| Rape Sclerotinia rot caused by Sclerotinia sclerotiorum (Lib) de Bary is the primary disease on rapeseed production. Rape Sclerotinia rot occurs during the whole rape growth period, leading to yield and quality decrease which will cause enormous economic losses. It is very difficult to control rape Sclerotinia rot due to the lacking of disease-resistant resources and the widely host range of the pathogenic fungus and their fungicide resistance. Mycoviruses are widely present in fungi, some of which can change the host phenotype including debilitation in their phytopathogenic fungal hosts. Therefore, Hypovirulence-associated mycoviruses isolated from S. sclerotiorum can provide new bio-control resource for biological control of sclerotial disease and studies on the interaction between virus and their fungal host.In this study, we have isolated 196 strains of S. sclerotiorum collected from Changde and Yiyang of Hunana province, among which there were 104 from Changde and 92 from Yiyang. Virulence test on the detached leaves of rapeseed showed that lesion diameters separately infected by 9 strains from Changde and 7 strains from Yiyang were greater than 60.0 mm, and 13 strains from Changde and 6 strains from Yiyang showed declined pathogenicity. The results indicated that the pathogenicity differentiation between these S. sclerotiorum strains from Hunan province of Changde and Yiyang is evident.DsRNA screening from 19 hypovirulent strains by cellulose chromatography were conducted. The hypovirulent strains CY019, CY080, YY077 and CY019 were confirmed to harbor dsRNA elements after being treated by DNAse I and S1 Nuclease, which was used to eliminate the contaminative DNA and ssRNA, and by gel-electrophoresed 1.0% agarose. Since the CY019 strain showed sparse hyphae and reduced sclerotia production, we selected this strain for further research.The dsRNAs in strain CY019 can be horizontally transmitted to the virulent strain CY013, CY021 and CY056 via the method of confronting incubation and can induce hypovirulence of these fungal strains. Virus-free derivative strain named CY019VF was obtained using the method of hypha tip isolation. The dsRNAs was confirmed to be thoroughly eliminated by dsRNA extraction. Virulence test on live rape stalks in the field and detached rape showed that the CY019VF strain has a stronger pathogenicity than that of strain CY019, indicating that the pathogenicity of strain CY019VF was recovered. These results preliminarily confirmed that the pathogenicity of strain CY019 was related to the dsRNA virus.The cDNA sequences in strain CY019 which has a molecular weight of 10 Kbp and 7 Kbp, respectively, were partial cloned, with the complete sequence of the 7kb dsRNA and partial sequences of the 10kb dsRNA being determined. Partial sequence of thelOKbp fragment had 99% sequence similarity to Sclerotinia sclerotiorum endornavirus-1 (SsEV1). We named the 7 Kbp dsRNA segment as SsBRV3-CY019 which actually composed of two dsRNA segments designed as dsRNA1 and dsRNA2. The two dsRNA segments were in length of 6196 bp and 5892 bp, respectively, each containing an ORF and sharing sequences identities of 97% and 98% to the respective dsRNAl and dsRNA2 of virus BpRVl isolated from Botrytis cinerea strain GarlicBc-72. These results mean that the SsBRV3-CY019 and BpRV1 are different strains of the same virus species.Strain GarlicBc-72 carrying BpRVl is characterized with sectors showed in colony morphology, slowly growth rate and reduced virulence. However, whether the exact reason in terms of hypovirulence of strain CY019 was caused by SsBRV3-CY019 or the combination of strain SsBRV3-CY019 and the endornavirus of 10 Kbp, which has sequence identity of 99% to SsEV1,is unclear and should be further studied. |
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