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5.1 Introduction

Alfalfa (Medicago sativa L.) is worldwide in distribution. It is the most important forage legume grown in the semiarid Argentinean Pampas because of the quality of the nutrients that it provides (Viglizzo 1995). Furthermore, its root system improves and conserves soil structure and consequently promotes soil fertility (Vance 1997). Soybean (Glycine max L.) cultivation is also continuously expanding in Argentina. In fact, its production has been accelerating during the last decade, making this country one of the main world exporters (Lattanzi 2002).

Interactions between plants and microorganisms and among rhizosphere microorganisms are largely unknown and available literature revealed that these interactions are complex and depend on multiple traits (Lugtenberg and Dekkers 1999). Stimulation of growth by plant growth-promoting rhizobacteria (PGPR) (Kloepper et al. 1989) has been attributed to an increase in the mobilization of insoluble nutrients and subsequent enhancement of nutrient uptake by plants (de Freitas et al. 1997; El-Komy 2005), production of plant growth regulators (Derylo and Skorupska 1993; Dubeikovsky et al. 1993) and suppression of deleterious soil bacteria and phytopathogenic fungi (Fridlender et al. 1993; Weller and Thomashow 1993; Liu et al. 1995). Several Pseudomonas strains display plant growth-promoting (PGP) activities and have been investigated due to their widespread distribution in soil, ability to colonize the rhizospheres of host plants, and capacity to produce a range of compounds that are antagonistic to a number of plant pathogens (Rodríguez and Pfender 1997; Ross et al. 2000).

Diversity studies of Pseudomonas spp. and their PGP potential is important not only for understanding their ecological role in the rhizosphere and the interaction with plants, but also for biotechnological applications (Berg et al. 2002).

Pseudomonas chlororaphis, Pseudomonas aureofaciens and Pseudomonas aurantiaca were considered as separate species until 1989, when P. aureofaciens was proposed as a later heterotypic synonym of P. chlororaphis with P. aurantiaca remaining as a separate species. More recently, Peix et al. (2007) reclassified P. aurantiaca as a synonym of P. chlororaphis based on analysis of the almost complete 16 S rRNA gene. These authors suggested the location of P. aurantiaca as a novel subspecies of P. chlororaphis.

P. aurantiaca SR1 (or P. chlororaphis subsps. aurantiaca SR1) strain was isolated from soybean rhizosphere in the area of Río Cuarto, Córdoba, Argentina, at the Universidad Nacional de Río Cuarto (UNRC) experimental field (33°07′ latitude S; 64°14′ longitude W; 421 m above sea level), from a typical sandy loam Hapludoll soil. This strain was initially classified as Pseudomonas aurantiaca by the BIOLOG (Biolog Inc., Hayward, CA) system (Rosas et al. 2001) and, more recently, by amplification and sequencing of a partial fragment from the 16 S rRNA gene.

5.2 Rhizosphere Colonization

The beneficial effect of the introduction in soil of a bacterium capable of promoting plant growth or exercising biological control of pathogens is related to its root colonization capacity (Benizri et al. 2001). It has been shown that P. chlororaphis mutants affected in root colonization are unable to eliminate the causal agent of the tomato foot and root rot disease (Chin-A-Woeng et al. 2000). On the other hand, coinoculation of P. fluorescens and Bradyrhizobium japonicum increased the colonization of rhizobia on soybean roots, nodule number and acetylene reduction activity (ARA) as reported by Chebotar et al. (2001). Moreover, addition of sterile spent medium of P. fluorescens increased the growth of B. japonicum in yeast mannitol broth (YMB, Vincent 1970), indicating that P. fluorescens may have released substances that increased the rhizobial populations.

The colonization of the soybean and alfalfa root system by P. aurantiaca SR1 is shown in Table 5.1. Mean comparisons were conducted using the least significant difference (LSD) test (P < 0.05). The density of adhering bacteria on soybean and alfalfa seeds was 5.8 × 105 CFU/seed and 1.2 × 105 CFU/seed, respectively. The tracking of the population density of P. aurantiaca SR1 was carried out by counting those colonies that expressed SR1’s characteristic orange pigment. Twenty-five days after seedling emergence, bacterial populations declined in both soybean and alfalfa root systems; however, the lower counts of P. aurantiaca SR1 suggested its establishment in the tested legume rhizosphere. The level of alfalfa roots colonization by P. aurantiaca SR1 was slightly lower than that observed in the same crop plant by Villacieros et al. (2003), who used P. fluorescens strain F113.

Table 5.1 Root colonization by Pseudomonas aurantiaca in soil at different days after planting
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In addition, P. aurantiaca SR1 possesses the capacity to colonize the root system of wheat and it behaves as an endophyte strain in several crops (Rosas et al. 2005; Carlier et al. 2008; Rosas et al. 2009).

5.3 Growth Promotion Mechanisms

Several direct and indirect mechanisms to promote plant growth are present in P. aurantiaca SR1 as follows:

5.3.1 Indole Acetic Acid Production

Phytohormones synthesis is the most important direct PGP trait, besides biological nitrogen fixation. There are reports that suggest that PGPR synthesize several different phytohormones that enhance various developmental stages of plants and most of the attention has been focused on the role of auxins. Auxins are a class of plant hormones; the most common and well characterized is indole-3-acetic acid (IAA), which is known to stimulate both rapid and long term responses in plants.

IAA produced by microbes colonizing the seed or root surfaces is proposed to act in conjunction with endogenous IAA to stimulate cell proliferation and/or elongation and enhance the host’s uptake of minerals and nutrients from the soil (Patten and Glick 2002; Suzuki et al. 2003). The growth of plants treated with IAA-secreting PGPR is affected by the amount of IAA that the bacterium produces. Thus, PGPR facilitate plant growth by altering the hormonal balance within the affected plant (Glick 1995; Vessey 2003; Asghar et al. 2004; Kang et al. 2006).

Figure 5.1 shows the growth curve and IAA production by P. aurantiaca SR1. The concentration of IAA in the supernatant began to increase consistently after 14 h of incubation and reached its highest level (11.7 μg/ml) at the end of the exponential growth phase or early stationary phase (24 h). Thereafter, there was a decrease in IAA synthesis by P. aurantiaca SR1.

Fig. 5.1
figure 1

Bacterial growth and IAA production by P. aurantiaca SR1 in 20% TSB medium. Culture was stopped after reaching the stationary phase

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In a recent study, Mehnaz et al. (2009) did not detect IAA production by P. aurantiaca PB-St2, a PGPR isolated from sugarcane stem. Our observations are in good agreement with other studies that suggested induction of IAA production during the stationary phase of culture, probably because of the induction of key enzymes involved in IAA biosynthesis (Oberhansli et al. 1990; Patten and Glick 2002; Ayyadurai et al. 2006).

5.3.2 Biocontrol Activity

Certain fluorescent pseudomonads are known to promote plant growth by producing metabolites able to inhibit bacteria and fungi that are deleterious to plants (Keel et al. 1990; Maurhofer et al. 1991; Hill et al. 1994). Some of these disease-suppressive antibiotic compounds have been chemically characterized and include phenazine-1-carboxylic acid (PCA), pyrrolnitrin, pyoluteorin, 2,5-dialkylresorcinol and 2,4-diacetylphloroglucinol (DAPG) (Thomashow et al. 1990; Keel et al. 1992; Kraus and Loper 1995; Nielsen et al. 1998; Dwivedi and Johri 2003). The results obtained by both the application of molecular techniques and direct isolation methods have demonstrated unequivocally that these antibiotics are produced in the spermosphere and the rhizosphere and are very important in suppressing soilborne plant pathogens (Bonsall et al. 1997; Ross et al. 2000; de Leij et al. 2002). DAPG produced by fluorescent pseudomonads is considered as a major determinant in the biocontrol activity of PGPR. The broad-spectrum antibiotic DAPG has wide antifungal, antibacterial, antihelminthic, nematicidal and phytotoxic activity (Cronin et al. 1997; Raaijmakers et al. 2002). Information of the production of antifungal metabolites by P. aurantiaca strains are scarce. Esipov et al. (1975) describe the production of “a new antibiotically active fluoroglucide” in P. aurantiaca. Then, a possible route of synthesis of 2,5-Dialkylresorcinol in P. aurantiaca was proposed by Nowak-Thompson et al. (2003). Mandryk et al. (2007) reported new antimicrobial compounds produced by P. aurantiaca, having molecular formulas of C18H36NO (molecular mass 282.8; antibacterial activity) and C20H31O3 (molecular mass 319.3; antifungal activity). Reports about PCA production by P. aurantiaca could not be found, although Peix et al. (2007) used the production of PCA as a characteristic feature of P. aureofaciens and P. aurantiaca when they reclassified these species. A new method for obtaining regulatory mutants of P. aurantiaca capable of overproduction of phenazine antibiotics was described by Feklistova and Maksimova (2008).

Quorum sensing is the major way by which many bacteria regulate production of antifungal factors and N-hexanoyl homoserine lactone (HHL) is a compound that indicates the presence of a quorum sensing mechanism. Feklistova and Maksimova (2008) and Mehnaz et al. (2009) reported the production of N-HHL by P. aurantiaca B-162 and P. aurantiaca PB-St2, respectively.

P. aurantiaca SR1 produces an orange pigment associated with a strong in vitro antifungal activity against different pathogenic fungi such as Macrophomina phaseolina, Rhizoctonia solani, Pythium spp., Sclerotinia sclerotiorum, Sclerotium rolfsii, Fusarium spp. and Alternaria spp. (Rosas et al. 2001). The antifungal compound is secreted by the bacterium when culture media such as tryptic soy agar, nutrient agar or minimum media supplemented with tryptone or peptone are used. Among the tested carbon sources, mannitol and saccharose have been found to induce pigment production while glucose acts as a repressor (Rovera et al. 2000).

In our study, an antifungal compound was isolated from P. aurantiaca SR1 grown on plates containing 25% tryptic soy agar (TSA-Britania Laboratory, Argentina), incubated at 30°C for 5 days. The antibiotic produced by P. aurantiaca SR1 was confirmed as DAPG using thin-layer chromatography (TLC), high performance liquid chromatography (HPLC) and spectrometric techniques, and tested for antibiosis toward the phytopathogenic fungi M. phaseolina.

To purify the antifungal compound, a 0.5 ml volume of crude extract was spotted on thin-layer plates coated with silica gel 60, 20 by 20 cm (Merck) and developed in chloroform/methanol (98:2 vol/vol). The thin-layer chromatography of the isolated compound was compared with standard DAPG (Toronto Research Chemicals Cat. No. 10365500). The fluorescing and absorbing bands developed on plates were observed under long- and short-wavelength UV light. All bands and blank areas were separately removed from the plates and eluted with 100% acetone (70 ml). The silica residue was removed by centrifugation and the supernatant was transferred to a second set of microcentrifuge tubes. Each fraction was concentrated by evaporating the acetone and tested in vitro for antifungal activity.

Aliquots of 0.1 ml of the acetone elutes were placed directly onto TSA plates and were maintained under a laminar air flow until the acetone evaporated. Potato dextrose agar (PDA) plugs (5 mm) of M. phaseolina were placed in the center of the plates and incubated at 28°C for 5–7 days. The control consisted of a M. phaseolina PDA plug onto the surface of plates containing TSA medium after 0.1 ml of acetone had evaporated. The spots were identified by irradiating plates with UV light (350 and 245 nm). Only one spot showed antifungal activity (Rf: 0.35 in chloroform/methanol 98:2 vol/vol) (Table 5.2). A similar behavior was observed using chloroform:acetone (2:1) as eluant, with a Rf of 0.54. The active fraction (dissolved in methanol) was further analyzed by HPLC (Fig. 5.2). The isolated active compound was also analyzed by spectrometric techniques. UV–visible absorption spectra were recorded on a Shimadzu UV-2401PC spectrometer. 1HNMR spectra were recorded on a Varian Gemini spectrometer at 300 MHz. Mass spectra were taken with a Varian Matt 312 operating in E.I. mode at 70 eV. IR spectra were recorded with a Nicolet Impact 400 FT-IR.

Table 5.2 Relative mobility (Rf) and antifungal activity against Macrophomina phaseolina of each band obtained from the TLC from Pseudomonas aurantiaca SR1 culture media
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Fig. 5.2
figure 2

HPLC chromatogram of DAPG (retention time 14.66 min) produced by P. aurantiaca SR1

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The UV–visible absorption spectrum of the active compound in methanol showed two bands at λ 267 and 328 nm. These bands are characteristic of π → π* and n → π* transitions of aromatic rings bearing a carbonyl chromophore, which is characteristic for DAPG (Bonsall et al. 1997).

The Fourier transform infrared (FTIR) spectrum showed the presence of a broad absorption at 3,421 cm−1, typically corresponding to a bound OH stretching, and bands at 2,923 and 2,852 cm−1, which can be attributed to the C–H stretching. A strong absorption band was observed at 1,689 cm−1 that is recognized as a stretching of the carbonyl group. Also, medium sharp bands were shown at 1,463 and 1,384 cm−1 and a strong band centered at 1,074 cm−1. Thus, the IR spectrum of the active sample was in agreement with the DAPG spectrum (Fig. 5.3). The mass spectrum and fragmentation of the isolated compound showed a match with that reported for DAPG, with peaks at 177, 195 (100%), 210 (M). Studies of 1HNMR in acetone confirmed the DAPG structure.

Fig. 5.3
figure 3

FT-IR spectrum of DAPG produced by P. aurantiaca SR1

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P. aurantiaca SR1, formulated as an inoculant that contains 2.4 × 109 CFU/g peat, was able to produce the antifungal compound in rhizosphere soil. Genetic studies demonstrated the role of DAPG in biological control, which is complemented by direct isolation of DAPG from the rhizosphere (Bonsall et al. 1997). This effect was deduced from the antifungal activity of the residue resulting from the extraction made with organic solvents from soybean and alfalfa root systems inoculated. The TLC that was performed with the extract showed the presence of an active “band” with a value of Rf: 0.35, visualized under an UV light illuminator. The chromatography revealed the same profile as that obtained in the in vitro assays. The purification of the compound by HPLC revealed a similar profile to that of the compound extracted from the bacterial growth in culture medium and it is comparable with the pure standard. The TLC control (soybean and alfalfa roots from uninoculated seeds) did not present the “band” with an Rf of 0.35, indicating that other native microorganisms did not produce the compound. The antifungal compound was still detected 25 days after seedling. This detection had correlation with P. aurantiaca SR1 viability in the rhizosphere.

In other studies, we showed that P. aurantiaca SR1 produces siderophores and hydrogen cyanide (HCN). One way that microorganisms can prevent the proliferation of phytopathogens, and thereby facilitate plant growth, is through the production and secretion of low molecular mass iron-binding molecules (siderophores) with a very high affinity for iron. These bind most of the Fe+3 that is available in the rhizosphere, and, as a result, effectively prevent any pathogens in the immediate vicinity from proliferating because of lack of iron (Glick 1995; Rachid and Ahmed 2005; Viswanathan and Samiyappan 2007). The bacterium that originally synthesized the siderophores takes up the iron-siderophore complex by using a receptor that is specific for the complex (O’Sullivan and O’Gara 1992). Furthermore, some plants have mechanisms for binding the bacterial iron-siderophore complex and then releasing the iron so that it can be used by the plant (Wang et al. 1993). We tested the production of siderophores by P. aurantiaca SR1 using the Chrome azurol S method described by Alexander and Zuberer (1991). Plates were incubated at 28°C for 5 days and colonies exhibiting an orange halo were considered to be siderophore producers.

Certain PGPR strains produce volatile antibiotics, of which HCN is the most important; this compound inhibits the cytochrome oxidase of many organisms. The producing strains possess an alternate cyanide-resistant cytochrome oxidase and are relatively insensitive to HCN (Voisard et al. 1989; Rudrappa and Baiss 2008). HCN production by P. aurantiaca SR1 was determined as described by Bakker and Schippers (1987) in 10% TSA amended with glycine (4.4 g/l). A change in color, from yellow to orange-brown, of filter paper impregnated with 0.5% picric acid – 2% sodium carbonate indicated production of cyanide.

5.4 Legume Responses to Inoculation with P. aurantiaca SR1

Bacteria belonging to the genera Rhizobium, Bradyrhizobium, Mesorhizobium, Sinorhizobium and Azorhizobium can interact with leguminous plant roots to form nodules, which function as sites for atmospheric nitrogen fixation (Relic et al. 1994). In addition, bacteria that promote nodulation of legumes by rhizobia are referred to as nodulation-promoting rhizobacteria (NPR) (Kloepper et al. 1988). However, these NPR include diverse groups of microorganisms such as Azospirillum (Hamaoui et al. 2001), Azotobacter (Tilak et al. 2006), Pseudomonas (Villacieros et al. 2003), Bacillus (Bai et al. 2003), Paenibacillus (Figueiredo et al. 2008), Streptomyces (Samac et al. 2003) and Serratia (Lucas García et al. 2004). Coinoculation studies with PGPR and Rhizobium/Bradyrhizobium spp. have been shown to increase root and shoot weight, plant vigor, N2 fixation and grain yield in various legumes including common bean (Grimes and Mount 1984), green gram (Sindhu et al. 1999), pea (Bolton et al. 1990) and soybean (Dashti et al. 1998; Rosas et al. 2006). Knight and Langston-Unkefer (1988) found that inoculation of nodulating alfalfa roots by a toxin-releasing Pseudomonas syringae pv. tabaci significantly increased plant growth, nitrogenase activity, nodule number, total nodule weight and nitrogen yield under controlled growth conditions. In other study, Pandey and Maheshwari (2007) reported cooperation between two rhizobacteria belonging to two distant genera, Burkholderia sp. and S. meliloti. They are rhizospheric isolates with the ability to produce IAA and solubilize inorganic phosphate. The two strains were tested on Cajanus cajan in sterile soil. Single inoculation of either resulted in significant increase in seedling length and weight, but an exceptional increase in seedling growth was recorded when coinoculating.

The P. aurantiaca and S. meliloti strains were maintained on 20% TSA medium. The coexistence of P. aurantiaca SR1 with S. meliloti 3Doh13 (Rosas et al. 2006) and B. japonicum E109 (Instituto Nacional de Tecnología Agropecuaria, Argentina) was tested. For this, P. aurantiaca SR1 was streaked along one side of Petri plates containing 20% TSA and parallel streaks of the respective rhizobia strains were made on the other side. Both strains were separated by a distance of 5 mm. Rhizobia was inoculated at the same time with P. aurantiaca and when P. aurantiaca growth was evident (normally after 48 h). Inhibition of rhizobial growth was measured after 5 days at 28°C. In a further experiment, we assessed the effects of the P. aurantiaca SR1 culture supernatant on S. meliloti 3Doh13 and B. japonicum E109 growth. P. aurantiaca SR1 was grown for 21 h in 20% TSB and the liquid culture was centrifuged at 6,000 rpm for 10 min at 4°C. The supernatant was filter-sterilized through a 0.22-μm Whatman filter and aliquots (0.2 ml) were placed directly onto 20% TSA agar plates. Then, S. meliloti 3Doh13 was streaked on this medium and it was incubated for 5 days at 28°C.

P. aurantiaca SR1 did not show any inhibitory effect neither on S. meliloti 3Doh13 nor on B. japonicum E109 in the coexistence assays performed at different times. Moreover, the exposure to the culture filtrate of strain SR1 did not affect the rhizobial growth.

Also, the combined effect of P. aurantiaca SR1 and S. meliloti 3Doh13 on growth and nodulation of alfalfa and P. aurantiaca SR1 and B. japonicum E109 on soybean growth under aseptic conditions was determined in this study. Rhizobia were routinely grown on yeast extract mannitol (YEM) solid medium (Vincent 1970) and P. aurantiaca SR1 on TSB medium. Bacterial cultures were raised to a level of 7×108 cfu ml−1 for S. meliloti 3Doh13, 1×109 cfu ml−1 for B. japonicum E109 and 4.5×108 cfu ml−1 for P. aurantiaca SR1.

First, soybean seeds were disinfected for 20 min with 0.4% calcium hypochlorite solution and alfalfa seeds were scarified by shaking for 15 min in concentrated sulfuric acid; then, seeds were disinfected with 70% ethanol for 3 min and washed with repeated changes of sterile, distilled water (Andrés et al. 1998; Rosas et al. 2006).

Second, seeds were treated with a culture of each rhizobial strain and P. aurantiaca by mixing culture broths of both the inoculant strains in a 1:1 ratio (v/v). One gram of alfalfa seeds and ten grams of soybean seeds were inoculated with 2 ml of the single or mixed bacterial suspension, and average populations of bacteria on inoculated seeds were in the order of 1×105 cfu alfalfa seed−1 and 3 ×105 cfu soybean seed−1, determined by dilution plate technique. Seeds (at least five seeds per treatment) were placed in tubes containing 10 ml of 0.1 M phosphate buffer (pH 7.4). Tubes were vortexed for 2 min and serial dilutions plated on 20% TSA and incubated at 28°C. Colonies were counted after 72 h.

Eight alfalfa or four soybean bacterized seeds were sown in plastic pots (15 cm diameter) filled with autoclaved perlite/sand (2:1). Plants were kept in a chamber under controlled conditions: 16 h light at 28°C and 8 h dark at 16°C, and a light intensity of 220 μE m−2 s−1. Pots were watered alternately with sterile, distilled water and an N-free Jensen solution (Vincent 1970). N-control was watered in the same manner, but with the addition of 0.5% KNO3 l−1 to the original Jensen solution. Each independent treatment was replicated three times.

Forty-five days after sowing, plants were harvested in order to evaluate shoot length, root length, shoot dry weight, root dry weight, nodule number and N content (only in alfalfa shoot). All samples were oven dried (at 70–80°C) for 48 h and weighed. The total N content was determined by a modified Kjeldahl method (Baker and Thompson 1992) by treating 100 mg sample with 1.25 g of catalytic mix (potassium sulfate:mercuric oxide, in a 24:1 relation) and 2.5 ml of conc. H2SO4. The mixture was digested for 40 min. Then, the ammonia liberated by alkalinization with NaOH solution was separated by distillation and collectical (as ammonium borate) in a 4% boric acid. Ammonium was determined by titration with a 0.02 N HCl. An automatic analyzer of N (Kjeltec Auto 1030, Tecator, Sweden) was used.

The composite inoculation of P. aurantiaca with S. meliloti increased the plant length, shoot and root dry weights and total N content in comparison to inoculation with S. meliloti alone or uninoculated control (Table 5.3). The dry matter accumulation in root and shoot and N-content in aerial parts increased significantly (p ≤ 0.05) with respect to the N-control.

Table 5.3 Effect of coinoculation of alfalfa with Pseudomonas aurantiaca SR1 and Sinorhizobium meliloti 3Doh13 on seedling growth and symbiotic parameters under sterile conditions
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Nodulation on root systems of alfalfa plants following coinoculation with S. meliloti 3Doh13 and P. aurantiaca SR1 was increased by 34% compared to single inoculation. However, this did not correlate with the dry weight of nodules. The shoot and root dry weights of coinoculated plants were 1.09 and 1.24 times higher, respectively, than those observed for S. meliloti-inoculated alfalfa plants and 1.64 and 1.7 times with respect to uninoculated controls. The coinoculation of P. aurantiaca SR1 and S. meliloti 3Doh13 significantly enhanced nodulation at 45 days of growth.

The coinoculation of B. japonicum E109 – P. aurantiaca SR1 promoted an increase in shoot and root dry biomass, but not in the number of nodules, compared with single inoculation with B. japonicum (Table 5.4). The shoot and root dry weight of coinoculated plants were 1.24 and 1.35 times, respectively, greater than that of B. japonicum-inoculated soybean and 1.85 and 1.93 times with respect to uninoculated controls. The coinoculation of seeds with antibiotic-producing bacteria and rhizobia strains resistant to these compounds were proposed as a way of reducing the detrimental interactions in the rhizosphere and thereby enhancing the colonization and nodulation of legume roots by rhizobia (Li and Alexander 1988).

Table 5.4 Effect of coinoculation of soybean with Pseudomonas aurantiaca SR1 and Bradyrhizobium japonicum E109 on seedling growth and symbiotic parameters under sterile conditions
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5.5 Conclusion

Extensive studies have demonstrated that PGPRs could have an important role in agriculture, improving crop productivity. In recent years, emphasis on the use of two or more microorganisms has been made with the aim of maximizing beneficial plant growth responses. It is important to identify the best strains of beneficial microbes, elucidate the mechanisms involved in growth promotion, to verify their compatibility and combined efficiency, both in vitro and in vivo, and employ such a combined inoculum in a real agricultural situation.

In this chapter, we describe the indole-3-acetic acid production by P. aurantiaca SR1 as well as the isolation and purification of a metabolite involved in the antibiosis capacity of this strain. The chemical characterization revealed that the cited compound is DAPG and its production was qualitatively detected in the in vitro assays and in the rhizosphere.

The P. aurantiaca strain was able to establish in the rhizosphere environment of the crop from which it was isolated (soybean) and also showed the capacity of colonizing the root system of alfalfa. It is interesting to note that although P. aurantiaca SR1 exerts a biocontrol effect on plant pathogenic fungi, probably due to the production of the secondary metabolites DAPG, HCN and siderophores, it does not interfere with rhizobial strains.

To summarize, we have shown that P. aurantiaca SR1 strain acted synergistically with S. meliloti 3Doh13 and B. japonicum E109 in promoting growth and symbiotic parameters of alfalfa and soybean under sterile conditions. It suggests a potential use of combinations of this strain and rhizobia in improving growth of these legumes. Our results are a contribution in the selection of bacterial strains for the formulation of new inoculants.