台灣地區松材線蟲新紀錄種Bursaphelenchus rainulfi（Nematoda: Parasitaphelenchidae）鑑定及其病原性探討

"Identification of Bursaphelenchus rainulfi (Nematoda: Parasitaphelenchidae), a new record pine wood nematode species in Taiwan and its pathogenicity"

本研究自2018年2月於金門地區死亡的松樹中，共分離了五個線蟲族群，其中由代號PKMNP線蟲族群中發現疑似松材線蟲屬（Bursaphelenchus spp.）之線蟲，建立純系族群PP2後觀察形態特徵及紀錄測量值。該線蟲體型細長，約505~585μm，口唇部高聳，口針短且口針結球不明顯，中部食道球佔體寬75%以上，熱殺固定後雌蟲微向腹側彎曲，少數個體尾部會呈鈎狀，雄蟲則呈J型。兩性皆具兩條側線，雌蟲陰門蓋短，雄蟲交接刺小，末端不具盤狀構造，交接刺頂盤明顯下凹，腹刺頂端（rostrum）與背刺頂端（condylus）發育良好，其形態特徵及測量值與B. rainulfi描述相符。增幅純系族群之D2D3、ITS區間及粒線體COI基因區間共三個片段（NCBI編號依序分別為MT373702，MT459324，MT643919），除與GenBank資料庫序列比對，也將相似種類序列經演化樹分析後確認該線蟲族群為B.rainulfi，是台灣的新紀錄松材線蟲種。國外先前研究僅描述其形態特徵、核酸序列資料及發現來源，因其形態與食菌性類群相似，皆未探究其對松樹是否具病原性。本研究以黑松（Pinus thunbergii Parl.）作為病原性測試供試松樹，共接種8株黑松，每株約接種2000隻線蟲，另三種對照組為僅接種Alternaria citri菌絲塊，僅接種無菌水，及未進行任何處理的松樹。接種35日後，15棵供試松樹皆仍保持健康狀態，8棵接種線蟲的松樹枝幹，剪碎經改良式柏門氏漏斗分離後，僅於一樣本（Br-7）中發現5隻雌蟲活體，據此結果推論B. rainulfi對黑松不具病原性，少量的個體可存活於活體松樹中。

"Five samples from dead pine trees located in Kinmen were collected in Feb. 2018 to isolate Bursaphelenchus spp. Sample PKMNP had individuals that might be Bursaphelenchus spp. and a pure line PP2 was established for the following morphological and molecular identification. Both female and male nematodes were slim with offset lip region, short stylet and indistinct stylet knobs. The width of medium bulb is more than 75% body width. Female relaxed and curled ventrally when heat killed, some individuals would have stronger curve at the end and formed a hook shape tail. Male is J shaped when heat killed. The body length is 505-585 um with 2 lateral incisures. The female has small vulva flap and the male has small spicule without cucullus. The capitulum of spicule is dented with well-developed rostrum and condyles. The morphometric data fits the description of Bursaphelenchus rainulfi. Three DNA regions, D2D3, ITS and COI genes were amplified and the sequences were submitted to NCBI with the accession number MT373702, MT459324 and MT643919, respectively. Sequences were processed for phylogenic tree analysis and the results confirmed that PP2 is B. rainulfi. Our study is the first attempt to conduct the pathogenicity test of B. rainulfi on pine trees, because according to morphometric data, this nematode was classified in fungal-feeding group and was always found in dead woods. B. rainulfi was inoculated onto 8 pine tree seedlings (Pinus thunbergii Parl.), approximately 2000 nematodes per seedling. The control treatments were inoculation of either A. citri only or sterilized water only, and one tree was without any treatment. 35 days after inoculation, all seedlings remain green and healthy. The branches that were inoculated with B. rainulfi were cut and processed for modified Baermann's funnel. Only 5 females were found in # Br-7 pine tree seedling sample. According to the results, we speculate B. rainulfi doesn't have pathogenicity to pine trees and few individuals are able to survive in living pine trees when introduced by wounds."