| Banana wilt disease was a destructive soil borne disease caused by Fusarium oxysporum f.sp.cubense(Foc)that destroyed the banana vascular bundle and caused plant death.Racel(Foci)and Race4(Foc4)were the most important pathogenicity species.The pathogenic fungi could survive in the soil for a long time and belong to typical latent infection in the soil.In actual production,there was a lack of effective methods for the prevention and control of Foc4.Chemical agents and biological control were used to control Foc4,which was not ideal.The resistant varieties were also easy to lose resistance due to the evolution of pathogens.It is the key to solve the problem that we should clarify the molecular mechanism of the Foc4.Benzimidazole fungicides have been widely used in the prevention and control of the disease caused by Fusarium,with the characteristics of wide spectrum,high efficiency and internal absorption.This kind of fungicide mainly acts on the beta-microtubulin of the pathogenic fungi.Fungicide inhibits mitosis by inhibiting the formation of spindle.However,pathogenic fungi were easily resistant to fungicide selection pressure due to the single location of fungicid,which leads to a decrease in the efficacy of fungicide.Another cyanoacrylate fungicide JS399-19 was a new type of fungicide which had good antifungal activity for most Fusarium.In recent years,studies had shown that the JS399-19 acted on the myosin I of pathogenic fungi.The JS399-19 inhibited the growth and development of pathogenic fungi by inhibiting the activity of ATPase enzyme.In order to reveal the specific function of the target gene of fungicide.The sensitivity of Foc4 to carbendazim and JS399-19 was determined in this study.The results showed that the Foc4 was sensitive to the two kinds of fungicides.The genes of ?1-tub(MF668108),?2-tub(MF668109)and myosin-1(MG601753)of Foc4 was cloned and identified.?1-tub gene encoded a protein containing 446 amino acids.?2-tub gene encoded a protein containing 448 amino acids.myosin-1 gene encoded a protein containing 1228 amino acids.We find that these 3 genes are highly conserved by homologous comparison.In this study,split-marker gene knockout technology was used,and protoplasts were transformed by PEG mediated method.The 3 genes were knocked out by homologous recombination,and the ?1-tub knockout mutant,?2-tub knockout mutant and myosin-1 knockout mutant of Foc4 were obtained.The functions of these 3 knockout mutants,including their growth phenotypes,sensitivity to fungicides and pathogenicity,were revealed respectively.The results were as follows.Compared with the Foc4 wild strain,the growth rate of the ?1-tub knockout mutant was obviously weakened and the mycelium growth was deformed.The sporulation increased and the sensitivity to carbendazim did not change significantly.The pathogenicity of potted plants banana seedlings was significantly reduced.The growth rate,mycelia growth morphology and sporulation amount of the,?2-tub knockout mutant did not change significantly,and the sensitivity to carbendazim increased,and the pathogenicity of potted plants banana seedlings decreased.Compared with Foc4 wild strain,the growth rate of myosin-1 knockout mutant was obviously decreased.The growth of mycelium was deformed and its branches increased.The amount of sporulation decreased significantly.The sensitivity to JS399-19 was obviously weakened.The pathogenicity of potted plants banana seedlings was significantly reduced.In addition,the sensitivity of the three knockout mutants to stress factors was detected,and compared with Foc4 wild strain,three knockout mutants were insensitive to cell wall selective pressure,oxidative stress and osmotic pressure.All the results showed that the ?1-tub,,?2-tub and myosin-1 genes play an important role in the growth,pathogenicity and sensitivity of fungicides to Foc4.These studays enrich the pathogenic mechanism of Foc4 and provide a reference for the development of the new fungicide target. |
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