A molecular tool for detection and tracking of a potential indigenous Beauveria bassiana strain for managing emerald ash borer populations in Canada

摘要

Emerald ash borer is an invasive species from Asia. Beauveria bassiana strain L49-1AA is being tested for the control of emerald ash borer in Canada, using an autocontamination trapping system. We have developed a simplified allele discrimination polymerase chain reaction (PCR) assay to screen B. bassiana strain, L49-1AA from other Beauveria species by targeting the inter-strain genetic differences in 5' end of EF1-alpha gene of the genus Beauveria. A single nucleotide polymorphism (SNP) site, T C was identified only in L49-1AA and was used to develop a simplified allele discrimination polymerase chain reaction (PCR) assay based on a modified allelic inhibition of displacement activity (AIDA) approach for distinguishing B. bassiana L49-1AA from all background Beauveria isolates. The SNP site was employed to design inner primers but with a deliberate mismatch introduced at the 3' antepenultimate from the mutation site in order to maximize specificity and detection efficiency. Amplification was specific to L49-1AA without cross-reaction with DNA from other Beauveria strains. In addition, the designed primers were also tested against environmental samples in L49-1AA released plots and observed to be highly efficient in detecting and discriminating the target strain, L49-1AA from both pure and crude DNA samples. This new method can potentially allow for more discriminatory tracking and monitoring of released L49-1AA in our autocontamination and dissemination projects for managing EAB populations. Additionally, the modified-AIDA format has potential as a tool for simultaneously identifying and differentiating closely related Beauveria species, strains/isolates as well as general classification of other pathogens or organisms. Crown Copyright (C) 2014 Published by Elsevier Inc. All rights reserved.