Investigation of Camellia Tree Diseases in Taiwan and Establishment of a Rapid Detection Method for Colletotrichum spp.

臺灣油茶病害調查及油茶炭疽病菌快速檢測方法之建立

劉則言(Tse-Yen Liu) ； 陳昭翰(Chao-Han Chen) ； 汪澤宏(Liang-Jong Wang) ； 洪挺軒(Ting-Hsuan Hung) ； 徐孟豪(Meng-Hao Hsu) ； 吳孟玲(Meng-Ling Wu)

《台灣林業科學》 36卷1期 (2021/03) Pp. 1-19

https://doi.org/10.7075/TJFS.202103_36(1).0001

摘要

Camellia is commonly used for afforestation and as an important oil-producing crop with high economic value. Diseases that occur on aboveground parts of Camellia are considered to be major reasons causing declines in the quality and quantity of tea oil, but little is known about them. In this study, we investigated disease occurrence of Camellia in Taiwan and discovered that blister blight, leaf spot, and soft rot were major symptoms on Camellia farmlands in the north, while leaf spot and anthracnose were found on Camellia farmlands in central Taiwan. Through tissue isolation, fungal pathogen candidates of Camellia diseases were identified; 15 fungal isolates were obtained, subjected to nucleic acid extraction, and sequenced. Sequence blast results showed that these isolates included Colletotrichum spp. (known as an anthracnose disease pathogen), Alternaria sp., Botryosphaeria dothidea, Botryotinia fuckeliana (Botrytis cinerea), Lasiodiplodia pseudotheobromae, Neofusicoccum parvum, Pestalotiopsis spp., Phomopsis sp., and Sclerotinia sclerotiorum. All isolates were reported to be plant pathogens, but Camellia-related research is still lacking. A phylogenetic tree constructed with internal transcribed spacers (ITSs) of the 15 isolates in this study showed that pathogens of Camellia anthracnose were clustered into 2 species complexes of C. gloeosporioides and C. acutatum. Colletotrichum spp. isolates from different regions were all classified into the same group and were evolutionally closer than other potential Camellia leaf pathogens. Anthracnose is considered to be the most important Camellia disease with 2 species complexes, and a rapid detection method was first established in this study. Three primer sets were designed for the rapid detection by a polymerase chain reaction (PCR) based on our ITSs to identify anthracnose disease on Camellia. The primer sets designed in this study showed high specificity and sensitivity and could successfully detect Colletotrichum spp. and distinguish between the C. gloeosporioides and C. acutatum species complexes. This will contribute to achieving prevention strategies and early disease prevention of Camellia anthracnose. In this study, Colletotrichum spp. and 8 other potential fungal pathogens were isolated from Camellia leaves, and results will be useful in understanding potential pathogens. With the establishment of a rapid detection method for serious anthracnose disease of Camellia, our study can be used as an important reference for early diagnosis, which will be valuable for disease control and management.

關鍵字

Camellia ； Colletotrichum spp. ； molecular rapid detection ； polymerase chain reaction

並列摘要

油茶為臺灣早期的造林樹種，也是近年重要油料作物。油茶地上部病害為造成油茶產量與品質下降的主要原因之一，但卻缺少相關之研究。本研究經由臺灣北部及中部油茶園的病害調查，發現在北部油茶園葉部病害以餅病、葉斑病和軟腐病為主，中部則以葉斑病和炭疽病最為常見。進一步將感病油茶葉片進行組織分離，獲得真菌分離菌株，並依菌落型態進行分群後，從中選取15株真菌分離株，萃取核酸並定序與序列比對，發現油茶葉片及果實的植物病原真菌，以炭疽病菌屬（Colletotrichum spp.）真菌所占比例最高。此外，自油茶罹病葉上分離而得的菌株還包含Alternaria sp.、Botryosphaeria dothidea、Botryotinia fuckeliana（Botrytis cinerea）、Lasiodiplodia pseudotheobromae、Neofusicoccum parvum、Pestalotiopsis sp.、Phomopsis sp.、Sclerotinia sclerotiorum。這些真菌分離株皆有被報導為植物病原菌，但在油茶上的研究仍相當缺乏。進一步將真菌分離株的ITS序列進行親緣關係分析，可以發現感染油茶的炭疽病菌，主要來自於C. gloeosporioides和C. acutatum這兩個種群，並與自其他作物上分離到的常見炭疽病菌歸為同一系群，與其他葉部潛在病原真菌在類緣關係上有所區別。有鑑於炭疽病菌的兩個種群為油茶主要病原菌，本研究首次針對油茶上此兩個重要炭疽病菌種群發展快速檢測方式。透過真菌核酸ITS區域，設計3組油茶炭疽病菌之專一性引子對，利用分子檢測技術（聚合酶鏈鎖反應，PCR）來進行快速檢測。結果顯示本研究設計之引子對油茶炭疽病菌具良好專一性及靈敏度，並可成功區分在油茶上主要造成炭疽病菌的不同種群。透過快速檢測方式的建立，未來將有助於病原菌在潛伏感染時期的檢測。本研究自臺灣油茶罹病葉片上分離出炭疽病菌屬及其他8種真菌，有助於了解油茶葉部之潛在病原菌，同時針對危害嚴重之油茶炭疽病發展快速檢測技術，可作為未來油茶病害防治與健康管理之用，提供病害早期發現早期防治的管理模式。

並列關鍵字

油茶 ； 炭疽病菌 ； 分子快速檢測 ； 聚合酶鏈鎖反應