First Report of Pine Wilt Disease Caused by Bursaphelenchus xylophilus on Pinus thunbergii in the Inland City of Zibo, Shandong, China.

The pinewood nematode (PWN) Bursaphelenchus xylophilus (Steiner & Buhrer 1934) Nickle 1970 is the causal agent of pine wilt disease. It is especially damaging in East Asian countries, including Japan, China, and Korea. In China, the nematode has been found in Anhui, Guangdoung, Guizhou, Chongqing, and Zhejiang Provinces since its discovery in Jiangsu Province in 1982 (1). China is confronted with an enormous threat to its pine forests. B. xylophilus is transmitted by the insect vector pine sawyer beetle (Monochamus alternatus). The main host trees are Pinus massoniana, P. thunbergii, and P. densiflora, which are the most common pine trees in China. Shandong Province, located north of Jiangsu Province, is a high-risk area because it was thought to be the northernmost suitable area for the pine wood nematode. P. tabulaeformis, P. densiflora, and P. thunbergii are the principal hosts. In 2010, a pine tree with suspected wilt disease was found in Lushang Forest (36°16'31.11″ N, 118°03'59.79″ E) of P. thunbergii located in Zibo city of Shandong Province. Symptoms were systemic, with almost all leaves brown or yellowish; the tree was nearly dead. Wood samples were collected and nematodes were extracted using a modified Baermann's funnel method. After 12 h, the nematodes were collected from the wood chips, and their morphology was observed with an inverted light microscope (Nikon 90i, Japan). Nematodes had a typical Aphelenchoid-type esophagus and female vulva flap. Females had subcylindrical tails, usually with broadly rounded terminus, some with a short mucro, and flat vulva, whereas males had large paired arcuate spicules with a sharply pointed prominent rostrum, and typical disc-like expansions on distal ends. Standard measurements of these nematodes were as follows: 25 females: body length = 960.9 ± 117.4 (791.5 to 1,265.2) μm, a = 32.1 ± 5.1 (23.7 to 44.5), b = 13.6 ± 1.4 (11.4 to 16.1), c = 28.3 ± 4.6 (21.7 to 42.2), V = 77.8 ± 2.0 (74.2 to 83.9), stylet length = 13.7 ± 1.6 (11.4 to 17.6) μm; 21 males: body length = 785.6 ± 103.2 (609.6 to 1,004.5) μm, a = 33.3 ± 4.4 (26.0 to 40.8), b = 11.9 ± 1.3 (9.0 to 14.6), c = 31.0 ± 2.7 (25.5 to 37.1), stylet length = 13.5 ± 1.9 (11.0 to 17.5) μm, spicule length = 18.8 ± 2.5 (14.9 to 23.9) μm. The morphometrics of this population, apart from body length and "a" value, which are shorter than the Portugal isolate measured by Mota et al. (3), are very much in the same range reported for B. xylophilus. For a more accurate identification, DNA was extracted from individual nematodes using a liquid nitrogen method. The internal transcribed spacers (ITS-1, ITS-2, 5.8S) were amplified by using PCR (2). Nucleotide sequences were compared with the sequences of B. xylophilus in GenBank, accession nos. JN684828 (Portugal), JN684829 (Portugal), JF826219 (Madeira Island) and JQ288086 (Japan). The ITS DNA sequences of the nematode from P. thunbergii were 99% identical to those of B. xylophilus in GenBank. A sequence of this nematode was submitted to the GenBank database and assigned the number KC460340. We have thus confirmed that B. xylophilus is now present north of Changjiang River in Zibo city, Shandong Province. This range expansion, perhaps the result of global warming, will affect both domestic and international quarantine efforts to control the further spread of pinewood nematode. References: (1) X. Y. Cheng et al. Heredity 100:356, 2008. (2) K. Metge and W. Burgermeister. J. Plant Dis. Protect. 113:275, 2006. (3) M. Mota et al. Nematology 1:727, 1999.