| Fusarium is a cosmopolitan genus of filamentous ascomycete fungi,causing many globally devastating diseases,including wilts,blights,root or stem rots and cankers.In addition to causing severe yield and economic losses in agricultural crops,Fusarium also produce a diversity of toxic secondary metabolites（mycotoxins）,such as trichothecenes and fumonisins,which can contaminate agricultural products,are a threat to human and animal health.Fusarium graminearum sensu lato（teleomorph Gibberella zeae（Schwein.）Petch）is the main pathogens of Fusarium head blight（FHB）of wheat and other small cereal grains.Fusarium spp.is a species complex.Different Fusarium spp.may be associated with FHB in different regions of the world because of variations in cropping systems and climate.So far,altogether 13 phylogenetic species were defined.The mainly causal pathogen of FHB are F.graminearum s.str.and F.asiaticum.The species Fusarium oxysporum is a major pathogen of many important crops over the world,causing root rots and crown as well as vascular wilts.F.oxysporum provokes vascular wilt disease in over 150 field and crop hosts,and also causes onychomycosis,keratitis and various cutaneous diseases in humans.Recently,the most efficient strategy for the control of Fusarium diseases,such as FHB,are through the application of fungicides.Phenamacril（experimental code JS399-19）,which is a Fusarium-specific fungicide that is used to control FHB and Rice bakanae disease,is a cyanoacrylate fungicide.Many cyanoacrylates that contain heterocycles exhibit excellent activity against weeds,insect pests,fungal pathogens,viruses,and cancer.Under phenamacril selection pressure,F.graminearum has frequently developed resistance mutations to phenamacril.And there is no cross-resistance between phenamacril and well-known fungicides,suggesting that the mode of action and resistance mechanisms of phenamacril are different from those of other fungicides.Elucidating the resistance and response regulation mechanism of Fusarium to phenamacril is important because it will increase our understanding of the fungicide’s control strategy and of ways to avoid the development of resistance.In our previous studies,we found that β2-tub was phenamacril-resistance related genes.Therefore,we screened and identified many genes related to β2-tub,and we determined the relationship between these genes and phenamacril-resistance by gene deletion strategy.In this study,we found that FgApsB is related to resistance in F.graminearum to phenamacril by constructing a deletion mutant of FgApsB,designated △FgApsB-28.And △FgApsB-28 showed enhanced resistance to phenamacril.The functions of FgApsB were furtherly evaluated.We found that conidiation and mycelial growth rate are reduced in △FgApsB-28.The hyphae of △FgApsB-28 are thinner than those the wild type and have a different branching angle.AFgApsB-28 exhibited reduced aerial hyphae formation but increased production of rubrofusarin.Whereas nuclei are evenly distributed in germ tubes and hyphae of the wild type,they are clustered and irregularly distributed in △FgApsB-28.The mutant exhibited increased resistance to cell wall-damaging agents but reduced virulence on flowering wheat heads,which is consistent with its reduced production of the toxin deoxynivalenol.All of the defects in △FgApsB-28 were restored by genetic complementation with the parental FgApsB gene.Taken together,the increased resistance to phenamacril of FgApsB deletion mutants might result from the enhanced ability of response to stress but not the regulation of phenamacril-resistance genes.FgFim from the wheat scab pathogenic fungus Fusarium graminearum strain Y2021A,which is highly resistant to the fungicide JS399-19,was identified by screening a mutant library generated by HPH-HSV-tk cassette-mediated integration.The functions of FgFim were evaluated by constructing a deletion mutant of FgFim,designated △FgFim-15.The deletion mutant exhibited a reduced rate of mycelial growth,reduced conidiation,delayed conidium germination,irregularly shaped hyphae,a lack of sexual reproduction on autoclaved wheat kernels,and a dramatic decrease in resistance to JS399-19.△FgFim-15 also exhibited increased sensitivity to diverse metal cations,to agents that induce osmotic stress and oxidative stress,and to agents that damage the cell membrane and cell wall.Pathogenicity assays showed that the virulence of the FgFim deletion mutant on flowering wheat heads was impaired,which was consistent with its reduced production of the toxin deoxynivalenol in host tissue.All of these defects were restored by genetic complementation of the mutant with the parental FgFim gene.Quantitative real-time PCR assays showed that basal expression of three Cyp51 genes,which encode sterol 14a-demethylase,were significantly lower in the mutant than in the parental strain.The results of this study indicate that FgFim plays a critical role in the regulation of resistance to JS399-19 and in various cellular processes in F.graminearum.To determine the mechanism of resistance to the fungicide phenamacril（JS399-19）in Fusarium graminearum,the causal agent of Fusarium head blight,we sequenced and annotated the genome of the resistant strain YP-1（generated by treating the F.graminearum reference strain PH-1 with phenamacril）.Of 1.4 million total reads from an Illumina-based paired-end sequencing assay,92.80%were aligned to the F.graminearum reference genome.Compared with strain PH-1,strain YP-1 contained 1,989 single-nucleotide polymorphisms that led to amino acid mutations in 132 genes.We sequenced 22 functional annotated genes of another F.graminearum sensitive strain（strain 2021）and corresponding resistant strains.The only mutation common to all of the resistant mutants occurred in the gene encoding myosin-5（point mutations at codon 216,217,418,420,or 786）.To confirm whether the mutations in myosin-5 confer resistance to phenamacril,we exchanged the myosin-5 locus between the sensitive strain 2021 and the resistant strain Y2021A by homologous double exchange.The transformed mutants with a copy of the resistant fragment exhibited resistance to phenamacril,and the transformed mutant with a copy of the sensitive fragment exhibited sensitivity to phenamacril.These results indicate that mutations in myosin-5 confers resistance to phenamacril in F.graminearum.In this study,we need to examine the relationship between FoMyo5 mutations and phenamacril-resistance in these difficult-to-control Fo isolates.Six kinds of Fo isolates were collected from different hosts.Fungicide susceptibility testing showed that these Fo isolates demonstrated different resistance or susceptibility to phenamacril,which is Fusarium-specific antifungal,compared with Fg species.When aligned these homologous myosin5 motor domains of these strains,we found that the resistance of Fo strains resulted from the mutations（Vall51 to Ala and Ser418 to Thr）in FoMyo5.And we confirmed this result by gene replacement stragety among three Fo isolates Fo47,Fol4287 and Fo32931.This is the first report of diverse plant-host fungi exhibiting target-site phenamacril resistance,which provide the basis for controlling vascular wilt disease in diverse crop hosts.To determine the response regulation mechanism of Fusarium asiaticum to the fungicide phenamacril（JS399-19）,we sequenced the RNA-seq of sensitive mutant Z0212 and high-resistance mutants K216E and S217P.Using Illumina sequencing technology,we generated over 2G clean bases of high-quality sequence data on each library（Z0212,Z0212js,S217P,S217Pjs,K216E,K216Ejs）.Of the clean reads,more than 58%were uniquely mapped to Fusarium asiaticum reference genome for each library.From all the libraries,13,993 genes were obtained in total,including 297 new transcripts.And we found that only 14.2%（1,992）of the total number of genes（13,993）were differently expressed（DEGs）.We performed Gene ontology（GO）enrichment analysis,the DEGs were assigned to 1,837 functional terms by enrichment analysis of GO assignments.When treated with phenamacril,functional groups of resistant mutants were enriched in the biological process and cellular component.Especially,the compound binding were the significantly enriched terms.In contrast,oxidoreductase activity and catalytic activity were only dominant in sensitive mutant Z0212.We performed KEGG pathway analysis and found that Cell cycle-yeast and DNA replication pathways were specific to sensitive mutant but ABC transporters were significantly enriched in resistant mutants.Finally,we found that phenamacril treatment affected transcripts by Alternative Splicing（AS）and furtherly inhibited the growth of Fusarium asiaticum. |
PDF Full Text Request