Keywords

7.1 Introduction

Soil adhered around root vicinity, called rhizosphere soil. This rhizosphere soil directly comes under influence of root exudates secreted from the plant root system (Badri and Vivanco 2009; Bais et al. 2006; Broeckling et al. 2008; Gransee and Wittenmayer 2000). In 1904, Hiltner first time described rhizosphere soil with the name of “rhizosphere effect.” He observed that rhizosphere soil has an intense microbial population and activity compared with nonrhizosphere soil, bulk soil. Root exudate has recorded with triggering effect on microbial population located on rhizoplane (root surface), rhizosphere zone (buffer zone), and nonrhizosphere zone (bulk soil) (Hirsch and Mauchline 2012; Kristin and Miranda 2013; Toju et al. 2018; Turner et al. 2013; Zhang et al. 2017). Other than bulk soil, rhizoplane and rhizosphere zone are colonized more intensively by beneficial microbes, plant growth-promoting rhizobacteria (PGPRs), or harmful microbes [disease-causing microorganisms (DCMs) or plant pathogenic microbes (PPMs)] (Chaparro et al. 2012, 2014; Mitter et al. 2013; Pii et al. 2015). The beneficial microbes perform plant growth-promoting activities and provide additional nutrients availability (Adesemoye et al. 2008; Ahemad and Kibret 2014; Babalola 2010; Berendsen et al. 2012; Chauhan et al. 2015) while protecting from pathogenic microbes (Compant et al. 2005) and also tolerance to abiotic stresses (Berg et al. 2014; Fahad et al. 2015). The PGPRs can fix atmospheric nitrogen and mineralize and solubilize phosphorus, potash, silica, zinc, and oxidizing sulfur to the plants. The PGPR strain includes Rhizobium, Azospirillum, Azotobacter, Gluconacetobacter, Bacillus, Pseudomonas, Burkholderia, Paenibacillus, Serratia, etc. (Gupta et al. 2015; Jha and Saraf 2015; Lugtenberg and Kamilova 2009; Nehra and Choudhary 2015). Rhizobium is a well-known example of nodule-forming bacteria in legume crops where it fixes atmospheric nitrogen. Atmospheric nitrogen fixation by Rhizobium is a very sensitive process to oxygen. In the presence of oxygen, the biological nitrogen fixation pathway inhibited. Azospirillum, Azotobacter, Gluconacetobacter, etc. are atmospheric nitrogen-fixing bacteria of other nonlegume crops. Bacillus PGPR strain has better survival under adverse climatic conditions, while Pseudomonas is considered as better root-colonizing bacteria. These PGPR strains mineralize and solubilize fixed plant nutrients into unfix form, which is absorbed by the plant roots during the water translocation process (Bulgarelli et al. 2013, 2015; Itelima et al. 2018; Nelson 2004). Some PGPRs have been identified for restricting or limiting the population of pathogenic microbes. The population of soil-borne plant pathogenic microbes is highly affected and reduced by the production of cell wall lytic enzymes, antimicrobial metabolites, and also competes for nutrients availability. Induced systemic resistance and phytohormone production have been reported as one of the mechanisms of PGPRs for managing plant diseases by manipulation in crop plants’ physical and chemical properties. Some PGPR strains have been identified for providing tolerance to drought and salt stress through the production of osmoprotectants metabolites (Baez-Rogelio et al. 2017; Cardinale et al. 2015; Wani et al. 2016). The interaction between plant and PGPR strain is complex (Gray and Smith 2005; Jones et al. 2004; Leach et al. 2017), and PGPRs trigger cumulative effects on plant growth and yield (Bashan 1998; Bender et al. 2016; Vessey 2003), while root exudate provides a congenial atmosphere for the beneficial microbial population (Beattie 2015; Berg et al. 2016; Bossio et al. 1998; Evangelou and Deram 2014).

7.2 Rhizosphere

The soil region directly comes under the influence of root and/or root exudates, called the rhizosphere. The rhizosphere zone covers around 20–30 cm top of the undisturbed soil and consists of plant roots, soil matrix, and soil microflora and fauna. The soil microflora and fauna are bacteria, fungi, cyanobacteria, nematodes, protozoa, and even mites too. The soil provides physical supports as well as water and minerals to the plants. Plant roots secrete low-molecular-weight organic compounds such as sugars, amino acids, organic anions (OAs), and phenolics. They can easily be disintegrated and assimilated by soil microorganisms and serve as a substrate for microbes. The high-molecular-weight organic compounds (proteins, pigments, mucilage, and miscellaneous other substances) secreted by plant roots require additional extracellular enzymatic activity to break down before assimilation. Mucilage is a mixture of organic substances, released proton, oxygen, and water. In addition to organic substances, some inorganic substances like inorganic ions, H+, electrons, water, and siderophores are produced. These released substances make soil physical and chemical structural changes (Fig. 7.1) (Garcia-Pausas and Paterson 2011). Almost 20–50% of total photosynthates of the crop are secreted in the rhizosphere. These root exudates are preferably utilized by soil microbiota, and this effect is called the “rhizosphere effect,” the first time described by Hiltner, 1904. The rhizosphere soil has 500 times more microbial load compared to the nonrhizospheric soil (bulk soil). The quality and quantity of secreted compounds depend on plant species cultivated and to a certain extent on soil physical and chemical properties (Andreote and Pereira 2017). Because of this reason, certain species of the bacteria and fungi can survive in this selective microenvironment, called phytomicrobiome (Badri et al. 2013). Surviving bacteria and fungi may have beneficial and harmful relationships to the crop plants. Some soil-borne plant pathogenic fungi are reported to cause economic damage to crops. The fungal genera are Fusarium, Pythium, Phytophthora, Alternaria, Rhizoctonia, etc. However, rhizosphere-residing bacterial population has a positive effect on growth and crop yield called plant growth-promoting rhizobacteria (PGPRs). So far, well-known examples of PGPRs are Rhizobium, Gluconacetobacter, Pseudomonas, Bacillus, Paenibacillus, Azotobacter, Azospirillum, Burkholderia, and Biocontrol agents (Babalola 2010). In the rhizosphere zone, a specific plant–soil–microbes interaction takes place. This interaction is mediated by chemical substances released by plants and microbes. In leguminous plants, some flavonoids are considered for playing a major role in nodule formation in plant roots (Dakora et al. 2015; Desbrosses and Stougaard 2011). Some researchers also emphasized that these flavonoids compounds may also be associated with vesicular-arbuscular mycorrhiza (VAM) colonization. Root colonization and the microbial population vary from crop growth stage. The grand growth and root elongation state of the crop are considered to have the greatest number of the rhizospheric microbial population. The ratio of the microbial population in the rhizosphere zone to bulk soil is always recorded 3–4 times more. Application of PGPR strain in the production of the agricultural crop through soil, seed, and root inoculation improves qualitative economic values and also avoids environmental risk efficiently (Fig. 7.2).

Fig. 7.1
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Root exudates change soil physical and chemical properties

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Fig. 7.2
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PGPRs mediated ecological cycle while sustaining soil fertility and enhancing crop productivity

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7.3 PGPRs in Rhizosphere: For Better Crop Growth and Yield

It has been estimated that almost more than a hundred million tonnes of nitrogen, phosphorus, and potassic fertilizers are applied in soil annually. Due to the nonjudicious application of these chemical fertilizers, tremendous detrimental effects on soil, water, and human beings besides the increased cost of crop production have been reported worldwide. Rhizosphere used to call root adhered soil region has intense microbial activity and also dynamic zone in the soil. In 1904, German agronomist Hiltner first time defined the term rhizosphere for the effect of legume roots on the surrounding soil. He recorded that more microbial activity at neighboring roots or root influenced soil. The reason for intense microbial activity is reported because 20% and 50% of their photosynthates released through the root (Bottner et al. 1988). The diverse group of low- and high-molecular-weight organic compounds are released in the rhizosphere. The soil microflora and fauna supported by these organic substances and microbial population in soil exert a beneficial effect on plant growth promotion and yield as well. Plant growth promotion itself describes the increased plant growth and crop yield occurred while treating seed or soil with certain plant growth-promoting bacteria. The plant growth-promoting rhizobacteria are free-living soil inhabitant bacteria. PGPRs improve seed germination, root formation, branching and tillering, fluorescence, fruit ripening in crop plants. Besides this, PGPRs also provide tolerance to biotic and abiotic stresses (Table 7.1). These plant growth-promoting attributes are finally visible in terms of increased seed germination, root formation, excessive branching and tillering, fluorescence, fruit ripening, and also tolerance to abiotic stresses. PGPRs are found in the rhizosphere of the crop plants (Kloepper et al. 1989). The effect of plant growth-promoting rhizobacteria on agricultural crops is reported by various researchers. Some PGPRs strain has been reported for plant growth-promoting attributes such as the cytokinin production by Pseudomonas fluorescens strain G20-18 (Bent 2006), biological nitrogen fixation by Rhizobium leguminosarum strain MNF 710 and P solubilization by Pseudomonas putida strain GR12-2, etc. The PGPRs promote plant growth and crop yield by facilitation of the nutrients from the soil environment or by producing inhibitory substances to restrict the growth and minimize plant pathogenic load in soil.

Table 7.1 Role of PGPRs in plant growth-promoting attributes of crop plants
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7.3.1 Atmospheric Nitrogen Fixation

Nitrogen is one of the most important macronutrients for the plant. Conversion of gaseous atmospheric di-nitrogen into nongaseous ammonium nitrogen compound by microbial intervention is called biological nitrogen fixation. Further, gaseous ammonium nitrogen is oxidized in the form of nitrate. Both nitrogen forms are absorbed by plants. Living entities depend on the availability of fixed nitrogen because nitrogen molecule is required for the biosynthesis of amino acids, proteins, nucleic acids, and other nitrogen-containing biomolecules. The plant makes different associations with beneficial microbes and symbiotic prokaryotic microorganisms like Rhizobium meliloti, R. leguminosarum, Rhizobium phaseoli, and Rhizobium japonicum in legume crops and asymbiotic microorganisms like Azotobacter chroococum, Azotobacter beijerinckii, Azotobacter vinelandii, Azospirillum brasilense, Azotobacter lipoferum, and Gluconacetobacter diazotrophicus in nonlegume crops. These rhizobacterial genera are identified as endophytic nitrogen fixers. The application of these endophytic nitrogen-fixing bacterial strains not only improves crop growth, yield, and crop productivity but also reduces chemical fertilizer’s load. Biological nitrogen fixation is highly sensitive to the presence of oxygen, intensive energy input process, and involves functional and regulatory gene products. The nitrogenase protein complex consists of two metalloprotein subunits. The first one is composed of two different dimers (MoFe protein) which are encoded by nifD and nifK genes. This nitrogenase protein complex performs an actual reduction of atmospheric di-nitrogen. The second protein subunit is made of two similar dimers (Fe protein) which are encoded by the nifH gene. This site ensures ATP hydrolysis and electron transfer between subunits. Thus, acetylene reduction assay (ARA) is used as an indirect method to study the efficiency of the nitrogenase enzyme. Among the various biological nitrogen fixers, bacterial group belonging to rhizobia is well established and well-known example of this. The biological nitrogen fixers are generally called “diazotrophs.” Rhizobium strain was used for the first time to develop the microbial product in the name of “Nitropin.” Subsequently, a number of symbiotic and nonsymbiotic bacterial strains are isolated, screened, and identified. Worldwide several researchers used these nitrogen-fixing bacterial strain and recorded that the application of nitrogen fixers in the crops at the sowing, planting, and transplanting could reduce the fertilizer load up to 25–50% without compromising crop growth and yields. Bhattacharjee et al. (2008) reported that species of Rhizobium in legume crops like pea, gram, cowpea, pigeon pea, lentil, and bean can supply nitrogen sufficiently. Gluconacetobacter, Azospirillum, Azotobacter, Bacillus, and Burkholderia inoculation have been reported to enhance crop growth and yields in nonlegume crops (Allen et al. 2017; Baldani et al. 2000). The atmospheric fixed nitrogen is calculated in terms of the percentage of total plant nitrogen, protein, and yield increased. As per reports available, about 30–40 kg/ha/year nitrogen is fixed by seed/planting material bacterized with diazotrophs.

7.3.2 Phosphate Solubilization

Phosphorus is the second major macronutrients for better crop growth and yields. It is a constituent part of phosphorylated sugar, phospholipid, phytin, nucleotide, nucleic acid, and coenzymes. Soil pH plays a major role in its absorption by the plant roots (soil pH <4.0—H3PO4, between pH 4.0 and 7.0—H2PO4, between pH 7.0 and 10.0—HPO4, and pH >10—PO4). Phosphorus is available in the form of orthophosphate, and plant takes phosphorus either in H2PO4 or in HPO4. Maximum phosphorus is available in soil pH ranging from 6.0 to 7.0. The mobility of available phosphorus is very limited. In soil, phosphorus is available in the organic and inorganic state. The total phosphorus content in arid soil in India is reported around 700 kg/ha, but the only plant accessible phosphorus quantity is very low, 15–25 kg/ha. Phosphorus contributes to a total plant dry weight of around 0.2%. The early stage of crop growth requires a sufficient supply of phosphorus for primordial development, tillers formation, and photosynthetic processes. Deficiency of P nutrient resulted in stunted plant growth, twisted and tilted petioles, and leaflets. Soil is rich in phosphatic components but its availability to the crop plants is less. Besides this, almost 75–90% of applied phosphatic fertilizers easily chelate with calcium (Ca) and form calcium phosphates, with iron (Fe) and form iron phosphate, and with aluminum (Al) and form aluminum phosphate. This phosphorus precipitation occurs in acidic soil associated with Al and Fe compounds and mono-, di-, and tricalcium phosphate in calcareous soil (Stevenson 1986). Organic matter plays a major role in making organic phosphorus available. Organic matter contributes around 50–80% of the total organic phosphorus. Phytate, phospholipids and nucleic acid are the mother of soil organic phosphorus and utilized by the soil microbial population. Soil microbial populations especially PGPRs are capable to mineralize insoluble mineral phosphate in the soil. Phosphate-solubilizing microbial population constitutes around 20–40% of the culturable microbial population of soil. Among the phosphate-solubilizing microbial population, phosphate-solubilizing bacteria are the major ones. The majority of soils have been reported to have PSB strains, but the population in arid and semiarid soils is very less. Besides this, climatic conditions regulate PSB strains in soil. However, a mild and moist climate is more congenial for population buildup than dry and flooded conditions. The rhizosphere zone is the best habitat for PSB strain multiplication. These P-solubilizing bacteria produce phosphatase, phytases enzyme, and organic acids in both liquid and solid medium which leads to a drop. Nearly all P bacteria strain has the potential to solubilize Ca-P complexes, and only a few of them can solubilize Fe-P and Al-P complexes. Acid phosphatase and phytases are considered major phosphate-solubilizing substances. Besides this, P-solubilizing bacterial strains have other plant growth-promoting attributes like the production of siderophore, phytohormone, antibiotics, antimicrobial substances, vitamins, and HCN. Inoculation with PSB strains like Pseudomonas fluorescens, Bacillus megaterium, and Bacillus polymyxa (Bhatti and Yawar 2010; Chhabra et al. 2013; Demissie et al. 2013) were done on various crop plants by various researchers and recorded significant results. It was recorded that additional phosphate solubilization, 15–30 kg/ha, has been reported. A cheap source of phosphate in arid soils is rock phosphate but becomes inaccessible in normal and alkali soils. P-solubilizing bacterial strains have greater potential to release insoluble and fixed forms of phosphates when seed or soil inoculated.

7.3.3 Production of Plant Growth Regulating Substances

A tripartite (plant–soil–microbes) interaction takes place in the rhizosphere. Phytohormone is a growth regulator produced by either plants or microbes which assists plant for seed germination, root development, cell elongation, cell division, primordial formation, and other several morphological and physiological changes. Besides this, phytohormone increases plant resistance to environmental conditions, suppresses the expression of undesired genes, and assists in the biosynthesis of pigments, enzymes, and metabolites. The phytohormone is auxins, cytokinins, gibberellins, ethylene, and abscisic acids. For root initiation, auxins play a major role. In plants, auxins are produced only by a tryptophan-mediated pathway. The tryptophan is the precursor of auxin production by the crop plants means it is a limiting factor for auxin production in plants. In PGPRs, various known pathways of auxin production especially dominating indole-3-acetic acid (IAA) biosynthesis pathway have been reported. IAA biosynthesis pathways in PGPRs are indole-3-pyruvic acid, indole-3-acetamide, tryptophan conversion into indole-3-acetic aldehyde, and tryptophan conversion into the indole-3-acetonitrile pathway. The majority of PGPRs studied for IAA biosynthesis have IAA formation either indole-3-pyruvic acid pathway or tryptophan conversion into indole-3-acetic aldehyde pathway. Almost 80% of rhizobacteria isolated from rhizosphere soil have the potential to produce auxins during in vitro screening. The predominant bacterial population in the soil is Pseudomonas, Bacillus, Rhizobium, Azotobacter, Azospirillum, Erwinia, Agrobacterium, Enterobacter, and Serratia. Moreover, the rhizospheric microbial community of the plants has great potential in the conversion of tryptophan into IAA. Similarly, PGPRs are identified for cytokinins, gibberellins, and abscisic acid synthesis. Pseudomonas fluorescens is an efficient PGPR strain in synthesizing phytohormone, mainly IAA, cytokinins, and gibberellic acid in inoculated wheat crop. PGPR strains like Bacillus pumilus, Herbaspirillum seropedicae, Acinetobacter calcoaceticus, and Promicromonospora produce gibberellins. Pieces of literature are available that Azospirillum brasilense and Bacillus megaterium are involved in the production and enlargement of the root by the production of cytokinins (Bottini et al. 2004; Cohen et al. 2015; de Santi Ferrara et al. 2012). Excess production of ethylene is a negative factor for plant growth promotion. For ethylene production, 1-aminocyclopropane-1-carboxylate (ACC) plays a major role in conversion into ethylene but ACC is metabolized by PGPR strains by producing ACC deaminase enzyme. The ACC deaminase enzyme breaks ACC into α-ketobutyrate and ammonium resulting in reduced ethylene concentration. PGPR strains like Bacillus subtilis, B. pumilus, Bacillus licheniformis, Achromobacter xylosoxidans, Lysinibacillus fusiformis, and P. putida are ABA-producing bacteria. Thus, PGPRs play a vital role in the production of plant growth regulators, and phytohormones which influence plant growth and development (Cohen et al. 2015; de Santi Ferrara et al. 2012; Etesami et al. 2015).

7.3.4 Mycelia Growth Restriction of Soil-Borne Plant Pathogens by PGPRs

Biological control agents are a method whereby an undesirable population of plant pathogenic microbes is reduced by the application of beneficial microbial populations rather than a man. To date, several researchers have defined the biological control. Besides this, during cultivating agricultural crops, it is noticed that several economically important diseases are caused by the fungal population. The soil-inhabiting fungal pathogenic population is more and very difficult to manage. The application of fungicides is not always effective and eco-friendly besides the huge cost involved. So plant growth-promoting rhizobacteria are considered an alternatively green approach for soil-borne plant pathogen management. Sanford (1926) first time used antagonists as a biological control for managing potato scab disease. He emphasized that increasing the population density of certain saprophytic bacteria on decomposing crop residues reduces potato scab disease. For abiotic management, the ice-minus strain of Pseudomonas syringae is used to exclude ice nucleation strains of P. syringae from the foliage of frost-sensitive plants. In the cross-protection defense mechanism, inoculation of mild strain against a virulent strain of the pathogenic virus induces a defense pathway in the crop plants. Besides this, there are some classical examples of biocontrol agents where pruning wounds to provide protection against Fomes and Armillaria caused disease in plants. Prominent PGPRs used in biocontrol agents are as follows: (1) Agrobacterium radiobacter against A. radiobacter pv. tumefaciens, (2) Bacillus subtilis, B. cereus, and Bacillus penetrans against Pythium, Phytophthora cinnamomi, Fusarium roseum, and Rhizoctonia solani, (3) Pseudomonas fluorescens, P. cepacia, and P. putida against Gaeumannomyces graminis, Fusarium oxysporum, Rhizoctonia solani, Sclerotium rolfsii, Pythium, etc., (4) Erwinia herbicola and Erwinia uredovora against Erwinia amylovora, (5) Streptomyces griseus, Streptomyces praecox, and Streptomyces lavendulae against Phomopsis, Fusarium, and Gaeumannomyces (Fravel 1988; Aliye et al. 2008; Beneduzi et al. 2012).

7.3.5 Production of Fungal Cell Wall Lysing Enzymes

The PGPRs besides enhancing growth of the crop are also potential biocontrol agents. These are usually isolated from the rhizospheric soils. Most of the PGPRs belong to the fluorescent Pseudomonas (P. fluorescens and P. putida), and also few are included in nonfluorescent Pseudomonas spp., like Bacillus subtilis and Serratia spp. These PGPR strains can parasitize fungi and kill them by secreting cell wall lytic enzymes like chitinase, B-1, 3-glucanases, proteases, and lipases. PGPR strain also produces low-molecular-weight fungi toxic compounds such as iturin and fengycin. For example, chitinase cell wall lytic enzyme is produced by Serratia marcescens, and RLOs have been associated with biocontrol of fungal kitinase of pea and bean. Besides this, chiA gene was cloned and expressed constitutively in PGPR culture Pseudomonas putida. This genetically engineered PGPR strain having chiA + recombinant provided increased protection of radish against Fusarium oxysporum f. sp. redolens.

7.3.6 Plant Defense System Activation

The plant growth-promoting rhizobacteria can suppress plant disease caused by plant pathogens by triggering plant-mediated resistance mechanisms called induced systemic resistance (ISR). The induced systemic resistance is almost similar to plant pathogen-induced systemic-acquired resistance (SAR). The induced resistance works both locally and systemically. These both enhance resistance against challenging pathogens. The ISR and SAR differ in their only signaling pathways. This is evident when plant growth-promoting bacteria and plant pathogens are applied at specially separated locations on the plant. In ISR, jasmonic acid is a signaling molecule, while in SAR, it is salicylic acid. SAR is associated with the accumulation of novel pathogenesis-related proteins (PR proteins), some of them also reported to have antifungal activity. In ISR, the systemic infection is not linked with necrosis (without necrosis), while SAR works as with the necrosis system. For SAR, a transgenic plant with the Nah G gene was developed from Pseudomonas putida, which codes enzyme salicylate hydroxylase. It causes the conversion of salicylic acid to catechol, and as a result, no SAR is developed in plants (Ryals et al. 1996). Thus, Nah G transformed plants have been used to determine whether ISR-inducing PGPRs can trigger the SAR pathway. PGPR-mediated ISR signaling pathway does not initiate biosynthesis of salicylic acid or pathogenesis-related proteins. Rather, it synthesizes jasmonic acid (jasmonate) and ethylene and just like SAR depends on the regulatory protein NPR1 which contains ankyrin repeats. Thus, NPR1 regulatory protein differentially regulates ISR- and SAR-related gene expression depending on the pathway that is activated. The jasmonic acid and ethylene dependency of ISR are based on the enhanced sensitivity of these phytohormones, rather than an increase in their production. However, the extent of induced resistance attained is similar in both ISR and SAR signaling pathway (Bakker et al. 2013; Bent 2006). It is yet not clear whether ISR is broad-spectrum like SAR, but there is no evidence available that these PGPRs stimulate to produce antimicrobial compounds such as phytoalexins. However, some PGPRs which produce salicylic acid as siderophore under iron-limiting condition have been reported to induce SAR. Similarly, Pseudomonas aeruginosa strain 7NSK2 has been found to induce SAR in bean and tobacco plants against Botrytis cinerea and tobacco mosaic virus (TMV), respectively.

7.3.7 PGPR-Mediated Drought and Salt Stress Management

A worldwide drought and salt stress are a serious concern for soil quality and soil fertility. Due to poor soil quality and fertility, crop growth and crop productivity are adversely affected. Almost 12 million ha of cultivated land in India is salt-affected. A major portion of salt-affected soil is found in semiarid and arid regions of the country. The pH of salt-affected soil is around 8.0 or more. In salt-affected soil, a mixtures of chlorides and sulfates of calcium, magnesium, and sodium are found. Among them, the concentration of sodium chlorides is dominant in nature. The ratio of sodium, calcium, and magnesium in most of the salt-affected soils has been recorded as 7:2:1. Due to the increased concentration of these mixtures of chlorides and sulfates, the availability of existed plant nutrients is affected. The reports are available that a large portion of applied inorganic phosphatic fertilizers in salt-affected soil usually precipitated in different forms such asmono-phosphates, di-phosphates, and tri-calcium phosphates. Precipitation of inorganic phosphates in the soil comes under soil salinization, where water-soluble salts accumulated in the soil. Similarly, it happens with other externally applied inorganic fertilizers. Due to deficiency of plant nutrients, crop growth and yield are badly affected. The reduction in crop growth and yield is due to a decrease in cell growth, leafsurface area, chlorophyll content, accelerated defoliation, and senescence. The soil salinity is measured by determining the conductivity of the saturation extract (dS/m). Among abiotic stresses, the drought stress is also the most destructive stress which causes a complete restriction on crop growth and yield. The severity of the damage depends on the drought period and crop stage. In drought conditions, the availability and transport of soil nutrients are affected. Besides, it induces free radicals which affect antioxidant defenses andreactive oxygen species mechanisms. Increased reactive oxygen species (ROS) causes various levels of plant physiological parameters. The decreased chlorophyll content is one of the major causes of drought stress. So in these prospects, a large group of plant growth-promoting rhizobacteria has been isolated, screened, characterized, and identified at the molecular level. The PGPR strain predominant in adverse climatic conditions is known to have a beneficial effect on drought and salt stress management in the cultivated crop. These PGPRs applied in drought and salt-affected soil performs different activities, such as phytohormone production (abscisic acid, gibberellic acid, cytokinins, indole—3 acetic acids), ACC deaminase production, induced systemic resistance, and exopolysaccharide production. Dimkpa et al. (2009) reported that IAA-producing Azospirillum strain enhances plant tolerance to drought stress. The effect of different PGPRs strain like Azospirillum brasilense, A. lipoferum, Bacillus subtilis, Pseudomonas fluorescens, and Bacillus thuringiensis has been reported on tomato, maize, wheat, soybean, etc., worldwide. Similarly, several researchers reported that the application of osmotolerance PGPR strain like Pseudomonas fluorescens, Pseudomonas pseudoalcaligenes, B. subtilis, and Azospirillum brasilense reduces osmotic pressure by the production of organic osmolytes (sugar and derivatives, amino acid and derivatives, polyols, betaines, and ectoines). Paul and Nair (2008) reported that P. fluorescens strain MSP-393 induces salt tolerance by the production of glycine betaine, alanine, glutamic acid, serine, threonine, osmolytes, and aspartic acid in their cytosol of wheat, rice, avocado, maize, chickpea, peanut, common bean, tomato, eggplant, cotton, radish, barley, lettuce, pea, groundnut, black pepper, kallar grass, etc.

7.4 Molecular Tools and Techniques for Identification of PGPRs

Rhizospheric soil is rich in microbial populations. A diverse group of beneficial plant growth-promoting microbes is reported worldwide. The beneficial plant growth-promoting microbes may be mainly bacteria and fungi. Few of them only 1–2% of the total soil microbial population are culturable in laboratory conditions. In laboratory conditions, phenotypic, biochemical, serological, and molecular-based identification can be done. Phenotypic, biochemical, and serological-based identification has certain limitations. So molecular-based identification provides correct identification, which can be correlated with a phenotypic-based identification system. The molecular-based identification of bacteria is done by the determination of base sequences of certain key nucleic acid genes such as 5S rRNA (120 bp), 16S rRNA (1600 bp), and 23S rRNA (2300 bp). In fungal identification, ITS region, TEF-α gene, ATP -6ATPase gene, and β-tubulin gene are targeted. This could be done due to the introduction of polymerase chain reaction, which allows for specific detection and investigation of even minor traces of genetic material. All these bases of nucleotide sequences of rRNA genes and their spacers can be used for phylogenetic analyses. Most commonly, 16S rRNA gene and ITS region are used for molecular identification of bacteria and fungi, respectively. After the sequencing of rRNA genes and spacer regions, sequencing data are blasted with GenBank-NCBI and deposited in public data banks (NCBI, EMBL, etc.). This approach provides information on isolated microbial culture and exact identification as well. Besides this, for the study of microbial diversity, community structure, and community response to environmental conditions in the soil, a meta-genomics approach can be used. Generally, the meta-genomics approach is adopted in the study of noncultural microbes. Further, soil meta-genomics study includes culture-dependent techniques (plate count, morphology analysis, community-level physiological profiling, CLPP) and culture-independent techniques—Microbial lipid-based (PLFA, FAME), non-PCR based (DNA reassociation, the G + C content of DNA, RSGP), PCR based (RAPD, RFLP, ARDRA, T-RFLP, RISA, ARISA, DGGE, TGGE, SSCP, HRP, qPCR, etc.), and sequencing based (clone library sequencing, amplicon sequencing, shotgun sequencing, etc.).

7.5 Bio-Formulation Development and Commercialization of PGPRs

In recent years, a number of entrepreneurs (small and medium levels) have entered commercial production of bio-formulations (biofertilizers and biocontrol agents) (Bhattacharjee and Dey 2014; Arora et al. 2016; Bashan and de Bashan 2015; Bashan et al. 2014). For bio-formulation development, it is a multistep process involving a wide range of activities. The first activity is to isolate potential microbial culture from the congenial natural or agro-ecosystems. In laboratory conditions, evaluation of isolated microbial culture is done for plant growth-promoting attributes. The potential microbial strain is further screened at glass-house conditions. After getting significant results, the microbial culture is validated in field conditions, at farmers’ fields, and/or by validating agencies. Besides this, microbial culture is also validated for shelf life, quality parameters, and population count in the eco-friendly and easily available multiplying substrate. After complete confirmation and validation, microbial culture is mass multiplied, formulations prepared, registration done, and released for application on farmer’s field (Table 7.2).

Table 7.2 List of some bio-formulations developed worldwide
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7.6 Conclusion

The rhizospheric zone of the plant has been characterized by intense microbial activity. It is also an interface between plant roots and bulk soil, the soil which is not under influence of root exudates. Root exudate is a mixture of the released low and high metabolites by the plant. The metabolites are synthesized during the photosynthesis pathway. They are a good food substrate for microbial population and exist around the root vicinity. Due to rich in carbon and nitrogen-based compounds, it creates and develops selective microbial community and structure called rhizomicrobiome. The beneficial microbial population in the rhizosphere zone is called plant growth-promoting rhizobacteria (PGPRs). The beneficial microbes increase tremendous and perform a number of plant growth-promoting attributes that are benefitted directly to crop plants, known as biofertilizers, or indirectly by restricting the population of plant pathogenic microbes, called biocontrol agents. Besides this, some PGPR strains induce systemic-acquired resistance and provide tolerance to abiotic stresses like drought and salt conditions. Today, molecular approaches are need-based and used for the detection and identification of microbes, microbial community and structure, and their response to environmental conditions. To date, a number of microbial formulations are being developed and commercialized worldwide. The use of these microbial inoculants is eco-friendly and safe and also increases crop growth and yields besides reduction in the cost of cultivation. A lot of research has been done on PGPRs and has characterized the plant growth-promoting traits of PGPRs. Still, the study of microbial community and structure has great scope for studying system biology. Soil microbial activity and distribution are directly affected by soil organic matter content and environmental conditions as well. So better understanding of the composition and distribution of microbes in nature can maintain the diversity of beneficial microbes and improve plant growth and productivity without developing any harmful environmental issues.