草莓灰黴病菌族群之分子特性

Molecular characterization of Botrytis cinerea populations on strawberry in Taiwan

灰黴病是臺灣地區草莓栽培最常見的病害之一，其病原菌Botrytis cinerea寄主範圍廣且遺傳變異大，對作物極具危害性。本研究自臺灣各草莓產區採取罹病果實以單孢分離法獲得菌株，選取其中36株供分子生物學及族群遺傳學分析之用。供試菌株以B. cinerea專一性引子對C729+/-進行聚合酶連鎖反應（PCR），皆可產生700 bp擴增子；而以Bc-hch基因座擴增子輔以HhaI限制酶截切，亦均於膠片產生屬第二群之517 bp條帶，確證供試菌株全為B. cinerea。運用聚合酶連鎖反應分析供試菌株之配對型及轉位子基因型獲知，供試菌株均屬異絲型，其中MAT1-1型有23株，MAT1-2型有13株；轉位子基因型則分別有transposa型20株、Boty型14株、Flipper型0株及vacuma型2株。整合菌株配對型、轉位子基因型及三種菌種鑑定用基因（RPB2, HSP60及G3PDH） 突變點構成之單核苷酸多態性 （SNPs）進行灰黴病菌族群遺傳分析，結果顯示，苗栗縣大湖及獅潭鄉作為草莓主要產區，其灰黴病菌族群的遺傳多樣性指數均顯著高於其他地區者。族群基因流動與遺傳距離成對評估表並揭示，苗栗縣與其他縣市族群間的遺傳分化程度不高，而表示基因流動的數值，則指族群間有很強的基因交流。上述兩地區灰黴病菌族群分子變異階層式分析 （AMOVA） 亦表明，有90.11%的變異源自族群內部，族群間僅佔9.89%。

Botrytis cinerea is a prevalent and devastating fungal pathogen on strawberry in Taiwan. Thirty-six selected single-conidium isolates collected from strawberry fields islandwide were identified as B. cinerea sensu stricto based on B. cinerea-specific primer pair C729+/- and belonged to Genetic Group II based on PCRRFLP of the Bc-hch locus. The 36 isolates were all heterothallic isolates. Twenty-three isolates belonged to MAT1-1 type and 13 were characterized as MAT1-2 type. The four transposon genotypes, transposa, Boty-only, Flipper-only, and vacuma, comprised 55.5%, 38.9%, 0.0%, and 5.6%, respectively, of all the isolates studied. Combined analyses of mating types, transposon genotypes and single nucleotide polymorphisms of RPB2, HSP60 and G3PDH genes of B. cinerea isolates were performed to assess their genetic population structure and diversity. Genetic diversity indexes within Dahu-Shihtan population were all higher than those of the other areas population. Pairwise estimates of Gst, Nm and Nei's unbiased genetic identity and genetic distance between these two geographical populations revealed that low genetic differentiation and high gene flow existed between them. Hierarchical analysis of molecular variance of these two populations further indicated that 90.11% of the variation occurred within populations, while the variation between populations was only 9.89%. This is the first report of the genotypic diversity of B. cinerea populations on strawberry in Taiwan.