First Report of Bursaphelenchus yongensis on Pinus rigida in Korea.

During a pinewood nematode survey in 2011, a dead pitch pine (Pinus rigida) was found in Daejeon city, Korea. Samples were taken from several regions of the trunk and nematodes were extracted from the wood samples by a modified Baermann funnel method (1). Recovered nematodes were then cultured on Botrytis cinerea grown in potato dextrose agar (PDA) medium. Males and females (n = 10) were examined under an inverted light microscope. Morphologically, these nematodes were identified as Bursaphelenchus yongensis sharing the characters of the genus Bursaphelenchus including a large median bulb, offset cephalic region, a well-developed stylet with small basal knobs, female with vulva at 70% of body length, and males with terminal bursa. Females of B. yongensis were similar to B. muconatus and B. fraudulentus by presence of tail mucron, but they were differentiated by a smaller vulval flap. Males had spicules with small cucullus, pointed rostrum, and with strongly dorsally bent condylus. The identity of the nematode was confirmed through molecular diagnosis. DNA was extracted from each of 10 nematodes using commercial DNeasy kits (Qiagen, Valencia, CA) and the ITS regions were amplified with a primer set by PCR (Bio Rad, US/PTC-0220) (3,5). Amplified ITS DNA was cloned and sequenced, and the sequence data was deposited to GenBank (Accession No. KJ857070). The ITS sequence of Korean isolate (KJ857070) showed 99% similarity to that from Chinese isolate in GenBank (AM180513). ITS-RFLP patterns using five different restriction enzymes, RsaI, HaeIII, MspI, HinfI, and AluI, matched 100% with those previously reported for B. yongensis (2,4). Host pathogenicity tests were conducted from June to August on saplings of five native and introduced conifer species found in Korea (Abies sp., Larix leptolepsis, P. koraiensis, P. strobus, and P. thunbergii). All saplings were 5 to 6 years old and 15 saplings were inoculated for each species. B. yongensis was cultures on B. cinerea for 10 days at 25°C and the inoculum concentration was calibrated at 5,000 nematodes/50 μl sterile water. Nematodes were artificially inoculated by making a 1-cm-long incision into the stem using a sterile razor blade. B. yongensis was pathogenic on L. leptolepsis, causing wilt and death in all inoculated saplings; however, the nematode had no effect on P. thunbergii, P. koraiensis, Abies sp., or P. strobus. To our knowledge, this is the first report of B. yongensis in Korea. This nematode is a potential threat to L. leptolepsis; however, the role of B. yongensis in the mortality of P. rigida is uncertain at this time. References: (1) S. M. Ayoub. Plant Nematology in Agricultural Training Aid. Department of Food and Agriculture, Division of Plant Industry, Sacramento, CA, 1977. (2) W. Burgermeister et al. Nematology 11:649, 2009. (3) V. R. Ferris et al. Fund. Appl. Nematol. 16:177, 1993. (4) J. Gu et al. Russian J. Nematol. 14:91, 2006. (5) T. C. Vrain. J. Nematol. 25:361, 1993.