生防菌盾壳霉作为载体传播核盘菌弱毒相关双分病毒SsPV1的研究

Sclerotinia sclerotiorum (Lib.) de Bary is a typical necrotrophic fungus that causes important diseases in a wide variety of broadleaf crops including soybean and rapeseed. The hypovirulence-associated mycoviruses (HAV) and Coniothyrium minitans are currently being exploited as means of biological control for combating plant diseases. However, how to overcome the vegetative incompatibility shown by many plant pathogens including S.sclerotiorum and how to release easily mycovirus into the field are two major barriers to their adoption as biological control agents. Previously research shows that the partitivirus SsPV1 is associated with the hypovirulent traits in S.sclerotiorum. Therefore, SsPV1 could be exploited as biocontrol agents in the management of Sclerotinia diseases. In present project, we plan to combine the mycovirus SsPV1 and a mycoparasite fungus C. minitans to control the disease caused by S.sclerotiorum. In this project, the particles of SsPV1 will be introduced into new host C. minitans using the method of PEG-mediated protoplast transfection and obtained C. minitans transfectants containing SsPV1. When hyphae of C. minitans infected by SsPV1 penetrates cell walls of the hyphal or sclerotium of S. sclerotiorum, the partitivirus SsPV1 has chances to transfer into the S.sclerotiorum and lead to convert the strong virulence S. sclerotiorum to hypovirulence. The results of our research may supply a novel way to spread the mycovirus to the field and breakthrough the barrage of vegetative incompatibility. Meanwhile, Combination of two excellent biocontrol resource(HVA and mycoparasite fungus) to control plant diseases also might provide new insights into theory and practice system of virocontrol plant disease with high efficacy of using mycovirus related hypovirulence.

核盘菌是多种重要农作物的主要病害。真菌病毒及盾壳霉都具有防治核盘菌的能力。如何有效使病毒突破寄主菌株间菌丝不亲和性障碍、有效将病毒释放到大田中，一直是真菌病毒应用中亟需解决的问题。研究表明双分病毒SsPV1与核盘菌毒力衰退等重要生物学特性紧密相关，具有控制菌核病的潜能。本项目拟以盾壳霉为载体传播核盘菌弱毒相关病毒SsPV1防治菌核病。即通过PEG介导重寄生菌盾壳霉原生质体的方法，将SsPV1病毒粒子转染至盾壳霉中，获得携带有真菌病毒且性状优良的工程菌株；而携带该病毒的盾壳霉工程菌株在寄生核盘菌过程中，将真菌病毒转染至核盘菌中。本研究期望有效克服田间应用中病毒释放及菌株间营养体不亲和性对病毒传播的限制，发挥真菌病毒在病害防治中的生防作用。本项目将盾壳霉及真菌病毒两种作用机理不同的生防资源"双剑合璧"，为高效利用毒力衰退相关真菌病毒及生防菌控制植物病害建立新的理论及实践体系。

核盘菌是引起作物菌核病重要植物病原真菌，利用真菌病毒防控菌核病是一种重要潜在生物防治策略，但是真菌复杂的营养体不亲和性是提高病毒防病效果的主要限制因子。盾壳霉是核盘菌重寄生真菌，试图以盾壳霉为传播载体，将低毒相关病毒传播至不同营养体亲和性核盘菌菌株中，达到控制病害的目的。通过PEG介导策略将低毒相关病毒SsPV1的病毒粒子转染ZS-1原生质体，获得携带SsPV1病毒的盾壳霉菌株。与出发菌株ZS-1相比，携带有SsPV1的盾壳霉在菌落形态、菌丝尖端、生长速度、孢子萌发及形成菌落能力上没有显著性差异，但病毒SsPV1不能通过盾壳霉寄生核盘菌的过程中传播至核盘菌中。在核盘菌中发现呼肠孤病毒SsMYRV4可以抑制寄主真菌的不亲和性反应从而促进异源病毒传播。核盘菌低毒菌株SX10菌株携带一种真菌呼肠孤病毒（Sclerotinia Sclerotiorum mycoreovirus 4, SsMYRV4）。感染SsMYRV4的菌株与不同营养体亲和群的菌株对峙培养时，SsMYRV4能抑制在非我识别初期的信号转导蛋白G-蛋白基因的表达，进而抑制het（Heterokaryon）基因及活性氧（Reactive Oxygen Species, ROS）相关基因的表达，降低菌丝体内ROS的水平，最终导致营养体不亲和性反应受到抑制。这种由SsMYRV4介导的对核盘菌非我识别的抑制作用，能作为异源病毒（SsDRV和SsMV1）传播的桥梁，突破营养体不亲和性的限制进行更广泛的水平传播。本研究揭示了病毒SsMYRV4与其他病毒及核盘菌存在的复杂互作关系，同时揭示真菌病毒在自然条件下的借助其他病毒的潜在传播途径。