拮抗鏈黴菌之篩選與Streptomyces sp. A272之抗菌物質分析

Abstract

土壤中的放線菌以鏈黴菌 (Streptomyces spp.) 為主，已有許多報告指出，鏈黴菌可拮抗多種植物病原菌，且對植物生長有促進的效果，具有多重的生物防治機制；而其中的二次代謝產物，包括可分解各種物質之酵素與抗生物質，使其被廣泛應用於醫藥、家畜飼料的添加物、殺蟲劑以及植物保護用途上。本研究自台灣各地區分離到 138 株放線菌，初步測試並篩選出 10 株具有拮抗活性的菌株，利用形態學與分子生物學鑑定得知皆為鏈黴菌屬，且其中有些菌株可分泌幾丁質與澱粉分解酵素。測試此 10 株鏈黴菌對植物病原真菌與細菌之生長抑制效果，得知 A272 與 A463 兩菌株對病原真菌之拮抗活性最佳，而 A337 菌株的抗細菌能力最強。此外，探討固態與液態培養基中添加不同碳素源對 A272、A337 及 A463 等 3 株菌株之生長與拮抗活性的影響，結果指出此 3 種鏈黴菌對碳素源的利用有明顯差異；以SEM 觀察固態培養，顯示此 3 株鏈黴菌於添加不同碳素源培養基上之分化形態不同，如於阿拉伯糖培養基中可大量產孢，在添加葡萄糖培養基上則有延遲產孢的傾向，而乳糖對供試菌株為利用速度緩慢的碳素源。測試添加不同碳素源對拮抗活性之影響，得知 A272 菌株除木糖外，於其他供試碳素源的培養基上皆可抑制立枯絲核菌之菌絲生長；A337 菌株僅於添加阿拉伯糖與葡萄糖培養基產生抑菌活性；而A463 菌株於阿拉伯糖培養基上的拮抗能力最強，然有些碳素源雖能促進菌株生長，但無法產生拮抗物質。本研究之結果證實形態分化與抗生物質之產生並無直接相關。 選取較具拮抗效果的 A272 菌株進行發酵與抗生物質分析試驗。在測試最佳培養條件試驗中，針對接種源濃度、pH、發酵時間及光照等因子對菌株生長量、pH變動及抗生活性的影響，進行測試。顯示 A272 菌株以 106 cfu/ml 接種於 pH5的PDB 液態培養基中震盪培養，培養至第 6 天時即表現高抑菌活性，其抑制範圍可達 1.2 cm。此外，培養濾液經不同熱處理後其抗生活性雖略為降低約30%，但顯示當溫度高於 40℃ 時，抑制活性並無明顯變化，此結果證實濾液中可能含有耐熱性抗生物質。利用 A272菌株發酵液進行溫室防治試驗，評估 A272 菌株防治白菜幼苗立枯病與白菜炭疽病的成效，針對幼苗立枯病的防治效果，以稀釋10倍之培養液較能增加葉用白菜三鳳與坂田交配品種播種於感染土後之存活率，各可提高 25.9% 及 41.6 %；而稀釋 100 倍之濾液則對移苗後的防治成效較佳，於三鳳與坂田交配品種各可提升 53.1% 與 50% 的存活率。根據防治白菜炭疽病的結果，顯示施用稀釋 100 倍培養液之效果最好，可降低其罹病度約 53%。 在鏈黴菌之拮抗性代謝產物分析結果中，A272 菌株培養液經乙酸乙酯進行液相萃取分割後，指出乙酸乙酯層與水層均具拮抗活性，乙酸乙酯層之抗菌活性於管柱層析 (Silica) 後的 100%甲醇沖提部中會出現，而水層中的主要抗菌物質則在60-70% 甲醇/水流動相進行管柱層析 (C18) 時可被帶出，因此，推測拮抗物質為偏高極性。以 HPLC 進一步分離並測試生物活性後，得知其抑菌效果可能來自多種拮抗物質結合所產生。分離出具抗菌活性之波峰 P14-4、P11 與 P1，經由1H NMR 與 GC/MS 分析後推測為混合物，包含核苷酸類、糖類、IAA、尿素、quinoline、cholestane及有機酸化合物等，然比對的結果中以糖類佔大多數，其次為長鏈化合物及核酸類，上述化合物或其衍生物均有抗真菌的相關報導，顯示 A272 菌株具有產生多種拮抗物質的特性。 本研究自台灣田間分離到 10 株具拮抗潛力之鏈黴菌，並了解其形態學與生理生化特性；此外，針對部份菌株探討碳素源對其生長與拮抗活性之影響，建立其液態培養所需最適條件，並於溫室條件下測試防治植物病害之成效；最後，分析鏈黴菌 A272 菌株之有效代謝產物的可能種類。本研究所得到的研究成果，祈能有助於未來進一步研發植物保護製劑之用。

Actinomycetes are widely distributed gram-positive filamentous bacteria with varied morphology. The genus Streptomyces is the main group among actinomycetes. Many reports indicate that Streptomyces can protect crops from pathogens and promote plant growth with multiple biocontrol mechanisms. The characters of numerous seconday metabolites biosynthesis, including enzymes and antibiotics, have been in commercial use in medicine, animal feeder additives, insecticides, and plant protection. This study was aimed to screen for antagonistic actinomycete strains with potential application as a biological agent. A total of 138 actinomycetes isolated from different areas in Taiwan were screened for their antagonistic activity against plant pathogens. Ten antagonistic strains were selected and identified as Streptomyces based on morphology, physiological properties and 16S rRNA. Some of them can secrete chitinase and amylase. According to the inhibition spectrum of Streptomyces sp. strains against plant pathogens, A35, A272 and A463 strains are superior in antifungal activity;A337, A377 and A454 strains have antibiotics against bacteria. The onset of antibiotic biosynthesis is determined and influenced by a variety of physiological and environmental factors. The most important medium component for growth and secondary metabolite formation is the carbon source. Investigating the influence of carbon sources on growth and antibiotic activity of Streptomyces sp. on medium and in submerged culture. According to the results, utilization of carbon sources depends on species. Morphological differeation and degrees of antifungal activity of A272 strain are varied on ISP9 basal medium and medium contained 1% arabinose, fructose, glucose, inositol, lactose, mannitol, sucrose, and xylose. One promising strain, Streptomyces sp. A272 with strong antifungal activity was selected for the further studies. After investigating the influence of initial inoculum, pH, time course, and light on the growth and antibiotic activity in liquid culture, the results revealed that the optimum condition for growth and antibiotic activity was added 106 cfu/ml inoculum in pH 5 PDB medium, and the antibiotic activity would be detected after 6 days cultured. The thermostability of the antibiotic activity in crude culture supernatant was examined. Although the antifungal activity are slightly decreased after heat treatments, the antifungal activity did not be affected between different heat treatments. A preliminary trial on the disease control efficacy indicated that the infection of R. solani AG4 and C. higginsianum on Brassica rapa L. (Chinese Group) was greatly reduced by drenching and spraying application of the broth culture. 100X-diluted A272 broth culture increased more percentage of survival of 7-days-old seedlings in infested soil;however,10X-diluted A272 broth culture was more effective on controlling damping-off disease after seed sowed. The application of 100X-diluted A272 broth culture reduced more disease index percentage of anthracnose infection than 10X-diluted broth, and the broth is much more effective than filtrate on disease controlling anthracnose and damping-off in green house. After analysis of antifungal metabolites against R. solani AG4 and C. gloeosporioides of A272 strain, the results indicated that A272 strains produced multiple water-soluble antibiotics with broad spectrum of activity, contained α-D-glucopyranoside, nucleoside antibiotics, urea, IAA, quinoline, cholestane and organic acid. Thus, the antagonistic activity of A272 strain is considered that all of secondary metabolites join in a common effect. It could decrease the risk of resistance in pathogen.