| Pear ring spot,caused by Botryosphaeria species,mainly damages branches and fruits in Rosaceae plants,and is a kind of stem disease commonly occurring in apple and pear orchards.Pear ring spot disease can cause tree decline and fruit rotten,which results in serious damage to fruit quality and yield,and brings serious economic losses to fruit farmers.Mycovirus-mediated hypovirulence can be used as a useful biological control way to control fungal diseases.BdCV1 induced to the hypovirulence of the phytopathogenic fungus B.dothidea,which will be used as an ideal candidate material for biological control.In this study,BdCV1 antibody was prepared and serological characteristics were analyzed.The transmission efficiency of BdCV1 in B.dothidea strains was comprehensively evaluated.Meanwhile the existence of mil RNA in B.dothidea was confirmed and the expression patterns of predicted target genes in response to BdCV1 were analyzed.The obtained results are as the followings: 1.BdCV1 antibody preparation and serological testsAntibody was separately prepared with the rabbits which was immunized by the purified BdCV1 particles.The antibody titer was 1:51,200,which reaction with BdCV1 isolates with good specificity was verified by western blot analysis.An indirect ELISA was effectively established to detect BdCV1 isolates in B.dothidea strains by screening and optimizing various parameters.The results provide materials for screening the BdCV1 strains from serological reaction,and lay the foundation for further exploring the interaction between BdCV1 and host.2.The determination of BdCV1 transmission to B.dothidea strainsBdCV1 was transmitted horizontally to the same genetic background compatible strains with LW-C,and different B.dothidea strains isolated from different regions,respectively.The results showed that BdCV1 could be successfully transmitted to the strains,and exhibited strong infectious capacity and influence on the biological characteristics of the recipient strains.Meanwhile,BdCV1 in LW-C transmission to LW-P with Bd PV1 may trigger the host gene silencing.The above results lay the foundation for the bio-control ring spot disease in practice based on BdCV1-mediated LW-C hypovirulence.3.The mil RNAs existence and m RNA gene expression profiles in B.dothidea stains in responsive to BdCV1BdCV1 transmission in B.dothidea strains is essentially the reaction result of interactions of BdCV1 and host.To further explore host genes interacted with BdCV1 at the molecular level,mil RNA and predicated target gene m RNA in combination with transcriptome,small RNA and whole genome sequencing results of B.dothidea strains are renewly analyzed by bioinformatics,which of important mil RNA and predicated target genes were selected to further analyzed.Four known Bd-mil RNAs and 2 novel Bdmil RNAs were verified by Stem-loop RT-PCR and poly(A)RT-PCR.It was the first report to verify the existence and expression of Bd-mil RNAs in B.dothidea strains.The results of twenty potential target genes expression patterns showed that BdCV1 in combination with Bd PV1 had different effects on gene expression levels in B.dothidea strains.The gene expression profiles were the results of the BdCV1 interaction with mycovirus and host,respectively.It revealed molecular information for exploration of mil RNA as a potential pathogenic factor involved in the interaction with B.dothidea and mycoviruses,which provided useful candidate genes for the bio-control fruit tree ring spot disease.In this research,we prepared BdCV1 antibody,analyzed its infectivity and influence on host biological characteristics and gene expression in B.dothidea strains.The obtained results provided important information for the effective detection BdCV1 isolates in B.dothidea strains and the comprehensive evaluation of the application potentiality of BdCV1-mediated hypovirulence for bio-control ring spot disease.It also provided a new way for the prevention and control of pear ring rot by exploring the potential genetic resources. |
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