Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31228
標題: 台灣灰黴病菌 (Botrytis cinerea) 對 Strobilurin 類殺菌劑 (QoIs) 感受性分析及抗藥機制探討
Investigation of sensitivities and resistant mechanisms of Botrytis cinerea to strobilurins in Taiwan
作者: 陳麗淑
Chen, Li-Shu
關鍵字: Botrytis cinerea
草莓灰黴病菌
strobilurin
QoI
resistant mechanism
strobilurin
QoI
抗藥性機制
出版社: 植物病理學系所
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摘要: 由 Botrytis cinerea 所引起的灰黴病 (gray mold) 為草莓重要病原真菌之一,除在田間可造成危害外,亦引可起貯藏期果實的腐爛。Strobilurin (QoI) 類為一種廣效性殺菌劑,具有抑制粒腺體電子傳遞鏈而降低 ATP 產生之作用機制,目前台灣尚未推薦用於防治 B. cinerea。本研究針對克收欣、亞托敏及百克敏三種 QoI 類殺菌劑進行不同有效濃度對 159 株 B. cinerea 菌株感受性測試。結果顯示所有菌株在百克敏 100 ppm (a.i.) 濃度下,菌絲生長明顯被抑制,然在 500 ppm (a.i.) 克收欣與亞托敏處理下,菌株呈現低感受性的比例則分別為 89 % 與 86 %,顯示草莓田間可能已出現抗 QoI 類殺菌劑的 B. cinerea 菌株。針對不同結構與殺菌作用的 benzimidazole 類殺菌劑中的免賴得進行 57 株菌株感受性測試,結果顯示有 24 株菌株之 EC50 濃度均大於 500 ppm,28 株菌株則介於100 ppm至500 ppm 之間,其餘 5 株菌株之 EC50 濃度均小於 100 ppm。進一步比較供試菌株對 QoIs 與免賴得之抗感性反應,顯示供試菌株對此兩類藥劑並無明顯產生交互抗性的現象。分析對 QoI 類藥劑表現抗感性之 7 株菌株的 cytochrome b (cyt b) 基因序列,得知 cyt b 基因中第 129 與 143 處密碼子並無發生核苷酸突變,推測 cytb 基因第 129 與 143 處密碼子與草莓灰黴病菌抗藥性產生之反應並不相關。利用 TEM 觀察 QoI 類藥劑對灰黴病菌株粒腺體形態與數量的影響,結果顯示抗感性兩菌株經100 ppm 亞托敏原體處理後,細胞內粒腺體的數目與形態均無明顯差異。將 salicylhydroxamic acid (SHAM) 藥劑添加於含亞托敏原體的 PDA 培養基中,然後進行抗感性菌株菌絲生長測試,得知抗藥性菌株對 100 ppm 亞托敏原體之感受性可明顯提高兩倍。由於 SHAM 的主要作用為抑制粒線體中 alternative oxidase (AOX) 之活性,顯示alternative pathway respiration 可能參與抗藥性產生之反應。進一步分析抗感性菌株 AOX 基因在添加亞托敏原體之表現,結果顯示抗性菌株 AOX 基因的表現並未因處理 100 ppm 亞托敏原體而誘導增加。利用亞托敏原體配合加入 SHAM 進行抗感性菌株氧氣消耗抑制測試,結果顯示抗性菌株之孢子懸浮液於添加 100 ppm 亞托敏原體後,呼吸作用受影響的程度隨供試菌株不同而有差異,而感受性菌株的呼吸作用則明顯受到抑制。且於反應中加入 0.5 mM SHAM 後,抗性菌株呼吸作用仍未完全受到抑制,然感受性菌株之呼吸作用則已完全停止。此外,對不同抗感性菌株處理 100 μM antimycin A 與 0.5 mM SHAM 後其耗氧作用則完全終止。根據耗氧測試結果顯示,灰黴病菌雖能進行 alternative pathway respiration 反應而增加對 QoI 類藥劑抗性,但仍有其他因素造成抗感性菌株間反應之差異。綜合本研究上述結果,推測造成灰黴病菌對 QoI 類藥劑產生抗性之機制仍與 cytochrome b 上之 Qo site 有關。
Gray mold, caused by Botrytis cinerea Pers.:Fr., is one of most important strawberry disease in the field and postharvest. Strobilurins (QoIs) are broad-spectrum fungicides for control crop diseases, however, strobilurins do not be recommended to control gray mold in Taiwan. The mode of action of strobilurins is that strobilurins are inhibitor of electron transportation at Qo site of cytochrome b and decrease the ATP production. In this study, sensitivity of 159 B. cinerea isolates was examined to kresoxim-methyl, azoxystrobin and pyraclostrobin by inhibition of mycelial growth and spore germination. These results showed that 100 ppm (a.i.) pyraclostrobin significantly inhibited the mycelial growth of all of B. cinerea isolates. However, 89 % and 86 % B. cinerea isolates were low sensitive to 500 ppm (a.i.) kresoxim-methyl and azoxystrobin, showed that strobilurins-resistant B. cinerea isolates from strawberry might exist in nature. For sensitivity to benomyl, 57 B. cinerea isolates were tested. The results indicated that 24 B. cinerea isolates were EC50>500 ppm, 28 isolates were 100<EC50<500 ppm, and 5 isolates were EC50<100 ppm. Consequently, the cross resistance between benomyl and strobilurins did not significantly be observed among B. cinerea isolates. For analysis the resistant mechanism of B. cinerea isolates to strobilurins, the cytochrome b gene were sequenced, however, the results did not show mutations at codon 129 or 143 which conferred resistance to strobilurins. Moreover, the morphology and number of mitochondria of B. cinerea conidiospores did not reveal variation based on transmission electron microscope (TEM) observation after treating with 100 ppm technical grade of azoxystrobin. In addition, adding alternative oxidase (AOX) inhibitor, salicylhydroxamic acid (SHAM), in 100 ppm technical grade of azoxystrobin added PDA medium could increase the inhibition rate of mycelial growth of strobilurins-resistant B. cinerea isolates. This result indicated that alternative pathway respiration could increase the resistance of B. cinerea isolates to strobilurins. However, the expression of AOX gene did not be induced significantly when the strobilurins-resistant B. cinerea isolates were treated by 100 ppm technical grade of azoxystrobin. According to the mode of action of strobilurins, the respiration of B. cinerea isolates would be reduced. For analysis the relationship between oxygen consumption and strobilurins-resistant and -sensitive B. cinerea isolates, we treated strobilurins-resistant and -sensitive isolates with 100 ppm technical grade of azoxystrobin. The result showed that oxygen consumption of strobilurins-sensitive isolates were reduced significantly after treating 100 ppm technical grade of azoxystrobin. However, the oxygen consumption of strobilurins-resistant B. cinerea isolates were variable. Furthermore, the oxygen consumption of strobilurins-sensitive isolates would be stopped completely after adding 0.5 mM SHAM. On the contrary, the oxygen consumption of strobilurins-resistant isolates did not show significantly variable after adding 0.5 mM SHAM. In this study, the Qi inhibitor, antimycin A, was used to treat B. cinerea isolates and compared the ratio of oxygen consumption with Qo inhibitor. The result revealed that 100 μM antimycin A combined with 0.5 mM SHAM could completely stop the oxygen consumption of strobilurins-resistant and -sensitive B. cinerea isolates. Thus, alternative pathway respiration is one of factors to increase resistance to strobilurins, and the major mechanism of strobilurins-resistant B. cinerea isolates might still correspond with cytochrome b gene. It is necessary to carry out the whole cytochrome b gene sequence and structure of strobilurins-resistant B. cinerea isolates in future.
URI: http://hdl.handle.net/11455/31228
其他識別: U0005-1908200817325100
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-1908200817325100
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