Genome Organizations And Evolution Of DsRNA Viruses In Rhizoctonia Cerealis

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.