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|標題:||拮抗性枯草桿菌Bacillus subtilis WG6-14菌株於柑橘潰瘍病防治應用
Control of citrus bacterial canker by antagonistic Bacillus subtilis WG6-14
|關鍵字:||citrus bacterial canker;柑橘潰瘍病;Bacillus subtilis;biocontrol;枯草桿菌;生物防治||出版社:||植物病理學系||摘要:||
拮抗性枯草桿菌Bacillus subtilis WG6-14菌株於柑橘潰瘍病防治應用
柑橘潰瘍病citrus bacterial canker(CBC)為世界性柑橘生產上最受重視的細菌性病害，此一病害對於柑橘產業破壞性極大，過去大多仰賴化學藥劑(多為銅劑)的施用，然近年來由於因抗藥性菌株的問題其應用效果常受質疑，又因永續生態意識抬頭，生物合理性製劑的發展蔚成趨勢，尋求一應用於防治柑橘潰瘍病之生物製劑是急迫需要的。本篇論文主要研究目的即為以枯草桿菌群(Bacillus subtilis group, BSG) WG6-14菌株之活體微生物製劑作防治應用，挑戰在全世界與台灣都危害相當嚴重的柑橘細菌性潰瘍病。研究中首先以研究室所保存七株本土性、對多種植物病原性真菌及細菌具優異拮抗性且產孢良好之BSG菌株為材料，進行防治柑橘細菌性潰瘍病篩選試驗。於本研究室先前之研究已知此七株BSG菌株對多種重要植物病原性Xanthomonads細菌具有相當優異的抗生活性，已經測試證實可於相關病害防治應用之實例包括由Xanthomonas campestris pv. manigiferae-indicae所引起的芒果細菌性斑點病(bacterial black spot)，X. axonopodis pv. vesicatoria所引起的番茄細菌性斑點病(bacterial spot of tomato)，與X. oryzae pv. oryzae引起的水稻白葉枯病(bacterial leaf blight)等等。就內生孢子量產特性而言，七支供試菌中以WG6-14內生孢子產量上的表現最高。接下來的溫室試驗中，經以所建立之多蟲針人工接種，可用以量化X. axonopodis pv. citri侵入感染與CBC病徵表現之模式系統，配合經專一性引子定性屬全台灣普遍存在之A病原型的Xac01菌株的人工接種模式，更進一步證明WG6-14菌株的防治效果，明顯優於其他供試菌株。於後續溫室及田間試驗中，利用本研究室既有先導液態發酵系統以及生產活體微生物製劑技術平台所產出之WG6-14菌株活體性內生孢子製劑（WG6-14’）為測試材料，溫室種植之臍橙(Citrus sinensis)，經以100倍稀釋的WG6-14’作葉面的噴霧處理24小時後，再人工接種Xac01（3.2×106 cfu/ml），試驗結果證實可使發病率降低94%，試驗中以水作前處理的對照組則發病率達97.7%，試驗中也證實所施用WG6-14’對病徵表現之抑制作用與施用劑量有關，且以接種前處理較接種後處理效果顯著較好。值得一提的是在較高濃度Xac01 (107 cfu/ml)接種下WG6-14’ 的防治效果明顯勝於波爾多液的前處理使用。在葉部組織中，拮抗菌WG6-14之族群在施用後七天呈波動起伏上升之勢，在施用十天後，則可見緩緩下降現象；利用抗rifampicin的病原菌突變株Xac01r，檢測其與拮抗菌WG6-14兩者在葉片表面族群消長與CBC病徵表現的相關性，試驗中證實此一突變株之致病力與野生型Xac01菌株並無差異，約可在接種後一星期表現典型突起的壞疽性潰瘍病斑；在無前處理WG6-14’的情況下，在接種一天後即可見Xac01r的族群在柑橘葉部穩定而快速的增加，在接種後10天病原菌Xac01r的族群成長近乎4個次元，而葉片上則在接種第4天即可觀察到典型CBC病斑的形成。而WG6-14’行前處理之植株，則拮抗菌明顯有效抑制病原菌Xac01r族群的爬升，且在接種後的5-10天才觀察到典型CBC病斑的形成。值得一提的是WG6-14’之抑制病原菌族群以及延後病徵表現的效果與施用劑量（dosage）間之相關性極為明顯。近一歩田間試驗中也可證實WG6-14前處理施用後在葉片上確可有效抑制病原菌Xac01族群的爬升，在接種後14天其Xac01族群降低達一個次元以上，在14天之後，才緩緩爬升；試驗中以波爾多液作前處理植株，其葉片上Xac01之消長則與以水做前處理之對照組類似，未見有病原菌族群衰退的情形。上述討論結果很明確指出B. subtilis WG6-14內生孢子活體製劑，於柑橘上對於CBC之防治確為一有用的微生物製劑。試驗中WG6-14的抑病效果，其作用機制顯然與抗生活性及其與病原菌間於葉片上，營養與生存空間之競爭作用有關；在研究過程中，在施用WG6-14’發酵液的植株上，皆可觀察植株生長促進之效果，包括新稍的生長茂密、樹冠增大與樹勢生長良好，生長勢及新梢發展均有顯著促進。在本研究中藉由一密閉系統測試 證實WG6-14’揮發性代謝物會促進對甘藍種子，生長包括發芽時間提前與根莖部生長發育的促進，此一生長促進作用除了在甘藍種子，見之於水稻(台農67號) 、洋香瓜、胡瓜(秀燕)、西瓜、番茄 (農友301)等的種子上皆可適用，而此類揮發性代謝物的產生顯然菌株之生長繁殖以及所應用之培養基質有關；本研究中藉由Voges-Proskauer法測試WG6-14 菌株活體生物製劑確可產生butanediol，此butanediol的存在是否與WG6-14’對於柑橘植株生長促成有關很值得未來繼續瞭解。
Control of citrus bacterial canker by antagonistic Bacillus subtilis WG6-14
The major objective of this investigation was to explore the antagonistic Bacillus resources as microbial fungicide for the control of citrus bacterial canker (CBC) disease-the most notorious bacterial disease on citrus in Taiwan and worldwide. The development of biofungicide effective for the control of CBC is in urgent need in Taiwan because the disease was devastative and the effectiveness of chemical control agent (mainly copper) was limited. In a screening trial for antagonistic Bacillus resources with application potential in plant disease control, the unusual antagonisity of Bacillus subtilis group (BSG, Priest, 1993.)against wide spectrum of plant pathogenic Xanthomonads was observed. By a pilot scale liquid fermentor system, technique platform for the production of BSG endospore formulation was established. The broth cultures obtained from the trial production have been shown effective in discouraging the infection caused by Xanthomonads; successful examples include bacterial black spot of mango (Xanthomonas campestris pv. manigiferaeindicae, Chou et al., 1997), bacterial spot of tomato (X. axonopodis pv. vesicatoria, Lee, 2002) and bacterial leaf blight of rice (X. oryzae pv. oryzae, Wang, 2002). To explore the use of BSG resources in the control of CBC in Taiwan, a total of 7 BSG strains known with superior antagonisity against wide spectrum of phytopathogenic fungi and bacteria were screened for the competence of endospore production and discouragement of CBC infection. Among them, strain WG6-14 was shown the best in endospore productivity. A followed greenhouse trial where that a multiple-pin artificial inoculation system was applied for quantitative assay of typical CBC infection, further demonstrated that strain WG6-14 was also among them the best in counteracting the infection by X. axonopodis pv. citri Xac01-a typical pathotype A strain of Xac which appeared to be prevalent all over the island. On the tested Navel orange (Citrus sinensis), the pretreatment by foliar spray a 100X diluted WG6-14 endospore formulation (2×109 endospores/ml) 24 hours before artificial inoculation of Xac01 (3.2×106 cfu/ml) resulted in 94% inhibition of the disease incidence. The disease incidence of the compared control which was pretreated by water spray was 97.7%. By greenhouse trial, the efficacy of disease control by WG6-14 was further shown to be dose dependent and the performance was apparently better as pre-inoculation treatment rather than post-inoculation treatment. The disease control efficacy appeared to persist in the case wherein inoculation concentration of Xac01 used for challenge inoculation was raised to 107cfu/ml. It was worth to mention that with the raised challenge inoculation pressure, the disease control efficacy by WG6-14 may exceed that by Bordeaux mixture (BM) application. On the foliar tissue, the antagonist (WG6-14) population showed a fluctuating increase dynamics about 7days. A slow decline of the population might be observed about 10 days after application. With the use of a rifampicin resistant mutant strain Xac01r, the interaction of antagonist (WG6-14) and pathogen (Xac01r) population in relating to CBC symptom development was examined. The virulence of the mutant strain appeared to be the same as the wild type strain Xac01 in that typical protruding necrotic CBC lesion developed within one week after artificial inoculation. Without pretreatment of WG6-14, a steady and rapid increase of Xac01r population was detected on the foliar tissue at the 1st day after the inoculation. In 10 days, the pathogen population increased by approximately 4 orders. And accompanied to that was the development of typical CBC symptoms which became prominent at 4th day after inoculation. Whereas with the pretreatment foliar application of WG6-14, the increase of challenge inoculated pathogen population was suppressed. And the development of CBC symptom appeared not detectable until 5-10 days after inoculation. It was worth noting that the suppression of the pathogen population and the associated symptom expression were apparently the function of the dosage of WG6-14 applied. The suppressiveness of WG6-14 on the pathogen population on the foliar tissue were further demonstrated in a field trial where that Xac01 in stead of Xac01r was applied for challenge inoculation. In field trial performed, the pretreatment of WG6-14 led to a substantial decrease of the afterward applied Xac01 population by more than 1 order at 14th day after inoculation, although the increase of the Xac01 was observed later on. The deteriorative effect in the pathogen population, however, was not observed in BM pretreated plants. The population density of Xac01 on BM pretreated plants remained at the same level as that of water treated control plants. The results discussed clearly indicated the developed endospore formulation of B. subtilis WG6-14 a useful microbial fungicide for the control of CBC on citrus. The effectiveness of disease control was apparently a function of the antagonisity of WG6-14 and its competition in available space and nutrient on the foliar tissue. During the course of the study, prominent growth promotion was consistently observed among plants pretreated with WG6-14.The growth promotion was manifested by the substantially increased new shoot development, canopy size and growth vigor. By a closed container system, we found that certain volatile metabolites produced by WG6-14 were stimulative to seed germination and seedling growth of cabbage (Brassica oleracea L. co. capitata). The bio-fumigation effect on growth promotion and seed germination appeared to work for cabbage, rice, muskmelon, cucumber, watermelon and tomato. The production of the growth promotive volatile metabolite was a function of the bacterial growth and medium used to culture the bacteria. By Voges-Proskauer test, the production of butanediol (approximately 0.0206μg/ml culture broth) was detected from the endospore formulation of WG6-14. Whether or not it may contribute to the growth promotive effect observed on citrus worth great attention.
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