Pathogenicity Differentiation Of Botrytis Cinerea In Hubei Province And The Mycoviruses Present In Strain HBstr-470

Gray mold disease caused by fungus of Botrytis cinerea is widely spread, and the geographical and climatic conditions in Hubei province is favorable for the occurrence of this disease. This dissertation studies the pathogenicity differentiation of B. cinerea in Hubei province and the mycoviruses present in strain HBstr-470. The main results are listed as follows:Firstly, gray mold incidence of greenhouse fruits and vegetables in all regions of Hubei province were investigated and sampled. We found that the gray mold disease of fruits and vegetables caused by B. cinerea is very common in Hubei province. A total of 1140 B. cinerea strains were isolated from the gray mold samples collected from Hubei province, except Shennongjia. The pathogenicity of 942 B. cinerea strains were tested through inoculation of mycelial plugs on detached Nicotiana benthamiana leaves, and the percentage of virulent B. cinerea strains(lesion diameter larger than 10 mm) is 93.94%, whereas the percentage of hypovirulent strains is 6.06%. Through the repeat pathogenicity test of the hypovirulent strains, 15 hypovirulent B. cinerea strains were finally determined.Secondly, the biological characteristics of one of the fifteen hypovirulent B. cinerea strains HBstr-470 were studied. This strain showed slow growth rate, and the colony morphology is abnormal. No sclerotia of strain HBstr-470 were produced after a long-term of cultivation, whereas lots of aerial hyphae and abundant conidia were found on the colony, and the conidia germination rate is high. The virulence of conidia of strain HBstr-470 was tested by inoculation of conidia suspensions on N. benthamiana leaves. The conidia of strain HBstr-470 also showed attenuated pathogenity to the leaves, and the lesion diameter caused by the mixed conidia suspensions of strains HBstr-470 and B05.10 is smaller than that of strain B05.10.Thirdly, five ds RNA segments were detected from the hypovirulent B. cinerea strain HBstr-470, named as A, B, C, D and E. The sequences of the five ds RNAs were partially or fully cloned and analyzed. Ds RNA-A is with the size of about 10,000 bp showed a high homologous to Sclerotinia sclerotiorum hypovirus 1. Ds RNA-B, ds RNA-C and ds RNA-D showed homologous to the segment 1, segment 3 and segment 4 of Rosellinia necatrix quadrivirus 1, respectively. Therefore, these three ds RNAs may be the different genome segments of the same mycovirus, of which ds RNA-C encoded the viral Rd Rp(RNA dependent RNA polymerase). In addition, the phylogenetic analysis based on the Rd Rp sequence also indicated this virus showed close relationship with viruses Rn QV-W1075 and Rn QV-W1118 in Quadriviridae, and Sc V-L-A and Sc V-L-BC in Totiviridae. Ds RNA-E with the size of about 2,000 bp showed homologous to Rosellinia necatrix partitivirus 5 and Sclerotinia sclerotiorum partitivirus S. Therefore, the hypovirulent B. cinerea strain HBstr-470 may be co-infected by several mycoviruses, which belong to viral families Hypoviridae, Quadriviridae and Partitiviridae, respectively.Finally, a derived strain B05.10(W1) was generated through the protoplast fusion of B. cinerea strain HBstr-470 and B05.10(01868). This strain showed abnormal culture morphology, slow growth rate, and produced large amount of aerial hyphae and conidia on the colony, which is similar to that of B. cinerea strain HBstr-470.