This study investigates the impact of Bursaphelenchus xylophilus infection on the gut bacterial communities of the pine sawyer beetle, Monochamus alternatus, with a distinction between male and female individuals. Utilizing specific primers, we determined the infection status and analyzed the bacterial composition across different taxonomic levels. Regardless of infection status or sex, Proteobacteria and Firmicutes were found to dominate the phylum level, with significant contributions from Actinobacteria and Bacteroidetes. At the class level, Bacilli, Gammaproteobacteria, Alphaproteobacteria, and Actinobacteria emerged as prevalent groups. Meanwhile, the genus level was characterized by a high abundance of Lactobacillus, Rickettsia, Bacillus, and Rahnella. Our analysis of alpha diversity metrics, including Observed Species, Shannon Index, Inverse Simpson Index, and Phylogenetic Diversity, revealed no significant differences attributable to B. xylophilus infection; however, notable variations were observed between sexes. Principal Coordinates Analysis and Non-metric Multidimensional Scaling further underscored that the differences in gut bacterial communities were more pronounced between male and female beetles than between infected and uninfected individuals. These findings highlight the influence of host sex over infection status in shaping the gut microbiome of Monochamus alternatus, providing new insights into the complex interactions between host biology, gut microbiota, and pathogen infection.

Monochamol is a male-produced aggregation pheromone of the Monochamus species, which are vectors of the pine wood nematode (PWN). This nematode is a fatal devastating pest, infecting pinewood trees and is one of the serious forest pest in Korea. The nematicidal activity of synthetic monochamol and its homologues (ROEtOH: R = C7-C13) were determinated to potential alternatives to the currently used PWN control agents. In this study, for elucidation of the lethal mechanism induced by monochamol, we performed Transcriptome analysis by next-generation sequencing (RNA-seq) from LC50 treated PWN and are characterizing some significant genes which were differentially expressed in PWN.

소나무재선충(Pine wood nematode, PWN)은 아시아와 유럽의 소나무류들에 침입하여 고사시키는 심각해 병원성 선충이다. 가장 완벽한 방제 방법은 감염목을 소각/분쇄의 방법으로 PWN 감염목을 제거하는 것이다. 현재 PWN의 다른 종과 차이를 가지는 유전자서열을 이용한 진단 방법이 다양하게 개발되어져서 실험실에서의 종 판명은 가능한 실정이다. 하지만, 대부분의 분자진단방법은 소나무 목편에서부터의 시료 추출, 그리고 분자진단 시약과 혼합하고, PCR을 이용하여 증폭을 진행 한 후, 결과를 확인하는 과정이 필요하다. 그러므로, 분자진단법을 현장에 적용하기 위해서는 향 후 많은 노력이 필요하다. 이에 반하여 항원/항체 반응을 이용한 진단 방법의 경우는 항원 추출 후, 일정량을 신속진단키트에 떨어 뜨려서 반응을 확인하는 과정을 거치므로, 전문적인 지식이나 기술이 필요하지 않다. 하지만, PWN과 PWN 감염목에 특이적인 항원이 밝혀지지 않은 상황이다. 최근에 우리는 PWN단백질 중에서 분비되는 Aspartic peptidase 1 (ASP1)을 항원으로 선정을 하고, 전장단백질을 Baculovirus Expression System으로 발현을 하였다. PWN-ASP1을 항원으로 단클론항체를 분비하는 세포 주를 확립하였다. 이러한 단클론항체는 향후, PWN의 소나무 침입에 관한 연구와 신속진단키트의 개발에 활용될 수 있을 것이다. (본 연구는 국립산림과학원의 연구비 지원으로 진행되었음)

Bursaphelenchus xylophilus, commonly known as pine wood nematode (PWN) and pathogen of pine wilt disease, is transmitted to new host trees through Monochamus spp.. In this study, five points were set 1 km apart placing the interface of the damaged area in the middle to measure density of the beetles and confirm the presence of PWN in Chungcheongbuk-do, Republic of Korea. The vectors collected from five traps on each point were sorted on weekly basis until the 12th week. We confirmed the presence of PWN in each collected vector by PCR with species-specific ITS primers.

Bursaphelenchus sinensis was first found in Austria from the wood packaging material imported from China. In Korea, B. sinensis was detected from dead branches of Pinus densiflora located in Jinju, Gyeongnam province. Morphology of B. sinensis was characterized by unique male spicule with weak rostrum and squared condylus, and female with small vulval flap and conical shaped tail. Genomic DNA of B. sinensis was extracted and ITS region was fully amplified by PCR. ITS-PCR product was analyzed by RFLP and also directly sequenced. Polymorphism by RFLP-ITS was matched with that of B. sinensis and ITS sequence data was identical to B. sinensis information in GenBank. The Korean isolate of B. sinensis, Bs-Jinju, was submitted to GenBank and acquired the accession number, #MG934676. Host pathogenicity of B. sinensis was tested by artificial inoculation of nematode (30,000/ sapling) on 5-year-old P. densiflora. There were no disease symptoms in all tested pines inoculated by B. sinensis, whereas 100% mortality showed on pine saplings by B. xylophilus inoculation. This is the first report of B. sinensis in South Korea.

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.

Japanese pine sawyer beetle, Monochamus alternatus Hope (Coleoptera: Cerambycidae) is considered as a serious pest in pine trees. To develop an eco-friendly strategy to manage this forest insect, we collected entomopathogenic fungi from Korean soil and assessed their virulence against the adults of the insect in laboratory conditions. As a result, two isolates with conidial suspension (1.0×107conidia/ml), showed 87% and 90% mortality 12 days after fungal treatment, respectively. We assessed the potential of the fungi-derived destruxin and protease as additives to the fungal isolates, and they showed insecticidal activity via feeding and spraying treatments. Finally, we produced fungal conidia in massive solid cultures and formulated wettable powders, and now studying optimal conditions of oil-based formulation with two isolates. Based on these results, we are evaluating the control efficacy of the fungal agents against M. alternatus in field conditions.

The nematode Bursaphelenchus xylophilus and its vector insect Monochamus alternatus are major quarantine pests of timber products in Korea. Due to the phaseout of methyl bromide as a fumigant for plant quarantine and pre-shipment treatments, the need for an alternative fumigant is essential. Based on preliminary studies on the efficacy of ethanedinitrile (C2N2) to these two pests, three quarantine trials on logs covered with PVC-tarpaulins were conducted at three dosages and three temperatures potential for inhalation exposure was assessed by monitoring atmospheric levels of C2N2 in relation to the threshold limit value (TLV, 10 ppm, v/v). Concentration × time products (Ct) of 398.6, 547.2 and 595.9 g h m-3 were obtained for each quarantine trial. A 100% mortality of B. xylophilus and M. alternatus larvae at 23±4°C and 10±4°C occurred with a load factor of pine logs of 46% and at 3±1°C with a load factor of 30%. During all fumigant procedures, atmospheric levels of C2N2 20m downwind from the PVC tarpaulin were below the TLV. During aeration, levels 10 m and 5 m downwind were below the TLV after a maximum of 0.4 h and 1 h, respectively. For the purpose of an emergent quarantine or phytosanitary treatment, specific doses of C2N2 at the temperatures that occurred during fumigation could control B. xylophilus and M. alternatus larvae without significant inhalation risk to workers.

Bursaphelenchus xylophilus (Bx) is the main plant-parasitic nematode of the Japanese Black Pine (Pinus thunbergii), Red Pine (Pinus densiflora) and Korean Pine (Pinus koraiensis) in the South Korea. Until now, the nematode morphological classification or PCR method using specific marker of Bx were used for the diagnosis of pine wilt disease. However, both methods have a disadvantage that these take a long time to confirm the result. Thus, these methods can not be used quickly at the newly damaged regions. For above the reasons, we had been developed the diagnostic method for Bx combining direct gDNA extraction buffer (DAP) with Recombinase Polymerase Amplification (RPA). This method is able to directly use mixed lysates extracted from Bx-infected pinewood by DAP buffer as gDNA template to RPA without another process for increase gDNA yield. Together, our method is able to detect Bx within 20 mins.

소나무재선충과 근연종인 Bursaphelenchus 속 2종간의 매개충 채내 침입력과 경쟁력을 비교 실험하였다. 공시충은 Bursaphelenchus xylophilus (이하 Bx), B. mucronatus (이하 Bm), B.thailandae (이하 Bt) 선충 3종과 솔수염하늘소 (Monochamus alternatus)와 북방수염하늘소 (M. saltuarius) 2종을 사용하였다. 매개충 번데기에 선충 3종을 10,000 마리씩 단독 또는 혼합접종을 하고, 우화 후 체내 선충 개체수를 조사한 결과, 솔수염하늘소에서는 Bx, Bm, Bt가 각각 2,283마리, 1,575마리, 3,083마리로써 Bt의 침입력이 가장 높게 나타났다. Bx+Bt 처리 시에는 Bx 24%, Bt 76%, Bx+Bm 처리 시, Bx 68%, Bm 32% 비율로 조사되어 Bt>Bx>Bm 순으로 침입력이 강한 것이 확인되었다. 북방수염하늘소 에서 Bx, Bm의 침입력을 비교한 결과 Bx는 2,120마리, Bm 1,730마리로 솔수염하늘소에서와 유사한 결과를 나타내었다. 이로써 선충 종간 침입력은 다양하며 이는 매개충 종과는 무관함을 알 수 있다.

재선충속(Bursaphelenchus) 선충의 식물 기주체내 증식 능력 및 병원성을 비교하기 위하여 선충 3종 (이하 Bursaphelenchus xylophilus: Bx, B. mucronatus: Bm, B. thailandae: Bt)과 해송(Pinus thunbergii), 잣나무(Pinus koraiensis) 2종을 실험에 사용하였다. 5가지 선충 처리 조합 (①Bx+Bt, ②Bx+Bm, ③Bx+Bt+Bm, ④Bt+Bm, ⑤Bm)으로 한 본당 10,000마리씩 접종 한 후 7주 동안 외부 병징 및 선충 밀도를 조사하였다. 그 결과, Bx가 포함된 대부분 처리구에서는 5주 차에 전체 고사가 진행 되었고, Bx가 포함되지 않은 처리구는 병징이 나타나지 않았다. 예외적으로, Bx+Bm, Bx+Bt+Bm 해송 처리구에서는 7주 후에도 전체고사가 나타나지 않았다. 이 경우 선충 밀도 조사 결과, Bm이 약 42%를 차지하였으며, 이는 소나무재선충병의 병징 발현을 약화를 유도하는 역할을 하는 것으로 추정된다. 모든 처리구에서 Bx의 밀도가 가장 높았으며, Bt는 전혀 검출되지 않았다.