Isolation, screening and identification of Bacillus thuringiensis
The Bt was isolated from 5 soil samples collected from each of the 15 different blocks of the north western zone of Tamil Nadu state, India, and stained with Coomassie Brilliant Blue (CBB) (0.133% CBB in acetic acid 50%) for identification of crystal morphology as described by (Ramalakshmi and Udayasuriyan 2010). Twenty-four hours old broth culture of crystal positive isolates were prepared as glycerol stocks and stored at − 70 °C for further studies. Molecular identification, using 16SrRNA gene amplification by PCR was performed to identify the crystal positive isolates. The phylogenetic relationships of isolates are given in Fig. (S3).
Preparation of spore-crystal mixtures (SCM) and SDS-PAGE
The spore-crystal mixtures were prepared from Bt isolates as described earlier (Ramalakshmi and Udayasuriyan 2010). Five microlitres of SCM were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli 1970).
Screening for nematicidal cry genes by PCR
The total genomic DNA from Bt isolates was extracted, using appropriate methods (Sambrook and Russell 2001). The nematode-specific cry gene primers were used to identify the cry5, cry6, cry14 and cry21 gene families in Bt isolates (Porcar and Juárez-Pérez 2003). The 25 μl PCR reaction mixture consisted of 1X Taq buffer, 2.5 mM MgCl2, 0.5 μM forward and reverse primers, 0.25 mM dNTP mixtures and 3 U of Taq polymerase (all from Fermentas, USA). PCR condition includes 95 °C for 5 min (initial denaturation), 35 cycles of 95 °C for 1 min (denaturation), 55 °C for 1 min (annealing), 72 °C for 1 min (primer extension), followed by 72 °C for 10 min (final extension). Amplification was performed in Biorad T100 PCR. Four reference strains of Bt, 4 M1, 4Q1, 4E1 and 4BG1 obtained from Bacillus Genetic Stock Centre (BGSC), the Ohio State University, Columbus, Ohio 43210, were used as a positive control for screening of native Bt isolates by PCR.
Root knot nematode and maintenance of egg masses
The egg masses of root knot nematode (RKN), M. incognita were obtained from the Department of Nematology, TNAU, Coimbatore, and maintained in pots having tomato plants.
In vitro inhibition of M. incognita egg masses by SCM of Bacillus thuringiensis
The Bt isolates were grown in T3 broth till more than 95% cell lysis. After lysis, the different crystal protein suspensions 0.1, 0.2, 0.3, 0.4 and 0.5% with sterile water were prepared. The egg masses were collected from the pot culture experiment and washed with sterile distilled water before exposure to the crystal protein suspension. Three egg masses were released into each concentration of suspension in 6 mm dia petriplates. The sterile water with egg masses was used as control. The number of hatched J2 was recorded every 24 h up to 3 days under binocular light microscope (model Olympus); the images were recorded in Cannon camera 20 MP colour CMOS attached to the Olympus microscope. All the treatments were carried out with three replications.
Scanning electron microscope analysis
The virulence of Bt isolates against M. incognita egg masses was confirmed by SEM analysis. The unhatched egg masses of M. incognita were observed under SEM after 3 days incubation with different concentrations of SCM of Bt isolates, reference strains and control. Sample preparation was made by the modified method (Orion et al.1994; Wergin and Orion 1981). Briefly, the egg samples were fixed at 4 °C in closed tubes containing 1.25% glutaraldehyde and 1.25% paraformaldehyde in 0.05 M cacodylate buffer (pH 7.2) for 4 h. The samples were treated serially for 15 min with 50, 70, 90 and 100% ethanol, and butanol. Then the samples are vacuum freeze-dried for 24 h. The samples were coated by carbon grid with platinum coating observed under a SEMFEI-Quanta 250.
In vitro inhibition of J2 juveniles by SCM of Bacillus thuringiensis
The spore-crystal mixtures from nematicidal positive Bt isolates were further tested against M. incognita (J2) stage. Known concentration of 5 μg/ml of SCM was transferred into glass plates containing 100 nos of surface sterilised infective juveniles (J2). Four replications of each treatment were made to assess the juvenile mortality. The dead juveniles were counted from each treatment up to 48 h. Juveniles not showed any movement upon addition of water were considered as dead ones. Juveniles released in sterile water served as control. The experiments were performed at 30 ± 0.5 °C.
A pot culture experiment was conducted under greenhouse conditions for testing the biocontrol efficacy of Bt against M. incognita. Sterilised cocopeat mixture obtained from Orchard, TNAU, Coimbatore, was prepared and filled in 96 well portrays. Tomato seeds (PKM 2 variety obtained from HC and RI, TNAU, Coimbatore) were planted in portrays and maintained for better establishment. Tomato seedlings after 25 days were transferred to earthen pots of 10 kg capacity which were filled by steam sterilised pot mixture. M. incognita was inoculated at 1 J2/g of soil around the roots of tomato plants by making 3 holes around the plant and covered with sterilised soil. Ten millilitres of spore-crystal mixture of isolates BC and BD along with reference strain (4 M1) were applied near root zone. As a standard check Carbofuran 3G at 1 kg a.i/ha and an untreated control were included. Regular watering was done with tap water passed through 325 mesh sieve. The experiment consisted of 4 treatments with 3 replications in a completely randomised block design. Pots were placed at room temperature of 35 ± 2 °C under controlled conditions in the greenhouse.
Observations on shoot and root length, shoot and root weight were recorded after 60 days of planting. Plants were carefully uprooted and observation on gall index, number of females, number of egg masses and number of eggs per egg mass were observed. Population of nematode in soil was assessed by (KOPPENHõFER et al. 1998; Schindler 1961).
Data were pooled and analysed in the SPSS 16 software. Duncan’s multiple range tests were performed for treatment with spore-crystal mixture. The P value < 0.005 was kept as cutoff value for significance. The LC50 values were determined by probit analysis using SAS 8.0.