Effects Of FUB4 And FUB11 Genes Of Fusarium Oxysporum F.sp.niveum On The Inhibition Of Fusaric Acid Toxin Synthesis By Fuminic Acid And Its Function

Cuminic acid is a natural small molecule organic acid extracted and isolated from the seed of Cuminum cyminum.Previous studies found that it has a strong inhibitory effect on a variety of plant pathogens,among which the antifungal activity against Fusarium oxysporum f.sp.niveum is very significant.It can not only inhibit the mycelial growth of Fusarium oxysporum f.sp.niveum,but also significantly reduce the content of fusaric acid toxin（FA）of Fusarium oxysporum f.sp.niveum.However,the mechanism of its inhibition of toxin synthesis is not clear.Therefore,in this study,the expression of fusaric acid toxin synthesis gene（FUB1-FUB12）was determined by q RT-PCR,the genes with large differential expression were screened for gene deletion and complement test,and their physiological and biochemical indexes were measured and compared with the wild strain,in order to verify the function of the above genes in the inhibition of fusaric acid synthesis by cuminic acid.At the same time,it provides a basis for further clarifying the action mechanism of cuminic acid.The main results are as follows:1.The effects of cuminic acid treatment on the expression of 12 genes in fusaric acid toxin synthesis gene（FUB）cluster were determined.The results showed that after treatment with cuminic acid,except that the specific transcription factor FUB10 gene in the toxin synthesis gene cluster was up-regulated by 1.68 times,the other FUB genes were down regulated in varying degrees,among which FUB4 and FUB11 genes were down regulated by0.24 times and 0.34 times,respectively.2.Taking Fusarium oxysporum f.sp.niveum as the test strain,FUB4 gene encoding serine hydrolase,FUB11 gene encoding MFS transporter and FUB10 gene encoding pathway specific transcription factor in fusaric acid toxin synthesis gene cluster were knocked out and complemented.The transformants were verified by PCR and Southern hybridization,and the deletion and complementation mutants were successfully obtained（ΔFUB4、ΔFUB11、ΔFUB4-C、ΔFUB11-C）,but no deletion mutant of FUB10 gene was obtained.3.The results of physiological phenotypes of FUB4 and FUB11 gene deletion and complement mutants showed that compared with wild strain,the growth rate and sporulation quantity ofΔFUB4 decreased,and the pathogenicity decreased.The pigmentation ofΔFUB11 colony almost disappeared,and the growth rate and sporulation of mycelia increased compared with wild strain,but the pathogenicity was weakened.The cell membrane permeability and POD activity ofΔFUB11 increased significantly,and the sensitivity ofΔFUB11 to osmotic stress factor and cell wall stress factor was increased,but the sensitivity ofΔFUB11 to oxidative pressure was decreased significantly.The above changes in all complement mutants returned to similar levels of wild strain.These results indicated that FA toxin affected the growth of pathogen and played a role in host defense.4.The FA toxin content in the mutant culture medium was analyzed by liquid chromatography,and it was found that the FA content of the gene deletion mutantΔFUB4was reduced by 98%compared with the wild strain.q RT-PCR results showed that the expression levels of other FUB genes inΔFUB4 decreased to varying degrees.The FA content ofΔFUB4 treated with cuminic acid decreased,but there was no significant difference compared withΔFUB4 without cuminic acid treatment.The sensitivity ofΔFUB4to cuminic acid decreased but was not significant,and the EC₅₀value increased to 1.55 times that of wild strain.It indicated that FUB4 gene had a positive regulatory effect on FA synthesis,but FUB4 gene was not directly involved in the inhibition of FA synthesis by cuminic acid.5.Liquid phase analysis found that FA was not detected in the culture medium ofΔFUB11.The results of q RT-PCR showed that,except for the significant increase in the expression of FUB12,which is involved in FA derivatization,the expression of other FUB genes inΔFUB11 did not change significantly.FA content in the corresponding complement mutant recovered to the level of wild strain,indicating that FUB11 gene plays an important role in regulating the extracellular transport of FA,but does not directly regulate the synthesis of FA.The accumulated FA in the cell may be converted into less toxic FA derivatives under the action of the FUB12 gene.The sensitivity ofΔFUB11 to cuminic acid was significantly reduced,and the EC₅₀value increased 2.84 times that of wild strain.These results suggest that FUB11 gene plays an important role in cuminic acid inhibition of FA toxin transport.In conclusion,FUB4 and FUB11 genes play important regulatory roles in FA synthesis and transport.FUB4 gene is not directly involved in the inhibition of FA synthesis by cuminic acid.The decrease of FA content after cuminic acid treated may be the after-effect caused by cuminic acid.In addition,cuminic acid may interact with FUB11 gene encoding toxin transporter to affect FA transport,and jointly reduce FA toxin content of Fusarium oxysporum f.sp.niveum after treatment,reducing the pathogenicity of the pathogen and the defense response to the host,so as to prevent and control Watermelon Fusarium wilt.