본 연구에서는 소나무재선충의 매개충인 솔수염하늘소와 북방수염하늘소에 대하여 무인항공기 (무인헬리콥터)를 이용하여 스피네토람의 약효 및 약해를 조사하였다. 하늘소를 대상으로 등록된 펜토에이트 유제, 비펜트린 액상수화제, 하늘소를 제외한 딱정벌레가 대상인 에토펜프록 스 유제, 디플루벤주론 수화제와 나방류에 방제 약제로 등록된 인독사카브 수화제, 스피네토람 액상수화제 6종을 ULV기로 살포하여 솔수염하 늘소에 대한 섭식독과 접촉독을 확인한 후 선발하였다. ULV 시험 결과, 펜토에이트, 비펜트린, 인독사카브, 스피네토람의 33배, 55배 희석배수 액은 처리후 3일차에 접촉독과 섭식독에서 100% 살충율을 보였으나, 에토펜프록스는 7일차 접촉독 살충율 88.9%(33배), 88.9%(50배), 섭식독 살충율 93.4%(33배, 50배), 디플루벤주론은 7일차 접촉독 살충율 83.3%(33배), 섭식독 80.3%, 53.9%(50배)로 조금 낮은 살충률을 보였다. ULV 시험에서 선발된 가장 적합한 스피네토람의 33배액을 무인항공기로 살포하여 솔수염하늘소와 북방수염하늘소에 대한 감수성을 평가한 결 과, 98.6% - 100%의 살충율을 보였다. 그러나, 해당 약제의 매개충 방제에 적용하기 전에 항공 살포에 의한 꿀벌에 대한 위해성 평가가 필요할 것 으로 판단되었다.

소나무재선충병의 예방을 위하여 실시하는 예방나무주사는 소나무재선충 피해를 예방할 수 있는 매우 효과적인 방제방법이지만 매개충에 대해서는 효과가 밝혀진 바 없다. 본 연구는 살선충제와 살충제가 혼합된 약제를 나무주사하여 소나무재선충병의 예방효과 뿐만 아니라 신초를 후식하는 하늘소류에 대해 소화중독에 의해 매개충살충까지 동시에 가능한 방제방법을 개발하기 위하여 실시하였다. Abamectin+Sulfoxaflor DC을 비롯한 3개 약종 2018년 3월 6일에 소나무에 나무주사 후 90여일 이 경과한 6월 5일에 소나무 가지에 망대를 설치하고 각 망대에 솔수염하늘소 1마리씩을 접종하여 7, 14, 21일의 살충율을 조사하였다. 나무주사된 3약종 모두 솔수염하늘소에 대하여 살충력이 우수한 것으로 나타났으며, 경기도 남양주시 잣나무림에 Abamectin+Sulfoxaflor DC를 나무주사 후 산란유인목을 설치하여 현장 실험을 실시한 결과 대조구와 비교하여 유충에 의해 발생된 목설흔이 매우 적게 나타나 소나무재선충병 방제에 적용 가능성을 확인할 수 있었다.

Avermectin produced by Streptomyces avermitilis is an anti-nematodal agent against the pine wood nematode Bursaphelenchus xylophilus. However, its potential usage is limited by its poor water solubility. For this reason, continuous efforts are underway to produce new derivatives that are more water soluble. Glycosylation is generally used to enhance the aqueous solubility and biological activity of natural compounds. Uridine diphosphate (UDP)-glycosyltransferase (BLC) from Bacillus licheniformis is involved in the biosynthesis of the bioactive compound by transferring UDP-activated sugar moieties to acceptor molecules. Here, the enzymatic glycosylation of avermectin was catalyzed by uridine diphosphate (UDP) glycosyltransferase from Bacillus licheniformis with various UDP-sugars. As a result, the following four avermectin B1a glycosides were produced: avermectin B1a 4’’-β-D-glucoside, avermectin B1a 4’’-β-D-galactoside, avermectin B1a 4’’-β-L-fucoside and avermectin B1a 4’’-β-2-deoxy-D-glucoside. The avermectin B1a glycosides were structurally analyzed based on HR-ESIMS, 1D and 2D nuclear magnetic resonance (NMR) spectra. The solubility of avermectin B1a4’’-β-D-glucoside and avermectin B1a4’’-β-D-galactoside in water are 49 and 21 times higher than that of avermectin B1a. Consistent with the improved water solubility of avermectin glycosides, the anti-nematodal effect of avermectin B1a4’’-β-D-glucoside was found to exhibit the highest activity,which was approximately 32 times greater than that of avermectin B1a, followed by avermectin B1a 4’’-β-2-deoxy-D-glucoside, avermectin B1a 4’’-β-L-fucoside, and avermectin B1a 4’’-β-D-galactoside. These results show that glycosylation of avermectinB1a effectively enhances itsin vitro anti-nematodal activity and that avermectin glycosides can be further applied for treating infestations of the pine wood nematode B. xylophilus.

소나무재선충병의 방제를 위한 나무주사는 살선충제를 사용하여 예방적인 측면에서 이루어 졌다. 본 연구는 소나무에 살충제를 나무주사하여 약제를 소나무 전체에 침투 이행시킨 후 우화한 매개충이 후식하는 과정에서 약제가 포함된 신초를 섭식하게 되면 소화중독으로 교미 및 산란 전에 살충시킴으로서 매개충을 방제하고자 실시하였 다. 이러한 살충제 나무주사 방법은 신초를 후식하는 하늘소류만을 소화중독에 의해 살충시키는 방법으로 기존의 항공방제에 비해 환경에 대한 영향을 최소화 할 수 있다. 살충제 나무주사에 가장 적합한 약제 선발을 위해 실내에서 Neonicotinoid계 살충제 7종을 각 2,000배로 희석한 후 솔수염하늘소에 2㎕를 경구투입하여 살충효과를 평가한 결과 Clotianidin SL, Thiamethoxam DC이 소화중독에 의한 살충효과가 가장 우수하였으며, 이들 살충제를 야외에서 소나무에 나무주사 후 가지에 망대를 설치하고 솔수염하늘소를 접종하여 살충력을 검정한 결과 Thiamethoxam DC가 살충력이 가장 우수한 것으로 판단되었다. 또한 현장적용을 위해 잣나무림에 나무주사후 산란유인목을 설치하여 무처리구와 비교한 결과 살충제 나무주사 시험지의 산란유인목에서는 산란이 이루어지지 않아 현장방제에 적용 가능성을 확인할 수 있었다.

소나무재선충은 현재까지 도입된 국가의 토착수종인 소나무류를 모두 치사시키는 것으로 알려져 있으나, 고사목으로부터 추출 배양된 계통 중에는 소나무에 피해를 주지 않는 계통이 존재한다. 이러한 저병원성 계통 선충의 사전접종이 고병원성 소나무재선충에 대한 소나무의 저항성에 미치는 영향을 조사하기 위하여 연구가 수행되었다. 치수에 저병원성 소나무재선충을 선접종한 결과, 선접종에 의하여 소나무재선충에 대한 저항력이 유도되었다. 그 유도저항성은 1년 후까지 유지되었으며, 저병원성 선충의 반복 감염은 보다 저항성 유도 효과가 큰 것으로 나타났다. 소나무재선충에 의하여 고사한 후 경과기간이 3개월 이하인 피해목 및 피해진행목에서 분리된 선충은 모두 고병원성이었으며, 고사 후 2-3년이 경과한 벌채목에서 분리된 선충 5계통 중 4계통은 저병원성이었다. 저병원성 선충이 소나무 치수에 인공접종 되었을 때 초기 정착률은 고병원성 선충과 차이가 없었으나, 접종 30일 후에 수목내의 부위별 개체수를 조사한바 저병원성 선충의 증식률은 고병원성보다 현저히 낮았다. 저병원성 선충의 잿빛곰팡이병 균사배지에서의 선충증식 속도는 계통에 따라 차이가 있었으며, 증식속도가 빠른 계통은 대체로 고병원성 선충의 증식속도와 차이가 없었다.

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 63 aliphatic compounds 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, C6, C9, C10 2E-alkenal C12 alkanoic acid were observed the > 45% BxACE inhibition rate and they were subsequently estimated the inhibition rate against three recombinant BxACEs. Whole compounds showed the high inhibition rate against BxACE-1 and BxACE-2. Interestingly, C6 2E-alkenal and C12 alkanoic acid exhibited the high inhibition rate against BxACE-3 which would be insensitive to ACE inhibitors.

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.

Injection of nematicides such as emamectin benzonate and milbemectin is the most common practice to control the pinewood nematode, Bursaphelenchus xylophilus, in Korea. These macrocyclolactone nematicides, however, are expensive, limiting their practicability despite of high efficacy. In an attempt to screen affordable alternative organophosphate (OP) and carbamate (CB) nematicides, we identified and characterized three acetylcholinesterases (ACE, EC 3.1.1.7) from B. xylophilus and functionally expressed them using baculovirus system. In inhibition assay using 11 OPs and 3 CBs, all the three ACEs were highly inhibited by paraoxon, DDVP, chlorpyrifos-oxon and mevinphos of OPs and carbofuran and carbaryl of CBs but not inhibited well by the others. Interestingly, inhibition assay revealed that BxACE-3 is less sensitive to all insecticides tested than other two ACEs. In additional bioassay, chlorpyrifos, DDVP and parathion showed a high LC50 but all CBs tested did a very low mortality. The inhibition kinetic data and bioassay data obtained in this study should provide essential information for the development of OP-based nematicidal agents against B. xylophilus. Availability of expressed ACE will also facilitate the development of in vitro screening system to develop potential OP nematicides.

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.

tasks that require nematode extraction and microscopic examination. To develop a more efficient detection method for Bursaphelenchus xylophilus, we first generated monoclonal antibodies (MAbs) specific to B. xylophilus. Among 2,304 hybridoma fusions screened, a hybridoma clone named 3-2A7-2H5 recognized a single protein from B. xylophilus specifically. We finally selected the MAb clone 3-2A7-2H5-D9-F10 (D9-F10) for further studies. To identify the antigenic target of MAb-D9-F10, we analyzed proteins in spots, fractions or bands via nano liquid chromatography electrospray ionization quadrupole ion trap mass spectrometry (nano-LC-ESI-Q-IT-MS). Peptides of galactose-binding lectin-1 of B. xylophilus (Bx-LEC-1) were commonly detected in several proteomic analyses, demonstrating that this LEC-1 is the antigenic target of MAb-D9-F10. The localization of MAb-D9-F10 immunoreactivities at the area of the median bulb and esophageal glands suggested that the Bx-LEC-1 may be involved in food perception and digestion. The Bx-LEC-1 has two non-identical galactose-binding lectin domains important for carbohydrate binding. The affinity of the Bx-LEC-1 to D-(+)-raffinose and N-acetyllactosamine were much higher than that to L-(+)-rhamnose. Based on this combination of evidences, MAb-D9-F10 is the first identified molecular biomarker specific to the Bx-LEC-1.

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.

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.

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.

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.

Pine wilt disease (PWD) is one of the most serious forest tree diseases and now the damaged area covers 7,877 ha and more than 60 cities in Korea. The causal agent of PWD, Bursaphelenchus xylophilus is transmitted by the insect vectors, pine sawyers (Monochamus spp.). Spread of PWD damage and control effect for PWD are directly related to dispersal potential of Monochamus spp. vectors. Recently, harmonic radar technology has been applied as a means for tracking individual dispersal of tagged insects. Tracking insects with harmonic radar system is similar to a mark-and-recapture technique, in which an organism is tagged, released, and recaptured. Our objective is to develop a harmonic radar tracking system by using lightweight tags to investigate dispersal of Monochamus spp., insect vectors. The harmonic radar system consists of two parts: a commercially available, light-weight (1.6 kg), handheld transceiver (RECCO Rescue System) and a small tag that reflects the transceiver signal (917 ㎒) at harmonic frequencies (1,834 ㎒). A tag consists of a miniature diode (Toshiba ISS350) with wires (36-gauge tinned copper) soldered using conductive epoxy on each side to produce a dipole antenna. These tags were evaluated in an open field to assess their maximum detection range, and at tag length of 13 ㎝, the tag was detected at >100 m.

Pine wood nematode(PWN), Bursaphelenchus xylophilus, is a causal organism to induce pine wilt disease in many varieties of pine trees. PWN is mainly distributed in the East Asia including Japan, China, and Korea, but it was originally imported from the North America of the West. Over 70 species of Bursaphelenchus have been reported, but they are morphologically similar to each other. In Korea, only two species of Bursaphelenchus xylophilus, B. mucronatus (both Asian type and European type) have been reported however, a recent survey showed the distribution of extra species of Bursaphelenchus in dead trees. Three isolates, BSPD-1, BSPD-2, and BSPL-1, were identified as Bursaphelenchus thilandae, B. hylobianum, and B. doui, respectively, which was determined by both morphological and molecular biological characteristics. Both BSPD-1 and BSPD-2 were originally collected from Pinus densiflora in Namyangju and BSPL-1 came from Liriodendron tulipifera in Wanju. The morphology of each species were compared from the original descriptions focusing on male spicule and female tail and reproductive organ. A molecular diagnosis method, ITS-RFLP was applied to confirm morphological identification. Genomic DNA was extracted from a single individual nematode and ITS DNA was amplified by PCR. Amplified ITS was digested by 5 different restriction enzymes (Rsa I, Hae III, Msp I, Hinf I, and Alu I) and provided a discriminatory profile for different species of Bursaphelenchus. The three species, B. thilandae, B. hylobianum, and B. doui, are all unrecorded species in Korea.

Pine wilt is the most important disease of pine trees in Korea, Japan and China. The pathogen causing this disease, the pinewood nematode (Bursaphelenchus xylophylus), is transmitted vectored by adults of some cerambycid beetle species and the Japanese pine sawyer, Monochamus alternatus, is the major vector species in Korea. Although chemical insecticides have been used to kill vector insect and thus prevent transmission of the pathogen, the efficacy is not good. In Japan, to control this insect, an entomopathogenic fungus was studied and developed as an insecticide. This is thought to be the convenient and effective method to control M. alternatus. Recently, there are several reports about the pinewood nematode is vectored by also the pine sawyer, M. saltuarius, in Korea. The objective of this study, therefore, was to isolate and identify entomopathogenic fungi from M. saltuarius cadaver to control it. We collected the cadaver of M. saltuarius and then screened several fungi colonies. The pathogenicity of each fungus was tested using oak longicorn beetle, Moechotypa diphysis, as substitutive insect. M. diphysis is also serious pest to various trees in forest. As the result, only one of them showed high pathogenicity against M. diphysis. Selected fungus was identified by microscopic examination and DNA analysis. Pathogenicity was also evaluated to M. saltuarius.