20.1 Introduction

Increasing agricultural productivity per unit of land and ensuring that agricultural growth responds to food security needs are the major concerns in agriculture of today. The fertilizer-based monocropping farming model that we have been following since long is not sustainable as it is harmful for human, plant, and soil health (Kumar et al. 2017a). Day by day, the food demand is increasing in the developing countries dramatically, and production of more food and fiber to feed a growing population and implementation of more efficient and sustainable production methods are challenges in today’s era. In the twenty-first century, loss of productivity in the agricultural trade is due to abiotic and biotic environmental stresses (Barnabas et al. 2008). Ecological stresses are the major limiting factors for plant metabolism, growth, and productivity, especially in the arid and semiarid zones of the world. Abiotic stresses associated with soil salinity, drought, pH of soil, environmental temperature, ozone, toxic metals, and low nutrient concentration, singly or in combination, can cause lethal effects in almost all phonological stages of plant, from germination to plant enlargement limiting factors for crop production (Rengasamy 2006; Ladeiro 2012; Ashraf and Harris 2013).

Reports have been revealed the crop yield loss (70%) may be attributed to abiotic stresses, like drought. Drought is one of the major checks in agriculture (Raju et al. 2014). Drought induces changes in physiological processes of plants, together with photosynthesis, membrane integrity, enzyme stability, proline, and ABA (Karim and Rahman 2015). Bacteria, viruses, fungi, nematodes, and herbivore insect-like living organisms are the causal factors of biotic stress (Fisher et al. 2012), and they reduce agricultural yield by 30% globally. They affect the natural habitat ecology. Healthy soil conservation is a strategic element of sustainable agriculture. The noticeable solutions that can yield more agricultural products are land management, use of renewal inputs, usage of transgenic crops, and expanded practice of plant growth-promoting rhizobacteria (PGPR) (Glick 2012). PGPR is a set of soil microbial flora. They abode in the rhizosphere and on the surface of the monocot and dicot plant roots (Vacheron et al. 2013). PGPR has shown the potential to be a promising technique in the practice of supportable agriculture and could play a key role in the mitigation of drought. The microbes colonize and impart drought by synthesizing exopolysaccharides (EPS), phytohormones, 1-aminocyclopropane-1-carboxylate (ACC) deaminase (Govindasamy et al. 2008), volatile compounds, antioxidants, inducing accumulation of osmolytes, up- or downregulation of stress-responsive genes, and changes in root morphology at the rhizosphere/endo-rhizosphere region of the affected plant roots (Vurukonda et al. 2016). The induced systemic tolerance (IST) system, the physiological state of beneficial microbes, elicits tolerance to drought stresses (Lim and Kim 2013). Inoculation of cytokine-producing PGPR helps on growth and water stress consistence of forest container seedlings under drought condition (Liu et al. 2013). Biotic stresses even can be prevented after the use of PGPR (Gupta et al. 2015).

Based on the colonization abilities of the bacterial members, PGP microbes are broadly classified into extracellular (ePGPR) and intracellular (iPGPR) colonizers. Extracellular PGP microbes belonging to the genera Bacillus, Burkholderia, Caulobacter, Chromobacterium, Pseudomonas, Agrobacterium, Arthrobacter, Azotobacter, Azospirillum, Flavobacterium, Micrococcus, Erwinia, and Serratia reside in the rhizosphere or spaces between cells of the root cortex and in the rhizoplane, while intracellular (iPGPR) bacteria such as species of Allorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, endophytes, and Frankia are mostly associated with the root nodules (Gupta et al. 2015; Gray and Smith 2005). Accepting and enumerating the impact of PGPR on the root system and the whole plant remain challenging (Gupta et al. 2000). Studies have confirmed that PGPR are perhaps plant-specific genotype and cultivar (Bashan 1998; Lucy et al. 2004). The molecular mechanisms of PGPR affect the architecture of the root system and interfere with the plant hormonal pathways (Vacheron et al. 2013). The two-way cross talk between microbes and plant host for plant growth promotion is presented in Fig. 20.1.

Fig. 20.1
figure 1

Multifaceted diagram of bidirectional response of PGPR and host for plant growth promotion

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20.2 Mechanisms of Plant Growth Promotion

The mechanisms of plant growth differ between species and strains; so, typically, not a single mechanism is accountable for plant growth promotion. PGPR enhances plant growth either by following direct or indirect mechanisms (Glick 1995; Gupta et al. 2000; Kumar et al. 2012, 2016a) or a combination of both (Fig. 20.2) corresponding to siderophore production, biological nitrogen fixation, phosphate solubilization (Richardson et al. 2009; Ortiz Castro et al. 2009; Hayat et al. 2010; Kumar et al. 2017b), rhizosphere engineering, production of 1-aminocyclopropane-1-carboxylate deaminase (ACC), quorum sensing (QS) signal interference and inhibition of biofilm formation, phytohormone production, antimicrobial activity (Yuwono et al. 2005), and volatile organic compound (VOC) production (Bhattacharyya and Jha 2012). Direct mechanisms, facilitating resource acquisition and modulating phytohormone levels, affect the plant’s metabolism and balance plant growth regulators by leading to an increase in its adaptive capacity and releasing hormones. Plants and colonization of bacteria have cohabited for millions of years. They live and promote the healthy growth of plant. Facilitating resource acquisition is categorized as nitrogen fixation, potassium solubilization, iron sequestering, and phosphate solubilization (Glick 2012).

Fig. 20.2
figure 2

Direct plant growth promotion by bacteria

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20.2.1 Nitrogen Fixation

Nitrogen, being the vital nutrient required for plant growth, and nitrogenase (nif) are the key players in providing available N (NH4 +) to the plant through biological nitrogen fixation. Nitrogenase includes structural genes that are involved in the initiation of the Fe protein, biosynthesis of the molybdenum cofactor, and electron donation and regulatory genes for the synthesis and function of the enzyme. The most critical fixation gene, Nif, is typically present in a cluster of around 20–24 kb with 07 operons encoding 20 different proteins (Ahemad and Kibret 2014). Nitrogen-fixing microbes are generally categorized as (a) symbiotic N2-fixing bacteria like species of Rhizobium, Bradyrhizobium, Sinorhizobium, Mesorhizobium, Azoarcus, Azotobacter and (b) nonsymbiotic N2-fixing bacteria, viz., species of Azospirillum, Diazotrophicus, Gluconacetobacter, Burkholderia, Acetobacter, and Enterobacter (Kumar et al. 2013a; Kumar 2017).

20.2.2 Phosphate and Potassium Solubilization

The phosphate solubilization mechanisms include the release of complexing or mineral-dissolving substances such as organic acid protons, anions, CO2, hydroxyl ions, and siderophores, the liberation of extracellular enzymes, and the emancipation substrate degradation (McGill and Cole 1981; Sahoo et al. 2017). Species of Bacillus, Burkholderia, Microbacterium, Rhizobium, Enterobacter, Rhodococcus, Beijerinckia, Arthrobacter, Serratia, Erwinia, Flavobacterium, and Pseudomonas are documented as phosphate solubilizers. Members of Pseudomonas, Paenibacillus, Burkholderia, Acidithiobacillus ferrooxidans, Bacillus edaphicus, and Bacillus mucilaginosus (Goswami et al. 2016) are standard potassium (K) solubilizers. These bacterial groups convert insoluble form of K in the soil to soluble forms, through various chemical reactions like exchange reactions, chelation, and acidification (Masood and Bano 2016).

20.2.3 Sequestering Iron (Siderophore)

Iron is an essential element and plays a key role in various physiological processes like DNA synthesis, respiration, and photosynthesis along with key factors of many enzymes and Fe–S cluster (Dellagi et al. 2009), but the availability of soluble Fe is limited because of its low solubility at neutral pH. Microorganisms secrete high-affinity iron-chelating compounds in low Fe environments which refer to siderophores as the strong iron-chelating agents. These are water-soluble, and extracellular and intracellular siderophores, which have greater affinity for Fe, are synthesized by almost all microbes under iron limitations. Siderophores produced by the same genus are homologous, while others that could utilize those produced by other rhizobacteria of various genera are heterologous siderophores. Loper and Buyer (1991) reported the production of siderophore by different bacterial genera, like pyoverdines by Pseudomonas spp., hydroxamates by Erwinia carotovora and Enterobacter cloacae, catechols by Agrobacterium tumefaciens and Erwinia chrysanthemi, and rhizobactin by Rhizobium meliloti. Species of Aeromonas, Streptomyces, Rhizobium, Bacillus, Azadirachta, Burkholderia, Serratia, Azotobacter, and Pseudomonas are grouped as iron-chelating bacteria. In these rhizobacteria, Fe3+ siderophore complex is reduced to Fe2+ which is further released into the cell from the siderophore via the inner and outer membrane linking (Parker et al. 2007). The siderophores are destroyed/recycled during the process. The microorganisms producing siderophores have also a major role in the disease suppression of soil-borne disease especially toward fusarium wilts by the action of siderophore-mediated iron competition as well as inducing systemic resistance in plants (Leeman et al. 1996; Meziane et al. 2005).

20.2.4 Modulating Phytohormone Levels

Plant growth-regulating hormones are called phytohormones, namely indole acetic acid (IAA), ethylene, cytokinins, and gibberellins (Glick 2012; Kumar et al. 2013b; Kumar and Mishra 2014). Auxin production is mediated by tryptophan (Trp)-dependent and Trp-independent pathways (Wani et al. 2016). Several beneficial effects have been documented for indole acetic acid, viz., regulation in plant cell division and differentiation; stimulatory effects on germination of seed and tuber; development of root and xylem; management of vegetative growth; formation of lateral and adventitious root; effective response to light, gravity, and fluorescence; affects photosynthesis; pigment formation; biosynthesis of various metabolites; and resistance to biotic/abiotic stresses (Glick 2012).

Members of the genera Pseudomonas, Rhizobium, Bradyrhizobium, Agrobacterium, Enterobacter, and Klebsiella are good at IAA production. Ethylene, a gaseous phytohormone, is biosynthesized from methionine via S-adenosyl-l-methionine (AdoMet) and the cyclic nonprotein amino acid ACC (Wani et al. 2016). ACC synthase converts AdoMet to ACC, while ACC oxidase catalyzes the conversion of ACC to ethylene. Species of Acinetobacter, Achromobacter, Alcaligenes, Azospirillum, Ralstonia, and Serratia are ethylene producers. Ethylene also plays a key role in the defense to heat stress. The cytokinins are master regulators during plant growth and development. They increase their endogenous levels via uptake and enhanced biosynthesis. The gibberellins are tetracyclic diterpenoid carboxylic acids, and few of them function as growth hormones in higher plants, of which GA1 and GA4 are the predominant ones. They are effective counters to seed germination, leaf expansion, stem elongation, flower and trichome initiation, and flower and fruit development. Members of the genera Azotobacter, Pantoea, Rhodospirillum, and Paenibacillus are effective cytokinin and gibberellin producers.

20.2.5 Induced Systemic Resistance

The ability of the plant to resist against the disease and develop a defense to overcome it is known as induced systemic resistance (ISR). ISR is directly linked to physiological tolerance with microbial antagonisms in the rhizosphere region as well as production of phytoalexins as a consequence of defense response. Metabolism of jasmonic acid is the major key player in the whole process. PGPR produce antagonistic substances like siderophores, antibiotics (Mageshwaran et al. 2010, 2012), antimicrobial peptides, acyl homoserine lactones, and volatile compounds (acetoin and 2,3-butanediol) that help plant resist against microbial pathogens, thus enhancing plant growth promotion (Weller et al. 2002). Several strains of Pseudomonas sp., Pseudomonas syringae, and Pseudomonas stutzeri have been applied effectively against phytopathogens like Colletotrichum and Fusarium wilt diseases (El-Badry et al. 2006). Application of several Bacillus species (B. amyloliquefaciens, B. mycoides, B. sphaericus, and B. subtilis) is reported to cause significant reduction in disease incidence (Ryu et al. 2004; Govindasamy et al. 2010) in varied field condition experiments. Productions of defense-related enzymes like peroxidase, polyphenol oxidase, β-1,3-glucanase, chitinases, and phenylalanine are the most primary mechanisms of PGPR for inducing SR against Fusarium oxysporum and Rhizoctonia solani (Dutta et al. 2008). There are reports describing many potential Pseudomonas strains (AN-1-UHF, AN-5-UHF, PN-7-UHF, and PN-13-UHF) to produce proteolytic enzymes which have a very pivotal role in plant growth promotion of apple and pear (Ruchi et al. 2008). Combinations of such strains with other biocontrol agents pose a potent synergistic inhibitory effect against pathogens and in the promotion of plant growth.

20.2.6 Volatile Organic Compound Production

Some specific PGPR strains are found to release some mixed chemicals also known as volatile organic compounds (VOCs) which have a noteworthy role in plant growth promotion. These volatile compounds have also an important role in the mechanism for the stimulation of growth of plants by rhizobacteria. These compounds have also a major task in ISR mechanisms (Ryu et al. 2004). Some major volatile compounds mostly produced by PGP microbes belong to the class of acetaldehyde, ethanol, hydroxyurea, cycloserine, butanal, ethoxyethene, 2-butanol, 1-butanol, 2-methyl,1-propanol, 2-pentanone, 3-hydroxy-2-butanone, 2-ethyl-1-butanol, methoxy-phenyl-oxime, benzaldehyde, dimethyl disulfide, 2-heptanone, dimethyl trisulfide, trimethyl pyrazine, 2-ethyl 1-hexanol, 2-phenyl ethanol, phenyl acetaldehyde, etc. There are some volatile organic compounds, viz., 2,3-butanediol and acetoin, which have been found to be released by certain PGPR strains like Bacillus subtilis GB03, Bacillus amyloliquefaciens IN937a, and Enterobacter cloacae JM22 that have a major role in plant growth promotion of Arabidopsis thaliana (Ryu et al. 2003). In Arabidopsis against Erwinia carotovora, the compounds secreted by these Bacillus species have also been able to induce ISR (Ryan et al. 2009). VOCs produced by the rhizobacterial strains can act as signaling molecules in the mediation of plant–microbe interactions as volatiles produced by PGPR colonizing roots are generated at adequate dose to activate the plant responses (Ryu et al. 2003). Some plant volatiles having low molecular weight, viz., jasmonates, terpenes, and green leaf components, as effective signal molecules for living organisms in different trophic levels have also been recognized (Farmer 2001) which have several roles in plant defense mechanisms.

20.2.7 Indirect Mechanisms

Plant growth-promoting microbes indirectly and effectively enhance the plant defense strategies against phytopathogens through several ways (Fig. 20.3), and these processes happen outside the plant, with the involvement of the plants’ defensive developments (Goswami et al. 2016). The defensive setups are maintained by the presence of the species of Bacillus, Streptomyces, Pseudomonas fluorescens, Pseudomonas putida and Stenotrophomonas, Bradyrhizobium, Rhizobium, Serratia, and Streptomyces. Productions of antibiotics (streptomycin, oligomycin A, butyrolactones, oomycin A, kanosamine, phenazine-1-carboxylic acid, pyrrolnitrin, pyoluteorin, xanthobaccin, viscosinamide, zwittermicin A, and 2,4-diacetylphloroglucinol) prevent the growth of plant pathogens in the vicinity of the plant root (Whipps 2001; Govindasamy et al. 2010; Kumar et al. 2016b), having a broad-spectrum activity. These antibiotics are effective against many phytopathogenic fungi belonging to Basidiomycetes, Deuteromycetes, and Ascomycetes, including Botrytis cinerea, Rhizoctonia solani, Sclerotinia sclerotiorum (Kumar et al. 2016b), and Verticillium dahliae (Raaijmakers et al. 2010).

Fig. 20.3
figure 3

Multifaceted diagram of indirect mechanisms of plant growth promotion by PGP microbe

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Secretion of microbial extracellular lytic enzymes including chitinases, cellulases, β-1,3-glucanases, proteases, and lipases can lyse a portion of the cell walls of many pathogenic fungi of Fusarium and Rhizoctonia member groups. Production of laminarinase and extracellular chitinase is produced by P. stutzeri lyse mycelia of F. solani. Pseudomonas strains, AN-1-UHF, AN-5-UHF, PN-7-UHF, and PN-13-UHF, were reported to produce lytic enzymes especially proteolytic enzymes which have a significant role in the plant growth promotion of apple and pear (Ruchi et al. 2008). Bacillus species isolated from different tomato rhizospheric soil are also found to secrete several hydrolytic enzymes such as β-1,3-glucanase, protease, chitinase, and cellulose which have a vital role in plant growth promotion and plant disease management (Kumar et al. 2012). Chitinolytic Pseudomonas isolate has also showed a pronounced antifungal activity (Velazhahan et al. 1999). PGP bacteria induce defense systems by inducing systemic acquired resistance and induced systemic resistance (López-Bucio et al. 2007).

The resistance mechanisms reduce the phytotoxic microbial communities and also elicit induced systemic tolerance to abiotic stress (Yang et al. 2009). Solubilization of minerals by PGP microbes (highly specialized lithoautotrophs) is one of the most interesting feature for the availability of inorganic nutrients like K, Na, Ca, and other trace elements by producing inorganic acids (HNO3, H2SO4) as an end product of their metabolism. Members belonging to the genus Thiobacillus (S metabolizing) and nitrifiers (Nitrosomonas and Nitrobacter) are the prominent bacterial members solubilizing rock minerals (K/Ca bearing or PO4 3− minerals). Thiobacilli members (T. thiooxidans, T. ferrooxidans) are acidophilic or acid tolerant (below pH 1–2), are able to fix CO2, and use reduced inorganic S compounds. Nitrifying bacteria use urea, ammonium compounds, nitrite, and NO as energy source and some organic compounds for the production of acid on mineral surfaces (concrete, natural stone, glass, feldspar minerals). Some microbial members are potent producers of CO2 as the major end product, where CaO, Ca(OH)2, and CaSiO2 react with CO2 to form CaCO3 in the process of carbonatization, resulting in the decrease of pH from 12.5 to around 8.5 and the subsequent iron/concrete corrosion. The organic acids produced by microbes are having two modes of action of minerals: (a) action of protons and (b) chelation of metal ions. Acids like acetic, gluconic, glucuronic, oxalic, oxaloacetic, succinic, malic, glyoxylic, and others are the most favorable for solubilization processes.

Along with these, other organic acids (amino acids) and polysaccharides are also excreted outside by the microbial cells as a result of unbalanced growth, metabolic bottlenecks, surplus of substrates, or limited supply of nutrients (P, N, K, etc.). Production of organic acids (acetic, butyric, formic, fumaric) and organic solvents (ethanol, butanol, propanol, lactate, acetoin, aldehydes, etc.) as a result of fermentation is also the potential contributor for partial dissolution, swelling, and wear-tear of minerals. Some plant growth-promoting microbes produce exopolymeric substances containing sugars, sugar acids, and amino acids that act as complexing agents and also as metal chelators facilitating reduced metal stress in root rhizosphere. Microbial action of the production of biotic elicitors is also promising in developing defense system of plants, where chemical stimuli activate the production of phytoalexin-type molecules, which elicit morphological and physiological responses in plants in opposition to phytopathogens (Sekar and Kandavel 2010). Compounds like serpentine, ajmalicine, crocetin, picrocrocin, scopolamine, hyoscyamine, and tanshinone are the major stimulatory chemicals produced by PGP microbes for plant defense against pathogenic organisms.

20.3 Taxonomy of Candidate PGP Microbes

Taxonomy, systematics, biosystematics, scientific classification, biological classification, and phylogenetics have allied meanings in records. Classification of small and simple shapes holding bacteria on the basis of morphological characterization is extremely difficult. Besides shape, bacteria are well identified and classified on the basis of their biochemistry and growth conditions. They take account of media, morphology, antibiotic sensitivity, biochemical tests, serological methods, and bacteriophage typing, together constituting the chemotaxonomic and physiological characterization. Recent developments in taxonomic studies including genotypic characters (G+C % content, DNA–DNA homology % based on HPLC and TM methods, whole genome-based average nucleotide identity, average amino acid identity, tetra correlation among nucleotides, pulse-field gel electrophoresis), chemotaxonomic characters (fatty acid methyl esters, cell wall polyamines, cellular sugars, polar lipids, respiratory quinones, cellular amines), characters (pigments, colony properties), numerical taxonomy (computer-assisted characterization like correlation based on Jaccard’s coefficient, simple matching coefficient, Spearman coefficient), and genomic (multilocus sequence typing, pan genomics ribosomal protein sequences, genome relatedness from whole genome) have revolutionized the characterization of many species. The details of the taxonomic markers and their resolution in bacterial systematics are presented in Fig. 20.4. Current strategies of integrating multiple omics technologies like whole genome sequencing (functional and comparative genomics), proteomics (whole-cell and membrane associated), transcriptomics (total RNA pool sequencing), along with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) have shown high potentiality in evolutionary biology to consider how differently bacteria are associated and evolved (Jia et al. 2015) and their complete physiological as well as genetic cataloging.

Fig. 20.4
figure 4

Schematic overview of taxonomic methods used for characterization of microbial candidates and their resolution

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20.4 Genus Rhizobium: Associative Symbiotic and Free-Living N2 Fixers

The genera Azorhizobium, Bradyrhizobium, Burkholderia, Devosia, Ensifer, Methylobacterium, Mesorhizobium, Microvirga, Ochrobactrum, Phyllobacterium, Rhizobium, Shinella of Alphaproteobacteria, and Cupriavidus of Betaproteobacteria and some Gammaproteobacteria form the set of rhizobia (Berrada and Fikri-Benbrahim 2014). Among all, the members of the genus Rhizobium are the most studied for its N2 fixation ability and supportive plant growth-promoting behavior. Members are Gram-negative, aerobic to facultative anaerobic, nonsporulating, motile rods of 0.5–0.9 × 1.2–3.0 μm (Zakhia and de Lajudie 2001; Willems 2006), mostly attributed to symbiotic N2 fixation as well as free-living forms (Mohapatra et al. 2016). Since its first description by Frank (1889), 94 validly named species (LPSN, http://www.bacterio.net/) were affiliated to the genus Rhizobium. G+C % is on average 59–64 mol%. Colonies are found circular, semitranslucent, raised, and 2–4 mm in diameter within few days of inoculation on solid medium. Turbidity develops in liquid medium after 2 or 3 days. They are chemoorganotrophic in nature. Optimum pH and temperature range between 6–7 and 25–30 °C, respectively. Rhizobium is often located in the nodules of beans, peas, and groundnuts. Strains seem host specific in many cases. The bacterial colonization is able to invade the root hairs naturally. In nodules, bacterial clusters fix atmospheric nitrogen into ammonia for plants (Frank 1889). Study shows Rhizobium resists chloramphenicol, polymyxin B, erythromycin, neomycin, and penicillin (Cole and Elkan 1979).

On the basis of scientific classification, Rhizobium comes under kingdom, Bacteria; phylum, Proteobacteria; class, Alphaproteobacteria; order, Rhizobiales; and family, Rhizobiaceae. For cultivation and isolation of Rhizobium species, yeast mannitol agar and Rhizobium medium are used (Gulati 1979). Yeast extract, mannitol, dipotassium phosphate, magnesium sulfate, sodium chloride, and agar are the key components of the medium. Rhizobium genus includes R. galegae (Mousavi et al. 2014) isolated from the nodules of wild Galega orientalis and Galega officinalis; R. gallicum (Amarger et al. 1997) cultivated in Europe and Tunisia from flat-podded variety of nodulating beans, i.e., Phaseolus vulgaris; R. indigoferae (Wei et al. 2002) isolated from Indigo fera shrubs; R. leguminosarum (Frank 1889; Noel et al. 1996) isolated from canola and lettuce; R. loessense (Wei et al. 2003) isolated from nodules of Astragalus and Lespedeza species; R. lusitanum (Valverde et al. 2006) isolated from Phaseolus vulgaris and Leucaena leucocephala; R. mongolense (van Berkum et al. 1996) isolated from Inner Mongolian Medicago ruthenica; R. bangladeshense ; and R. binae (Rashid et al. 2015) isolated from root nodules of lentils in Bangladesh. The members are well distributed in soil with immense ecological as well as agricultural significance for their ability to fix nitrogen (N2) in legume crops for their ability to form root nodules on legumes and fix N2 (Viteri and Schmidt 1987; Young et al. 2001), with 94 species being in standing nomenclature (http://www.bacterio.net/rhizobium.html). In recent years, new members have been isolated from diverse nonlegume niches including sand dunes, effluent treatment plant, activated sludge, bioreactor, pesticide-contaminated sites, freshwater river, and sea water. New members are also described to degrade various pollutants, heavy metals, and hydrocarbons like naphthalene (R. naphthalenivorans; Kaiya et al. 2012), selenite reduction (R. selenitireducens; Hunter et al. 2007), exopolysaccharide production (R. alamii; Berge et al. 2009), aniline (R. borbori; Zhang et al. 2011), use of PAH (R. petrolearium; Zhang et al. 2012), and triazophos (R. flavum; Gu et al. 2014).

20.5 Genus Pseudomonas: Plant Beneficial, Pollutant Degrader

In 1894, the Pseudomonas group was depicted as the most assorted and ever-present bacterial genera like Antarctica to the tropics and described to include Gram-negative, strictly aerobic rods that are motile by polar flagella (Skerman et al. 1980). Pseudomonas species have been cultured from all kinds of environments worldwide, in sediments, water, soil, the sea, deserts, the plant rhizosphere, fungi, diseased animal specimens, and human clinical samples. Pseudomonas strains can linger their constancy in diverse habitats and under very unpleasant circumstances. Over decades, the taxonomy of the Pseudomonas genus has been controversial for other bacterial taxa (Peix et al. 2009). Based on the 16S-rRNA similarity, currently there are 140 species belonging to the genus Pseudomonas which are termed as sensu stricto group I with names that have standing in nomenclature in LPSN (http://www.bacterio.net/pseudomonas.html).

The members are aerobic, Gram-negative, straight or slightly curved rods, 0.5–1.0 μm in diameter, and 1.5–5.0 μm in length. Pseudomonas are motile with one or several polar flagella. Some species are found well particular in forming poly-β-hydroxybutyrate as the carbon-storage granule, which appears as sudanophilic inclusions. No resting stages are documented. Pseudomonas is not fussy in general. They can grow up on protein hydrolysate, magnesium chloride, and potassium sulfate kind intermediates containing agar media. Species-specific Pseudomonas isolation agars also contain cetrimide, nalidixic acid, cephaloridine, penicillin G, pimaricin, malachite green, and glycerol. According to biochemical characterization, Pseudomonas shows catalase positive, Voges–Proskauer, and indole and methyl red negative in general. An additional attribute associated with Pseudomonas species is that they ooze a yellowish green fluorescence, called pyoverdine, pyocyanin as a blue pigment, a reddish pigment called pyorubin, and pyomelanin as brown function under iron-limiting conditions, as a siderophore, but few secrete quinolobactin as yellow/dark green in the presence of iron. Pseudomonas strains are reported to produce IAA, HCN, siderophores, phenazines, cyclic lipopeptides, pyoverdine, and quorum-sensing signaling compounds (Gupta et al. 2014; Kumar et al. 2016b). On the other hand, Pseudomonas strains have been executed using MALDI-TOF-MS for excellent identification results (Pineda et al. 2010).

According to the scientific classification, Pseudomonas comes under kingdom, Bacteria; phylum, Proteobacteria; class, Gammaproteobacteria; order, Pseudomonadales; family, Pseudomonadaceae; genus, Pseudomonas; and species, P. fluorescens, P. aurantiaca, and P. putida. Pseudomonas fluorescens strains play a major role in plant growth promotion, induction of systemic resistance, and action as bacterial antagonist to control pathogenic bacteria and fungi. It is a potential biopesticide for augmentative biological control of several diseases and bioremediation of various unrefined compounds in agriculture and horticulture (Ganeshan and Kumar 2005). Pseudomonas aurantiaca strains are generally orange-colored soil bacterial members. Rhizosphere soils of sugarcane, soya bean, canola, and potatoes are the customary habitats of such species. The bacterium produces di-2,4-diacetylfluoroglucylmethan. Di-2,4-diacetylfluoroglucylmethan is a natural phenol compound, which inhibits the growth of phytopathogens and promotes plant growth indirectly. Based on 16S rRNA analysis, Pseudomonas aurantiaca is a subspecies of Pseudomonas chlororaphis (Peix et al. 2007). Pseudomonas putida strains harbor multi-plasmid hydrocarbon-degrading genes (called degradative plasmids). They are the first patented organisms in the world. P. putida has been confirmed as a potential biocontrol agent with effectual antagonist activity on damping off diseases such as Pythium (Amer and Utkhede 2000) and Fusarium (Validov et al. 2007).

20.6 Genus Bacillus: Dominant Cum Abundant Members

The majority of Bacillus edaphicus, Bacillus mucilaginosus, Bacillus amyloliquefaciens, Bacillus subtilis, Bacillus cereus, Bacillus megaterium, Bacillus lipopeptides, Bacillus pasteurii, Bacillus pumilus, Bacillus mycoides, and Bacillus sphaericus are distributed globally with the extensive amount of ability to promote plant growth and have been widely recognized (Govindasamy et al. 2010). The growth promotion includes production of siderophore, phytohormones and antibiotics, solubilization and mobilization of phosphate, inhibition of plant ethylene production, and induction of efficient pathogen resistance (Whipps 2001; Gutiérrez-Mañero et al. 2001; Idris et al. 2007; Richardson et al. 2009). Multilayered chambers of cell wall, secretion of peptide signal molecules and peptide antibiotics, with extracellular enzymes, contribute to survival under unfavorable conservation for extensive periods of time. Repressing capability of plant pathogens by Bacillus subtilis and Bacillus cereus has been widely recognized. Genus Bacillus was named in 1835 by Christian Ehrenberg. By Ferdinand Cohn, Bacillus was further characterized as most ubiquitous, spore-forming, Gram-positive, aerobic/facultative anaerobic bacteria. Bacillus has expanded to extreme phenotypic variety and heterogeneity. Today, Bacillus holds 243 types of species with cultivable isolates (16S rRNA gene sequences >1200 bp) from varied environments (https://rdp.cme.msu.edu/hierarchy/hierarchy_browser/Bacillus), where only 19 types of strains have been reported to be from plants or plant-associated niches.

20.7 Genus Azotobacter: Free-Living N2 Fixers

Azotobacter is a motile, free-living aerobic bacterium with a genomic content of G−C of 63–67.5% (T m) (Becking 1981). This heterotrophic group of bacteria has thick-walled cysts which may produce large quantities of capsular slime. The particular genus plays an important role in nitrogen cycle as nonsymbiotic nitrogen fixer and acts as PGPR. The bacterial group makes possible the root expansion, improves nutrient uptake potentiality, protects from plant diseases, and increases biomass production in the rhizosphere region of nearly every one of the crops (Kasa et al. 2015). They are distributed in soils, water, and sediments. Azotobacter chroococcum, an oval or a spherical kind of Gram-negative bacterium, was revealed and explained by Martinus Beijerinck in 1901 for the first time (Beijerinck 1901; Mrkovacki and Milic 2001). Lipman stated about Azotobacter vinelandii in 1909 and in 1904 on the subject of Azotobacter beijerinckii, which he named in the admiration of Beijerinck. In 1949, Russian microbiologist Nikolai Krasilnikov identified the species of Azotobacter nigricans. Azotobacter nigricans was divided into two subspecies—Azotobacter nigricans subsp. nigricans and Azotobacter nigricans subsp. achromogenes in 1981 by Thompson Skerman. Again, in the year 1981, Thompson and Skerman described Azotobacter armeniacus.

In 1991, Page and Shiv Prasad informed concerning Azotobacter salinestris—a micro-aerophilic and air-tolerant bacterium. According to the taxonomical division, Azotobacter comes near the domain, Bacteria; phylum, Proteobacteria; class, Gammaproteobacteria; order, Pseudomonadales; and family, Pseudomonadaceae/Azotobacteraceae (Becking 1999), with most members reported to be described as A. vinelandii or A. chroococcum. Morphological similarity and biochemical uncertainty with FNFB like Derxia, Azomonas, and Beijerinckia are the difficulties in characterizing Azotobacter species. In 2004, a phylogenetic study has shown that Azotobacter vinelandii evolved from Pseudomonas aeruginosa. After years, in 2007, the genera Azotobacter, Azomonas, and Pseudomonas were publicized as allied or might be synonyms.

20.8 Genomic Insight and Behavior of Some Plant Growth-Promoting Microbes

Of today, 20,584 eubacterial and 907 archaebacterial candidates have been described, out of which 9966 non-type bacterial, 3890 type bacterial, and 210 archaebacterial genomes have been sequenced. The use of genome sequencing through next-generation sequencing (NGS) approach with massively parallel sequencing capacity, high depth coverage, and cost-effective features has moved the basics of bacterial species designation, taxonomy, and phylogeny to a next level termed as “taxonogenomics or phylogenomics.” Complete genome projects are enabling the researchers to study the genetic and functional relatedness between organisms at the whole-cell level, thus far beyond conventional 16S rRNA-based phylogeny system. Genetic events such as horizontal gene transfer (HGT), gene rearrangements, plasmid functions in species evolution, and niche adaptation, have become a newer attraction for the geneticists with the high affordability and accessibility to general microbiology laboratories. Completed genome projects with genome features of some candidate PGPR strains are presented in Table 20.1. Recently, NGS has been used to study genomes of different PGPR (free-living and endophytic strains) mainly isolated from crop species such as rice, maize, wheat, potato, sugarcane, barley, coffee, tea, soybean, etc. and are presented in Table 20.2, with their plant-beneficial properties.

Table 20.1 Genomic properties of PGPR bacteria as obtained through whole genome sequencing from JGI-IMG database
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Table 20.2 Genomic perspective of some plant-beneficial PGP microbes
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The microbiology of the rhizosphere has been thoroughly studied for more than 100 years, but study on endosphere and the organisms associated (endophytes) remains largely unexplored. Endophytic microbes reside within various tissues of the host plant in a commensal or beneficial manner, and endophytic microbiome is known for its antagonistic activity against pathogens (Berg et al. 2013). They are found to be the promising source of natural metabolites with potential benefits to plant as well as other animals because of their significant bioactivities and medical importance (Kaul et al. 2012; Premjanu and Jayanthy 2012; Mousa and Raizada 2013; Kusari et al. 2014). Endophytes are also beneficial for the host plants with biotic and abiotic stress tolerance, nutrient acquisition, and plant growth promotion (Rodriguez et al. 2008; Kumar et al. 2013c). Genome sequencing has revealed the genetic inventory of these organisms with capability for various plant growth-promoting properties like nitrogen fixation, production of phytohormone (IAA, GA, etc.), mineral acquisition (Fe, P, K), biotic/abiotic stress tolerance, and other nutrient cycling processes (Fouts et al. 2008; Firrincieli et al. 2015; Martinez-Garcia et al. 2015). Recent studies have provided greater understanding on the mode of endophytism in plant root and other plant hosts through gene coding for N-acyl homoserine lactone synthases, hydrolases, adherence factors, and fusaric acid resistance in Pantoea ananatis (Megias et al. 2016). Genomes of such entophytes (Gluconacetobacter diazotrophicus Pal5, Stenotrophomonas maltophilia R551-3, Pseudomonas fluorescens PICF7, Kosakonia oryzae K0348, Raoultella terrigena R1Gly, Bacillus thuringiensis KB1, Pseudomonas putida W619, Azospirillum sp. B510, Variovorax paradoxus, Herbaspirillum seropedicae strain SmR1, Burkholderia phytofirmans strain PsJN, Burkholderia sp. strain KJ006, Pseudomonas poae RE∗1-1-14, Paenibacillus sp. P22, Pantoea agglomerans, Pseudomonas sp. strain RIT288, Janthinobacterium lividum) are served to be the model systems for studying entophytic plant–microbe interactions. The concept of PGPR-mediated plant growth promotion is gaining worldwide importance and acceptance and has been applied on a wide range of crops including cereals, pulses, vegetables, oilseeds, and plantation crops. Combination of the use of these microbes in plant disease management and the solutions of soil nutrient management might provide ample advantages to agriculture.

20.9 Conclusions and Future Prospects

To avert the lack of sufficient amount of one or more nutrient sources such as nitrogen, iron, and phosphorus and also to obtain higher crop yields, it would obviously be advantageous if efficient biological resources of providing nitrogen, iron, and phosphorus to plants could be commercialized to substitute inexpensive chemical nitrogen, iron, and phosphorus that are currently used. Plant growth-promoting bacteria (PGPB) modulates plant stress indicators under environmental stresses. PGPB helps in mounting niche in the expansion of organic agriculture. The benefits done by PGP bacteria to the agriculture are enormous. Numerous genetically engineered PGP bacteria are already being used successfully in a number of countries in the developing world commercially as adjuncts to agricultural practice. The use of detailed molecular techniques and next-generation OMICS-based tools is still to be implemented to study elaborate biochemical and molecular functions of the plant-beneficial microbes. Integrated use of genomics, proteomics, transcriptomics, metabolomics, and secretomics might help biologists to gain better insight into the ecophysiological aspects and niche adaptation strategies of PGP microbes. In spite of all odds, commercialized and more efficacious strains of Azotobacter, Bacillus, Paenibacillus, Pseudomonas, and various Rhizobia sp. are showing promising development in the field of inoculation. So, study on microbes and their interaction with plants on commercial scale is still required to make PGPB an efficient technique in agricultural sustainability and intensive production practices.