【作者】 李伟；

【导师】 王克荣；

【作者基本信息】 南京农业大学 ， 植物病理学， 2013， 博士

【摘要】 禾谷丝核菌Rhizoctonia cerealis Van der Hoeven (=Ceratobasidium cereale Murray and Burpee)是指能够引起禾谷类作物纹枯病(Sharp eyespot of cereals)及禾本科草坪草或牧草黄斑病(Yellow patch)的一类双核丝核菌,属于AG-D融合群。由禾谷丝核菌侵染小麦引起的小麦纹枯病,是一种世界性小麦病害,几乎遍布世界各温带小麦种植区,在欧洲、北美洲、非洲、大洋洲和亚洲都有报到。近30年来,禾谷丝核菌引起的小麦纹枯病已经成为世界小麦生产中危害最严重的病害之一。在我国,纹枯病成为长江中下游和黄淮麦区小麦增产的重要制约因素。目前生产上缺少抗纹枯病的小麦品种,改变耕作措施等方法虽然可减轻病害的危害,但往往难以实现。化学防治是目前主要的防治方法,但防效差及潜在的病原菌抗药性风险等是纹枯病化学防治面临的最主要问题,因此有必要开发新的防治措施。从病原真菌中发掘弱毒真菌病毒从而减轻病害的危害是目前生物防治的新思路。本实验室之前的研究发现在小麦纹枯病菌禾谷丝核菌中能够检测到dsRNA,而目前多数的真菌病毒的基因组都为dsRNA,本研究进一步系统研究了禾谷丝核菌中的dsRNA的多样性,从中发现了一些新的真菌病毒并研究了其系统进化,为以后开发具有生防潜力的真菌病毒奠定了基础。在多核的立枯丝核菌(R. solani)的AG-1到AG-13融合群中,普遍存在dsRNA病毒,并且其中的一些病毒能够引起寄主真菌的致病力减弱。但是在双核的禾谷丝核菌中是否存在dsRNA病毒至今未有报导。本研究从我国的山东、河南、江苏和安徽四省收集了21株禾谷丝核菌菌株,提取了其中的dsRNA,并与菌株的菌落形态、生长速度、对小麦的致病力等进行了相关性研究。结果表明,禾谷丝核菌中普遍存在dsRNA,不同菌株中dsRNA的条带大小和数量存在丰富的多样性。目前暂未发现禾谷丝核菌中dsRNA的类型、数量与菌株的菌落形态、生长速度及致病力等之间的相关性,但致病力较弱的菌株中dsRNA条带的多样性更丰富。禾谷丝核菌中普遍存在一条接近17kb的大片段dsRNA序列,本研究对该片段进行了切胶回收、反转录及克隆测序,分析了其基因组的结构。结果从菌株R0959和R10125中发现了3个完整的内源RNA病毒(Endornavirus)基因组序列及一个不完整的序列,暂时将其定名为禾谷丝核菌内源RNA病毒(R. cerealis endornavirus,RcEV),将完整的3个基因组编号为RcEV1-R0959-1, RcEV2-R10125-1和RcEV3-R10125-2,不完整的序列编号为RcEV-R0959-2.3个完整的病毒基因组都只有一个大的开放阅读框(ORF),编码1段多肽,包含3个保守结构域,分别是甲基转移酶(viral methyltransferase, MTR)、解旋酶(helicase1, Hel-1)和依赖于RNA的RNA聚合酶(RNA-dependent RNA polymerase, RdRp)结构域。其中RcEV2-R10125-1还编码一段呼肠孤病毒S7核心蛋白(Phytoreovirus S7protein, S7)结构域。本研究首次发现在禾谷丝核菌中存在内源RNA病毒,并解析了其基因组结构。真菌寄主被两个基因组大小非常相似的内源RNA病毒同时侵染的现象也是首次发现。本研究对从禾谷丝核菌中发现的3个内源RNA病毒的新种RcEV1、 RcEV2和RcEV3与已经报导的该属其它病毒的基因组结构进行了比较。并根据其编码的甲MTR、Hel-1和RdRp结构域氨基酸序列进行了系统进化分析。结果表明,根据不同结构域构建的系统进化树的拓扑结构有所差别,病毒的聚类关系与寄主来源具有一定的相关性,同类寄主(丝状真菌或植物)的病毒有聚在一起的趋势。因此,内源RNA病毒的不同种可能具有各自独立的进化途径。内源RNA病毒科与dsRNA病毒及ssRNA病毒代表科的系统进化分析表明,内源RNA病毒科属于ssRNA病毒,并不是之前普遍认为的dsRNA病毒,因此在新的分类系统中需要更正。多种病毒共同侵染同一个真菌菌株的情况比较少见,但是在禾谷丝核菌R10125菌株中,除了存在两种内源RNA病毒外,在小于3kb的位置还有3条dsRNA条带,本研究对这些条带进行了分析。结果从这些条带中测序获得了4条完整的病毒基因组序列,将其命名为禾谷丝核菌dsRNA病毒(R. cerealis dsRNA virus, RcRV)4条序列分别编号为RcRV-1、 RcRV-2, RcRV-3和RcRV-4。四条序列的长度分别为2239bp、2238bp、2168bp和1737bp,都编码一个ORF,其中前3条各含有一个复制酶结构域,RcRV-4编码的蛋白序列功能未知。系统发育分析表明,从禾谷丝核菌中分离的RcRV-1、 RcRV-2和RcRV-3与所有已分类的dsRNA病毒区别明显,与另外几种真菌病毒同属于一类暂时未分类的dsRNA病毒。这是首次从禾谷丝核菌中发现该类病毒,其对寄主真菌有何影响暂时还不清楚。真菌病毒没有体外传播途径,只能通过有性或无性繁殖孢子进行垂直传播或者通过菌丝融合进行水平传播。禾谷丝核菌不产生有性或无性的孢子,因此禾谷丝核菌中的病毒若能进行水平传播则只能通过菌丝融合进行。本研究首先研究了菌株R10125的菌尖分离后代菌株中dsRNA的存在情况,结果表明菌尖分离的继代培养菌株可能会丢失一些dsRNA条带。强致病力菌株R0301与弱致病力菌株R10125对峙培养后,未发现其可获得dsRNA条带,其致病力也没有改变,因此不同菌株间dsRNA病毒是否能够进行水平传播、若能传播其传播途径和条件还需要进一步的研究。对禾谷丝核菌一个群体的56个菌株进行的营养体亲和性测定表明,该真菌的营养体亲和性的划分界限不是非常清晰,同一个菌株可能被划分到不同的亲和群中。对6个菌株中的总dsRNA的高通量测序结果表明,禾谷丝核菌中有很非常丰富的病毒资源,有望从中发掘有生防潜力的病毒。更多还原

【Abstract】 The binucleate species Rhizoctonia cerealis Van der Hoeven (=Ceratobasidium cereale Murray&Burpee, Basidiomycota) belongs to the Rhizoctonia AG-D anastomosis group, and is the causal pathogen of sharp eyespot in wheat and yellow patch in grasses. Sharp eyespot has been reported in many countries in Europe, North America, Africa, Oceania and Asia, and is potentially an economically important disease of wheat in the temperate regions of the world. In the last two decades, R. cerealis has become a major fungal pathogen and a new threat to wheat production in the world. In China, sharp eyespot has been one of the most economically important diseases of wheat. Because lack of resistant wheat variety to sharp eyespot in production at present, change tillage systems can reduce disease development degree, but sometimes difficult to implement, chemical control is the most applied method. The lower efficacy and risk of fungicide resistance are the major problems of chemical control, so it is necessary to raise new control strategies.Mycoviruses are widespread in all major groups of plant pathogenic fungi. There is convincing evidence that mycoviruses are responsible for debilitation or hypovirulence phenotypes in some pathogenic fungi. From agricultural perspectives, mycoviruses may contribute to sustainable agriculture as biological control agents. Our previous study had detected dsRNA in some isolates of R. cerealis. In this study, we investigated the diversity of dsRNA viruses in R. cerealis, and found some new mycoviruses species. The evolution of these new viruses was also studied. This research is to lay a foundation of further study on the biocontrol fungal viruses.Double stranded RNA (dsRNA) viruses are commonly detected in natural populations of multinucleate R. solani isolates AG-1to-13, some of the dsRNA will suppress the virulence of their host. However, it is still not clear whether dsRNA mycoviruses are also present in binucleate R. cerealis. In this tudy, we gathered21isolates of R. cerealis from Shandong, Henan, Jiangsu and Anhui province in China. The dsRNA of these isolates was extracted, and the relationship with the colony morphology, growth rate and pathogenicity was studied. The results indicated that the dsRNA is common among R. cereals isolates. The dsRNA’s size and quantity have abundant diversity in different isolates. There is no relationship between dsRNA and the colony morphology, growth rate and pathogenicity in these isolates, but the diversity of the dsRNA is much higher in less pathogenic isolates.In this study, we report the complete genomic sequences of three novel species in the genus Endornavirus from R. cerealis isolates R0959and R10125. Accordingly, we propose the name R. cerealis endornavirus (RcEV), and name the three viruses are RcEV1-R0959-1, RcEV2-R10125-1and RcEV3-R10125-2. The genomes of endornaviruses from R. cerealis consist of linear dsRNA with a characteristic, single ORF of up to18kbp in length. The viral ORF contains the viral methyltransferase (MTR), viral RNA helicases (Hel), and RNA-dependent RNA polymerase (RdRp) domains, specially the viruse RcEV2-R10125-1also contain a Phytoreovirus S7proteinm (S7) domain. This is the first report of the full-length genomic sequence of a dsRNA mycovirus in R. cerealis, and two evolutionally distinct endornaviruses co-infecting one R. cerealis isolate is also found.The phylogenetic tree based on the MTR, Hel-1and RdRp amino acid sequences suggests that RcEV1, RcEV2and RcEV3are new distinct species within recognized endornaviruses species. Previous studies have shown that the topology of the phylogenetic tree does not follow the relationships of the host, while in this study performed with a larger number of species, the phylogenetic tree showed that the viruses of the same host (fungi or plants) have the tendency to cluster together with some occasional exception. Further sequence analysis of more endornavirus genomes may provide more evidence and elucidate the evolution of this interesting virus family. Therefore, different species of Endornavirus may have different evolutionary pathways. The evolutionary analysis also indicated that the Endornaviridae belongs to ssRNA virus, is not dsRNA virus as previous reports.There are few reports, verified by sequencing, on the occurrence of multiple viruses in single fungal isolates. In R. cerealis isolate R10125, besides the two endornavirus RcEV2and RcEV3, there are another three dsRNA segments less than3kb. From these segments, we obtained four complete genome sequences, named R. cerealis dsRNA virus (RcRV), and the four sequences numbered RcRV-1, RcRV-2, RcRV-3and RcRV-4respectively. The length of these four sequences were2239bp,2238bp,2168bp and1737bp, encoding one ORF respectively. Of which the first three sequences each contain a replicase domain, the function of protein code by RcRV-4was unknown. Phylogenetic analysis indicated that, the viruses of RcRV-1, RcRV-2and RcRV-3from R. cerealis belong to a temporary unclassified dsRNA virus, and whether there have some impact to their fungal host need to further study.Mycoviruses are widespread in fungi, they are not known to have natural vectors and are transmitted in nature intracellularly by hyphal anastomosis and heterokaryosis (lateral transmission), and are disseminated via spores (serial transmission). R. cerealis does not produce sexual or asexual spores, so the transmition of the virus in this fungus only through hyphal fusion. In this study, we found that there may lose some dsRNA segments in the isolates obtained from hyphal tip of R10125. The virulent isolate R0301will not obtained any dsRNA segments, after confrontation cultured with weak pathogenic isolate R10125, and its pathogenicity is not changed, so the ways and conditions of dsRNA virus transmission between different isolates still need further research. The vegetative compatibility tests in a R. cerealis population contain56isolates indicated that, the vegetative compatibility groups is not clear, the same isolate could be divided into different groups. The high-throughput sequencing results of total dsRNA extracted from6isolates showed that there contained very rich virus resouses in R. cerealis, and expected to find a potential biocontrol virus.更多还原