【作者】 阿拉坦夫；

【导师】 斯琴巴特尔； 张克诚；

【作者基本信息】 内蒙古师范大学 ， 植物学， 2008， 硕士

【摘要】 本文从样品采集、菌株分离保存、活性筛选、菌种鉴定、生物学特性、基础发酵培养基的选择、作用机理、小麦白粉病防治效果等多个方面对分离自保护性耕作农田土样的菌株A2进行了研究。主要结论如下:⑴从保护性耕作农田采集土样,选用不同的分离培养基,采用土壤稀释及平板划线法分离出38个单菌落;以番茄灰霉病菌和水稻稻瘟病菌为指示菌,对峙生长法初筛获得具有拮抗作用的菌株12株。10代继代后,重新测定活性并结合管碟法进行复筛,对指示病原菌仍具有较强抑菌作用且较稳定的只有细菌菌株A2。⑵室内平板对峙结果表明,菌株A2抑菌谱广,对20种病原真菌均表现出抑制作用。但对不同的植物病原真菌抑菌效果不同,抑制率在29.00%～86.00%之间,其中对番茄灰霉病菌、水稻稻瘟病菌的抑菌效果最好,抑制率达85.61%、81.82%;抑菌带宽平均可达17.00mm、15.83mm。⑶运用形态、生理生化以及分子生物学方法对菌株A2进行了鉴定。根据A2菌株的形态、生理生化特性以及16S rDNA的序列比对结果,鉴定菌株A2为枯草芽孢杆菌(Bacillus subtilis)的一株菌株。其主要特性为:菌体杆状,内生芽孢、芽孢卵圆形,无伴孢晶体,具鞭毛,革兰氏染色阳性;在LB平板培养基上菌落呈淡黄色、无规则、突起、边缘不整齐、表面粗糙有褶皱、不透明、黏性;兼性厌氧,接触酶反应、V-P(Vogos-Proskuer)测定为阳性、能利用柠檬酸盐生长。PCR扩增获得菌株A2的16S rDNA序列全长为1437b(pGenBank序列登录号为EU567068)。利用BLAST软件将测得的基因序列与GenBank数据库进行序列比对分析,利用DNAMAN软件进行同源性比较和进化树绘制。结果表明菌株A2与B.subtilis(AY881643)序列相似性最高、遗传距离最小,分别为99.7%和0.003,并且在系统进化树当中与枯草芽孢杆菌处于一个分支。⑷菌株A2在牛肉膏蛋白胨培养基中生长迅速,14h即达到稳定期(28℃,180rpm)。最适生长pH为7,在26-35℃条件下,生长良好,最适生长温度为28℃,较适宜的摇瓶装液比为1:10(250mL三角瓶装25mL培养液)。50mL装液量、28℃,180rpm摇瓶培养最适基础发酵培养基配方:蔗糖30g,蛋白胨10g,酵母膏5g,KH2PO4 4.5g,CaCO3 2g,(NH4)2SO4 2.5g,蒸馏水1000mL。⑸在离体条件下,对水稻稻瘟、苹果树腐烂、小麦根腐、棉花枯萎病菌菌丝生长、致畸作用等进行了研究。结果发现,菌株A2发酵上清液对4种病菌表现出了强烈的抑制作用,平均抑菌圈直径分别为30.50mm、30.00mm、29.76mm、21.20mm。其中对苹果树腐烂病菌的抑制率最高83.60%,随着发酵上清液稀释倍数的增加,抑制作用下降。但是直到7d仍具有20.00%以上的抑制率。证明其代谢产物也具有较强的抑菌效果。该菌可以致使病原菌菌丝生长形态异常,出现菌丝分支加剧、膨大、生成圆珠形囊状体和细胞质溢出而使菌丝体崩溃染色较深等现象。⑹温室小麦活体生物测定中,菌株A2的发酵液及发酵上清液均表现出较好的防治效果(29.00%以上);当菌液与上清液混合(发酵液)后其防治效果有加和作用。本文主要是研究了该菌对小麦白粉病的预防作用,发酵原液防治效果可达72.22%,发酵上清原液也有29.24%的防治效果。更多还原

【Abstract】 The dissertation describes the isolation of a Bacillus strain A2 from the soil of protected farmland, as well as the identification of the strain based on its morphological, physiological characteristics and the homological analysis of 16S rDNA sequence, the optimization of fermentation conditions, and the screening for its biological activities. The main conclusions as follow:⑴Using different media, 38 colonies were obtained by the infinite dilution on plate followed by 12 colonies with antagonistic activity screened out using the Botrytis cinerea and Magnaporthe grisea inhibition model. After 10 generations, A2 was only strain which still keeping up its inhibitory ability to phytopathogenic fungi.⑵Antagonistic Spectrum of Bacillus strain A2 was studied by means of Duel-culture method. The results showed that Bacillus strain A2 had a wider antimicrobial spectrum, it exhibited an inhibitory ability to 20 species of phytopathogenic fungi (29.00%-86.00% inhibition ratio) and its inhibition zone of Botrytis cinerea and Magnaporthe grisea achieved 17.00mm、15.83mm respectively.⑶A2 strain was identified as Bacillus subtilis based on the results of morphological, physiological characteristics and the homological analysis of 16S rDNA sequence. The phylogenetic tree built on the 16S rDNA sequence indicated A2 strain was the nearest to B.subtilis. The study of morphology, physiological and biochemical characteristics showed it can produce amylase, fluidity glutin, and showed the positive reaction in the litmus milk test, suggesting A2 strain had the common characteristics of Bacillus subtilis.⑷Fermentation conditions were reasonably optimized in the research, including fermentation temperature, fermentation time, inoculation dose, the makeup and pH of culture medium and so on. The strain grows well in much wider temperature and pH ranges, with 28℃and 7as the optimum. The optional ratio in liquid fermentation is 3:1 with cane sugar as carbon source and beef peptone as nitrogen source.⑸A2 strain showed strong inhibitory activity against the hypha growth and spore germination of Magnaporthe grisea、Valsa mali、Pythium graminicola and Fusarium oxysporum f.sp.vasinfectum. A series of diluted culture supernatant of A2 strain had been tested and the results showed that the hypha can hardly grow,and spore germination was completely suppressed. The growth of hypha was deformed by A2 strain. With their tips bulged or attenuated, the hypha grew twisty, ramulose, yielded a large number of round cystids and their protoplasm agglomerated.⑹In pot plant experiments, A2 strain and its culture supernatant showed significant control efficacies (over 29.00 % ) against Blumeria graminis f. sp. tritici, and the control efficacy of admixture of them was much better. The control efficacy of fermentation broth of A2 strain achieved 72.22%, when the control efficacy of culture supernatant of A2 strain was 29.24%.更多还原