Bio-efficacy and molecular charaterization of the native isolates of Bacillus thuringiensis Berliner

Abstract

Bacillus thuringiensis Berliner shortly known as B.t., is a gram-positive, spore forming bacterium that produces proteinaceous crystal containing insecticidal toxins. The host range of B.t. has expanded considerably due to extensive screening programmes. By virtue of its lack of toxicity towards other species of animals and human beings, B.t. has emerged as a safe candidate in the IPM programmes of various agroecosystems. Frequent exposure to one type of B. thuringiensis toxins can develop resistance in insects. Isolation of novel strains is important to overcome the onset of insect resistance. The present study on “Bio-efficacy and molecular characterization of native isolates of Bacillus thuringiensis Berliner” was conducted at the Department of Agricultural Entomology and CPBMB, College of Horticulture, Vellanikkara during the period from 2006 to 2008 with an objective to study the pathogenicity of 20 B. thuringiensis isolates collected from the Western Ghats of Kerala, one of the well known hot spots of biodiversity. The tobacco caterpillar, Spodoptera litura Fb. which was used as the test insect was reared in semi-synthetic diet (Ballal, 2004) and also in natural diet on castor leaves. The preliminary screening was performed for lepidopteran specificity with the ideal dose of 1 x 109 spores per ml showed that three isolates were toxic to S. litura. The per cent mortality caused by the lepidopteran effective isolates, namely, KAU-11, KAU-51, KAU-166 and the reference strain, HD-1 were 76.7 per cent, 80.0 per cent, 86.7 percent, 96.7 per cent respectively. A standardized bioassay was carried out with five different concentrations of all the selected isolates namely, KAU-11, collected from Thusharagiri (Calicut Dt.), KAU-51 from Kolahalamedu (Idukki Dt.), KAU-166 from Nelliyampathi

(Palakkad Dt.) and the reference standard HD-1 (B. thuringiensis subsp. kurstaki). The mortality ranged from 63.3 to 100.0 per cent in various isolates and there was no significant difference between isolates. This indicated the equal effectiveness of the native isolates with the standard strain, HD-1. The LC50 value for the isolates was calculated by Finney’s Method of Probit Analysis. The lowest LC50 was obtained in KAU-51, with 6.3095 x 104 spores per ml and highest in KAU-11, with 1.2589 x 106 spores per ml. The lethal time to cause 50 per cent mortality (LT50) ranged from 44.4 h. to 96.0 h. in different isolates. The lowest LT50 was recorded in HD-1, which shows the ability of the standard to cause mortality slightly earlier compared to other isolates. The molecular characterization of the selected isolates was performed with RAPD-PCR technique. RAPD-PCR is a simple and rapid method for determining genetic diversity in various organisms and is a means of creating a biochemical finger print of an organism. Out of the 20 primers screened, 10 primers which produced more than five bands were selected for RAPD analysis. The RAPD data was used to generate a similarity matrix using the NTSyS programme. Clustering was done and dendrogram was drawn using Unweighted Pair Group Method of Arithematic Averages (UPGMA). The results showed that high variability exists between the selected isolates. Further studies are required to identify the subspecies of the efficient B.t. isolates to evaluate the field effectiveness against S. litura and other major lepidopteran pests for its utilization in pest management programmes. More primers need to be screened to study the genetic diversity of the isolates.