Laboratory Evaluation of Beauveria bassiana Isolates on Red Flour Beetle Tribolium castaneum and Their Characterization by Random Amplified Polymorphic DNA

Abstract

Beauveria bassiana (Balsamo) Vuillemin is an important natural regulator of insect populations. Identification of a suitable molecular marker for detecting a virulent phenotype on a target pest would be useful in screening for effective isolates against the pest. Nine isolates of B. bassiana were tested for their virulence to adults of Tribolium castaneum (Herbst) in laboratory bioassay with 1×108 conidia mL-1. DNA markers provide more detailed genomic information. DNA fingerprints were generated by RAPD markers. Fungal DNA was extracted by CTAB. Twelve random oligonucleotide primers were used for amplification. After bioassay, three arbitrary categories of isolates were chosen i.e. isolates that caused > 45%, 45-30% and < 30% mortality, and were classified as highly (H), moderately (M), and less (L) virulent isolates based on average mortality, respectively. Also, based on LT50 values, three arbitrary categories were chosen i.e. isolates with < 80 h, 80-100 h and > 100 h LT50 values, and were classified as highly (H), moderately (M), and less (L) virulent isolates, respectively. The results of bioassay showed that isolates IRAN 440C and DEBI 004 were the causative agents of mycoses with the highest and lowest lethal effect, respectively. The lowest LT50 value was related to DEBI 014. Cluster analysis of the RAPD data showed four clusters according to similarity, following cluster analysis using the Jaccard similarity coefficient and clustering was done using un-weighted pair group method with arithmetic (UPGMA). The results showed that there was genetic diversity between these isolates, but the groups based on virulence rating and LT50 values did not match with the RAPD clusters completely. Keywords: Entomopathogenic fungus, Virulence, Bioassay, Tribolium castaneum, RAPD analysis