Study Of The Infection Process Of Botryosphaeria Dothidea On Apple Shoots And Identification Of A Mycovirus Infect Botryosphaeria Dothidea

Apple ring rot caused by Botryosphaeria dothidea is one of the most important diseases of apple in China.Infected apple shoots showed wart or canker symptom.However,the infection process and pathogenesis of B.dothidea have not been fully understood.In this study,we use scanning electron microscope(SEM)and laser scanning confocal microscopy(LSCM)to monitor the infection process of B.dothidea on apple shoots.Then,the genome of B.dothidea and transcriptome of both pathogen and host during infection were sequenced via next-generation sequencing.At last,a strain of dsRNA mycoviruses was identified from the hypovirulent B.dothidea strain Gl.The main results were as follows:1.B.dothidea conidia were inoculated on nonwounded apple shoots.At 24 hours post inoculation(hpi),most of germinated hyphae were spotted entered into the shoots through lenticels,cracks and wounds without appressorium formation.A few of infection hyphae were found in the epidermis and suberisation cells were come into existence at 7 days post inoculation(dpi).The symptom of wart appeared at 30 dpi.Infected apple tissues were surrounded by suberisation cells during the whole process.B.dothidea conidia were inoculated on wounded apple shoots.At 24 hours post inoculation(hpi),most of germinated hyphae were spotted entered into the shoots through wounds.Lots of hyphae were found in the wound and suberisation cells were come into existence at 7 dpi.The symptom of canker emerged at 20 dpi.2.The final assemblied B.dothidea genome size is 45.17 Mb.In total 14191 protein-coding genes were predicted,which fall into 6035 gene families.Phylogenetic analysis base on the orthologous genes shows B.dothidea and Pyrenophora teres f.teres were the most closely related.By analysis of disease-related genes,333 carbohydrate-active enzyme(CAZymes)genes,61 secondary metabolite biosynthesis backbone genes and falls into 59 gene clusters,309 cytochrome P450 genes,and 1411 transporter genes were predicted.In the meantime,a total of 308 candidate effectors and 561 pathogen-host interaction genes were identified.Twenty-five PKS encoding genes,seventeen NRPS and one PKS-NRPS encoding genes have similar domain structures with known PKS,NRPS PKS-NRPS genes which involved in melanin or toxin biosynthesis.Phylogenetic analysis also clustered them in close clades.Those results suggest B.dothidea possess the ability of melanin or toxin biosynthesis.B.dothidea were found containing less cellulose but sufficient pectinase and cutinase by genome comparation with other representative fungi.3.RNA-seq was applied to comparatively analyze transcriptional profiles of nonwounded and wounded inoculated apple shoots with B.dothidea at 24 hpi,7 dpi 20 dpi and 30 dpi.A total 1156 differentially expressed genes(DEGs)were identified which 517 genes up-regulated.GO enrichment shows the metabolic process and catalytic activity functioned which were significant enriched at 7 dpi for nonwounded B.dothidea inoculation samples.A part of predicted secondary metabolite backbone genes were up regulated during the infection.Cazyme encoding genes involved in infection process were up-regulated at 24 hpi in the sample of wounded inoculated shoots and were up-regulated at 7 dpi in the sample of nonwounded inoculated shoots.Candidate effectors encoding genes were up regulated at 7 dpi in the sample of nonwounded inoculated shoots.4.Transcriptional profiles of nonwounded and wounded inoculated apple shoots samples at 24 hpi,7 dpi,20 dpi,and 30 dpi showed a total 3333 DEGs in which 1542 genes were up-regulated.Abscisic acid(ABA)signaling pathway related genes were up-regulated at 24 hpi seduced high gene expression of callose synthesis related genes at 20 dpi and 30 dpi during infection.Genes involve in the cork biosynthesis pathway were differentially up-regulated during infection.The gene expression profiles samples of nonwounded inoculated apple shoots with B.dothidea showed genes involve in the cork biosynthesis pathway were differentially up-regulated at 7 dpi,20 dpi and30 dpi.The gene expression profiles samples of wounded inoculated apple shoots with B.dothidea showed genes involve in the cork biosynthesis pathway were up-regulated 20 dpi and less up-regulated genes at 30 dpi.5.A new strain of Botryosphaeria dothidea chrysovirus 1,BdCVl-G1,was identified in strain G1.The complete nucleotide sequences of the four dsRNAs were determined to be 3,885,2,816,2,583,and 2,574 nucleotides(nt)long,respectively.BdCV1-G1 has a shortened RNA-dependent RNA polymerase(RdRp),and a 20-aa-longer coat protein(CP).To produce virus-free isolates from the hypovirulent strain G1,single-hypha isolates were obtained using a hypha tipping method.Campare with strain G1,Virus-free-strains are significant strong in virulence and significant faster at growth.Virus-free-strains colony morphology became regular.Double strands RNA mycovirus BdCVl-G1 is the reason of hypovirulence of B.dothidea strain G1.In summary,the results mentioned above demonstrated B.dothidea initiate the infection process with entering lenticels,cracks and wounds,24 hpi,7 dpi,20 dpi and 30 dpi are the key time-points of infection.Wart symptoms were the results of defense response to pathogen infection.A mass of pathogenesis-related genes were predicted in B.dothidea genome.Different kind of pathogenesis-related genes expressions were up-regulated during infection.Disease-resistant related genes expressions were also up-regulated during infection.The expression changes of the disease-resistant related could an explanation at transcriptome level of the formation of cork cells around infected shoots tissue.