| Fungal diseases seriously affect agriculture production and potentially damage food safety and human health.Citrus are prone to many fungal diseases during postharvest storing,transportation and marketing processes.Penicillium digitatum(green mold),P.italicum(blue mold)and P.crustosum lead to decay of citrus fruits and result in considerable losses in postharvest products,thus all being predominant fungal pathogens to limit the development of citrus industry.In contrast to blue and green molds,P.crustosum species have more extensive hosts,e.g.,infecting not only citrus but also melons,fruits and many food raw materials,bringing about more adverse effects.The sterol demethylation inhibitor(DMI)fungicides such as prochloraz are widely applied to control fungal diseases but have resulted in highly frequent emergence of resistant isolates that gradually reduce the fungicide efficacy.The prochloraz resistance developed by P.crustosum species is significantly higher than blue and green molds,and there are rare reports of P.crustosum isolates with relatively lower prochloraz-resistance.The mechanisms underlying prochloraz resistance of blue and green molds have been reported by comparative transcriptome analysis but not for P.crustosum due to the absence of control strain with suitable resistance.Mycoviruses inhabit various blue and green molds,reducing host virulence or fungicide-resistance,i.e.,resulting in fungal strains with hypovirulence or'fungicide-conditioned hypovirulence'.Such virus-infected fungal strains with reduced fungicide-resistance can be desirable materials for resistance-mechanism studies.Studying on P.crustosum virus and the related regulation on host fungicide-resistance can provide new mycovirus knowledge and more insight into molecular mechanisms underlying Penicillium pathogens fungicide-resistance as well as fungicide-conditioned hypovirulence.Based on two P.crustosum strains(CQ1 and CQ15)with contrast in high prochloraz-resistance,the present study applied transcriptome in combination with RT-qPCR to identify prochloraz-resistance genes and mechanism underlying their effects.The present study isolated and characterized a new P.crustosum virus and investigated its effect on the host prochloraz-resistance.In this study,the virus-infected P.crustosum strain CQ1V was constructed,and transcriptome in combination with RT-qPCR were applied to further compare gene expression profiles between CQ1V,CQ1,and the virus natural-host CQ15 at prochloraz treatments,in order to identify gene targets and their functions in the virus regulation of host fungicide-resistance.The main results are listed below.1.Transcriptome interpretation of P.crustosum prochloraz-resistance mechanism(s)Using two P.crustosum strains(i.e.,CQ1 and CQ15 with significant contrast in prochloraz-resistance)as materials,RNA-sequencing in combination with bioinformatics interpreted prochloraz-resistance genes and their multiple-metabolism background.According to different gene expression profiles between the two strains induced or no induced by prochloraz,the present study applied COG,KOG,GO and KEGG enrichments and classifications to identify P.crustosum prochloraz-resistance genes,including CYP51 target-enzyme genes,MFS and ABC drug-pump protein genes,cytochrome b5 gene,key enzyme genes of sterol biosynthesis(e.g.,ERG3,ERG4,ERGS,ERG11,ERG24,and ERG27),key enzyme genes of fatty acid metabolism,cell wall polysaccharide metabolism-related genes,GSH anti-oxidation metabolism-related enzyme genes,and key enzyme genes involved in cell respiration and mitochondrial oxidative phosphorylation.RT-qPCR results validated the different gene expression profiles.The obtained results indicated that P.crustosum prochloraz-resistance mechanisms included not only traditional target genes(P450 genes,drug-pump protein genes,etc)but also cell membrane anti-stress reactions(e.g.,GSH anti-oxidation)-centered multiple-metabolism regulation including carbon,nitrogen,lipid,and oxidative phosphorylation metabolisms.The present transcriptome evidences provided important cues for biological function experiments to further study Penicillium pathogens DMI-resistance mechanisms.2.Isolation and characterization of P.crustosum virus and prochloraz-resistance regulationThe present study isolated a double stranded(ds)RNA virus from P.crustosum strain CQ15 with relatively lower prochloraz-resistance,and applied high throughput sequencing in combination with random primer PCR to clone the complete virus genomic dsRNAs.The virus genome is comprised of 4 segments(dsRNA1,dsRNA2,dsRNA3,and dsRNA4)with 3600,3177,3078,and 2808 bp in full-length,respectively.Each of them contains one open reading frame encoding RNA-dependent RNA polymerase(RdRp with 1113 amino-acid(AA)residues),coat protein(CP with 981 AA residues),and two function-unknown proteins(with 912 and 847 AA residues,respectively).Phylogenetic analysis based on RdRp sequences classified this virus into genus Chrysovirus and family Chrysoviridae,thus designated as Penicillium crustosum chrysovirus 1(PcCV1).This is the first P.crustosum dsRNA virus and a new member to the chrysovirus family.The present study cured PcCVl from CQ15 and verified that the virus-cured strain(CQ15C)showed higher prochloraz-resistance(EC50=4.6±0.3 mg·L-1)than CQ15(EC50=2.5±0.4 mg·L-1).The present study constructed PcCV1-infected strain(CQ1V)by artificial transfection and verified that CQ1V showed lower prochloraz-resistance(EC50=5.9±0.6 mg·L-1)than CQ1(EC50=10.5±1.9 mg·L-1).The results indicated the ability of PcCVl to induce its host fungal fungicide-conditioned hypovirulence,i.e.,the mycovirus infection can improve prochloraz control efficacy to the P.crustosum pathogens.3.Transcriptome interpretation of mechanisms underlying PcCVl regulation of host prochloraz-resistanceThe present study applied RNA-sequencing in combination with bioinformatics to interpret gene expression profiles of CQ1V and its virus-free parental strain CQ1 at prochloraz treatment,and compared gene expression profiles between CQ1V and virus natural host CQ15 at prochloraz treatments and the background expression profiles of related resistance-genes at prochloraz-free conditions.Based on the DEG profiles,COG,KOG,GO and KEGG enrichments and classifications in combination with RT-qPCR indicated the reduction of PcCVl on host adaptation to fungicide stress.PcCV1 significantly antagonized the fungicide induction of various resistance genes including CYP51 target-enzyme genes,MFS and ABC drug-pump protein genes,cytochrome b5 gene,key enzyme genes of sterol biosynthesis,cell membrane anti-stress-related multiple-metabolism(e.g.,carbon,nitrogen,lipid,and oxidative phosphorylation metabolisms)enzyme and transporter protein genes.The obtained evidences indicated the PcCVl regulation of P.crustosum prochloraz-resistance genes to result in host fungicide-conditioned hypovirulence.In summary,the present study applied transcriptome in combination with mycovirus to interpret P.crustosum prochloraz-resistance mechanisms and revealed the multiple-metabolism regulations underlying ubiquitous P.crustosum high-resistance,providing new transcriptome evidences to better understand Penicillium pathogens fungicide-resistance mechanisms and the involvement of mycovirus in host anti-fungicide regulation,as well as essential theoretical basis to find novel target(s)for fungicide design. |
PDF Full Text Request