Functional Analysis Of 3 Target Genes Of Phenamacril In Fusarium Oxysporum F.sp.Cubense Race 4

Banana wilt is a vascular systemic disease caused by Fusarium oxysporum f.sp.cubense（Foc）.Among them,Race1（Foc1）and Race4（Foc4）are the most harmful.At present,there is no complete and effective control method.Chemical control lacks a good control effect,and will cause many problems such as serious loss of chemicals in the field,drug resistance and harm to human health.Due to defects such as long breeding cycle and loss of disease resistance,it is difficult to break through breeding of disease-resistant varieties.The control effects of biological control in field production systems are not ideal.A more advanced,efficient and stable method for controlling banana wilt disease is yet to be studied.In recent years,scholars have made some progress in the research on the disease-causing genes of banana wilt and the pathogenesis of these genes.Therefore,by studying the genomics of banana wilt pathogen,it is clear that the pathogenic mechanism of banana wilt is the key to effectively solve the control problem.Phenamacril（Test No:JS399-19）is a new type of pesticide with specific antibacterial activity against Fusarium.It has a good inhibitory activity on most Fusarium,effectively preventing wheat scab and inhibiting DON toxin production.It has the advantages of high activity,strong specificity,and without drug resistance,which is expected to replace other types of fungicides.In order to reveal the specific functions of the pesticide target genes,based on Fusariurn graminearum,which was resistant to Phenamacril,the genes with a single mutation were analyzed and identified,including AAPINDA1,NTH1 and ACO,Subsequently,sequence analysis and comparison were performed in Fusarium oxysporum GenBank,and three genes AAPINDA1,NTH1 and ACO of Fusarium oxysporum（Foc4）were obtained.In this study,the gene expression levels of three genes were measured under the treatment of Phenamacril at concentrations of 0 μg/mL,0.5 μg/mL and 50 μg/mL respectively.AAPINDA1 gene（XM₀31203202）encoding amino acid permeable enzyme protein,NTH1 gene（XM₀31206259）encoding neutral trehalase protein,and ACO gene（XM₀31215663）encoding aconitate hydratase protein were cloned and identified.The three mutantsΔaapindal,Δnth1 and Δaco were obtained by knockout technique.The pathogenicity and biological characteristics of these three mutants were analyzed and identified.and their pathogenicity,biological characteristics and resistance to Phenamacril were analyzed.The results are as follows:the expression levels of AAPINDA1 and NTH1 genes were significantly decreased with the increasing concentration of cyanocinate treatment.At first,the expression level of ACO gene was not increased significantly with the increase of the treatment concentration of Phenamacril,while then significantly decreased.The results are as follows:The expression levels of AAPINDA1 and NTH1 decreased significantly with the increase of the concentration of Phenamacril,and the expression levels of ACO decreased significantly with the increase of the concentration of Phenamacril.The pathogenicity of the Aaapindal mutant decreased,and the colony growth rate did not change significantly,but on the PDA medium,the colony growth size was significantly reduced,the colony color was pink,and the aerial hyphae and conidia production were significantly reduced,while There is no difference in spore morphology and spore germination,and it has obvious resistance to Phenamacril（EC50=9.77 μg/mL）;the pathogenicity and colony growth of the Δnth1 mutant both decreased,and the spore morphology and number did not change significantly.,While the germination rate of spores decreased significantly,and the resistance level to Phenamacril did not change significantly（EC50=6.07 μg/mL）;the pathogenicity of the Δaco mutant,the growth state of the strain,the germination and number of spores did not change,And the spore morphology has changed,and it has obvious resistance to Phenamacril（EC50=8.19 μg/mL）.The resistance adaptation analysis of the three mutants showed that Aaapindal did not participate in cell wall synthesis and response to oxidative stress,but increased sensitivity to high glucose;Δnth1 was involved in cell wall synthesis and oxidative stress response,but did not respond to osmotic pressure.,High sugar,high temperature and low temperature stress factors have no significant difference with the wild type;Δaco does not participate in the response to osmotic pressure,high sugar and high and low temperature stress.This study provides scientific basis for enriching the specific functional knowledge of three resistance-related genes and developing target genes for pesticide control.