连作田大豆胞囊线虫自然衰退土壤微生物群落结构多样性和作用

Soybean cyst nematode (SCN) is one of the most serious soil-borne diseases in the world, which is difficult to be controlled and reduce soybean production. The severity of SCN is usually increased in a soybean field with continuous cropping. The phenomenon of natural population decline of SCN appeare after long-term contious cropping has been reported in the past decades. However, the study of such phenomenon were performed by using some conventional and simple methods, e.g. the investigation and isolation of microbial parasites of SCN, which limiting the understanding of the natural population decline of SCN. To discuss the composition and function of natural population decline factor of SCN, a 26-years continuous cropping field with typical natural population decline of SCN are chosen in this study to reveal the relationship between long-term continuous cropping of soybean and formation of decline factors. The contribution of bacterial and fungal community, as the biotic factor, on the natural population decline of SCN are determined in a lab-scale experiment by using substrate-induced respiration inhibition method. The bacterial and fungal community size and composition in decline and non-decline soil of the soybean field are analyzed and compared by using real-time quantitative PCR and Miseq high-throughput sequencing. In contast, the community composition and diversity of dominant bacterila and fungal parasites in cysts are determined by using DGGE and clone sequencing. Finally, the suppression of parasitic microorganism on SCN is comfirmed in a greenhouse experiment.There will be an important academic value and practical significance in this study. The result will contribute to reveal the mechanism of decline soil of SCN, and enrich the theory of nematode ecology and SCN controlling with their natural enemy.

大豆胞囊线虫病是一种极难防治的世界性土传病害，对大豆产量影响很大，大豆连作使该病危害加重。近年学者们相继报道大豆经过一段时间连作后会出现“大豆胞囊线虫自然衰退”现象，但以往研究多局限于大豆生产田的线虫寄生菌调查、分离和生防效果研究。为明确引起大豆胞囊线虫自然衰退的因子及组成和作用，揭示连作大豆与胞囊线虫病衰退因子形成的关系，本研究拟以26年以上连作大豆田土壤为对象，通过模拟试验确定引起线虫衰退的生物因子种类；应用实时荧光定量PCR和Miseq高通量测序技术，分析大豆胞囊线虫衰退和非衰退土壤中细菌、真菌群落丰度差异和结构多样性；采用变性梯度凝胶电泳DGGE和克隆测序技术，分析线虫胞囊优势寄生菌组成及多样性；实验室条件下测定对大豆胞囊线虫寄生能力，盆栽试验明确引起线虫衰退优势菌种类及作用。该结果对揭示大豆胞囊线虫病自然衰退机制、丰富线虫生态学理论和利用天敌控制大豆胞囊线虫具有重要的学术价值。

大豆胞囊线虫病是一种极难防治的世界性土传病害，对大豆产量影响很大，大豆连作使该病危害加重。近年学者们相继报道大豆经过一段时间连作后会出现“大豆胞囊线虫自然衰退”现象，为明确引起大豆胞囊线虫自然衰退的因子及组成和作用，揭示连作大豆与胞囊线虫病衰退因子形成的关系。本项目开展了以下研究：1、大豆胞囊线虫衰退土壤中微生物抑制因子的作用；2、大豆胞囊线虫衰退土壤中微生物群落特征解析；3、大豆胞囊线虫优势寄生微生物种群的组成和多样性分析；4、大豆胞囊线虫衰退因子对胞囊线虫的作用。.通过模拟试验明确抑制性土壤中对大豆胞囊线虫的抑制作用是生物因素，且真菌为比细菌对大豆胞囊线虫的抑制作用强。提出了不同轮作系统土壤中大豆胞囊线虫自然衰退因子种群结构特征。明确了大豆胞囊线虫衰退土壤可以改变大豆根际土壤微生物，尤其是真菌的群落组成，使其演替成了不同于短期连作的微生物群落结构。通过高通量测序检测，进行长期和短期连作种植方式下的大豆根际土壤微生物群落结构特征的初步解析，构建了大豆根际土壤真菌ITS序列和细菌16S rRNA基因的高通量测序文库。明确了长期连作大豆田根际土壤真菌和细菌群落的物种丰富度和多样性与长期轮作方式相似，且均显著高于短期3年连作方式。该结果也暗示了随着连作年限的增加，大豆根际土壤微生物群落结构发生了明显的演替变化。研究确定了引起大豆胞囊线虫衰退的优势真菌主要包括厚垣轮枝菌（Verticillium chlamydosporia）、淡紫拟青霉（Paecilomyces lilacinus）和镰孢菌（Fusarium spp.）等常见大豆胞囊线虫优势寄生真菌，此推断这3个属真菌是东北黑土区大豆胞囊线虫抑制性土壤中重要因子。这些寄生真菌对大豆胞囊线虫自然衰退的作用方式是抑制胞囊和卵孵化，使大豆胞囊线虫二龄幼虫致死。其中厚垣轮枝菌作用更为突出。接种寄生真菌和浇灌真菌发酵液可有效地控制土壤中大豆胞囊线虫。该结果对揭示大豆胞囊线虫病自然衰退机制、丰富线虫生态学理论和利用天敌控制大豆胞囊线虫具有重要的学术价值和实践意义。