【作者】 陈一夫；

【导师】 丑敏霞；

【作者基本信息】 西北农林科技大学 ， 微生物学， 2023， 硕士

【摘要】 大豆玉米的间作模式是当前应用十分广泛的农业种植体系,其中根际微生物和根系分泌物所在的根际微生态系统中发生着十分频繁的物质和信息交流。本研究以大豆、玉米为材料,对不同生育期二者单作、间作模式下根际微生物群落组成、功能及植物根系代谢组进行了研究。利用微生物组高通量测序技术,分析大豆、玉米单、间作种植模式下的根际微生物群落差异,并获得核心微生物组;同时,分别采集了不同栽培方式下的玉米、大豆的根系分泌物并加以测定,找寻根系分泌物和根际微生物菌群之间的关系;最后,以核心微生物菌群为基础,结合菌株特性测定结果,筛选出促生效果显著的益生菌株并加以优化组合,以构建合成菌群。主要结果如下:(1)播种后15 d、45 d、65 d分别对单、间作模式下的大豆、玉米取样分析,表型及生理指标检测结果表明,与单作相比,间作模式下植物普遍长势更好。特别是65d时,间作模式提高了植物的生物量,促进了大豆根瘤的形成。(2)植物及土壤营养元素含量的测定结果表明,间作模式促进了大豆、玉米对土壤营养元素的吸收利用,间作植株体内磷、钾含量相比单作总体上表现出上升的趋势;但是就氮素而言,间作大豆的氮含量相比单作呈现下降的趋势,特别是在45 d时,玉米则表现出相反的趋势,说明间作体系中玉米对土壤氮素的争夺能力强于大豆,这可能造成大豆氮素匮乏,从而促进了其共生根瘤的形成。土壤氮磷钾含量总体上随植物生长时期而下降,说明期间被植物吸收利用;与单作相比,间作土壤的硝态氮、速效钾、有效磷及有机碳含量在相同取样时期都有不同程度的降低,说明间作模式促进了大豆、玉米对土壤营养元素的吸收利用。(3)对不同种植模式下的根际土壤样品进行高通量测序分析得到24320个土壤ASV。经过微生物多样性、微生物组成分析发现不同种植模式和不同取样时间点对土壤微生物群落具有显著影响。在单作模式下,大豆和玉米的根际都显著富集Proteobacteria(变形菌门)和Acidobacteria(酸杆菌门)。同时,在45 d和65 d间作体系下的大豆玉米Chloroflexi(绿弯菌门)和Bacteroidota(拟杆菌门)显著高于单作。根际差异微生物分析结果显示,大豆在间作模式下根际富集了Sphingobacterium、Pseudoxanthomonas、UTCFX1等菌属,在单作模式下富集了Lysobacter、Sphingomonas、Pseudarthrobacter等菌属;玉米在间作模式下根际富集了Flavisolibacter、Stenotrophomonas、Variovorax,在单作模式下富集了Bryobacter、Sphingomonas、Arenimonas。根际微生物共发生网络分析结果显示,无论是大豆还是与玉米,在间作模式下植物根际微生物网络都比单作更加复杂。通过共发生网络中各节点的模块内连通度(Zi)和模块间连通度(Pi)分析,筛选出了核心微生物,分别属于放线菌门、变形菌门、拟杆菌门。(4)土壤N循环功能基因分析表明,与单作相比,间作下根际土中硝化作用相关基因丰度上调,而反硝化作用相关基因丰度大多下调,推测间作提高了土壤中亚硝酸盐向硝酸盐的转化,并可能抑制了反硝化过程。(5)通过代谢组学分析,从根系分泌物中共检测到2249种物质,主要为糖、有机酸、氨基酸类。对不同种植模式下差异代谢物注释之后,在间作模式下,大豆根系富集了Enoxolone、Ademetionine、methadone-d9等代谢物;而玉米根系富集了Acetophenone、Daidzein、Heptamethoxyflavone等代谢物。将同一取样时期下同种植物单作和间作对比所得差异代谢物和差异微生物进行关联分析后,发现在大豆中可以看到Sphingobacterium、Bacillus等属的微生物与N-Acetyl-DL-valine、Polygalic acid等多种根系代谢物具有显著的相关性。而玉米根际土壤中Flavisolibacter、Pseudorhodoferax等属的微生物与Nicotianamine、Bruceine D等多种根系代谢物具有显著的相关性。(6)从上述根际土壤中分离获得284株菌并进行了初步种属鉴定,以不同种植模式下根际微生物组分析结果为指导,并结合菌株促生性能检测,筛选得到8株促生菌,分别属于Stenotrophomonas、Ensifer、Kosakonia、Pseudomonas、Leclercia、Bacillus等属,并将其组合为两个合成菌群。将两种合成菌群在自然土壤条件下接种大豆、玉米和小麦,结果显示,两个菌群对三种作物均有不同程度的促生效果。综上所述,本研究通过对不同种植模式下大豆玉米生长指标、元素含量、根际微生物组和根系分泌物的分析,初步探究了大豆玉米间作模式提升作物生长的微生物学机制,为全面解析间作模式下的根际微生态过程提供了借鉴,也为如何提升根际微生态功能从而改善单一种植模式下的土壤健康提供了参考。更多还原

【Abstract】 The intercropping pattern of soybean and maize is a widely used agricultural cropping system,in which the rhizosphere microorganisms and root secretions are located in the rhizosphere micro-ecosystem where frequent material and information exchange is occurring.In this study,we investigated the composition and function of the rhizosphere microbial community and the metabolome of the plant root system under monoculture and intercropping patterns of soybean and maize at different fertility stages.At the same time,root secretions of corn and soybean under different cultivation methods were collected and measured to find the relationship between root secretions and the rhizosphere microflora.Finally,the probiotic strains with significant growth-promoting effects were selected and optimized to build a synthetic flora.The main results are as follows:(1)The results of phenotypic and physiological indexes of soybean and maize sampled and analyzed in monoculture and intercropping patterns at 15 d,45 d and 65 d after sowing showed that plants generally grew better in the intercropping pattern compared with monoculture.In particular,at 65 d,the intercropping pattern increased plant biomass and promoted the formation of soybean root nodules.(2)The results of plant and soil nutrient content measurements showed that the intercropping pattern promoted the uptake and utilization of soil nutrients by soybean and maize,and the phosphorus and potassium contents of intercropped plants showed an overall increasing trend compared with monocropping;however,in terms of nitrogen,the nitrogen content of intercropped soybean showed a decreasing trend compared with monocropping,especially at 45 d,while maize showed the opposite trend,indicating that the intercropping system This may have caused nitrogen deprivation in soybean,thus promoting the formation of symbiotic root nodules.Soil N,P,and K contents generally decreased with plant growth period,indicating that they were absorbed and utilized by plants during the period;compared with monoculture,nitrate N,fast-acting potassium,effective phosphorus,and organic carbon contents of intercropped soils decreased to different degrees during the same sampling period,indicating that the intercropping pattern promoted the uptake and utilization of soil nutrients by soybean and corn.(3)High-throughput sequencing analysis of rhizosphere soil samples from different cropping patterns yielded 24,320 soil ASVs.microbial diversity and microbial composition analysis revealed that different cropping patterns and different sampling time points had significant effects on soil microbial communities.In the monoculture model,both soybean and corn were significantly enriched in Proteobacteria and Acidobacteria in the inter-rhizosphere.Meanwhile,Chloroflexi and Bacteroidota were significantly higher in soybean and corn under45 d and 65 d intercropping systems than in monoculture.The results of rhizosphere differential microbial analysis showed that soybean was enriched with Sphingobacterium,Pseudoxanthomonas,and UTCFX1 genera in the intercrop mode and Lysobacter,Sphingomonas,and Pseudarthrobacter in the monocrop mode;corn was enriched with The results of the rhizosphere microbial co-occurrence network analysis showed that both soybean and maize were enriched in the intercropping mode with Flavisolibacter,Stenotrophomonas,and Variovorax,and Bryobacter,Sphingomonas,and Arenimonas in the monocropping mode.The results of the rhizosphere microbial network analysis showed that the rhizosphere microbial network was more complex than that of the monocrop in both soybean and maize.The core microorganisms were screened by intramodule connectivity(Zi)and inter-module connectivity(Pi)analysis of each node in the cogenerative network and belonged to the phyla Actinomycetes,Aspergillus,and Phytophthora,respectively.(4)The analysis of soil N cycle functional genes showed that the abundance of nitrification-related genes was up-regulated in intercropping compared with monocropping,while the abundance of denitrification-related genes was mostly down-regulated in intercropping,presumably enhancing the conversion of nitrite to nitrate in soil and possibly inhibiting the denitrification process.(5)A total of 2249 substances,mainly sugars,organic acids,and amino acids,were detected from root secretions by metabolomics analysis.After annotation of differential metabolites in different cropping patterns,soybean roots were enriched in metabolites such as Enoxolone,Ademetionine,and methadone-d9 in the intercrop pattern,while maize roots were enriched in metabolites such as Acetophenone,Daidzein,and Heptamethoxyflavone.After correlation analysis of differential metabolites and differential microorganisms obtained by comparing monocultures and intercrops of the same plant species under the same sampling period,it was found that microorganisms of genera such as Sphingobacterium and Bacillus were significantly correlated with various root metabolites such as N-Acetyl-DL-valine,Polygalic acid,etc.in soybean.And microorganisms of genera such as Flavisolibacter and Pseudorhodoferax in maize inter-rhizosphere soil were significantly correlated with various root metabolites such as Nicotianamine and Bruceine D.(6)284 strains were isolated from the above-mentioned inter-rhizosphere soil and preliminary species identification was performed.Using the results of inter-rhizosphere microbiome analysis under different cropping patterns as a guide and combined with the strain growth-promoting performance testing,eight strains of growth-promoting bacteria were screened,belonging to the genera Stenotrophomonas,Ensifer,Kosakonia,Pseudomonas,Leclercia,Bacillus and other genera,and combined them into two synthetic bacterial groups.The two synthetic colonies were inoculated with soybean,corn and wheat under natural soil conditions,and the results showed that both colonies had different degrees of growthpromoting effects on all three crops.In summary,this study provides a preliminary investigation into the microbiological mechanisms of soybean and corn intercropping patterns to enhance crop growth through the analysis of growth indicators,elemental content,rhizosphere microbiome and root secretions under different cropping patterns,and provides a reference for the comprehensive analysis of rhizosphere microecological processes under intercropping patterns,as well as how to enhance rhizosphere microecological functions to improve soil health under monoculture patterns.It also provides a reference for improving soil health under monoculture by enhancing rhizosphere microecological functions.更多还原