本文選題：不同綠肥輪作　切入點：水稻種子　出處：《東北農業(yè)大學》2015年碩士論文

【摘要】：本研究以湖南祁陽農田系統(tǒng)國家野外科學研究站長期稻-稻-紫云英、稻-稻-油菜、稻-稻-黑麥草、稻-稻-冬閑四種綠肥輪作下的水稻種子、根為材料,采用可培養(yǎng)方法對水稻種子、根中的內生細菌進行分離,研究微生物的多樣性,并篩選出具有促生功能的菌株。對分離出的潛在新菌進行多相分類。通過稀釋涂布平板法對不同綠肥輪作下的水稻種子、根內生細菌進行分離、純化,然后利用細菌通用引物,對純化后的菌株進行基于16S rDNA基因序列的PCR擴增。結果水稻種子中共獲得246株內生細菌,其中稻-稻-冬閑、稻-稻-紫云英、稻-稻-油菜、稻-稻-黑麥草輪作下分離出的內生細菌分別為42、102、58、44株,它們分別屬于23個屬中的39個種,其中以假單胞菌(Pseudomonas)數目最多,屬于優(yōu)勢菌;水稻根中共獲得360株內生細菌,其中稻-稻-冬閑、稻-稻-紫云英、稻-稻-油菜、稻-稻-黑麥草輪作下分離出的內生細菌分別為55、173、72、60株,它們分別屬于31個屬中的50個種,其中以草螺旋屬(Herbaspirillum)數目最多,屬于優(yōu)勢菌。用大米湯、阿須貝、TSA和1/4R2A四種不同的培養(yǎng)基對水稻種子內生細菌進行分離,結果表明稻-稻-冬閑輪作下內生細菌的數量為大米湯阿須貝TSA1/4R2A,稻-稻-紫云英輪作下為1/4R2ATSA阿須貝大米湯,稻-稻-油菜輪作下為1/4R2A阿須貝大米湯TSA,稻-稻-黑麥草輪作下為1/4R2A大米湯阿須貝TSA。通過定性或定量的研究方法對挑選出的179株優(yōu)勢種進行促生功能初篩,共篩選出具有溶解無機磷活性的有80株,具有分泌鐵載體能力的有64株,具有分泌IAA功能的有93株。對40株初篩結果較好的菌株進行復篩,結果顯示,具有抗串珠鐮刀菌(Fusarium moniliforme)的有28株,固氮功能的有5株。對17株經初篩和復篩結果中促生功能較好的菌株進行大試管促生能力評價,結果表明,17株經過促生菌株處理的水稻株高、鮮重和干重都高于對照組,經過SPSS軟件對其差異性分析,有4株水稻的株高、鮮重和干重和對照組有顯著的差異。并對這些促生功能明顯的菌株進行下一步的田間試驗。對從稻-稻-紫云英輪作下的水稻根內、水稻根際土中和松嫩平原鹽堿地土壤中分離出的四株潛在新種ZYY112,1ZS3-5,NEAU-ST5-5和NEAU-ST5-21,從表型特征、化學分類特征和基因型特征方面進行多項分類鑒定。其中包括形態(tài)學特征,生理學特征,生化特征,營養(yǎng)要求,脂肪酸分析,呼吸醌分析,極性脂分析,16S rDNA和持家基因序列的系統(tǒng)發(fā)育分析,基因組DNA G+C mol%,DNA-DNA雜交同源性等。結果表明菌株ZYY112屬于Novosphingobium屬,菌株1ZS3-5屬于Paenibacillus屬,NEAU-ST5-5和NEAU-ST5-21均屬于Pseudomonas屬。并且它們與其各自屬中最近緣種存在著顯著的差異,最后確定它們?yōu)楦髯詫僦械男鲁蓡T。
[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.
【學位授予單位】：東北農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S511;S182

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