Isolation and molecular characterization of bacillus thuringiensis from different habitats of Nepal and potato tuber moth (phthorimaea operculella)

Abstract

Potato tuber moth (PTM), Phythorimae operculella (Zeller) is a pest of many solanaceous crops including potatoes. It is one of the major constraints to potato production worldwide. Farmers rely extensively on broad spectrum chemical pesticides to check the pests but this has serious side effects on human health, ecosystem and the beneficial insects. Therefore, for sustainable agriculture, the use of biopesticides is very critical. Biopesticides based on Bacillus thuringiensis (Bt) have been very popular and successful. Bt is a sporulating, Gram – positive facultative aerobic bacterium. Its principal characteristic is the synthesis of a crystalline inclusion containing the protein known as cry protein during sporulation. These proteins have insecticidal properties. This research was focused on the isolation and molecular characterization of Bt from wide range of habitat of Nepal. A total of 28 different samples were collected from Sauraha/Chitwan, Tulsipur/Dang, lalitpur, Beni/Myagdi, Ghasa/Myagdi, Bhairahawa/Rupandehi and Bandipur/Tanahun. Sodium acetate and quick isolation methods were used to isolate Bt. Gram staining was done to select Gram – positive bacteria. Bt was confirmed by Commassie Brilliant Blue (CBB) staining which stains the crystal into purple blue. A total of nine Bt strains were isolated. Most of them were bipyramidal. The types of cry gene of the isolates were determined by PCR using universal primers specific to cry1 and cry2 genes. Five of the isolated strains were positive for cry1 gene only and three strains were positive for both cry1 and cry2 genes. Cry1 subgrouping reveals the presence of cry1Aa gene in five strains, cry1Ab gene in seven strains and cry1Ac gene in six strains and absence of cry1B, cry1C and cry1D genes in all the isolated and reference strains. The bioassay of isolated strains against PTM showed their effectiveness. The lowest LDvalue was 6.67±3.024 µg/ml crude protein for strain d1 and highest LD 5050value was 36.71±5.68 µg/ml crude protein for reference strain B. thuringiensis kurstaki. These isolated strains showed high promise for biopesticides production. Key words: Bt, biopesticides, crystal proteins, PTM, bioassay.