English

Ngo Dinh Binh,Asano Shin Ichiro,Tang Thi Chinh,O O,Nguyen Thi Hoa,B Hisanori,Le Thi Minh Thanh

doi:10.5897/ajmr2012.2340

Abstract

Bacillus thuringiensis strains were isolated from sugarcane farming soil in three provinces of northern Vietnam. Of these, 24 strains were identified as serotype H5a, 5b, B. thuringiensis serovar galleriae. A strain, namely MHB11.3 (deposited in GenBank: FN796428) belonging to the serovar galleriae, was highly toxic to larvae of the scarab beetles, Anomala cuprea and Cotinis nitida (Coleoptera: Scarabaeidae). A 2-kb DNA fragment, obtained by PCR from the strain MHB11.3 using cry8 specific primers, namelycry8Dat (truncated cry8Da gene), was cloned and expressed in Escherichia coli. The sequence of the cry8Dat from B. thuringiensis serovar galleriae MHB11.3 showed almost complete identity (99%) to that of B. thuringiensis serovar galleriae SDS502 (GenBank: AB089299), differs only by two nucleotides (positions A26G; A1493T), leading to 2 amino acid changes (Y9C and N498I). The recombinant protein pET32a(+)-Cry8Dat of 96 kDa molecular weight was purified by Ni(2+) affinity chromatography. This polypeptide was tested to be toxic to A. cuprea and C. nitida. The amino acid change (Y9C) is located in a region responsible for the host specificity of the Cry8Da toxin. The strain B. thuringiensisserovar galleriae MHB11.3 harboring cry8Da gene as characterized in this study is a promising candidate as a biopesticide and suggested as an important genetic material for gene transfer into sugarcane for scarab beetle control.   Key words: Bacillus thuringiensis serovar galleriae, Coleoptera, cry8Da gene, Anomala cuprea, Cotinis nitida.

Highlights

One of the most important findings in Bacillus thuringiensis research, after their establishment as alternative for control of insect pests in agriculture (Burges, 1981; Roh et al, 2007) was the discovery of Cry proteins with Coleopteran specificity. Krieg et al (1983) first cloned a novel gene from B. thuringiensis serovar tenebrionis encoding the Cry3A protein that kills Colorado potato beetle
Soil samples collected from three provinces of sugarcane planting areas contained many B. thuringiensis isolates, supporting our previous data, that B. thuringiensis is widely distributed in Vietnam (Martin and Travers, 1989; Binh et al, 2005)
Of the 39 isolates with spherical crystal proteins, 24 (61%) reacted with H5a, 5b, serovar galleriae antiserum, while the average frequency of B. thuringiensis serovar galleriae is only 1.5% in the Vietnam B. thuringiensis Collection (Binh et al, 2007). The reason for this is that the high frequency of B. thuringiensis serovar galleriae in the current study may be due to the location of soil sampling, sugarcane planting areas

Summary

Introduction

One of the most important findings in Bacillus thuringiensis research, after their establishment as alternative for control of insect pests in agriculture (Burges, 1981; Roh et al, 2007) was the discovery of Cry proteins with Coleopteran specificity. Krieg et al (1983) first cloned a novel gene from B. thuringiensis serovar tenebrionis encoding the Cry3A protein that kills Colorado potato beetle. Soon after, Herrnstadt et al (1986) isolated a similar strain, B. thuringiensis serovar morrisoni san diego, and its insecticidal crystal protein (ICP) gene was sequenced. Among B. thuringiensis ICPs, Cry, Cry, Cry, Cry, Cry, Cry and Cry are known to be active against coleopteran species. Asano et al (2003) reported a new B. thuringiensis strain, called serovar galleriae SDS-502, that showed high insecticidal activity against Anomala cuprea, Anomala orientalis and Popillia japonica, which are species of scarab beetles known as the Japanese beetles. The ICP gene responsible for the highly activity of SDS-502 against Japanese beetles has been isolated and was designated cry8Da. The cry8Da gene was transferred into turf grass, which showed strong resistance against feeding attack by the Japanese beetle (Asano et al, 2005)

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Conclusion