Introduction

Charcoal rot disease, caused by the fungus Macrophomina phaseolina (Tassi) Goidanich, is the most common root disease of soybeans (Wrather et al. 1997). It was previously considered to be a secondary pathogen since its effects were mainly observed at the final stages of the crop; however the disease can appear at any stage of plant growth (Machado 1987). Soybean [Glycine max L. Merril] is a significant source of income for farmers in many countries. The major soybean-producing states in India are Madhya Pradesh, Maharastra, Rajasthan, Gujarat, and Uttar Pradesh. About 90% of India’s soybean crop is planted during July, and the remainder is planted during the spring only in Madhya Pradesh. Factors that reduce soybean production, such as diseases, insects, weeds, and weather, can influence the economic or general welfare of many countries and individuals. The fungus is considered to be a soil-borne pathogen and is able to infect several plants viz., cotton (Gossypium hirsutum L.), corn (Zea mays L.), sunflower (Helianthus annuus L.), and sorghum [Sorghum bicolor (L.) Moench]. Because of the wide host range of M. phaseolina and the long survival times of the microsclerotia, crop rotation would probably have little benefit in reducing charcoal rot (Almeida et al. 2003).

Despite inconsistency in field performance, biological control is considered as an alternative or a supplemental way of reducing root diseases in agro-ecosystem (Sharma and Johri 2003). The widely recognized mechanism of biocontrol mediated by plant growth-promoting rhizobacteria (PGPR) is competition for an ecological niche/substrate, production of inhibitory allelochemicals, and induction of systemic resistance (ISR) in host plants to a broad spectrum of pathogens (Hass et al. 2002). Induced resistance is a state of enhanced defensive capacity developed by a plant when appropriately stimulated (van Loon et al. 1998). A large number of enzymes have been associated with induced resistance that includes peroxidase (EC 1.11.1.7), phenylalanine ammonia-lyase (EC 4.3.1.5), lipoxygenase (EC 1.13.11.12), β-1,3-glucanase (EC 3.2.1.6), and chitinase (EC 3.2.1.14) (Yan et al. 2002).

The objectives of this study were to evaluate plant growth-promotion activities of rhizobacterial strains for soybean plant against charcoal rot pathogen and their molecular characterization by using Amplified rDNA restriction analysis.

Materials and methods

Fungal culture and spore suspension

An isolated culture of anamorphic ascomycetous pathogen, M. phaseolina, was maintained on potato/dextrose agar. A suspension of 108 microsclerotia per ml in 10 mM MgSO4 was prepared from a culture grown for 7 days at 25°C on PDA. This sclerotial suspension (100 μl/g) was added to soil and allowed to germinate and grow for 2 days at 25°C. The final density of colony-forming units (cfu) in soil was determined by plating dilutions of the soil suspension on PDA: 1.2 × 106 microsclerotia/ml was used for inoculation.

Bacterial cultures

The two isolates employed in this study were selected among 189 bacterial isolates screened on the basis of in vitro antibiosis dual plate assay against test pathogen (M. phaseolina) together with important plant growth promoting properties viz., P-solubilization, IAA, secretion of hydrolytic enzymes and siderophore production. In this study an ISR producing strain (Pathak et al. 2004; Sharma et al. 2007b), Pseudomonas sp. strain GRP3, which was recovered from the rhizosphere of soybean that showed plant growth-promoting properties (Sharma et al. 2003, 2007a), was considered as reference strain (Table 1).

Table 1 Plant growth-promotion (PGP) and antagonistic properties of bacterial isolates
Full size table

Seed microbiolization

Cultures of FPT721 and A5F and Pseudomonas sp. strain GRP3, grown for 24 h, were used to treat soybean seeds. Seed ‘microbiolization’ was performed according to Sharma et al. (2003). Seeds were surface-sterilized with 0.1% HgCl2 (acidified with H2SO4) and washed repeatedly with sterile triple-distilled water prior to sowing. Overnight grown bacterial cultures were centrifuged at 11,000×g for 20 min and the pellets were washed with 10 mM phosphate buffer saline (PBS, pH 7.2). Pellets were resuspended in PBS and the OD540 adjusted to 0.2 (108 cfu ml−1). Surface-sterilized seeds were placed in PBS containing 0.1% carboxymethylcellulose (CMC) as a binder for 1 h, followed by drying under air. Sterile conditions were maintained throughout. Seeds immersed only in water (non-microbiolized) treated as control.

Effect of bacterial strains on disease reduction

Microbiolized soybean seeds were planted in plastic pots containing unsterilized soil and maintained in the greenhouse (25 ± 2°C). Seven days after planting, the plants were inoculated by spraying with the microsclerotial suspension (1.2 × 106 microsclerotia/ml) in the rhizosphere area. Each treatment was run with five replications and each replication consisted of four plants per pot. The uninoculated plants were sprayed with water. Thus four treatments were evaluated for each bacterial strain (FPT721 and A5F and Pseudomonas sp. strain GRP3): inoculated (T1) and uninoculated (T2) plants from microbiolized seeds; inoculated (T3) and uninoculated (T4 control) plants from non-microbiolized seeds. Disease severity was assessed and analyzed statistically at the time of nodulation (on 45th day). The root rot severity index was recorded at the end of the experiments from two independent assessment and the scores were averaged for statistical analysis.

Chlorophyll estimation

Chlorophyll estimation was performed according to Hiscox and Israelstam (1979). Fresh leaves were removed and chopped into small pieces and 7 ml dimethylsulfoxide (DMSO) added to 100 mg tissue. The tubes containing leaf pieces and DMSO were incubated at 65°C for 3 h; extract was made up to 10 ml with DMSO and the A645 and A663 were measured employing DMSO as blank.

Enzyme activity detection bioassay

Enzyme assays were performed on leaf extracts. Soybean leaves were collected seven days after pathogen inoculation and stored at 4°C until plant extracts were prepared according to Lanna et al. (1996). Leaves were collected from three plants for each repetition in each treatment. Three repetitions per treatments were employed for each enzyme analyzed. Leaf tissue extracts of about 1 g fresh wt were ground in a mortar and pestle containing liquid N2. The powder was macerated for 3 ml 50 mM sodium phosphate buffer, pH 6.5, containing 1% polyvinylpyrrolidone and 1 mM PMSF (extraction buffer), and then centrifuged at 15,000×g for 30 min at 4°C. The supernatants were kept on ice and used for determination of total protein, lipoxygenase (LOX), peroxidase (POD), and phenylalanine ammonia-lyase (PAL) activities. One unit represents the amount of enzyme that produces one μmol product per min.

Protein concentration

Total soluble protein content of the samples was estimated by using the Lowry method with BSA as standard.

LOX, PAL and POD activities

LOX activity was detected employing method described by Axelrod et al. (1981), which is based on the increase in A234 that result from the conjugated double bond system in the hydroperoxide produced from the substrate, linoleic acid. The reaction mixture consisted of 3 ml 50 mM sodium phosphate buffer, pH 6.0, 60 μl linoleic acid (10 mM), and 30 μl plant extract. Absorbance readings were made every 30 s for 10.5 min at 25°C. A value of 25,000 M−1 cm−1 was employed for the molar extinction coefficient (є) of linoleic acid. LOX activity was expressed as μmol product formed min−1 mg protein−1.

PAL activity was determined according to Pascholati et al. (1986). Conversion of l-phenylalanine to cinnamic acid at 37°C was followed at 290 nm reading every 30 s for 5.5 min after the start of the reaction. The reaction mixture contained 30 μl leaf extract and 3 ml 0.2% phenylalanine; the reference used 30 μl extraction buffer. PAL activity was expressed as μmol cinnamic acid min−1 mg protein−1.

POD activity was determined (Allain et al. 1974) in the clear extract by monitoring the increase in A436 at 30°C. The reaction mixture consisted of 3 μl leaf extract and 3057 μl 20.1 mM guaiacol, 12.3 mM H2O2 and 10 mM sodium phosphate buffer pH 6). Absorbance readings were taken every 30 s for 15.5 min. Enzyme activity was calculated employing molar extinction coefficient (є) of 2.47 mM−1 cm−1. POD activity was expressed as μmol H2O2 min−1 mg protein−1.

Statistical analysis

The effects of the treatments on plant growth and disease suppression was analyzed using multiple comparisons of means according to Duncan’s multiple range tests (DMRT) at the 5% level. Critical deviation (CD) and standard error of mean (SEM) for each treatment was calculated.

Recovery of genomic DNA and PCR amplification of 16S rDNA

Total DNA from bacterial isolates was prepared according to the procedure of Bazzicalupo and Fani (1994). The amplified 16S rRNA gene was obtained from each bacterial isolate by PCR amplification employing the eubacterial universal primers (Weisburg et al. 1991); fDI (5′-AGAGTTTGATCCTGG-3′) and rP2 (5′-TACCTTGTTACGACTT-3′) which was targeted at universally conserved regions and permitted amplification of approximately 1,500-bp fragment. PCR amplification was carried out in a thermocycler. Reaction tubes contained 25 ng (5 μl) DNA extract, 1 U Taq polymerase (Genei, India), 1 X buffer (10 mM Tris/HCl [pH 9.0], 1.5 mM MgCl2, 500 mM KCl), 10 mM dNTPs and 0.25 mM of each primer. Initial DNA denaturation and enzyme activation steps were performed at 95°C for 7 min, followed by 25 cycles of denaturation at 94°C for 1 min, annealing at 51°C for 1 min and extension at 72°C for 1 min, and a final extension at 72°C for 10 min. The presence and yield of specific PCR product (16S rRNA gene) was monitored on 0.8% agarose (wt/vol.); gel electrophoresis was carried out at 100 V for 30 min in 1 × Tris–acetate–EDTA buffer and visualization by ethidium bromide staining and viewing on a UV transilluminator (GelDoc, Vilber Lourmat, CN08, France).

Amplified rDNA restriction analysis (ARDRA)

16S rRNA amplification product of bacterial genomic DNA was digested with HaeIII and TaqI. 15 μl of (120 μg) 16S rDNA amplification product was digested with restriction endonuclease. For 30 μl reaction mixture, added the following: restriction enzyme (10 U), 1 U (Genei, India); restriction buffer (10 X), 1 X (Genei, India); 16S rDNA amplicon 120 μg, and Milli-Q water. The reaction mixture was incubated at 37°C for HaeIII, and at 65°C for TaqI for 4 h. Restriction product was resolved on 2.5% agarose gel in 1 × TAE at 60 V for 5 h and visualized on a UV transilluminator.

Phylogenetic analysis

Phylogenetic tree was constructed on the basis of restriction fragment pattern of the amplicon of 16S rDNA using the software NTSYS pc version 2.02i. The clustering was done using Jaccard’s similarity coefficient based on presence and absence of band ignoring their intensities. The dendrogram was constructed employing the unweighted pair group means average (UPGMA) methods according to Sneath and Sokal (1973).

Results

Plant growth promotion in greenhouse bioassay

Plant–microbe interactions play an important role in soil and plant health. In the artificial (greenhouse) trial, strain FPT721 showed the largest increase in shoot length and total chlorophyll content for treatment T2 compared to other treatments. A similar result was obtained for strain GRP3 compared to strain A5F. The plants which were inoculated with pathogen alone (T3) showed reduction in shoot length and total chlorophyll content compared to control (T4) (Table 2). In addition, strain FPT721 afforded the greatest protection against pathogen compared to strain A5F and GRP3 (Table 2). Maximum root-rot severity index was observed for the strain A5F (Table 2).

Table 2 Effect of promising PGP and antagonistic isolate on root-rot index, shoot length and chlorophyll content of soybean under artificial (greenhouse) infestation, on 45 days after sowing
Full size table

Bacterial treatments change in LOX, PAL and POD activity

Higher LOX, PAL and POD activity was observed in soybean plants inoculated with pathogen (treatments T1 and T3) as seen in Table 3. Plants grown from microbiolized seeds presented the highest LOX activity (T1) compared to control treatment (T4). The presence of the pathogen/rhizobacterium alone resulted in similar increase in enzymes activity. Strain FPT721 and GRP3 exhibited maximum increase in LOX, PAL and POD activity compared to strain A5F (Table 3).

Table 3 Effect of promising PGP and antagonistic isolate on LOX, PAL and POD activity in soybean under artificial (greenhouse) infestation, on 45 days of sowing
Full size table

Amplified rRNA restriction analysis

The amplification of 16S rRNA of the two selected isolates and reference strain GRP3 along with standard Pseudomonas fluorescens biovar. I, III, and IV (ATCC 13525, ATCC 17400 and ATCC 12983, respectively), Sinorhizobium meliloti 102F34, Mesorhizobium ciceri G-3-97, Bradyrhizobium japonicum SB102 and Rhizobium gallicum R602 SPT, each generated a band of 1 kb. Restriction with HaeIII yielded a banding pattern which was comparable to that obtained with Taq1. Restriction digestion of the amplicons with HaeIII and Taq1 resulted in generation of fragment of size, 400-1000 bp, varying from two to six in number (Fig. 1a, b). A composite ARDRA profile generated with two restriction endonucleases (HaeIII and TaqI) indicated that the three isolates and standard reference bacteria grouped into three clusters (cluster 1 contains isolate A5F; cluster 2 contain P. fluorescens biovars I, III, and IV, and strain GRP3; cluster 3 contain Sino-, Meso-, Brady-, and Rhizobium along with strain FPT721) and indicated strains A5F, FPT721 and strain GRP3 made distinct group with standards. Strain GRP3 showed 58% similarity with P. fluorescens biovars I, III, and IV whereas strain FPT721 exhibited 45% similarity with Sinorhizobium and Mesorhizobium. Strain A5F was least similar (24%) to other two clusters and was present on an entirely separate lineage (Fig. 2).

Fig. 1
figure 1

Molecular fingerprinting analysis of bacterial isolates. a, b demonstrate ARDRA pattern of PCR amplified 16S rDNA with restriction endonucleases i.e., HaeIII and Taq1. Lane M Marker; Lane 1 A5F; Lane 2 P. fluorescens bv. IV; Lane 3 P. fluorescens bv. I; Lane 4 P. fluorescens bv. III; Lane 5 GRP3; Lane 6 FPT721; Lane 7 Bradyrhizobium strain SB102; Lane 8 Rhizobium strain R602SPT; Lane 9 Mesorhizobium strain G-3-97; Lane 10 Sinorhizobium strain 102F34

Full size image
Fig. 2
figure 2

Phylogenetic tree based on ARDRA pattern of PCR amplified 16S rDNA with two restriction endonucleases i.e., HaeIII and Taq1 showing relatedness among the isolates

Full size image

Discussion

We selected two rhizobacterial isolates along with reference strain Pseudomonas sp. strain GRP3, which were screened on the basis of in vitro antibiosis assays against the test pathogen M. phaseolina, together with other important plant microbe interaction properties such as P-solubilization and siderophore production. Kloepper (1991) has also suggested the use of rapid screening techniques for the selection of effective isolates. In the bacterial selection process, a similar emphasis was also given (Table 1). The production of siderophores occurs only under iron-limited conditions. Such conditions are likely to prevail in the rhizosphere, and competition for iron through the production of siderophore is one of the mechanisms of bacterial antagonism against soilborne pathogens. Thus, siderophore production by specific ISR-eliciting rhizobacteria can play a dual role in disease suppression by depriving resident pathogens from iron locally and by inducing resistance in the plant systemically. High-affinity iron uptake systems of the PGPRs that have promiscuous siderophores may interfere with iron nutrition of many pathogenic bacteria and fungi leading to their suppression (Sharma et al. 2003). Sharma et al. (2003) described the role of siderophores which is one of the determinants of ISR in effecting plant nutrition wherein they overcome problem of iron non availability in calcareous soils by incorporation of siderophore producing strains of fluorescent pseudomonads (FLPs). The sidrophore-producing bacterium, Pseudomonas strain GRP3, was employed in a pot experiment to assess the role of microbial siderophores in the iron nutrition of mung bean employing Fe-citrate, Fe-EDTA, and Fe(OH)3 in different concentrations. The plant showed a reduction of chlorotic symptoms and enhanced chlorophyll level in bacterized plant. Bacterization with GRP3 increased peroxidase activity and lowered catalase activity in roots. There was also a significant increase in total and physiologically available iron. Such siderophores producing systems have the potential of improving iron availability to plants and a reduction of fertilizer usage. Sharma et al. (2007c) have described molecular characterization of rhamnolipid that recovered from Pseudomonas sp. strain GRP3, considered as an inducing determinant of biocontrol activity. Since rhamnolipids are involved in the lysis of the plasma membrane of zoosporic fungi, Pythium and Phytophthora strains, application of such rhamnolipid producing rhizobacteria could facilitate control of damping-off of plant pathogens.

Genetic profiles generated by ARDRA, strains GRP3 and FPT721 were predictive of genus Pseudomonas and Rhizobium, respectively, whereas strain A5F was different. Because ARDRA detects interspecies and inter strain as well as interoperon variability and enables a relatively fast multiple strain analysis per taxon, this technique is appropriate to obtain indicative phylogenetic and taxonomic information. This information can be used to select strains for further detailed taxonomic studies. ARDRA fingerprinting also allows the construction of a database for identification purposes (Sneath and Sokal 1973).

PGP bacteria-triggered ISR fortifies plant cell wall strength and alters host physiology and metabolic responses, leading to an enhanced synthesis of plant defense chemicals upon challenge by pathogens and/or abiotic stress factors (Broetto et al. 2005). In the present study, an enhanced level in the amount of JA/ET-dependent enzymes was observed. In seeds that were treated with rhizobacterial strains together with spore suspension showed significant increase in activity of LOX, PAL, and POD. The observed increases in LOX activity in plant tissue expressing resistance in diverse pathosystems have been reported (Croft et al. 1990). The increase in LOX activity over the control observed in treatment T2 (uninoculated microbiolized seeds) may be explained by the recognized growth promoting effect of rhizobacteria (Gutierrez-Manero et al. 2001). The difference in LOX activities between treatments T3 and T4 was due to enzyme induction in the plant caused by the pathogen that, together with constitutive production (see treatment T4), apparently afforded an increase in enzyme activity in treatment T3.

The activity of plant POD and PAL enzymes are stress indicators. POD participates in the cell wall polysaccharides processes such as oxidation of phenols, suberization, and lignification of host plant cells during the defense reaction against pathogenic agents (Ray et al. 1998). The high peroxidase activities detected in treatments are linked to lignification and generation of peroxides that inhibit pathogens directly or generate other free radicals with antimicrobial effects (Hammerschmidt 1999). The peroxidase activity results reported in this study are in agreement with those of Podile and Lakshmi (1998), who observed an increase in POD activity in pea plants treated with B. subtilis, 7 days after inoculation with Fusarium udum.

PAL plays a key role in the phenylpropanoid pathway wherein lignin is one of the major products. Deposition of lignin is an inducible defense mechanism employed for protection against pathogen invasion (Liang et al. 1989). The type of bacterized plant response induced after challenge with a pathogen resulted in the formation of structural barriers, such as thickened cell wall because of the deposition of callose and the accumulation of phenolic compounds at the site of pathogen attack (Benhamou et al. 1998). PAL activity was higher in plants grown from microbiolized seeds that were challenge inoculated with charcoal rot pathogen. PAL activity is reflective of disease proliferation and consequent stress. Studies with different plant species showed that PAL activity increases with the biotic and abiotic stresses including bacterial infection (Hammerschmidt 1999; Yan et al. 2002).

To combat invasions by micro-organisms, plants have evolved several lines of defense. Besides pre-existing physical and chemical barriers, inducible resistance mechanisms can be activated upon pathogen infection. The increases in the activities of these enzymes (LOX, PAL, and POD) indicate that they may be important defense responses to pathogen infection. Plants that respond with higher activities of these enzymes may have enhanced resistance to pathogen attack. The data presented in this study described PGPR-phytopathogen-plant interactions in terms of rhizobacteria-mediated ISR. Besides inducing ISR, PGPR can exert a protective action against those soilborne pathogens that are particularly prone to attack emerging seedlings. ISR-eliciting rhizobacteria can be applied on seeds and then will readily colonize emerging plant-roots. Thus, seedlings can be better protected at an earlier growth stage (Kloepper et al. 1989). These properties make ISR-inducing PGPR a useful tool to reduce diseases caused by pathogens that are sensitive to JA- and ET-dependent defenses. Integrating ISR-triggering PGPR into disease management programme in conjunction with other strategies will be a worthwhile approach to explore. Understanding the diversity within populations of biocontrol agents holds the promise of pairing specific genotypes with their most supportive plant hosts or soil environments to maximize root colonization and disease suppression. Finally, establishing the presence and functionality of individual populations within a particular soil is just one first step toward fully understanding the nature of suppressiveness within that soil. The future studies of biologically based soil suppressiveness will present new insights into the microbial ecology of agricultural soils and lay the foundation for the development of creative management strategies for the suppression of soilborne diseases.