Expression Of Carbendazim Resistance In Fusarium Graminearum Heterokaryon And Resistance Monitoring Of Fusarium In Sichuan And Chongqing

Fusarium head blight（FHB）is a common soil-borne disease caused by Fusarium in cereal crop production.Carbendazim,tebuconazole and phenamacril（JS399-19）are effective in preventing and controlling FHB in production.However,long-term and large-scale use of fungicides has led to the development of resistance problems in Fusarium.The development of some fungal resistance is related to the variation caused by heterokaryosis and parasexuality.Heterokaryons formed by carbendazim-resistant and sensitive strains of Fusarium graminearum in the field demonstrated resistance to carbendazim.Therefore,exploring the expression pattern of carbendazim resistance in F.graminearum heterokaryons and monitoring the resistance of Fusarium in the field are of paramount theoretical and practical significance for the prevention and control of FHB.In this study,green fluorescent protein（GFP）and red fluorescent protein（RFP）were used to labelβ₂-tubulin of carbendazim-resistant and sensitive strains of F.graminearum.Then different marker strains were fused by protoplast fusion to generate carbendazim-sensitive heterokaryons and resistant heterokaryons.The morphology of heterokaryons and the fluorescence phenotype ofβ₂-tubulin were observed to elucidate the expression pattern of carbendazim resistance in heterokaryons of F.graminearum.In addition,field resistance monitoring of Fusarium in Sichuan and Chongqing in 2022was conducted to enhance the management of FHB.The main findings are as follows:1.Acquisition and biological characteristics ofβ₂-tubulin fluorescent labeled strains of F.graminearum.In this chapter,fluorescent proteins were inserted intoβ₂-tubulin of F.graminearum by the principle of homologous recombination to obtain fluorescently labeled strains.GFP or RFP gene was inserted into the terminal coding region ofβ₂-tubulin gene of carbendazim-resistant and sensitive strains by protoplast transformation.And fusion mutants were screened with hygromycin and geneticin as resistance tags.The results of PCR and fluorescence microscopy showed that GFP and RFP had successfully labeledβ₂-tubulin.The biological characteristics of the fusion mutant strains were determined.The results showed that the biological characteristics of the mutant strains after the fusion ofβ₂-tubulin with GFP or RFP were not significantly different from those of the parent strains.That is,the biological function of the fusedβ₂-tubulin gene was not affected.2.Acquisition of heterokaryons of F.graminearum and expression of carbendazim resistance.In this chapter,heterokaryons of F.graminearum were obtained by protoplast fusion so as to explore the expression pattern of carbendazim resistance in heterokaryons.The fusion mutant strains with different fluorescent tags were used as the starting strains to obtain carbendazim-resistant and sensitive heterokaryons using protoplast fusion techniques.The results of resistance screening and PCR verification showed that two different types of heterokaryons were successfully screened.The results of biological determination showed that the total growth level of carbendazim-resistant heterokaryons was higher than that of the original strains,and the total growth level of sensitive heterokaryons was similar to that of the original strains.Fluorescence observation of heterokaryotic hyphae showed that the reticular microtubule structure emitting green fluorescence could be observed in both heterokaryons,but no red fluorescence was observed.The results showed that carbendazim-resistance mutation ofβ₂-tubulin gene in heterokaryon of F.graminearum inhibited the expression of wild type,and heterokaryon alleles could not be expressed at the same time.3.Monitoring of resistant isolates of Fusarium in Sichuan and Chongqing.In 2022,the field resistance of Fusarium collected from Sichuan and Chongqing was monitored,and the Fusarium from Jiangsu was used as control.The results of species identification showed that all strains in Sichuan and Chongqing belonged to F.graminearum and F.asiaticum in the Fusarium graminearum complex species（FGSC）,and F.asiaticum was the dominant species.By measuring the sensitivity of Fusarium to carbendazim,tebuconazole and JS399-19,the sensitivity baseline was established according to the distribution of EC₅₀values.Statistical analysis showed that the EC₅₀values of Fusarium to carbendazim was between 0.0567 and 0.688μg/m L,and the sensitivity baseline was0.2857±0.138μg/m L.The EC₅₀values of Fusarium to tebuconazole was 0.0645～1.1756μg/m L,and the sensitivity baseline was 0.5357±0.253μg/m L.The EC₅₀values of Fusarium to JS399-19 was 0.1082～0.5031μg/m L,and the sensitivity baseline was0.3231±0.084μg/m L.The field resistance of Fusarium to the above three fungicides in Sichuan and Chongqing was determined.The results showed that carbendazim resistant strains appeared in Chongqing and Sichuan,the resistance frequency was 4.37%,and the resistance factor was between 5 and 9.All resistant strains were low resistant.The resistance frequency of the control group in Jiangsu was 52.63%,and the resistance factor was between 5 and 14.All resistant strains were low resistant.No tebuconazole-resistant strains and JS399-19-resistant strains were found in Sichuan,Chongqing and Jiangsu.By identifying the mutation types of carbendazim-resistant strains,the results showed that no mutation sites were detected in carbendazim-resistant strains in Sichuan and Chongqing.While six of the resistant strains in Jiangsu showed F167Y point mutation and one showed F200Y point mutation.The rest resistant strains did not have any mutation loci detected.In summary,this study obtained carbendazim-resistant and sensitive heterokaryons with fluorescent tags by protoplast fusion.It was revealed that the carbendazim-resistant mutation ofβ₂-tubulin gene could inhibit the expression of wild type in heterokaryons of F.graminearum,and the alleles could not be expressed simultaneously.The field resistance of F.graminearum to carbendazim,tebuconazole and JS399-19 was monitored.It lays a foundation for studying the expression pattern of heterokaryon alleles of homothallic fungi and preventing and controlling Fusarium head blight.