Use of a Microsatellite Assay to Differentiate Strains of the Entomopathogenic Fungus Beauveria bassiana




Cassie Fairchild

Introduction

Entomopathogenic fungi are naturally occurring pathogens of arthropods which have gained recognition as useful biological control agents for a variety of arthropod pests. Highly specific genetic markers would greatly facilitate research on these fungi by allowing for the differentiation of naturally present strains and strains that are artificially applied. Beauveria bassiana is one species of entomopathogenic fungus that has been pursued for its biocontrol potential.

Microsatellite loci, first discovered in 1982 (Hamada et al., 1982), are tandemly repeated di- or trinucleotide sequences known to exhibit high rates of evolution and therefore a relatively high degree of intraspecific variability. Rehner and Buckley (2003) characterized eight highly polymorphic microsatellite loci for B. bassiana. Here I examined four of these microsatellite loci (Ba02, Ba06, Ba08, and Ba12) for two strains of B. bassiana.

The objective of this study was to assess the capacity of these four microsatellite markers to differentiate between strains of B. bassiana that are relevant to ongoing research on the biocontrol potential of this fungus. Hence, the two strains tested were the strain naturally present in the soil at the Louis Calder Biological Field Station in Armonk, NY and the strain present in Botanigard ES, a commercially available suspension of B. bassiana spores.

Figures


Figure 1-Size Fractionation of PCR Products on 1.5% Agarose Gel. DNA purified from mycelia of 2 fungal isolates (labeled 1 & 2 in the figure) was PCR-amplified for each of 4 microsatellite loci. Products were run on a 1.5% agarose gel stained with EtBr.


Figure 2-Size Fractionation of Loci Ba02, Ba06, & Ba12 on a Sequencing Gel. DNA purified from mycelia of 2 fungal isolates (labeled 1 & 2 in the figure) was PCR-amplified for each microsatellite locus in the presence of 33P-dATP. Products were run in a polyacrylamide (sequencing) gel to assess small size differences between isolates. Size polymorphisms were detected at loci Ba06 & Ba12 but not at locus Ba02. Locus Ba08 is not shown because the fractionation of isolates 1 & 2 at this locus could not be clearly interpreted.


The ability to differentiate strains of B. bassiana with highly specific molecular markers would facilitate several aspects of the ongoing research on this fungus. First, accurate assessments of the rate at which the fungus contacts organisms when applied in the field would be made possible. molecular markers to make these assessments would improve evaluations of the efficacy of the fungus in controlling pest arthropods as well as increase our understanding of the effects of the fungus on non-target organisms. Further, researchers could use these markers to track the fate of the fungus when it is applied and, thus, examine the spatial and temporal impacts of applying the fungus in a given system.

Here four microsatellite loci were successfully PCR-amplified for 2 isolates of B. bassiana using the primers designed by Rehner and Buckley (2003). Sequencing of the resultant PCR products yielded inconclusive results due to apparent replication errors during amplification. Thus, the numbers of repeats could not be determined using the DNA sequence data. However, size fractionation on a sequencing gel revealed possible polymorphisms between isolates 1 and 2 for loci Ba06 and Ba12. No polymorphism is apparent for locus Ba02. Thus, loci Ba06 and Ba12 are likely candidates for molecular markers capable of differentiating between applied and natural strains of B. bassiana. Improvements in the PCR protocol (e.g. use of a higher fidelity DNA polymerase) may permit more conclusive determination of the numbers of repeats at these loci in the future.

Full Paper

Acknowledgments

I would like to thank Dr. Rubin, Joe Frezzo, and Leleesha Samaraweera for all their assistance and guidance on this project. Thanks also to all my classmates for sharing their knowledge and good company in the lab.


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