台灣草莓萎凋病菌之生理生化特性分析

Physiological and biochemical analyses of Fusarium oxysporum Schl. f. sp. fragariae in Taiwan

"西元2012年，在苗栗獅潭、大湖等栽培區，發現草莓植株出現矮化、新葉偏上生長、黃化及葉片萎凋乾枯等症狀；切開病株莖基部則可見到維管束褐變的病徵。由田間取回罹病植株，進行莖基部組織分離可疑病原菌，依柯霍氏法則測試後，確定各分離株的致病毒性後，選取毒力較強之Fofb 01-2與Fofb 4-13兩菌株進行草莓萎凋病菌之鑑定。首先將Fofb 01-2與Fofb 4-13菌株分別培養於半醣馬鈴薯瓊脂平板（Potato Dextrose Agar, PDA）上，菌落顏色呈白色至灰色，中央有紫色色素沉積。病原菌有三種形態的孢子，小孢子呈現卵圓狀或臘腸狀；大孢子呈直至彎曲鐮刀狀，具有3-5個隔膜；厚膜孢子呈圓形。利用前人發表針對Fusarium oxysporum Schl.所設計的專一性引子對FnSc-1（5'-TACCACTTGTTGCCTCGGCGGATCAG-3'）/FnSc-2（5'-TTGAGGAACGCGAA TTAACGCGAGTC-3’）進行polymerase chain reaction（PCR），確定Fofb 01-2與Fofb 4-13兩菌株歸屬於F. oxysporum。進一步將病原菌株接種於胡瓜、西瓜、萵苣、白菜、番茄及草莓等不同寄主植物，結果僅有草莓植株受感染罹病，隨後以前人發表針對尖鐮孢菌草莓分化種（Fusarium oxysporum Schl. f. sp. fragariae Winks & Williams）所設計的專一性引子對FofraF（5'-CAGACTGGG GTGCTTAAAGTT-3'）/FofraR（5'-AACCGCTAGG GTCGTAACAAA-3'）進行Fofb 01-2與Fofb 4-13的分子生物學分析，並與NCBI資料庫進行比對，確認兩菌株的學名為F. oxysporum Schl. f. sp. fragariae Winks & Williams。Fofb 01-2與Fofb 4-13菌株的菌絲最適生長溫度及分生孢子與厚膜孢子最適發芽溫度皆為28℃。分生孢子最適發芽pH值則介於4-8，發芽率皆可達80 %以上。利用不同碳氮素源培養本病原菌，發現兩菌株最適生長於麥芽糖（maltose）及山梨糖醇（sorbitol）、天門冬醯胺（asparagine）、纈氨酸（valine）及硝酸鈉（NaNO3）等碳氮素源。"

"Since 2012, a new disease of strawberry has been found in DaHu, Shih-Tan, and other cultivation areas in Taiwan. The symptoms consisted of stunting, wilting of foliage, necrotic crowns and roots, discoloration of the internal vascular tissue, and eventually plant death. The isolates of the causal agents were obtained from diseased plants. Among them, two isolates named Fofb 01-2 and Fofb 4-13 showed high virulence to strawberry plants and were selected for further analyses. Both isolates were subcultured on half sugar potato dextrose agar (PDA) and formed white-togray colonies with purple pigment in the middle. The isolates could produce microconidia, macroconidia, and chlamydospores. Microconidia were oval to ellipsoid. Macroconidia were straight to sickle shaped, 3 to 5 septate. Chlamydospores were round. A 327bp DNA fragment was successfully amplified from both fungal isolates by polymerase chain reaction (PCR) using two primers, FnSc-1 (5'-TACCACTTGTTGCCTCGGCGGATCAG-3')/FnSc-2 (5'-TTGAGGAACGCGAATTAACGCGAGTC-3'), specific for Fusarium oxysporum Schl. Virulence assays revealed that the two isolates could only infect strawberry showing similar symptoms as observed in the diseased plants in the field. No symptoms were observed after they were inoculated onto cucumber, watermelon, lettuce, Pak choi, or tomato. Further PCR analysis using primers FofraF (5'-CAGACTGGGGTGCTTA AAGTT-3')/FofraR (5'AACCGCTAGG GTCGTAACAAA-3') identified the two tested isolates as F. oxysporum Schl. f. sp. fragariae Winks & Williams. T h e o p t i m a l t e m p e r a t u r e f o r my c e l i a l g r o w t h , c o n i d i a l a n d chlamydospore germination of isolates Fofb 01-2 and Fofb 4-13 was at 28℃. For both isolates, the pH values between 4-8 were optimal for conidial germination (germination > 80%). The mycelial growth for the two tested isolates were better in media with maltose and sorbitol as carbon sources or with asparagine, valine, and NaNO3 as nitrogen sources."