偵測潛伏感染期草莓炭疽病病原菌技術之研發

Development of technology for detecting pathogens of strawberry anthracnose during latent infection

鐘珮哲(Chung, Pei-Che) ； 吳竑毅(Hung-Yi Wu) ； 曹嘉惠(Jia-Huey Tsao)

《苗栗區農業改良場研究彙報》 9期 (2020/06) Pp. 46-57

摘要

草莓為臺灣具高經濟價值之作物，栽培面積約為500公頃。近10年來炭疽病成為草莓頭號殺手，由於本病原菌能潛伏感染寄主，種植健康不帶菌的草莓苗，將可大幅降低本田期病害的發生，同時減少化學藥劑的使用。而為生產健康的草莓苗，準確、靈敏、快速又合乎成本效益的檢測技術便是其中最重要的關鍵，因此以現有的炭疽病菌基因體資料庫，搜尋適合的目標區域，並以臺灣草莓炭疽病菌的序列進行引子設計，開發巢式聚合酶連鎖反應技術。本技術可以偵測最主要的炭疽病菌Colletotrichum. siamense與C. fructicola，但不會偵測到其他草莓病原菌或土壤中常見的腐生菌，可偵測到低至100 fg之C. siamense DNA（約2個分生孢子），代表具有高度專一性及靈敏度。結合本場草莓炭疽病巢式PCR檢測技術，進行核酸探針修飾與快篩檢驗試紙製作。目前已開發完成之技術尚需配合本團隊正在設置之植物病害檢測工作站，才能達到田間快速檢測之效果，檢測工作站包含植物樣本核酸萃取、PCR及核酸檢測試紙判讀等部分，完整結合後將有助於田間病害檢測工作及病害之判斷。

關鍵字

巢式聚合酶連鎖反應 ； 核酸檢驗試紙 ； 健康種苗

並列摘要

Strawberry (Fragaria × ananassa Duch.) is a high economic-value crop with an average annual cultivated area of 500 ha in Taiwan. Over the past decade, anthracnose has become the most destructive disease because of the latent infectious characteristics of the pathogens. It would reduce not only the occurrence of anthracnose rot but also the usage of fungicides in the fields by healthy and pathogen-free strawberry runner plants. To produce healthy runner plants, it is important to diagnose anthracnose at the stage of latent infection. We conducted comparative genomics analysis of known Colletotrichum spp. genomes to search for ideal regions suitable for design of specific primers, and developed a nested- PCR assay. The predominant pathogens of strawberry anthracnose in Taiwan include C. siamense and C. fructicola could be specifically detected, but not other pathogens or saprophytes associated with strawberry plants. This new detection technique with high sensitivity could detect as low as 100 fg genomic DNA of C. siamense, which corresponds to 2 cells of C. siamense. The detection techniques, however combined with the nested-PCR assay, nucleic acid probe modification and nucleic acid strip, but also needs to cooperate with the plant disease detection workstation being developed by our team to achieve the effect of rapid field detection. The detection workstation includes nucleic acid extraction of plant sample, PCR assay, and interpretation of nucleic acid test strip. The complete combination will held disease detection applied in the field and disease analysis.

並列關鍵字

nested-PCR ； nucleic acid strip ； healthy runner plants