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.

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 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.

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.

In order to develop solid mass cultivation technology that maximizes insecticide of entomopathogenic nematode(EPN) isolated from Korea, we have studied the optimization of solid culture medium and production conditions. The optimized conditions from the harvest yields and the insecticidal activity against the Galleria mellonella larvae were yeast extract 2.0%, soybean flour 20%, whole milk powder 3%, olive oil 5% and egg yolk 5%. It was also found that the optimum condition of the carrier(polyurethane) content was about 8-12%, the culture time of symbiotic bacteria was about 48 hours, and the inoculation concentration of nematode was 4,000-5,000 per g medium. And the optimal harvesting point in solid culture conditions was confirmed after 12 days of inoculation.

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.

사탕무씨스트선충(Heterodera schachtii)은 최근 우리나라의 배추 재배지의 문제선충이다. 본 연구는 사탕무씨스트선충의 밀도와 배추의 묘 령이 사탕무씨스트선충의 증식에 미치는 영향을 알아보기 위하여 수행하였다. 접종농도가 높은(4마리 유충 또는 알/g 토양) 처리에서 씨스트 수나 암컷 또는 알 수가 유의하게 높았다. 40일 묘령 배추에 고밀도 선충 접종 시 30일이나 60일 후 다른 어린 묘령 배추(파종처리, 20일묘, 30일묘)에 비하여 증식 선충 밀도가 높았다. 높은 선충 밀도 조건에 묘령이 오래 된 모종 식재 시 선충 증식율이 높기 때문에 사탕무씨스트선충 관리를 위해서 는 정식 모종의 묘령 고려가 필요하다.

We reported a new white soybean cyst nematode, Heterodera sojae in 2016. This species is closely related to H. glycineswhich has the same host plant. Morphological observations of the cyst, female, male and second-stage juveniles indicatedthat this species is differed from those of H. glycines. Cysts of H. sojae appeared more round, shining and darker thanthat of H. glycines. The distribution of this new cyst nematode in soybean field in Korea is unknown yet. In 2016, 270soil samples were collected from soybean fields and examined the existence of H. glycines and H. sojae. Total of 111samples (41.1%) contained cysts. Among them 77% were H. glycines and 23% were H. sojae. Our results suggest thatextensive researches are required for white soybean cyst nematode.

Plant-parasitic nematodes causes yield losses of more than $100 million per year in the world. Among them, soybeancyst nematode (Heterodera glycines Ichinohe) causes the greatest yield loss to soybean compared to any other pest worldwide.SCN causes yield reductions by feeding on plant nutrients, retarding root growth and inhibiting Bradyrhizobium japonicum(Kirchner) Buchanan nodulation. The primary methods for controlling SCN include planting resistant cultivars and rotationwith nonhost crop. In order to control SCN, we should be breeding resistant soybean cultivars which has resistant genefor this pest. The research that can minimize the damage of the soybean yield is still insufficient, comprehensive andsystematic research is urgently required. Current researches in our lab are discussed.

Sugar beet cyst nematode (SBCN), Heterodera schachtii, is one of the important pests of sugarbeet growing area inthe world, especially in Europe, Canada and USA. SBCN has host in 23 plant families and the important crop host occursin two families; the Chenopodiaceae and Brassicaceae. Host crops include beets, mangolds, cabbage, spinach, brassicas,radish, mustard, and broccoli etc. In korea, SBCN was first detected in the highland Kimchi cabbage fields of easternGangwon-do, 2011. The obvious foliar symptom is poor plant stands and growth, wilting and yellowing. The most easilyrecognized sign of infection is the white to pale yellow and lemon-shaped adult female attached to roots. A survey ofBrassica spp. fields was conducted to determine the distribution of SBCN in Korea. The cyst nematode was only foundedin a restricted highland Kimchi cabbage fields in Gangwon-do. Damage from SBCN may be reduced by employing acombination of various management practies such as prevention of nematode infestation, crop rotation, SBCN resistanttrap crop, weed control and chemical control.

Cyst nematodes are well known to cause serious yield losses in many important crops producing in Korea such assoybeans and Chinese cabbage. Recently, a new cyst nematode Heterodera trifolii has been recorded in the highland vegetablesgrowing area occurring severe yield losses. As a newly recorded pest, the severity of the nematode is still not clear tomost of the farmers. Identification of this nematode is another big issue as the nematode existed in the field with Heteroderaschachtii. Recent reports revealed that the pest is steadily spreading within the highland areas and has a huge possibilityto spread over Korea. Therefore, it is the high time to set a monitoring or warning systems and test more feasible managementstrategies to prevent the dispersal of this nematode.

An increased use of organic phosphorous based chemical pesticides in domestic and overseas agriculture has resultedin emergence of tolerant strains of insect pests. These pests have spread throughout South Korea and are responsible forsignificant reduction in crop yields. Furthermore, as yet there is no effective way of controlling these insects or the damageresulting from pest infestation. To this end, we are attempting to develop biological control based methods to protectcrop plants against insect pests. A screen for biocontrol agents conducted in five farms that were heavily infested bynematodes in 2015-2016 identified Aspergillus niger F22 strain that was very effective against root-knot nematode. Afour-time treatment with A. niger F22 at an internal of ten days was able to reduce nematode population by 70-90%compared to untreated controls. The biocontrol agent was also more effective compared to ograno-phosphorus based pesticide,which caused a 30% reduction in nematode population. Notably, the extract (suspension concentrate) prepared from A.niger culture was equally effective in controlling the root-knot nematode and is currently being developed as a marketableproduct under the brand name “NAMAFREE”.

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%.

식물 기생성 선충은 토양 병해의 중요한 요인 중 하나로 작물 생산에 심각한 피해를 일으키지만 토양 속에서 일어나므로 그 피해를 판단하기 어렵다. 식물 기생성 선충은 주로 오이나 당근, 상추 등 많은 작물의 뿌리를 통하여 침입하는데 이때 생긴 상처를 통하여 토양 전염 병원균의 감염이 발생되고 또한 바이러스도 전파하여 작물의 많은 수확 손실을 가져온다. 본 연구에서는 뿌리혹선충 방제를 위한 친환 경적인 방법을 찾고자 유기성 부산물의 퇴비 처리에 의한 상추의 뿌리혹선충 방제 효과와 상추 수확량, 토양 화학성분 및 미생물밀도를 분석하였다. 퇴비를 처리하지 않은 무처리구와 퇴비 처리량(3, 10kg/3.3m2) 별 효과를 비교한 결과, 퇴비 처리량의 증가에 따라 상추의 뿌리혹선충 발병이 유의성 있 게 감소하였으며(R2=0.230, p=0.022) 따라서 수확량은 증가하였다(R2=0.360, p=0.004). 상추 근권 토 양 중 방선균의 밀도도 퇴비 처리에 의하여 약 10배 정도 유의성 있게 증가하였으며(R2=0.276, p=0.013), 방선균 밀도와 수확량 간에 높은 정의 상관관계가 확인되었다(R2=0.232, p=0.021). 각 처리 구의 토양 성분을 분석 비교한 결과 퇴비를 처리한 토양에서 유기물 함량과 양이온 교환용량이 높게 나 타났다. 이러한 결과로 볼 때 충분히 완숙된 유기성 부산물 퇴비 처리는 상추의 뿌리혹선충의 발병율을 낮추고 좋은 미생물인 방선균 밀도를 증가시킴과 동시에 토양의 지력도 향상시켜 상추 생산량을 증가 시킬 수 있는 친환경적이고 효과적인 뿌리혹선충 방제법 임을 알 수 있었다.

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.

Since first reported in 1988 the Pine Wilt Disease has been established and spreaded nearly nationwide in South Korea, causing tremendous economic damage in pine forests. The pine wilt nematode (Bursaphelenchus xylophilus), causing the Pine Wilt Disease, in Korea is known to be transported by two insect vectors that are Monochamus alternatus Hope and Monochamus saltuarius Gebler. Currently the pest management strategy largely relies on pesticide application and infected tree clearing. The aim of the present study was to search for early larval parasitoids of M. alternatus using the sentinel log infested with the cerambycid eggs. Only one braconid species was collected and identified as Spathius verustus Chao. Two to ten wasp larvae attached and fed on one host larva. Parasitism rate was 55.5 to 59 % in Jinju site, while 1 to 18.1 % the other sites. Sex ratio was female-biased (female : male = 18.9 : 1). The S. verstus has potential to be use as parasitic natural enemy of M. alternatus.