“健康型-连作障碍型-严重连作障碍型-抑病型”大蒜根际微生物群落结构解析及关键病原菌/拮抗菌的收集鉴定

After garlic continuous cultivation for many years, continuous cropping obstacles questions such as imbalance of soil microbial community, frequent occurrence of soil borne diseases, and so on have become increasingly prominent, which leaded to increase the fertilizers and pesticides usage. Systematic study on microbial community structure of garlic soil, collection and identification of key pathogenic and antagonistic microbial resources of garlic, and development of garlic specific microbial fertilizer accord with the national strategic demand of reducing chemical fertilizer and reducing pesticide. This project takes the healthy type (continuous cropping 1~10 years), continuous cropping obstacle type (continuous cropping 15~25 years), severe continuous cropping obstacle type (continuous cropping 15~25 years) and disease suppressive type (more than 35 years of continuous cropping) of four kinds of soil as the research object. Using 16S rDNA/ITS two generation and Pacbio Sequel three generation high-throughput sequencing technology and combining with the field disease condition and soil factors, we will systematically research on garlic rhizosphere microbial community structure and dynamic changes, and explored the key pathogens / antagonistic microbes causing / controlling the continuous cropping of garlic. Guided by high throughput data, possible key microorganisms were directionlly separated using very-low nutrient technology. We studied on their classification status, pathogenic / antagonistic abilites, and other characteristics, and determined the key pathogenic / antagonistic microbes associated with the continuous cropping disorder of garlic to provide microble resources for the development of special type multifunctional microbial fertilizer.

大蒜连续多年种植后，土壤微生物群落结构失衡、土传病害频发等连作障碍问题日益凸显，导致化肥农药用量不断增多。系统研究大蒜土壤微生物群落结构，收集鉴定大蒜关键病原微生物和拮抗微生物资源，开发大蒜专用型微生物肥料符合我国农业减肥减药的国家战略需求。本项目以健康型（连作1~10年）、连作障碍型（连作15~25年）、严重连作障碍型（连作15~25年）和抑病型（连作35年以上）四类土壤为研究对象，采用16S rDNA/ITS二代和三代Pacbio Sequel高通量测序技术，结合田间发病情况、土壤因子等，系统研究大蒜根际微生物群落结构及动态变化，预测引起/防控大蒜连作障碍的关键病原菌和拮抗菌；以高通量数据为指导，采用寡养分离技术，对可能的关键微生物进行定向分离，研究菌株分类地位、致病性/拮抗效果及其他功能特性，确定大蒜连作障碍相关的关键病原菌和拮抗菌，为开发专用型多功能微生物肥料提供微生物资源。

我国是世界上大蒜种植面积最大、产量最高的国家，大规模种植大蒜达40余年。我国出口大蒜主产区山东及周边地区大蒜连作障碍严重，病害频发，微生物群落结构失衡。本论文利用高通量测序和生物信息学分析技术，研究健康型、严重感病型、恢复型大蒜田的根际土与根内在不同生长期的细菌和真菌群落结构，界定大蒜核心微生物，解析大蒜根部关键病原菌和潜在的有益微生物类群，开展大蒜根茎腐烂病病原菌和拮抗促生菌的筛选和评价，为改善大蒜连作障碍提供理论和技术支撑。主要结果如下：.（1）明确大蒜根部细菌多样性及核心病原菌和有益菌。针对健康型、严重感病型、抑病型三种不同类型以及大蒜生长的三个不同时期的根际土和根样品，基于宏基因测序和高通量生物信息学分析技术，发现健康型和抑病型细菌和真菌的互作更为相似，严重感病型根际土细菌互作关系最为复杂，而根内最为简单。大蒜根部的核心病原菌包括镰刀菌属、土栖棘壳孢属、链格孢菌属；核心有益菌包括芽孢杆菌属、溶杆菌属、链霉菌属、蓝状菌属等。相关内容文章还在撰写中。 .（2）大蒜根茎腐烂病主要病原菌的分离、鉴定及致病能力研究。在大蒜根茎腐烂样品中获得大蒜根茎腐烂病病原真菌106株，属于5个属7个种，镰刀菌属为优势菌属。其中首次发现的大蒜根茎腐烂病病原菌3种，分别为土栖棘壳孢属（Setophoma terrestris）、角担菌属（Ceratobasidium sp.）、根霉属（Rhizopus arrhizus）。该部分内容发表SCI文章3篇，中文核心论文2篇。.（3）有益微生物的分离鉴定及评价。基于高通量数据分析结果，分离获得优势细菌293株和优势真菌13株，筛选防控大蒜连作障碍的拮抗微生物，开展温室和田间试验；针对获得的潜在微生物新种，通过多项分类鉴定技术鉴定微生物新种1株。该部分内容发表SCI论文1篇，中文核心论文2篇，授权国家发明专利2件。