Single-Strand Conformation Polymorphism Analysis of Ribosomal DNA for Detection of Phytophthora ramorum Directly from Plant Tissues

Ping Kong, Patricia A. Richardson, Chuanxue Hong, Virginia Polytechnic Institute and State University, Hampton Roads Agricultural Research and Extension Center, 1444 Diamond Springs Road, Virginia Beach, VA 23455, (757) 363-3908, chhong2@vt.edu; and Thomas L. Kubisiak, USDA Forest Service, Southern Institute of Forest Genetics, Saucier, MS 39574s

At the first science symposium of sudden oak death, we reported use of a single strand conformation polymorphism (SSCP) analysis for rapid identification of Phytophthora ramorum in culture (Kong and Hong 2002). We have since assessed and improved the fingerprinting technique for detecting this pathogen directly from plant tissues. The improved SSCP protocol uses a single run PCR reaction with the same primer pair (ITS6/7) and consistently detects P. ramorum at 10 fg per reaction or above. It provides reliable diagnoses of sudden oak death no matter whether it is a single infection or dual infection (a second Phytophthora species involved). This technique also can provide accurate diagnoses of diseases caused by 12 other species of Phytophthora without additional work. These species (P. cactorum, P. cambivora, P. cinnamomi, P. cryptogea, P. citricola, P. citrophthora, P. gonapodyides, P. lateralis, P. megasperma, P. nemorosa, P. nicotianae, and P. pseudosyringae) are common in ornamental plant and forest tree nurseries as well as in natural forested environments. The through-put capacity of this technique can be greatly improved by use of fluorescence-based technologies such as those common to most commercially available DNA sequencers. This study provides an alternative protocol with increased detection scope and accuracy at a reduced cost for future surveys of nurseries, parks, and forests for Phytophthora spp.

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