Analysis of genetic diversity and host preference in Colletotrichum gloeosporioides

炭疽病菌Colletotrichum gloeosporioides (Penz.) Penz. & Sacc.為重要植物病原真菌，寄主範圍廣泛，可侵染芒果、酪梨、杏仁、草莓和木瓜等多項經濟作物，常在世界各地造成嚴重損失。為探討自不同寄主所分離之C. gloeosporioides菌株的遺傳變異情形，本研究以inter-simple sequence repeats (ISSR)分析技術針對各分離株進行基因型分析。研究結果顯示，分離自相同寄主的菌株常呈現相似或相同的ISSR圖譜；進一步以未加權算數平均法(unweighted pair group method with arithmetic mean, UPGMA)針對各菌株的ISSR圖譜進行群叢分析，並繪製親緣樹狀圖也發現，來自相同寄主的分離株有明顯群聚現象，且以芒果分離株的群聚現象最為顯著─ 雖然於不同時間採集自不同地點，且所採自的芒果品種也不一樣，多數芒果分離株會落在同一群內。相對的，即令木瓜分離株多數收集自同一地區，其ISSR圖譜卻顯現出相當程度的差異性。本研究亦嘗試以expressed sequence tags-SSRs (EST-SSRs)進行C. gloeosporioides遺傳變異性的分析，但發現各菌株之EST-SSR圖譜的差異性極為有限，無法成為有效的分子標記。此外，為探討C. gloeosporioides 各分離株的病原特性，將自木瓜、酪梨和芒果所收集的分離株分別針對木瓜、酪梨和芒果果實進行接種測試。結果顯示，雖然多數分離株具有交叉感染的能力，但仍以接回原寄主時所造成的病斑最大(P < 0.05)，顯示這些菌株具有顯著的寄主偏好性。不過，所測試的芒果分離株中，C157及MAC060在酪梨與木瓜果實所造成的病斑都相當小，甚至無法侵染，而分離株C442及C447則可在酪梨與木瓜果實造成相當大的病斑，顯示縱使來自相同寄主的分離株，其病原特性仍有所不同。Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. is known to infect a wide variety of crop species including mango avocado, almond, strawberry and papaya, and they cause significant economic damage to crops worldwide. To investigate genetic variation among these fungal isolates from different hosts, we developed a genotyping method based on inter-simple sequence repeat (ISSR), and used it for the analysis of our fungal isolates. The results obtained by ISSR-PCR indicated that, in most cases, C. gloeosporioides obtained from the same host species displayed similar gel patterns and were grouped in the same cluster by the UPGMA-based cluster analysis. This phenomenon was most predominant for isolates collected from mango; irrespective of the collection time, location, and mango varieties, C. gloeosporioides collected from this host tends to exist in the same cluster. Isolates from papaya, in contrast, were highly diverse in the ISSR patterns, despite that they were collected from the same area. We tried to develop expressed sequence tags-SSR markers, but less polymorphism was observed. Therefore, the EST-SSR markers which we analyzed are not the effective markers. To investigate the pathogenicity of isolates, we conducted inoculation tests of mango, avocado and papaya isolates. The results showed the potential of cross-infection among the C. gloeosporioides isolates from different host. However, isolates showed host preference since they were more pathogenic in their natural hosts (P < 0.05). The mango isolate C157 and MAC060 produced small lesions on avocado and papaya fruit, whereas isolates C442 and C447 produced large lesions. It indicates that even from the same host, isolates showed different pathogenicity.誌謝.......................................................i文摘要..................................................ii文摘要................................................ iii、前言...................................................1、前人研究...............................................1、炭疽病菌簡介...........................................1、炭疽病菌的病原特性與影響因子...........................3、炭疽病菌遺傳變異性之探討...............................5、SSR 簡介...............................................7、材料與方..............................................10、菌株收集與核酸抽取....................................10、EST-SSR 之分析........................................10、引子設計..............................................11、ISSR 與EST-SSR 之PCR 與電泳反應條件...................11、親緣樹狀圖之繪製......................................12、序列分析..............................................12、南方雜合分析..........................................13、接種試驗..............................................15、結果..................................................17、C. gloeosporioides 各分離株菌落型態變異性大...........17、ISSRs 圖譜和寄主來源有關..............................18、(AAC)6 放大的片段多位於基因間區段.....................23、芒果分離株基因體中並無缺少(AAC)6 於其他寄主放大之片段.24、EST-SSR 於C. gloeosporioides 的種內遺傳變異性.........25、炭疽病菌接種測試結果..................................27、討論..................................................31、ISSRs 圖譜分析之結果討論..............................31、EST-SSR 於C. gloeosporioides 基因體內的多型性較低.....34、接種測試之結果討論....................................35、未來展望..............................................38、參考文獻..............................................39、表....................................................46、圖....................................................61、附錄..................................................7