Functional Analysis Of Three Genes Related To Phenamacril Susceptibility In Fusarium Oxysporum F.sp.cubense Race 4

Banana wilt is a devastating soil-borne disease caused by Fusarium oxysporum f.sp cubense（Foc）.The most common pathogens in China are race 1（Focl）and race 4（Foc4）,which belong to the typical latent infections.They have saprophytic ability and can survive in the soil for a long time.The traditional medicaments are not ideal for the control of banana wilt in the actual production.The widespread use of pharmaceutical agents increased the resistance of pathogens.Phenamacril（JS399-19）is a new fungicide with unique action mode,novel structure,low price and no stimulation to human skin.It has strong specific activity against Fusarium,mainly acting on the myosin I of pathogenic bacteria.It can inhibit the growth and development of pathogenic bacteria by inhibiting the activity of the ATPase.However,Fusarium is easily resistant to it in vitro1.In order to reveal the specific function of the fungicide target gene,three genes tha t could be related to phenamacril resistance were obtained based on the mutation site associ ated with phenamacril resistance in Fusarium graminearum.The homologous genes Smy1,FOIG 05203 and FOIG₁5754,were obtained by alignment with Fusarium oxysporum in NCBI2.This experiment cloned and identified the smyl gene（ESU11006）of Foc4,encoding 954 amino acids;FOIG 05203（XM₀11323613.1）,encoding protein containing 924 amino acids;FOIG₁5754（XM₀11325720.1）,encoding protein containing 526 amino acids After homologous alignment,these three genes were highly conserved3.The Split-marker knockout technique was used to knock out smyl,FOIG₀5203.and FOIG₁5754,and the growth state,growth rate,spore yield,the Phenamacril sensitivity,and abiotic stress to Brazilian banana.The specific results were as follows（1）Compared with wild Foc4,the growth rate of △smy1 knockout mutant mycelium decreased significantly,mycelial malformation,sporulation increased,and the sensitivity to Phenamacril was not significantly reduced.（EC50 6.41 μg/mL）,the phytotoxicity to Brazilian banana was significantly reduced.In the abiotic stress test,the mutant was not sensitive to osmotic pressure and cell wall selective pressure,sensitive to SDS and oxidative stress.（2）Knockout mutant of △FOIG₀5203 mycelium grew slightly slower than the wild type,the sporulation decreased,the mycelial growth status did not change significantly,and the sensitivity to Phenamacril was slightly reduced（EC50 8.73 μg/mL）.There was no effect on the change of pathogenicity,it was sensitive to oxidative stress,and it was not sensitive to osmotic pressure,cell wall selection pressure and SDS.（3）The growth rate of △FOIG₁5754 knockout mutant mycelium did not change significantly compared with wild-type strain,and sporulation increased.There was no significant change in mycelial growth status,and the sensitivity to phenamacril was reduced（EC50 7.52 μg/mL）.The pathogenicity of Brazilian banana was not significantly reduced.In the abiotic stress test,the mutant was sensitive to oxidative stress,osmotic pressure,cell wall selection pressure and Insensitive to SDS;but the growth rate of wild-type and mutant strains was increase under stress sorbitol.The above results indicate that the smy1、FOIG₀5203 and FOIG₁5754 genes play an important role in the growth and development of Foc4,pathogenicity and sensitivity to phenamacril.This study enriched the pathogenesis of banana and Fusarium oxysporum and provided a reference for the development of new fungicide targets.