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研究生:邱硯詩
研究生(外文):chiou yann-shy
論文名稱:最近為害台灣中部馬鈴薯之青枯病菌菌系
論文名稱(外文):Characteristics of Strains of Ralstonia solanacearum Recently Affecting Potatoes in Central Taiwan
指導教授:徐世典
指導教授(外文):Shih-Tien Hsu
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物病理學系
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
中文關鍵詞:馬鈴薯青枯病青枯病菌菌系殘存遺傳多型性
外文關鍵詞:bacterial wilt of potatoRalstonia solanacearumbiovarsurvivalgenetic diversity
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在台灣,馬鈴薯青枯病在冬天多在南部發生,然民國88年冬天於台中神岡鄉卻大發生,為了解此次引起中部病害大量發生之青枯病菌是否和以往台灣所發生之青枯病菌有所不同,本研究探討了兩者菌株之特性比較。供試52株菌株包括最近(民國89年)由台中縣神岡及后里地區罹病馬鈴薯分離的33株及往年由台灣各地馬鈴薯上分離的19株。生物型 (biovar) 測定之結果,顯示最近分離的33株中,除一株為第四生物型 (biovar 4) 外,其餘均屬第二生物型 (biovar 2A) ,而以往分離的19個菌株中,除一株為第三生物型 (biovar 3) ,其餘均屬於第四生物型 (biovar 4) 。 Biovar 4菌株在溫度較高之環境下,其病原性一般較 biovar 2A 菌株為強,而biovar 2A菌株在溫度較低之環境下,只對馬鈴薯與番茄有強病原性,且致病力高於biovar 4 菌株,符合第三生理小種之特性。利用可區分青枯病菌演化群之引子組 (OLI1 + Y2 + BV345) 進行聚合酵素連鎖反應, biovar 3 菌株與 biovar 4 菌株皆可產生292、409與 840 bp 之DNA條帶,而 biovar 2A 菌株只可產生292 bp 的條帶,顯示最近分離的 biovar 2A 菌株可歸類於第二演化群 (division Ⅱ) ,而其餘供試菌株屬於第一演化群 (division Ⅰ) 。此外,利用前人所設計之引子對 630 與 631,在較高黏合溫度下,可從所有 biovar 2 菌株增幅出307與440 bp之DNA 條帶,而 biovar 3 與 biovar 4菌株則無任何條帶產生。以 BOX-PCR 與 REP-PCR 分析台灣馬鈴薯青枯病菌菌株間之差異,可分成二個主要的類群,一群包含 biovar 3 和 biovar 4 的菌株,而另一群僅有 biovar 2 的菌株。由上述各項結果,顯示最近引起中部地區馬鈴薯嚴重發生青枯病之青枯病菌屬於第三生理小種,第二生物型,與台灣以往存在的菌系不同,此亦為台灣出現第三生理小種,第二生物型的首次報告。利用青枯病菌突變種之rifampin抗性作為標記,由人工混菌土壤中測定結果, biovar 4 菌株在土壤中之存活能力較 biovar 2A 菌株強,且存活之時間亦較長。
Potato is usually grown in the winter season in Taiwan. When bacterial wilt of potato caused by Ralstonia solanacearum occured, it was almost observed in the southern part of Taiwan. However, in 1999 winter an outbreak of the bacterial wilt was found in Shengang of Taichung county, the central part of Taiwan. This study was to determine the characteristics of the strains of R. solanacearum involved in this outbreak. Fifty-three strains of R. solanacearum from potato including 33 strains isolated from the outbreak areas (Shengang and Houli, Taichung) in 2000 and 19 strains collected in various areas of Taiwan in earlier years (1971-1997) were compared. The 33 bacterial strains isolated in 2000 were identified as biovar 2A with the exception of one strain, which was biovar 4, whereas the 19 strains isolated in earlier years belonged to biovar 4 with one exception that was biovar 3. In the high temperature, the virulence of the biovar 4 strains was generally higher than the biovar 2A strains, however, in the lower temperature, the biovar 2A strains was highly pathogenic only to potato and tomato, and they were more virulent than the biovar 4 strains. Therefore, the pathogencity of the biovar 2A strains was similar to that of strains belonging to race 3 according to the classification of Buddenhagen et al.. Multiplex PCR using primers OLI1 + Y2 + BV345 to differentiate the two evolutionary divisions of R. solanacearum was used. All of the biovar 3 and 4 strains yielded products of 292, 409, and 840 bp, and were identified as division Ⅰ, whereas all of the biovar 2A strains only yielded product of 292 bp and were identified as division Ⅱ. Using reported primers 630 and 631 for identifying biovar 2A strains, amplification products of 307 and 440 bp were found in all of the biovar 2A strains when the annealing temperature was changed to 65 ℃, but no any products were observed in all of the biovar 3 and 4 strains. Analyses of the BOX-PCR and REP-PCR data revealed that there were two main clusters of potato strains existing in Taiwan. Cluster 1 comprised the biovar 3 and 4 strains and cluster 2 consisted of only biovar 2A strains. The above results indicated that the recent outbreak of bacterial wilt in central Taiwan was caused by a strain of R. solanacearum different from the strains previously reported in Taiwan. This is the first report of biovar 2A/race 3 of the bacterium in Taiwan. The soil survival studies employing rifampin resistance as a marker showed that the biovar 4 strain (Pss259r) had a greater survivability in the soil than the biovar 2 strain (Po1r).
目錄
壹、前言………………………………………………………………………1
貳、材料與方法………………………………………………………………5
一、供試青枯病菌菌株之來源………………………………………………5
二、接種源之製備……………………………………………………………5
三、抗生素抗性突變菌株的選取……………………………………………5
四、菌落形態與生理生化特性………………………………………………5
(一)菌落形態………………………………………………………………5
(二)碳水化合物之氧化作用………………………………………………6
(三)其他生理生化測試……………………………………………………6
五、病原性之測定……………………………………………………………7
(一)供試寄主植物 …………………………………………………………7
(二)接種方式………………………………………………………………7
(三)病徵之等級區分(Disease rating)及發病指數(Disease index)7
六、應用聚合酵素連鎖反應區分青枯病菌菌株……………………………8
(一)DNA模板之製備………………………………………………………8
(二)利用引子 OLI1+BV345+Y2 區分菌株之演化型…………………8
(三)利用引子對 630/631 鑑定 biovar 2A菌株…………………………8
七、以 BOX-PCR 與 REP-PCR 分析菌株差異性……………………………10
八、樹狀圖分析………………………………………………………………10
九、馬鈴薯青枯病菌於土壤中之存活測定…………………………………11
(一)土壤溫度對存活之影響 ……………………………………………11
(二)土壤含水量對存活之影響……………………………………………11
參、結果 ……………………………………………………………………12
一、馬鈴薯青枯病菌之特性…………………………………………………12
(一)菌落形態………………………………………………………………12
(二)碳水化合物氧化作用…………………………………………………12
(三)其他生理生化測試……………………………………………………12
二、青枯病菌對六種供試植物之病原性……………………………………13
(一)溫室內 (25~35 ℃) 接種之結果……………………………………13
(二)可降溫之溫室中 (17~28 ℃) 接種之結果…………………………13
三、應用聚合連鎖反應區分馬鈴薯青枯病菌菌株………………………13
(一)利用引子 OLI1+BV345+Y2 區分菌株之演化型…………………13
(二)利用引子對 630/631 鑑定 biovar 2A菌株………………………13
四、以 BOX-PCR 與 REP-PCR 分析青枯病菌之遺傳差異性………………14
五、馬鈴薯青枯病菌於土壤中之存活………………………………………14
(一)土壤溫度對存活之影響………………………………………………14
(二)土壤含水量對存活之影響……………………………………………14
肆、討論………………………………………………………………………15
伍、參考文獻…………………………………………………………………18
陸、中文摘要…………………………………………………………………25
柒、英文摘要…………………………………………………………………26
捌、圖表………………………………………………………………………28
玖、附錄………………………………………………………………………45
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