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9.1 Introduction

The rhizosphere around the growing plant roots is a very dynamic environment and harbors a large number of total microorganisms, especially bacteria, greater than root-free soil. The heterogenous microbial populations interact with each other and with the plant through symbiotic, associative, neutralist or antagonistic effects. The outcome of colonization and penetration of the plant tissue with a microorganism varies from asymptomatic to disease and from associative to symbiosis, depending upon the mutual perception or recognition between the interacting cells. Such interactions are influenced greatly by the environment. The microbes that penetrate and colonize plants have evolved an elaborate system for subverting the plant defense system. The group of beneficial, root associative bacteria that stimulate the growth of a plant is termed as plant growth-promoting rhizobacteria (PGPR). Fluorescent pseudomonads and bacilli comprise the major group among PGPR along with other bacteria, like, Acetobacter, Agrobacterium, Alcaligenes, Arthrobacter, Azospirillum, Azotobacter, Cellulomonas, Clostridium, Enterobacter, Erwinia, Flavobacterium, Pasteuria, Serratia and Xanthomonas. The beneficial rhizosphere microorganisms also include rhizobia and bradyrhizobia, which establish symbiotic relationship with leguminous plants. In the absence of appropriate microbial populations in the rhizosphere, plant growth may be impaired (Sturz et al. 2000).

Legumes are widely used for food, fodder, fuel, timber, green manure, and as cover crops in different agricultural systems. In developing countries, legumes are often an integral part of forest, pastures and agricultural ecosystems. On global scale, nitrogen-fixing legumes are the major source of soil N pool. Legume crops meet their N requirement through symbiotic N2-fixation by forming nodules with rhizobia. Legumes and the rhizosphere provide most of the nutritional requirements of nodule bacteria and enhances the Rhizobium population several folds during plant growth. Rhizobium–legume associations are usually host specific, and a given rhizobial strain can infect only a limited number of hosts. Most of the characterized rhizobial strains have been isolated from the limited range of cultivated legume species.

The high-input agricultural practices of the more industrialized nations of temperate zones are rarely suitable for tropical conditions in most developing countries. Therefore, emphasis on biological processes which are able to improve agricultural productivity, while minimizing soil loss and ameliorating adverse edaphic conditions, are essential. A better understanding of rhizobial ecology, optimization of N2-fixing conditions in legume-Rhizobium symbiosis and selection of rhizosphere bacteria having synergistic interactions with Rhizobium leading to growth-promoting effects on legumes are crucial for improving and sustaining agricultural ecosystems. The inoculation effects of diverse bacterial groups possessing plant growth-promoting traits on the performance of legumes are discussed. The fundamentals of the different processes involved in plant growth promotion are briefly introduced. The different strategies or biotechnological approaches adopted for enhancing biological N2-fixation (BNF), P-solubilization, auxins production and improving biocontrol activity are also described. Various constraints involved in crop improvement following inoculation with genetically engineered bacterial strains and the possibilities of deriving desired benefits by ensuring the establishment and survival of introduced microbial inoculants in soil are explored.

9.2 Mechanisms Involved in Plant Growth Promotion

Microbial ecology of the rhizosphere includes the study of the interactions of microorganisms with each other and the environment surrounding the plant root (Weyens et al. 2009). Rhizosphere microorganisms are of major interest due to their beneficial or detrimental effects on plant growth. It is therefore, important to understand the mechanism by which rhizosphere microorganisms impact plant growth in order to develop technologies that could enhance their activities. Microbial populations present in the rhizosphere of legumes have shown substantial effects on nodulation by Rhizobium spp. and on subsequent growth and yield of leguminous crops (Kloepper et al. 1989; Glick 1995). Microorganisms inhabiting rhizosphere of legumes may benefit plants in a variety of ways, like increased recycling, mineralization and uptake of nutrients; synthesizing vitamins, amino acids, auxins, gibberlins and plant growth regulating substances; reducing metal toxicity (bioremediation) in contaminated soils; antagonism with potential plant pathogens through competition and development of amensal relationships based on production of antibiotics, siderophores, and/or hydrolytic enzymes (Stockwell and Stack 2007; Sindhu et al. 2009c).

9.2.1 Increased Recycling, Mineralization and Uptake of Nutrients

Microorganisms in the rhizosphere influence the availability of mineral nutrients to the plants, sometimes by increasing the availability of inorganic nutrients to the plant, and in other cases, using limiting concentrations of inorganic nutrients before they could reach plant roots. Some rhizosphere bacteria, i.e., rhizobia, azotobacters, and azospirilla have the ability to fix atmospheric N into plant utilizable form, ammonia (Franche et al. 2009). Other microorganisms help plants by solubilizing bound P (Vessey 2003) and potassium or by providing iron and sulfur (Crowley et al. 1991; Scherer and Lange 1996; Crowley and Kraemer 2007).

9.2.1.1 Biological Nitrogen-Fixing Bacteria and Inoculation Responses

Sustainable agriculture involves the successful management of agricultural resources to satisfy the changing human needs, while maintaining or enhancing the environmental quality and conserving natural resources. Consequently, sustainability considerations demand that alternatives to nitrogen fertilizers are sought. In this context, BNF offers an alternative in farming practices as it exploits the capacity of certain N2-fixing bacteria to reduce atmospheric nitrogen into a compound (ammonia) mediated by enzyme nitrogenase (Bohlool et al. 1992; Burris and Roberts 1993). Legume crops meet their N requirement through symbiotic nitrogen fixation by forming root nodules with rhizobia (Brewin 2002; Gage 2004) which in turn reduce the dependency of agricultural crops on fossil fuel-derived nitrogenous fertilizers. Additionally, biologically fixed N is bound in soil organic matter and thus is much less susceptible to soil chemical transformations and physical factors that lead to volatilization and leaching. Therefore, BNF has an important role in sustaining productivity of soils.

Only some prokaryotes, a few bacteria and cyanobacteria, have acquired the ability to reduce atmospheric dinitrogen and add this essential nutrient to agricultural soils. Biological N2 fixation occurs in a free-living state, in association with or in symbiosis with plants. Different N2-fixing bacteria have been used to improve the supply of fixed N as nutrient to crop plants. Among the nitrogen-fixing systems, the legume-Rhizobium symbiosis alone accounts for 70–80% of the total N fixed biologically on global basis per annum and one-third of the total N input needed for world agriculture. The symbiotic rhizobia have been found to fix N ranging from 57 to 600 kg ha−1 annually (Elkan 1992). Annual inputs of fixed nitrogen are calculated to be 2.95 million tonnes (Tg) for the pulses and 18.5 Tg for the oilseed legumes (Herridge et al. 2008).

Rhizobium includes the fast-growing species, Bradyrhizobium includes slow-growing species and Azorhizobium includes those fast-growing species capable of forming both stem and root nodules on tropical water-logged legume, Sesbania. Chen et al. (1995) proposed a separate genus, Mesorhizobium, to indicate a growth rate intermediate between that of Bradyrhizobium strains and typical fast-growing Rhizobium strains. Subsequently, it was used to denote a phylogenetic position for rhizobia intermediate between these two genera. According to current taxonomic classification of root-nodule bacteria, 11 genera and 45 species have been defined (Sahgal and Johri 2006; Wiliems 2006).

In the rhizosphere of legumes and cereals, other diazotrophic bacteria could also contribute N to plants. Free-living diazotrophic bacteria contribute upto 15 kg ha−1 year−1 fixed N and the root-associative bacteria fix N to a level of 15–36 kg ha−1 year−1. Similarly, cyanobacteria, the free-living nitrogen fixers contribute about one-third of the N requirement of the crop and add about 15–80 kg ha−1 year−1 to the rice cropping system (Elkan 1992) (Table 9.1). The free-living/associative diazotrophs, although have limited potential in terms of average N input on acreage basis but inhabit almost all ecological environments and contribute more in nutrient use efficiency and improvement in crop physiology (Pandey and Kumar 1989; Wani 1990; Fujiata et al. 1992).

Table 9.1 Estimated average rates of biological nitrogen fixation by diazotrophs and associations
Full size table

The symbiotic effectiveness of different legume species and their microsymbionts has been found to be variable. In general, faba bean (Vicia faba), and pigeonpea (Cajanus cajan) have been found to be very efficient; soybean (Glycine max), groundnut (Arachis hypogaea) and cowpea (Vigna sinensis) to be average; common bean and pea poor in fixing atmospheric N (Hardarson 1993). Among the legumes, soybean is the dominant crop legume, representing 50% of the global crop legume area and able to fix 16.4 million tones N annually, representing 77% of the N fixed by the legumes (Herridge et al. 2008). Inoculation of legumes with efficient strains of rhizobia has often resulted in significant increases in yields of various legume crops (Thies et al. 1991; Wani et al. 2007; Franche et al. 2009). Elsiddig et al. (1999) studied the inoculation effect of Bradyrhizobium strains TAL 169 and TAL 1371 (introduced) and strains ENRRI 16A and ENRRI 16C (local) on five guar (Cyamopsis tetragonoloba) cultivars in a field experiment. Most of the Bradyrhizobium strains significantly increased yield, protein, crude fiber and mineral content. The locally-isolated strains affected these parameters more than the introduced ones. Karasu et al. (2009) observed that inoculation of chickpea (Cicer arietinum) seeds with R. ciceri isolate had a significant effect on seed yield, plant height, first pod height, number of pods per plant, number of seeds per plant, harvest index and 1,000 seed weight. But, nitrogen doses (applied at 0, 30, 60, 90, and 120 kg ha−1 level as ammonium nitrate) had no significant effect on yield and yield components. Local population genotype as crop material gave the highest yield (2,149.1 kg ha−1) among three chickpea genotypes used.

Sindhu et al. (1992) compared the potential of N fixed by Rhizobium strains in chickpea using non-nodulating genotype PM233 derived from normal nodulating genotype ICC640. The N fixed by the Rhizobium strains Ca534 and Ca219 in parent cultivar gave the plant dry weights more than those obtained by applying urea (80 kg N ha−1) in the non-nodulating mutant PM233, suggesting that in chickpea effective symbiosis with rhizobia provides more than 80 kg N ha−1. The benefits of N fixed in legumes to subsequent cereal crops are substantial and persist for several years due to progressively slow mineralization. The benefits obtained were of higher magnitude with green manuring crops and upto 532 kg N could be incorporated by 60 days green manuring crops where the rate of N accumulation were rapid upto 10.8 kg N ha−1 day−1 (Peoples and Herridge 1990).

In coinoculation experiments of N2-fixing Azotobacter vinelandii with Rhizobium spp., it was found that coinoculation increased the number of nodules on the roots of soybean, pea (Pisum sativum) and clover (Trifolium pratense) (Burns et al. 1981). Increased nodulation of soybean also occurred in field trial. Similarly, coinoculation of Azospirillum brasilense with Rhizobium strains showed synergistic effect on soybean and groundnut (Iruthayathas et al. 1983; Raverkar and Konde 1988). Compared to single Rhizobium inoculation, coinoculation of Rhizobium spp. and Azospirillum spp. was found more effective in enhancing the number of root hairs, the amount of flavonoids exuded by the roots and the number of nodules (Itzigsohn et al. 1993; Burdman et al. 1997; Remans et al. 2007, 2008b). The effect of Azospirillum on the legume-Rhizobium symbiosis was found to depend on the host genotype used. It was observed that Azospirillum–Rhizobium coinoculation increased the amount of fixed N and the yield of DOR364 genotype of common bean (Phaseolus vulgaris L.) across all sites on-farm field experiments, whereas a negative effect of Azospirillum–Rhizobium coinoculation on yield and N2-fixation was observed in BAT 477 genotype on most of the sites as compared to sole application of Rhizobium (Remans et al. 2008b).

Field and greenhouse data indicated that increased nodulation of beans (Phaseolus vulgaris) by R. phaseoli occurred with coinoculation of Pseudomonas putida (Grimes and Mount 1984). However, bean yield and shoot weight were not significantly affected by coinoculation, demonstrating that increasing nodule number or infection by Rhizobium spp. may not affect plant productivity. Bolton et al. (1990) also demonstrated that nodulation of pea increased following the inoculation of mixtures of R. leguminosarum and a deleterious toxin-releasing Pseudomonas sp. However, nodules and dry matter accumulation in shoots were the same whether or not the Pseudomonas sp. was coinoculated. On the other hand, enhancement in nodulation, root length, plant biomass and yield by mixed inoculation of rhizobia with other rhizobacteria in different legumes have been reported. For example, Chanway et al. (1989) tested nine PGPR strains against single cultivar of lentil and pea in the field. None of the strains stimulated the growth of pea, but in lentil plots inoculated with one or more rhizobacterial strains, there were significant increase in emergence, vigor, nodulation, acetylene reduction activity and root weight. The enhanced nodulation and growth of chickpea along with reduction in wilt incidence was observed on coinoculation of rhizobacteria obtained from chickpea rhizosphere when these strains were coinoculated with an effective R. ciceri strain Ca181 (Khot et al. 1996).

Coinoculation of the five plant growth-promoting fluorescent pseudomonad strains, isolated from Indian and Swedish soils, and R. leguminosarum bv. viceae strains, recovered from Swedish soils, improved growth of pea cv. Capella (Dileep Kumar et al. 2001). In a similar study, Goel et al. (2002) observed that coinoculation of chickpea with Pseudomonas strains MRS23 and CRP55b, and Mesorhizobium sp. cicer strain Ca181 increased the formation of nodules by 68.2–115.4%, at 80 and 100 days after planting as compared to single inoculation of Mesorhizobium strain under sterile conditions. The shoot dry weight ratios of coinoculated treatments at different stages of plant growth varied from 1.18 to 1.35 times that of Mesorhizobium-inoculated and 3.25–4.06 times those of uninoculated plants. Similar synergistic effects on nodulation and plant growth have also been observed for other legumes by dual inoculation of B. japonicum and P. fluorescens in soybean (Li and Alexander 1988; Nishijima et al. 1988; Dashti et al. 1998), R. meliloti with Pseudomonas in alfalfa (Li and Alexander 1988; Knight and Langston-Unkeffer 1988), R. leguminosarum with an antibiotic-producing P. fluorescens strain F113 in pea (Andrade et al. 1998) and Mesorhizobium/Bradyrhizobium strains with Pseudomonas sp. in greengram [Vigna radiata (L.) wilczek] and chickpea (Sindhu et al. 1999; Goel et al. 2000, 2002).

Similarly, bacterization of Bacillus species to seeds or roots altered the composition of rhizosphere, leading to increase in growth and yield of different legume crops (Holl et al. 1988). For instance, Halverson and Handelsman (1991) observed that seed treatment with B. cereus UW85 had 31–133% more nodules than untreated soybean plants after 28 and 35 days of planting in the field. In the growth chamber, in sterilized soil-vermiculite mixtures, UW85 seed treatments enhanced nodulation by 34–61% at 28 days after planting. It was suggested that UW85 affected the nodulation process soon after planting by stimulating bradyrhizobial infections or by suppressing the abortion of infections. In a follow up study, Turner and Backman (1991) reported that coating of peanut seeds with B. subtilis improved germination and emergence, enhanced nodulation by Rhizobium spp., enhanced plant nutrition, reduced levels of root cankers caused by Rhizoctonia solani AG-4 and increased root growth. In a similar study, Srinivasan et al. (1997) reported enhanced nodulation in Phaseolus vulgaris when coinoculated with R. etli strain TAL182 and B. megaterium S49. The mixed inoculation increased root hair proliferation and lateral root formation. The potential of Bacillus sp. to enhance nodulation, plant dry matter and grain yield on coinoculation with rhizobia has also been reported for pigeonpea (Podile 1995) and white clover (Holl et al. 1988). Sindhu et al. (2002a) found that coinoculation of Bacillus strains with effective Bradyrhizobium strain S24 caused enhancement in shoot dry mass of green gram ranging from 1.28 to 3.55 at 40 days of plant growth. Nodule promoting effect and increase in nitrogenase activity was also observed with majority of Bacillus strains at 40 days of plant growth.

Mishra et al. (2009a) showed that plant growth-promoting bacteria (PGPB) strain B. thuringiensis-KR1, originally isolated from the nodules of Kudzu vine (Pueraria thunbergiana), promoted plant growth of field pea and lentil (Lens culinaris L.) when coinoculated with R. leguminosarum-PR1 under Jensen’s tube, growth pouch and non-sterile soil, respectively. Coinoculation with B. thuringiensis-KR1 (at a cell density of 106 c.f.u. ml−1) had the highest and most consistent increase in nodule numbers, shoot weight, root weight, and total biomass, over rhizobial inoculation alone. The enhancement in nodulation due to coinoculation was 85 and 73% in pea and lentil, respectively, compared to R. leguminosarum-PR1 treatment alone. The shoot dry-weight gains on coinoculation with variable cell populations of B. thuringiensis-KR1 varied from 1.04 to 1.15 times and 1.03–1.06 times in pea and lentil, respectively to those of R. leguminosarum-PR1 inoculated treatment at 42 days of plant growth. The cell densities higher than 106 c.f.u. ml−1 had an inhibitory effect on nodulation and plant growth whereas lower inoculum levels resulted in decreased cell recovery and plant growth performance. Similarly, enhanced nodule number and biomass yield were achieved after coinoculation of soybean with the B. japonicum SB1 and the plant growth-promoting B. thuringiensis-KR1 (Mishra et al. 2009b).

Inoculation of legumes with different rhizobial strains in general results in a 10–15% increase in yield of legumes. However, the desired impact of biofertilizer on legumes is usually not achieved under certain field conditions. The inoculation with commercial inoculants often fails to improve crop productivity (van Elsas and Heijnen 1990) probably due to the inability of rhizobial species to compete with the indigenous, ineffective and built in populations, which presents a competitive barrier to the introduced strains (Sindhu and Dadarwal 2000). In contrast, production of bacteriocins by rhizobia have been shown to suppress growth as well as nodulation by the indigenous non-producer strains, thus improving nodulation competitiveness of bacteriocin-producing inoculant strains (Goel et al. 1999; Sindhu and Dadarwal 2000). Transfer and expression of tfx genes (involved in trifolitoxin production) in various rhizobia showed stable trifolitoxin production and restricted nodulation by indigenous trifolitoxin-sensitive strains on many leguminous species (Triplett 1988, 1990). However, attempts to manipulate certain rhizobial genes in specific legume rhizosphere niches for improving competition have not been impressive (Nambiar et al. 1990; Sitrit et al. 1993; Krishnan et al. 1999).

Biotechnological approaches used to enhance N2 fixation and crop productivity (Pau 1991; Hardarson 1993; Sindhu et al. 2009a) under field conditions have been of limited use. For example, recombinant constructs of R. meliloti and B. japonicum having increased expression of nifA and dctA genes although showed increase in the rate of N fixed but under field conditions, the same constructs did not show any significant increase in N2-fixation or yields (Ronson et al. 1990). Manipulations of common nodulation genes to improve the bacterial competition have usually resulted in either no nodulation, delayed nodulation or inefficient nodulation (Devine and Kuykendall 1996). Mendoza et al. (1995) enhanced NH +4 assimilating enzymes in R. etli through genetic engineering, by inserting an additional copy of glutamate dehydrogenase (GDH), which resulted in total inhibition of nodulation on bean plants. However, nodule inhibition effect was overcome when gdhA expression was controlled by NifA and thereby, delaying the onset of GDH activity after nodule establishment (Mendoza et al. 1998). Similarly, attempts to engineer hydrogen uptake (Hup+) ability by cloning hydrogenase genes into Hup- strains of Rhizobium resulted in experimental successes only in areas where soybeans are cultivated and where the photosynthetic energy is limited (Evans et al. 1987). Attempts to develop self-fertilizing crops for N have also been a failure, mainly because of the complexity of the nitrogenase enzyme complex to be expressed in the absence of an oxygen protection system in eukaryotes (Dixon et al. 1997). Moreover, induction of nodule-like structures or pseudonodules using lytic enzymes or hormones treatment in wheat (Triticum aestivum) and rice (Oryza sativa) though showed nitrogenase activity and 15N2 incorporation, but the activity expressed was >1% of the value observed for legumes (Cocking et al. 1994).

9.2.1.2 Phosphate Solubilization and Mobilization and its Agronomic Significance

In agriculture, phosphorus (P) is second only to N in terms of quantitative requirement for crop plants (Goldstein 1986; Fernandez et al. 2007). It is found in soil, plants and microorganisms in both organic and inorganic forms. However, the total P content in an average soil is 0.05% and only a very small fraction (∼0.1%) of the total P present in the soil is available to the plants because of its chemical fixation and low solubility (Stevenson and Cole 1999). The pool of immediately available P is thus, extremely small and must be supplied regularly to offset plant demands (Bieleski 1973). Phosphorus may be added to soil either as chemical fertilizers or as leaf litter, plant residues or animal remains. The P fertilizers are the world’s second largest bulk chemicals used in agriculture and therefore, the second most widely applied fertilizer (Goldstein et al. 1993; Goldstein 2007). However, 75% of phosphate fertilizers applied to soil are rapidly immobilized and thus become unavailable to plants (Rodriguez and Fraga 1999). Therefore, P deficiency is a major constraint to crop production and under such conditions, the microorganisms offer a biological rescue system capable of solubilizing the insoluble inorganic P of soil to make it available to plants and thus maintain the soil health and quality (Rodriguez and Fraga 1999; Richardson 2001; Deubel and Merbach 2005; Chen et al. 2006; Khan et al. 2007).

Phosphate solubilizing (PS) and mobilizing microorganisms include bacteria, actinomycetes as well as the fungi. The most important PS bacteria (PSB) belong to genera Bacillus and Pseudomonas, though species of Achromobacter, Alcaligenes, Brevibacterium, Corynebacterium, Serratia and Xanthomonas have also been reported as active P solubilizer (Venkateswarlu et al. 1984; Cattelan et al. 1999; Khan et al. 2007). In a study, Naik et al. (2008) screened 443 fluorescent pseudomonad strains for the solubilization of tricalcium phosphate(TCP) and reported that 18% formed visible dissolution halos on Pikovskaya agar medium plates. Based on phenotypic characterization and 16S rRNA gene phylogenetic analyses, these strains were identified as P. aeruginosa, P. mosselii, P. monteilii, P. plecoglosscida, P. putida, P. fulva and P. fluorescens. The P-solubilizing Bacillus species isolated from the rhizosphere of legumes and cereals included B. subtilis, B. circulans, B. coagulans, B. firmus, B. licheniformis, B. megaterium and B. polymyxa (Gaind and Gaur 1991; Rajarathinam et al. 1995). Other PSB include species of bacteria like, A. chroococcum, Burkholderia cepacia, Erwinia herbicola, Enterobacter agglomerans, E. aerogenes, Nitrosomonas, Nitrobacter, Serratia marcescens, Synechococcus sp., Rahnella aquatilis, Micrococcus, Thiobacillus ferroxidans and T. thiooxidans (Banik and Dey 1983; Kim et al. 1998; Bagyaraj et al. 2000). Rhizobium and Bradyrhizobium strains have also been found to solubilize rock phosphate (RP) or organic P compounds effectively through the production of organic acids and/or phosphatases (Halder et al. 1991; Abd-Alla 1994). The various fungi having efficient PS ability belong to genera Aspergillus, Fusarium, Penicillium and Trichoderma (Rashid et al. 2004; Khan et al. 2010). Ahmad et al. (2008) reported that out of 72 isolates obtained from rhizosphere soil and root nodules, solubilization of P was commonly detected in Bacillus (80%) followed by Azotobacter (74%), Pseudomonas (56%) and Mesorhizobium (17%). The principal mechanism of increasing P availability is the microbial production of organic acids that may dissolve appetite, releasing soluble forms of P through acidification of rhizosphere soil. Additionally, the acidification of the rhizosphere environments through metabolic production of hydrogen ions alters the pH sufficiently to mobilize soil minerals (Rodriguez et al. 2006).

Phosphatic biofertilizers were first prepared in USSR, using B. megaterium var. phosphaticum as PSB and the product was named as “phosphobacterin.” It was extensively used in collective farming for seed and soil inoculation, and reported to give 5–10% increase in crop yields. Subsequently, inoculation experiments using phosphobacterin and other PSM for legumes like, groundnut, peas, and soybean showed an average 10–15% increase in yields in about 30% of the trials (Agasimani et al. 1994; Dubey 1997; Vessey 2003). The variations under field conditions are expected due to the effect of various environmental conditions and survival of the inoculant strains in soil. The inoculation of PSB along with Rock phosphate (RP) also resulted in increased availability of P for plant utilization (Jisha and Alagawadi 1996). It was observed that inoculation of mineral phosphate solubilizing bacteria (MPSB) along with 17.5 kg P ha−1 as Massourie rock phosphate (MRP) increased dry matter in chickpea and was as effective as single super phosphate (Prabhakar and Saraf 1990). Saraf et al. (1997) showed that PSB inoculation increased seed yield (10.3 q ha−1) of chickpea as compared to control (8.8 q ha−1). Increased grain yield (14%) and uptake of N and P was reported in chickpea by inoculation of PSB along with P fertilizers. The grain and straw yield of chickpea was found to increase with increasing levels of P (0–60 kg P2O5 ha−1) which further improved by inoculation of PSB (Sarawgi et al. 1999, 2000). Plant growth-promoting fluorescent pseudomonad isolate PGPR1, which produced siderophore and indole acetic acid, and solubilized TCP under in vitro conditions, significantly enhanced the pod yield (23–26%, respectively), haulm yield and nodule dry weight over the control during 3 years.

Phosphorus deficiency has a negative effect on BNF and the impaired BNF in P-deficient plants is usually explained by an effect of the low P supply on the growth of the host plant, on the growth and functioning of the nodule, or on the growth of both plant and the nodule (Christiansen and Graham 2002). Some particular strategies have been adopted for the adaptation of nodulated legumes to limited P supply, such as the maintenance of concentrations of P in nodules much higher than in other organs (Pereira and Bliss 1987), higher absorption of P from the solution directly by the nodules and bacteroids (Al-Niemi et al. 1998), increased N2-fixation per unit of nodule mass to compensate for reduced nodulation, (Almeida et al. 2000) and higher accumulation of soluble sugars in nodules than in roots and shoots (Olivera et al. 2004). Araujo et al. (2008) observed an increase in the activities of acid phosphatases and phytases in nodules of common bean genotypes at different levels of P supply indicating that this increase in activities may constitute an adaptive mechanism for N2-fixing legumes to tolerate P deficiency. Similarly, plants grown at limited P supply can increase the activities of phosphatases and phytases in roots to hydrolyze organic-P compounds in the soil, thus improving plant P acquisition.

Synergistic effect was observed after coinoculation of N2-fixing bacteria with PSB. For example, the composite application of P. putida and R. phaseoli increased P availability to common bean plants and enhanced nodulation of common bean (Grimes and Mount 1984). The seed inoculation with thermo-tolerant PSB, viz. B. subtilis, B. circulans and A. niger was found to improve nodulation, available P2O5 content of soil, root and shoot biomass, straw and grain yield, P and N uptake by mungbean (Gaind and Gaur 1991). High pod yield and P uptake in groundnut due to inoculation of P. striata were also recorded (Agasimani et al. 1994). Increased nodulation, yield attributes, seed index and seed yield of rainfed soybean were also reported with combined inoculation of P. striata and B. japonicum (Dubey 1997). Similarly, a significant increase in nitrogenase activity, plant growth and grain yield of pea was found following dual inoculation of R. leguminosarum and PSB (Srivastava et al. 1998).

Attempts to express the mineral phosphate solubilization (MPS) genes in a different host were found to be influenced by the genetic background of the recipient strain, the copy number of the plasmids present and metabolic interactions. Thus, genetic transfer of any isolated gene involved in MPS to improve P-dissolving capacity in PGPR strains, is an interesting approach. An attempt to improve mps in PGPR strains, using a PQQ synthase gene from E. herbicola was carried out (Rodriguez et al. 2000). This gene was subcloned in a broad-host range vector pKT230. The recombinant plasmid was expressed in E. coli and transferred to PGPR strains of Burkholderia cepacia and P. aeruginosa, using tri-parental conjugation. Several of the exconjugants showed a larger clearing halo on medium plates containing TCP as the sole P source. This experiment indicated the heterologous expression of this gene in the recombinant strains and improved MPS ability of PGPR.

9.2.1.3 Mineralization of Potassium, Iron and Sulfur Nutrients in the Rhizosphere

Potassium (K) is the third major essential nutrient for plant growth. It plays an essential role in enzyme activation, protein synthesis and photosynthesis. Potassium in soil is present in water-soluble (solution K), exchangeable, non-exchangeable and structural or mineral forms. Of these, water-soluble and exchangeable pools are directly available for plant uptake. At low levels of exchangeable K in certain soils, non-exchangeable K can also contribute significantly to the plant uptake (Memon et al. 1988). India ranks fourth after USA, China and Brazil in terms of total consumption of K-fertilizers. Some microorganisms in the soil are able to solubilize “unavailable” forms of K-bearing minerals, such as micas, illite and orthoclase, by excreting organic acids which either directly dissolves rock K or chelate silicon ions to bring the K into solution (Barker et al. 1998; Bennett et al. 1998). These microorganisms are commonly known as potassium-solubilizing bacteria (KSB) or potassium-dissolving bacteria or silicate-dissolving bacteria whose application is termed as “biological potassium biofertilizer (BPF)”. It was shown that KSB increased K availability in soils and increased mineral uptake by plant (Sheng and Huang 2002; Sheng et al. 2002, 2003). Therefore, application of KSB holds a promise for increasing K availability in soils.

In Egypt, some studies were conducted on potassium-dissolving bacteria which were mainly concentrated on their K releasing capacity along with their effects on growth and K uptake of the treated plants. In a trial conducted by Balabel-Naglaa (1997), there were positive responses of broad bean to inoculation with some species of Bacillus (K releasing bacteria). These positive responses were obvious on dry weight of shoot and root, nodule number and dry mass of nodules, nitrogenase activity, N, P, K contents of foliage, number as well as dry weight of pods, seed and straw yields. Hu et al. (2006) isolated two phosphate-and potassium-solubilizing strains, KNP413 and KNP414 from the soil of Tianmu Mountain, Zhejiang Province (China). Both isolates actively dissolved mineral P and K, while strain KNP414 showed higher dissolution capacity even than Bacillus mucilaginosus AS1.153, the inoculants of potassium fertilizer widely used in China. In another study, Lian et al. (2008) studied the mechanism for the release of mineralic potassium using a thermophilic fungus Aspergillus fumigatus. The thermophilic fungus A. fumigatus promoted potassium release by means of at least three likely routes, firstly, by complexing soluble organic ligands, secondly, appealing to the immobile biopolymers such as the insoluble components of secretion and thirdly, involving mechanical forces in association with the direct physical contact between cells and mineral particles.

Iron is yet another essential nutrient and is abundant in soil but most of it is found in the insoluble form, ferric hydroxide. Thus, iron is only available to organisms at concentrations at or below 10−18 M in soil solutions at neutral pH. To cope up with its solubility, many microorganisms synthesize extracellular, low molecular weight, high affinity Fe3+ chelators commonly referred to as siderophores, in response to iron stress (Neilands 1981; Neilands and Nakamura 1991) that transport iron into bacterial cells. Fuhrmann and Wollum (1989) detected a decrease in the number of taproot nodules and in seedling emergence of soybean and altered nodulation competition among B. japonicum strains when coinoculated with Pseudomonas spp. Iron availability was implicated as a factor involved in the plant-B. japonicum-rhizosphere microflora interactions. Thus, rhizobacteria help plants in absorbing iron from the soil. The metal-chelating agents produced by rhizobacteria also play an important role in the acquisition of heavy metals (Leong 1986). These organic substances scavenge Fe3+ and significantly enhance the bioavailability of soil bound iron (Kanazawa et al. 1994) and regulate the availability of iron in the plant rhizosphere (Bar-Ness et al. 1992; Loper and Henkels 1999). The competition for iron in the rhizosphere is controlled by the affinity of the siderophore for iron, and the probable availability of iron to the microorganisms ultimately decides the rhizosphere population structure. The concentration of various types of siderophores, kinetics of exchange and availability of Fe-complexes to microbes as well as plants has been found to control the binding affinity of siderophore (Loper and Henkels 1999). Interestingly, the binding affinity of phytosiderophores for iron is less than the affinity of microbial siderophores, but plants require a lower iron concentration for normal growth than microbes do (Meyer 2000).

Masalha et al. (2000) reported that plants grown under non-sterile soil systems were better in terms of iron nutrition than those grown under sterile conditions. Their data emphasized the role of microbial community on the iron nutrition of plants. It has been demonstrated that plants grown in metal-contaminated soils are often iron deficient and the production of siderophores by plant growth-promoting bacteria may help plants to obtain sufficient iron (Wallace et al. 1992; Burd et al. 2000). In fact, there is evidence that at least part of the toxic effects of some heavy metals in plants results from an induced iron deficiency and since bacterial siderophores could provide iron to various plants (Bar-Ness et al. 1991; Wang et al. 1993), therefore, siderophores produced by rhizobacteria may reduce nickel toxicity by supplying the plant with iron and hence reduce the severity of nickel toxicity (Bollard 1983; Bingham et al. 1986).

Another plant nutrient, sulfur (S), is the ninth and least abundant essential macronutrient. Its uptake and assimilation is crucial because of the key role played by the S containing aminoacids, methionine and cysteine in maintaining protein structure and because of its role in plant defense (Rasch and Wachter 2005). Sulfur atoms are widely distributed in soil and are found in a wide variety of organic and inorganic forms. These atoms are an integral component of soil humus, plants, microbial biomass and minerals. Scherer (2009) reported that sulfate (SO 2−4 ), which is a direct source of sulfur for plants, contributed up to 5% of total soil S; generally more than 95% of soils S are organically bound. Sulfur containing minerals include pyrite (FeS2) which occurs in ingenious rocks, gypsum (CaSO4.2H2O) and epsonite (MgSO4.7H2O). Despite its abundance in the earth’s crust, S is often present in suboptimal quantities in soil or either in unavailable states. Moreover, the decrease of S input from atmospheric depositions has led to S deficiency of crops over the past two decades on a worldwide scale that reduced yield and affected the quality of harvested products. Especially in Western European countries, incidence of S deficiency has increasingly been reported in oilseed rape, which is an S demanding plant (Fismes et al. 2000). Therefore, more attention should be paid to the optimization of S fertilizer application, in order to cover plant S requirements whilst minimizing environmental impacts.

Sulfur turnover involves both biochemical and biological mineralization (Gharmakher et al. 2008). Biochemical mineralization, which is the release of SO 2−4 from the ester sulfate pool through enzymatic hydrolysis, is controlled by S supply, while the biological mineralization is driven by the microbial need for organic C to provide energy. The biological oxidation of elemental S and inorganic S compounds such as H2S, sulphite and thiosulphate is brought about by chemoautotrophic bacteria and photosynthetic bacteria. Sulfur-oxidizing bacteria include Beggiotoa, Chromatium, Chlorobium, Thiobacillus, Sulfolobus, Thiospira and Thiomicrospira. The species of Bacillus, Pseudomonas, Arthrobacter and Flavobacterium are also reported to oxidize elemental S or thiosulphate to sulfate. Under anaerobic conditions, sulfate is reduced to H2S by sulfate-reducing microorganisms, mostly the bacteria. Many bacteria including species of Bacillus and Pseudomonas are known to reduce S or sulfate to H2S, but among these Desulphovibrio desulfuricans and Desulfotomaculum spp. are important.

Nitrogen fixation appears to be affected by S fertilization in faba bean, lucerne, pea and in red clover (DeBoer and Duke 1982; Scherer and Lange 1996; Habtemichial and Singh 2007). An important link between S and N nutrition was found in white clover, lucerne and pea (Zhao et al. 1999; Varin et al. 2009) and sulfur fertilization was found to stimulate N2 fixation strongly. Scherer et al. (2008) observed that the amount of leghaemoglobin was reduced by S deficiency in peas and alfalfa, when no S was added and nodules devoid of leghaemoglobin were more numerous. Varin et al. (2008) analyzed a set of functional traits in three white clover lines along a gradient of N and S fertilization on a poor soil. Nitrogen was found to be the most limiting factor for the VLF (very low fixation) line. S was the element that modulated the most traits for the nitrogen fixing lines NNU (normal nitrate uptake) and LNU (low nitrate uptake). Nitrogen fertilization was found to inhibit N2 fixation in clover but N2 fixation was enhanced when S was added. S fertilization also increased nodule length, as well as the proportion of nodules containing leghaemoglobin. Thus, sensitivity of white clover to S nutrition would be a disadvantage for competition in a situation of sulfur impoverishment.

9.2.2 Synthesis of Auxins, Cytokinins, Gibberlins and Vitamins

Microbial communities of soil and rhizosphere have been found to synthesize auxins, cytokinins, vitamins and gibberellin-like compounds (Arshad and Frankenberger 1991; Derylo and Skorupska 1993; Patten and Glick 1996; Gutierrez-Manero et al. 2001). These compounds increase the rate of seed germination and stimulate the development of root tissues leading to an increase in the capacity of the root system to provide nutrients and water to above ground organs of plants (Arkhipova et al. 2007), and also help the plants to tolerate abiotic stress (Yang et al. 2009). Derylo and Skorupska (1993) reported that stimulation of clover plant growth under gnotobiotic conditions resulted from the secretion of water-soluble B vitamins by fluorescent Pseudomonas sp. strain 267. This was demonstrated by enhancement of clover growth by naturally auxotrophic strains of R. leguminosarum bv. trifolii in the presence of the Pseudomonas sp. strain 267 supernatant. The addition of vitamins to the plant medium increased symbiotic N2-fixation by the clover plants.

Indole acetic acid (IAA) is known as the main auxin in plants and has been implicated in all aspects of plant growth and development (Taele et al. 2006). The exposure of plant roots to exogenous microbially produced IAA can affect plant growth in diverse ways, varying from pathogenesis and growth inhibition to plant growth stimulation (Spaepen et al. 2007). In fact, low levels of IAA released by rhizobacteria has been found to promote primary root elongation, whereas, high levels of IAA stimulated lateral and adventitious root formation (Glick 1995) but inhibited primary root growth (Xie et al. 1996). Thus, plant growth-promoting bacteria can facilitate plant growth by altering the hormonal balance within the affected plant (Lambrecht et al. 2000; Kamnev 2003). Such relationships of rhizobacteria between different crop species could be cultivar or genotype-specific (Cattelan et al. 1998). For example, the rhizosphere of wheat seedlings harbors a significant proportion of bacteria that produce phytohormone, indole acetic acid (IAA), known to increase root growth (Patten and Glick 2002). Moreover, differential response of inoculation was observed in two genotypes of common bean with A. brasilense Sp245 mutant strain having reduced auxin biosynthesis or to addition of increasing concentrations of exogenous auxin (Remans et al. 2008a). Genetic analysis of recombinant inbred lines revealed two quantitative trait loci (QTLs) associated with basal root responsive to auxin in common bean.

Although significant and consistent yield increases of rhizobia-inoculated crops have been attributed to N2 fixation, plant growth regulators may also be involved (Mayak et al. 1999; Malik and Sindhu 2008). For instance, the rhizobial species are known to produce IAA in vitro (Bandenoch-Jones et al. 1982; Wang et al. 1982; Boiero et al. 2007) and nodulated roots often contained substantially greater auxin concentrations than non-nodulated roots (Dulhart 1967, 1970). Inoculation of soybeans with spontaneous mutants of Rhizobium japonicum that overproduced IAA (30-fold more auxin than the wild-type strain) showed a three-fold increase in the number of root nodules (Kaneshiro and Kwolek 1985). Mutants of B. elkanii strain deficient in IAA production induced fewer nodules on soybean roots in comparison to the parental strain and the normal numbers of nodules were reestablished following application of exogenous IAA (Fukuhara et al. 1994). IAA derived from B. elkanii has been implicated as a causative agent in the swelling of outer cortical cells of soybean roots and was suggested to provide a competitive advantage for nodulation (Yuhanshi et al. 1995). However, enlargement of cortical cells was not observed after inoculation with either IAA-deficient mutants of B. elkanii (Yuhanshi et al. 1995) or wild-type B. japonicum strains that do not produce IAA (Minamisawa and Fukai 1991). Prinsen et al. (1991) demonstrated that flavonoids released from legume plant roots, which also act as inducers of Rhizobium nodulation genes, stimulated the production of IAA, suggesting that nodule morphogenesis could be controlled by the highly specific nodulation signal in combination with phytohormones such as auxins, released by rhizobia.

Coinoculation of legumes with Rhizobium and free-living IAA-producing bacteria such as Azospirillum brasilense (Yahalom et al. 1990) and several Bacillus species (Srinivasan et al. 1996) significantly increased the number of nodules on the host roots and increased nodule fresh weight and nitrogenase activity, compared to inoculation with Rhizobium alone. Zhang et al. (1996) reported that Serratia stimulated soybean growth through the production of a plant growth-regulating compound, which stimulated overall plant vigor and growth, resulting in subsequent increase in nitrogen fixation. Mayak et al. (1999) showed that an IAA over producing mutant of P. putida caused extensive development of adventitious roots on mung bean cuttings. It was suggested that inoculation with these free-living bacteria increase the number of infection sites on roots for attachment and nodulation by Rhizobium. In addition, enhanced production of flavonoid-like compounds or phytoalexins in roots of several crop plants by inoculation of Pseudomonas sp. (Parmar and Dadarwal 1999; Goel et al. 2001) could induce the transcription of nodulation (nod) genes (Peter and Verma 1990), leading to increase in nodulation. In contrast, similar experiments using mutants of B. megaterium with altered IAA production levels had a negative effect on symbiotic parameters (Srinivasan et al. 1996). Mutants of Pseudomonas strains altered in IAA production were derived by Tn5 mutagenesis (Malik 2002; Malik and Sindhu 2008). Coinoculation studies of wild-type Pseudomonas strains with Bradyrhizobium strain S24 and IAA over-producer Pseudomonas mutants resulted in more nodules in green gram compared to wild type Bradyrhizobium strain at 50 days of growth. Camerini et al. (2008) introduced iaaM gene (involved in IAA biosynthesis) from Pseudomonas savastanoi and the tms2 gene from A. tumefaciens into R. leguminosarum bv. viciae LPR1105. Free-living bacteria harboring the promoter iaaM-tms2 construct (strain RD20) released 14-fold more IAA in the growth medium than the wild-type parental strain and elicited the development of vetch root nodules containing up to 60-fold more IAA than nodules infected by the wild-type strain LPR1105. The root nodules elicited in vetch by RD20, were heavier and had an enlarged and more active meristem, and showed a significant increase in acetylene reduction activity (ARA).

9.2.3 Effect of Rhizobacteria on Phytoremediation in Metal Stressed Soil

Pollution of biosphere by toxic metals has accelerated dramatically since the beginning of the industrial revolution (Kabata-Pendias and Pendias 1989). Heavy metal pollution of soil is a significant environmental problem and has its negative impact on human health and agriculture. The most common heavy metal contaminants are Cd, Cr, Cu, Hg, Pb, and Ni. Heavy metals ions, when present at an elevated level in the environment, are excessively absorbed by roots and translocated to shoot, leading to impaired metabolism and reduced growth (Bingham et al. 1986). In addition, excessive metal concentrations in contaminated soils resulted in decreased soil microbial activity and soil fertility, and yield losses (McGrath et al. 1995). Phytoremediation has been reported to be an effective, in situ, non-intrusive, low-cost, ecofriendly, socially accepted technology to remediate polluted soils (Garbisu et al. 2002). Another alternative is to provide them with an associated plant growth-promoting rhizobacteria, which also is considered an important component of phytoremediation technology (Glick 2003; Jing et al. 2007). Therefore, the use of rhizobacteria to enhance phytoremediation of soil heavy metals pollution has recently received more attention (Weyens et al. 2009).

The functioning of associative plant-bacterial symbioses in heavy-metal-polluted soil can be affected from the side of both the micropartner (plant-associated bacteria) and the host plant (Glick 1995). Chaudri et al. (1992) found that Rhizobium populations were reduced at concentrations >7 mg kg−1 soil in their Cd treatments. Field studies of metal contaminated soils have similarly demonstrated that elevated metal loadings can result in decreased microbial community size (Chander and Brookes 1991). Some rhizobacteria can exude a class of rhizobacterial secretion, such as IAA, siderophores, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase which increased bioavailability and facilitated root absorption of heavy metals, such as Fe (Crowley et al. 1991), enhanced tolerance of host plants by improving the P absorption (Liu et al. 2000) and promoted plant growth (Burd et al. 2000; Ellis et al. 2000). Rajkumar et al. (2005) isolated Pseudomonas sp. strain RNP4 from tannery waste contaminated soil which tolerated concentrations up to 450 mg Cr6+ L−1 on a Luria-Bertani (LB) agar medium and reduced a substantial amount of Cr6+ to Cr3+ in LB liquid medium. The strain also produced substantial amount of IAA, exhibited the production of siderophores and solubilized phosphorus. The strain was found to promote the growth of black gram, Indian mustard and pearl millet in the presence of Cr6+, suggesting the innate capability of the Pseudomonas isolate for parallel bioremediation and plant growth promotion. In another study, Safronova et al. (2006) found that pea plants inoculated with root-associated bacteria containing ACC deaminase activity produced longer roots, greater root density and improved nutrient uptake by pea genotypes cultivated in cadmium supplemented soil. Inoculation of pea plants with a poplar endophyte that degraded 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in increased removal of 2,4-D from the soil (Germaine et al. 2006). Moreover, the plants did not show toxic responses and did not accumulate 2,4-D in their tissues.

Liu et al. (2007) demonstrated that inoculation of alfalfa with Comamonas sp. strain CNB-1 not only removed 4-chloronitrobenzene (4-CNB) completely within 1 or 2 days from soil but also eliminated the phytotoxicity of 4-CNB to alfalfa plants. Tank and Saraf (2009) selected five plant growth-promoting bacterial strains based on their P solubilization ability, IAA production and biocontrol potentials. These isolates were also able to grow and produced siderophores in presence of heavy metals like Ni, Zn, and Cd. A positive response of bacterial inoculants was observed in chickpea plants towards toxic effect of nickel present in soil at different concentrations (0, 1 and 2 mM) and bacterial inoculants enhanced fresh and dry weight of chickpea plants even at 2 mM nickel concentration. The accumulation of nickel plant−1 was just 50% in Pseudomonas-inoculated plants as compared to uninoculated plants with 2 mM nickel concentration along with increased biomass. The development of engineered endophytic bacteria that improved the phytoremediation of volatile organic compound trichloroethylene (TCE) was found to protect host plants against the phytotoxicity of TCE and contributed to a significant decrease in TCE evapotranspiration (Barac et al. 2004). Similarly, the genetic modification of the polychlorinated biphenyls (PCB)-degrading bacteria Pseudomonas fluorescens F113, to improve its performance in the rhizosphere, could be manipulated by improving symbiotic microorganisms. Thus, the rhizoremediation of PCBs by P. fluorescens was improved in which biphenyl degradation is regulated using a system that responds to signal from alfalfa roots (Villacieros et al. 2005).

9.2.4 Rhizobacteria as Biocontrol Agents

The suppression of growth of soil-borne plant pathogens by the use of microorganisms, natural or modified, genes or gene products to reduce the effects of undesirable organisms (pests) is referred to as biocontrol. Rhizobacteria inhibit the growth of various pathogenic bacteria and fungi resulting in suppression of the diseases caused by such pathogens (Weller 1988; Thomashow and Weller 1996). Disease suppression by biocontrol agents involves a sustained manifestation of interactions among the plant, the pathogen, the biocontrol agent, the microbial community on and around the plant, and the physical environment (Pierson and Weller 1994). Strains of Pseudomonas fluorescens, P. putida, P. aureofaciens, P. cepacia and P. aeruginosa have been found to antagonize the growth of pathogens leading to substantial disease control (Chandra 1997; Weller 2007). Different strains of Bacillus thruingiensis, B. sphaericus, B. cereus and B. subtilis are also used as biocontrol agents (Asaka and Shoda 1996; Hervas et al. 1997).

Trapero-Casas et al. (1990) reported that coating of chickpea seeds with the P. fluorescens (strain Q29z-80) increased the yield which was comparable to those obtained with any of the fungicide seed treatments used to control seed rot and preemergence damping-off disease caused by P. ultimum in the field. Hervas et al. (1997) observed that treatment of B. subtilis, nonpathogenic F. oxysporum and/or T. harzianum, when applied alone or in combination, to chickpea cultivars “ICCV 4” and “PV 61” could effectively suppress the disease caused by the highly virulent F. oxysporum f. sp. ciceris. In comparison with the control, the final disease incidence was reduced by B. subtilis (18–25%) or nonpathogenic F. oxysporum (18%). The extent of disease suppression was higher and more consistent in cultivar “PV 61” than in “ICCV 4” whether colonized by B. subtilis, non pathogenic F. oxysporum or T. harzianum. Nautiyal (1997) found that among 478 bacteria obtained from roots of chickpea rhizosphere by random selection, 44 rifampicin resistant strains showed biocontrol activity against F. oxysporum f. sp. ciceri, R. bataticola and Pythium sp. under in vitro studies. In a greenhouse test, seed bacterization of chickpea with P. fluorescens NBRI 1303 increased the germination of seedlings by 25% reduced the number of diseased plants by 45% as compared with non-bacterized controls. Significant growth increases in terms of shoot length, dry weight, and grain yield, averaging 11.6, 17.6, and 22.61%, respectively, above untreated controls were obtained in field trials.

Plant growth-promoting fluorescent pseudomonad isolate PGPR1, which produced siderophore and IAA, and solubilized TCP under in vitro conditions, also suppressed the soil-borne fungal diseases like collar rot of peanut (caused by A. niger) in field trials (Dey et al. 2004). Jamali et al. (2004) studied effect of seven antagonistic bacteria on control of Fusarium wilt under green house conditions. Isolates B-120, B-32, B-28 and B-22 were identified as B. subtilis and isolates Pf-100, Pf-10 and CHAO were identified as P. fluorescens. Results revealed that only isolate B-120 reduced Fusarium wilt of chickpea in both seed and soil treatments. Soil treatment of bacteria showed better effects on plant growth than that of bacterial seed treatment. Statistically significant biocontrol effects were observed when lettuce seedlings were inoculated into naturally Rhizoctonia solani-infested lettuce fields with bacterial suspensions of two endophytic strains, Serratia plymuthica 3Re4-18 and P. trivialis 3Re2-7 with rhizobacterium P. fluorescens L13-6-12, 7 days before and five days after planting in the field (Scherwinski et al. 2008). Usually, no general relationship was observed between the ability of a bacterium to inhibit a pathogen under in vitro and in situ disease suppression (Schroth and Hancock 1982; Wong and Baker 1984). Bacterial strains producing the largest zones of inhibition on agar media do not always make the best biocontrol agents. Therefore, some in vitro conditions have been modified to more closely simulate natural conditions (Randhawa and Schaad 1985). Among the numerous examples of biocontrol agents reported for disease control of soil-borne pathogens, only few studies provide mechanistic information for the activities of these agents. Recently, the use of mutants that lack certain in vitro and in situ activities have provided strong evidence for the involvement of specific molecules in biocontrol.

9.2.4.1 Mechanisms Involved in Biocontrol

For effective biocontrol of plant disease, the rhizobacteria must establish and grow in an ecological habitat that includes indigenous pathogenic microorganisms. Thus, root colonization by rhizobacteria appears to be an important factor in biological control and plant growth promotion. In recent years, tremendous progress has been made in characterizing the process of root colonization by biocontrol agents, the biotic and abiotic factors affecting colonization, bacterial traits and genes contributing to pathogen suppression (Benizri et al. 2001; Sindhu et al. 2009b). Rhizobacteria inhibit the growth of phytopathogenic microorganisms by various mechanisms.

9.2.4.1.1 Competition for Nutrients and Infection Sites

The rhizosphere microflora directly or indirectly inhibit the invasion of pathogen on plant tissue. Root-inhabiting microorganisms and plant pathogens could compete for space, nutrients or even for binding sites on the root surface. Space and nutrients competition could result in failure of the pathogen to develop critical population densities for disease initiation, whereas, the competition for specific binding sites would reduce the capability of plant pathogen to initiate the infection process. Pseudomonads possess the capacity to catabolize diverse nutrients and have fast generation time in the root zone, and hence, they are logical candidates for competition for nutrients against the slow growing pathogenic fungi and could result in biological control of pathogens (Weller 1985). Elad and Chet (1987) carried out a study to evaluate the antagonistic mechanism of rhizobacteria against damping off disease caused by Pythium. The competition for nutrients between germinating oospores of Pythium aphanidermatum and biocontrol rhizobacteria was unique and was correlated significantly with disease suppression.

9.2.4.1.2 Interference in Chemotactic Attraction

Crop rotations and tillage management have been shown to influence specific microbial populations (Sturz et al. 1997). Rhizobacteria could spur a root exudation response in plants that is species specific (Merharg and Killham 1995). The close interactions between plants and rhizobacteria encourage the establishment of specific and beneficial rhizosphere, and such associations between different crop species could be cultivar-specific. Thus, certain cultivars of clover can foster the development of rhizo- and endophytic bacteria that favor the growth and development of specific cultivars of potatoes (Sturz and Christie 1998). An additional role of rhizosphere microbes in reducing root disease incidence is in interfering with chemotactic attraction of the pathogen to root receptor sites. Scher et al. (1985) suggested that chemotaxis might be the first step in root colonization. A variety of compounds as components of root exudates may serve as attractants for plant pathogens. Growth of root inhabitants (including mycorrhizal fungi) necessarily reduced both the quantity and diversity of organic compounds diffusing from the root, thereby, diminishing the probability of encounter by a plant pathogen (Davis et al. 1979).

9.2.4.1.3 Antibiotic Production

Antibiotic production by rhizobacteria is one of the major mechanisms postulated for antifungal activity to suppress pathogens in the rhizosphere and to promote plant growth. The role of antibiotics in disease suppression has been demonstrated in many biocontrol systems by mutant analyses and biochemical studies using purified antibiotics (Stockwell and Stack 2007). These antimicrobial compounds may act on plant pathogenic fungi by inducing fungistasis, inhibition of spore germination, lysis of fungal mycelia or by exerting fungicidal effects. A large number of antibiotics including phenazine carboxylic acid, diacetyl phloroglucinol, oomycin A, pyocyanine, pyrroles, pyoluteorin, pyrrolnitrin, iturin A, surfactin, etc. are produced by rhizobacteria (Bender et al. 1999; Sindhu et al. 2009b), which help in the suppression of pathogen growth. The first antibiotic clearly implicated in biocontrol by fluorescent pseudomonads was the phenazine derivative that contributed to disease suppression by Pseudomonas fluorescens strain 2-79 and P. chlororaphis strain 30–84 (formerly P. aureofaciens), which were suppressive to the take-all disease of wheat roots caused by Gaeumannomyces graminis var. tritici (Gurusiddaiah et al. 1986). The antibiotic was found active against several fungi including G. graminis var. tritici, R. solani and P. aristesporum. Pseudomonas fluorescens strain CHAO was found to produce a variety of secondary metabolites, i.e., 2,4-diacetyl phloroglucinol, pyoluteorin, hydrogen cyanide, salicylic acid, pyochelin and pyoverdine, and protected various plants from diseases caused by soil borne pathogenic fungi (Stutz et al. 1986). Anjaiah et al. (2003) found that an isolate of P. aeruginosa PNA1, obtained from chickpea rhizosphere, protected the plants from Fusarium wilt until maturity in moderately tolerant genotypes of pigeonpea and chickpea. Root colonization of pigeonpea and chickpea, which was measured using a lacZ-marked strain of PNA1, showed ten-fold lower root colonization of susceptible genotypes than that of moderately tolerant genotypes, indicating that this plant-bacteria interaction could be important for disease suppression in this plant. Its Tn5 mutants (FM29 and FM13), which were deficient in phenazine production, caused a reduction or loss of wilt disease suppression in vivo. Similarly, B. cereus strain UW85 suppressed the diseases caused by the oomycetes. Analysis of B. cereus mutants showed a significant quantitative relationship between disease suppressiveness and the production of two antibiotics, zwittermicin A and kanosamine (Silo-Suh et al. 1994; Milner et al. 1996). The purified antibiotics suppressed the disease and inhibited the development of oomycetes by stunting and deforming germ tubes of germinating cysts. Bacillus subtilis RB14, which produced antibiotics iturin A and surfactin, was found to suppress damping off disease caused by Rhizoctonia solani (Asaka and Shoda 1996).

9.2.4.1.4 Production of Siderophores

Iron is an essential element for all living organisms and most of it is found in the insoluble form at neutral pH. To cope up with low solubility of iron, many microorganisms synthesize extracellular Fe3+ chelators i.e., siderophores, in response to low iron stress (Neilands and Nakamura 1991) that transport iron into bacterial cells. Plant growth promoting rhizobacteria (PGPR) produced different types of siderophores, which were involved in disease suppression and plant growth promotion (Leong 1986). The various categories of siderophores produced by PGPR include catechol, hydroxamate, pyoverdine, pyochelin, cepabactin, schizokinen and some other types like azotochelin, rhizobactin, anthranilic acid and azotobactin.

Kloepper et al. (1980) were the first to demonstrate the importance of siderophores production in biocontrol of plant pathogens with pseudobactin, a siderophore produced by Pseudomonas strain B10. The addition of 1.0 μM ferric chloride to an iron-deficient medium abolished the antagonism under in vitro conditions and the fluorescence by the PGPR were not observed. Studies with various siderophore-negative Tn5 mutants showed that pseudobactin of either pyoverdine and pyochelin type was necessary to achieve wild-type levels of protection against Pythium-induced damping off disease (Buysens et al. 1996). Goel et al. (2000) isolated pigment overproducer mutant MRS16M-1 from Pseudomonas strain MRS16, that was more inhibitory to the fungal pathogens, whereas, non-producer mutant MRS16M-5 was less inhibitory on nutrient agar medium. Addition of 100 μM ferric chloride to the medium decreased inhibition of fungal growth, suggesting the involvement of siderophores and other antifungal secondary metabolites. Dileep Kumar et al. (2001) found that both the fluorescent pseudomonads and Rhizobium strains exhibited a wide range of antifungal activity against pathogens specific to pea. In a synthetic culture medium, all the plant growth promoting fluorescent pseudomonad strains produced siderophores, which expressed antifungal and antibacterial activity. Seed bacterization with plant growth-promoting strains, alone and together with a Rhizobium leguminosarum biovar viceae isolate, 361–27 reduced the number of infected peas grown in Fusarium oxysporum infested soils. Seed bacterization with siderophore-producing P. fluorescens isolates, viz. PGPR1, PGPR2 and PGPR4, suppressed the soil-borne fungal diseases like collar rot of peanut caused by A. niger and isolate PGPR4 also suppressed stem rot caused by S. rolfsii (Dey et al. 2004).

9.2.4.1.5 Production of Hydrolytic Enzymes

Some cell wall lysing enzymes produced by rhizobacteria have been found to cause the destruction of pathogens. For example, Chet et al. (1990) cloned the gene encoding chitinase enzyme from S. marcescens and transferred it into E. coli. The partially purified chitinase caused extensive bursting of the hyphal tips. This chitinase preparation was effective in reducing disease incidence caused by R. solani under greenhouse conditions. In other study, Chet et al. (1993) isolated three different chitinase genes from Serratia, Aeromonas, and Trichoderma. The cloned genes were expressed in E. coli and subsequently introduced into R. meliloti, P. putida, and Trichoderma strains resulting in increased chitinolytic activity of transformants against Sclerotium rolfsii and R. solani. Recombinant strains of R. meliloti were constructed which carried chiA genes to produce chitinase. The recombinant strain expressed chitinase during symbiosis in alfalfa roots (Sitrit et al. 1993). Khot et al. (1996) reported that certain isolates of Pseudomonas and Bacillus produced chitinase, β-1,3 glucanase (laminarinase) and siderophores. Seed inoculation of these bacteria or application of cell free extract on seed resulted in 48.6 and 31.6% reduction of the wilt incidence of chickpea under field conditions in a wilt sick nursery. Pseudomonas strains isolated from the rhizosphere of chickpea and green gram were also found to produce chitinases and cellulases in culture-free supernatants and inhibited growth of P. aphanidermatum and R. solani on potato dextrose agar medium plates (Sindhu and Dadarwal 2001).

9.2.4.1.6 Production of Secondary Metabolites

Among other metabolites, hydrogen cyanide (HCN) is produced by many rhizosphere bacteria and has been demonstrated to play a role in biological control of pathogens (Voisard et al. 1989). HCN over-producing bacterial strains resulted in small but statistically significant increase in the suppression of symptoms caused by Mycophaerella graminicola and Puccinia recondita f. sp. tritici on wheat seedling leaves. Pseudomonas aeruginosa strain zag2 was reported to produce pyocyanin, siderophore and hydrogen cyanide (Hassanein et al. 2009). The minimum inhibitory concentration of the extracted pigmented compound against Candida albicans was 40.69 µg ml−1 and the antifungal activity of the compound was remarkable at 100°C for 20 min. The toxic volatile compound HCN produced by the bacteria was found to reduce the growth of both F. oxysporum and Helminthosporium sp. whereas A. niger was not affected.

The fungal pathogens also cause the plant to synthesize stress ethylene (van Loon et al. 2006) and much of the damage sustained by plants infected by phytopathogens occurs as a result of the response of the plant to the increased levels of ethylene (van Loon 1984). It is well known that exogenous ethylene often increases the severity of fungal infection, while some ethylene synthesis inhibitors significantly decrease the severity of a fungal infection (Elad 1990; Robinson et al. 2001). A number of PGPR, which stimulated root growth of different plant species were found to contain the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which hydrolysed the ethylene precursor ACC to ammonia and α-ketobutyrate, and as a result, decreased ethylene biosynthesis by plants (Glick et al. 1994; Hall et al. 1996; Belimov et al. 2001). The ACC deaminase-containing biocontrol bacterial strains were also found more effective than biocontrol strains that did not possess this enzyme (Wang et al. 2000).

9.2.4.1.7 Induction of Systemic Resistance

Some biocontrol agents induced a sustained change in the plant and increased its tolerance to infection against fungal and bacterial pathogens (Maurhofer et al. 1998), a phenomenon known as induced systemic resistance (ISR). Various non-pathogenic rhizobacteria have the ability to induce a state of systemic resistance in plants, which provided protection against a broad spectrum of phytopathogenic organisms including fungi, bacteria and viruses (Bakker et al. 2007). Induced resistance brought about by prior inoculation of the host by a pathogen, avirulent or incompatible forms of a pathogen, or heat killed pathogens has been attributed to induce physiological response of the host plant against subsequent inoculation by the virulent pathogens (Hoffland et al. 1996). Induced systemic resistance in plants has been demonstrated in over 25 crops, including legumes, cereal crops, cucurbits, solanaceous plants and trees against a wide spectrum of pathogens. The mechanism of ISR has also been studied in plant growth-promoting Bacillus spp. (Kloepper et al. 2004). Bacterial production of the volatile 2,3-butanediol triggered the expression of Bacillus-mediated ISR in Arabidopsis. The signaling pathway that is activated in this case depended on ethylene but was independent of salicylic acid and jasmonic acid signaling (Ryu et al. 2004). Whether or not biocontrol agents suppress disease by inducing resistance, it is essential that ISR and biocontrol strategies be compatible because future agricultural practices are likely to require the integration of multiple pest control strategies.

9.2.5 Selection of Rhizobacteria with Multiple Plant Growth-Promoting Traits

The use of beneficial soil microorganisms as agricultural inputs for improved crop production requires selection of rhizosphere-competent microorganisms with plant growth-promoting attributes. The selection of PGPR strains depends largely on their growth promoting activities, such as, production of IAA and siderophores, P solubilization and inhibition of pathogenic microorganisms. However, the presence of one or all of these traits does not qualify them to be a PGPR for one particular crop or spectrum of crops. For example, Cattelan et al. (1999) reported one rhizobacterial isolate which did not share any of the PGPR traits tested in vitro except antagonism to Sclerotium rolfsii and Sclerotinia sclerotium, but it promoted soybean growth. This indicates that besides such growth promoting traits, there are unexplained mechanisms, which also influence the growth of plants and requires the close proximity of PGPR to the roots of plants. Hence, there is no clear separation of growth promotion in plants and biological control induced by bacterial inoculants (Lugtenberg et al. 1991; Bloemberg and Lugtenberg 2001). Bacterial strains selected initially for in vitro antibiosis as part of evaluating biological control activity frequently demonstrated growth promotion in the absence of target pathogen (Sindhu et al. 1999; Goel et al. 2002). Similarly, PGPR selected initially for growth promotion in the absence of pathogens, may demonstrate biological control activity when challenged with the pathogens, presumably by controlling deleterious microorganisms or non-target pathogens.

Direct growth promotion occurs when a rhizobacterium produces metabolites that directly promote plant growth without interactions with native microflora (Kloepper et al. 1991). Dileep Kumar and Dube (1992) reported that fluorescent siderophore-producing Pseudomonas strain RBT13, originally isolated from the tomato roots, enhanced seed germination of chickpea and soybean, and resulted in increased root and shoot weight as well as yield of the crops. Sindhu et al. (2002b) reported plant growth promoting effects of fluorescent Pseudomonas sp. on coinoculation with Mesorhizobium sp. Cicer strain under sterile and “wilt sick” soil conditions in chickpea. The coinoculation resulted in enhanced nodulation by Mesorhizobium sp. and increased shoot dry weight by 3.92–4.20 times in comparison to uninoculated controls. Under indirect growth promotion mechanism, the production of antibiotics, siderophores and HCN by microorganisms decreased the population and activities of pathogens or deleterious microorganisms and thereby, increased the plant growth (Pierson and Weller 1994).

Nine different isolates of PGPR (Pseudomonas sp.) were selected from a pool of 233 rhizobacterial isolates obtained from the peanut rhizosphere based on ACC-deaminase activity (Dey et al. 2004). Four of these isolates, viz. PGPR1, PGPR2, PGPR4 and PGPR7 produced siderophore and IAA. In addition, P. fluorescens PGPR1 also possessed the properties like, P-solubilization, ammonification and inhibited A. niger and A. flavus under in vitro conditions. In addition to the traits exhibited by PGPR1, the strain PGPR4 showed strong in vitro inhibition to S. rolfsii. In field trials on peanut, plant growth-promoting fluorescent pseudomonad isolates, viz. PGPR1, PGPR2 and PGPR4, significantly enhanced the pod yield (23–26, 24–28, and 18–24%, respectively), haulm yield and nodule dry weight over the control in 3 years. Inoculation with plant growth-promoting P. fluorescens isolates, viz. PGPR1, PGPR2 and PGPR4, was found to suppress the soil-borne fungal diseases like collar rot of peanut caused by A. niger and isolate PGPR4 also suppressed stem rot caused by S. rolfsii. Hynes et al. (2008) screened 563 bacteria originating from the roots of pea, lentil and chickpea for the suppression of legume fungal pathogens and for plant growth promotion. Screening of bacteria showed that 76% isolates produced siderophore, 5% isolates showed ACC deaminase activity and 7% isolates were capable of indole production. Twenty-six isolates (5%) suppressed the growth of Pythium species strain p88–p3, 7% suppressed the growth of Fusarium avenaceum and 9% suppressed the growth of R. solani CKP7. Four isolates promoted the growth of lentil and one isolate promoted the growth of pea. Fatty acid profile analysis and 16S rRNA sequencing of the isolates showed that 39–42% were the members of Pseudomonadaceae and 36–42% of the Enterobacteriaceae families.

In search of efficient PGPR strains, 72 bacterial isolates were obtained from different rhizospheric soil and plant root nodules (Ahmad et al. 2008). Of these, more than 80% of the isolates belonging to genera Azotobacter, Pseudomonas, and Mesorhizobium produced IAA, whereas, only 20% of the Bacillus was IAA producer. Solubilization of P was commonly detected in the isolates of Bacillus (80%) followed by Azotobacter (74%), Pseudomonas (56%) and Mesorhizobium (17%). All tested isolates produced ammonia. Siderophore production and antifungal activity of these isolates except Mesorhizobium were exhibited by 10–13% isolates. HCN production was more common trait of Pseudomonas (89%) and Bacillus (50%). Pseudomonas Ps5 and Bacillus B1 isolates showed broad-spectrum antifungal activity against Aspergillus, Fusarium and Rhizoctonia bataticola.

9.3 Biotic and Abiotic Factors Affecting Rhizosphere Colonization

It is well established that root colonization by biocontrol agent and beneficial microorganisms is a prerequisite to suppress the plant disease and to enhance plant growth. Root colonization by introduced bacteria could be improved by increasing the population size, distribution or survival of bacteria, along with manipulation of soil factors that may positively or negatively affect colonization. Bacterial traits such as growth rate, cell surface properties, motility (Boelens et al. 1994), chemotaxis to root exudates, production of secondary metabolites and tolerance to stressed environment (e.g., dehydration and temperature) also contributes to rhizosphere competence. Plant characteristics, like root structure, age and plant genotype as well as physico-chemical properties of soil, application of pesticides etc. were found to affect rhizosphere colonization by the beneficial rhizobacteria. Use of green fluorescent protein (gfp) and in situ monitoring based on confocal laser scanning microscope (CLSM) could be used to understand the rhizosphere competence and root colonization (Johri et al. 2003). Using this technique, it was found that the Pseudomonas (biocontrol strains) colonized the seed and root surface at the same position, as did the pathogenic fungi that they controlled (Bloemberg et al. 2000). Another promising option considered important for understanding colonization is to screen mutants directly. Mutants of Pseudomonas strains of both phenotypes have been identified and analysis of these mutants indicated that prototrophy for amino acids and vitamins, rapid growth rate, utilization of organic acids and lipopolysaccharide properties contributed to colonization ability. Modification of genes involved in the biocontrol activity of biological control agents also played a key role in improving the potential rhizosphere competence as well as antifungal activity of biological control agents (Carroll et al. 1995). Moreover, biocontrol activity of P. fluorescens carrying PCA coding mini-Tn5 vector was enhanced by introducing phzH gene from Pseudomonas chlororaphis PCL1391 (Timmis-Wilson et al. 2000).

9.4 Development of Bacterial Inoculants and Constraints in Their Use

Rhizobium and Bradyrhizobium inoculants have been marketed with success for over a century. Releases of these nodule-forming microorganisms into soils have been successful. Inoculation with such inoculants has resulted in their establishment into soil and onto plant roots to a level sufficiently higher for the intended purpose. However, the desired impact of biofertilizer application under field conditions has been variable and inoculation of legume plants with commercial inoculant strains often fails to improve crop productivity (van Elsas and Heijnen 1990; Akkermans 1994). The problem is of the survival of inoculant diazotrophic bacteria under field conditions. For each introduction, abiotic soil factors such as texture, pH, temperature, moisture content and substrate availability need critical assessment since these factors largely determine the survival and activity of the introduced microorganisms (van Veen et al. 1997). In addition, the response of the inoculant to the prevailing soil conditions also depends on its genetic and physiological constitution (Brockwell et al. 1995). The use of genetic markers like resistance to antibiotics or introduction of metabolic markers from other bacterial species could help in tracing the introduced strains, whether it is rhizobia, cyanobacteria, azotobacters or azospirilla (Wilson et al. 1995). Another important reason for the inconsistency observed due to inoculation of PGPR could be the coating of seeds by low number of rhizobial cells. Higher or lower dosage of PGPR may have a detrimental effect on nodulation and growth of plant as demonstrated by Plazinski and Rolfe (1985). On the commercial front, approximately 20 bacterial biocontrol products based on Pseudomonas, Bacillus, Streptomyces and Agrobacterium strains have been marketed. The discovery of many traits and genes involved in the beneficial effects of PGPRs has resulted in a better understanding of the performance of bioinoculants in the field.

Some of these strains may provide effective control of diseases in certain soils, in certain geographic regions or on particular crops. Generally, microorganisms isolated from the rhizosphere of a specific crop are better adapted to that crop and may provide better control of disease than organisms originally isolated from other species (Cook 1993). Despite the extensive research where biological agents have been used to control plant diseases, there have been limited commercial success. Many biological agents do not perform better in the field due to the complexity and variability of physical, chemical, microbiological and environmental factors in the field. Therefore, applications of a mixture of biocontrol agents may be a more ecologically sound approach because it may result in better colonization and enhance the level and consistency of disease control by providing multiple mechanisms of action, a more stable rhizosphere community and effectiveness over a wide range of environmental conditions occurring throughout the growing season. In addition, the genetic diversity of these strains may be tapped by combining them in mixed inoculants. Certain mixtures of fluorescent pseudomonads suppressed disease more effectively than did single-strain inoculants (Pierson and Weller 1994; Duffy et al. 1996).

Spadro and Cullino (2005) concluded that the use of genetically modified microorganisms could play an important role in crop production and protection. Genetic manipulation could result in new biocontrol strains with increased production of toxic compounds or lytic enzymes, improved space or nutrient competence, wider host range or enhanced tolerance to abiotic stress (Glick and Bashan 1997). Thus, biocontrol performance of soil pseudomonads may be improved by the introduction of antibiotic biosynthetic genes (Maurhofer et al. 1992; Haas and Keel 2003). Recombinant DNA strains with greatly increased diacetyl phloroglucinol (DAPG) and phenazine-1-carboxamide (PCN) antibiotics production have been constructed (Mavrodi et al. 1998, 2001). The production of DAPG and PCN could be placed under the control of strong promoters or of exudate-induced or rhizosphere-induced promoters (Mavrodi et al. 2006). Genes and enzymes involved in the biocontrol mechanism could also be applied directly or transferred to crops.

From the perspective of developing nations, these are exciting strategies that may help to increase yield while reducing the inputs and environmental problems. However, most of the microbial biodiversity in soil remains unexplored and much work remains to be done to first identify and then characterize microorganisms that could be used in such applications. Furthermore, such approaches require a detailed knowledge of the molecular signaling that takes place between plants and microbes to drive expression of desirable traits and to suppress unwanted effects in a controlled manner. Future strategies are required to clone genes involved in the production of antibiotics, siderophores and other metabolites, and to transfer these cloned genes into the rhizobacterial strains having good colonization potential along with other beneficial characteristics such as N2 fixation, P-solubilization and/or hormone production. Exploiting plants and microbes by using such an integrated approach requires a coordinated strategy to understand the degree and complexity of plant-microbe interactions employing modern “genomics/proteomics” technologies. The generation of complete genomic sequences for plant-associated bacteria, including pathogens and symbionts is already increasing our knowledge of these organisms. The increasing amount of genomic data available for the model plant species and their associated microorganisms, will assist in determining the most suitable beneficial bacterial strains for inoculation. In the near future, the molecular tools adopted in manipulation of bacterial traits are likely to improve the availability of nutrients, efficiency of phytoremediation and enhancement of biocontrol activity that will consequently improve the crop productivity and also protect the food chain by reducing levels of agrochemicals in food crops.

9.5 Conclusion

Although striking advances have been made in understanding the molecular and biochemical mechanism regulating N2 fixation, P solubilization and hormone production, this has yet to be translated into applied environments. To overcome the problem of establishment of inoculated microbes, the beneficial bacteria intended for inoculation should be selected from local ecological niches and reinoculated into the same environment to ensure the desired benefits. The effects of soil and environmental factors on the physiology and ecology of introduced microorganisms are still poorly understood. Research is therefore, needed to understand the in situ physiology of inoculant cells and strategies must be developed as to how such microbes could be manipulated for desired performance. For example, the use of reporter genes inserted either randomly or directly into the bacterial genome may allow the specific detection and possible enhancement of in situ gene expression in inoculant cells.

The complex interactions among the PGPR, the pathogen, the plant and the environment are responsible for the variability observed in disease suppression and plant growth promotion. However, genetic manipulation of PGPR has the potential to construct significantly better strains with improved biocontrol efficacy (Trevors et al. 1990; Chet et al. 1993). Further, the efficacy of biocontrol bacteria can be improved by developing better cultural practices and delivery systems that favor their establishment in the rhizosphere. From the application point of view, consortia of ecologically diverse strains for N2 fixation, P-solubilization, root growth promotion among others, should be practiced instead of single strain. In near future, both traditional and biotechnological approaches could be employed to increase rates of N2 fixation, P solubilization, hormone production and increase in efficiency of biocontrol activity along with bioremediation of contaminants, leading to increase in crop yield under sustainable agricultural crop production system.