Authors
Tyler J. Dreaden and
John M. Davis, School of Forest Resources and Conservation, University of Florida, Gainesville 32611;
Carrie L. Harmon, Department of Plant Pathology, University of Florida, Gainesville 32611;
Randy C. Ploetz and
Aaron J. Palmateer, Tropical Research and Education Center, University of Florida, Homestead 33031;
Pamela S. Soltis, Florida Museum of Natural History, and
Jason A. Smith, School of Forest Resources and Conservation, University of Florida, Gainesville
Abstract
Laurel wilt, caused by the fungus Raffaelea lauricola, is an exotic disease that affects members of the Lauraceae plant family in the southeastern United States. The disease is spreading rapidly in native forests and is now found in commercial avocado groves in south Florida, where an accurate diagnostic method would improve disease management. A polymerase chain reaction (PCR) method based on amplifying the ribosomal small-subunit DNA, with a detection limit of 0.0001 ng, was found to be suitable for some quantitative PCR applications; however, it was not taxon specific. Genomic sequencing of R. lauricola was used to identify and develop primers to amplify two taxon-specific simple-sequence repeat (SSR) loci, which did not amplify from related taxa or host DNA. The new SSR loci PCR assay has a detection limit of 0.1 ng of R. lauricola DNA, is compatible with traditional and real-time PCR, was tested in four labs to confirm consistency, and reduces diagnostic time from 1 week to 1 day. Our work illustrates pitfalls to designing taxon-specific assays for new pathogens and that undescribed fungi can limit specificity.
