A survey of plant-parasitic nematodes (PPNs) was carried out in medicinal crop cultivated fields from July to August in 2023. Three-leaf ladybell, Adenophora triphylla var. japonica is a highly valued medicinal plant that is used to treat or prevent bronchitis, cough, cancer, and obesity in Korea. A. triphylla plants with small root-galls were observed in a field of Yeongju Agricultural Technology Center, which were identified as a root-knot nematode. Additional morphological and molecular analyses studies were performed and identified as Meloidogyne hapla, Northern root-knot nematode. Population densities of M. hapla ranged from 20~30 nematodes per 100 cm3 of soil. M. hapla was detected at lower densities in soil compared to other infected host crops, but there are concerns about damage to M. hapla since A. triphylla is cultivated for more than two years once planted. Our results indicate that A. triphylla roots damage by M. hapla were identifed, it is necessary to prepare control methods such as registration of applicable nematicides and crop rotation.

In order to identify the specific antigens for pine wood nematode (PWN), we confirmed that one of the genes commonly found in the transcriptome, proteome and secretory proteins of PWN belonged to the Aldose Reductase (AR) family protein. 36.5 kDa PWN-AR1 was expressed and purified using Baculovirus Expression System. Total 1,546 hybridoma fusion library was generated and screened for specificity to PWN-AR1 by Enzyme Linked Immunosorbent assay (ELISA). Nine clones showed strong immunoreactivity to PWN-AR1 were limited-diluted. Total 864 limited-diluted clones were further screened using PWN-AR1 by ELISA and 34 monoclonal antibody (Mab) clones were selected. 34 Mab clones were further screened using PWN extracts and a standard PWN-infected pine tree extract by ELISA. Finally nine clones were selected and their immunoreactivities to 4 different nematodes were examined by ELISA. Seven clones pecifically recognized PWN while two clones recognized 4 nematodes. Our data suggested that PWN-AR1 is a PWN secretory enzyme while PWN is invading pine trees, Thus, PWN-AR1-Mabs could be used to develop diagnosis tools for PWN and its infected pine trees. (This work was supported by National Institute of Forest Science)

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 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.

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.

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.

Pine wood nematode, Bursaphelenchus xylophilus, is a causative organism to induce pine wilt disease in many varieties of pine trees. Until 2006, Monochamus alternatus had been known as the only insect vector of pine wood nematode in Korea which targeted on Pinus densiflora (Japanese red pine) and P. thunbergii (Japanese black pine). However, pine wilt disease was also reported from Korean pine tree (Pinus koraiensis) in 2006 and we found another insect vector, M. saltuarius, was involved to transmit pine wood nematode. Both Monochamus species were confirmed to transfer pine wood nematode to their hosts but, there is no detail information about other transmitted nematode. Especially Bursaphelenchus mucronatus is common species transmitted by Monochamus species which is morphologically closed to B. xylophilus. Moreover B. mucronatus have two genotypes; one is East Asian type and the other is European type. Both genotypes of B. mucronatus were found in Korea but, the host and vector information related to the genotypes of B. mucronatus was not clear. Monochamus saltuarius was collected from three different geographical locations and nematodes were extracted and identified. For the identification of the juveniles, nematode DNA was extracted and ITS-RFLP analysis was done by PCR and gel electrophoresis. The selected enzymes were Hinf I, Alu I, Msp I, Hae III, Rsa I. Most of Bursaphelenchus species carried by M. saltuarius, which collected from pine wilt disease-free area, was determined as European type of B. mucronatus. We will compare the nematode species and genotypes carried by M. alternatus and M. saltuarius. In addition the rate of nematode carrying insect and the average number of nematode per single insect will be counted and compared.

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.