台灣茶白紋羽病之研究

Abstract

於台灣主要茶區進行茶白紋羽病採樣，結果於南投5處出現立枯茶樹之高山茶園根腐檢體中，分離出11株隔膜具有白紋羽菌梨形膨大菌絲特徵之病原菌，分離率為總檢體數之2.41 %，罹病茶園比例為5.38 %。另外，由其他寄主檢體中亦分離出25株具相同菌絲特徵之病原菌。由菌株R203誘導產生7個成熟子囊果進行單孢分離後，共收集到48株子囊孢子菌株。所收集之田間菌株，接種於茶苗上皆可產生黑色剛毛狀、頭部白色膨大之分生孢梗束無性世代，其分生孢子呈透明，單胞、橢圓形截頭狀，大小為2.9-5.8 × 1.9-3.5 μm。菌株R203產生之有性世代子囊果為單生或聚生、深褐色至黑褐色、次球狀至球狀、內含單子囊殼，子囊為單套膜、棒狀至圓柱狀，內含8個單層排列之子囊孢子，其頂栓與Mezler試劑反應呈藍色。子囊孢子為單細胞、褐色至深褐色、呈不對稱窄梭狀、二端尖，大小為37.0-55.0 × 5.4-7.9 μm，內有一條平行於縱軸、小於其一半長度之短線狀發芽縫。收集之36株白紋羽田間菌株經ITS DNA定序後，進行序列比對及親緣性分析之結果，顯示其中35菌株為Rosellinia necatrix Berl. Prill；而另外一菌株R701之ITS DNA序列相較於其他菌株，有較多變異性，然其序列與保守性序列（consensus sequence）間之相似度仍高達97 %，因此，推定其為近似R. necatrix之Rosellinia sp.。所收集台灣菌株R101、 R203、 R210、 R301、 R401、 R701 及 R801等7條 ITS DNA 序列已傳送至美國國家生物技術資訊中心GenBank資料庫。茶白紋羽菌最適生長溫度範圍為25 ℃，高於35 ℃ 則不生長，而溫度低至5 ℃ 時，7天後仍可有0.1 mm之生長，其最適pH 為5.0。茶白紋羽菌在葡萄糖碳素源上生長最快，而在木糖碳素源上生長較慢；有機氮素源如大豆水解胜肽及酪蛋白，對促進菌絲生長之效果佳，而於硝酸鈣或尿素二種無機氮源上，白紋羽菌絲有生長緩慢之現象。本研究發現台灣地區白紋羽菌株之親和群相當分歧：36田間菌株，共可分成18個菌絲親合群，其中來自紅榨槭之14菌株－R101至R114，皆屬於菌絲親和群1；來自六月雪之菌株R202和R204屬於菌絲親和群2；來自六月雪之4菌株R203、R205、R206、R208及來自福建茶之1菌株R209屬於菌絲親和群3，其餘之田間菌株及子囊孢子菌株，彼此間皆不親和，而子囊孢子菌株與其親代菌株R203間也不親和。AFLP群聚分析之結果，顯示同一親和群內之菌株無法予以區別，表示同一親和群內之菌株皆源自同一基因體，顯示其病害可能肇因於相同菌株來源之無性傳播。此外，50 %子囊孢子菌株與其親代菌株R203彼此遺傳相似度極高，顯示AFLP群聚分析適用於台灣白紋羽菌族群結構之探索以及具有追溯白紋羽病害傳播之潛能。病原性測試結果，顯示人工接種之仟插茶苗可產生與田間觀察到類似之茶白紋羽病徵，從罹病死亡茶苗殘株中，可再度分離出茶白紋羽菌而完成柯霍氏法則驗證。致病性方面，發現除來自紅榨槭之菌株對茶苗之致病性較低以外，其他寄主來源之白紋羽菌株及其子囊孢子菌株對茶苗之致病性皆相當高，而菌株R701 Rosellinia sp.對茶苗之致病性亦相當高。上述結果顯示台灣地區白紋羽病之傳播機制除前述之無性傳播方式以外，可能另有藉由有性子囊孢子散佈之傳播方式存在。病害防治方面，本研究結果顯示二種藥劑－亞托敏及百克敏對白紋羽菌亦具有抑制效果，其EC50與貝芬替、撲克拉及植物保護手冊梨白文羽病推薦藥劑扶吉胺等皆位於ppb水準，顯示白紋羽菌對各藥劑之感受性相當高。進行白紋羽菌接種枝條藥劑浸泡試驗之結果，統計分析發現亞托敏、百克敏與扶吉胺對白紋羽菌之抑制效果並無差異（α=0.05）。人工接種茶白紋羽病化學藥劑灌注防治試驗之結果，經統計分析顯示1000倍稀釋之亞托敏、百克敏、貝芬替、撲克拉及扶吉胺等藥劑具有相同的茶白紋羽病害防治效果且無藥害產生（α=0.05），相對地，疾病對照組茶苗於4個月內已全數死亡。因此，日後或可將亞托敏、百克敏、貝芬替、撲克拉及扶吉胺等藥劑列入我國茶白紋羽病防治時之考量。

Eleven isolates, appearing the typical hyphal characteristic of septa adjacent pear-shape swellings of Rosellinia necatrix Hartig, were isolated from white root rot samples of Camellia sinensis (L.) O. Kuntze, gathered from five tea gardens that suffered from the dieback disease in mountainous Nantou during the study. The isolation rate was 2.41 % for tea samples and 5.38 % for tea gardens. In addition, 25 pathogen isolates originated from other hosts were also collected from the central and northern areas of Taiwan. 48 ascospore isolates were also collected from 7 mature ascomata produced by isolate R203. The synnemata (anamorph) produced by the field isolates were black and bristle-like, tapering toward the enlarged whitish gray heads. Conidium was hyaline, one-celled, subglobal, and base truncated, 2.9-5.8 × 1.9-3.5 μm. The ascoma was single or gregarious, dark brown to black, uniperitheciate, subglobose to globose. Ascus was unitunicate, clavate to cylindrical, contained 8 uniseriate ascospores, with a basally conical, apically enlarged and strong blue amyloid apical apparatus. Ascospore was one-celled, brown to dark brown, asymmetrically narrow fusiform with two acute ends, 37.0-55.0 × 5.4-7.9 μm, with a less than a half, short straight germ slit parallel to its longitude. Based on the result of ITS DNA sequencing comparison and phylogenetic analysis, 36 isolates of white root rot pathogens collected from Taiwan except R701 were identified as Rosellinia necatrix Berl. Prill. Isolate R701, which was relatively more polymorphic in ITS DNA sequence than others but still had high similarity with the consensus sequence (over 97 % similarity), was temporarily regarded as R. necatrix related pathogenic Rosellinia sp. The ITS DNA sequences of isolates R101, R203, R210, R301, R401, R701 and R801, have been deposited in NABI GenBank data library. The optimum temperature for radial growth of R. necatrix mycelium was 25 ℃. No growth was observed when temperature over 35 ℃. On the contrary, there was still 0.1 mm mycelial growth when incubated at 5 ℃ after 7 days. The optimum pH for radial growth was around pH 5.0. Glucose was the best carbon source to enhance the radial growth of the pathogen, while xylose was the inferior one. The organic nitrogen sources, peptone and casein, could enhance the mycelial growth of the pathogen, while inorganic nitrogen sources such as calcium nitrate or urea seemed to retard radial growth. The VCGs of white root rot pathogen, 18 vegetative compatible groups existing among 36 field isolates, were quite complex. All the 14 isolates from Acer morrisonense belonged to VCG1, isolate R202 and R204 from Serissa japonica belonged to VCG2, and isolate R203, R205, R206 and R208 from S. japonica and isolate R209 from Ehretia microphylla belonged to VCG3. The other field isolates and ascospore isolates were mycelial incompatible with each other, even the ascospore isolates and the parental isolate R203 were mycelial incompatible. The result that isolates within the same VCG were genetically undistinguished by AFLP analysis indicated they were originating from a genet. AFLP analysis also showed that 50 % ascospore isolates were highly genetically related to parental isolate R203. It demonstrated that AFLP analysis could suitably be applied to explore the genetic structures and trace the dispersal of R. necatrix. When artificially inoculated to tea cuttings, the pathogen isolates, which could be re-isolated from dead infected tea cuttings to fulfill Koch’s postulates, produced tea white root rot symptoms similar to those observed in diseased fields. Pathogenicity determination indicated that all the field isolates including R701 and ascospore isolates were highly virulent except those originated from A. morrisonense. Therefore, it suggested the possible ways of dissemination of white root rot disease in Taiwan including mycelial strands transmittion as well as ascospores dispersal. The EC50s of azoxystrobin, pyraclostrobin, carbendazim, prochloraz, and fluazinam against the pathogen were quite sensitive at the ppb level. Drug dipping tests for the previous chemicals against inoculated twigs showed no statistically significant difference (α=0.05) in inhibitive effects. The result of chemical drenching for tea white root rot disease in greenhouse demonstrated that the control effect of the previous 5 chemicals at 1000X dilution had no statistically significant difference (α=0.05). No harmful drug side-effects were observed for tea cuttings of treatments, whereas those of disease control died within 4 months. Therefore, azoxystrobin, pyraclostrobin, carbendazim, prochloraz, fluazinam could be taken into consideration for chemical control of white root rot disease of Camellia sinensis in the future.