臺灣博碩士論文加值系統

瓜類細菌性果斑病菌 (Acidovorax citrulli) 為重要的植物病原細菌，可引起 瓜類細菌性果斑病 (Bacterial fruit blotch of cucurbits)，造成瓜類作物產量嚴重損 失。此病菌可經由種子傳播，為重要的植物檢疫病原細菌。開發其高度專一及靈 敏度之檢測技術為重要之項目。選擇性培養基為瓜類細菌性果斑病菌常被使用之 檢測技術，此方法之操作較血清學及分子檢測技術簡單，且成本花費較低。目前 常被使用之瓜類細菌性果斑病菌之半選擇性培養基有 AacSM、 WFB68 及 WFB68MA。然而，部分台灣瓜類細菌性果斑病菌株於此些培養基上生長不良， 其中為改進 AacSM 選擇性培養基對台灣瓜類細菌性果斑病菌之檢測能力，本研 究以 succinic acid 、 L-asparagine 、 L-aspartic acid 、 L-glutamic acid 及 L-pyroglutamic acid 等五種氮素源分別取代 AacSM 培養基中之 ammonium adipate，並去除其中碳素源酵母萃取物及微量無機鹽類進行測試。結果顯示，添 加 L-glutamic acid 之 AacG 培養基較其它之培養基可促進台灣瓜類細菌性呆 斑病菌菌株之生長。以 1% 帶菌種子進行果斑病菌之回收，並比較 Aac31、 Aac102 及 Aac159 三菌株分別以 AacSM、WFB68MA 與 AacG 三種培養基作 比較。結果顯示，Aac31、Aac102 及 Aac159 菌株於 AacG 培養基中之回收率 為 45~100%﹔Aac31 菌株於 AacSM 及 WFB68MA 培養基之回收率則分別為 10% 及 100%﹔而 Aac102 及 159 菌株於此三種培養基之回收率則皆為 0%， 顯示菌株間其有差異性。進一步以 0.5% 及 0.2% 帶菌種子檢測果斑病菌之回 收率，使用 AacG 培養基回收果斑病菌 Aac31 菌株之效率分別為 246.97% 及 46.34%，非目標菌回收率為 5.22% 及 2.8%，與 AacSM 及 WFB68MA 培養基 相比能有效抑制非目標菌生長，且其明顯提高果斑病菌之檢測效率。根據以上結 呆，本研究所開發之 AacG 選擇性培養基可明顯提高瓜類細菌性果斑病菌之回 收率，有效改善台灣瓜類細菌性果斑病生長不良之情形。

Bacterial fruit blotch of cucurbits, caused by Acidovorax citrulli (Ac) is a devastating disease of cucurbit crops. Ac, seed-borned pathogen, is a quarantine pest to avoid pecuniary losses. Hence, it is important to develop a rapid, accurate detection technique for this pathogen. The commonly used method is a selective medium to incubate Ac which is easier to use and less costly than the immunological or molecular methods. The semiselective media, e.g. AacSM, WFB68 and WFB68MA are currently used to detect Ac. However, the sensitivity and selectivity of the these media were not suitable for Ac strains in Taiwan. Therefore, the objective of this study is to modify the formula of AacSM medium by allocating the composition of carbon and nitrogen sources. In this study, five nitrogen sources including succinic acid, L-asparagine, L-aspartic acid, L-glutamic acid and L-pyroglutamic acid were tested, respectively, and the yeast extract and inorganic salts were also tested to remove from AacSM. These results were showed that the developed AacG medium which adding L-glutamic acid can enhance the growth of the tested Ac strains than the other semiselective media. Furthermore, comparison of the recovery of A. citrulli from the seed infestation rate at 1% on AacSM, WFB68MA and AacG media, the recovery of Ac strains Aac31, Aac102 and Aac159 on AacG medium was 45%-100%, but Aac102 and Aac159 could not recover on AacSM or WFB68MA. When the seed infestation rates were 0.5% or 0.2%, the recovery of A. citrulli from on AacG media was higher than that of AacSM and WFN68MA media. The results reveal that the recovery of Ac on AacG semiselective medium are significantly improved growth of Ac strains isolated from Taiwan, and also reduced the growth saprophytic bacteria present in seed lots. In future, it is potential for that the AacG medium could apply the process of seed detection of Ac.

中文摘要 1

英文摘要 ii

目次 111

表目次-----------------------------------------------1v

圖目次 v

前言 1

材料與方法 6

一、菌株來源 6

二、瓜類細菌性果斑病菌堵養於AacSM、WFB68 及WFB68MA 三種半選擇性培養基 6

三、改進 AacSM 培養基對瓜類細菌性果斑病菌之生長影響 7

四、L-glutamic acid 濃度對瓜類細菌性果斑病菌於 AacG 培養基中之生長 及回收率 7

五、酵母萃取物對瓜類細菌性果斑病菌及非目標菌於 AacG 培養基中之生 長情形 8

六、離子對瓜類細菌性果斑病菌於 AacG 培養基之生長影響 8

七、非目標菌於 AacG 培養基之生長情形及回收率 9

八、瓜類細菌性果斑病菌對抗生素之敏成性測試 9

九、不同種類及濃度之抗生素於 AacG 培養基對瓜類細菌性果斑病菌、西 瓜種子及土壤表面非目標菌之回收率影響 10

十、種子帶菌於 AacG 培養基回收率與 PCR 偵測 11

結果 13

一、瓜類細菌性果斑病菌堵養於 AacSM、WFB68 及 WFB68MA 三種半選 擇性培養基之生長情形 13

二、改進 AacSM 培養基對瓜類細菌性果斑病菌之生長影響 13

三、L-glutamic acid 濃度對瓜類細菌性果斑病菌於 AacG 培養基中之生長 及回收率 14

四、酵母萃取物對瓜類細菌性果斑病菌及非目標菌於 AacG 培養基中之生 長情形 14

五、離子對瓜類細菌性果斑病菌於 AacG 培養基之生長影響 15

六、非目標菌於 AacG 培養基之生長情形及回收率 15

七、瓜類細菌性果斑病菌對抗生素之敏成性測試 16

八、不同種類及濃度之抗生素於 AacG 培養基對瓜類細菌性果斑病
菌、西 瓜種子及土壤表面非目標菌之回收率影響 16

九、種子帶菌於 AacG 培養基回收率與 PCR 偵測 17

討論 19

參考文獻 25

圖表 30

附錄 53

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