臺灣牛樟樹內生真菌之多樣性調查與防治植物病害潛力評估

Diversity of fungal endophytes in Cinnamomum kanehirae in Taiwan and control assessment on crop diseases

"牛樟樹（Cinnamomum kanehirai; Syn. C. micranthum）為台灣本土特有之闊葉樹種。本研究之目的為調查牛樟樹中內生真菌之多樣性、篩選具拮抗能力之菌株，並分析有效物質成分與種類。自惠蓀林場內牛樟樹分離到分屬13科22屬共189株內生真菌菌株；自國立自然科學博物館內牛樟樹則分離到7科7屬共35株。內生真菌菌相的族群分布與密度會因枝條部位、氣候環境之不同而有所差異。測試224株內生真菌對植物病原真菌之拮抗能力，僅有6株對供試病原真菌具有不同程度之抑制作用，其中CKL005菌株可抑制8種植物真菌病原，一種卵菌病原及4種病原細菌。根據形態及分子親緣性之特性，CKL005菌株與Colletotrichum屬種類較相似。CKL005菌株發酵液經乙酸乙酯萃取後，不具有效抗生物質；然而，菌絲萃取液對草莓灰黴病菌、十字花科黑斑病菌、芒果及白菜炭疽病菌的抑制效果顯著。菌絲萃取物經管柱層析、高效能液相層析（HPLC）分離、純化之後，進行NMR與GC/MS分析，得知具有活性的主要成分包括β-胡蘿蔔素、單棕櫚甘油酸、乙酸去氫皮質醇、麥角固醇、蟾蜍他靈、皮質類固醇、洋地黃毒苷及吉勃素，其中以酯類蟾蜍他靈的相對含量最高。CKL005菌株亦能產生揮發性氣體以抑制白菜炭疽病菌菌絲生長。GC/MS分析結果得知：可能有抗菌作用的氣體成分為二種BHT（Butylated hydroxytoluene）類脂溶性抗氧化劑（2,5-Cyclohexadiene-1,4-dione,2,6-bis（1,1-dimethylethy）-methylphenol與2,6-Bis（1,1-dimethylethyl）-4-methylphenol及3,6,7-thimethoxy- isobenzofuran-1（3H）-one。CKL005菌絲萃取液稍可提高白菜種子發芽率及幼苗鮮重量。於溫室防治試驗中得知，無論於接種白菜炭菌病孢子2小時之前或之後，施用菌絲萃取液均可有效降低炭菌病罹病度。"

"Cinnamomum kanehirai (Syn . C. micranthum) i s a n indigenous arbor in Taiwan. The objectives of this study are to investigate the distribution and diversity of fungal endophytes in C. kanehirai and environmental factors influencing their diversities, and to identify strains that have bioactivity against plant pathogens. Of 224 fungal endophytes isolated from C. kanehirai trees grown in two different locations, 189 were from Hui-Sun Forest, Nantou, Taiwan, belonging to 13 families and 22 genera and 35 from inside the National Museum of Natural Science, Taichung, Taiwan, belonging to 7 families and 7 genera. It appears that fungal endophytes have a higher diversity in trees grown in natural habitats. The diversity was also observed in the samples collected from different tissues of the same tree. Of 224 isolates examined, only 6 displayed antagonistic activities against Colletotrichum gloeosporioides, Botrytis cinerea, and Fusarium oxysporum f. sp. lilii. Among them, CKL005 had the most impressive ability to suppress additional pathogens, including Phytophthora capsici, Alternaria brassicicola, F. oxysporum f. sp. lycopersici, F. oxysporum f. sp. tracheiphilum, C. higginsianum, Rhizoctonia solani, Erwinia carotovora subsp. carotovora, Xanthomonas campestris p v . campestris, a n d Ralstonia solanacearum. Judging from morphological and phylogenetic analyses, CKL005 was likely a Colletotrichum sp. Ethyl acetate extracts derived from mycelia but not culture filtrate displayed strong antagonistic activities. Purification and analysis through silica gel, HPLC, NMR and GC/MS revealed that the bioactive components contained β-carotene, glycerol 1-palmitate, prednisolone acetate, ergosterol, hydrocortilsore acetate, gammabufotalin, digitoxin, and gibberellin with gammabufotalin being the highest. In addition, CKL005 could produce volatile 2,5-cyclohexadiene-1,4-dione,2,6bis(1,1-dimethylethy)-2,6-Bis(1,1-dimethylethyl)-4-methylphenol and 3,6,7-thimethoxy-isobenzofuran-1(3H)-one, which were likely toxic to plant pathogens. CKL005 mycelial extracts could promote the germination of cabbage seeds and increased seedling fresh weight. Most importantly, spray application of CKL005 mycelial extracts onto cabbage seedlings before or after inoculation of C. higginsianum effectively reduced disease severity."