【作者】 刘妍；

【导师】 沈其荣；

【作者基本信息】 南京农业大学 ， 植物营养学， 2022， 博士

【摘要】 农业生产中以提高农产品产量为目的的化肥、农药等化学产品的长期大量投入,给人畜健康和生态环境等带来了一系列的问题。以环境友好的植物根际促生菌(Plant-growth promoting rhizobacteria,PGPR)为核心的生物肥料,可以缓解生态压力,减少化学品投入,促进农业的绿色发展。目前关于PGPR自身在根际促进植物生长和健康的机制研究较为透彻,但PGPR如何影响根际微生物并改善植物生长的研究还鲜有报道。研究根际微生物的生态学行为有助于明晰生物肥料中功能菌株进入根际后的生态过程,探索合成菌群构建的理论与方法对于合成菌群生物肥料的研发有重要意义。芽孢杆菌(Bacillus spp.)因其根际生物活性高,能形成芽孢利于贮存,常被应用于农业生产和生态系统。本研究以一株根际促生模式菌株—贝莱斯芽孢杆菌(Bacillus velezensis)SQR9为研究对象(菌株SQR9分离自黄瓜种植发病区的健康植株根际,在盆栽和大田试验中表现出卓越的植物促生和生防效果),研究其以接种剂的形式进入黄瓜根际后,对根际土著细菌群落(芽孢杆菌属内)产生的生态影响,解析了造成此影响背后的芽孢杆菌亲缘识别行为的分子机制,并综合以上研究结果提出了植物促生芽孢杆菌菌群构建的新模式。主要研究成果如下:1.芽孢杆菌管家基因引物的设计与评估微生物生态学研究中,16S rRNA基因在属水平及其以下分类层级的分辨率非常低。与16S rRNA基因相比,管家基因gyrA的高通量扩增子进化速度更快、多样性更高,且多数为单拷贝,比16S rRNA基因更能准确地描述细菌群落中属内物种的多样性。上述两个基因在20个芽孢杆菌物种(Bacillus spp.)中的种间核苷酸多样性和在解淀粉芽孢杆菌(Bacillus amyloliquefaciens)、地衣芽孢杆菌(Bacillus licheniformis)、短小芽孢杆菌(Bacillus pumilus)三个物种中的种内核苷酸多样性的分析结果证明,gyrA基因在芽孢杆菌种间和种内的变异程度均显著高于16S rRNA基因。针对芽孢杆菌管家基因gyrA,设计了一对可以用于Illumina二代高通量测序的引物─gyrA3,并通过传统菌株PCR扩增和计算机模拟PCR分析结合的方法,评估了引物gyrA3对不同芽孢杆菌gyrA基因的扩增能力,结果显示gyrA3可以潜在地检测76个芽孢杆菌物种和16个芽孢杆菌近源属物种。此外,基于解淀粉芽孢杆菌、短小芽孢杆菌、巨大芽孢杆菌(Bacillus megaterium)和炭疽芽孢杆菌(Bacillus anthracis)四个物种中16S rRNA基因和gyrA基因高通量扩增子测序区(16S rRNA基因V3-V4和gyrA3扩增区)的寡核苷酸多态性(Single nucleotide polymorphisms,SNPs)的分析和对比,发现gyrA3扩增区的SNPs频率始终远高于16S rRNA基因的V3-V4区,证明gyrA3扩增区在某些芽孢杆菌亚种鉴定和分型上具有应用优势。最后,基于引物gyrA3在人工模拟群落(包含8株菌)上的高通量测序结果,证明其在芽孢杆菌群落的扩增子测序方面同样具有较大的应用潜力。2.接种植物益生菌SQR9对根际细菌群落(重点关注芽孢杆菌属)的影响基于16S rRNA和gyrA两基因的高通量测序结果,分别表征根际有益菌SQR9接种到黄瓜根际后根际细菌和芽孢杆菌群落的多样性。结果显示,接种菌株SQR9处理的香农指数显著低于未接种处理,表明菌株SQR9的引入降低了细菌和芽孢杆菌群落的α多样性。非度量多维尺度分析显示,接种和未接种菌株SQR9处理的细菌群落差异不显著(基于16S rRNA基因测序结果),但基于gyrA基因测序结果表征的芽孢杆菌群落差异显著,表明利用gyrA扩增子测序结果能更精细区分芽孢杆菌群落差异。接种菌株SQR9可以改变根际土著细菌群落的组成,而且这些变化在较低的分类水平上展现得更显著。共现性网络分析结果显示,接种菌株SQR9的处理与未接种相比,网络总节点数减少,总边数增加,平均度、平均聚类系数、模块化程度和网络密度等都得到了不同程度地增加,与菌株SQR9亲缘关系极近的物种(系统发育距离为98%～100%)和亲缘关系较近的物种(70%～80%)数量增加,而亲缘关系远的物种(50%～60%)数量减少,表明菌株SQR9的接种改变了根际芽孢杆菌群落的组成和结构,使与菌株SQR9系统发育距离相近菌株的比例得到提升,菌株间关联性增强。进一步分离了上述两处理(接种和未接种菌株SQR9处理)根际土壤的芽孢杆菌,分析了分离菌株群游对峙表型,发现接种菌株SQR9处理的根际分离株的群游对峙边界表型比例比未接种处理降低了34.5%、融合和半融合表型的比例分别提高了29.2%和5.3%,潜在提高了根际土著芽孢杆菌的菌群兼容性和合作程度。基于分离菌株的系统发育关系和群游对峙表型联合分析结果,证明根际芽孢杆菌群落中成员菌株的亲缘关系变化与群游对峙表型反应一致,推测这种亲缘菌株之间的相互作用的改变受到亲缘识别机制的调控。3.贝莱斯芽孢杆菌SQR9亲缘识别位点研究利用菌株SQR9的突变体文库结合群游对峙实验研究了菌株SQR9根际亲缘识别的分子机制。突变体文库中包含以下突变体:拮抗物质相关突变体(Δwap I、Δbmy、Δfen、Δdhb、Δsrf、Δbae、Δdfn、Δmln、Δsfp和ΔGI),细胞表面分子相关突变体(Δdlt A、Δlyt C和Δtua D),细胞免疫反应调控相关突变体(Δyvr H、Δlyt ST和Δsig W),群体感应和生物膜相关突变体(Δlux S和Δeps D),以及细胞鞭毛相关突变体(Δflh O和Δhag)等。突变体与野生型SQR9及SQR9的kin(亲缘)菌株的对峙表型为边界,说明该基因参与了菌株SQR9与kin菌株的亲缘识别过程;突变体与野生型SQR9的non-kin(非亲缘)菌株的群游对峙边界减弱或消失,则说明该基因参与了菌株SQR9与non-kin菌株的亲缘识别过程。研究结果表明,抗生素类物质合成相关基因(sfp和基因岛GI3)、细胞表面分子相关基因(dlt A、lyt C、tua D)、调控免疫反应的双组份调控系统相关基因(yvr H和lyt ST)和鞭毛相关基因(flh O和hag)等都参与了贝莱斯芽孢杆菌SQR9的亲缘识别,称为菌株SQR9的亲缘识别位点,推测这些基因参与了菌株SQR9亲缘识别的不同过程。4.鞭毛丝蛋白参与贝莱斯芽孢杆菌亲缘识别的机制研究菌株SQR9中hag基因(鞭毛丝蛋白合成基因)的缺失会导致细胞鞭毛胞外细丝部分无法正常组装,菌株SQR9缺失鞭毛丝的突变体丧失了其在半固体表面上的群游能力,且与野生型SQR9的群游对峙表型由融合表型变成了边界表型。通过对菌株SQR9的鞭毛丝进行改装,发现回补了亲缘菌株鞭毛丝蛋白的菌株SQR9与野生型SQR9在群游对峙时形成融合表型,回补了非亲缘菌株鞭毛丝蛋白的菌株SQR9与野生型SQR9在群游对峙时形成边界表型。通过对野生型SQR9、鞭毛突变体和回补菌株抗生素类物质(surfactin或bacillunoic acid)的产量测定,证明鞭毛的缺失和回补并不会影响次级代谢产物surfactin和bacillunoic acid的分泌。以上结果暗示了鞭毛影响菌株SQR9亲缘识别的主要原因是在于鞭毛本身,而非抗生素类物质的分泌改变。研究结果表明鞭毛丝蛋白介导了菌株SQR9亲缘识别过程中的初级识别。此外,综合分析了贝莱斯芽孢杆菌种内鞭毛丝蛋白序列,发现鞭毛丝蛋白中央可变区变异度较高,除鞭毛帽外的其它鞭毛结构蛋白种内变异度较低,推测鞭毛结构中的鞭毛帽蛋白有可能参与菌株SQR9亲缘识别过程中的初级识别。5.基于菌株SQR9对根际芽孢杆菌生态影响的促生型合成菌群综合构建模式上述研究结果表明,黄瓜根际接种植物益生菌SQR9后,根际土著芽孢杆菌群落多样性发生改变,土著芽孢杆菌菌群内部菌株的亲缘度和兼容性得到提升,从植物促生效应推测菌株SQR9带来的生态影响潜在提高了菌群的植物促生能力。菌株碳源利用测试结果显示,菌株的碳源利用模式与其亲缘关系密切相关,亲缘关系越近的菌株碳源利用模式也越相似。同时考虑菌株碳源利用和自身促生能力,通过学习菌株SQR9影响下形成的根际促生芽孢杆菌菌群,人工构建了三类亲缘度(高、中、低亲缘度)不同丰富度(包含1、2、3、4、8株菌)的菌群,利用水培、土培两种体系测试两类菌群对黄瓜植株的促生效果。研究发现低亲缘度菌群的促生效果优于高、中亲缘度菌群,且会随着菌群丰富度的增加而增强。综上所述,本研究设计并评估了用于芽孢杆菌生态研究的高通量测序引物gyrA3,发现外源接种菌株SQR9可以改变根际芽孢杆菌群落的组成和结构,使芽孢杆菌及其相关群落向低拮抗、高融合的合作趋势转变;同时鉴定了菌株SQR9的亲缘识别位点,证明鞭毛丝蛋白参与菌株SQR9的亲缘识别,并为芽孢杆菌群落的智能组装提供了一个通用的生态框架。更多还原

【Abstract】 The long-term and massive investment of chemical products such as fertilizers and pesticides in agricultural production to increase agricultural product yield has brought problems to human and animal health and the ecological environment.Bio-fertilizers with environmentally friendly plant growth-promoting rhizobacteria(PGPR)as the core can relieve ecological pressure,reduce chemical input and promote the green development of agriculture.At present,the mechanism of PGPR itself promotes plant growth and health in the rhizosphere is relatively well known,but there are few reports on how PGPR affects the rhizosphere microorganisms and improves the growth of plants.Studying the ecological behavior of microorganisms in the rhizosphere helps clarify the ecological process after the functional strains contained in biofertilizers enter the rhizosphere.It is of great significance to explore the theory and method of synthetic community construction for researching and developing synthetic community biofertilizers.Bacillus spp.is often used in agricultural production and ecosystems because of its high rhizosphere biological activity and the ability to form spores for storage.In this study,a rhizosphere growth-promoting model bacteria,Bacillus velezensis SQR9(strain SQR9 was isolated from the roots of healthy plants in the diseased area of cucumber cultivation and showed outstanding plant growth-promoting and biocontrol effects in pot and field trials),was used to study the effect of the rhizosphere indigenous bacterial community(within the Bacillus genus),and the molecular mechanism of Bacillus kin discrimination behavior behind this effect was analyzed.Combined with the results,a new model of plant growth-promoting Bacillus community construction was proposed.The main results are as follows:1.Development of house-keeping gene primers for BacillusIn microbial ecology research,the resolution of the 16S rRNA gene at the genus level and below the taxonomic level is very low.The genes sequenced in high-throughput amplicon from another housekeeping gene gyrA have a faster evolution rate and higher diversity than those from 16S rRNA gene,and most of them are single copies,which can describe the diversity of species within the genus in bacterial communities more accurately than 16S rRNA gene.The analysis results of the interspecific nucleotide diversity of the above two genes in 20 Bacillus species and the intraspecific nucleotide diversity in three species of B.amyloliquefaciens,B.licheniformis,and B.pumilus proved that the inter-species and intra-species variability of gyrA gene in Bacillus was significantly higher than that of 16S rRNA gene.For the housekeeping gene gyrA of Bacillus,we designed a primer pair,gyrA3,that could be combined with the Illumina second-generation high-throughput sequencing,and evaluated the primer pair gyrA3 by combining traditional strain PCR amplification and computer simulation PCR analysis to evaluate amplification capacity of the primer pair gyrA3 pair of different Bacillus gyrA genes.The results of the ability of primer pair gyrA3 to detect the gyrA gene of Bacillus showed that gyrA3 could potentially detect 76 Bacillus species and 16 Bacillus-related species.In addition,the single nucleotide polymorphisms(SNPs)analysis of the high-throughput amplicon sequencing regions of 16S rRNA and gyrA genes(16S rRNA V3-V4 and the gyrA3 amplification region)showed that the SNPs frequency of the gyrA3 amplification region was always much higher than that of the 16S rRNA V3-V4 region,which proved the application advantages of the gyrA3 amplification region in the identification and typing of certain Bacillus subspecies.Finally,the high-throughput sequencing results of the primer pair gyrA3 on the synthetic microbial community(containing 8 strains)proved that the primer pair gyrA3 had great potential for sequencing the amplicon of Bacillus colonies.2.Effects of plant probiotic strain SQR9 inoculation on bacterial community(focus on Bacillus genus)in the rhizosphereBased on the high-throughput sequencing results of 16S rRNA and gyrA genes,the diversity of rhizosphere bacterial and Bacillus communities was characterized after inoculating the beneficial strain SQR9 into the cucumber rhizosphere.The results showed that the Shannon index of the SQR9-inoculated treatment was significantly lower than that of the uninoculated treatment,indicating that the inoculation of strain SQR9 reduced the alpha diversity of bacterial and Bacillus communities.Non-metric multidimensional scaling analysis showed that bacterial communities of SQR9-inoculated treatment and uninoculated treatment were not significantly different(16S rRNA-based sequencing results),but gyrA-based sequencing results showed a significant difference in Bacillus communities,indicating that the gyrA amplicon sequencing results can more finely distinguish the Bacillus community differences.inoculation with strain SQR9 can alter the composition of indigenous bacterial communities in the rhizosphere,and these changes were manifested at lower taxonomic levels more significantly.The results of co-occurrence network analysis showed that the total number of nodes in the network decreased;the total number of edges increased;and the average degree,average clustering coefficient,modularity,and network density were all improved to different degrees in the treatment of inoculation with strain SQR9 compared with the treatment without inoculation.The number of very closely related species of strain SQR9(phylogenetic distance:98%～100%)and closely related species of strain SQR9(70%～80%)increased,and the number of very distantly related species(50%～60%)decreased,indicating that the inoculation of strain SQR9 changed the composition and structure of the Bacillus rhizosphere community,increased the proportion of strains with similar phylogenetic distances to strain SQR9,and enhanced the correlation between strains.Bacillus-related strains from the rhizosphere soil of the above two treatments(inoculated and uninoculated with strain SQR9)were further isolated,and the swarm boundary assay phenotype of the isolated strains was analysed.It was found that rhizosphere isolates treated with inoculated strain SQR9 had a 34.5%reduction in the proportion of boundary phenotype and a 29.2%and 5.3%increase in the proportion of merge and intermediate phenotypes,respectively,compared with the results of the uninoculated treatment,potentially increasing the compatibility and degree of cooperation in indigenous Bacillus community in the rhizosphere.Joint analysis of phylogenetic relationships and swarm boundary phenotype of strains proved changes in the relationships of member strains in a Bacillus rhizosphere community were consistent with swarm boundary phenotype.We speculate that this altered interaction between relative strains is regulated by a kin discrimination mechanism.3.Research of kin discrimination gene site of the B.velezensis SQR9We used mutant libraries of strain SQR9 combined with a swarm boundary assay to research further study the molecular mechanism of strain SQR9 kin discrimination in the rhizosphere.The library of mutants contains the following mutants:antagonist-associated mutants(Δwap I,Δbmy,Δfen,Δdhb,Δsrf,Δbae,Δdfn,Δmln,Δsfp,andΔGI),cell surface molecule-associated mutants(Δdlt A,Δlyt C,andΔtua D),mutants associated with regulation of cellular immune responses(Δyvr H,Δlyt ST,andΔsig W),quorum sensing and biofilm-associated mutants(Δlux S andΔeps D),and cell flagella-associated mutants(Δflh O andΔhag),etc.If the swarm boundary phenotype between the mutant and wild-type SQR9(and kin strains of wild-type SQR9)is the boundary,it indicates that the gene is involved in the kin discrimination of strain SQR9 and kin strains;Attenuated or disappeared off the boundary,indicated that the gene is involved in the kin discrimination process of strain SQR9 and non-kin strains.The results showed the following genes were involved in the kin discrimination of B.velezensis SQR9:the genes related to the synthesis of antibiotics(sfp and GI3 gene island),the genes related to cell surface molecules(dlt A,lyt C,tua D),the genes related to the two-component regulatory system that regulates the immune response(yvr H and lyt ST)and the genes related to flagella(flh O and hag).they are called the kin discrimination site of strain SQR9.We propose that these genes are involved in distinct processes of strain SQR9 kin discrimination.4.The involvement of flagellin in kin recognition of B.velezensisDeletion of the hag gene(flagellin synthesis gene)in strain SQR9 resulted in the abnormal assembly of the extracellular filament part of the cell flagellar filament,and the mutant of strain SQR9 lacking flagellar filaments loses its ability to swarm on semi-solid surfaces,and the swarm boundary phenotype with wild-type SQR9 changed from the merge to a"detour"boundary.By complementing the hag genes of kin and non-kin strains,it was found that strain SQR9 with the hag genes of kin strains and wild-type SQR9 would form a merge phenotype,and strain SQR9 with the hag genes of non-kin strains and wild-type SQR9would form a boundary phenotype.By measuring the production of antibiotics(surfactin or bacillunoic acid)in wild-type SQR9,flagellar mutants,and complementary strains,it was proved that the absence and complementation of flagella did not affect the production of secondary metabolites surfactin and bacillunoic acid.The above results suggested that the main reason for the influence of flagella on kin discrimination of strain SQR9 was the flagella itself rather than the altered secretion of antibiotics.The findings suggest that flagellin mediates primary recognition during kin discrimination of strain SQR9.In addition,we comprehensively analyzed the structural evolution of flagellin proteins within B.velezensis species and found that the central variable region of the flagellin in B.velezensis species has a large degree of variation,and other flagellin proteins have low intraspecies variability,except the flagellar cap.Therefore,they are less likely to be involved in kin discrimination.In addition,combined with the analysis of the intraspecific flagellin protein sequences of Bacillus velezensis,it was found that the central variable region of the flagellin protein had a high variability.The intraspecific variability of other flagellar structural proteins except the flagellar cap was low.It is speculated that the flagellar cap protein may be involved in the primary recognition of the kin discrimination of strain SQR9.5.Comprehensive construction model of growth-promoting synthetic consortia based on the ecological impact of strain SQR9 on Bacillus community rhizosphereThe above research results showed that after the cucumber rhizosphere was inoculated with the plant probiotic strain SQR9,the diversity of the indigenous Bacillus rhizosphere community was changed,and the affinity and compatibility of strains within the community were improved.We speculate that the ecological impact of the plant growth-promoting strain SQR9 potentially enhances the plant growth-promoting ability of the indigenous Bacillus community in the rhizosphere.The results of the strains’carbon source utilization test showed that the strains’carbon source utilization patterns were closely related to their relationships,and the carbon source utilization patterns of the strains with the closer relationship were more similar.Taking into account the carbon source utilization and growth-promoting ability of the strains,and the rhizosphere-promoting Bacillus community formed under the influence of the strain SQR9,we constructed three consortia(high,moderate,and low related)with different richness(containing 1,2,3,4,and 8 strains).The growth-promoting ability of cucumber plants of bacterial consortia was tested using two systems of hydroponics and soil culture.The results showed that the growth-promoting ability of low related consortia was better than that of high and moderate related consortia,and it would be enhanced with the increase of consortia richness.In conclusion,this study designed and evaluated the high-throughput sequencing primer pairs gyrA3 for Bacillus ecological research,and by using this new method,we found that inoculation of strain SQR9 could alter the composition and structure of the Bacillus community in the rhizosphere,shifting Bacillus and its related communities toward a low-antagonism,high-integration cooperative trend.And some kin discrimination sites of strain SQR9 were identified,which proved that flagellin is involved in the kin discrimination of strain SQR9,and provided a general ecological framework for the intelligent assembly of Bacillus communities.Besides,a general ecological framework was provided for the intelligent assembly of Bacillus consortia.更多还原