臺灣草莓炭疽病之探討

草莓 (Fragaria × ananassa Duch.) 為具高經濟價值且深受消費者喜愛之作物，其栽培面積於臺灣約為500公頃。在臺灣炭疽病為草莓生產最大的限制因子之一，尤以感病品種‘桃園1號’受害最為嚴重。調查國內草莓主要產區，本病害除可感染草莓全株各部位外，高達50%以上之病株呈現典型冠腐病徵。本研究於2010至2018年由新竹縣、苗栗縣、南投縣及嘉義縣等地區之草莓罹病植株分離52株菌，透過病原菌型態及internal transcribed spacer (ITS)、glyceraldehyde-3-phosphate dehydrogenase (GAPDH)、chitin synthase (CHS1)、actin (ACT)、β-tubulin (TUB2)、calmodulin (CAL) 及intergenic region between Apn2 and MAT1-2-1 (ApMAT) 等序列進行多基因親緣分析，確認造成臺灣草莓炭疽病之病原菌包含Colletotrichum siamense、C. fructicola (C. gloeosporioides species complex)、C. karstii、C. boninense (C. boninense species complex) 及一新種C. miaoliense (C. acutatum species complex)。C. siamense及C. fructicola為主要致病菌，在25°C或30°C下不論葉片有無傷口皆產生較大之病斑，而其他三種病原菌僅在有傷口條件下產生微小病斑。由於炭疽病菌能潛伏感染寄主，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。本研究分析已發表之29種炭疽病菌菌株之全基因體序列，找到位於L-arabinitol 4-dehydrogenase (ladA) 及NAD(P)H-dependent D-xylose reductase (xyl1) 兩基因間之非保守序列進行引子設計，開發出巢式聚合酶鏈鎖反應技術。本技術可以偵測最主要的炭疽病菌C. 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1 pg之C. siamense DNA (約15個細胞)，代表具有高度專一性及靈敏度。本研究釐清臺灣草莓炭疽病菌種類及特性，有助於防治策略之擬定與執行，及未來草莓抗病育種之篩選。本研究所開發之檢測技術已直接應用於草莓種苗病害驗證作業，期望加速推廣草莓健康種苗之使用。

關鍵字

炭疽病；多基因親緣分析；潛伏感染；健康種苗；比較基因體學；巢式聚合酶鏈鎖反應

並列摘要

In Taiwan, strawberry (Fragaria × ananassa Duch.) is a high-value and popular crop with an average annual cultivated area of ~500 ha. Anthracnose disease is one of the key limiting factors of strawberry production in Taiwan, especially for the susceptible cultivar ‘Taoyuan no.1’. We surveyed anthracnose in major strawberry cultivation areas, and observed that it could infect all parts of the strawberry plant and more than 50% of diseased plants showed typical anthracnose crown rot symptoms. A total of 52 isolates were collected from Hsinchu County, Miaoli County, Nantou County and Chiayi County during 2010 to 2018. Based on morphological characterization and multi-gene phylogenetic analysis using the sequences of transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase (CHS1), actin (ACT), β-tubulin (TUB2), calmodulin (CAL) and intergenic region between Apn2 and MAT1-2-1 (ApMAT), we revealed that Colletotrichum siamense, C. fructicola (C. gloeosporioides species complex), C. karstii, C. boninense (C. boninense species complex) and C. miaoliense sp. nov. (C. acutatum species complex) were associated with strawberry anthracnose in Taiwan. The predominant species C. siamense and C. fructicola caused larger lesions at 25°C or 30°C on leaves with or without wounds, whereas the other three species caused tiny lesions only on wounded leaves. Because Colletotrichum spp. could cause latent infections, use of healthy and pathogen-free strawberry runner plants will greatly reduce the occurrence of anthracnose rot in the field and the usage of fungicides. To produce healthy runner plants, it is important to diagnose anthracnose at the stage of latent infection. We conducted comparative genomics analysis of 29 known Colletotrichum spp. genomes to search for non-conserved regions suitable for the design of specific primers which are located between L-arabinitol 4-dehydrogenase (ladA) and NAD(P)H- dependent D-xylose reductase (xyl1). We developed a nested PCR assay which can specifically detect the predominant anthracnose pathogens C. siamense and C. fructicola, but not other pathogens and saprophytes associated with strawberry plants. It could detect as low as 1 pg genomic DNA of C. siamense, which corresponds to 15 cells of C. siamense, suggesting the high sensitivity of this new detection technique. This study clarified the species identity and characteristics of strawberry anthracnose pathogens in Taiwan, which can help develop and apply effective control strategies as well as disease resistance breeding in the future. The detection method developed in this study has been applied to the voluntary pathogen-free certification system for strawberry propagation, which is expected to accelerate the promotion of healthy strawberry runner plants.