黄瓜疫霉菌拮抗菌株的筛

发布时间：2018-04-28 14:54

  本文选题：黄瓜疫霉菌 + 拮抗菌　； 参考：《湖南农业大学》2015年硕士论文

【摘要】：黄瓜疫病(Cucumber phytophthora blight)是黄瓜疫霉菌(Phytophthora melonis)引起的一种对黄瓜影响较大的土传病害,严重影响了黄瓜的生长、产量及品质。目前化学防治效果不佳且存在生态安全问题。因此,筛选、利用有益微生物及其代谢产物来防治黄瓜疫病是实施绿色植保的有效措施。本研究将从土壤中分离到的细菌和放线菌与黄瓜疫霉菌进行对峙培养,筛选到2株对黄瓜疫霉菌有明显拮抗作用的生防菌株,对这2株菌株进行了培养性状、生理生化和分子生物学鉴定,并对其中1株拮抗菌进行发酵优化和抑菌活性物质初步分析。主要研究和结果如下：(1)采用土壤稀释法,从采自湖南、北京等地的土壤样品中分离得到269株放线菌和细菌。以黄瓜疫霉菌为靶标菌,通过平板对峙培养法,初步筛选到7株对黄瓜疫霉菌有明显拮抗作用的微生物菌株。经过发酵液稀释5倍后复筛,发现放线菌X54菌株和细菌P3菌株对黄瓜疫霉菌菌丝生长的抑制率达到70%以上。此外,放线菌X54对辣椒炭疽病菌、烟草黑胫病菌、水稻纹枯病菌、小麦赤霉病菌、柑橘炭疽病菌、黄瓜枯萎病菌等都有一定的抑制作用。(2)通过对拮抗菌株X54和P3的培养性状、生理生化特性进行研究,以及利用PCR技术从基因组DNA中扩增16SrRNA序列,并结合生物信息学方法进行分析,初步认为,放线菌X54为弗吉尼亚链霉菌(Streptomyces virginiae),其16SrRNA的登录号KM078926；细菌P3为地衣芽孢杆菌(Bacillus licheniformis),其16SrRNA的登录号KJ872538。(3)对拮抗菌X54进行发酵条件优化,首先对培养基优化,确定其碳氮源的配方：麦芽糖20.0g/L、蛋白胨3.0g/L。再对发酵条件进行单因素以及响应面试验,最终确定对放线菌X54的发酵条件为：种龄37h、温度28℃、转速230r/min、接种量8%、装液量50m1、pH值7.0、发酵时间5d。(4)本研究对放线菌X54产生的抑菌活性物质的传代、酸碱、光照、温度、紫外线这5方面的稳定性进行初步研究,又对其分泌类型、硫酸铵沉淀蛋白质及有机试剂萃取等进行初探。结果表明,活性物质具有较高的遗传稳定性；光照稳定性；能耐高温,可能是非蛋白类物质；在酸碱环境下,稳定性较差,在后期的物质分离纯化等研究中尽量保持pH为7.0；其对紫外线照射不敏感,抑制作用相对稳定。通过物质分泌测试,抑菌活性物质属于胞外分泌型。对X54发酵液做硫酸铵沉淀结果表现抑菌活性物质可能是非蛋白类物质。通过不同极性有机试剂的萃取,发现能易溶于极性较强的甲醇,不溶于氯仿。根据“相似相溶”原理,推断出抑菌活性物质具有一定的极性。
[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，

本文编号：1815740