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標題: 內生真菌Lasmenia sp. CB10菌株之特性與防治番茄萎凋病效果評估
Characteristics of endophytic fungi Lasmenia sp. CB10 isolate and evaluation of its efficacy for control of tomato Fusarium wilt
作者: Pei-Hsin Lo
關鍵字: 內生真菌;Lamenia sp.;番茄萎凋病;米糠;菌絲萃取液;endophytic fungi;Lasmenia sp.;Fusarium wilt;rice bran;mycelia extract
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內生真菌 (endophytic fungi) 為纏據於植物組織,且不會造成植物產生病徵之真菌。前人研究指出,某些內生真菌可分泌二次代謝產物或誘導系統性抗病反應,進而提供植物抗病蟲害的能力。此外,內生真菌亦可產生與寄主植物結構相同或相似之化合物,特別是中草藥內生真菌,已被視為萃取有效代謝物或產物之重要來源。本研究測試自芸香科中草藥植物佛手柑 (Citrus medica var. sarcodactylis) 所分離,具抑制多種植物病原真菌菌絲生長效果之內生真菌CB10菌株的生物特性與應用於番茄萎凋病的防治,並分析具抑菌活性物質之成分。CB10菌株經形態與分子親緣性分析,鑑定為Lasmenia屬;最適生長溫度為20-24℃,與寄主植物佛手柑具有相同之生長溫度,且於不同溫度下,CB10菌株具不同菌落形態。CB10菌株培養於小麥粒會產生類似柑橘之香氣,經氣相層析 (GC/ MS) 分析結果顯示,揮發性氣體中有多種柑橘上常見具芳香活性之成分,其中又以柑橘類作物產生之單萜類與倍半萜類物質為主要成分。於胞外酵素測試結果顯示,CB10菌株具有廣泛之養分利用能力,可分泌澱粉酵素 (amylase)、木聚糖酵素 (xylanase)、纖維素酵素 (cellulase)、漆化酵素 (laccase)、木質素酵素 (ligninase)及解脂酵素 (lipase),可於植物組織老化時,扮演腐生菌 (saprophyte) 的角色。以不同基質嘗試培養CB10菌株,得知該菌株可生長於榖類作物基質,如小麥粒、燕麥粒及燕麥片;農業廢棄資材基質,如米糠、豆粕、芝麻粕;太空包木屑。進一步測試拮抗能力,指出培養於燕麥、米糠及豆粕基質上的CB10菌株,對番茄萎凋病菌Fol146具最佳生長抑制效果,顯示高氮含量基質可促進CB10菌株產生有效二次代謝產物。以抑制效果較佳之燕麥與米糠基質培養CB10菌株,並與番茄萎凋病菌 (Fol146) 帶菌土混合,得知以1: 5 (w/ w) 比例混合含CB10菌株米糠 (簡稱RBCB10) 時,具降低土壤中番茄萎凋病菌族群量之效果;而混合含CB10菌株燕麥 (簡稱OMCB10) 則無降低番茄萎凋病菌族群量的效果。將0.5% (w/ v) RBCB10與使用未滅菌土混合,可降低番茄萎凋病罹病度達29.6%,並抑制Fol146菌株之族群量;反之混合0.5% (w/ v) RBCB10於滅菌過土所配製之Fol146帶菌土時,則無防治番茄萎凋病與抑制Fol146生長之效果。於混合不同比例RBCB10對番茄植株生長影響結果中,指出混合1% (w/ v) 比例之米糠基質會影響番茄生長,而0.5% (w/ v) 比例則不會影響番茄生長。另比較不同RBCB10基質施用方式對防治番茄萎凋病之影響,得知先行以混合RBCB10 0.5% (w/ v) 之泥炭土育苗的植株,可降低萎凋病的罹病度達26.7%;而以0.5% (w/ v) RBCB10直接混合於帶菌土中的結果,可降低萎凋病罹病度達40.7%。測試CB10菌株有效抗菌物質類似物diosmin標準品之拮抗效果,證實diosmin不具抑制番茄萎凋病菌生長與誘導抗萎凋病效果。將CB10菌株菌絲萃取液有效成分配合diosmin再分析結果指出,以高效能液相層析法 (HPLC) 所得圖譜與diosmin圖譜不同,證實CB10菌株之抑菌有效物質並非diosmin。

Endophytic fungi are the microorganisms which live inside of plant tissue without causing symptoms. Endophytic fungi can protect host against pathogens based on their secondary metabolites or induced systemic resistance. Previous studies demonstrated that certain endophytic fungi can produce similar metabolites as host plants. Especially, the endophytic fungi from medicinal plants have been considered as the important source of the novel metabolites. In this study, the endophytic fungi CB10 isolate from medicinal plant fingered citron, Citrus medica var. sarcodactylis, of Rutaceae have been reported to show the ability against several plant fungal pathogens. The CB10 isolate was identified as Lasmenia sp. based on morphology and molecular characters. Moreover, the temperature test indicated that 20-24℃ was optimum for CB10 isolate growth and same with fingered citron. The colonies morphology were variable with different temperatures. The analysis result showed that CB10 isolate cultured on wheat grain could produce volatile compounds of citrus-like aroma based on GC/ MS, especially, aroma monoterpenes and sesquiterpenes existed in the citrus. The extracellular enzyme test revealed that CB10 isolate could produce amylase, cellulase, lipase, xylanase, laccase and liginase. The results indicated that CB10 isolate might play a role of saprophyte in plant during the host died. Solid culture test indicated that CB10 isolate could grow on cereal, such as wheat, oat grain, oatmeal; the agriculture waste, such as rice bran, soybean meal, sesame meal, mushroom sawdust. Moreover, CB10 isolate cultured on oatmeal, rice bran, and soybean meal showed high efficacy on inhibiting the mycelia growth of Fusarium oxysporum f. sp. lycopersici (Fol146). The result revealed that substrates with high nitrogen could promote the production of the active compounds. CB10 isolate cultured on rice bran (RBCB10) mixed 1: 5 (w/ w) ratio with infested soil had efficacy on inhibiting the growth of Fol146. However, CB10 isolate cultured on oatmeal (OMCB10) didn't show the efficacy. Mixed 0.5% (w/ v) RBCB10 with the Fol146 infested soil made from non-sterilized soil could decrease the population of Fol146 in the soil and delayed 29.6% disease severity of tomato. In other hand, the RBCB10 mixed with the Fol146 infested soil made from sterilized soil didn't show the control efficacy. Moreover, mixed 1% (w/ v) rice bran with peat moss as seedbed soil had negative efficacy on the growth of tomato seedlings in the greenhouse. However, mixed 0.5% (w/ v) did't show negative efficacy. In addition, mixed 0.5% (w/ v) RBCB10 with peat moss as seedbed soil could decrease 26.7% disease severity of tomato Fusarium wilt; meanwhile, mixed 0.5% (w/ v) RBCB10 with infested soil decreased the 40.7% disease severity. Evaluation of the efficacy of diosmin on control Fusarium wilt showed that diosmin didn't has ability to inhibit Fol146 or induce resistance of tomato against Fusarium wilt. Moreover, comparing the profile of mycelia extract of CB10 isolate with diosmin based on HPLC (high performance liquid chromatography) analysis indicated that the active compound did not show same profile with diosmin. Thus, the active compounds are not diosmin analogue.
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