MOLECULAR AND PHENOTYPIC CHARACTERIZATION OF BACILLUS-THURINGIENSIS ISOLATED FROM LEAVES AND INSECTS

Bacillus thuringiensis isolates from the phylloplane of organically cu ltivated cabbage were characterized using molecular and phenotypic met hods. Of the 58 isolates under study, 31 belonged to serovar kurstaki, 16 did not react with any of the currently recognized antisera, 7 rea cted with known antisera, and 4 could not be serotyped as they were no nmotile. Round crystals were found in 26 isolates, while bipyramidal c rystals mere found in the remaining 32 isolates, all of which had acti vity to lepidopteran larvae. Further, one isolate with unknown serotyp e and round crystals had lepidopteran activity. Colony hybridization w as found to be a useful tool for screening the isolates for specific g ene homologies and showed good correlation with the phenotypic observa tions. Polymerase chain reaction (PCR) was used for confirmation of th e colony hybridization data, in most cases with concordant results. Ho wever, in one case some of the colony hybridization data could not be confirmed by PCR, due to DNA sequence variations in the binding area o f one of the primers. The random amplified polymorphic DNA (RAPD) anal ysis showed that isolates otherwise indistinguishable could be disting uished by this method. However, the method was not able to distinguish the 31 kurstaki isolates. Further, the kurstaki isolates could not be distinguished from the B. thuringiensis serovar kurstaki HD-1 strain used in commercial products for lepidopteran control. One of the isola tes was a serovar israelensis, but no genes encoding dipteran activity could be detected, and the RAPD analysis revealed that the DNA finger print of this israelensis isolate deviated from the israelensis ONR60A isolate used in commercial products. In conclusion we find that a mol ecular method like colony hybridization is suitable for screening larg e collections of bacteria. When colony hybridization data are combined with RAPD analyses isolates can be grouped based on genetic potential and DNA fingerprint, whereby further characterizations by PCR and the more labourious phenotypic methods can be performed more effectively. (C) 1998 Academic Press.