Effect of Botryosphaeria dothidea Chrysovirus 1 isolate belonging to the Chrysoviridae family on growth and pathogenicity of the B. dothideastrain infection in pears

Abstract:【Objective】To identify LW-C infection by only Botryosphaeria dothidea Chrysovirus 1 (desig⁃nated as BdCV1), an attenuated strain, and evaluate the effect of BdCV1 on the biological features of thephytopathogenic fungus of B. dothidea. This study aims to answer if BdCV1 is responsible for the attenuated hypovirulence of the phytopathogenic fungus B. dothidea.【Methods】In this study, the suspectedstrain (designated as LW-C) only infected with BdCV1 was obtained as the test material, from the hypovir⁃ulence isolate of LW-1 coinfected with BdCV1, a member of the family of Chrysoviridae, and B. dothideaPartitivirus 1 (designated as BdPV1), and a member of the Partitiviridae by the hyphal tipping technique.In order to confirm LW-C only infection with BdCV1, isolated from the LW-1 strain, dsRNA detection, RT-PCR and sequence analysis were used. To assess the biological features of LW-C strain, mycelialagar plugs from the colony margin of a freshly cultured LW-C strain, LW-1, LW-P and HL-1 as controls,respectively, were placed on a PDA in petri dishes (9 cm in diameter) and incubated at 25 °C in the dark⁃ness for determination of the mycelial growth rate and for phenotype observation. The virulence was detect⁃ed by inoculating detached fruits of Pyrus pyrifolia‘Huangguan’. The lesions developed from the strainswere measured and recorded at 7 days post-inoculation（dpi）for the inoculated fruits. The horizontaltransmission of BdCV1 infecting the LW-C strain was assessed by LW-C and mock dually cultured. Thederivates from Mock were biologically characterized and their virulence was determined as described.【Results】BdCV1 only infection with LW-C was confirmed by the detection of dsRNA patterns, RT-PCRfor RdRp genes and sequencing analysis from dsRNA2 of BdCV1. The biological characteristics of theLW-C strain were analyzed in comparisons with B. dothidea strong virulent strains of LW-P and HL-1,and the attenuated strain LW-1, respectively. The results showed that LW-C exhibited an abnormal phe⁃notype with irregular and sectored colony margins. The growth rate of strain LW-C cultured on the PDA (25 ℃ in darkness) was slower with 4.29 mm·d^-1, almost equal to that of LW-1, whereas the growth ratesfor LW-P and HL-1 were 22.05 and 15.5 mm·d^-1, respectively. In addition, it was difficult to producespores for the LW-C. More importantly, the LW-C strain exhibited no or very weak virulence on‘Huangguan’fruits, equal to that of LW-1 with a lesion size of less than 5.0 mm, respectively, whereas the lesionsizes were 38.65 mm and 34.56 mm on fruits for LW-P and HL-1 of the B. dothdiea strains. The hypovirulent characterization of the LW-C and the horizontal transmission of dsRNA with LW-C to mock (25 ℃ indarkness) were determined. In contact cultures between strains LW-C and Mock, the Mock grew rapidlyand covered the entire plates after 3 days, while the LW-C strain grew much slower. Five mycelial deriva⁃tive subisolates selected randomly (designated as M-13, M-16, M-17, M-21 and M-22) were obtainedfrom five derivates of the Mock in five contact cultures of LW-C/mock at 3 dpi. Isolates of M-13, M-16,M-17 and M-22 were similar to the LW-C strain as the donor in the mycelial growth on the PDA (1.1 to3.9 mm·d^-1), morphological features of abnormal phenotype with sectored regions, and weak pathogenicityon the pears (fruits of‘Kuerle Xiangli’and‘Huangguan’had lesions of 1.3 to 5.1 mm and 3.1 to 5.9 mmin diameter, respectively), which was apparently different from that of Mock in the growth rate of 18.5 mm·d^-1, and the lesions of 29.7 and 32.3 mm on fruits of ‘Kuerle Xiangli’and ‘Huangguan’, respectively.The presence of dsRNAs patterns in derivates of M-13, M-16, M-17 and M-22 were detected by 1.2%agarose gel electrophoresis, which were in accordance with that of the BdCV1 from LW-C. Therefore,BdCV1 has inhibitory effects on LW-C, which can be horizontally transmitted to mock through a hyphalcontact culture. However, the other subisolate of M-21 in comparison with the parental strain Mockshowed no significant difference in the growth rate of the 13.6 mm·d^-1 or virulence on the pear fruits of‘Kuerle Xiangli’at 5 dpi and ‘Huangguan’at 8 dpi with lesions of 23.6 and 31.6 mm, respectively. Lotsof conidia were observed on the PDA palate at 7 d for the M-21 strain. Examination by agarose gel electro⁃phoresis disclosed no dsRNA existence in the M-21, revealing that BdCV1 from LW-C could not transmitto Mock.【Conclusion】It was demonstrated that LW-C isolated from the LW-1 strain infected only byBdCV1 resulted in significant alteration in the growth rate, virulence and phenotype of the phytopathogen⁃ic fungus B. dothidea. The obtained results support the suggestions that BdCV1 is responsible for the at⁃tenuated hypovirulence of the phytopathogenic fungus. Till now, this is the first report to confirm that BdCV1, belonging to a new member of the Chrysoviridae family, induced the hypovirulence of the phyto⁃pathogenic fungus B. dothidea. Therefore, the BdCV1 of Chrysovirus is a good candidate for the biologicalcontrol of apple and pear ring spot diseases. In the future, it will be necessary to theoretically evaluate thetransfection effect of BdCV1 infecting LW-C on the other B. dothidea strains and identify the molecularmechanism of the host hypovirulence associated mycovirus BdCV1. It will aim to provide new ideas andstrategies for safe biological control of ring rot disease induced by B. dothidea strains in practice.