FomSIX基因在尖孢镰刀菌侵染紫花苜蓿过程中的功能分析

Root rot disease caused by Fusarium oxysporum is one of the major constraints in alfalfa (Medicago sativa) production in our country and the world. This pathogen mainly relies on chlamydospores as the survival structure in soil to overcome stress conditions and is the primary inocula next year in the fields, but the pathogenicity mechanisms of this pathogen to alfalfa are still not reported. Studies have shown that F. oxysporum-specific Secreted-In-Xylem (SIX) proteins were involved in the infection of this pathogen to alfalfa, but this was reported only in annual and non-leguminous plants such as tomato. The applicant has got the F. oxysporum strain (FomLZ2017-30) with high pathogenicity to alfalfa, developed the method for the production of chlamydospores, and screened six SIX homolog genes. Based on these, this project will first define the infection process of F. oxysporum to alfalfa through morphology and histology methods; and then analyse the expression patterns of FomSIX genes during infection process by qRT-PCR, and screen the gene with strongest expression; afterwards, study the role of FomSIX gene in the infection process through methods such as gene knock-out and gene complementation. This project will preliminarily reveal the pathogenicity mechanisms of F. oxysporum to alfalfa, and thus provide theoretical basis for developing new ways to manage root rot in alfalfa through the inhibition of pathogenicity factors.

尖孢镰刀菌引致的根腐病是我国和世界紫花苜蓿生产上的主要限制因素之一。该病原菌主要以厚垣孢子形式在土壤中度过逆境，翌年在田间成为初侵染源，但对紫花苜蓿的致病机理尚无报道。研究表明，尖孢镰刀菌特有木质部分泌蛋白(SIX)参与对植物的侵染，但仅在番茄等一年生非豆科植物中有所报道。申请人已获得对紫花苜蓿具有高致病力的尖孢镰刀菌菌株(FomLZ2017-30)、建立了产生厚垣孢子的方法，并筛选出6个SIX同源基因。在此基础上，本项目拟用厚垣孢子接种紫花苜蓿幼苗，通过形态组织学方法明确尖孢镰刀菌对紫花苜蓿的侵染过程；进而通过qRT-PCR分析不同FomSIX在侵染过程中的表达模式，并筛选出表达最强烈的FomSIX；然后通过基因敲除和基因互补等方法研究FomSIX在侵染过程中的作用。以期初步揭示尖孢镰刀菌对紫花苜蓿的致病机理，从抑制病原菌致病因子表达角度，为开发紫花苜蓿根腐病防治新方法提供理论依据。

紫花苜蓿(苜蓿)是我国和世界种植面积最大最重要的豆科牧草，但其生产深受根腐病的制约。由于根腐病病原菌的土传特性且能在土壤中长期存活，加上苜蓿为多年生作物，一旦感染尚无有效防治方法。本项目明确了我国苜蓿根腐病的发生和流行规律，发现土壤真菌群落结构及多样性与根腐病严重程度相关；首次对我国苜蓿根腐病病原菌进行了系统的分离鉴定，明确了土传病原真菌尖孢镰刀菌为苜蓿根腐病的优势病原菌，并建立了根腐病病原菌菌种保藏库。系统评价了我国目前主要苜蓿品种对根腐病病原菌的抗感病性，筛选出对尖孢镰刀菌具有较高抗病性的品种。明确了尖孢镰刀菌对苜蓿不同抗感病性品种的侵染过程及其生物量表达模式。揭示了FoSIX基因在尖孢镰刀菌侵染苜蓿过程中的作用及与尖孢镰刀菌其它植物专化型的进化关系。研究结果从抑制土传病原真菌致病因子表达角度，为苜蓿根腐病防治新方法的开发提供理论依据，并从多年生豆科牧草角度拓展对尖孢镰刀菌致病机理的认识。研究结果还为苜蓿根腐病监测预警和土传病原菌检测定量，以及进一步研究尖孢镰刀菌和苜蓿互作体系从而全面了解尖孢镰刀菌对作物的致病机理建立了基础。