Specific PCR assays for the detection of DNA from Beauveria bassiana F-263, a highly virulent strain affecting the Japanese pine sawyer, Monochamus alternatus (Coleoptera: Cerambycidae), by a sequence-characterized amplified region (SCAR) marker

Abstract

To monitor the Beauveria bassiana F-263 strain, which is under commercial development for controlling the Japanese pine sawyer Monochamus alternatus, I developed molecular markers based on a sequence-characterized amplified region (SCAR). A diagnostic fragment generated from random amplified polymorphic DNA analysis (RAPD) was converted into two SCAR markers designated as C81068 and C8352 using sequence information from the fragment. The two primer pairs for the SCAR markers were sensitive, but the primer pair for C8352 had superior sensitivity, capable of detecting as little as 10 pg B. bassiana F-263 strain DNA, regardless of whether the sample contained pure F-263 DNA or was contaminated with DNA from another strain. The markers were used in combination with dilution plating on selective medium, which allowed for both detection of B. bassiana F-263 presence and indirect density estimation of the strain. In addition, I developed a simple DNA extraction method by boiling fungi directly from colonies on medium. This DNA extraction method avoids laborious DNA extractions and expedites SCAR assays for monitoring the B. bassiana F-263 strain in the environment.