Risk Assessment And Resistance Mechanism Of Rhizoctonia Solani To Thifluzamide

Rhizoctonia solani is an important soil-born pathogen.It can infect rice in whole growth period and cause serious losses.Thifluzamide is a succinate dehydrogenase inhibitor(SDHI)fungicide and has excellent control effect to rice sheath blight.However,there are few reports about its resistance risk or resistance mechanism.In this study,we investigated the resistance risk,resistance mechanism and evolution of resistance to thifluzamide in R.solani.The activity of thifluzamide to different fungi or oomycetes was determined by mycelia grown inhibition and spore germination assay.Thifluzamide exhibited good inhibition effect on Ustilago maydis,Sporisorium holci-sorghi,Ustilago nuda,Rhizoctonia solani and Rhizoctonia cerealis.The EC50 values were lower than 0.5μg/mL.It can also inhibit Sclerotinia sclerotiorum,Alternaria solani,Pyricularia oryzae and Gaeumannomyces gramims.There was no activity on the other ten tested pathogens.The baseline sensitivity of 226 isolates ranged from 0.0041～0.1549 μg/mL with a mean EC50 value of 0.0351 μg/mL.There was no significant shift in sensitivity of isolates which collected from different years and locations.These data can be used for resistance monitoring in fields.A series of thifluzamide-resistant mutants were acquired.One thifluzamide-resistant isolate was obtained from the field with resistance factor of 24.66.Sixteen laboratory mutants were generated via exposure of sensitive isolates to thifluzamide-amended media and to UV radiation.The mutant T2 exhibited the highest resistant factor of 272.32,and others were 14.54-62.79.The resistance mechanism of R.solani to thifluzamide was clarified.Four succinate dehydrogenase genes(sdhA,sdhB,sdhC and sdhD)in both wild-type isolates and resistant mutants were cloned and analyzed.Two types of thifluzamide-resistant mutants were observed in SDHB subunit.Type Ⅰ mutants were heterokaryons with both histidine(H249)and tyrosine(Y249),type Ⅱ mutants were homokaryons with one allele of Y249 in the SDHB subunit.Relative to thifluzamide EC50 values of sensitive parental isolates,EC50 values were 100-fold or less higher for type Ⅰ mutants and 250-fold higher for type Ⅱmutants.No overexpression was found in sdhB gene under thifluzamide treatment.Docking studies indicated that the point mutation at position 249 reduced the affinity of the SDH pocket for thifluzamide.One of seven heterokaryotic mutants evolved into a homokaryon when repeatedly cultured on agar media or on rice plants with and without thifluzamide treatment;the genotypes of sensitive isolates and homokaryotic mutants,however,did not change in response to these treatments.The resistant mutants had no less fitness than the sensitive isolates,which means the resistant mutants had the ability to become dominant subpopulation.Cross resistance was found between thifluzamide and boscalid,penflufen,carboxin,fenfuram,but not between thifluzamide or difenoconazole,propiconazol,carbendazim,SYP-2815,iprodione,fluazinam,pyrimorph,jinggangmycin.Negative cross resistance was found between thifluzamide and mepronil.Fluopyram had no inhibitory activity against R.solani.Based on the point mutation on sdhB gene,CAPS and AS-PCR methods were established for resistant mutant molecular detection.The fungicide combination containing thifluzamide,jinggangmycin and prochloraz as a ratio of 1:5:2 was screened.16%suspension concentrate showed good field control effects on rice sheath blight,rice blast and false smut.