| Fusarium head blight(FHB),caused by Fusarium species complex is an important fungal disease,which can infect various cereal crops including wheat and barley.The major physiological race of Fusarium are Fusarium asiaticum and Fusarium graminearum in China.The FHB can happen during the completely growing cycle of host plants causing the yield of grain decreased and the pathogens produce trichothecene mycotoxins seriously influence the quality of grain and food.The toxin can inhibit protein synthesis;destroy immunity system in eucaryon,which is the potential threat to the health of human and animals.Application of the fungicide is the efficient way to control the FHB but people pay less attention to the influence of fungicide application on trichothecene mycotoxins accumulation in grain.The previous study in our lab showed that the carbendazim and azoxystrobin are the effective fungicide to control FHB,the two fungicides can induce the trichothecene mycotoxins accumulation in the wheat seeds,however,and the new type fungicide phenamacril has unique action mechanism and can effectively inhibit the Fusarium.The phenamacril can effectively control the FHB in the field and decrease the toxin accumulation in the wheat seeds.Therefore,the inhibition mechanism of trichothecene mycotoxins production by phenamacril has important role in formulating and implementing the management strategy of trichothecene mycotoxins contamination in cereal grain and ensuring the sustainable management of food and grain security.In vitro,the sub-lethal concentration of phenamacril can decrease the expression of Tri4,which catalyze four steps oxidation reaction in the trichothecene mycotoxins synthesis pathway and inhibiting the accumulation of trichothecene mycotoxins in F.asiaticum,but sub-lethal concentration of phenamacril has no effect on the Tri5 expression which gene involved in start reaction in the the trichothecene mycotoxins synthesis pathway.However,the sub-lethal concentration carbendazim can induce various toxin synthesis relation genes expression up-regulation including Tri4、Tri5、Tri6、Tri12 and obviously induce the abundant accumulation of trichothecene mycotoxins in F.asiaticum.In addition,phenamacril or carbendazim can effect on the chemotype differentiation of trichothecene mycotoxins of F.asiaticum.Durning secondary metabolism stage,the sub-lethal concentration of phenamacril up-regulate the expression of catalase genes in the antioxidant system.Maintain the low level of reactive oxygen(ROS)which has the negative effect on the toxin accumulation in F.asiaticum;however,under the sub-lethal concentration carbendazim condition,the expression of catalase gene obviously down-regulation and inducing the substantial accumulation of ROS,which leads to trichothecene mycotoxins excessively,accumulate in F.asiaticum.In addition,phenamacril and carbendazim have no effect on the expression of genes are related to the superoxide dismutase(SOD),glutathione(GSH)and Ferredoxin(FD)from the primary metabolism stage to the senior metabolism stage.The results indicated that phenamacril and carbendazim regulates the accumulation of trichothecene mycotoxins in F.asiaticum via affecting on the activity of catalase and superoxide dismutase(SOD)influencing the intracellular ROS level and inducing the expression of Tri4 up-regulate or down-regulate and then inhibit or stimulate the toxin systhesis.Base on the genomic re-sequencing results of phenamacril-resistant strain YP-1 in our lab,we screen and identify the extracellular secretion protein catalase-peroxidase coded by FaCcp1 gene.Which has single point mutations in the amino acid sequence:valine(GTC)was replaced by isoleucine(ATC)at 557-position codon(V557L)in this phenamacril-resistant strain.In this study,to investigate the role of FaCcp1 and this single mutant in the toxin synthesis in F.asiaticum,we generate the FaCcp1 deletion mutant(△FaCcp1)and the V557L single point mutant(2021F-FaCcp1)using the protoplast transformation method.These results indicated that sensitivity of △FaCcp1 to H2O2 increased but the sensitivity of 2021F-FaCcp1 to H2O2 obviously decreased.The expression of FaCat4 and FaCat6 were obviously up-regulation in △FaCcp1 and 2021F-FaCcp1.The toxin production of △FaCcp1 was increased,and 2021F-FaCcp1 was decreased,in addition,under the phenamacril condition,the toxin production ability of △FaCcpl and 2021F-FaCcp1 were obvious lower than that of control treatment,the results indicates that FaCcp1 involved in the regulation of trichothecene mycotoxins production inhibited by phenamacril in F.asiaticum.The phenamacril has no effect on the Tri5 expression but can induce the expression of Tri4 down-regulate in △Facp1和 2021F-FaCcp1 Therefore,we inferred that phenamacril obviously induce the activity of catalase in antioxidant system by directly or indirectly effecting on FaCcpl which can maintain low level of ROS,keep the expression of Tri4 down-regulation and then reduce the trichothecene mycotoxins accumulation in F.asiaticum.In S.cerevisiae,the Smi1 is a core protein for regulating the formation of cell wall,the protein plays role in β-1,3-glucan synthesis.Base on the provious result,we found that there are fourteen single amino acid mutant in the FgSmil of phenamacril-resistant strain,meanwhile these amino acid are the same as that in FaSmil of F.asiaticum wild-type strain 2021,in addition,five phenamacril-resistant strains domesticated from the parental strain 2021 have no any amino acid mutant in the FaSmi1.After treatment with phenamacril for 12h or 72h,the expression of FaSmil obviously down-regulate,indicating while the phenamacril run antifungal activity the FaSmil involved in this fungicide stress response.In our study,we generated the FaSmil deletion mutant in F.asiaticum using the protoplast transformation method to investigate whether the FaSmil involved in the inhibition of trichothecene mycotoxins synthesis by phenamacril.The FaSmil deletion mutant show decreased sensitivity to various fungicides,antioxidant system activity increased,the trichothecene mycotoxins production decreased,but under the sub-lethal concentration treatment,the toxin production ability has no difference between the FaSmil deletion mutant and the 2021 strain.Therefore,we infer that phenamacril inhibit the expression of FaSmil which lead to the low level of ROS there by leading to the trichothecene mycotoxins accumulation decreased,rather than direct action on the FaSmil in F.asiaticum.In addition,the deletion of FaSmil leads to the biological fitness of F.asiaticum decreased showing impaired in the mycelial growth rate of,asexual reproduction,and pathogenicity.The mutant showed increased sensitivity to osmotic stresses generated by NaCl and KCI was and decreased sensitivity to cell wall inhibitor(Congo red and Caffeine)and lyase.Septins are a highly conserved family of GTP-binding proteins is contribute to many cellular and metabolic functions including cell polarity,cytokinesis,cell morphogenesis,pathogenesis and interaction with protein coded by myosin 5.In this study,we characterized the septins FaCdc3 and FaCdc12 in the filamentous fungus F.asiaticum.In this study,The requirement of phenamacril regulate trichothecene mycotoxins production and function research of FaCdc3 and FaCdc12 were evaluated by constructing deletion mutants of FaCdc3 and FaCdc12,designated △FaCdc3 and △FaCdcl2,respectively.The results indicate the sensitivity of △FaCdc3 or △FaCdcl2 to phenamacril has no difference.The deletion mutants exhibited decreased accumulation of trichothecene mycotoxins in the sub-lethal condition.The toxin production between mutant and wild-type strain have no difference,which inferring that FaCdc3 and FaCdc12 indirectly involved in the inhibition of trichothecene mycotoxins production by phenamacril.In addition,△FaCdc3 and△FaCdc12 exhibiting a reduced rate of mycelial growth;increased aerial hyphae formation;irregularly shaped hyphae;reduced conidiation and lose the ability of sexual reproduction.The amount of large lipid droplets(LDs)in conidia and hyphae were increased,exhibited decreased sensitivity to various environmental stresses,the amount of septa decreased,aberrant nuclear distribution,cytoplasm divide abnormal,decreased virulence on wheat spikelets.All of these defects were restored by genetic complementation of the two mutants with the parental FaCdc3 and FaCdc12.These results indicate that FaCdc3 and FaCdc12 play critical role in various cellular processes and are essential for vegetable growth,reproduction development and pathogenicity in F.asiaticum. |
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