Introduction

As Actinomycetes produce important bioactive compounds of high commercial value, they are being routinely screened for new bioactive substances. About 61% of all bioactive microbial metabolites were isolated from actinomycetes (Moncheva et al. 2002). Most common antibiotic producing soil microorganisms belong to the genus Streptomyces; about two-third (>4,000) of naturally occurring antibiotics, such as aminoglycoside, anthracyclines, chloramphenicol, β-lactams, macrolides, and tetracyclines, among others, has been discovered from Streptomyces strains (Goodfellow et al. 1988). The production of large number of antimicrobial compounds by Streptomyces spp. (Berdy 2005) and antagonistic impacts of actinomycetes on pathogenic fungi are well documented, but few species have been implicated in biological control of plant diseases (Bressan 2003).

Except a few deeply studied actinomycetes taxa, others are difficult to be isolated from soil by common isolation techniques because of its slow growth and specific requirements (Kim et al. 1995). Pre-treatments like enrichment, heat treatment, chemical treatment, baiting, and others, may be used to isolate specific actinomycetes from natural environments. Mycophagy may also be applied selectively for the isolation of actinomycetes antagonistic to soil borne plant pathogens and plant growth promotion (El-Tarabily and Sivasithamparam 2006; Leveau and Preston 2008). However, none of the reported methods is able to isolate antagonists from natural environments, because the factors required for optimal growth and predatory activities are different for every species (Tsay et al. 2006). Some rare actinomycetes were reported to be isolated using different baits other than fungal mycelia, such as pinus pollen grain for selective isolation of Actinoplanes spp. (Masayuki 2008). However, the fungal baiting technique mimics natural habitat, therefore selective isolation of aggressive antagonists from soil may be achieved to ensure its efficiency in vivo (Leveau and Preston 2008). Biocontrol offers special significance in disease management, being an ecofriendly and cost effective strategy. This study describes selective isolation of actinomycetes antagonistic to R. solani, their evaluation, characterization and diversity using polyphasic approach and mechanism of antagonism using scanning electron microscopy (SEM).

Materials and methods

Selective isolation of actinomycetes

Mycelial mat of R. solani from 1 week old broth culture (approx. 10 gm) was recovered, washed twice with sterile distilled water and wrapped in a 50-mesh nylon cloth. These bags were buried into soil at 7–10 cm depth. After 2 weeks, the bags were retrieved and 1 g of soil sample was collected from the vicinity of the bags. Putative antagonistic microorganisms were isolated using serial dilution spread plate method. Selective isolation of actinomycetes was done on different specific media viz. starch casein nitrate agar (SCA; 10 g of starch, 2 g of K2HPO4, 2 g of KNO3, 2 g of NaCl, 0.3 g of casein, 0.05 g of MgSO4·7H2O, 0.02 g of CaCO3, 0.01 g of FeSO4·7H2O, 15 g of agar and 1 l of distilled water), actinomycetes isolation agar (AIA; 4 g of sodium propionate, 2 g of sodium caseinate, 0.5 g of K2HPO4, 0.1 g of aspargine, 0.1 g of MgSO4·7H2O, 1 g of FeSO4·7H2O, 5 ml of glycerol, 15 g of agar and 1 l of distilled water), ISP-3 medium (oat meal agar; 20 g of oat meal powder, 20 g of agar, 1 ml of trace salt solution in 1 l of distilled water; trace salt solution contains FeSO4·7H2O, MnCl2.4H2O and ZnSO4·7H2O 0.1 g each in 100 ml distilled water) and humic acid-vitamin B medium (HV; 1 g of humic acid, 0.5 g of Na2HPO4, 1.7 g of KCl, 0.5 g of MgSO4.·7H2O, 0.01 g of FeSO4.7H2O, 0.02 g of CaCO3, 20 g of agar in 1 liter of distilled water and 500 μl of vitamin-B complex was added from 0.5 mg ml−1 stock) (Hayakawa and Nonomura 1987; Cho et al. 1994). Randomly picked colonies were transferred on SCA media and subcultured for getting axenic culture. Preliminary identification of the isolates was done on the basis of growth characteristics, then pure isolates were kept in refrigerated condition. Pathogenic strain of R. solani (NAIMCC F- 01971) was obtained from the culture collection of NBAIM, India, and maintained on potato dextrose agar (PDA; 200 g of peeled potato, 18 g of dextrose and 15 g of agar in 1 l of distilled water).

In vitro screening for antagonism

Actinomycetes isolates were screened for their antagonistic potential against widely spread filamentous fungus R. solani by dual culture and agar well diffusion plate (Subramani and Narayanasamy 2009) on PDA. The percent growth inhibition was calculated as described by Nourozian et al. (2006). Effect of volatile compound on growth of R. solani was evaluated by a modified sealed plate assay, in which actinomycetes isolates and test pathogen were separately inoculated on SCA and PDA plates, respectively. The lid of plates was removed and R. solani plate was kept inverted above the antagonist’s plate, closed tightly. After 1 week incubation at 26 °C, the radial growth of R. solani was observed and compared with control. All antagonistic assays were conducted in three replicates. Isolates were also tested for their plant growth promoting traits viz. phosphate solubilization, siderophore and HCN production using Pikovskaya’s medium (10 g of glucose, 0.5 g of yeast extract powder, 0.5 g of (NH4)2SO4, 0.2 g of KCl, 0.2 g of NaCl, 0.1 g of MgSO4·7H2O, 5.0 g of Ca3 (PO4)2, traces of FeSO4·7H2O, MnSO4·7H2O and 15 g of agar in 1 l of distilled water), chrome azurol sulfonate (CAS) agar plate (60.5 mg CAS was dissolved in 50 ml water and mixed with 10 ml iron solution (1 mM FeCl3·6H20, 10 mM HCl). Under stirring this solution was slowly added to 72.9 mg HDTMA dissolved in 40 ml water. It was mixed with Nutrient agar (2% agar) in 1:3 proportion at the time of pouring) and picric acid dipped strip method, respectively. The qualitative assay for chitinase and protease production was performed on colloidal chitin agar (0.7 g of K2HPO4, 0.3 g of KH2PO4, 0.5 g of MgSO4·7H2O, 0.1 g of FeSO4·7H2O, 0.001 g of ZnSO4·7H2O, 0.001 g of MnCl2·7H2O, 1% glycerol, 5 ml of colloidal chitin in 1 l of distilled water and pH was adjusted 8.0 with 5 N NaOH) and skimmed milk agar (10 g of dehydrated skimmed milk and 20 g of agar in 1 l of distilled water), respectively. Colloidal chitin was prepared from crab shell chitin using concentrated HCl as described by Hsu and Lockwood (1975). Isolates were spot inoculated, then plates were incubated at 32 °C and clearance halos around and beneath the growth indicating the enzymatic degradation was observed and measured after 8–10 days.

Scanning electron microscopy

The interaction of the test fungi with antagonistic isolate (S. toxytricini vh22) was studied by scanning electron microscopy (SEM). The hyphae from the interaction zone was transferred on glass cover slips, then fixed with 1.5% glutaraldehyde and dehydrated with graded series of ethanol washes followed by drying in desiccator (Walter and Crawford 1995). Samples were affixed to SEM stubs using carbon tape followed by thin coating with gold: palladium (60:40) and examined by SEM (JEOL, JSM-63804).

Evaluation of biocontrol activity of isolates under green house condition

Disease suppression capability of effective antagonists was determined in pot (soil, sand and compost in 6:3:1) experiment under pathogenic stress (3% inoculums of R. solani, solid state fermentation in wheat bran) in a greenhouse, using a complete randomized design with five replicates. Seedlings were treated with liquid inoculum of antagonists (2 h dip in inoculum, 3.6 × 106). Seedlings without treatment and with non-antagonist (isolate vh51) served as control and negative control, respectively. Disease severity was recorded 45 days after transplanting.

Characterization of antagonistic isolates

Morphological characterization of the potential antagonists was done on ISP-4 medium (10 g of soluble starch, 2 g of CaCO3, 2 g of (NH4)2SO4, 1 g of K2HPO4, 1 g of MgSO4·7H2O, 1 g of NaCl, 1 mg of FeSO4·7H2O, 1 mg of MnCl2·7H2O, 1 mg of ZnSO4·7H2O in 1 l of distilled water) after 1 week incubation, while ISP-2 medium (10 g of malt extract powder, 4 g of yeast extract powder, 4 g of glucose and 20 g of agar in 1 l of distilled water) was used for microscopic analysis. Basal medium (2.64 g of (NH4)2SO4, 2.38 g of KH2PO4, 5.65 g of K2HPO4·3H2O, 1.0 g of MgSO4·7H2O, 6.40 mg of CuSO4·5H2O, 1.10 mg of FeSO4·7H2O, 7.90 mg of MnCl2·4H2O and 15 g of agar in 1 l of distilled water and pH was adjusted to 6.8–7.0) (Promnuan et al. 2009) was used for studying sugar utilization pattern with various carbon sources (Table 1). The medium with d-glucose and without any carbon source were used as positive and negative control, respectively, where cell suspension (prewashed thrice with sterile distilled water) was spot inoculated. According to the methods of the International Streptomyces Project (ISP) (Shirling and Gottlieb 1966) and Bergey’s Manual of Systematic Bacteriology (Williams et al. 1989), isolates were differentiated at generic level on the basis of thin layer chromatography. Through this method, acid hydrolysate of the biomass was chromatographed on cellulose coated TLC plates (Merck KGaA TLC cellulose plastic sheets 20 × 20 cm) to detect 2,6- diaminopimelic acid, one of the cell wall component of actinomycetes mycelia and developed as per protocol by Staneck and Roberts (1974).

Table 1 Phenotypic characteristics of selected antagonistic actinomycetes
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Genetic diversity analysis of antagonistic actinomycetes

Nine potential antagonistic actinomycetes isolates were selected for characterization using molecular tools. Genomic DNA of isolates was extracted through enzymatic lysis (Pospiech and Neumann 1995) with little modification of lysozyme and proteinase K concentration. PCR (Peltier Thermal Cycler, BIORAD) amplification was performed using 16S universal primer pA (5′-AGAGTTTGATCCTGGCTCAG-3′) and pH (5′-AAGGAGGTGATCCAGCCGCA-3′) (GeNei, India) (Edwards et al. 1989). The amplified product was subjected for restriction digestion using five restriction enzymes viz. TaqI, MboI, MspI, AluI and HaeIII (Promega, USA). Band pattern was observed on agarose gel (2.5%) electrophoresis as well as native polyacrylamide gel (8%) electrophoresis (PAGE). All nine isolates were sequenced to get partial 16S rDNA sequence using dideoxy sequencing (BDT version 3.1 ABI cycle sequencing) with same primer.

Data analysis

The sequences of the isolates were searched in NCBI GeneBank database using BLASTn program. An entry with the highest score was downloaded and alignment was performed using CLUSTALX (version 1.81) (Thompson et al. 1997). Phylogenetic analysis was conducted using MEGA version 4 (Tamura et al. 2007). The data of disease suppression potential of selected isolates were analysed using the Tukey test (GraphPad InStat®). The differences in the mean values were compared at P < 0.05.

BOX-PCR fingerprinting

The single primer corresponding to BOX (BOXA1R, 5′-CTA CGG CAA GGC GAC GCT GAC G-3′) was obtained from Integrated DNA technology (Coralville, USA). PCR amplification was performed in 24 μl volume consisting 5× Gitschier buffer, 0.2 μl (20 mg/ml) nuclease free BSA, 2.5 μl (100%) DMSO, 1.25 μl ultra pure dNTP set 100 mM mix, 1 μl BOX A1R primer (0.3 μg/μl), 0.4 μl Taq DNA polymerase (5 U/μl) (GeNei, India) and reaction mixture was balanced with autoclaved MQ water. One microliter (50 ng ml−1) genomic DNA was used for fingerprinting analysis followed by initial denaturation at 94 °C for 1 min and 39 cycles of 94 °C for 30 s, 53 °C for 1 min, 72 °C for 8 min, with final extension at 72 °C for 16 min. Amplicons were separated on 1.3% agarose gel electrophoresis (60 V/3 h) with low range DNA ladder (100 bp–3 kb).

Results

Isolation and screening of antagonistic actinomycetes

One hundred ten actinomycetes were isolated, of which nine were found to be potential antagonists against R. solani after in vitro screening. Maximum growth was inhibited by Actinomycetales bacterium vh41 in dual culture (52.6%) and by Streptomyces toxytricini vh22 in agar well diffusion (50.0%), whereas S. toxytricini vh22 and vh55 performed well in dual culture as well as in the agar well diffusion method (Fig. 1). Out of these isolates, nine appeared to be promising antagonists and hence were selected for further evaluation and characterization. Some of the selected isolates were found to be potential for phosphate solubilisation (55.5%), siderophore (77.8%) and protease (55.5%) production, while few were capable to produce chitinase (22.2%) and only one was able to produce HCN (Table 1).

Fig. 1
figure 1

Antifungal activity of selected actinomycetes isolates against R. solani

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Scanning electron microscopy

Scanning electron microscopy of antagonistic interaction between growing R. solani mycelia and S. toxytricini vh22 exhibited reduced apical growth, curling of hyphal tips and irregular distortions in the fungal hyphae (Fig. 2b). The pore formation on mycelial surface and cytoplasmic extrusion (Fig. 2c, d) was compared with healthy mycelia in absence of antagonists (Fig. 2a) and in presence of non-antagonists (Fig. 2e).

Fig. 2
figure 2

Scanning Electron Micrograph of S. toxytricini vh22 and R. solani interaction after 4 days of interaction. a Characteristic perpendicular branching hyphae in the absence of an antagonist (control) b Loss of apical growth, curling of hyphal tips and mycelial deformation c Hyphal destruction and pore formation on mycelial surface d Cytoplasmic extrusion from hyphae e Intact mycelia on interaction with vh51 showing non-antagonism

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Evaluation of biocontrol activity of isolates under green house condition

The nine isolates significantly suppressed the disease as compared to the control as well as negative control. The maximum disease suppression (53.3%) was recorded on the inoculation of S. tricolor vh85 followed by S. tricolor vh84 and S. toxytricini vh55, which were statistically at par (Table 1).

Characterization of antagonistic isolates

The data presented in Table 1 reveals that the antagonistic isolates had spiral sporophores while colonial morphology showed a powdery growth, irregular shape and elevation with pink, cream, white and grey coloured growth, which are features of group Streptomyces; we also found a tendency to group our isolates within Streptomyces. Carbon utilization profile and presence of L-A2AP in cell wall hydrolysate gave an indication of Streptomyces group, which supports morphological as well as biochemical characterization.

ARDRA was poorly discriminative with all five restriction enzymes, hence all 9 isolates were further characterized using 16S rDNA partial sequencing. Based on this partial sequencing, similarity values ≥97% suggested that all the isolates belongs to genus Streptomyces, viz. S. toxytricini vh17, S. globosus vh18, S. toxytricini vh22, S. avidinii vh32, S. rochei vh52, S. toxytricini vh55, S. tricolor vh84 and vh85, except isolate vh41, which was identified at order level as Actinomycetales bacterium. Partial 16S rDNA sequences of the isolates were submitted to NCBI GeneBank and the following accessions were obtained: GQ262795 to GQ262800 and GQ282615 to GQ282617. A corresponding phylogenetic analysis of the isolates based on NJ method with 1000 bootstrap sampling resulted into 2 major clades (Fig. 3). In BOX-PCR fingerprinting, all 9 isolates were found to have a characteristic band pattern, where genetic relatedness was more convincing with the most of the major bands obtained above 600 bp, while secondary bands appeared through the range (Fig. 4). The dendrogram resulted into 8 distinct BOX profiles including 3 heterogeneous groups and one homogeneous group (Fig. 5).

Fig. 3
figure 3

Phylogenetic tree of partial 16S rDNA sequences of Streptomyces isolates with those of maximum similar entries from database. The tree is created by neighbor-joining method with 1,000 bootstrap re-samplings; values lower than 50 are not shown. Scale bar represents the number of changes per base position

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Fig. 4
figure 4

BOX-PCR fingerprint of antagonistic Streptomyces isolates

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Fig. 5
figure 5

BOX-PCR fingerprinting: Dendrogram is obtained from similarity coefficient and clustering was done using UPGMA algorithm using NTSYS software

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Discussion

There has been increasing interest in the discovery of natural products from rare and uncommon actinomycetes for their exploitation in agriculture and medicine (Long and Wildman 1992; Lazzarini et al. 2000). Some members of streptomycetes which are potentially sound in producing bioactive compounds have been promising in the past (Goodfellow and Williams 1986), but those prospective entrants against phytopathogens are still less explored.

Results indicate that selective isolation of aggressive antagonistic actinomycetes may be achieved using fungal mycelial baiting from the soil and that may enhance natural screening and improve isolation procedure. The advantage of using native antagonists for biocontrol was elucidated by Alvindia and Natsuaki (2008). We found that mycelia of phytopathogenic fungus (R. solani) as bait uphold the endorsement for selective isolation of potent antagonistic actinomycetes (Singh et al. 1987; Masayuki 2008), however, reports on the effectiveness of any such baiting technique with fungal mycelia in the soil system is lacking.

The mode of action following antagonism by S. toxytricini vh22 was studied by SEM, which shown gradual destruction of mycelia leads to the death due to cytoplasmic extrusion validate potency to inhibit R. solani. It is well known that Streptomyces species are capable of antagonising fungal spores and hyphal structures (Walter and Crawford 1995), producing extracellular cell wall degrading enzymes, such as chitinase, cellulase, amylase, and 1,3-β-glucanase (Hopwood and Chater 1990) and growth inhibiting compounds (Houssam 2009). Although the exact mode of action is not clear at present, from SEM analysis it may be concluded that antagonism may be offered by antimicrobial compound as destruction of hyphae, which has been observed without any physical contact between antagonists and target pathogen.

Even though it is always difficult to extrapolate the biocontrol activity of a given strain from the laboratory to natural environments (Hermosa et al. 2000), the results of pot experiment under greenhouse condition suggest the efficiency of the actinomycetes isolates that showed aggressive in vitro antagonism, in controlling root rot caused by R. solani. Although S. toxytricini vh22 was found to be an effective antagonist under in vitro condition, comparable results were not observed under greenhouse condition, which may be due to less rhizospheric competitiveness and root colonization ability. Disease suppression ability of the antagonists may remain low, as it is evident that a certain threshold population of antagonists must be attained to achieve significant level of disease suppression (Mazzola 2007).

Morphological, microscopic, chemotaxonomic and physiological profile of selected isolates confirmed them as Streptomyces spp. (Thomas 1965; Matsukawa et al. 2007); these findings were supported by 16S rDNA partial sequencing, useful also for confirming the identification of isolates as Streptomycetes spp. Isolate vh55 shares 99% similarity with Streptomyces toxytricini, Streptomyces globosus and Streptomyces flavotricini, however microscopic, biochemical and cluster analysis confirmed it as S. toxytricini.

16S rDNA sequence as well as BOX-PCR based genotypic analyses suggested a high degree of diversity among the antagonistic actinomycetes isolates. The BOX fingerprinting generated cluster analysis revealed three heterogeneous and one homogeneous group, where three isolates of S. toxytricini (vh17, vh22 and vh55) appeared very distant (Fig. 5), while in the same heterogeneous group based on 16S rDNA generated dendrogram. The BOX element has discriminating power up to species level (Lanoot et al. 2005) and represents whole genomic DNA. Similarly, isolate S. tricolor vh84 and vh85 also grouped in two different heterogeneous groups in BOX fingerprinting, sharing more than 98% similarity in 16S rDNA sequence based analysis.

Present study fetches us to conclude that potential antagonistic actinomycetes may be isolated using baiting, being superior over conventional non-selective isolation. BOX fingerprinting was a rapid and highly discriminating tool for typing of Streptomyces, as compared to other DNA dependent approach used in the study.