真菌病毒ArHV及其与Amphobotrytis ricini耐高温的关系研究

Amphobotrys ricini, a Botrytis-like fungus, is the causal agent for the gray mold disease of castor bean, resulting in severe economic losses. A. ricini usually occurs in summer and autumn. Our previous studies indicated that A. ricini was commonly infected by a mycovirus named Amphobotrys ricini Hypovirus (ArHV). ArHV-infected A. ricini tolerated to 30-35 C for mycelial growth and conidial germination, secreted toxins and caused infection on plant tissues. Persistent infection of A. ricini by ArHV and the associated biological characteristics in A. ricini imply that ArHV might be associated with the tolerance of A. ricini to high temperature (HT). In order to prove this hypothesis, we proposed to study the mycovirus ArHV and to characterize the relationship between ArHV infection in A. ricini and its tolerance to HT. The project includes: (1)to clone and analyse the genome of the mycovirus ArHV;(2)to determine the causal effect of ArHV infection on HT tolerance in A. ricini by elimination and introduction of ArHV in A. ricini, and by construction of infectious clone; (3)to mine HT tolerance-associated genes in A. ricini by comparing the gene expression profiles in the presence of ArHV and in the absence of ArHV; (4) to confirm the function of HT-tolerance genes in A. ricini using the routine gene manipulation techniques such as gene knock-out, gene complementation and gene over-expression. The project will provide experimental evidence to show the first case of the mutualistic interaction between the mycovirus infection and HT tolerance in fungus and to understand the molecular mechanisms involved in the interaction. The research results will lay a solid foundation for exploitation of some novel strategies to control A. ricini and other Botrytis spp. in the future.

葡萄孢类似真菌Amphobotrys ricini（Ar）在夏秋季引起蓖麻灰霉病，造成严重的经济损失。前期研究结果表明：Ar普遍遭到真菌病毒ArHV侵染，被ArHV感染的Ar较耐高温、分泌毒素，并能正常致病。ArHV与Ar之间的稳定依存关系及其相关联的生物学特性暗示：ArHV可能与Ar耐高温及致病相关。因此本项目提出研究ArHV的基因组结构及与Ar耐高温的关系，并解析其中的分子机理。研究内容包括：克隆及分析ArHV基因组结构；脱除ArHV、导入ArHV及构建侵染性克隆，以确定ArHV对Ar耐高温、分泌毒素和致病的影响；比较在ArHV存在和不存在的情况下Ar基因表达差异，并筛选ArHV诱导的耐高温相关基因；采用敲除、互补和过表达等基因操作技术对Ar耐高温相关基因进行功能验证。研究结果将为首例真菌病毒促使宿主真菌耐高温提供证据。明确ArHV与Ar耐高温之间的关系及机理将为灰霉病防控提供新线索。

蓖麻葡萄孢Amphobotrys ricini（Ar）主要侵染大戟科植物，造成严重的经济损失。本项目从真菌病毒的角度出发，研究真菌病毒基因组结构及其寄主Ar耐受高温的关系。结果表明：在蓖麻葡萄孢中普遍存在1种新的真菌病毒Amphobotrys ricini hypovirus 1（ArHV1）。ArHV1基因组全长9232 bp，含有1个大的ORF，编码RdRp，与Betahypovirus属病毒CHV3、CHV4、PlHV1、SsHV1和VcHV1的RdRp序列一致性较高，且含低毒病毒科和Fusariviridae科病毒RdRp的保守模体。系统发育分析表明ArHV1是Betahypovirus的一个新成员。ArHV1在蓖麻葡萄孢菌株中分布广泛，不同菌株中ArHV1的积累量和序列存在差异。ArHV1能通过蓖麻葡萄孢产生分生孢子，垂直传播到后代菌株中。菌株WHA-1的菌丝侵染叶片时，病健交界处ArHV1的积累量高于腐烂病斑处的积累量，且在侵染叶片的菌丝中ArHV1的积累量高于腐生生长菌丝中的积累量。ArHV1在蓖麻葡萄孢中存在DNA形态，DNA形态序列与cDNA序列高度保守。上述研究结果丰富了感染植物灰霉病菌的真菌病毒种类，加深了对真菌病毒生物学的认识。比较了蓖麻葡萄孢和灰葡萄孢菌丝和分生孢子对温度的适应性，发现蓖麻葡萄孢较灰葡萄孢更耐高温。蓖麻葡萄孢菌丝生长最适温度范围为20~30°C，灰葡萄孢菌丝生长最适温度为20°C；在30°C下，蓖麻葡萄孢菌丝生长速度显著高于灰葡萄孢；在32°C高温下，蓖麻葡萄孢可以缓慢生长而灰葡萄孢不能生长。蓖麻葡萄孢分生孢子萌发最适温度（26~30°C）高于灰葡萄孢（20~26°C）；在32°C高温下，蓖麻葡萄孢分生孢子萌发率为94%，而灰葡萄孢分生孢子萌发率为2.3%；在36°C高温下，蓖麻葡萄孢仍有5.3%的分生孢子能够正常萌发，而灰葡萄孢分生孢子不能萌发。在田间调查了Ar引起的铁苋菜灰霉病的流行情况，发现该菌在夏末秋初高温条件下流行，在日平均气温25°C以上的条件下，Ar能在铁苋菜上完成分生孢子产生、传播再侵染和菌核形成这一病害流行阶段。项目实施3年来发表SCI论文2篇。在国内学术会议上作专题报告2次，培养研究生3人（2人毕业）及本科生2人（2人毕业）。