水稻内生细菌的分离及促生功能研究和几株新菌的多相分类

发布时间：2018-04-05 12:15

本文选题：不同绿肥轮作　切入点：水稻种子　出处：《东北农业大学》2015年硕士论文

【摘要】：本研究以湖南祁阳农田系统国家野外科学研究站长期稻-稻-紫云英、稻-稻-油菜、稻-稻-黑麦草、稻-稻-冬闲四种绿肥轮作下的水稻种子、根为材料,采用可培养方法对水稻种子、根中的内生细菌进行分离,研究微生物的多样性,并筛选出具有促生功能的菌株。对分离出的潜在新菌进行多相分类。通过稀释涂布平板法对不同绿肥轮作下的水稻种子、根内生细菌进行分离、纯化,然后利用细菌通用引物,对纯化后的菌株进行基于16S rDNA基因序列的PCR扩增。结果水稻种子中共获得246株内生细菌,其中稻-稻-冬闲、稻-稻-紫云英、稻-稻-油菜、稻-稻-黑麦草轮作下分离出的内生细菌分别为42、102、58、44株,它们分别属于23个属中的39个种,其中以假单胞菌(Pseudomonas)数目最多,属于优势菌;水稻根中共获得360株内生细菌,其中稻-稻-冬闲、稻-稻-紫云英、稻-稻-油菜、稻-稻-黑麦草轮作下分离出的内生细菌分别为55、173、72、60株,它们分别属于31个属中的50个种,其中以草螺旋属(Herbaspirillum)数目最多,属于优势菌。用大米汤、阿须贝、TSA和1/4R2A四种不同的培养基对水稻种子内生细菌进行分离,结果表明稻-稻-冬闲轮作下内生细菌的数量为大米汤阿须贝TSA1/4R2A,稻-稻-紫云英轮作下为1/4R2ATSA阿须贝大米汤,稻-稻-油菜轮作下为1/4R2A阿须贝大米汤TSA,稻-稻-黑麦草轮作下为1/4R2A大米汤阿须贝TSA。通过定性或定量的研究方法对挑选出的179株优势种进行促生功能初筛,共筛选出具有溶解无机磷活性的有80株,具有分泌铁载体能力的有64株,具有分泌IAA功能的有93株。对40株初筛结果较好的菌株进行复筛,结果显示,具有抗串珠镰刀菌(Fusarium moniliforme)的有28株,固氮功能的有5株。对17株经初筛和复筛结果中促生功能较好的菌株进行大试管促生能力评价,结果表明,17株经过促生菌株处理的水稻株高、鲜重和干重都高于对照组,经过SPSS软件对其差异性分析,有4株水稻的株高、鲜重和干重和对照组有显著的差异。并对这些促生功能明显的菌株进行下一步的田间试验。对从稻-稻-紫云英轮作下的水稻根内、水稻根际土中和松嫩平原盐碱地土壤中分离出的四株潜在新种ZYY112,1ZS3-5,NEAU-ST5-5和NEAU-ST5-21,从表型特征、化学分类特征和基因型特征方面进行多项分类鉴定。其中包括形态学特征,生理学特征,生化特征,营养要求,脂肪酸分析,呼吸醌分析,极性脂分析,16S rDNA和持家基因序列的系统发育分析,基因组DNA G+C mol%,DNA-DNA杂交同源性等。结果表明菌株ZYY112属于Novosphingobium属,菌株1ZS3-5属于Paenibacillus属,NEAU-ST5-5和NEAU-ST5-21均属于Pseudomonas属。并且它们与其各自属中最近缘种存在着显著的差异,最后确定它们为各自属中的新成员。
[Abstract]:In this study, the rice seeds and roots of four green manure rotation crops, rice, rice-rice, ryegrass, rice-rice-rice-rape, rice-rice-rice, ryegrass, rice-rice-rice-winter leisure, were used as materials.The endophytic bacteria in rice seeds and roots were isolated by culturable methods, the diversity of microbes was studied, and the strains with growth-promoting function were screened out.Polyphasic classification of potential new bacteria was carried out.Rice seed and root endophytic bacteria under different green manure rotation were isolated and purified by dilution and coating plate method. The purified strains were amplified by PCR based on 16s rDNA gene sequence using universal primers.Results A total of 246 endophytic bacteria were obtained from rice seeds, of which 42102 and 5844 endophytic bacteria were isolated from rice, rice and winter, rice / rice, rice / rape, rice / ryegrass and ryegrass, respectively.They belong to 39 species of 23 genera, among which Pseudomonas is the most abundant and dominant, and 360 endophytic bacteria were obtained from rice roots, including rice-rice-winter idle, rice-rice-purple, rice-rice-rice-rapeseed.The number of endophytic bacteria isolated under rice-rice-ryegrass rotation was 55173 and 722 respectively. They belong to 50 species of 31 genera, among which Herbaspirillum is the most abundant and dominant.Endophytic bacteria were isolated from rice seeds in four different media, rice soup, TSA and 1/4R2A.The results showed that the number of endophytic bacteria in rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice, rice,Under rice-rice-rapeseed rotation, 1/4R2A Asube rice soup was used as TSA, and rice-rice-ryegrass rotation was used as 1/4R2A rice soup.By qualitative or quantitative methods, we screened 179 dominant species for promoting growth, 80 of them had dissolved inorganic phosphorus activity, 64 of them had the ability to secrete iron, and 93 of them had the function of secreting IAA.The results showed that 28 strains had resistance to Fusarium moniliforme and 5 strains had nitrogen-fixing function.The growth promoting ability of 17 strains were evaluated in vitro. The results showed that the plant height, fresh weight and dry weight of 17 strains treated with the strain were higher than that of the control group.The difference of plant height, fresh weight and dry weight of 4 rice plants was significantly different from that of the control group by SPSS software.The next field experiment was carried out on these strains with obvious growth promoting function.These include morphological, physiological, biochemical, nutritional requirements, fatty acid analysis, respiratory quinone analysis, phylogenetic analysis of 16s rDNA and housekeeping gene sequences, homology of genomic DNA G C mol-DNA hybridization, and so on.The results showed that ZYY112 belongs to Novosphingobium genus, 1ZS3-5 belongs to Paenibacillus genus NEAU-ST5-5 and NEAU-ST5-21 belongs to Pseudomonas genus.And there are significant differences between them and the most recent marginal species in their respective genera. Finally, they are identified as new members of their genera.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S511;S182

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