineralization [84]. Application of phosphate solubilizing bacteria belonging to following genera*: Achromobacter, Agrobacterium, Bacillus, Pseudomonas, Erwinia, Flavobacterium, Microbacterium and Rhizobium* has resulted in increased phosphorus uptake and eventually higher yields.

### *4.1.3 Potassium solubilization*

A diverse range of soil microorganisms such as saprophytic bacteria, fungi, and actinomycetes show potential to solubilize potassium effectively converting soil K to plant-available forms [87–90]. Among these, solubilizing bacteria (KSB) can dissolve K-rich materials and convert insoluble K to soluble forms that plants can absorb. Although some KSB can work anaerobically, the majority of these are aerobic. The potassium solubilizing rhizobacteria (KSR) use a number of ways to make the K available to plants. Mechanisms such as Acidolysis, chelation, exchange reactions, complexolysis, and the production of organic acids are few well known alternatives. The acidolysis (organic and inorganic acids, as well as the synthesis of protons) is the main mechanism of K mineral solubilization [87, 91–95]. Formation of organic acids by KSB that are useful in releasing K from K-bearing minerals include oxalic acid, tartaric acids, gluconic acid, 2-ketogluconic acid, citric acid, malic acid, succinic acid, lactic acid, propionic acid, glycolic acid, malonic acid and fumaric acid [96–103]. Tartaric acid, citric acid, succinic acid, ketogluconic acid, and oxalic acid are the most effective acids secreted by KSB among the several organic acids involved in the solubilization of insoluble K. *Acidothiobacillus ferrooxidans, Paenibacillus spp., Bacillus mucilaginosus, Bacillus edaphicus,* and *Bacillus circulans* are among the bacteria that can solubilize K minerals such as biotite, feldspar, illite, muscovite, orthoclase, and mica [96, 104]. It has been observed that *B. mucilaginosus, B. circulanscan, B. edaphicus, Burkholderia, A. ferrooxidans, Arthrobacter sp., Enterobacter hormaechei, Paenibacillus mucilaginosus, Paenibacillus frequentans, Cladosporium, Aminobacter, Sphingomonas, Burkholderia, and Paenibacillus glucanolyticus* solubilize K from silicate rocks. Further, *B. mucilaginosus, B. edaphicus,* and *B. circulanscan* have been identified

### *DOI: http://dx.doi.org/10.5772/intechopen.102657 Potential Applications of Rhizobacteria as Eco-Friendly Biological Control, Plant Growth…*

as excellent K solubilizers in soil bacterial populations [88, 89]. Furthermore, microbial degradation of organic materials produces ammonia and hydrogen sulphide, both of which can be oxidized in the soil to make powerful acids like nitric acid (HNO3) and sulfuric acid (H2SO4). Consequently, K+, Mg2+, Ca2+, and Mn2+ are displaced from the cation-exchange complex in soil by hydrogen ions [105]. Organic acids produced by KSB can liberate K ions from the K mineral via complexing agent Si4+, Al3+, Fe2+, and Ca2+ ions (chelating) linked with K minerals, additional to decreasing soil pH [106, 107]. In addition, accumulation of diverse extracellular polymers (mainly proteins and polysaccharides) has also been linked to the release of K from K-bearing minerals [99, 103, 108]. Such substances act as adhesive structures to the surface of minerals or rocks. Fresh microbial EPS (exopolysaccharides) solution, for example, accelerates the dissolution rate of feldspars by forming complexes with framework ions in solution (Welch and Vandevivere 1994). Other PGPRs (for example, IAAproducing bacteria) may also play a role in delivering K to plants via boosting root exudates [109].

Under greenhouse and field circumstances, studies have demonstrated that inoculating seeds and seedlings of many plants with KSB improves germination percentage, seedling vigor, plant development, yield, and K uptake [87, 88, 110–115]. Several studies show that KSB inoculation improves the growth of a variety of crops [101, 103, 112, 116–125]. Overall, studies indicate application of KSB as bio-fertilizers for agriculture development can reduce the usage of agrochemicals while also promoting sustainable crop production

### *4.1.4 ACC deaminase production*

The infection caused in the roots by rhizobium bacteria during nodule formation results in stress conditions. Consequently ethylene, a stress regulating hormone, inhibits the infection put forth by the bacteria, besides restricting nodulation and root growth [126]. Specific genes are involved in the interaction mechanisms of Rhizopheric bacteria with the plants by means of which they influence their growth. One of these genes encoding for the enzyme ACC deaminase, is involved in cleaving ACC, the precursor of ethylene biosynthesis produced by plants. ACC deaminase degrades ACC into ammonium and ketobutyrate and prevents ethylene biosynthesis [127]. Under limited ethylene concentration, rhizobial colonization of the roots is enhanced which result in the formation of a greater number of nodules on the host plant. Horizontal Gene transfer allows the spread of acdS within the species [128]. However, the genetic analysis carried out by Nascimento et al. [129] revealed that acdS are inherited vertically during evolution. Glick, [22], confirmed that the bacteria which produce IAA synthesize high level of ACC deaminase which inhibits ethylene biosynthesis and promote plant growth, root nodulation and increase uptake of minerals from the soil. Rhizobial strains including *R.leguminosarum, R. hedysari, R. gallicum, B. elkani and S. meliloti* have been reported to synthesize ACC deaminase [3].

#### **4.2 Plant growth promotion by indirect promotions**

#### *4.2.1 Salt stress and osmotic stress*

Plant growth improvement has been of great concern since the beginning of agriculture. There are various abiotic factors including temperature, pH, heavy metal toxicity, salt stress which obstruct plant growth and crop productivity [130]. Among them salinity stress is a real hazard for plant growth and production. Under saline

conditions plants uptake high amounts of salt which interferes with their physiological and metabolic processes which hampers their growth and makes their survival difficult. Reclamation of saline soils by conventional methods i.e., adding soil amendments like gypsum, calcium etc. do not help to overcome salinity stress completely, moreover they adversely affect the ecosystem. Therefore, for the enhancement of plant growth and productivity, development of sustainable and safer methods is of utmost importance [131]. Large number of microbes belonging to different genera of salt tolerant plant growth promoting rhizobacteria (ST-PGPR), present in the soil are able to tolerate salinity stress as well as promote plant growth [132]. These rhizobacteria (ST-PGPR) include genera *Pseudomonas, Enterobacter, Agrobacterium, Streptomyces, Bacillus, Klebiella* and *Ochromobacter* [133, 134]. Salt-tolerant rhizobium isolated from legumes growing in sand dune sand tree legume [135] were able to tolerate upto 2.5–3% of NaCl concentration. In 2018, Zhang et al. [136] isolated 305 bacterial strains and found that 162 out of 305 could grow in NaCl concentration of 150 g/l. For boosting nitrogen fixation and productivity in high salt containing soils co-inoculation of legumes with salt tolerant rhizobial bacteria is a sustainable solution. Under non saline and saline condition silicon was found to enhance growth and nitrogen fixation in leguminous plants [137].

#### *4.2.2 Temperature stress*

Worldwide climate change had led to an increase in temperature, which adversely effects plant growth and development. Elevated temperatures result in decreased rate of photosynthesis, negatively influence plant water relations, flower and fruit development. Soil rhizobia indirectly help plants to combat heat stress. Most rhizobia prefer an optimum temperature range of 25–30°C for their growth, however, during their life cycle they experience a temperature out of this range. The growth promotion effect of different PGPR strains in plants was attributed to their nitrogen fixing ability but these effects were noticed prior to the beginning of nitrogen fixation [138]. This shows that the favorable effects of rhizobium in alleviating temperature stress does not depend on nitrogen status. It is due to stimulation of genes to express under high temperature stress conditions. The expression of these genes is regulated by heat stress transcription factors (Hsfs) [139]. HSPs are a family of proteins that are induced by a sudden temperature rise, they include chaperones and proteases, which confer high temperature tolerance to bacteria and thus contribute to the tolerance mechanism [140]. A microarray study conducted in *Sinorhizobiummeliloti* showed that 169 genes, which included the genes coding for HSPs and chaperones, were up regulated under high temperature conditions. Chaperones, like DnaK–DnaJ and GroEL–GroES, form an important component of the heat shock response. After heat shock, the hydrophobic domains of proteins are exposed, and they get denatured. These chaperons help the denatured proteins to get back to their original conformation [141]. The increased expression of chaperone genes was induced in heat tolerant strains compared to the strains of the same species that were sensitive to heat. Under high temperature stress HSPs increase the stability of cell membrane, thereby conferring heat tolerance to both, rhizobacteria as well as the plant under stress. Breeding of heat tolerant or development of transgenic heat tolerant cultivars is a laborious and less economic method. Hence, the application of rhizobacterial inoculants to plants under temperature stress should be preferred as it is relatively cheaper and less time consuming. Various physiological and biochemical changes in plants, are induced by low temperature resulting in poor plant growth and low crop survival rates [142].

## *DOI: http://dx.doi.org/10.5772/intechopen.102657 Potential Applications of Rhizobacteria as Eco-Friendly Biological Control, Plant Growth…*

Rigidification of membranes due to the decreased fluidity of cell membrane is one of these changes that plants experience when exposed to chilling stress [143]. Response to cold shock results in the synthesis of cold shock proteins (CSPs). Rhizobia strains isolated from the wild relative of chickpea at low temperatures (9–15°C), successfully nodulated chickpea, indicating that it could serve as a potential microbial inoculant under low temperature conditions to maintain the normal functioning of plants. Symbiotic association of rhizobium with alfalfa enhances its tolerance to low temperature by regulating important physiological and metabolic processes. The oxidative enzymes were more active in AN (active nodules) and IN (inactive nodules) groups, providing higher cold tolerance to these plants [144].

### *4.2.3 Oxidative stress*

Plants, in response to various kinds of environmental stresses such as biotic and abiotic stress produce reactive oxygen species (ROS). Examples of ROS are singlet oxygen (1 O2 ), superoxide anion (O2−), hydrogen peroxide (H2O2) and hydroxyl radical (OH-). Accumulation of reactive oxygen species (ROS) as a result environmental stress is detrimental for plant growth as they modify the primary cell constituents like DNA, lipids, proteins etc. [145]. PGPR reduce the deleterious effects of ROS by producing antioxidant enzymes [146, 147] which include peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), nitrate reductase (NR) and glutathione reductase (GR) and thus help in maintaining plant growth and crop productivity [148]. Based on the results of Shen et al. [149] it could be concluded that due to the activation of antioxidant machinery by the rhizobium inoculants, their use is the most effective way for enhancing plant growth and mitigating stress induced by ROS.
