First Report of Bursaphelenchus pinophilus on Korean pine (Pinus koraiensis).

Abstract

The genus Bursaphelenchus Fuchs, 1937 contains approximately 90 species (3) that are morphologically similar. Pine wood nematode, Bursaphelenchus xylophilus (4) Nickle, 1970, is the causal organism of pine wilt disease and accurate identification is essential for diagnosis of the disease. In Korea, pine wilt disease was first reported in 1988 and devastated 6,800 ha of pine forest through 2008. For a survey of trees with pine wilt disease, wood samples were taken randomly from dead Pinus koraiensis in Namyangju, Gyeonggi Province in Korea. The extracted nematodes from dead trees were maintained in culture on Botrytis cinerea and morphological characteristics were observed with an inverted light microscope (Leica DE/DMI 3000B). Identification of Bursaphelenchus spp. based on morphological characteristics is difficult, especially for identification of juveniles that carry few morphological features for species identification. The internal transcribed spacer (ITS) region in ribosomal DNA provides useful molecular diagnostic markers for this genus (1). The nematodes were provisionally identified as Bursaphelenchus pinophilus based on the characteristic long and arcuate body shape, male spicule with distinctive rostrum and small cucullus, female vulval flap, and mucronate conical tail. Other Bursaphelenchus spp. with vulval flaps and spicules with cucullus are B. xylophius, B. mucronatus, B. abruptus, and B. pinophilus. For molecular diagnosis, DNA was extracted from more than 30 individual nematodes with a DNeasy Kit (Qiagen, Valencia, CA) and ITS regions 1, 2, and 5.8S in rDNA were amplified by PCR (US/PTC-0220; Bio Rad, Hercules, CA). The ITS-restriction fragment length polymorphism pattern was consistent with that of B. pinophilus (2). The ITS rDNA sequence of B. pinophilus from Korean pines had a 98% sequence homology to that of B. pinophilus in GenBank (Accession No. AM160664). The pathogenicity of B. pinophilus has not been determined. To our knowledge, this is the first report of B. pinophilus on P. koraiensis, but it was previously reported from Poland, Germany, and Portugal on P. sylvestris and P. pinaster (1).