Isolation and characterization of New Bacillus thuringiensis strains with insecticidal activity to difficult to control lepidopteran pests

Abstract

Strains were isolated from soil samples collected from mountains and fields of Yeong-Dong and Ok-cheon, ChungBuk province in order to select B. thuringiensis strains with high insecticidal activities against lepidopteran pests. Eight B. thuringiensis strains producing spores and endotoxin proteins were selected from a total of 26 soil samples. Tests of biological activities in Spodoptera litura, Spodoptera exi-gua, Plutella xylostella, and Aedes aegypti were performed with 8 isolates of B. thuringiensis. CAB565 and CAB566 strains were selected, because such strains showed more than 80% of insecticidal activities against four kinds of pests. Selected two strains formed endotoxin proteins in a typical bipyramidal type. CAB565 and CAB566 strains were identified as thuringiensis subsp. kurstaki, and thuringiensis subsp. aizawai, respectively. LC50, value of CAB566 strain against larvae of S. litura at the fourth stage was 7.7×105 (cfu/ml), which was the highest activity. LC50 value of CAB565 strain against larvae of S. exigua at the fifth stage was 3.0×106 (cfu/ml), which was high activity. Through SDS-PAGE, CAB565 and CAB566 strains showed a band at 130 kDa. After it reacted with midgut juice of S. litura and S. exigua, a band of toxin protein was shown at 65 kDa. According to results of PCR analysis, it was found that cry1Aa, cry1Ac and cryII genes were present in CAB565 strain and it was different from B. thuringiensis subsp. kurstaki. cry1Aa, cry1C, cry 1D and cry1I genes were present in CAB566 strain and it was different from B. thuringiensis subsp. aizawai. As a result of plasmid DNA patterns, B. thuringiensis subsp. kurstaki KB099 strain and CAB565 strain had 5 and 10 plasmid DNAs, respectively. B. thuringiensis subsp. aizawai KB098 strain and CAB566 strain had 9 and 7 plasmid DNAs, respectively.