Three acetylcholinesterases (ACEs) were identified from the pinewood nematode, Bursaphelenchus xylophilus, and named BxACE-1, BxACE-2, and BxACE-3. Sequence comparison with known ACEs in conjunction with three-dimensional structure analysis suggested that all BxACEs share typical characteristics of ACE but show some differences in the peripheral anionic site. BgACE-3 was most predominantly transcribed, followed by ACE-1 and ACE-2. Immunohistochemistry using anti-BxACEs antibodies revealed that BxACE-1 is most widely distributed whereas BxACE-2 exhibits more localized distribution in neuronal tissues. BxACE-3 was detected from entire body together with some limited tissues, and determined to be soluble. Kinetic analysis of in vitro expressed BxACEs revealed that BxACE-1 has the highest substrate specificity whereas BxACE-2 has the highest catalytic efficiency with BxACE-3 having the lowest catalytic efficiency. Interestingly, presence of BxACE-3 in the pool of BxACEs significantly reduced the inhibition of BxACE-1 and BxACE-2 by inhibitors. Knockout of BxACE-3 by RNAi significantly increased the toxicity of nematicides, supporting the protective role of BxACE-3 against these toxicants. Taken together, BxACE-1 appears to be the major ACE with the function of postsynaptic transmission whereas BxACE-3 has been evolved to acquire the function of chemical defense. BxACE-2 appears to play a role in post-synaptic transmission in specialized neurons.

The pinewood nematode, Bursaphelenchus xylophilus is a serious forest pathogen in many countries including Japan, China and Korea. To minimize the environmental problems caused by synthetic chemicals broadly utilized in the control of B. xylophilus, we estimated the nematicidal potency of 97 phytochemicals (49 monoterpenes, 17 phenylpropenes, 16 sesquterpenes and 15 sulfides) against B. xylophilus by measuring their inhibition activity against acetylcholinesterases (ACE, EC 3.1.1.7) of B. xylophilus (BxACEs). In the primary inhibition assay using B. xylophilus crude protein, (+)-α-pinene, (-)-α-pinene and 3-carene in the monoterpenes, ο-anisaldehyde and coniferyl alcohol in the phenylpropenes and cis-nerolidol in the sesquterpenes were observed the > 50% BxACE inhibition rate and they were subsequently estimated the inhibition rate against three recombinant BxACEs. The (+)-α-pinene and coniferyl alcohol showed the high inhibition rate against BxACE-1 and BxACE-2. Interestingly, coniferyl alcohol also exhibited the high inhibition rate against BxACE-3 which would be insensitive to ACE inhibitors.

A survey was conducted in the northern conifer forests of Korea from February to May in 2009 for the Monochamus species vectored Bursaphelenchus species. Dead pine trees infested with bark beetle larvaes were collected from Pinus densiflora and P. koraiensis located in the following target areas: Pocheon, Yeongi, Jincheon, Chuncheon, and Bonghwa. A cerambycid beetle, M. saltuarius was only found from sampled log piles in target areas. Bursaphelenchus species carried by M. saltuarius was only isolated in Pocheon, Gyeonggi province. The nematodes recovered from 17 (71%) of 24 beetles from P. koraiensis and 12 (75%) of 16 beetles from P. densiflora. The average number of nematode per adult was 1,974 and 11,823 from P. koraiensis and P. densiflora, respectively. The nematode was also recovered from the inner bark of both Pinus species infested with M. saltuarius larvae. Bursaphelenchus species identification was performed by morphological characters and molecular analysis with ITS-RFLP patterns and sequences of ITS and D2D3 region. Both morphological and molecular characters match well with the original description of the European type of B. mucronatus. This is the first report of M. saltuarius species as a vector of the European type of B. mucronatus on both P. densiflora and P. koraiensis in Korea.

Since pine wilt disease was first reported in Busan in 1988, this disease has become a serious threat to pine forests in Korea. A general survey for Bursaphelenchus xylophilus group (B. xylophilus, B. mucronatus) nematodes were carried out to determine their geographic range, host tree species and insect vectors, Monochamus species. Based on the survey results, B. xylophilus were found over 57 cities or district and a similar nematode, B. mucronatus also has been isolated over 30 cities or district including Jeju and Pocheon. The Asian type of B. mucronatus was founded from Pinus thunbergii and P. densiflora, generally associated with M. alternatus as its insect vector. The European type of B. mucronatus which was limited distribution to the central areas, such as Yeongwol, Bonghwa, Yeongdeok, was also found on P. koraiensis and P. desiflora associated with M. saltuarius. Both Monochamus species transmitted both Bursaphelenchus species and co-existed in Muju and Yeongdeok predicted as their range margin. Their range margins shifted northwards compared to 2006, seemingly as a result of climate change. Recently, the European type of B. mucronatus has been identified in other several areas since it was first reported in Pocheon in 2008. Several reports indicate the potential pathogenicity of European type of B. mucronatus and other species, B. sexdentati and B. lenoni in Europe. Therefore, sustainable survey and the study for the newly invading Bursaphelenchus species as well as the member of B. xylophilus group should be performed to protect Korean forests.