咖啡葉片內生炭疽菌種類之研究

Abstract

本研究由南投縣仁愛鄉、雲林縣古坑鄉、嘉義縣中埔鄉及竹崎鄉、嘉義農業試驗分所、台南市東山區、台東縣泰武鄉等地7 個咖啡園，採集健康咖啡葉進行表面消毒及組織分離，分離到之內生真菌共計有859 株炭疽菌 (Colletotrichum spp.)、475 株葉點黴屬真菌 (Phyllosticta spp.)、505 株炭角菌科 (Xylariaceae) 真菌及214 株其他真菌。炭疽菌在7 個咖啡園中所占比例最高。為進一步瞭解內生炭疽菌種類，利用actin、calmodulin、chitin synthase、glyceraldehyde 3-phosphate dehydrogenase 以及β-tubulin 等序列， 以貝氏演算法 (Bayesian inference) 建構類緣樹，結果顯示分離的炭疽菌種類包含Colletotrichum gloeosporioides 複合種 (species complex)：Colletotrichum cordylinicola、Colletotrichum fructicola、Colletotrichum gloeoporioides、Colletotrichum jiangxiense、Colletotrichum siamense、Colletotrichum theobromicola、Colletotrichum tropicale及1 個尚未命名可能為新種的Colletotrichum sp.，此外尚有Colletotrichum boninense 複合種：C. boninense 及Colletotrichum karstii 等共10 種。針對南投仁愛、嘉義中埔、嘉義竹崎3 個咖啡園進行葉片內生炭疽菌的種類及比例分析，發現南投仁愛及嘉義中埔之菌相種類組成較相近，其中C. cordylinicola 在2 園的比例分別占83% 及27%，且2 園皆有C. siamense 之存在。而嘉義竹崎咖啡園之炭疽菌則以C. karstii 與C. fructicola 比例最高，各占45%，且未分離到C. cordylinicola 及C. siamense，而C. karstii 則僅在嘉義竹崎咖啡園分離獲得，顯示咖啡葉內生炭疽菌種類具有豐富多樣性，且所組成之菌相依果園不同亦有所差異。本研究為國內外首篇利用多基因類緣分析方法解析咖啡葉片內生炭疽菌種類之報告，其結果可作為後續咖啡病害或內生菌相關研究之參考。 In this study, healthy coffee leaves from seven coffee orchards were collected. The seven orchards were located in different areas, namely Ren-ai Township, Nantou County, Gukeng Township, Yunlin County, Chiayi Agricultural Experiment Station, Jhongpu, and Zhuqi Township, Chiayi County, Dongshan District, Tainan City, and Taiwu Township, Pingtung County. Coffee leaves were surface-disinfected and incubated on acidified potato dextrose agar to obtain endophytic fungi. Colletotrichum spp., Phyllosticta spp. and Xylariaceous fungi were isolated from these seven orchards with a high frequency, and Colletotrichum spp. were the most frequently isolated fungi in six of the seven orchards. According to the results of phylogenetic analysis based on actin, calmodulin, chitin synthase, glyceraldehyde 3-phosphate dehydrogenase, β-tubulin sequences constructed by Bayesian inference, the endophytic Colletotrichum spp. in coffee leaves contained nine species belonging to Colletotrichum gloeosporioides species complex, namely Colletotrichum cordylinicola, Colletotrichum fructicola, Colletotrichum gloeosporioides, Colletotrichum jiangxiense, Colletotrichum siamense, Colletotrichum theobromicola, Colletotrichum tropicale and an unknown new Colletotrichum sp., and two Colletotrichum boninense complex species, C. boninense and Colletotrichum karstii. Isolation frequency and species composition of endophytic Colletotrichum spp. from three orchards were compared. The Colletotrichum spp. composition of the orchard located in Ren-ai Township, Nantou County resembled that located in Jhongpu Township, Chiayi County. The isolation frequencies of C. cordylinicola were 83% and 27% in orchards of Ren-ai and Jhongpu, respectively. C. siamense was also isolated from both of the two orchards. In contrast, no C. cordylinicola or C. siamense was isolated from the orchard in Zhuqi Township, Chiayi County. The most frequently isolated Colletotrichum spp. in Zhuqi orchard were C. fructicola (45%) and C. karstii (45%). The result suggested that endophytic Colletotrichum spp. in coffee leaves are highly diverse, and the fungal composition is different among orchards. This is the first intensive study of endophytic Colletotrichum spp. in coffee leaves using multi-locus sequence analysis to resolve the species, and the result provides information for further study on coffee pathogen or endophyte.