Pine tree, a dominant species in the flora of Korea is the most beloved tree in Korea. However, recent outbreak of pine wilt disease caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus threatens the vegetation of Pinus species seriously. Furthermore, Pinus densiflora and P. thunbergii, the predominant species in Korea forest are highly susceptible to PWN. Therefore, there has been an urgent demand for the development of a new nematocidal compounds to control PWN, which spurred the national research for the development of new compounds. To find novel sources for nematocidal agents, we used various plant resources for the development. In addition to the foreign plant extracts themselves, we also used the extracts of endophytes composed of over 300 endophytic fungi and 1,000 endophytic bacteria from plants which was reported to contain nematocidal activity. Several extracts of endophytes and plant extracts contained strong nematocidal activity, and the resources are analyzed to identify the active nematocidal compounds. These integrated approach of finding effective nematocidal compounds from plants could be a novel way to elucidate the sources for brand-new nematocidal agents.

Pine trees are ecologically important in Korea. They are seriously imperiled by Pine wilt disease (PWD), by pine wood nematode (PWN, Bursaphelenchus xylophilus). Here, we isolated and characterized bacterial endophytes (BEs) from pine trees in Korea for biological control of PWN using BE metabolites. Using culture-dependent approach BE isolates were extracted from three tissues (needles, stems, and roots) of four pine species across 18 sampling sites in Korea. Bacterial isolates were characterized into 389 distinct isolates based on 16S rDNA sequencing. Ethyl acetate crude extracts (CEs) of bacterial liquid cultures were prepared using ethyl acetate and screened for nematicidal activity against PWN. BEs (1,622 isolates) were isolated; their taxonomic binning resulted in 215 operational taxonomic units (OTUs). Analysis of species richness and Shannon’s diversity of the three tissues revealed that BEs colonized the needles more than the stem and root tissues. Furthermore, based on nematicidal activity screening of 389 isolates, 44 BEs were identified, with two isolates exhibiting a significant inhibitory activity against PWN. Taken together, these data revealed numerous nematicidal BEs in pine trees, providing new insights that can serve as an effective and promising alternative approach to combat PWD.

Pine wood nematode (PWN), Bursaphelenchus xylophilus causes devastating damage to pines worldwide. To develop new agent for controlling PWN, alkyloxyalcohols(A-H) were synthesized and nematicidal activity was tested against PWN. As a control, monochamol, which reported to have nematicidal activity was also tested. The nematicidal activity was diffferent according to carbon chain length in compounds. LC50 values of F, G and H were as same as that of monochamol. However A-E which have shorter carbon length than F-H showed weak nematicidal activity.

소나무재선충(pine wood nematode, PWN) 감염 소나무를 현장에서 신속하게 진단을 할 수 있는 방법은 현재 없다. 본 연구에서는 PWN특이적인 항원으로 알려진 PWN-GaLectin을 baculovirus발현체계로 발현시켜서 총 1,464개의 fusion hybridoma 세포 주 라이브러리 를 제작 하는 항원으로 이용하였다. 총 1,464개의 fusion hybridoma 세포 주 중, PWN-GaLectin에 대한 높은 반응을 보이는 62개의 fusion hybridoma 세포 주를 선별했다. 이들 중, 표준 PWN 감염소나무 PBS추출물과 PWN 단백질 추출물에 강한 반응을 보이는 세포 주 12개를 선 별하여 단클론 항체(monoclonal antibody, Mab) 분비세포 주 확립을 위한 limited dilution을 실시하였다. Mab분비세포 주 확립을 위해서 표 준 PWN 감염소나무 추출물에 대한 반응이 표준 정상 소나무 추출물 보다 높은 세포 주들을 선별했다. 그리고 추가로 PWN 단백질 추출물에 대 해서 3종의 비 병원성 선충 단백질 추출물 보다 높은 반응을 보이는 세포 주들도 선별, 확립했다. 본 연구에서 확립된 Mab들을 우리는 현장과 실 험실에서 사용할 수 있는 신속진단키트의 개발에 이용할 수 있을 것이다.

In this study, we isolated and identified an aggregation-sex pheromone from Monochamus saltuarius, the major insectvector of the pine wood nematode in Korea. Adult male of M. saltuarius produces 2-undecyloxy-1-ethanol, which is knownto be an aggregation-sex pheromone in other Monochamus species. We performed field experiments to determine the attractivenessof the pheromone and other synergists. More M. saltuarius adult beetles were attracted to traps baited with the pheromonethan to unbaited traps. Ethanol and (-)-α-pinene interacted synergistically with the pheromone. Traps baited with pheromone+(-)-α-pinene+ethanol were more attractive to M. saltuarius adults than traps baited with pheromone, (-)-α-pinene, or ethanol alone.Ipsenol, ipsdienol, and limonene were also identified as synergists of the aggregation-sex pheromone for M. saltuarius adults.

We evaluated the larvicidal and nematicidal activities of 48 3-acylbarbituric acids analogues against the Asian tigermosquito, Aedes albopictus and the pine wood nematode, Bursaphelenchus xylophilus, organisms of increasing global concern.Among 48 3-acylbarbituric acids analogues, four compounds 10, 14d, 14g, and 19b showed >90% larvicidal activity againstAe. albopictus at 10μg/mL concentration, and one (compound 10) showed the strongest larvicidal activity against Ae. albopictus,with a LC50 value of 0.22μg/mL. Only compound 18 showed strong nematicidal activity against pine wood nematode.Most active compounds possessed similar physicochemical properties; thus, actives typically had ClogP values of around1.40-1.50 and rel-PSA values of 16-17%.

Pine wilt disease is currently the most deadly forest disease in the world and is known to be caused by infection of Bursaphelenchus xylophilus. Until now, no method has been developed to confirm the pine tres infected with pine wood nematodes. In order to develop a method to diagnose pine wood nematode infection, we produced a monoclonal antibody against Expansin B3, which was found to be secreted by pine wood nematodes. ELISA assay using various monoclonal antibody confirmed that the pine trees infected with the rewarming can be detected. Further studies using our antibodies may help to develop a diagnostic method that can quickly confirm infection of pine wood nematodes in the field.

Monochamus alternatus (M. alternatus) and Monochamus saltuarius (M. saltuarius) are major vectors for Bursaphelenchus xylophilus in South Korea. When an adult, they are easily distinguishable by several morphological classification. However, it is difficult to identification between M. alternatus and M. saltuarius when they are larvae as they have very similar morphological characters. Thus, they are not easily distinguishable without expertise about Cerambycidae taxonomy. Furthermore, during epidemiological investigation, sometimes, adults or larvae would not be founded in death pine trees. For these reasons, in this experiment, we are able to identified between M. alternatus and M. saltuarius by mitochondrial 12S rRNA gene primers that are specific to 12S rRNA gene fragment of M. alternatus using larvae tissue and frass. Moreover, we had examined whether vectors that were already escaped from dead pine tree have Bursaphelenchus xylophilus or not by multiplex PCR using larva frass that was remained in dead pine tree.

It is difficult to identification between Bursaphelenchus spp. and Pine Wood Nematode (PWN) by morphological characteristics without expertise about nematode taxonomy. Furthermore, Baermann funnel method, which is nematode extraction method from wood chips or soil, requires at least 24 hours to extract nematode that is unsuitable to rapid diagnose the Pine Wilt Disease (PWD). For these reasons, the aim of this experiment is not only to improve accuracy of a PCR based method but also to reduce total experiment time for detection Bursaphelenchus spp. and PWN in the wood chips of PWD infected pine tree. In this experiment, we had been employed two PCR primer sets, which were originated from PWN specific Internal Transcribed Spacer (ITS) sequence region and Bursaphenchus spp. universal mitochondrial Cytocrome Oxidase subunit I (mtCOI) sequence region in order to discrimination between Bursaphelenchus spp. and PWN at the same PCR reaction. This experimental procedure was able to reduce experiment time and cost as well as to improve accuracy of detection than previous PCR based detecting method by not using Baermann funnel method and commercial genomic DNA extraction kit but using direct pine wood chips lysis method.

Thermal death kinetics was examined to develop a heat treatment method to control the Pine wood nematode, Bursaphelenchus xylophilus, in wood packing materials for export goods. To determine the feasibility of microwave irradiation as an alternative treatment, we performed laboratory experiments on the blocks (200x200x250mm) of Douglas-fir artificially infested with live adults with subsequent 2.45GHz microwave energy irradiation. The 100% mortality of treated Pine wood nematode was achieved above 64℃ of the measured wood temperature, regardless of irradiated time. All measurement points in the blocks reached 64℃ within 84-312 seconds with microwave irradiation by wood weight. Likewise, the 100% mortality of nematodes were observed by hot water treatment on 62℃. We need further experimental verification, but commercial microwave (2.45GHz) treatment is a feasible alternative to conventional heat treatment to control Pine wood nematode on the wood packing materials for export goods.

The ovarian development and oviposition response of two vector beetles of pine wood nematode, Monochamus alternatus and M. saltuarius, was investigated. The ovary structure of two beetles was investigated by means of light microscopy. The ovary of both beetles was composed of two ovaries that are connected by a common oviduct. Each of the two ovaries was consisted of twelve ovarioles, the functional units of female oogenesis. The ovary type was meroistic telotrophic with nurse cells and oocytes in the tropharium. Ovarian development of two vector beetles was completed at 12-14 days after maturation-feeding pine twigs. Aspect of accumulation of vitellogenins in the ovary of two vectors showed difference in developmental stages and major yolk proteins, differently from vitellogenin. To investigate oviposition performance of two vector beetles, we provided P. thunbergii and P. koraiensis bolts for egg laying and collected emerged adults from each pine bolts in the next year. M. saltuarius females made more oviposition wounds and entrance hole of larvae than M. alternatus on pine bolts. We also investigated whether two beetles can transmission of Bursaphelenchus xylophilus and the European type of B. mucronatus via oviposition to noninfected dead pines. 48-83% from newly emerged adults of two beetles were carried two species nematode. This results suggested that two species beetles can transmit nematodes through oviposition performance and transmitted nematodes successfully propagate in non-infected dead pines.

Pine wood nematode (PWN), Bursaphelenchus xylophilus is associated with the pine wilt disease and transmitted by pine sawyer, Monochamus alternatus. Because pine sawyer has one-year life cycle, one natural infection of PWN is occurred a year. Therefore, artificial propagation method of PWN is needed to improve experiment associated with PWN. In this study, effect of diameter, paraffin sealing of twig and dosage on pine wood nematode reproduction in Japanese black pine, Pinus thunbergii. PWN reproduction was compared in twigs of P. thunbergii and P. densiflora. Numbers of reproduced PWN were higher with decreasing diameter of twig. Distance (5 and 10 cm) from inoculation site of PWN did not influence reproduction of PWN. Reproduced numbers of PWN were higher in the paraffin-sealing twig than non-sealing twig. Dosage of PWN influenced reproduction of PWN. Reproduction rate was the highest at the rate of 10 IJs (13.7 and 61.1-fold increasing in P. densiflora and P. thunbergii, respectively 30 days later) whereas lowest at the rate of 1000 Ijs (1.1 and 0.7-fold increasing in P. densiflora and P. thunbergii, respectively 30 days later). Numbers of reproduced PWN were more in P. thunbergii than P. densiflora.

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.

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.

There are increasing interests in developing methods specifically detecting pathogenic Bursaphelenchus xylophilus. In order to develop a detecting method for B .xylophilus, at first we generated monoclonal antibodies (MAbs) specific to B. xylophilus, discriminating from other pine tree resident nematodes. Among 2304 hybridoma fusions screened. We finally selected a MAb clone, 9F10 and used for further study. To identify the antigenic target of MAb-9F10, we employed several biochemical methods such as SDS-PAGE, 2 dimensional electrophoresis, anion exchange chromatography, and immunoprecipitation to separate and isolate an antigenic target. Proteins from above methods were analyzed via nano-LC-ESI-Q-IT-MS. Peptides of GaLECtin were always detected from several proteomic analyses, suggesting that GaLECtin is the antigenic target of MAb-9F10.

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

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.

부산, 경남, 경북, 전남, 전북 22개 지역 19수종 238본의 고사목에서 1989년 4월부터 9월까지 선충류와 곤충류를 조사한 결과, 소나무 재선충은 부산지역에서만 검출되었으며, 어리소나무 재선충은 진주와 진해에서 분포가 확인되었다. 고사목에서 분리.동정된 선충류는 9속 13종이었으며 6속은 미동정되었다. 동정된 선충 중 Diplogasteroides dimidius, Rhabdontolaimus adephagus, R.janae, Mikoletzkya diluta, M. ruminis, m. langcauda, Parasitorhabditis hylurgi, Panagrolaimus concolor, Panagrodontus dentatus, Prothallonema intermedium, Macrolaimus canadensis는 우리나라 미기록종이다. 한편, 고산목에서 채집된 곤충은 5목 9과 25속 27종이었는데 딱정벌레목이 3과 19속 22종으로 가장 많았다. 그중 나무종이 10속 12종으로 빈번히 채집되었으며, Hypothenemus eruditus가 꽃싸리, 싸리, 조록싸리, 만리화, 닥나무에서 채집되어 새 기주로 추가되었다. 소나무 재선충의 매개충인 솔수염하늘소는 복부에서의 선충 검출수가 가장 많았으며, 성충 한마리당 선충 보유수는 최대 127,535마리, 최소 2,616마리, 정균 42,817마리였다.