Title: Quantitative detection of Beauveria bassiana GHA (Ascomycota: Hypocreales), a potential microbial control agent of the emerald ash borer, by use of real-time PCR Authors
Submitted to: Biological Control
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 7, 2008
Publication Date: January 17, 2008
Citation: Castrillo,, L.A., Griggs, M., Vandenberg, J.D. 2008. Quantitative detection of Beauveria bassiana GHA (Ascomycota: Hypocreales), a potential microbial control agent of the emerald ash borer, by use of real-time PCR. Biological Control. 45:163-169. Interpretive Summary: Control of invasive insect pests cost municipalities, land managers and farmers billions of dollars every year. The emerald ash borer is an invasive insect that does not yet have a natural suite of enemies, and it must be managed using existing tools until longer term control can be achieved. We are testing the use of an insect pathogenic fungus, Beauveria bassiana, which is already registered for use as a commercial biological insecticide. We wish to determine the persisence of fungal spores in the field. For this study we developed a method to quantify the amount of fungal DNA from the strain that is the active ingredient in the commercial fungal insecticide. We showed that the method is sensitive enough to distinguish DNA from the target fungal strain from the DNA of other strains and other organisms. The method allows us to predict the number of fungal spores present, on leaves, bark or in soil, following application for emerald ash borer control. These findings improve our understanding of the possible impacts of fungal insecticides and will help us design effective strategies for their use as safe biological control agents.
Technical Abstract: Accurate monitoring of an introduced, mass-released microbial control agent is essential in evaluating its persistence and in designing application strategies for insect pest control. As part of our multi-year study on the development and use of the entomopathogenic fungus Beauveria bassiana against the emerald ash borer, a major invasive pest of ash trees, we are determining persistence of the fungus sprayed on ash trees and leached onto soil. We developed a real-time PCR assay to detect and quantify B. bassiana strain GHA, the active ingredient in the mycoinsecticide utilized in our field studies. Real-time PCR primers and probe, based on a 445-bp characterized amplified region fragment unique to GHA, generated a 96-bp fragment that was also specific to this strain. We also developed a DNA extraction method to maximize accuracy of quantification from environmental samples. DNA from fungal conidia in soil samples or recovered from leaf and bark washings was extracted using bead mill homogenization followed by purification of the crude extract using Sephadex-polyvinylpolypyrrolidone micro columns. Sensitivity assays comparing quantity of GHA DNA detected between pure and mixed samples showed that background genomic DNA from bark and leaf did not significantly affect detection sensitivity. However, background soil DNA and likely remaining co-extracted contaminants reduced sensitivity of assays at 400 pg or less of GHA DNA. Comparison of various conidial titer on ash bark and leaves versus predicted values based on real-time PCR showed that estimates were best obtained from samples with high titer of the fungus.