This study was undertaken at the Department of Plant Pathology and Department of Plant Nematology, Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India.
Bacillus DNA isolation
Authenticated B. subtilis strain Bbv 57 (KF718836) was obtained from the culture collection center of the abovementioned department. Bbv 57 was grown in nutrient broth at 28 °C. Total DNA that included chromosomal and plasmid DNA was extracted (Robertson et al., 1990). Cultures grown for 18 h in nutrient broth were centrifuged into a pellet, washed in TE buffer (10 mM Tris pH 7.5/1 mM EDTA pH 8.0), and suspended in 10% sucrose. Cells were incubated at 37 °C in lysozyme solution (5 mg/ml lysozyme, 50 mM Tris pH 7.5, 10 mM EDTA pH 8.0), followed by the addition of 20% SDS containing 0.3% beta-mercaptoethanol. DNA was purified and quantified.
Identification using genus-specific primers
For Bacillus sp., confirmation 16S rRNA intervening sequence-specific BCF1 (5′-CGGGAGGCAGCAGTAGGGAAT-3′) and BCR2 (5′-CTCCCCAGGCGGAGTGCTTAAT-3′) primers were used to get an amplicon size of 546 bp (Cano et al., 1994). A 20-μl reaction mixture containing 10× buffer (with 2.5 mM MgCl2, 2 μl; 2 mM dNTP mixture, 2 μl; 2 M primer, 5 μl; Taq DNA polymerase, 3 U; H2O 8 μl and 50 ng of template DNA samples) were amplified on DNA thermal cycler using the PCR conditions 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 1 min. The total number of cycles was 40 with the final extension time of 10 min. The PCR products were resolved on 2% agarose and sequenced.
Lipopeptide antibiotic biosynthesis genes iturin (ItuD gene) and surfactin (srfA gene; sfp gene) amplification
ITUD F (5′-GATGCGATCTCCTTGGATGT-3′) forward and ITUD R (5′-ATCGTCATGTGCTGCTTGAG-3′) reverse primers were used for amplification of ItuD gene (1203 bp) (Ramarathnam, 2007). The 20-μl mixture contained approximately 50 ng of total DNA, 5 mM each of dNTPs, 20 pmol each of forward and reverse primers, and 0.5 U of Taq DNA polymerase. PCR amplification was performed in a thermocycler (Eppendorf Master cycler, German), using the following conditions: initial denaturation at 94 °C for 3 min, 30 cycles consisting of 94 °C for 1 min (denaturation), 50 °C for 1 min (annealing), 72 °C for 1 min 30s (primer extension), and final extension 72 °C for 10 min.
The forward primer SRFA-F1 (5′-AGAGCACATTGAGCGTTACAAA-3′) and reverse primer SRFA-R1 (5′-CAGCATCTCGTTCAACTTTCAC-3′) were used for amplification of srfA gene (626 bp) (Hsieh et al., 2004). The 20 μl PCR reaction mixture contained DNA template 50 ng, 1× Taq buffer, 0.2 mM each of dNTP mixture, 1 μM of each primer, 1.5 mM MgCl2, and 2 U of Taq DNA polymerase. The PCR conditions are as follows: an initial denaturation at 95 °C for 15 min; 40 cycles of 95 °C for 1 min, annealing at 62 °C for 1 min, and 72 °C extension for 1.5 min; and a final extension at 72 °C for 7 min.
The forward primer SFP F (5′-ATGAAGATTTACGGAATTTA-3′) and reverse primer SFP R (5′-TTCCGCCACTTTTTCAGTTT-3′) were used for amplification of sfp gene (675 bp) (Hsieh et al., 2004). The 20-μl PCR reaction mixture contained DNA template 50 ng, 1× Taq buffer, 0.2 mM each of dNTP mixture, 1 μM of each primer, 1.5 mM MgCl2, and 2 U of Taq DNA polymerase. The PCR conditions are as follows: an initial denaturation at 95 °C for 15 min; 40 cycles of 95 °C for 1 min, annealing at 55 °C for 1 min, and 72 °C extension for 1.5 min; and a final extension at 72 °C for 7 min.
Mycolytic enzyme β-1,3-glucanase detection
The forward primer β-GLU F (5′-AATGGCGGTGTATTCCTTGACC-3′) and reverse primer β-GLU R (5′-GCGCGTAGTCACAGTCAAAGTT-3′) were used for amplification of glucanase gene (400 bp) encoding PR2 protein (Baysal et al., 2008). The 20-μl PCR mixture contained approximately 50 ng of total DNA, 5 mM each of dNTPs, 20 pmol of each forward primer and reverse primer, and 0.5 U of Taq DNA polymerase. The PCR conditions are as follows: an initial denaturation at 94 °C for 5 min; 40 cycles consisting of 92 °C for 1 min (denaturation), 34 °C for 2 min (annealing), and 72 °C for 2 min (primer extension); and final extension 72 °C for 2 min.
Thin-layer chromatography (TLC)
Extraction of lipopeptide biosurfactants
Cultures of B. subtilis Bbv 57 were grown separately in 20 ml of pigment production broth (peptone, 20 g; glycerol, 20 ml; NaCl, 5 g; KNO3, 1 g; distilled water, 1 l; pH 7.2) for 4 days on a rotary shaker at 30 °C. The fermentation broth was centrifuged at 15,000 rpm for 30 min in a tabletop centrifuge, and the supernatant was collected. It was acidified to pH 2.0 with 1 N HCl and then extracted with an equal volume of ethyl acetate. The ethyl acetate extract was reduced to dryness in vacuo. The residues were dissolved in methanol and kept at 4 °C until used for TLC (Rosales et al., 1995).
Surfactin and iturin identification
For the identification of surfactin and iturin, a volume of 4 μl of sample was spotted on to the aluminum-coated sheets with silica gel TLC plates. Separation was performed with chloroform/methanol/distilled water (8:1:1) as a solvent system for surfactin and iturin. After separation, the spots were visualized under short wavelength (245 nm). For surfactin, the spots were visualized after spraying with 0.1% ninhydrin ethanol solution. Rf values for the spots confirming surfactin and iturin were calculated.
In vitro bioassay against F. oxysporum
A 9-mm mycelial disc of the wilt pathogen F. oxysporum f. sp. gerberae (KM523669) was placed in the center of the Petri plate. Sterile Whatman No. 40 filter paper discs with 6 mm dia were placed 1 cm away from the edge at four sides centering on the fungal disc. Different increments (10–100 μl) of crude extract of B. subtilis Bbv 57 were dropped over the sterile filter paper discs. The plates were incubated at room temperature (28 ± 1 °C) and were scored when the mycelium grew over the control disc. Control was maintained with the sterile distilled water instead of crude extract.
In vitro bioassay against M. incognita
Crude antibiotic extract of B. subtilis Bbv 57 was taken at different concentrations of 25, 50, and 100% in a 50-mm Petri dish, and five egg masses of M. incognita were placed in each dish and incubated at 28 ± 1 °C. Egg masses placed in distilled water without crude antibiotic extract served as control. The experiment was replicated four times. Observation on the number of hatched juveniles was made after 24, 48, and 72 h of exposure.
Crude antibiotic extract of B. subtilis Bbv 57 at concentrations of 25, 50, and 100% were poured into separate Petri dishes. The second stage juveniles of M. incognita were introduced at the rate of 100 juveniles in each dish and incubated at 28 ± 1 °C. Juveniles placed in dishes containing distilled water served as control. Each treatment was replicated four times. Observations were recorded on the mortality of juveniles after 24, 48, and 72 h of exposure period, and percent mortality was calculated. The inactive nematodes were transferred separately from each dilution into sterile distilled water and kept overnight to check whether mortality was permanent or temporary.
The liquid formulation of B. subtilis Bbv 57 was assessed for its efficacy against wilt-nematode disease complex in Gerbera cv. Palm Beach under greenhouse conditions. A pot culture study was undertaken, using the completely randomized design (CRD), with three replications. The different treatments are listed below:
T1 = Seedling dip in liquid formulation of B. subtilis (Bbv 57) at 500 ml/ha (3.2 × 109 cfu/ml)
T2 = Soil drenching with B. subtilis (Bbv 57) 1000 ml/ha, at monthly interval (once in every month)
T3 = Soil drenching with B. subtilis (Bbv 57)1000 ml/ha at bimonthly interval (once in 2 months)
T4 = Seedling dip + soil drenching with B. subtilis (Bbv 57) at monthly interval
T5 = Seedling dip + soil drenching with B. subtilis (Bbv 57) at bimonthly interval
T6 = Soil application of carbendazim 0.05% + carbofuran (1 kg ai/ha)
T7 = Control
Observations on plant growth parameters like shoot length (cm), fresh shoot weight (g), root length (cm), fresh root weight (g), stalk length (cm), and flower stalk girth (cm) were recorded at 240 days after planting (DAP). For yield parameters, total number of flowers per plant, flower diameter (cm), disc diameter (cm), total number of normal flowers, total number of bent neck flowers, and vase life (days) were recorded till 240 DAP. Soil population of root-knot nematode was recorded. In roots, a number of females per gram of root, egg mass per gram of root, gall index, and percent wilt incidence were recorded at 240 DAP.
The data were analyzed statistically using the IRRISTAT version 92 (Gomez and Gomez, 1984). The percentage values of the disease index were arcsine transformed. Data were subjected to analysis of variance (ANOVA) at two significant levels (P < 0.05 and P < 0.01), and means were compared by Duncan’s multiple range test (DMRT) (Duncan, 1955).