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研究生:林立心
研究生(外文):Lih-Shin Lin
論文名稱:茄科細菌性斑點病菌 Xanthomonas perforans 之拮抗細菌篩選及其外泌抗生物質之探討
論文名稱(外文):Characterization of the Secreted Substances from Antagonistic Bacteria which can Inhibit the Growth of Xanthomonas perforans
指導教授:曾國欽曾國欽引用關係
指導教授(外文):Kuo-Ching Tzeng
口試委員:鄧文玲王惠亮鄭安秀
口試委員(外文):Wen-Ling DengHui-Liang Wang
口試日期:2016-07-13
學位類別:碩士
校院名稱:國立中興大學
系所名稱:植物病理學系所
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:2016
畢業學年度:104
語文別:中文
論文頁數:53
中文關鍵詞:茄科細菌性斑點病Xanthomonas perforans生物防治Paenibacillus
外文關鍵詞:Bacterial spot of solanaceous plantsXanthomonas perforansBiocontrolPeanibacillus
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茄科細菌性斑點病廣泛分布於世界各地,並造成番茄產量嚴重損失。在台灣, Xanthomonas perforans 為造成茄科細菌性斑點病之主要病原。為了尋找可抑制X. perforans之拮抗細菌,由農用土壤分離出 240 個菌株與 X. perforans 於平板上進行對峙試驗,其中 SAn-03 菌株對 X. perforans 具有最佳的抑制能力。利用脂肪酸圖譜、ITS 及 rpoB 基因之序列比對,將 SAn-03 鑑定為 Paenibacillus sp.。接著以 Imaging mass spectrometry (IMS) 之技術偵測此拮抗菌外泌之拮抗物質,結果顯示可能的拮抗物質分布在 500-600 m/z、800-950 m/z 及 1,000-1,100 m/z 之間,據前人文獻報導,來自 Paenibacillus 屬、符合上述分子量之抑菌物質有 paenimacrolidin、fusaricidins、paenilamicins、battacin 及pelgipeptins。分別使用硫酸銨沉澱法及 C18 column 收集 SAn-03 培養液中的親水性蛋白類及疏水性抗菌物質,與 X. perforans 對峙後顯示抗菌物質屬於親水性的蛋白類,進一步測試其物理及化學特性,證實此類拮抗物質具熱穩定性,且其分子量大於 14 kDa,並可被 proteinase K 所分解。以番茄植株進行生物試驗,調查 SAn-03 在有接種及沒有接種 X. perforans 的情形下,是否會對植物生長造成影響。結果顯示將番茄種子直接播種於混拌有 SAn-03 之栽培介質,不會影響種子之發芽率,且繼續於該介質中栽培 4 週後亦不會影響番茄植株之生長。以葉噴方式施用拮抗菌之試驗,同時接種 X. perforans 與 SAn-03 的懸浮液,或是於接種 X. perforans 前預先於葉面噴灑 SAn-03 懸浮液或培養稀釋液,皆可降低番茄細菌性斑點病之罹病度。以澆灌方式施用拮抗菌之試驗,於接種 X. perforans 前一週澆灌 SAn-03 懸浮液或培養稀釋液,均可降低番茄細菌性斑點病之罹病度,而於接種 X. perforans 的前三週每週澆灌一次 SAn-03 懸浮液,則可同時降低細菌性斑點病之罹病度,並減輕該病害對於植株營養生長之危害。綜合以上結果,顯示 SAn-03 防治番茄細菌性斑點病之能力,具有發展為生物防治製劑之潛力。

Bacterial spot disease is a worldwide-distributed plant disease on solanaceous plants and reduces plant production. Xanthomonas perforans, one of the pathogenic bacteria causes the bacterial spot disease on tomato in Taiwan. The SAn-03 strain, isolated from agricultural soil, was confirmed to have inhibitory ability against X. perforans using confrontation assays. According to the fatty acid profiles and similarity analysis of 16S rDNA and rpoB gene sequences, the SAn-03 strain was identified as Paenibacillus sp. Imaging mass spectrometry (IMS) analysis was employed to investigate the candidate antagonistic compounds, revealed that the candidate substances secreted by SAn-03 in the spectra of 500-600, 800-950, and 1,000-1,100 mass to charge ratio (m/z) might participate in antagonizing X. perforans. Moreover, to elucidate the effects on plant growth upon the inoculation of SAn-03 alone or together with X. perforans, tomato seeds were sown in the soil mixed with SAn-03, the result indicated that germination of seeds and vegetative growth of tomato seedlings are similar to the results performed by water control. Co-inoculation with SAn-03 and X. perforans on tomato by leaf spraying method, exhibited mild symptoms of bacterial spot disease, while the pre-treatment of cell suspension and the diluted culture broth of SAn-03 using leaf spraying method reduced the disease severity of bacterial spot elicited by X. perforans. On the other hand, the weekly drenching inoculation of cell suspension of SAn-03 for 3 weeks prior to the inoculation of X. perforans could increase the length and weight of stem and root of tomato. Moreover, the pre-treatment of cell suspension and the diluted culture filtrate of SAn-03 by drenching method could reduce the disease severity of bacterial spot disease. Altogether, this study revealed that the SAn-03 strain is potential to be a biocontrol agent for controlling bacterial spot diseases on tomato.

摘要-i
Abstract-iii
目錄-v
表目次-vii
圖目次-viii
壹、前言 -1
貳、材料與方法-5
一、供試菌株之來源、保存及培養條件-5
二、拮抗細菌之篩選-5
三、拮抗細菌之鑑定-6
五、拮抗物質之初步純化及特性分析-9
六、拮抗細菌對番茄生長之影響-11
七、生物防治試驗-12
參、結果-17
一、拮抗細菌之篩選-17
二、拮抗細菌之鑑定-17
三、以 Imaging MALDI-TOF MS 進行拮抗物質之偵測-18
四、拮抗物質之初步純化及特性分析-18
六、生物防治試驗-19
肆、討論-24
伍、參考文獻-32
陸、圖表-39


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