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Plant Growth Promoting Rhizobacteria: A Novel Source in Plant Growth Promotion

The zone surrounding the plant roots called as rhizosphere is a region of maximum microbial activity compared to surrounding soil (Hiltner 1904). This environment is a favorable habitat for microbial growth that exerts a potential impact on plant health as well as soil fertility (Podile and Kishore 2006). A number of beneficial microorganisms are associated with the root system of higher plants which depend on the exudates of these roots for their survival (Whipps 1990). In soil environment, particularly in rhizosphere, plants are mostly colonized by microbes (Berg et al. 2005). A variety of compounds present in root exudates including polysaccharides and proteins enable the bacteria to colonize plant roots (Somers et al. 2004; Rodriguez-Navarro et al. 2007). Due to competition for nutrients, those microbial populations having better ability to degrade complex compounds like chitin, cellulose, and seed exudates can survive better in such environment (Baker 1991). Among the diverse microbial population, bacteria are the most abundant microorganisms that competitively and progressively colonize the plant roots. Among this large bacterial population, a number of bacterial strains are considered as very important owing to their metabolically and functionally diverse characteristics. These are free-living plant growth promoting rhizobacteria (PGPR) that promote plant growth by root colonization (Kloepper et al. 1989) and have been studied extensively due to their optimistic effect on plant growth and development. These PGPR belonging to some important genera include Serratia, Bacillus, Pseudomonas, Burkholderia, Enterobacter, Erwinia, Klebsiella, Beijerinckia, Flavobacterium, and Gluconacetobacter (Podile and Kishore 2006; Dardanelli et al. 2009; Nadeem et al. 2010b). These PGPR enhance plant growth through various mechanisms like synthesizing a compound essential for plant and facilitating the host in nutrient uptake and also through disease prevention (Glick 1995). The major mechanisms used by PGPR can be divided into two categories, i.e., direct and indirect mechanisms. Phosphate solubilization and phytohormone and siderophore production are some examples of direct growth promotion (Kloepper et al. 1989; Glick et al. 1995; Ayyadurai et al. 2007), while indirect growth promotion occurs by inhibiting the growth of plant pathogens (Glick and Bashan 1997; Persello-Cartieaux et al. 2003; Ravindra Naik et al. 2008). In addition to these general growth promotion mechanisms, PGPR also protect the plant from the deleterious effects of environmental stresses by some particular mechanisms. These include lowering of stress-induced ethylene, production of exopolysaccharides, regulating nutrient uptake, and enhancing the activity of antioxidant enzymes (Sandhya et al. 2009; Glick et al. 2007). There are a number of reports that show outstanding role of this natural microbial population for improving plant growth and development in normal as well as stress environment (Zahir et al. 2004; Glick et al. 2007; Jha et al. 2009; Tank and Saraf 2010; Nadeem et al. 2010b).

Better plant growth promotion depends upon positive plant–microbe interactions. Belowground plant–microbe interactions are more complex than those occurring above the soil surface (Bais et al. 2004), and understanding of these interactions is crucial for maintaining plant growth and health (Barea et al. 2005). The plant–microbe interactions as well as interactions between other rhizosphere microorganisms are still not much clear, and literature shows that most of these interactions are complex in nature. An understanding about microbial ecology, their growth-promoting traits, mechanisms of action, and their application for plant growth stimulation is of pivotal importance for maximum utilization of this naturally occurring population. The diverse study of PGPR is important not only for understanding their ecological role and interactions with plants but also for biotechnological applications (Berg et al. 2002).

Plant Growth Promotion Mechanisms

Plant growth promotion by PGPR is a well-known phenomenon, and this growth enhancement is due to certain traits of rhizobacteria. Some of these traits are very common among certain bacterial species; however, other traits might be specific with some particular species. There are a number of mechanisms used by PGPR for enhancing plant growth and development in diverse environmental conditions (Fig. 2.1). In general, PGPR work as phytostimulators, biofertilizers, biocontrol agent, root colonizers, and environmental protectors (Vessey 2003; Zahir et al. 2004). Some of the important and valuable mechanisms are discussed in the following sections.

Fig. 2.1
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Mechanisms used by PGPR for enhancing plant growth

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Phytostimulation

One of the direct growth promotion mechanism used by PGPR is the production of phytohormones including indole acetic acid, abscisic acid, cytokinins, gibberellins, and ethylene. There are a number of reports which advocate the effectiveness of these growth regulators for enhancing plant growth and development (Zahir et al. 2004; Glick et al. 2007). These phytohormones enhance the plant growth by virtue of their positive effect on cell division, cell enlargement, seed germination, root formation, and stem elongation (Taiz and Zeiger 2000; Khalid et al. 2006). Phytohormones influence the physiological processes of plants and facilitate plant growth by altering the hormonal balance (Asghar et al. 2004; Kang et al. 2006). These phytohormones are equally effective in normal and stress conditions. For example, ABA abscisic acid (ABA) helps plant in stress conditions (Zhang et al. 2006) and plays an important role in the photoperiodic induction of flowering (Wilmowicz et al. 2008). Patten and Glick (2002) observed 35–50 % longer roots in canola inoculated with wild-type GR12-2 compared to IAA-deficient mutant and uninoculated control. Fassler et al. (2010) demonstrated the importance of IAA in stress alleviation of sunflower. Seed inoculation with wild-type GR12-2 induced the formation of tap roots that were 35–50 % longer than the roots from seeds treated with the IAA-deficient mutant and the roots from uninoculated seeds. Similarly, many Pseudomonas, Bacillus, and Azospirillum spp. produce cytokinin and gibberellins (Gamalero and Glick 2011), and positive effects on plant biomass have been reported by these hormones (Gutierrez-Manero et al. 2001; Arkhipova et al. 2005; Spaepen et al. 2009). Steenhoudt and Vanderleyden (2000) demonstrated that the main mechanism used by Azospirillum for enhancing plant growth is the production of phytohormones. Although commercially available phytohormones are also used for promoting plant growth, however, microbially produced phytohormones are more effective due to the reason that the threshold between inhibitory and stimulatory levels of chemically produced hormones is low, while microbial hormones are more effective by virtue of their continuous slow release (Khalid et al. 2006).

Biofertilization

The potential of PGPR to enhance plant growth and their participation in carbon, nitrogen, sulfur, and phosphorous cycling increase the effectiveness of PGPR in sustainable agriculture. The application of PGPR for increasing nutrient availability for plants is an important and necessary practice (Freitas et al. 2007) and is very helpful for increasing the nutrient concentration of certain essential elements like N, P, K, Ca, Mg, Zn, Fe, and Mn (Dursun et al. 2010). Inoculation of cotton with PGPR showed enhanced uptake of N, P, K, and Ca (Yue et al. 2007), and similarly PGPR inoculation also enhanced the nutrient content of salinity-stressed maize (Nadeem et al. 2006).

The conversion of insoluble form of phosphorus to make them plant-available form is a common mechanism of various PGPR strains and plays important role to fulfill the phosphorus requirement of plant. Phosphate-solubilizing bacteria are common in the rhizosphere (Ravindra Naik et al. 2008; Jha et al. 2009) that solubilize inorganic phosphate by various mechanisms like production of organic and inorganic acids, release of H ions, and production of chelating substances and through enzymes like phosphatase (Rodriguez et al. 2004; Gamalero and Glick 2011). Also, the exopolysaccharides produced by these bacteria have indirect effect on phosphate solubilization by binding free phosphorus (Yi et al. 2008). It was also observed that cold-tolerant species were able to solubilize P at low temperature (Selvakumar et al. 2008). The application of P-solubilizing bacteria can solve the problem of P precipitation in the soil and therefore increase its availability to plants (Lin et al. 2006). The role of PGPR to improve the uptake of other macronutrients has also been established. Inoculation of Pseudomonas sp. having the ability to stimulate calcium (Ca) uptake caused significant improvement in tomato growth and also reduced blossom-end rot of tomato fruits that generally occurs due to Ca deficiency (Lee et al. 2010). Similarly, the solubilization of biotite by silicate mineral-solubilizing bacteria like Bacillus sp. can enhance the availability of K+ to plants (Sheng et al. 2008).

The production of low-molecular-weight ferric-chelating compound siderophores directly increases the iron availability for plant (Robin et al. 2008) and indirectly protects the plant from pathogenic organisms (Singh et al. 2010b). Siderophores play important role in iron nutrition of plants (Jin et al. 2006). Vansuyt et al. (2007) reported that Fe–pyoverdine complex synthesized by Pseudomonas fluorescens C7 was efficiently taken up by the Arabidopsis thaliana that resulted in enhanced iron content in plant tissue and better growth. Similarly, bacterial strains improved maize growth through biofertilization and phytostimulation mechanisms (Marques et al. 2010).

Certain bacteria can fix atmospheric nitrogen and make it available for plant. The symbiotic relationship between legumes and nitrogen-fixing bacteria and nitrogen fixation by free-living bacteria without forming association is a source of nitrogen for plant (Carvalho et al. 2010). Co-inoculation of PGPR with rhizobia caused positive effect on nitrogen fixation, plant biomass, and grain yield in various crops like alfalfa, soybean, and pea (Bolton et al. 1990; Dashti et al. 1998; Tilak et al. 2006). Similarly, Azospirillum sp. have the potential to increase nitrogen fixation (Rai and Hunt 1993) which can contribute about 70 % of the total nitrogen requirement of the host plant (Malik et al. 1997). The presence of such bacteria also enhances ability of plant to use nitrogen efficiently and minimizes its leaching and denitrification losses. Some important genera of such bacteria include Enterobacter, Klebsiella, Pseudomonas, and Rhizobium (James 2000).

Zinc is also an essential nutrient and in deficient soils the solubilization of Zn near the root zone can alleviate the deficiency for plants. The Zn solubilization by sugarcane-associated Gluconacetobacter diazotrophicus has been demonstrated by Saravanan et al. (2008). The inoculation with Burkholderia cepacia enhanced Zn uptake, its translocation from root to shoot, and improved plant growth (Li et al. 2007).

Due to high price and certain environmental concerns about the chemical fertilizers, the use of PGPR in the form of biofertilizers is an effective supportive strategy to provide crop nutrition (Cakmakci et al. 2006). The use of PGPR inoculants as biofertilizer provides a promising support to chemical fertilizers. Moreover the use of PGPR with inorganic fertilizer can increase the availability of nutrients to the crops (Kumar et al. 2009) and therefore could be useful for increasing efficiency of these fertilizers in one hand and also reducing their quantity on other.

Root Colonization and Rhizosphere Competence

Rhizosphere is a complex habitat with temporal and spatial changes where plant and microbial populations interact with each other and are affected by a number of biotic and abiotic factors. The success of bacteria to enhance plant growth depends on its potential to colonize the plant root. The significant effects of microbial inoculation cannot be obtained unless the environment supports growth and survival of these introduced microorganisms (Devliegher et al. 1995). The ineffectiveness of PGPR, particularly in field conditions, is due to their inability to colonize plant root properly (Bloemberg and Lugtenberg 2001). One of the aspects of better root colonization is the ability of the bacteria to compete with the indigenous microbial populations. Being the most abundant microorganisms, it is very likely that bacteria can cause great effect on plant physiology owing to their better competitiveness for root colonization (Barriuso et al. 2008). Literature shows that certain PGPR strains have ability to tolerate unfavorable environment (Paul and Nair 2008; Malhotra and Srivastava 2009) and therefore can be considered as the best population for promoting crop production.

The microbes use different strategies for their survival in the environment. The success of these strategies depends upon their ability to adapt to the nutrient-limited conditions, efficient utilization of root exudations, as well as their interaction with plants (Devliegher et al. 1995; Van Overbeek and Van Elsas 1997). In soil environment, the survival of the inoculated bacteria depends on the availability of an empty niche, so that they can compete effectively with better adopted native microbial population (Rekha et al. 2007). It has been observed that PGPR which possess some particular traits like ACC-deaminase activity and the production of antioxidant enzymes, exopolysaccharides, and organic solutes have some selective advantages over other bacteria under stress environment (Mayak et al. 2004a, b; Saravanakumar and Samiyappan 2007; Sandhya et al. 2009). A variety of compounds, like surface proteins and polysaccharides, have a good role in adherence of bacteria to plant root (Dardanelli et al. 2003; Rodriguez-Navarro et al. 2007), and such bacteria have competitive advantages to colonize plant roots because these exopolysaccharides help them to attach and colonize the roots due to fibrillar material that permanently connects the bacteria to root surface (Sandhya et al. 2009).

Enzymatic Activity

Growth enhancement through enzymatic activity is another mechanism used by PGPR. Bacterial strains can produce certain enzymes such as cellulase, ACC-deaminase, and chitinase. Through the activity of these enzymes, bacteria play a very significant role in plant growth promotion particularly to protect them from biotic and abiotic stresses. For example, the reduction of elevated level of ethylene under stress by ACC-deaminase activity and disease suppression by chitinase activity are common mechanisms used by PGPR (Glick et al. 2007; Nadeem et al. 2010b). Similarly, the enhancement of nodule formation by rhizobia might be due to the production of hydrolytic enzymes such as cellulase which could make penetration of rhizobia into root hairs leading to increased numbers of nodules (Sindhu and Dadarwal 2001).

Growth Enhancement Through Vitamins

Vitamins are organic nutritional factors that influence the growth of living organisms. In addition to the vitamins present in root exudates as a source for bacterial growth (Mozafar and Oertli 1993), certain bacterial species also produce vitamins (Dahm et al. 1993). Like other growth promoting traits of PGPR, the production of vitamins also causes positive effect on plant growth and development (Derylo and Skorupska 1993; Azaizeh et al. 1996; Dakora 2003). More root colonization ability of vitamin-producing Pseudomonas fluorescence has been observed (Marek-Kozaczuk and Skorupska 2001). Similarly, co-inoculation of vitamin-producing P. fluorescence and Rhizobium stimulated the growth and symbiotic nitrogen fixation in clover plants (Marek-Kozaczuk et al. 1996).

Biocontrol Activity

Biocontrol mechanisms for diseases suppression are an important strategy against a number of plant pathogens that cause reduction in crop yield. PGPR also act as effective biocontrol agents by suppressing the effect of diseases (Kotan et al. 2009) and provide protection to the plants against harmful pathogens. The PGPR use certain mechanisms including competition, antibiotic production, degradation of fungal cell wall, and sequestering iron by the production of siderophores (Velazhahan et al. 1999; Siddiqui 2006; Ramyasmruthi et al. 2012).

Cell wall degrading enzymes are very important for controlling the phytopathogenic fungi (Picard et al. 2000). Chitinase, cellulase, and lyases are well-known fungal cell wall degrading enzymes (Inbar and Chet 1991; Lorito et al. 1996; Ayyadurai et al. 2007). These enzymes play very important role by suppressing the onset of diseases. The presence of chitinase enzyme in Pseudomonas sp. inhibits the growth of Rhizoctonia solani by degrading the cell wall (Nielsen et al. 2000). A volatile antibiotic hydrogen cyanide produced by certain bacterial strains also plays role in disease suppression. Suppression of black rot of tobacco by HCN producer Pseudomonas strain was observed by Voisard et al. (1981). The production of siderophores by the bacteria reduces the availability of iron to fungi (Sayyed et al. 2008), therefore causing negative impact on its growth (Arora et al. 2001). Matthijs et al. (2007) reported the suppression of disease caused by Pythium sp. owing to siderophores that decreased the availability of iron for fungal growth. It is also an evident fact that fungi are unable to absorb the iron–siderophore complex that causes unavailability of iron to pathogenic fungus (Solano et al. 2009). Bacterial siderophores are also suggested to be involved in inducing systemic resistance (ISR) that enhances plant’s defensive capacity against pathogens. Enhanced ISR in tomato has been reported by siderophores, pyochelin, and pyocyanin (Audenaert et al. 2002). Similarly, a number of reports have shown the effectiveness of PGPR for enhancing ISR against various fungal and viral diseases (Radjacommare et al. 2002; Saravanakumar et al. 2007). Systemic resistance can also be induced by a mechanism where inducing bacteria and pathogen remain separated without showing any direct interaction (Ryu et al. 2004).

The disease suppression by PGPR also occurs by the production of antibiotics. The antibiotics in addition to suppressing the pathogen also induce systemic resistance in the plant. The synergistic interaction between antibiotics and ISR further increases resistance against pathogens (Jha et al. 2011). The Bacillus thuringiensis, having the ability to produce insecticidal protein (Singh et al. 2010a), can be used as biocontrol agent.

In addition to above-discussed mechanisms, certain environmental factors like water, soil pH, temperature, nutrient contents, and competition for root exudates as well as indigenous microbial population affect the ability of an organism to colonize the plant root. The exclusion of pathogenic organisms from the rhizosphere is one of the significant mechanisms to protect the plant from deleterious effect of such disease-causing organisms. Above discussion shows that owing to their number of mechanisms, PGPR have great competitive advantages over pathogens and could be very effective for protecting the plant from their attack by suppressing their growth.

Removal/Detoxification of Organic and Inorganic Pollutants

Plant growth promotion by PGPR inoculation is also due to reduction and improving plant tolerance against heavy metals (Belimov et al. 2005; Sheng et al. 2008). Bacteria use different intra and extra mechanisms to detoxify the adverse effects of heavy metals in their tissues. These mechanisms include production of proteins which absorb heavy metals and detoxification by taking them in vacuoles (Gerhardt et al. 2009; Giller et al. 2009). The mechanisms used by PGPR for tolerating and detoxifying of heavy metals may also vary among bacterial species and also for different metals. For example, microbes can detoxify zinc (Zn) by binding it in the outer membrane, by producing Zn-binding protein, and/or by complexation of organic acids (Appanna and Whitmore 1995; Choudhury and Srivastava 2001). Bacterial inoculation resulted in degradation of chlorobenzoates and pesticides (Crowley et al. 1996; Siciliano and Germida 1997) and the enhancement of plant growth by PGPR inoculation in highly contaminated soils (Gurska et al. 2009).

The production of siderophores by metal-resistant bacteria plays an important role in the successful survival and growth of plants in contaminated soils by alleviating the heavy metal stress-imposed impact on plants (Belimov et al. 2005; Braud et al. 2006; Rajkumar et al. 2010). Also, the production of enzymes and certain hormones which mobilize heavy metals and plant–microbe interactions affects the process of bioremediation (Abbas-Zadeh et al. 2010). For example, the inoculation of Lupinus luteus with genetically engineered nickel-resistant B. cepacia showed high nickel concentration that was approximately 30 % more than uninoculated control (Lodewyckx et al. 2001). The application of such bacteria could be helpful for the removal of heavy metals from the environment.

Enhancement of Photosynthetic Activity

Photosynthesis is considered as one of the very important reactions in plant growth and development. Under stress environment, reduction in photosynthesis occurs that might be due to decrease in leaf expansion, premature leaf senescence, impaired photosynthetic machinery, and associated reduction in food production (Wahid and Rasul 2005). PGPR enable the plants to maintain their growth by causing positive effect on photosynthesis. Drew et al. (1990) reported that reduction in photosynthetic activity might be due to osmotic stress and closing of stomata; however, the application of PGPR minimized this negative impact and caused significant increase in photosynthesis (Golpayegani and Tilebeni 2011). Heidari and Golpayegani (2011) observed enhancement in chlorophyll contents in drought stress basil (Ocimum basilicum L.) by PGPR application. More improvement in chlorophyll content was observed where PGPR were applied in combination than alone. The increase in shoot length, chlorophyll content, and dry weight was observed when banana plants were inoculated with PGPR (Mia et al. 2010a). According to them, this growth enhancement in addition to other factors was likely to be due to the higher accumulation of nitrogen that contributed to chlorophyll formation which consequently increased the photosynthetic activity. While Xie et al. (2009) demonstrated that enhanced photosynthetic activity in Arabidopsis by volatile emission from Bacillus subtilis might be due to accumulation of iron, because iron is often a limiting ion in photosynthesis. They also observed that when bacterial volatile signal was withdrawn, the photosynthetic capacity and iron content returned to untreated levels. The importance of iron has already been documented by Spiller and Terry (1980) who demonstrated that biogenesis of the photosynthetic apparatus makes heavy demands of iron availability.

Stress Tolerance

Due to sophisticated signaling system, microbes develop high degree of adaptability to environmental stresses. Bacteria are well known for their ability to tolerate the stress conditions due to their exceptional genetic makeup. The PGPR strains have showed tolerance against stress conditions like salinity and drought (Sandhya et al. 2009; Tank and Saraf 2010). Andre’s et al. (1998) demonstrated great resistance ability of Bradyrhizobium japonicum against high doses of thiram. Although the microbial adaptations to such situations are difficult to understand (Spaepen et al. 2009), however, it might be due to some of their particular traits which enable them to survive under unfavorable conditions. For example, production of exopolysaccharides (EPS) by the bacteria protects them against unfavorable conditions and enhances their survival (Sandhya et al. 2009; Upadhyay et al. 2011b). In an earlier study, Hartel and Alexander (1986) also showed a significant correlation between the amount of EPS produced by the bacteria and their desiccation tolerance. The accumulation of poly-β-hydroxybutyrate during saline conditions and other osmoprotectants like proline and ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) are protective measures taken by bacteria to survive under stress conditions (Bernard et al. 1993; Arora et al. 2006). The occurrence of such stress-tolerant strains could be very effective for improving soil fertility and enhancing plant growth (Mayak et al. 2004a; Egamberdieva and Kucharova 2009), and application of such stress-resistant strains could also be very useful for enhancing plant growth under stress environment (Glick et al. 2007; Nabti et al. 2010). The above-discussed mechanisms not only show the abilities of bacterial strains to withstand in variable soil environmental conditions but also enable them to compete effectively with the other microbial population. These mechanisms could be very useful for maintaining proper soil conditions and promoting sustainable agriculture.

Application of Rhizobacteria for Plant Growth Promotion

Owing to their well-established growth promoting abilities, PGPR are being used effectively for enhancing crop production. The growth promoting abilities of PGPR have been observed in laboratory under control conditions as well as in natural greenhouse and filed conditions. The crop improvement by PGPR inoculation under normal and stress environment has been reviewed by various workers (Zahir et al. 2004; Glick et al. 2007; Nadeem et al. 2010b; Ahemad and Khan 2011).

Growth Promotion Under Normal Conditions

The use of PGPR is an effective biological approach to increase crop yield and is applied to a wide range of agricultural species. Inoculation with PGPR promotes plant growth through phytohormone production, phosphate solubilization, siderophore production, regulation of hormonal level, and certain other mechanisms which have been discussed in the previous section. The root length of canola, lettuce, tomato, barley, wheat, and oats increased when seeds of these crops were treated with PGPR (Hall et al. 1996). Qiaosi et al. (2005) also reported that the roots of inoculated plants were more in number and longer than untreated control. This growth enhancement is due to common and some particular trait of bacteria, as is evident from the work of Cattelan et al. (1999) who tested eight strains of PGPR for their growth-promoting activity in soybean. They examined that six strains promoted growth more as compared to other, and they observed that these strains contained ACC-deaminase activity in addition to other characteristics. The growth enhancement by the PGPR has also been reported under natural field conditions. Inoculation with PGPR increased the dry weight of leaf, stem, and grain of maize (Gholami et al. 2012). They observed that inoculation caused significant effects on leaf area index and crop growth index. A number of other studies have also shown the importance of PGPR for improving plant growth and development, and some selected examples have been mentioned in Table 2.1.

Table 2.1 Plant growth promotion by PGPR inoculation under normal conditions
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Considerable work conducted by different researchers shows that PGPR can be used as biofertilizers, and, thus, the use of chemical fertilizer can be reduced (De Freitas et al. 1997; Rabouille et al. 2006). Work of Godinho et al. (2010) showed that application of four PGPR strains having various growth-promoting traits enhanced biomass of eggplant due to balanced nutrient availability and uptake. This growth promotion was also associated with other growth-promoting traits especially indole acetic acid and siderophores. Similarly in a greenhouse study, the application of six bacterial strains on maize plant promoted root and shoot growth and the nutrient status of plant particularly nitrogen and phosphorus (Marques et al. 2010). Such findings have confirmed the perspectives of PGPR as phytostimulators and biofertilizer for agricultural crops. These microbes are also equally effective for promoting growth of fruit trees like apple, apricot, strawberry, plum, and mulberry (Sudhakar et al. 2000; Esitken et al. 2006, 2010; Karakurt and Aslantas 2010; Erturk et al. 2012). Early studies conducted by most of the workers show growth-promoting activity of the PGPR by some common direct and indirect mechanism; however, the production of volatile compound by the bacteria is another growth-promoting mechanism. Zou et al. (2010) found that volatile compounds produced by Bacillus megaterium had great growth promotion activity in A. thaliana. The fresh weight of inoculated plants was twofold more than uninoculated. They suggested that 2-pentylfuran is a compound that plays an important role in the plant growth promotion activity of this bacterial strain. Prior to this work, Ryu et al. (2003) showed the growth promotion of A. thaliana by the volatile compounds 2,3-butanediol and acetoin.

Effectiveness in Stress Agriculture

Environmental stresses are the most limiting factors for crop productivity. Both biotic and abiotic stresses including salinity, drought, extreme temperature, chilling, heavy metals, and insect and pathogen attack are the most detrimental and common stresses plants face in the natural environments. These stresses affect the normal plant processes in one or other way and therefore cause significant reduction in crop yield. PGPR inoculation also proved effective for alleviating the negative impact of these stresses. In addition to improved plant growth under normal conditions, PGPR have great potential for enhancing plant growth under adverse conditions. PGPR use various mechanisms to combat these stresses and enable the plant to maintain their growth under stress environment (Fig. 2.2). There are a number of reports elaborating the effectiveness of PGPR for improving plant growth under stress environment (Glick et al. 2007; Nadeem et al. 2010b; Nabti et al. 2010). The PGPR strains were found equally effective for this growth promotion in variable stress environment like salinity, drought, heavy metal, nutrient stress, and pathogen. Some of the selected examples have been discussed in this section and also listed in Table 2.2.

Fig. 2.2
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Impact of environmental stresses on plant growth and effectiveness of PGPR for mitigating this negative impact

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Table 2.2 Plant growth promotion by PGPR inoculation under biotic and abiotic stress conditions
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Abiotic Stress Tolerance

Among various stresses, salinity and drought are the most common that cause adverse effects on crop production in most of the arid and semiarid regions of the world. Salinity limits the production of nearly over 6 % of the world’s land and 20 % of the irrigated land (Rhoades et al. 1992; Munns 2005). The changes in environmental scenario result in increasing aridity due to decrease in annual rainfall and because of agriculture under sustained pressure to feed an ever-increasing population. Water limitation in the growing medium reduces diffusion, nutrient uptake by roots, and transport of nutrients from roots to shoots due to restricted transpiration rate, impaired active transport, and altered membrane permeability (Sardans et al. 2008a, b). Similarly, under salinity stress, increasing Na+ contents cause an increase in Na+ uptake and, in general, decrease in K+ and Ca2+ contents of plant. Moreover, under stress conditions, plants produce significant quantity of ethylene which can damage them due to negative impact on roots, and it can also cause epinasty, premature senescence, and abscission (Nadeem et al. 2010b). Many efforts have been made to understand the adaptive mechanisms of stress tolerance. These include the reduction of stress ethylene, reduction of toxic ion uptake such as Na+, and formation of stress-specific protein in plants. Microbial inoculation to alleviate stresses in plants could be a more cost-effective and environment-friendly option which could be available in a shorter time frame.

Stress environment can also make physicochemical and biological properties of soil unsuitable for microbial and plant growth. However, particular characteristics of certain bacteria enable them to survive under such harsh environments. For example, certain bacterial strains have the ability to tolerate high salinity, and, similarly, the production of exopolysaccharides by the bacteria protects them from water stress. Besides developing mechanisms for stress tolerance, microorganisms can also impart some degree of tolerance to plants toward abiotic stresses like drought, salinity, metal toxicity, and high temperature (Grover et al. 2011). The exopolysaccharides released into soil can be adsorbed by clay particles and form a protective layer around soil aggregates (Tisdall and Oades 1982) and, therefore, protect the plant from desiccation. Moreover, exopolysaccharide production increases root colonization of microbes (Santaella et al. 2008), improves soil aggregation (Sandhya et al. 2009), channelizes water and nutrients to plant roots (Tisdall and Oades 1982; Roberson and Firestone 1992), and forms biofilm (Seneviratne et al. 2011) which is beneficial to plant growth and development. Alami et al. (2000) observed a significant increase in root-adhering soil per root tissue (RAS/RT) ratio in sunflower rhizosphere inoculated with the EPS-producing rhizobial strain YAS34 under drought conditions. The inoculation with ACC (1-aminocyclopropane-1-1carboxylic acid)-deaminase-containing bacteria can reduce negative impact of stress-induced ethylene (Mayak et al. 2004a, b). The elevated level of ethylene caused negative impact on plant growth by inhibiting the root growth particularly. These microorganisms secrete enzyme ACC-deaminase that hydrolyses ACC into ammonia and a-ketobutyrate. The rhizobacteria bound to plant roots act as sink for ACC (immediate precursor of ethylene) and thereby lower the level of ethylene in a developing seedling or stressed plant. Therefore, the inoculation of seeds with such strains containing ACC-deaminase would be very useful for enhancing plant growth under stress conditions by diluting the negative impact of stress-induced ethylene on root growth (Glick et al. 2007). As is evident from one of our greenhouse study conducted under salinity-stressed conditions, that application of PGPR strains having ACC-deaminase activity significantly enhanced the root length of maize compared to uninoculated control (Fig. 2.3). The work of Mayak et al. (2004a) shows that bacterial strain (Achromobacter piechaudii) containing ACC-deaminase conferred tolerance to water deficit in tomato and pepper. Ethylene production was reduced in inoculated plants, resulting in significant increase in fresh and dry weights compared to uninoculated controls. Pseudomonas spp. also improved the growth of pea (Pisum sativum) under drought stress in axenic conditions as well as in potted soil (Zahir et al. 2008). They concluded that inoculation might have reduced the ethylene synthesis, which resulted in better plant growth under drought stress. Similar results were also obtained by Arshad et al. (2008) while studying the effectiveness of Pseudomonas spp. for eliminating the drought effect on growth, yield, and ripening of pea. It has been observed that the presence of elevated levels of ethylene in the vicinity inhibits the nitrogen fixation by rhizobia. However, the co-inoculation of Rhizobium with PGPR having ACC-deaminase activity can minimize this negative impact of ethylene and enhance nodulation (Ahmad et al. 2011). Stimulation of root elongation and biomass production of different plant species by inoculation with PGPR having ACC-deaminase activity has been repeatedly documented, particularly when the plants were subjected to stressful growth conditions (Nadeem et al. 2007, 2010a; Saravanakumar and Samiyappan 2007; Tank and Saraf 2010; Siddikee et al. 2012). Similarly, the presence of other growth-promoting characteristics like indole acetic acid (IAA), siderophore production, phosphate solubilization, and phytohormone production may provide extra benefits for stress tolerance in plants and improve their growth. The production of antioxidant enzymes protects the plant from the harmful impact of reactive oxygen species. The reactive oxygen species (ROS) as singlet oxygen (O−), hydrogen peroxide (H2O2), and hydroxide ions (OH−) are developed in the photosystem of plants. These ROS denature cell membranes, proteins, and DNA through oxidation reaction. To combat/reduce the impact of these ROS, plant’s immune system generates antioxidant enzymes such as superoxide dismutase, peroxide dismutase, catalase, and glutathione reductase (Arora et al. 2002). The PGPR inoculation also enhances the activity of these enzymes and helps them to reduce the negative impact of stress (Fu et al. 2010). Similarly, enhanced production of osmoprotectants by bacterial inoculation under stress enables the plant to maintain their internal water potential for better uptake of water and nutrients.

Fig. 2.3
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Effect of PGPR containing ACC-deaminase on root growth of maize in a pot trial at 12 dS m−1 salinity level

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Rhizobacteria as Biocontrol Agent

In soil environment, there are a number of plant pathogens that reduce crop yield. Although these plant pathogens can be controlled by the application of chemicals and growing disease-resistant varieties, however, there are certain environmental concerns about the use of such chemicals like their persistent nature in the soil as well as accumulation of toxic residues of these chemicals in the food parts. Some of these toxic chemicals have been banned due to their persistent nature. Similarly in certain cases, the resistance of genetically resistant crops is often broken by the pathogen that results in reduction in crop yield (Fry 2008). An alternative strategy to overcome this problem is the use of PGPR that act as biocontrol agent by virtue of their certain biocontrol mechanisms like production of antibiotics, production of antifungal metabolites, decreasing availability of iron for pathogenic organisms, production of fungal cell wall-degrading enzymes, and through induced systemic resistance. Number of reports have shown the effectiveness of PGPR for enhancing plant growth by protecting them from pathogens (Siddiqui et al. 2005; Ayyadurai et al. 2007; Ravindra Naik et al. 2008; Srinivasan and Mathivanan 2009). PGPR have competitive advantage over fungi for iron uptake due to production of siderophores. These siderophores have very high affinity for iron, and bacteria can take up iron–siderophore complex. By using this mechanism, PGPR retard the pathogen growth by reducing the availability of iron and therefore providing protection to the plant against diseases (Penyalver et al. 2001).

The above-discussed review and number of examples mentioned in Tables 2.1 and 2.2 show the effectiveness of PGPR for enhancing plant growth and development under normal as well as stress environment. Such growth promotion was due to certain direct and indirect mechanisms used by PGPR. It was also evident from discussion that inoculation of plant seed or seedlings with most promising strains having best growth-promoting traits not only enables the plant to maintain their proper growth but also causes positive impact on soil health.

Role of Bacterial Consortium in Advance Agriculture: Effectiveness and Challenges

Although above-discussed review highlights the effectiveness of rhizobacteria for enhancing plant growth under stress environment, however, under certain cases, the results obtained in the laboratory could not be reproduced in the field (Zhender et al. 1999; Smyth et al. 2011). This might be due to the low quality of the inocula and/or the inability of the bacteria to compete with the indigenous population under adverse environmental conditions (Brockwell and Bottomley 1995; Catroux et al. 2001). Great variations in the plant response to PGPR in laboratory and field assays demonstrate that the full potential of rhizobacteria to promote plant growth should be more extensively investigated. It is necessary to develop efficient inocula that can perform better under field conditions (Ahmad et al. 2008). The application of multistrain PGPR in combination could be more beneficial than a single strain. It has been reported that co-inoculation and coculture of microbes have better ability to fulfill the task in an efficient way than single-strain inoculation (Guetsky et al. 2002). Each strain in the multistrain consortium can compete effectively with the indigenous rhizosphere population and also enhance plant growth with its partners (Shenoy and Kalagudi 2003). The two strains used in a consortium showed that each strain not only competed successfully for rhizospheric establishment but also promoted plant growth (Shenoy and Kalagudi 2003). The co-inoculation of Rhizobium with PGPR proved useful for promoting growth and increasing nodulation (Tilak et al. 2006). The use of multistrain inoculants could be a good strategy that enables organisms to successfully survive and maintain themselves in communities (Andrews et al. 1991). Van Veen and others (1997) critically reviewed the reasons for the poor performance of agricultural bioinocula in natural environments and in the rhizosphere of host plants and suggested that instead of using a single strain for a single trait, multiple microbial consortia could be used for multiple benefits. Microbial studies performed without plants indicated that some combinations allow the bacteria to interact with each other synergistically, provide nutrients, remove inhibitory products, and stimulate growth of each other through physical and biochemical activities that may have beneficial impacts on their physiology (Bashan 1998). Rajasekar and Elango (2011) studied the effectiveness of Azospirillum, Azotobacter, Pseudomonas, and Bacillus sp. separately and in combination on Withania somnifera for two consecutive years. They observed that PGPR consortia significantly increased plant height, root length, and alkaloid content in W. somnifera when compared to the uninoculated control and single inoculation. Similarly, dual inoculation with Azotobacter and Azospirillum significantly increased total dry weight, leaf area index, and crop growth index (Gholami et al. 2012). Jha and Saraf (2012) observed that growth of Jatropha (Jatropha curcas) plant improved maximally in greenhouse and field experiments when three strains were applied together. Co-inoculation provided the largest and most consistent increases in shoot weight, root weight, total biomass, shoot and root length, total chlorophyll, shoot width, and grain yield. Similarly, the consortia of three strains gave the best performance in terms of growth parameters of Lycopersicum esculentum (Ibiene et al. 2012). They demonstrated that the use of combined biofertilizers containing consortia of bacteria is an excellent inoculant for growth performance of plants.

As far as growth under stress environment is concerned, Annapurna et al. (2011) studied the effectiveness of PGPR separately and in combination for reducing the impact of salinity on wheat growth. They found that single and dual inoculations of PGPR strains showed variations in their effect to enhance the crop tolerance to salts. The bacterial consortium was more effective for inducing salinity tolerance in wheat plants. They considered it as an acceptable and environment-friendly technology to improve plant performance and development under stress environment. In another study, Upadhyay et al. (2011a) evaluated the growth-enhancing potential of single and dual inoculation of B. subtilis SU47 and Arthrobacter sp. SU18 on wheat under saline conditions. They observed that in addition to enhancing dry biomass, soluble sugars, and proline content, wheat sodium content was reduced under co-inoculated conditions but not after single inoculation with either strain or in the control. The results indicate that co-inoculation with B. subtilis and Arthrobacter sp. could alleviate the adverse effects of soil salinity on wheat growth. The bacterial consortium is also effective for protecting the plant from disease under field condition. It is evident from the work of Srinivasan and Mathivanan (2009) that effective control of necrosis virus in sunflower can be obtained by the application of powder and liquid formulations of PGPR consortia. They applied two bacterial consortia consisting of Pseudomonas, Bacillus, and Streptomyces spp. along with farmer’s practice, i.e., imidacloprid + mancozeb. They observed a significant reduction in disease with an increase in seed germination, plant height, and crop yield. They demonstrated that PGPR consortia show high benefit–cost ratio compared to farmer’s practice and untreated control.

Inoculant Technology: Formulation and Commercialization

The application of PGPR for improving crop production is becoming an emerging technology owing to their environmental friendly traits. For that purpose various microbial inoculants have been formulated and are being marketed. A number of strains having ability to protect plant from pathogens belonging to genera Bacillus, Pseudomonas, and Agrobacterium are being used as biopesticides (Fravel 2005).

Formulation of Microbial Inoculants

A number of PGPR strains have great potential to be formulated as biofertilizer for improving plant growth and development under normal and stress environment. Successful inoculation of PGPR can result in better plant growth and therefore higher economic return to the farmers. For effective transfer of research findings from laboratory to field, an excellent formulation technology has great advantages. Various microbial inoculants have been formulated, marketed, and applied successfully (Reed and Glick 2004). Commercial bioinoculants prepared from Bacillus spp. are used widely as biocontrol agents (Ongena and Jacques 2007). B. thuringiensis, which is used to control insect pest, is estimated having sale of >70 % (Ongena and Jacques 2007; Sanchis and Bourguet 2008). Pseudomonas putida, Paenibacillus, and Bacillus sp. are formulated and have successfully enhanced the growth and yield of wheat (Cakmakci et al. 2007). Similarly, field application of salt-tolerant bioformulation of certain bacteria enhanced plant growth under salinity stress (Paul et al. 2006).

The major bottleneck to the commercial use of PGPR as biofertilizers is their inconsistent performance in the field. In certain cases, plant growth promotion due to microbial inoculation is not so effective in terms of investment applied and net return when compared with chemical fertilizers (Lucy et al. 2004). The development of valuable formulation is a challenging task for improving the efficacy of microbial inoculants. Actually, formulation is one of the crucial steps that determines the success or failure of a PGPR strain. However, this important step is generally neglected which results in less efficient outcome. The reason of this failure is the preparation of microbial formulation under lack of quality control and proper guidelines (Paau 1988; Berg 2009). The active ingredient in a microbial formulation is its viable culture. Regardless of the organism used, the success of bioagent depends upon the preparation of such inoculum having high level of viability and vigor (Jones and Burges 1998). In microbial formulation, the maintenance of bacterium in metabolically and physiologically active state is an important aspect for gaining maximum advantage (Paau 1998). In certain environmental conditions, where single-strain inoculum is unable to perform better, the development of multistrain inoculum can be very effective (Domenech et al. 2006). Such multistrain inoculum would be more effective for enhancing plant growth and development due to the presence of more growth-promoting traits which might not be possible in single strain.

Another important aspect regarding formulation is carrier material, which plays active role in shelf life of formulation. It aids in the stabilization and protection of the microbial cells during storage and transport (Xavier et al. 2004). It also protects the active ingredient, i.e., microbe from environmental conditions, and enhances its activity in field (Deaker et al. 2004). Various organic and inorganic carrier materials are used for formulation development (Bashan and Levanony 1990; Bashan 1998). Organic carriers like peat have some advantages due to their higher nutrient content, and, however, complete sterilization by steam is difficult, and also during sterilization, toxic by-products are produced that may cause decrease in bacterial population (Weiss et al. 1987). Therefore, the use of inorganic carrier may be a good strategy for enhancing the effectiveness of the microbial formulation. However, the effectiveness of these inorganic carrier materials may also be different, as it is evident from the work of Saharan et al. (2010) who used talc and aluminum silicate powders to develop inorganic carrier-based formulation. They observed that the shelf life of talc powder-based formulation was higher compared to aluminum silicate-based formulation. It was also observed that both sterile and nonsterile carrier formulations significantly enhanced the growth of Vigna mungo and Triticum aestivum. The application of microbial inoculants in the form of granular or liquid form is also attaining much attention nowadays. For optimizing nodulation, granular inoculants particularly rhizobia can be placed below or at the side of seeds with appropriate equipment according to seeding depth and moisture availability (Stephens and Rask 2000). On the other hand, due to easy application of liquid inoculants, liquid formulation has also achieved much popularity (Xavier et al. 2004). However, both types of formulations have shown their effectiveness for enhancing the biomass yield of soybean (Atieno et al. 2012). They have also demonstrated that formulation of rhizobia and PGPR gave better response.

Bacterial Characters for Formulation Development

Although a good number of microbial strains are used for formulation development and also their performance is observed, however, there are various constraints for commercialization of microbial inocula. One of the challenges for developing PGPR inoculants on commercial basis is the selection of such strains which could have competitive advantage over indigenous population and also have the ability to maintain their growth under unfavorable environment. The most important aspect in this regard is the selection of such strains which have host plant specificity as well as adaptation to soil and climatic conditions (Bowen and Rovira 1999). An organism with properties like phosphate solubilization, phytohormone production, root colonization, siderophore, and indole acetic acid production is thought to be an ideal bioinoculant.

To develop a successful PGPR formulation, in addition to above-mentioned growth-promoting traits, bacteria should have the ability to tolerate harsh environmental conditions like drought, heat, salinity, and toxic metals. It should have high rhizosphere competence and compatibility with other rhizobacteria. Such bacteria should also have capability of multiplication and broad spectrum of action. In addition to possessing a number of other characteristics, a PGPR should also have great viability and good shelf life (Lianski 1985). Cost-effectiveness, shelf life, and delivery systems are very important aspects that should be kept in mind while preparing the microbial formulation.

Concluding Remarks and Future Prospects

The above discussion showed the effectiveness of PGPR for enhancing the growth and development of plants. These beneficial effects are obtained owing to a number of direct and indirect mechanisms including phosphate solubilization, production of plant growth regulators, iron sequestration by siderophores, production of antibiotics, synthesis of antifungal metabolites, production of fungal cell wall degrading enzymes, inducing systemic resistance, reducing deleterious effects of stress-induced ethylene by ACC-deaminase activity, and production of vitamins. These plant growth promoting abilities of microbes under normal as well as stress conditions have certified their role in sustainable agriculture. For better performance, the PGPR strain must be rhizosphere competent that should be able to survive and colonize (Cattelan et al. 1999). In addition to rhizosphere competency, the compatibility between the rhizodeposition of compounds by the plant host and the ability of the inoculated bacteria to utilize them are also very important (Strigul and Kravchenko 2006). However, there is still lack of evidence about the consistent performance of these microbes, particularly under field conditions. In certain cases, the results obtained in laboratory are not reproduced in the field (Zhender et al. 1999; Smyth et al. 2011). This may occur due to the low quality of the inoculum and/or the inability of the bacteria to compete with the indigenous population (Brockwell and Bottomley 1995; Catroux et al. 2001). Therefore, the use of such technologies that enhance the agriculture production is indispensable to feed the burgeoning population. The application of multistrain bacterial consortium over single inoculation could be an effective approach for reducing the harmful impact of stress on plant growth. Strains that have the ability to protect the plant from diseases through biocontrol mechanisms may also be included in the formulation. The efficacy of such strains may be enhanced by ACC-deaminase gene (Hao et al. 2007). Therefore, the application of such strains which have multitraits for growth promotion should be preferred for inoculant formulation. It is also necessary to understand the interactions between microbial consortium and plant system. Understanding of such interactions could be very effective for improving plant growth (Raja et al. 2006).

It has been seen that certain growth-promoting traits may interact with each other and have influence on plant growth. For example, in one of our studies (submitted for publication), the strain having high ACC-deaminase activity and low IAA and/or high ACC-deaminase and high IAA performed better compared to a strain having high IAA and low ACC-deaminase. Therefore, such aspects need further research so that most effective strains or combinations of strains can be selected. Other beneficial aspects of bacterial inoculation also need special attention. For example, the addition of ice-nucleating bacteria to agriculture has potential benefits of protecting crops from frosts dropping below freezing, which might contribute to a solution of the worldwide problem of starvation and chronic hunger. Therefore, the application of these bacteria could be an effective technology for enhancing plant growth at low temperature. Similarly, cyanide-producing bacteria can be used effectively for disease suppression. Certain Pseudomonas strains produce allelochemicals that can be used as bioherbicides to minimize the use of chemicals and therefore eliminate environmental hazards.