Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31749
標題: 鏈黴菌Streptomyces griseobrunneus S3菌株作為植物真菌性病害應用生物防治製劑之發展
Development of Streptomyces griseobrunneus S3 as a Bioagent for the Control of Plant Fungal Diseases
作者: 賴文瑞
Lai, Wen-Ruey
關鍵字: biological control;生物防治;plant fungal diseases;Streptomyces griseobrunneus S3;screening;植物真菌性病害;鏈黴菌Streptomyces griseobrunneus S3菌株;篩選
出版社: 植物病理學系
摘要: 
生物防治為對環境友善、親和的一種植物病害防治方法,其在未來它具有潛力減少,或替代化學藥劑的使用。於自然界中,鏈黴菌 (Streptomyces spp.) 為普遍存在於根圏土壤的腐生菌,它們同時也是主要的抗生素來源,且具有良好的產生胞外水解酵素之特性,此些水解酵素可用來分解存在於自然環境中的各種生物性聚合物,諸如幾丁質、纖維素和 β-1,3 葡萄聚醣等,此外,鏈黴菌亦可產生對環境逆境具有良好抗性的孢子,由於此些特性使得鏈黴菌被認為是很適合發展為生物防治劑的良好物種。因此,本論文研究的主旨乃是利用幾丁質培養基做為半選擇性培養基,由採自田間的根圏土壤、各種泥碳土與有機堆肥中大量分離篩選菌落生長形態類似鏈黴菌之菌株,檢測其發展作為生物性殺菌劑之應用潛力,本研究中,總共分離出219株具幾丁質分解能力類似鏈黴菌之菌株,藉由對峙培養檢測其生體外拮抗活性 (in vitro antagonism),證實其中72株對Pythium aphanidermatum具有抗生活性,同時,153株對Rhizoctonia solani AG4具抗生活性,上述菌株經綜合其生體外抗生活性與幾丁質分解活性的結果,挑選出S1、S2、S3與S7等4株具有應用潛力之菌株,進一步於50公升的醱酵醩中進行試醱酵量產,結果顯示S1與S3可快速且大量地生產以孢子為主要有效生質體的醱酵液,進一步測試比較此2個菌株與S30於溫室的條件下在已滅菌過的泥碳土中,對於腐霉菌猝倒病的防治效果 (S30為本研究室發展已久之已達商品化量產標準的鏈黴菌菌株),試驗結果証實此3個菌株皆可明顯地提昇幼苗的存活率,其中以分離自柑桔根圏土壤的S3效果最佳,與對照組相比較可提昇約40% 的存活率。S3經鑑定為Streptomyces griseobrunneus。在已滅菌過的泥碳土中測試S3對立枯絲核菌 (R. solani AG4) 所造成在甜椒的根腐與猝倒之防治效果。結果亦証實S3可明顯地減少死亡率達25%,同時亦有效地減少植株的罹病程度。另外,於炭疽病菌 (Colletotrichum gloeosporioides) 接種前1天與接種後4天噴霧處理S3,對於炭疽病的病斑形成與其擴展皆具明顯的抑制效果。再者,於8片葉片大的甜椒植株上先行噴霧處理S3,仍然還是有效地減少炭疽病所造成的罹病程度。在後續利用未經滅菌過的泥碳土進行對胡瓜之腐霉菌猝倒病的防治試驗中,利用S3醱酵液稀釋400倍2次 (播種後立即施用及播種後2天施用),結果顯示與對照組相比較處理S3可提昇約15% 的存活率,而澆灌處理S30與滅達樂 (metalaxyl) 則分別提昇約8%與40% 的存活率。測試4種穀物培養基所生產的醱酵液對腐霉菌猝倒病的防治效果,結果顯示此4種醱酵液皆可明顯地抑制該病害,同時在6 ℃下貯藏時間長達7與10個月的醱酵液仍然還可有效地發揮抑病效果,其防治效果與只貯藏2星期者並無統計分析上之差異,進一步的試驗亦証實施用醱酵液的稀釋液可有效減少腐霉菌猝倒病,其主要作用來自於醱酵液中之菌體的作用。再者,藉由種子浸泡處理更加提昇了S3對猝倒病的防治效果,與原先的澆灌處理相比較還可提昇約20% 的存活率,同時,浸泡處理S3於5種測試的栽培介質中亦皆明顯地減少猝倒病的發生。最後,本研究亦探討營養因子對S3的生長,抗生物質、胞外分泌性幾丁質分解酵素與β-1,3 葡萄聚醣分解酵素產生之影響,以期能應用於未來量產技術的改進,利用Czapek’s培養液作為基礎培養基進行搖瓶培養,供試碳、氮素源中以幾丁質和硝酸鈉最有利於其生長與抗生物質的產生,同時,除了以幾丁質為碳素源之處理外,在Czapek’s培養液中無法偵測到胞外分泌性幾丁質分解酵素。測試6種天然穀物煎汁培養基,其中以黃豆、粉頭與燕麥對促進S3生長的效果最佳,於培養後第7天,此3種培養基的log CFU/ml 值皆可大於9.0,同時,此3種培養基亦較有利於抗生物質、胞外分泌性幾丁質分解酵素及β-1,3 葡萄聚醣分解酵素的產生,進一步選用對供試菌生長較佳的粉頭培養基,與對抗生物質和胞外分泌性水解酵素之產生較具優勢的燕麥與玉米培養基,以5公升醱酵醩進行初步量產,結果証實與搖瓶培養的趨勢相似。由上述結果所提供的証據顯示,S. griseobrunneus S3具有潛力發展為廣譜性的生物性殺菌劑,以應用於永續性植物健康管理體系中來減少多種的真菌性病害。

Biological control is an environmentally friendly alternative for reducing the deteriorative effect on the ecosystem due to the prolonged and over use of chemical pesticides in crop production system. As a saprophyte and common resident in rhizosphere, Streptomyces species are well recognized for their prominent antagonistic activity against wide-spectrum of plant pathogens, and the profound lytic enzyme activity for decomposing various biopolymers including chitin, lignocellulose, and β-1,3-glucan etc. The use of Streptomyces spp. as biocontrol agents against soil-borne fungal pathogens has been actively researched for long in plant protection. The major focus of this investigation was initiated to screen for natural Streptomyces resources with potential application as a biocontrol agent for important plant fungal diseases. A total of 219 chitinolytic strains with characteristic streptomycetes colonial morphology were obtained by use of a semi-selective isolating medium which consisting colloidal chitin. Among these Streptomyces strains, results obtained from dual culture assay indicated that 72 (approximately 33%) were antagonistic against Pythium aphanidermatum, and 153 (approximately 70%) were antagonistic against Rhizoctonia solani AG4. For exploring the potential of practical application, 4 strains, namely S1, S2, S3, and S7 were chosen among them for further evaluation based on overall performance of antagonisity and chitinolytic activity. By liquid fermentation using 50-liter fermentors, broth cultures with spore mass as major constituent were successfully produced for strain S1 and S3, respectively. A preliminary trail on the disease control efficacy indicated that by drenching application of the broth culture, the infection of P. aphanidermatum on cucumber (Cucumis sativus L.) was greatly reduced. The efficacy of disease control was equivalent to or even superior than that by Streptomyces sp. S30, a strain under development for commercialization, included in the trial as a comparison. The survival stand of strain S3 treatment was about 62%, the compared S1and S30 treatment were 53% and 47% respectively, whereas that of the compared water treated control was only 23%. Strain S3 which showed best performance in this trial test was thus chosen as a target for the attempted biofungicide development. The test strain was originally isolated from citrus rhizosphere soil and has been identified as a member of Streptomyces griseobrunneus. In addition to Pythium damping-off, strain S3 was also tested and shown to be effective in controlling damping-off and root rot caused by R. solani AG4 and anthracnose caused by Colletotrichum gloeosporioides on sweet pepper (Capsicum annuum L. cv. Trim Star). By drenching treatment of S3 broth culture at proper dilution, Rhizoctonia damping-off incidence of greenhouse grown sweet pepper was greatly reduced. The disease control efficacy was manifested by the significantly increased survival percentage and the greatly reduced average disease severity. Likewise, for pepper subjecting to artificial inoculation of C. gloeosporioides, spraying application of a 200X-diluted S3 broth culture 1 day before and 4 days after inoculation, significantly reduced the percent lesion formation and average disease severity (DSI). The spraying application of strain S3 also effectively reduced the anthracnose infection and disease severity percentage (DS %) on sweet pepper. For greenhouse cucumber grown on non-sterilized peat moss, drenching application of strain S3 suppressed Pythium damping-off infection by 15 ± 5%, whereas that by S30 and metalaxyl was 8 and 40%, respectively. The application of broth culture derived from oat, corn, wheat bran, and soybean showed equal effectiveness on the control of Pythium infection. The effectiveness of the disease control was apparently a function of the cellular composition rather than the metabolites in the broth culture. The efficacy was greatly improved by soaking treatment. A 2-hour soaking of primed seeds by S3 before seeding reduced the disease incidence by 35%; and what's more encouraging was that the disease control efficacy appeared to work disregard the cultivation substrates applied. By modified sand tube assay, soaking treatment of primed cucumber seeds with strain S3 appeared to not only stimulated deeper colonization of chitinolytic streptomycetes on rhizoplane, but also promoted growth of the roots. The nutritional requirements for the growth, production of anti-fungal metabolites, and production of extracellular chitinase and β-1,3-glucanase were tested, thus to provide useful information for the mass production of strain S3. In a shaking flask culture system (125 rpm and 30 ℃), in which Czapek's broth was applied as the basal medium, chitin and calcium nitrate were found among 8 carbon sources and 7 nitrogen sources, respectively, the best to support the growth and production of antifungal metabolites. Besides chitin treatment, the production of extracellular chitinase was hardly detectable from these Czapek's broths tested. In addition, a total of 6 grain decoction broths were screened for the effectiveness in supporting the growth and production of antifungal metabolites, extracellular chitinase and β-1,3-glucanase. Among them, soybean, wheat bran, and oat broths were in the order the best to growth of strain S3, in which their values of log CFU/ml broth culture were all attained more than 9.0 after incubation for 7 days. While these broths also resulted in greater production of antifungal metabolites and extracellular chitinase and β-1,3-glucanase. The superior efficacy of wheat bran decoction broth in supporting the growth and production of extracellular chitinase, and that of oat and corn decoction broth in supporting the production of antifungal metabolites was further demonstrated by scale up production trails in which a series of 5-liter liquid fermentor system were used. The accumulated results indicated the potential of S. griseobrunneus S3 as a broad-spectrum bioagent for the control of plant fungal diseases.
URI: http://hdl.handle.net/11455/31749
Appears in Collections:植物病理學系

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