Kyushu Research Center, Forestry and Forest Products Research Institute, Ministry of Agriculture, Forestry and Fisheries, Kurokami 4-11-16, Kumamoto 860-0862, Japan

Background and objectives
Pine wilt disease is the most serious disease of forest trees in Japan. Pine wood nematode, Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle, the causal agent of this disease, differ in pathogenicity among isolates [1]. However, there are few studies on the genetic variation of B. xylophilus isolated from some stands in Japan. In this study, we report intraspecific genetic variation of B. xylophilus by randomly amplified polymorphic DNA (RAPD).

Materials and methods
Thirteen isolates of B. xylophilus and one isolate of B. mucronatus were used for this study. Ten isolates of B. xylophilus were isolated from Japanese pine sawyer (Monochamus alternatus) or wilted Japanese black pine (Pincis thunbergii) at three forest stands in Kyushu district, southwestern Japan. Three isolates of B. xylophilus were isolated in Kanto district, eastern Japan. All of these isolates were cultured monoxenically on Botrytis cineria grown on PDA. Genomic DNA was extracted from nematodes and hyphae of B. cineria. The DNA fragments were amplified by PCR with twenty 10-mer oligonucleotide primers (Primer kits B and X, Operon Technologies Inc.). The reaction mixture (15 l) contained 50 mM Tris HCI at pH 8.5, 5 mM MgCI2, 0.01 mM EDTA, 4 mM Tartrazine, 2.0% Ficoll 400, 7. 5 230 g of BSA, 200 M each of dNTP, 0.5 M primer, 2-5 ng of template DNA and 0.3 units of TDNA polymerase (TOYOBO). The amplification was carried out in the Rapidcycler (Idaho Technology Inc.), programed for 1 min at 94C, 60 cycles of 10 sec at 94C, 30 sec at 36C, 1 min at 72C, and 5 min at 72C. The products were electrophoresed in 1% agarose gel in TAE buffer and visualized with ethidium bromide and UV illumination.

Results and conclusion
DNA fragments specific to B. xylophilus were amplified with nine primers. Banding patterns between B. xylophilus and B. mucronatus were completely different with most of the primers. Banding patterns among isolates of B. xylophilus were relatively simmilar. Some different DNA fragments were amplified among the isolates of B. xylophilus from different stands and even a single stand. The resuIts show that two morphologically similar species of Bursaphelenchus are dinstinguishable by RAPD and intraspecific genetic variation of B. xylophilus exists in Japan.

1. Kiyohara T, 1989. Bulletin of the Forestry and Forest Products Research Institute 353, 127-176 (in Japanese with English summary).