连作黄瓜根际枯萎病病原菌与拮抗微生物菌群互作关系研究

The deterioration of soil microbial properties is one of the causes of soil sickness (replant disease). The elucidation of the interrelationship between rhizosphere microbial communities in continuous monocropping systems, and researches on the mechanism of soil degradation caused by continuous monocropping have become a major subject for investigations on the sustainable development of agriculture. This research aims to analyze the interrelationship between rhizosphere Fusarium wilt pathogen and antagonistic microbial communities in continuous cucumber. (1) In both pot and field experiments, the community structure, abundance and composition of cucumber rhizosphere specific microbial communities, which include cucumber Fusarium wilt pathogen (Fusarium oxysporum f.sp. cucumerinum Owen), potential antagonistic microorgasms (Bacillus, Pseudomonas and Trichoderma), and antibiotic-producing microorgasms (PRN, DAPG, PCA), are analyzed with PCR-DGGE, qPCR and clone library methods. (2) A plant-soil feedback experiment is used to validate the effects of continuous monocropping induced changes in microbial communities on cucumber and Fusarium oxysporum f.sp. cucumerinum growth. Overall, this project plans to reveal the relationship between changes in specific soil microbial communities, and cucumber growth and incidence of Fusarium wilt; and to explore the role of these changes in microbial communities in cucumber soil sickness, which can further our understanding of the soil microbiology mechanism involved in the soil sickness of cucumber. This project is expected to provide theoretic basis for the construction of healthy rhizosphere microbial community and relieving the soil sickness.

土壤生物学性状的恶化是连作障碍发生的重要原因之一，探讨连作体系中作物根际微生物菌群之间的相互关系，研究连作情况下土壤退化的机理，成为农业可持续发展研究的重大课题。本项目拟研究连作黄瓜根际枯萎病病原菌与拮抗微生物菌群的互作关系。(1) 通过盆栽和田间试验，利用PCR-DGGE、qPCR和克隆文库技术分析不同连作茬次黄瓜根际土壤中黄瓜枯萎病病原菌(尖镰孢菌黄瓜专化型)、拮抗微生物(芽孢杆菌、假单胞菌、木霉菌)、产抗生素(PRN、DAPG、PCA)微生物菌群的群落结构、丰度及组成，明确连作对黄瓜根际特异微生物菌群的影响；(2) 通过植物-土壤反馈试验研究连作诱导的土壤微生物菌群的变化对黄瓜和枯萎病病原菌生长的影响，探明土壤微生物菌群的变化与黄瓜生长、枯萎病病害发生的关系，进一步揭示黄瓜连作障碍发生的土壤微生物学机理，并为相关作物根际健康微生物区系的构建以及连作障碍的消减技术提供理论基础。

连作体系中作物长势变弱、土传病害加剧与土壤生物学性状的变化密切相关，本项目通过盆栽和田间试验研究了连作黄瓜根际枯萎病病原菌与拮抗微生物菌群的互作关系。盆栽条件下，黄瓜在第7茬生长状况明显变差，在第9茬又明显好转；田间条件下，黄瓜从第7茬开始生长状况明显变差、枯萎病发病率增加，在第13、15茬黄瓜生长状况变好、枯萎病发病率降低。高通量测序及PCR-DGGE分析表明盆栽和田间条件下黄瓜连作改变了黄瓜根际总细菌、总真菌、假单胞菌和芽孢杆菌群落结构，盆栽条件下黄瓜连作也改变了木霉菌群落结构；盆栽条件下在第7茬、田间条件下的第13茬的细菌、真菌、芽孢杆菌、假单胞菌群落结构与其它茬次存在差异。盆栽条件下，细菌和真菌α多样性指数在第7茬最低。黄瓜生长较差的茬次(盆栽第7茬、田间第9茬)具有较高丰度的枯萎病病原菌FOC、较低丰度的产DAPG菌。盆栽条件下，黄瓜连作改变了芽孢杆菌、假单胞菌、木霉菌群落组成；田间条件下，黄瓜连作改变了芽孢杆菌群落组成。植物-土壤反馈试验表明，黄瓜生长较差茬次的土壤微生物抑制了黄瓜幼苗生长、促进了FOC的繁殖，长期连作后黄瓜长势好转茬次的土壤微生物对黄瓜幼苗和FOC则具有相反的作用。总之，黄瓜连作改变了土壤微生物群落结构、组成和丰度，连作诱导的土壤微生物菌群的变化对黄瓜和枯萎病病原菌生长产生了反馈作用，土壤微生物菌群的变化是黄瓜连作障碍发生的重要因子。研究进一步揭示了黄瓜连作障碍发生的土壤微生物学机理，为促进设施黄瓜的优质高效生产和设施土壤的可持续利用提供了理论依据。

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