由Botryosphaeriaceae引起之檬果果腐病研究

Abstract

檬果果腐病為近年來，嚴重影響臺灣檬果經濟價值的重要採收後病害之一，本論文針對其田間發生率、病原菌種類鑑定、分子檢測技術開發以及防治方法進行一系列的研究。首先於 2009-2011 年間，由屏東枋山、台南玉井及官田等檬果主要產區進行果腐病害調查及病原分離，發現其病害發生率自 18.7% 至 58.1% 不等，其中官田地區之發生率更高於玉井及枋山。自果腐病病斑分離之病原菌，分別以菌落及分生胞子形態特性及核糖體非轉錄區間 (ribosomal internal transcribed spacer, ITS)、微管蛋白 (β-tubulin, TUB) 與 Elongation factor 1-α (EF1-α) 等序列之特性分析後，共發現四種 Botryosphaeriaceae 病原菌，包括 Lasiodiplodia theobromae、Neofusicoccum mangiferae、N. parvum 及 Fusicoccum aesculi。此四種病原菌不論是接種於採收後未製造傷口或先製造傷口之果實，都可成功感染，造成果腐病斑，顯示其皆具有病原性，而且 L. theobromae 之致病力明顯較其它三種病原菌強。另於田間直接將四種 Botryosphaeriaceae 病原菌分生胞子接種於檬果樹上未成熟果實，發現不需製造傷口，病原菌即可直接感染果實，並於採收後之果實上出現果腐病斑。由於以上四種真菌之培養形態彼此相近，因此增加了菌種鑑別上之困難度，為此本研究藉由比對四種菌之核糖體非轉錄區間 (ITS) 序列，設計了四組種專一性引子對，並且建立 nested multiplex PCR 方法，藉以快速檢測果腐病原種類，其檢測靈敏度可達 100 fg-1 pg。此方法也可以成功檢測病原在接種檬果之存在情形。為進一步瞭解果腐病原之田間感染途徑，本研究利用枝條、果梗、花穗之病原分離與果實發病之比較分析，發現檬果果腐病之感染源可能來自枯枝上產生之胞子飛濺至果身而造成感染，因此未來田間進行清園以防止胞子累積及飛濺，應為防治本病害之首要工作。此外，本研究利用亞托敏、賽普護汰寧、貝芬依滅列、貝芬菲克利及依普同等五種化學藥劑進行檬果果腐病之田間防治測試，結果顯示僅亞托敏可以降低果腐病發生率至 10% 以下，其他藥劑則無明顯防治效果。另一方面，本研究利用不同溫度熱水處理生長在玻璃紙之上述四種果腐病菌，發現除了 L. theobromae 在 58℃ 處理 2 分鐘後仍有 10% 之存活率外，其餘病原之菌絲一經溫水處理，均無法再生長，顯示 L. theobromae 是最耐熱的果腐病原。應用 58℃/ 2 分鐘、60℃/ 20 秒及 62℃/ 30 秒等三種溫水處理條件，分別進行玉井及官田產區之檬果果腐病防治評估，結果僅顯著降低官田地區之果腐病，對於玉井地區之果腐病並無採後防治之效果，其原因為何仍有待進一步闡明。

Fruit rot disease was one of the most important postharvest diseases on mango, and severely influenced the economic value of mango production in Taiwan. The objectives of the present study were to characterize the causal agents of mango fruit rot disease and to develop the control strategies, including fungicide application and hot water treatment. A field survey was performed in Guntian, Fanshan, and Yujing area during 2009-2011. The results showed a disease incidence ranging from 18.7% to 58.1%, with those of Guntain significantly greater than the incidence found in Yujing and Fanshan. Based on morphological characteristics and nucleotide sequences of the internal transcribed spacer (ITS), β-tubulin gene (TUB) and elongation factor 1-alpha (EF1-α) gene, four species of Botryosphaeriaceae, including Lasiodiplodia theobromae, Neofusicoccum mangiferae, N. parvum, and Fusicoccum aesculi were identified. Pathogenicity tests indicated that all of these fungal species were pathogenic to harvested mango fruits, and L. theobromae was the most aggressive pathogen. Moreover, when attached, immature mango fruits were inoculated with conidia of Botryosphaeriaceae species, disease symptoms of fruit rot appeared on the fruits after harvest and ripening. To aid reliable identification of Botryosphaeriaceae species associated with mango fruits, four pairs of species-specific primers (Lt397F/Lt397R, Bd318F/Np479R, Np479F/Np479R and Bd318F/Bd318R) were designed according to sequences of the ribosomal internal transcribed spacers (ITS), and a rapid method was established based on nested multiplex polymerase chain reaction (PCR) in this study. With this method, a low limit of 100 fg-1 pg of purified fungal DNA was detectable. It could also successfully detect L. theobromae, N. parvum, N. mangiferae and F. aesculi in total DNA extracted from inoculated mango fruits. In order to understand the infection pathway, the corelation between pathogen isolation frequency from twigs panicle and the incidence of fruit rot disease was survayed. The results showed that inoculum of fruit rot could originate from conidia produced on dead twigs, and spreaded to the fruit body, suggesting that orchard cleaning was the priority for the control of fruit rot disease in field. Besides, Azoxystrobin, Cyprodinil+Fludioxonil, Carbendazim+ Imazalil, Carbendazim+Hexaconazole, Iprodione were tested. The results reveled that Azoxystrobin was the only effective chemical agent, and could reduce the disease incidence to 10%. As for the hot water treatment against mycelial growth of Botryosphaeriaceae species, most pathogens were killed under 58℃ and 60℃ treatment. L. theobromae remained 10% survival rate as treating under 58℃ for 2 min, revealing that L. theobromae was the most heat-resistant pathogen. Moreover, hot water treatments by 58℃/2 min, 60℃/20 sec, 62℃/30 sec were effectively controlled fruit rot of mango collected from Guntian, but not for mango collected from Yujing. The difference of control results between two areas were needed to be further investigated.