本文選題：黃瓜疫霉菌 + 拮抗菌　； 參考：《湖南農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：黃瓜疫病(Cucumber phytophthora blight)是黃瓜疫霉菌(Phytophthora melonis)引起的一種對黃瓜影響較大的土傳病害,嚴(yán)重影響了黃瓜的生長、產(chǎn)量及品質(zhì)。目前化學(xué)防治效果不佳且存在生態(tài)安全問題。因此,篩選、利用有益微生物及其代謝產(chǎn)物來防治黃瓜疫病是實(shí)施綠色植保的有效措施。本研究將從土壤中分離到的細(xì)菌和放線菌與黃瓜疫霉菌進(jìn)行對峙培養(yǎng),篩選到2株對黃瓜疫霉菌有明顯拮抗作用的生防菌株,對這2株菌株進(jìn)行了培養(yǎng)性狀、生理生化和分子生物學(xué)鑒定,并對其中1株拮抗菌進(jìn)行發(fā)酵優(yōu)化和抑菌活性物質(zhì)初步分析。主要研究和結(jié)果如下：(1)采用土壤稀釋法,從采自湖南、北京等地的土壤樣品中分離得到269株放線菌和細(xì)菌。以黃瓜疫霉菌為靶標(biāo)菌,通過平板對峙培養(yǎng)法,初步篩選到7株對黃瓜疫霉菌有明顯拮抗作用的微生物菌株。經(jīng)過發(fā)酵液稀釋5倍后復(fù)篩,發(fā)現(xiàn)放線菌X54菌株和細(xì)菌P3菌株對黃瓜疫霉菌菌絲生長的抑制率達(dá)到70%以上。此外,放線菌X54對辣椒炭疽病菌、煙草黑脛病菌、水稻紋枯病菌、小麥赤霉病菌、柑橘炭疽病菌、黃瓜枯萎病菌等都有一定的抑制作用。(2)通過對拮抗菌株X54和P3的培養(yǎng)性狀、生理生化特性進(jìn)行研究,以及利用PCR技術(shù)從基因組DNA中擴(kuò)增16SrRNA序列,并結(jié)合生物信息學(xué)方法進(jìn)行分析,初步認(rèn)為,放線菌X54為弗吉尼亞鏈霉菌(Streptomyces virginiae),其16SrRNA的登錄號KM078926；細(xì)菌P3為地衣芽孢桿菌(Bacillus licheniformis),其16SrRNA的登錄號KJ872538。(3)對拮抗菌X54進(jìn)行發(fā)酵條件優(yōu)化,首先對培養(yǎng)基優(yōu)化,確定其碳氮源的配方：麥芽糖20.0g/L、蛋白胨3.0g/L。再對發(fā)酵條件進(jìn)行單因素以及響應(yīng)面試驗(yàn),最終確定對放線菌X54的發(fā)酵條件為：種齡37h、溫度28℃、轉(zhuǎn)速230r/min、接種量8%、裝液量50m1、pH值7.0、發(fā)酵時(shí)間5d。(4)本研究對放線菌X54產(chǎn)生的抑菌活性物質(zhì)的傳代、酸堿、光照、溫度、紫外線這5方面的穩(wěn)定性進(jìn)行初步研究,又對其分泌類型、硫酸銨沉淀蛋白質(zhì)及有機(jī)試劑萃取等進(jìn)行初探。結(jié)果表明,活性物質(zhì)具有較高的遺傳穩(wěn)定性；光照穩(wěn)定性；能耐高溫,可能是非蛋白類物質(zhì)；在酸堿環(huán)境下,穩(wěn)定性較差,在后期的物質(zhì)分離純化等研究中盡量保持pH為7.0；其對紫外線照射不敏感,抑制作用相對穩(wěn)定。通過物質(zhì)分泌測試,抑菌活性物質(zhì)屬于胞外分泌型。對X54發(fā)酵液做硫酸銨沉淀結(jié)果表現(xiàn)抑菌活性物質(zhì)可能是非蛋白類物質(zhì)。通過不同極性有機(jī)試劑的萃取,發(fā)現(xiàn)能易溶于極性較強(qiáng)的甲醇,不溶于氯仿。根據(jù)“相似相溶”原理,推斷出抑菌活性物質(zhì)具有一定的極性。
[Abstract]:Cucumber phytophthora blightis a soil-borne disease caused by Phytophthora melonis (Phytophthora melonis), which seriously affects the growth, yield and quality of cucumber. At present, the effect of chemical control is not good and there are ecological safety problems. Therefore, screening and using beneficial microorganisms and their metabolites to control cucumber blight is an effective measure to implement green plant protection. In this study, the bacteria and actinomycetes isolated from soil were cultured in confrontation with Phytophthora cucumbers, and two biocontrol strains with obvious antagonistic effect against Phytophthora cucumbers were screened, and the two strains were cultured. Physiological, biochemical and molecular biological identification, and one of the antagonistic bacteria was optimized for fermentation and preliminary analysis of antimicrobial active substances. The main results are as follows: (1) 269 strains of actinomycetes and bacteria were isolated from soil samples from Hunan and Beijing by soil dilution method. Using Phytophthora cucumbers as the target bacteria, 7 strains with obvious antagonism to Phytophthora cucumbers were screened by means of plate confrontation culture. After the fermentation broth was diluted for 5 times, it was found that the inhibition rate of actinomycetes X54 and bacteria P3 on the hypha growth of Phytophthora cucumbers was more than 70%. In addition, actinomycetes X54 had some inhibitory effects on pepper anthracnose, tobacco black shank, rice sheath blight, wheat scab, citrus anthracnose and cucumber wilt. The physiological and biochemical characteristics were studied, and the 16SrRNA sequence was amplified from genomic DNA by PCR technique, and analyzed with bioinformatics. Actinomycetes X54 is Streptomyces virginiaeae of Virginia, its 16SrRNA accession number is KM078926, bacteria P3 is Bacillus licheniformis, and its 16SrRNA accession number is KJ872538. 3) the fermentation conditions of antagonistic bacteria X54 are optimized. The formula of carbon and nitrogen source was determined as follows: maltose 20.0g / L, peptone 3.0 g / L. The fermentation conditions of actinomycetes X54 were determined by single factor test and response surface test. The fermentation conditions were as follows: seed age 37 h, temperature 28 鈩，

本文編號：1815740