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Title: STRAIN-SPECIFIC DETECTION OF INTRODUCED BEAUVERIA BASSIANA IN AGRICULTURAL FIELDS BY USE OF SEQUENCE-CHARACTERIZED AMPLIFIED REGION MARKERS

Author
item Castrillo, Louela
item Vandenberg, John
item Wraight, Stephen

Submitted to: Journal of Invertebrate Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/23/2002
Publication Date: 1/1/2003
Citation: CASTRILLO, L.A., VANDENBERG, J.D., WRAIGHT, S.P. STRAIN-SPECIFIC DETECTION OF INTRODUCED BEAUVERIA BASSIANA IN AGRICULTURAL FIELDS BY USE OF SEQUENCE-CHARACTERIZED AMPLIFIED REGION MARKERS. JOURNAL OF INVERTEBRATE PATHOLOGY. 2003. v. 82. p. 75-83.

Interpretive Summary: Biological insecticides based on strains of the fungus Beauveria bassiana are registered for use in the United States and many other countries against a variety of insect pests. Strains of the fungus also occur naturally in the soil in many habitats. Our objective was to develop a method to identify a particular strain of the fungus (strain GHA) that is used in a commercial product available for pest control in the U.S. We wished to be able to distinguish this strain from others that may occur in soil in agricultural fields. We used a modification of a DNA fingerprinting technique (the polymerase chain reaction, or PCR) that involved using a small portion of the DNA unique to strain GHA. We discovered 3 unique DNA fragments and showed we could detect quantities of DNA as low as 10 picograms (10 one-trillionths of a gram) from strain GHA. This method will allow us to determine whether introduced strains (such as GHA) become established in soil following their use as microbial control agents. We will also be able to determine whether indigenous strains are displaced by application of other strains used in insect control.

Technical Abstract: Field studies on the efficacy and persistence of an introduced strain of Beauveria bassiana for insect control require detection assays to differentiate the non-native strain from indigenous populations. In this study we developed strain-specific molecular markers based on PCR amplification of sequence-characterized amplified regions (SCAR) in combination with dilution plating on semi-selective medium to detect and estimate B. bassiana strain GHA propagules from field samples. Using random amplified polymorphic DNA (RAPD) analysis, unique fragments that distinguished GHA from other strains of B. bassiana were obtained. Three amplicons, OPA-140.44, OPA-150.44, and OPB-90.67, generated with RAPD primers were cloned and sequenced and used as bases for designing SCAR primers OPA14 F/R445, OPA15 F/R441, and OPB9 F/R677, respectively. All three SCAR primers were highly sensitive, capable of detecting 10 to 100 pg gof B. bassiana GHA genomic DNA, and thus could be used to detect varying levels of the fungus in the field.