選殖褐根腐病菌 Phellinus noxius 之錳過氧化酵素(PnMnP)及其 與其病原性之相關性

Cloning and characterization of Phellinus noxius manganese peroxidase (PnMnP) gene and its relatedness with virulence

黃子葳(Tzu-Wei Huang)

指導教授 ： 曾顯雄

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.01432

摘要

褐根病菌(Phellinus noxius)為白腐病，其所造成之褐根病，再近幾年漸趨嚴重。自1980年起，在世界各地都有報導過此一病害，如馬來西亞、印度、非洲等熱帶、亞熱帶國家。褐根腐病主要侵染闊葉喬木，在經濟或觀賞作物上造成相當的損害，如果樹、橡膠樹及常見之行道樹或建材用樹種，皆是其侵染之目標。褐根腐病最明顯之病徵，即是在受到侵染的樹木，其樹勢會急速衰退，末端枝葉黃化或小葉化，其根部或近基部之表面，會有褐色或深褐色之菌絲覆層。在樹皮下之木質部，會有褐色之網狀紋路。褐根腐病在長時間受到侵染的樹木上，則會發育出不規則層疊狀之子實體。為了侵染植物組織，褐根病菌等白腐菌會分泌一群可分解木質素之酵素，如木質分解素(lignin peroxides, LiP)或是錳過氧化酵素(Manganese peroxidase, MnP)等。在本研究中，將探討錳過氧化酵素與褐根腐病在病原性上之關係。研究中以基因靜默(RNA silencing)之技術，並以農桿菌轉殖Agrobacterium-mediated transformant)之方式，導入一段與過氧化酵素互補之DNA片段，以誘發其基因靜默，降低錳過氧化酵素之表現量。在轉殖成功的六個轉型株中，其錳過氧化酵素之表現量降低50%~90%，其中有兩個轉型株之錳過氧化酵素表現量有更明顯之減少。之後將轉型株接種於枇杷樹(Eriobotrya japonica)上，以探討其錳過氧化酵素與病原性之關係。結果顯示其基因靜默效果越好之轉型株，其病原性會越差，換言之錳過氧化酵素與其病原性確實有一正向之相關性，而錳過氧化酵素也可作為設計防治褐根腐病之系統性殺菌劑目標之一。

關鍵字

褐根腐病 ； 錳過氧化酵素 ； 木質素分解酵素 ； 木質素

並列摘要

Brown root rot disease, caused by basidiomycetous fungus Phellinus noxius, becomes more noticeable from 1980s. The pathogen primarily attacks board-leave trees, fruit trees, and ornamental plants, such as rubber, coffee, Ficus and Cinnamomum, etc. The most pronounced symptom and sign of the infection are the massive yellowing and defoliating of the leaves, retarding the twig growth, and in the later infection stage, the butt and root near the ground covered with brown to dark mycelial mat, accompanied with internal wood decay and occasional formation of resupinate or sessile pileate fruiting body. The dense mycelium and deep brown to black rhizomorphs under the bark also are the indication of infection. In order to penetrate and colonize the host to acquire the essential nutrients, rationally, the brown root rot pathogen has to produce an array of tissue degrading enzymes, i.e., cellulase, hemicellulase, and lignin peroxidase, etc. Therefore, the magnitude of such enzymic activity might relate to its virulence. In this study, we explored the relevance of MnP to virulence. A gene silencing strategy is initiated to down regulate the expression of manganese peroxidase which is usually encoded by multiple copy gene family. The RNA silencing cassette was constructed based on the skeleton of pCAMBIA plasmid, and introduced into P. noxious genomic DNA by Agrobacterium tumefaciens-medicated transformation (ATMT) to obtain stable transformants. Subsequently, bioassay and pathogenicity test of the transformants have been performed in vitro and in vivo, to determine the relatedness between lignin-degrading manganese peroxidase and the virulence of P. noxious. The results reveal that the virulence of P. noxius declined considerably on loquat (Eriobotrya japonica) seedlings relevant to the down regulation of MnP gene expression by gene silencing. Prospectively, the MnP can be envisaged as the selection target for designing systemic fungicide in control P. noxius brown rot.

並列關鍵字

phellinus noxius ； Brown root rot ； Manganese peroxidase ； lingin

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