【作者】 李洁；

【导师】 徐凌川；

【作者基本信息】 山东中医药大学 ， 中药鉴定学， 2023， 硕士

【摘要】 目的:研究泰山白首乌根际微生物多样性和菌群分布规律,分析微生物群落组成和丰度差异,筛选具有不同生理功能的根际促生菌,探索土壤-根际微生物-植物微生态环境的相互联系与作用,为更好的创造仿野生种植条件、培育品质优良的泰山白首乌提供科学且有效的理论基础。方法:本文以不同产地(邹平、临沂、徂徕山前、徂徕山后、泰山、莱芜、历下、长清)和不同地下根茎深度(地下10 cm和地下20 cm)的泰山白首乌根际土壤为研究对象,利用高通量测序技术分析根际微生物的多样性和群落分布规律;采用传统分离鉴定法从不同样本中纯化土壤根际微生物,通过生药学形态和分子基因比对确定菌株种类;利用特定培养基对纯化得到的菌株进行功能筛选和活性测定,确定有益根际促生菌群。结果:(1)高通量测序结果表明泰山白首乌根际真菌多样性受产地因素影响大于地下根际深度的影响,根据样品曲线图和多样性指数显示根际真菌物种丰度依次为泰山、徂徕山后、徂徕山前、临沂、邹平、长清、历下、莱芜莲花山;优势菌门为子囊菌门Ascomycota、担子菌门Basidiomycota和被孢霉门Mortierellomycota。土壤理化因子显示担子菌门与p H成负相关,被孢霉门与铵态氮、速效钾成负相关,子囊菌门与有机质、铵态氮和有效成负相关;随着地下根茎深度的加深,根际真菌微生物的多样性和物种丰度增大。(2)高通量结果显示产地对泰山白首乌根际细菌的多样性和分布组成有显著影响,根茎深度在优势菌群多样性上具有相似性,但在丰度含量方面存在差异性;样品曲线图和多样性指数表明根际细菌的丰度依次为泰山、邹平、临沂、长清、莱芜、历下、徂徕山后、徂徕山前;优势菌门均为疣微菌门Verrucomicrobia和酸杆菌门Acidobacteria;土壤理化因子显示疣微菌门与p H成正相关性,酸杆菌门与有机质SOM和铵态氮AN成正相关性;随着地下根茎深度增大,根际细菌微生物的多样性和物种丰度减小。(3)传统分离鉴定法得到4门、9纲、11目、13科、16属、39种根际真菌;产地对根际真菌多样性和优势菌群分布存在显著影响,地下根茎深度在真菌属分离水平和优势菌分布上具有相似性,但优势菌群的丰度具有显著差异;可培养根际真菌的优势菌属为镰刀菌属Fusarium、木霉属Trichoderma、被孢霉属Mortierella和冠革胞菌属Lindtneria;优势菌种为深绿木霉T.atroviride、T.Irregularis、拟康宁木霉T.koningiopsis、绿木霉T.virens、L.trachyspora、尖刀镰孢菌F.osysporu、腐皮镰孢菌F.solani、M.ambigua和球詹金氏被孢霉M.cystojenkinii。(4)传统分离鉴定得到5门、8纲、10目、15科、22属、54种根际细菌,产地因素对根际细菌多样性的影响强于根际深度因素;优势菌属为类芽胞杆菌属Paenibacillus、芽孢杆菌属Bacillus、Lysinibacillus、假单胞菌属Pseudomonas和根霉菌属Rhizomicrobium,优势菌种为氮还原类芽孢杆菌P.azoreducens、多粘芽孢杆菌B.polymyxa、凝结芽孢杆菌B.coagulans、A.piechaudii、L.fusiformis、路邓葡萄球菌S.lugdunensis、荧光假单胞菌P.fluoyescens、绿脓杆菌P.aeruginosa、单孢根霉菌R.azygosporus。(5)通过特定培养基筛选得到21株固氮菌、12株解有机磷菌、18株解无机磷菌和17株解钾菌,Trichoderma真菌属和Pseudomonas、Bacillus细菌属是在四种功能筛选中同时存在的菌属。结论:通过高通量测序技术研究泰山白首乌根际微生物多样性,表明产地因素对根际微生物的影响强于地下根茎深度,不同菌群受理化因子影响的程度不同,说明产地土壤环境的差异与根际微生物的定殖有密切相关性;传统分离鉴定法只得到了部分根际微生物,结合两种方法确定根际优势真菌门为被孢霉门Mortierellomycota和子囊菌门Ascomycota,根际优势细菌门为酸杆菌门Acidobacteria、芽单胞菌门Gemmatimonadetes、疣微菌门Verrucomicrobia和变形菌门Proteobacteria;筛选得到68株根际功能促生菌,其中Trichoderma、Pseudomonas、Bacillus菌属的促生效果最显著。研究结果为分析泰山白首乌根际微生物多样性和筛选有益功能促生菌提供了基础数据,为更好的利用根际微生物资源、开发有益生物菌肥、创造优良的仿野生种植条件奠定了基础。更多还原

【Abstract】 Objectives:To study the diversity and distribution of microorganisms in the rhizosphere of Cynanchum bungei Decne,Screening rhizosphere growth-promoting bacteria with different physiological functions,In order to explore the interaction and function of soil,rhizosphere microorganisms and plant micro-ecological environment.It provides a scientific and effective theoretical basis for better creating conditions for imitating wild planting and cultivating high-quality Cynanchum bungei Decne.Methods:Taking the rhizosphere soil of Cynanchum bungei Decne as the research object,the diversity and community distribution of rhizosphere microorganisms were analyzed by high-throughput sequencing technology;The soil rhizosphere microorganisms were purified from different samples by traditional separation and identification methods,the strains were determined by pharmacognostic morphology and molecular gene comparison;The functional screening and activity determination of the purified strains were carried out using specific culture medium to determine the beneficial rhizosphere growth promoting bacteria.Results:(1)The results of high-throughput sequencing showed that the diversity of fungi in the rhizosphere of Cynanchum bungei Decne was more affected by the factors of origin than by the depth of underground rhizosphere,according to the sample curve and diversity index,the species abundance of rhizosphere fungi is in the order of TS,CLH,CLQ,LY,ZP,CQ,LX and LW;The dominant bacteria are Ascomycete,Basidiomycota and Mortierellomycota.The soil physical and chemical factors showed that Basidiomycetes had negative correlation with p H,Mortierella had negative correlation with ammonium nitrogen and available potassium,and Ascomycetes had negative correlation with organic matter,ammonium nitrogen and available potassium;With the deepening of underground rhizome depth,the diversity and species abundance of rhizosphere fungi and microorganisms increased.(2)The high-throughput results showed that the origin had a significant impact on the diversity and distribution of bacteria in the rhizosphere of Cynanchum bungei Decne,the rhizome depth is similar in the diversity of dominant flora,but different in the abundance content;The sample curve and diversity index showed that the abundance of rhizosphere bacteria was in the order of TS,ZP,LY,CQ,LW,LX,CLQ and CLH;The dominant bacteria are Verrucomicrobia and Acidobacter;Soil physical and chemical factors showed that there was a positive correlation between verruca microflora and p H,and a positive correlation between acidobacteria and organic matter and ammonium nitrogen;With the increase of underground rhizome depth,the diversity and species abundance of rhizosphere bacteria and microorganisms decreased.(3)39 species of rhizosphere fungi belonging to 4 phyla,9 classes,11 orders,13 families,16 genera were obtained by traditional isolation and identification methods;The place of origin has a significant impact on the diversity of rhizosphere fungi and the distribution of dominant bacteria.The depth of underground rhizome is similar in the level of fungi isolation and the distribution of dominant bacteria,but the abundance of dominant bacteria has a significant difference;The dominant genera of culturable rhizosphere fungi are Fusarium,Trichoderma,Mortierella and Lindtneria;The dominant strains are T.viride,T.Irregularis and T.Koningiopsis,T.virens,L.trachyspora,F.oxysporum,F.osysporum,F.solani,M.ambigua,and M.bulgensis,M.cystojenninii.(4)54 species of rhizosphere bacteria belonging to 5 phyla,8 classes,10 orders,15 families,22 genera,were isolated and identified by traditional methods;The dominant bacteria are Paenibacilli,Bacillus,Lysinibacilli,Pseudomonas and Rhizomycobium,and the dominant bacteria are P.azoleducens,B.polymyxa,B.coagulans,A.piechaudi,L.fusiformis,S.lugdensis,P.fluorescens,P.aeruginosa,R.azygosporu s.(5)21 strains of nitrogen-fixing bacteria,12 strains of organophosphorus bacteria,18 strains of inorganic phosphorus bacteria and 17 strains of potassium bacteria were screened through specific culture medium,Trichoderma fungi and Pseudomonas and Bacillus are the bacteria that exist simultaneously in the four functional screening.Conclusions:Study the microbial diversity in the rhizosphere of Cynanchum bungei Decne,the results showed that the influence of the factors of origin on rhizosphere microorganisms was stronger than that of the depth of underground rhizome,and the degree of the influence of the acceptance factors of different bacterial groups was different,indicating that the difference of soil environment in the origin was closely related to the colonization of rhizosphere microorganisms;Only some rhizosphere microorganisms were obtained by traditional isolation and identification methods,combining the two methods,the dominant rhizosphere fungi are Mortierellomycota and Ascomycota,and the dominant rhizosphere bacteria are Acidobacter,Germatimonadetes,Verrucomicrosbia and Proteobacteria;68 strains of rhizosphere functional growth-promoting bacteria were screened,among which Trichoderma,Pseudomonas and Bacillus had the most significant growth-promoting effect.The research results provide basic data for the analysis of rhizosphere microbial diversity and screening of beneficial functional growth-promoting bacteria of Cynanchum bungei Decne,and lay a foundation for better utilization of rhizosphere microbial resources,development of beneficial biological fertilizer,and creation of excellent conditions for imitating wild planting.更多还原