Distribution of Bacillus Thuringiensis in Gunung Jerai Forest (Malaysia)with Insecticidal Activity Against Lepidopteran and Dipteran Insects

Abstract

Globally there has been a concerted effort to isolate Bt strains from different environments for use as microbial pesticides and in transgenic plants. In our study, 106 soil samples were collected from different altitude in Gunung Jerai forest, an undisturbed rain forest ecosystem, Kedah Darul Aman State, Malaysia. Preliminary screening included temperature selection methods to isolate Bacillus-like colonies, followed by phase contrast microscopy to screen presumptive Bt isolates. Twenty three isolates were selected for PCR analysis with Cry1 general primer (gcry1), PCR to identify the presence of cry1-type genes. Out of 23 isolates, only 17 isolates showed the presence of cry1-type genes with the product size of 558 bp. Subsequently, the 17 Bt isolates which showed the presence of cry1-type genes were subjected to pathotype PCR to predict the insecticidal activity against lepidopteran pests. The results of the pathotype PCR revealed that out of 17 Bt isolates, only 11 isolates showed PCR product indicating their possible anti lepidopteran activity. Plasmid profiling studies indicated the presence of a major plasmid of size 22.5 to 28.5kb.Then the hemolytic positive strains were subjected to one step bioassay against the second instar larvae of Aedes aegypti. One Bt isolate, Bt 02, showed elevated larvicidal activity, which was compared to that of the reference strain Bt.subsp. israelensis. The crystal protein studies of the Bt isolate 02 indicated the presence of cuboidal crystals. SDS-PAGE analysis of the Bt isolate 02 showed a major protein of 101.77 kDa. However, presence of a protein of 32.68 kDa, indicted the possible presence of cytolytic toxin. Finally to study the diversity, two Bt isolates, Bt 22 and Bt 42 were subjected to partial DNA sequencing of the PCR amplicon. The results indicated that these two strains have different nucleotide sequences and each strain showed homology with the reported Bt strains which are also different from each other, thus indicating the presence diverse Bt strains in this environment. The Bt isolates showing anti lepidopteran and anti dipteran activity can be used in integrated pest management to control agricultural pests and to control the selected mosquito vectors.