【作者】 王燕；

【导师】 赵宝华； 董志平；

【作者基本信息】 河北师范大学 ， 微生物学， 2008， 硕士

【摘要】 本试验旨在从谷子纹枯病根际土壤中分离并筛选出对谷子纹枯病具有良好拮抗作用的生防菌,并对其发酵条件和产生的活性物质进行初步研究。主要结果如下:(1)由全国各谷子产区采集谷子纹枯病根际土壤样品,通过梯度稀释法对土壤样品进行分离,共分离到包括细菌、链霉菌、真菌在内的土壤微生物1346株。通过对峙培养法,共筛选到拮抗细菌16株,链霉菌1株,木霉菌3株。其中链霉菌SS56抑菌活性最高,发酵液平皿和盆栽试验抑菌活性测定显示了较强的抑菌活性。SS56菌株对所有作物的纹枯病菌及某些主要作物的土传病害都有很好的抑制作用,抑菌谱较广。(2)根据SS56菌株的菌丝和孢子形态及其培养特征和生理生化特性试验,初步判定其为灰绿链霉菌。SS56对纹枯病菌气生菌丝具有明显的抑制作用,通过显微镜观察,初步明确了SS56主要通过分泌活性物质抑制谷子纹枯病菌菌丝的生长和伸长,造成菌丝节间缩短,分枝增多,弯曲变形;菌丝体原生质凝集,顶端生长点膨大,内容物外泄。(3)对SS56发酵条件进行了初步研究,并对21种常用链霉菌发酵培养基进行了筛选,结果显示,PD培养液为最适发酵培养基;以PD为发酵培养基对菌株发酵的接种菌龄、接种量、发酵温度、摇床转速和发酵时间等条件进行了选择,结果显示,SS56在接种菌龄4天,接种量为10%,发酵温度为31℃,摇床转速160rpm,培养4天,初始pH8为最适发酵条件。(5)对SS56发酵液中活性物质的的理化性质进行了初步研究,结果表明:活性物质在弱酸和中性pH条件下稳定;较耐高温,在80℃仍稳定存在,100℃时活性有所降低,121℃活性完全丧失。有效期较长,6个月后仍具有活性。(6)对SS56抑菌活性物质提取和分离方法进行了初步研究。结果显示,菌体和发酵液均有活性物质存在,采用乙酸乙酯静置萃取的方法可将活性物质分离,活性物质主要存在于有机相中,推测链霉菌SS56所产活性物质为脂溶性物质。静置吸附试验显示,强极性大孔吸附树脂NKA-9可将活性物质吸附。将洗脱液分段收集,并对各组洗脱液性进行活性测定,对活性组分利用TLC法分离制备。将TLC制备结果条带分别回收测活,经高压液相色谱分析,显示为一纯度较高单峰,可进一步进行结构鉴定。更多还原

【Abstract】 This study aimed at separating and screening the antagonistic bacteria from rhizosphere soil samples of foxtail millet that Rhizoctonia solani infected, which had a good antagonistic role to Rhizoctonia Solani, then preliminary researches were carried out for fermentative conditions and active productions.Rhizosphere soil samples of R.solani infected foxtail millet were collected from all production areas in China, from which, a total of 1346 microbial strains were separated, including bacteria, streptomyces, fungi, etc. By confrontation culture, antifungal activities to R.solani of these microorganism were determinated, as a result, 16 bacteria, 1 streptomyces and 3 trichoderma that had antagonism to R.solani were screened out. The streptomyces strain“SS56”, out of the 20, had shown the highest antifungal activity. At the same time, SS56 had performed higher antifungal activity according to the examination in petri dish and pot culture of fermentation broths. Antifungal spectrum was detected through confrontation culture, the results showed that SS56 had wide antifungal spectrum, which had well inhibition to all R.solani and most soil-borne diseases in crops, it suggested that SS56 had research value in-depth.SS56 strain was preliminarily identified as Glaucum streptomyces according to physiological and biochemical property experiments along with morphologic and cultural characteristics of mycelia and spores, referring to“Classification and Identification of Actinomyces”and“Berger bacterial identification manual”as well.The observation through microscope primarily definitized that SS56 restrained the growth and elongation of R.solani mycelia in foxtail millet by secreting active substances which could cause shortening, branches increasing, bending and metamorphosing, cytoplasmic agglutination of mycelia, topmost growing point expanding and the contents leakage. At the same time, SS56 has obvious inhibition for aerial mycelium of R.solani.The pilot studies of fermentation conditions for SS56, which screened 21 kinds of normal fermentation medium, showed as a result that PD medium was the optimal one. The bacterial age, inoculum doze, rotation speed, temperature and time of fermentation with PD medium were selected, the results demonstrated that the optimal fermentation conditions of SS56 were 4 days of the bacterial age, 10% inoculum doze, 31℃of fermentation temperature, 160rpm of shaking speed and 4 days of cultivation.The preparatory researches made on physical and chemical properties of the active substances existing in fermentation broth for SS56, which suggested that the active substances were stable in weak acid and neutral pH conditions, high temperature-endured, remained stable existence in the 80℃, little decreaced activities in 100℃, activity completely lost in 121℃; The activity still existed in a longer valid period of six month.The preliminary studies on extraction and separation methods of antibacterial active substances for SS56. The outcome showed that active substances existed in both thalli and fermentation broths, which could be separated with extraction by acetic ether and existed mainly in organic phase, it confered that the active substances produced by SS56 was a kind of fat-soluble. Static adsorption experiments revealed macropore polymeric adsorbent NKA-9 with intensive polarity could adsorb the active substance. Concentrated the elutions by subsection and determined their activities, then separated the active components by Thin-Layer Chromatogram and reclaimed preparative bands respectively. The high performance liquid chromatographic analysis showed that it was single peak with the higher activity and purity whose construction could be identified after preparation.更多还原