寡核苷酸微陣列同時快速診斷鑑定多種重要防檢疫森林病原真菌之研發

植物病原真菌可引起農藝、園藝或森林等植物各種不同類型之病害，危害嚴重時可引起糧食飢荒、林木大量死亡，故如何快速、精確、靈敏診斷植物真菌性病原，以提早採取適當有效之防制管理措施，以減少生命財產之損失，並提升全民之福址，實刻不容緩。近年來由於氣候變化，許多社區之綠化樹木、公園行道樹、造林樹以及海岸防風林普遍發生褐根病造成嚴重損失與公共危害，樹木病害才逐漸受到重視，而在樹木病害上木材腐朽菌最為常見，因此在本研究中收集了國內最常見的腐朽病菌，以及國外報導最嚴重之樹木病原真菌，共9科22屬67種，包含木層孔菌屬(Phellinus)之多種成員，如Phellinus pini及P. werii、隸屬於假蜜環屬Armillariella之A. mellea(假蜜環菌)、蜜環菌屬Armillaria之A. ostyae以及靈芝屬(Ganoderma spp.)成員等重要林木真菌病原，這些病原菌也曾報導於美加、紐澳、歐洲等地侵染許多針葉林木或濶葉樹，造成生長不良、弱化、落葉、提早開花、腐朽、萎凋、倒伏等病徵，成為林業經營莫大限制因子和潛在嚴重威脅。本研究開發寡核苷酸生物晶片，係利用細胞核內轉錄區(Internal transcribed spacer, ITS)基因序列上具有保守性區域經增幅、定序、並比分析多種目標病原菌之基因，設計寡核苷酸探針(oligonucleotide probe)，目標基因經毛地黃素(DIG)或生物素(Biotin)標定後再進行反轉式點膜雜合(reverse dot blot hybridization)。此檢測技術可同時、快速且精確鑑定重要林木病原真菌。實驗結果顯示本生物晶片具專一性、精準性，於尼龍膜晶片上其靈敏度可達1 pg/μl，於塑膠晶片可達100 fg/μl。此晶片可於7小時內完成精確診斷鑑定受病原感染之林間罹病組織或病原證據標本，可應用於果樹、林木、苗木之植物病原真菌早期檢測認證，以及在生態、防治、造林決策等多方面。

關鍵字

細胞核內轉錄區；真菌病原；木層孔菌屬；蜜環菌；寡核苷酸探針；反轉式點膜雜合；生物晶片

並列摘要

Plant fungal pathogens can cause various types of diseases in agronomical, horticultural and forestry plants, which can lead to widespread plant mortality and famine. Therefore, use of time-saving, accurate, and sensitive diagnosis methods coupled with appropriate and effective control measures, is essential in reducing economic loss and enhancing public well-being. In recent years, due to climate change, an outbreak of brown root disease among community greenery street trees, artificial forests, and windbreak plants has caused serious losses, but has also forced the public to take a serious view of fungal pathogens. Attempt to rapid and accurate diagnosis, totally 67 species of wood rot fungal pathogens were collected, including many crucial pathogens, i.e. Phellinus noxius, P. pini and P. werii, Armillaria mellea, Armillaria ostyae, and several Ganoderma species. These pathogens, which have also been reported in the United States, Canada, New Zealand, Australia and Europe, have infected many coniferous and broadleaf forests, resulting in the growth decline; defoliating, early flowering, decay, wilting, lodging and other symptoms, and become a great limiting factor and a potentially serious threat in forest management. The oligonucleotide microchip developed for simultaneous rapid identification of the 67 crucial forest pathogens was based on the DIG or biotin-labeled specific probes derived forms ribosomal DNA gens (ITS1-5.8S-ITS2) by using reverse-dot hybridization. These chips can precisely and accurately identify and diagnose many Armillaria, Antrodia, Antrodiella, Heterobasidium, and Phellinus species, including A. mellea, A. ostoyae, H. annosum, P. noxius, and P. weirii, etc. with a sensitivity of 1 pg DNA/μl on nylon membrane chip, and 100 fg DNA/μl on plastic chip, respectively. And the verification and identification of forest Phellinus pathogens in authentic samples or voucher specimens can be accomplished within 7 hrs. The chip can be applied in inspection and quarantine, also for healthy free, seedling and nursery certification, and in ecology and silviculture management, as well.

並列關鍵字

fungal pathogens ； Antrodia ； Armillaria ； Heterobasidium ； Ganoderma ； Phellinus ； Oligonucleotide probe ； reverse dot hybridization ； microchip

參考文獻

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寡核苷酸微陣列同時快速診斷鑑定多種重要防檢疫森林病原真菌之研發

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