真菌病毒FoOuLV1引致苦瓜枯萎病菌致病力衰退机制解析

Fusarium wilt caused by Fusarium oxysporum f.sp. momordicae (FoM) is an important fungal disease in the production of bitter gourd. Hypovirulence-associated mycoviruses have great potential and application prospects for controlling this disease. We have previously found a stain HuN8 infected by an ourmia-like mycovirus, FoOuLV1, which is closely associated with hypovirulence of FoM. This virus and its hypovirulence could be transmitted horizontally among the FoM strains. The FoOuLV1-infected strain is a promising biocontrol resource against fusarium wilt on bitter gourd. In this project, the direct correlation between FoOuLV1 and its host virulence decline will be clarified by constructing the full-length infectious cDNA clone. Meanwhile, other techniques such as pull-down, yeast two hybridization and immunoprecipitation will be used to screen the FoM protein interacted with the ploymerase (RdRp) of FoOuLV1, and the results will be used to identify the potential key genes responsible for hypovirulence of FoM by combination with transcriptomic sequencing analysis. The gene function in infection and pathogenesis of FoM will be analyzed using the gene knockout and complementary techniques. The project will clarify the mechanisms for the mycovirus-conferrred hypovirulence in FoM. It will provide the theoretical support for the fusarium wilt prevention with FoOuLV1.

由尖孢镰孢菌苦瓜专化型引起的苦瓜枯萎病是苦瓜生产上重要的土传真菌病害。利用病毒介导的弱致病力尖孢镰孢菌防治该病害是一种潜在的应用策略。申请者前期筛选到一株致病力严重衰退的苦瓜枯萎病菌HuN8，携带欧尔密病毒FoOuLV1，此病毒和弱致病力特性可以在苦瓜枯萎病菌株间水平传播，具有良好的生防潜力。本项目拟通过构建FoOuLV1侵染性克隆，验证FoOuLV1与苦瓜枯萎病菌致病力衰退的相关性；利用pull-down、酵母双杂交和免疫共沉淀等方法筛选并验证苦瓜枯萎病菌中与FoOuLV1的RdRp互作的蛋白；结合转录组分析FoOuLV1对苦瓜枯萎病菌基因表达的影响；筛选寄主中与FoOuLV1侵染相关的关键基因；进而通过敲除和互补等技术分析关键基因在苦瓜枯萎病菌侵染及致病过程中的功能；项目的完成将明确FoOuLV1引致苦瓜枯萎病菌致病力衰退的机制，为利用FoOuLV1防治苦瓜枯萎病提供理论支撑。

近年来，利用真菌病毒防治植物病害的研究以及病毒与寄主真菌互作的研究都取得了很大的进展。尖孢镰孢菌引起的瓜类枯萎病为一种典型的土传的维管束真菌病害，致病机理非常复杂，导致其防治十分困难。利用真菌病毒防治枯萎病是一种值得探讨的课题。本研究利用首次从实验室前期从尖孢镰刀菌中发现的一株对苦瓜枯萎病具有良好生防潜力的弱毒欧尔密病毒（FoOuLV1）为材料，构建了FoOuLV1与苦瓜枯萎病菌的互作研究体系，对FoOuLV1弱毒特性以及引致苦瓜枯萎病菌致病力衰退的机理进行了研究。研究结果首先验证了FoOuLV1可以引起苦瓜枯萎病菌致病力衰退的特性，且弱毒特性是由FoOuLV1病毒存在直接引起；验证了病毒FoOuLV1对苦瓜枯萎病具有良好的防治效果（田间防效达80%），同时发现弱毒菌株对苦瓜根内尖孢镰刀菌的定殖影响不大，但是可以明显降低苦瓜植株根际土壤中尖孢镰刀菌的数量。其次通过转录组、代谢组和互作等分析病毒FoOuLV1的弱毒机制一方面是由于病毒FoOuLV1降低了苦瓜枯萎病菌的侵染力；另一方面是因为病毒FoOuLV1侵染后，苦瓜枯萎病菌中涉及过敏反应（过氧化物酶反应和活性等）、细胞壁成分（葡聚糖代谢和多糖代谢）以及跨膜转运等生物学过程和氨基酸、糖类和脂肪酸等的生化代谢途径的相关功能基因出现的差异性表达而导致。此外，通过对相关基因的功能进行了验证，证明了过氧化氢酶基因和Ⅱ型卤酸脱卤酶以及YejF等基因参与了FoOuLV1介导的苦瓜枯萎病菌的致病衰退作用。本研究结果为利用真菌病毒FoOuLV1防治苦瓜枯萎病的防治提供理论和实践支撑。