Venom allergen-like protein 2 (Vap2) was characterized from the pinewood nematode, Bursaphelenchus xylophilus, which is a destructive pathogen in several countries including Japan, China and Korea. Among three vaps of B. xylophilus (Bx-vap)reported in GenBank, Bx-vap2 showed the highest transcript level in both pine-grown propagative stage (PGPS) and media-grown propagative stage (MGPS). Bx-vap2 also was revealed that its transcript level over 10-fold increased in PGPS. In addition, western blot using BxVap2-polyclonal antibody showed that expression level of BxVap2 was significantly increased in PGPS. In immunohistochemistry, moreover, strong signals were detected around putative subventral gland in PGPS, whereas weak signals were observed in MGPS. These experimental results suppose pathogenic function of BxVap2 and migration assay using Bx-vap2 knock-down worms by RNA interference supports this postulation.

Bursaphelenchus xylophilus is known to be a major pathogen of the pine wilt disease (PWD). The pathogenecity of PWD is considered to be related to cell wall-degrading enzymes such as endoglucanases, expansins and pectate lyases (PELs). Two PELs, Bx-PEL1 and Bx-PEL2 are known to be expressed in B. xylophilus and regarded as a putative pathogenic factor. Recently, we developed stage-specific expressed tag library of B. xylophilus and identified a novel Bx-PEL3. We cloned Bx-PEL3 gene with RT-PCR, which showed high similarity to previously reported Bx-PELs. Phylogenetic analysis revealed that PEL3 was much closer to Bx-PELs than any other PELs. PEL3 has a conserved intron site as found in other Bx-PELs in the genomic DNA. Quantitative real-time PCR analysis revealed that Bx-PEL1 and Bx-PEL2 were more predominant in B. xylophilus than the Bx-PEL3. Recombinant Bx-PEL3 showed the activity for polgalacturonic acid and its physical conditions such as PH and Ca2+concentration for optimized activity were 9.0 and 0.5 mM, respectively. The localization of PEL3 transcript is the anterior body of B. xylophilus, near the esophageal gland. Taken together, these results suggest that a novel PEL3 gene is biochemically functional and can play a role as a putative pathogenic factor like other PELs.

In this study, we investigated the nematicidal activities of 102 Korean domestic plant extracts which have not been tested before against B. xylophilus. Nematicidal activity of plant extracts were varied according to plant species and extraction part. Among 102 plant extracts, 42 plant extracts in 22 families showed >50% nematicidal activity against pine wood nematode at 10 mg/mL concentration. Among 31 wood extracts, very strong nematicidal activity (100%) was produced from extract of Neolitsea aciculate, Prunus sargentii and Rhus chinensis at 10 mg/mL concentration. Extracts from Camellia japonica, Carpinus cordata, Daphniphyllum macropodum, Eucommia ulmoides, Ilex cornuta, Myrica rubra, Vaccinium bracteatum, and Zelkova serrata showed strong nematicidal activities (80-99%). The other samples exhibited activities <80%. In a test with leaf extracts, strong neamticidal activity was observed in extract of Machius japonica (100%) followed by Picea koraiensis (99.4%), Meliosma oldhamii (82.7%), and Zelkova serrata (81%). The other 37 plant species revealed <80% mortality. Strong nematicidal activity (>80%) was observed from bark extract of Carpinus cordata, Comus kousa, Cryptomeria japonica, Machius japonica, Quercus glauca and Zelkova serrata. Nematicidal activity of other 19 bark extracts was less than 80%. Twig extract of Smilax china and fruit extract of Forsythia koreana showed 86.6 and 81.4% nematicidal activity at 10 mg/mL concentration, respectively.

The pinewood nematode (PWN, Bursaphelenchus xylophilus) is known to be a major pathogen of the pine wilt disease (PWD). However molecular pathology of B. xylophilus is not completely understood, the pathogenecity of PWD is related to cell wall-degrading enzymes such as endoglucanases, expansins and pectate lyases (PELs). Recently, we developed stage-specific expressed tag library of B. xylophilus and identified a novel PEL, Bx-PEL3. We cloned Bx-PEL3 gene with RT-PCR, which showed high similarity to previously reported Bx-PELs. Phylogenetic analysis revealed that PEL3 was much closer to PELs of B. xylophilus than any other PELs. PEL3 has a conserved intron site as found in Bx-PEL2 in the genomic DNA analysis. Quantitative real-time PCR analysis revealed that Bx-PEL1 and Bx-PEL2 were more predominantly expressed than the Bx-PEL3 in B. xylophilus. The difference of expression level among Bx-PELs according to growth condition suggests that each Bx-PEL plays different biochemical role in the pathogenesis of the PWD.

Three acetylcholinesterases (AChEs) were identified from the pinewood nematode, Bursaphelenchus xylophilus. Sequence comparison with known AChEs in conjunction with three-dimensional structure analysis suggested that all BxAChEs share typical characteristics of AChE at the major catalytic structures. BgAChE3 was most predominantly transcribed and then followed by AChE1 and AChE2. Immunohistochemistry using anti-BxAChEs antibodies revealed that BxAChE1 is most widely distributed whereas BxAChE2 exhibits more localized distribution in neuronal tissues. BxAChE3 was detected from entire body together with some limited tissues, including mouth parts and alimentary lining, and determined to be the only soluble AChE, suggesting its localization in hemolymph or/and extracellular space. Kinetic analysis of in vitro expressed BxAChEs revealed that BxAChE1 has the highest substrate specificity whereas BxAChE2 has the highest catalytic efficiency with BxAChE3 having the lowest catalytic efficiency. Interestingly, presence of BxAChE3 in the pool of BxAChEs significantly reduced the inhibition of BxAChE1 and BxAChE2 by inhibitors. Knockout of BxAChE3 by RNAi significantly increased the toxicity of nematicides, suggesting the protective role of BxAChE3 against these toxicants. Based on several features, including tissue distribution, expression level, substrate kinetics and inhibition property, it appeared that BxAChE1 is the major AChE with the function of postsynaptic transmission whereas BxAChE3 has been evolved to acquire the function of chemical defense, perhaps intrinsically against secondary toxic compounds from host pine trees, such as α-pinene and limonene. BxAChE2 appears to play a role in post-synaptic transmission in specialized neurons but its detailed physiological function still remains to be elucidated.

This study was performed to investigate the escape of pine wood nematode (PWN), Bursaphelenchus xylophilus, from two vector species (Monochamus alternatus and M. saltuarius) through oviposition and feeding behavior. First, we checked number of PWNs escaped from M. alternatus emerged from three different cases of pine logs. In case A, healthy pine trees were cut into logs and left in pine forest infected with PWN. In case B, healthy pine trees were cut into logs, left in large screen cage, and let them oviposited by M. alternatus emerged from pine trees infested with PWN. In case C, pine trees which were harboring M. alternatus were cut into logs, and PWN was inoculated artificially. The M. alternatus adults emerged from the above three cases of pine logs were checked in the next year to know how many PWN they were harboring in their bodies. The percentages of M. alternatus harboring PWN (18.3 and 15.6%, respectively) and number of nematodes per vector (5,713.1±9,248.3 and 2,034.1±4,746.8 PWNs, respectively) in case A and B logs are similar to each other. However, the percentage and the number in case C (38.3% and 20,083.1±32,188.3 PWNs) were higher than those of case A and B. Among 52 M. alternatus adults harboring PWN from all the three cases, 20 adults (38.5%) were harboring more than 5,000 PWNs per beetle. And these 20 adults were harboring 97.9% of the total PWNs in 52 adults. Second, we checked the daily escape of PWNs from M. alternatus and M. saltuarius collected at pine forest infested with PWN. The PWN escaped from their vector body for 34.9±12.4 days for M. alternatus, and for 23.9±16.2 days for M. saltuarius, reaching at peak escape during the 2nd week of emergence of the two vector species. A 44.5 and 47.2% to the total PWNs escaped from vector body within 2 weeks of vector emergence for M. alternatus and M. saltuarius, respectively. The number of PWNs escaped from each vector was 3,570.6±5,189.2 and 1,556.2±1,710.3 for M. alternatus and M. saltuarius, respectively.

The most effective methods against pine wilt disease (Bursaphelenchus xylophilus, PWD), black pine bast scale (Matsucoccus thunbergianae), pine needle gall midge (Thecodiplosis japonensis) is the trunk injection of insecticides. The period of trunk injection that was considered with ecology and physiology of pests such as pine wilt disease, black pine bast scale and pine needle gall midge, was applied from Dec. to next Feb. and June, Dec. respectively. And there are differences in quantities of being injected chemicals to pine trees by the period of trunk injection. Thus, we investigated to prevent the effect of insecticides, to estimate the effect under the trunk injection of insecticides for pine trees against pests by the injecting period and time, boring site, opening and shutting of injection site. On Pinuns thunbergii, it was examined to figure out the effect of insecticides by the injecting period, twice a month on a sunny day around 10 AM from January to September. Injecting of insecticides was tested at a dose of 5ml per cm dbh of a pine tree after boring with hand drill at 50 cm high from the ground. As a result, abmectin 1.8% EC and phospamidon 50% SL were injected over 90% of mortalities from the end of January to the middle of March but as time goes by, injecting insecticides tended to decrease. To compare the volume of injecting insecticides in a day, between 10 AM and 2 PM on February 23rd, abamectin 1.8% EC was injected at a dose of 5ml per pine trees with micro pipette after boring with a drill machine at 50 cm high from the ground. As a result, injecting insecticide of a pine which was injected around 10 AM was completely injected and about 90% of the insecticide was injected in case of a pine that was injected around 2 PM. Trunk injection of insecticides was examined to figure out differences of the volume of injected insecticides before and after rain. There was no difference not only the rainfall but also the bored direction.

Three acetylcholinesterase (ace) genes were identified from Bursaphelenchus xylophilus and named as Bxace1, Bxace2 and Bxace3. Open reading fragments of Bxace1, Bxace2 and Bxace3 were composed of 622, 625 and 588 amino acids, respectively. Sequencing comparison with Torpedo ace gene identified cholinebinding site, catalytic triad functional site, three internal disulfide bonds and aromatic residues for the catalytic gorge. Transcriptional profiling by quantitative real-time PCR revealed that Bxace3 is more actively transcribed than Bxace1 (2-3 times) and Bxace2 (9-18 times) in both propagative and dispersal stages. The three ace genes were functionally expressed using baculovirus system. Kinetic analysis using three choline substrates demonstrated that BxAce2 has higher maximum velocity than BxAce1 (ca. 2 times) and BxAce3 (ca. 100 times) whereas BxAce3 shows lower Michaelis constant than BxAce1 (12.8 times) and BxAce2 (13.6 times). In inhibition assay using five organophosphates (OPs) and three carbamates (CBs), all the three BxAces were highly inhibited by paraoxon, DDVP and chlorpyrifos-oxon but not inhibited well by fenamiphos and fosthiazate. Interestingly, inhibition assay revealed that BxAce3 is less sensitive to all insecticides tested than other two BxAces. The inhibition kinetic data obtained in this study should provide essential information for the development of OP- and CB-based nematicidal agents againt B. xylophilus.

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.

The pinewood nematode (PWN, Bursaphelenchus xylophilus) is known as a virulent factor of the pine wilt disease, transmitted to pinewoods by the pine sawyer beetle, Monochamus alternatus. It is very hard to discriminate B. xylophilus from B. mucronatus because these Bursaphelenchus species are genetically and biochemically very close. Therefore, it has been necessary to detect PWN-infected trees for the prevention of pine wilt disease transmission in a short time. We developed polyclonal antibodies against B. xylophilus in BalbC mice and primarily screened with ELISA. Positive clones releasing polyclonal antisera revealed B. xylophilus-specific immuno-reactivity, which were at least two times higher than that of B. mucronatus. Two clones, D9-F10 and 1F3, were finally selected and exhibited specific immuno-reactivity for B. xylophilus, not for B. mucronatus in Western blot analysis. D9-F10 clone was reactive with a 43-kDa whereas 1F3 clone with two proteins, 40- and 45-kDa. Their isotypes against mouse Ig family were identical, kappa-light chain. These results suggest that these monoclonal antibodies can be useful for the development of diagnostic kit for the pine wilt disease.

Nematicidal activity of aliphatic compounds was tested against pien wood nematode, Bursaphelenchus xylophilus. There was a significant difference in nematicidal activity among function groups. In a test with alkanols and 2-alkenols, compounds with C8-C11 chain length showed 100% nematicidal activity at 0.5 mg/mL concentration. C6-C10 2-alkenals exhibited >95% nematicidal activity, but the other compounds with C11-C14 chain length showed weak activity. Nematicidal activity of alkanoic acids with C7-C11 chain length was strong. Whole compounds belonging to hydrocarbons, alkanals and alkanoic acetate showed weak nematicidal activity at 0.5 mg/mL concentration. Nematicidal activity of compounds which showed strong nematicidal activity at 0.5 mg/mL concentration was tested at a lower concentration. At 0.25 mg/mL concentration, whole compounds except C8 alkanol, C8 2-alkenol and C7 alkanoic acid showed >80% nematicidal activity. C9-C11 alkanols, C10-C11 2-alkenols, C8-C9 2-alkenals and C9-C10 alkanoic acids showed >80% nematicidal activity at 0.125 mg/mL concentration. Only C11 alkanol exhibited strong nematicidal activity at 0.0625 mg/mL concentration

The pine sawyer beetle, Monochamus alternatus, transmits the pinewood nematode (PWN, Bursaphelenchus xylophilus), causing the pine wilt disease (PWD), which gives rise to enormously economic as well as forest damage. However, PWN has been identified as a pathogen of PWD, it is very difficult to discriminate B. xylophilus from B. mucronatus in a short time, which are genetically and morphologically very similar. Therefore, it has been necessary to detect and eliminate PWN-infected trees in the forest area for the prevention of PWD transmission. Up to date, there is no report on biomarkers such as DNA and protein for the diagnosis of B. xylophilus. In this study, we produced a B. xylophilus monoclonal antiserum (D9-F10) from BalbC mice and screened its specificity with various proteins extracts. Western blot analysis revealed that the D9-F10 is only reactive with B. xylophilus protein extract among other tested protein extracts, indicating that the D9-F10 is specific for a B. xylophilus protein. Furthermore, two-dimensional electrophoresis showed the D9-F10 detects a very highly acidic protein, pI≒3.5. These results suggest that the D9-F10 monoclonal antibody is useful for the development of a diagnostic kit for the pine wilt disease.

The pinewood nematode, Bursaphelenchus xylophilus (Steinner & Buhrer) Nickle, has two different life stages according to several environmental factors: dispersal stage and propagative stages. The dispersal stage is closely related to the migration to other host pines, whereas the propagative stage is coupled to the direct cause of pine wilt. To establish expressed sequence tag (EST) database of two life cycles of B. xylophilus, subtractive EST libraries were constructed using suppressed subtractive hybridization (SSH). From 3,072 and 3,840 sequences of dispersal- and propagative-specific stage cDNA libraries, 1,927 and 2,604 clusters were generated, respectively, which were annotated by BLASTx and Gene ontology (GO). A total of 1,112 (57.7%) and 1,215 (46.7%) clusters from the dispersal- and propagative-specific stage cDNA libraries respectively had the matched BLASTx hits (E≤10-2), among which 913 (47.4%) and 960 (36.9%) were classified into three categories in Gene ontology. From GO database, some respective stage-specific genes were searched and estimated the relative transcripts level according to stages using the quantitative real-time PCR.

The pinewood nematode (PWN, Bursaphelenchus xylophilus) causes the pine wilt disease, transmitted to pinewoods by the pine sawyer beetle, Monochamus alternatus. It is very difficult to discriminate B. xylophilus from B. mucronatus. Therefore, it has been necessary to detect PWN-infected trees for the prevention of pine wilt disease transmission in a short time. The development of biomarkers such as DNA and protein is important for diagnosis of B. xylophilus. However, there have been no reports regarding biomarker identifications of B. xylophilus. In this study, polyclonal antisera were raised against whole proteins of B. xylophilus in BalbC mice and were primarily screened with ELISA. Twenty five among over 500 cell lines releasing polyclonal antisera revealed B. xylophilus-specific immuno-reactivity, which was at least three times higher than that of B. mucronatus. Three cell lines among them were secreting monoclonal antibody through further screening. These cell lines only detect about a 33-kDa protein in B. xylophilus in the western blot. These results suggest that these monoclonal antibodies will be useful for the development of diagnostic kit for the pine wilt disease.

Commercial plant essential oils from 27 plant species were tested for their nematicidal activities against the pinewood nematode, Bursaphelenchus xylophilus. Good nematicidal activity against B. xylophilus was achieved with essential oils of coriander (Coriandrum sativum), styrax (Liquidamber orientalis) and valerina (Valeriana wallichi). Analysis by gas chromatography-mass spectrometry led to identification of 26, 10 and 4 major compounds from coriander (Coriandrum sativum), styrax (Liquidamber orientalis) and valerina (Valeriana wallichi) oils, respectively. These compounds from three plant essential oils were tested individually for their nematicidal activities against the pine wood nematode. Among compounds, benzaldehyde, trans-cinnamyl alcohol, cis-asarone, octanal, nonanal, decanal, trans-2-decenal, undecanal, dodecanal, decanol, and trans-2-decen-1-ol showed strong nematicidal activity. The essential oils and their described herein merit further study as potential nematicides against the pine wood nematode.

소나무재선충의 주 매개충인 솔수염하늘소의 우화생태를 연구하기 위하여 1999년부터 2002년까지 경남 진주의 소나무재선충병 피해임지에서 죽은 소나무를 대상으로 조사하였다. 양지에서 솔수염하늘소 성충의 우화시기를 조사한 결과 4개년 평균 매년 5월 15일을 전후하여 우화하기 시작하였고, 50% 우화일은 6월 중순경이었다 우화 종료일은 7월 상순-하순으로 해에 따라 차이가 있었으며, 수컷의 50% 우화일은 암컷보다 해에 따라 1-4일이 빨랐다. 그러나 음지에서의 50% 우화일은 양지에서보다 17일이 늦어졌다. 2001년 2월에서 4월까지 벌채한 공시목으로부터 우화한 성충 중에서 동년에 우화한 1년 1세대 충의 비율은 97.6%이었고, 이듬해인 2002년에 우화한 2년 1세대 충의 비율은 2.4%이었다. 솔수염하늘소 성충은 암수 구분 없이 24시간 내내 우화하였지만, 오전 8시부터 12시 사이에 우화하는 성충의 비율이 32.3%로 가장 높았으며, 새벽 2시부터 오전 8시까지는 우화율이 낮았다. 번데기방에서 성충이 우화한 후 나무 밖으로 탈출하는 데 소요되는 시각은 68.0초이었다.