来源于产黄青霉病毒科成员的分离物对梨轮纹病菌生长及其致病力的影响

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

【目的】明确产黄青霉病毒科成员(Botryosphaeria dothidea Chrysovirus 1,BdCV1)对寄主梨轮纹病菌(Botryosphaeria dothidea)菌株生物学性状的影响,证实单独感染BdCV1梨轮纹病菌分离株是弱毒菌株;以期确定BdCV1是否为引起梨轮纹病菌弱致病力的单一因子。【方法】从前期弱致病力复合感染双分体病毒科成员Bd PV1和BdCV1的梨轮纹病菌LW-1中经多代菌丝分离获得的疑似仅单独感染BdCV1分离株(命名为LW-C)为材料;采用dsRNA检测、RTPCR,鉴定LW-C菌株中仅携带BdCV1。采用菌丝块接种于PDA培养基以及3针针刺法接种梨果实,观察其菌落形态以及发病情况,测定菌落生长速度和致病性,明确LW-C生物学特性。将LW-C对无毒菌株Mock进行水平转染,检测其传染后衍生菌株的dsRNA并测定其生物学特性。【结果】证实了LW-C仅感染BdCV1,具有弱毒特性;水平转染结果明确了BdCV1因子对梨轮纹病菌的菌落形态、生长速度有抑制作用,对寄主有弱致病力。【结论】确认了BdCV1是引起梨轮纹病菌致病力衰退的主要因子,为真菌病毒防治果树轮纹病害提供了新颖的生防材料。

[Objective] To identify LW-C infection by only Botryosphaeria dothidea Chrysovirus 1 （designated as BdCV1）, an attenuated strain, and evaluate the effect of BdCV1 on the biological features of the pbytopathogenic fungus of B. dothidea. This study aims to answer if BdCV 1 is responsible for the attenuated hypovirulence of the phytopathogenic fungus B. dothidea. [Methods] In this study, the suspected strain （designated as LW-C） only infected with BdCV 1 was obtained as the test material, from the hypovirulence isolate of LW- 1 coinfected with BdCV 1, a member of the family of Chrysoviridae, and B. dothidea Partitivirus 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-I strain, dsRNA detection, RT-PCR and sequence analysis were used. To assess the biological features of LW-C strain, mycelial agar 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℃ in the dark- ness for determination of the mycelial growth rate and for phenotype observation. The virulence was detected by inoculating detached fruits of Pyrus pyrifolia ＇ Huangguan＇. The lesions developed from the strains were measured and recorded at 7 days post-inoculation （dpi） for the inoculated fruits. The horizontal transmission of BdCV1 infecting the LW-C strain was assessed by LW-C and mock dually cultured. The derivates 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-PCR for RdRp genes and sequencing analysis from dsRNA2 of BdCV1. The biological characteristics of the LW-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 phenotype 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 rates for LW-P and HL- 1 were 22.05 and 15.5 mm· d-1, respectively. In addition, it was difficult to produce spores for the LW-C. More importantly, the LW-C strain exhibited no or very weak virulence on ＇ Huang- guan＇ fruits, equal to that of LW- 1 with a lesion size of less than 5.0 mm, respectively, whereas the lesion sizes 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 ℃ in darkness） were determined. In contact cultures between strains LW-C and Mock, the Mock grew rapidly and covered the entire plates after 3 days, while the LW-C strain grew much slower. Five mycelial derivative subisolates selected randomly （designated as M-13, M-16, M-17, M-21 and M-22） were obtained from 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 to 3.9 mm· d-1）, morphological features of abnormal phenotype with sectored regions, and weak pathogenicity on the pears （fruits of ＇ Kuerle Xiangli＇ and ＇ Huangguan＇ had lesions of 1.3 to 5.1 mm and 3.1 to 5.9 mm in 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 BdCV 1 from LW-C. Therefore, BdCV1 has inhibitory effects on LW-C, which can be horizontally transmitted to mock through a hyphal contact culture. However, the other subisolate of M-21 in comparison with the parental strain Mock showed 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. Lots of conidia were observed on the PDA palate at 7 d for the M-21 strain. Examination by agarose gel electrophoresis disclosed no dsRNA existence in the M-21, revealing that BdCV1 from LW-C could not transmit to Mock. [Conclusion] It was demonstrated that LW-C isolated from the LW-1 strain infected only by BdCV1 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 biological control of apple and pear ring spot diseases. In the future, it will be necessary to theoretically evaluate the transfection effect of BdCV1 infecting LW-C on the other B. dothidea strains and identify the molecular mechanism of the host hypovirulence associated mycovirus BdCV1. It will aim to provide new ideas and strategies for safe biological control of ring rot disease induced by B. dothidea strains in practice.