水稻徒長病菌 Fusarium fujikuroi 致病過程探討

Abstract

水稻徒長病由 Fusarium fujikuroi Nirenberg所造成，其廣泛發生於全球水稻種植地區。本病於幼苗期引起葉片褪綠、植株纖細徒長等病徵，造成成株節上長出不定根，並使稻穗不稔，因而導致 30-95% 的減產。由於目前對於 F. fujikuroi 在水稻上的致病過程所知有限，本研究目的為深入瞭解徒長病菌在水稻不同生育期的侵染與拓殖情形。本研究使用人工接種之抗病 (臺農67號，TNG67) 與感病 (Zerawchanica karatals, ZK) 品種幼苗，以及收集自田間之自然感染成株 [高雄139 號 (KS139), 台梗2號 (TK2) 及臺南11號 (TN11)]，透過徒手切片配合顯微觀察，和即時定量PCR (real-time quantitative PCR, qPCR) 技術進行菌量測定。人工接種試驗使用帶有綠色螢光蛋白 (green fluorescent protein, GFP) 基因之 F. fujikuroi 轉殖菌株，其為本研究以原生質體 - polyethylene glycol (PEG) 轉殖法所建立。利用螢光顯微鏡進行觀察，發現徒長病菌於侵染初期，由胚逐漸侵入根基部及莖基部，以菌絲直接穿透莖和根的表皮層進行感染，菌絲在細胞間及細胞內延伸。人工接種之幼苗以每 3 公分為一段，以 qPCR 測定其中的菌量，發現徒長病菌集中在胚、根基部與莖基部；接種 14 天後，在胚中之菌量遠較根及莖基部多。TNG67 及 ZK 於根及莖組織所帶菌量皆相似，顯示初期抗、感病品種在巨觀病徵上的差異，與實際侵染之菌量無直接關聯。以 0.5 mg/L 之 gibberellic acid-3 (GA3) 處理發芽水稻種子後，ZK 較 TNG67 幼苗有更明顯之徒長現象，表示 ZK 對徒長病的高感病性可能與其對 GA3 之高感受性相關。於分蘗及孕穗期之罹病植株中，病原菌在高雄139號及台梗2號品種幾乎僅嚴重纏據維管束，而在臺南11號則亦觀察到少量纏據維管束、並擴展至鄰近薄壁細胞之情形。此外病原菌主要分布於 50 公分以下之莖桿 (~60%植株高度)，葉片中則並未測得病原菌。徒長病菌對於稻株的莖節或節間組織並無偏好性，而莖節上長出不定根之病徵，不代表該莖節已被病原菌所侵染，不定根的出現亦無法反映纏據於植株中之病原菌量。期望透過本研究，為 F. fujikuroi 與水稻之交互作用提供新資訊。

Bakanae disease of rice is caused by Fusarium fujikuroi Nirenberg. The disease occurs worldwide in rice-growing areas. The infected seedlings are thinner, taller, and paler than healthy ones. The infected adult plants show adventitious roots on the node or empty panicles, leading to yield losses ranging from 30-95%. Due to the lack of knowledge on the pathogenesis of bakanae disease of rice plant caused by F. fujikuroi, this study aims to explore the infection and colonization patterns of F. fujikuroi during different rice growth stages. Artificially inoculated rice seedlings [a resistant cultivar Tainung 67 (TNG67) and a susceptible cultivar Zerawchanica karatals (ZK)] and naturally infected adult plants [Kaohsiung 139 (KH139), Taikeng 2 (TK2), and Tainan 11 (TN11)] were investigated by hand sectioning and microscopic observation as well as real-time quantitative PCR (qPCR). A green fluorescent protein (GFP)-tagged F. fujikuroi transformant generated by protoplast-polyethylene glycol (PEG) transformation method was used for artificial inoculation. By fluorescence microscopy, we observed direct penetration of F. fujikuroi hyphae through the epidermis of stem and roots. F. fujikuroi infected intra- and intercellularly from the embryo to the basal part of rice stems and roots during early infection stage. qPCR analysis of consecutive 3-cm segments of the whole seedlings revealed that F. fujikuroi was mainly detected in the embryo, basal stem, and basal roots. Since 14 days post inoculation, the biomass of F. fujikuroi in the embryo became much higher than in the stem and roots. TNG67 and ZK contained similar amounts of F. fujikuroi in the stem and root tissues, implying that the macroscopic bakanae phenotypes were not necessarily correlated with the pathogen quantity. Treatment of germinated seeds with 0.5 mg/L of gibberellic acid-3 (GA3) resulted in significantly more elongated seedlings of ZK than TNG67, suggesting that the bakanae susceptibility in ZK is likely associated with its higher sensitivity to GA3. In the infected plants at the tillering and booting stage, the hyphae were found heavily colonizing almost entirely in vascular bundles in cultivars KH139 and TK2 but in TN11 hyphae colonizing less abundantly in vascular bundles as well as the surrounding parenchyma cells. Moreover, F. fujikuroi was mainly detected in the stem below 50 cm (~60% of plant height), but undetectable in the leaves. F. fujikuroi showed no preference for nodes or internodes, and the emergence of adventitious roots from a certain node did not indicate node invasion of F. fujikuroi. In addition, the appearance of adventitious roots did not reflect abundant colonization of F. fujikuroi in the plant. We hope this study can provide new insights into the interactions between F. fujikuroi and rice.