| Pear ring spot disease, caused by Botryosphaeria species, has led to substantial economic losses in recent years by creating a large numbers of fruit rots and stem canker,and causing severe recession of tree growth in the main pear producing areas of China. It is urgent to find new, safe and effective measures to control pear ring rot disease Mycovirus-mediated hypovirulence in plant pathogenic fungi became an important biological strategy to control of fungal diseases.In this study, the isolated stain (designated as LW-C) only infected with Botryosphaeria dothidea chrysovirus 1 (BdCV1) was obtained as the material, from the hypovirulence isolate of LW-1 coinfected with BdCVl and Botryosphaeria dothidea partitivirus 1(BdPV1). The biological characteristics of LW-C will be analyzed to know if BdCV1 is responsible for the attenuated hypovirulence of the phytopathogenic fungus. In addition, the B. dothidea strains collected from Hubei province are isolated and identified,from which the dsRNAs are detected. The above strains will serve as bio-control resources to prevention and control of pear ring spot disease. On the other hand, we conducted a comprehensive genome-wide transcriptome analysis to identify potential mRNA and miRNAs genes associated with the interaction of mycovirus and host, by using the De novo sequencing technology and small RNA sequencing from constructed four libraries of LW-CP (LW-1 strain infected with BdCV1 and BdPV1 as material),LW-C (BdCV1), LW-P (BdPV1) and Mock (free virus). The signal transduction pathways and key candidate genes from B. dothdiea related to mycovirus and pathogenicity will be obtained. The performance of the project will reveal the molecular mechanism of host hypovirulence associated mycovirus theoretically, and provide new ideas and strategies for safe controlg pear ring rot disease in practice. The detailed results are shown as the followings:1. Thirteen strains of Botryosphaeria dothidea were isolated from sandy pear(Pyrus pyrifolia) trunks collected at the Fruit and Tea Research Institute,Agricultural Scientific Academy, Wuhan, Hubei Province, China. Six strains selected randomly were determined from purification, molecular identification,growth rate and virulence assay, meanwhile the dsRNA patterns were detected.The results showed that the six strains with virulence and infected with dsRNAs,which provided materials with the biocontrol of pear ring spot disease, and exploration the classification and evolution of mycovirus.2. It was demonstrated that LW-C isolated from LW-1 strain infected only by BdCVl, and BdCV1 resulted in significant alteration in growth rate, virulence and phenotype of the phytopathogenic fungus B. dothidea, which supported the suggestions that BdCV1 was responsible for the attenuated hypovirulence of the phytopathogenic fungus. This was the first report that BdCV1, belonging to a new number of Chrysoviridae family, induced the hypovirulence of the phytopathogenic fungus B. dothidea. Therefore, the BdCV1 was a good candidate for the biological control of apple and pear ring spot diseases. In future, it is necessary to evaluate the transfection effect of BdCV1 with LW-C on the other B. dothidea strains and further explore the potential biological control for fruit tree ring spot diseases.3. The relative expression levels of BdCV1 and BdPV1 cp, RdRp mRNAs in LW-1,LW-C and LW-P cultured in 5 d, 10 d, 15 d, respectively, were detected by the RT-qPCR analysis. The results showed the relative expression levels of BdCV1 and BdPV1 cp, RdRp mRNAs were expressed differentially in B. dothdiea strains of LW-C, LW-P, LW-1 infection with single and mixed mycovirus,respectively, which revealed the interaction of BdCV1 and BdPV1 in B.dothdiea strains.4. In total of 30058 Unigenes with average length of 2128 bp were obtained from 4 libraries of B. dothdiea strains by De novo sequencing, which were annotated to know the function from the seven databases. The differentially expressed genes were screened in responsive to mycovirus, whose expression profiles were analyzed. In total of 5605 genes were up-regulated and 3301 down-regulated from LW-CP/Mock, 4478 up-regulated and 4311 down-regulated from LW-C/Mock, 2596 up-regulated and 4232 down-regulated from LW-P/Mock,respectively. The above results showed that the numbers of differentially expressed genes from B. dothdiea strains were more in response to BdCV1 infection, speculation that the BdCV1 had obvious effect on gene expression of B. dothdiea strains infection with pear ring spot disease. The relative expression levels of nine candidates differentially expressed genes were analyzed by RT-qPCR, whose results were consistent with that of the transcriptome sequencing to verify the reliability of the RNA-seq sequencing results. In addition, several key genes (Ago, Dicer and RdRp) participated in the gene silencing pathway from 4 libraries of B. dothdiea strains were detected and up-regulated in responsive to mycovirus. Gene ontology (GO) enrichment and KEGG analysis revealed that the differential expression of genes involved in metabolic pathways, the biosynthesis of secondary metabolic pathways, signal transduction of cell replication and disease resistance.5. MicroRNAs from B. dothdiea strains were analyzed in responsive to mycovirus by the small RNA high-throughput sequencing: Based on the sizes of miRNAs,the most abundant were 21 nt and 22 nt in length. To Characterize of known and novel miRNAs in B. dothdiea strains, 63 known miRNA and 20 novel miRNA were identified from LW-CP library, 48 known miRNA and 37 novel miRNA were identified from LW-C library, 83 known miRNA and 20 novel miRNA were identified from LW-P library, 86 known miRNA and 19 novel miRNA were identified from LW-Mock library, respectively. The expression levels were lower for novel miRNAs than that of known miRNAs.In responsive to mycovirus, 110 known miRNAs and 19 novel miRNAs were significant differentially expressed in LW-CP/Mock, 120 known miRNA and 15 novel miRNA in LW-C/Mock, 88 known miRNA and 14 novel miRNA in LW-P/Mock, respectively based on the Venn diagram analysis. 57 know miRNAs and 11 novel miRNAs were differentially expressed at the same time in LW-CP/Mock, LW-C/Mock, and LW-P/Mock libraries. In addition, the results showed that the majority of these predicated novel miRNA were mainly a 5' uridine (U) for the base bias in the first position.6. The comprehensive analysis of miRNA/mRNA of B. dothdiea strains in responsive to mycovirus were performed. We conducted GO enrichment analysis to obtain insight into essential gene functions regulated by mycovirus infection, which involved in metabolism, cell, catalytic reaction, combination,transporter and other components. KEGG function analysis showed that the target genes negatively regulated by miRNA involved in metabolism, RNA degradation, signag pathways, such as ABC transport. It reveled that miRNA involved in the interaction of host and mycovirus. |
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