Biological Properties And Genotypes Of Boscalid-and Pyrimethanil-Resistance In Sclerotinia Sclerotiorum Collected From Lettuce Field In Jiangsu Province

Sclerotinia stem rot disease caused by sclerotinia sclerotiorum is a serious fungal disease on lettuce,causing serious losses.S.sclerotiorum infects a wide range of important vegetables and oil crops,such as rapeseed,cabbage,carrots,soybeans,and sunflowers.Benzimidazole fungicides,such as carbendazim,and dicarboximide fungicides,such as dimethachlon,are widely used to control this disease for decades.However,resistant isolates to two fungicides above have been monitored in the field.Therefore,the diseased samples were collected from Changshu,Wuxi,Xuzhou,Yancheng and other regions of Jiangsu Province,were isolated and purified.A total of 172 S.sclerotiorum isolates were separated to detect resistance frequencies to six groups of fungicides,including boscalid,pyrimethanil,azoxystrobin,fluazinam,procymidone and tebuconazole.The biological properties and molecular mechanism of resistant isolates to boscalid and pyrimidine were studied.Double-resistant(boscalid and pyrimethanil)populations S.sclerotiorum were established in Jiangsu Province,with resistant frequency of less than 40%.The sensitivity and resistance frequency of 172 S.sclerotiorum to six fungicides were determined.Of the 172 S.sclerotiorum,76.99%was highly resistant to boscalid,whereas 41.62%was moderately resistant to pyrimethanil.No resistant isolates against azoxystrobin,tebuconazole,procymidone or fluazinam were detected.Three resistant phenotypes were founded,including 68 single-resistant(boscalid)isolates with a frequency of 39.53%,12 singleresistant(pyrimethanil)isolates with a frequency of 6.98%,and 64 double-resistant(boscalid and pyrimethanil)isolates with a frequency of 37.21%.Boscalid-resistant isolates of S.sclerotiorum exhibited high biological fitness.Most resistant isolates exhibited higher mycelial growth and stronger sclerotium production than the sensitive isolates,and similar virulence to the sensitive isolates.There was significant difference in intracellular glycerol contents between resistant and sensitive isolates.The EPS content of resistant isolates was higher than that of the sensitive isolates,while the cell membrane permeability was lower than that of sensitive isolates.After treated with boscalid,the oxalic acid content of all isolates decreased.The sensitivities of all isolates to different stress factors differed markedly.No cross-resistance between boscalid and thifluzamide,pydiflumetofen,fluazinam,or tebuconazole.SDH gene sequencing showed that 3 mutation types of eight resistant isolates were found,including single point mutation(A11V)at SDHB,single point mutation Q38R at SDHC,and double point mutations A11V(at SDHB)+Q38R(at SDHC).Docking total score(DTS)of the sensitive isolates was higher than that of the resistant isolates,suggesting that the sensitive isolates had a higher affinity of boscalid than resistant isolates.Biological fitness varied significantly with the pyrimethanil-resistant isolates of S.sclerotiorum.The mycelial growth and sclerotium production of the pyrimethanil-resistant isolates were similar to that of the sensitive isolates.The virulence result showed that resistant isolates exhibited significantly high pathogenicity on lettuce stem than that of sensitive isolates,but not on lettuce or rapeseed leaves.The EPS content of resistant isolates was lower than that of sensitive isolates.When treated with different stress agents,the inhibitive mycelial growth rates of the pyrimethanil-resistant isolates varied.The intracellular glycerol contents and membrane permeability of pyrimethanil-resistant isolates increased when treated with pyrimethanil.No significant difference was observed in the oxalic acid contents of all the isolates after treatment with pyrimethanil.A single-point mutation F780S was detected at MDL of the resistant isolates.The docking total score of the sensitive isolates was higher than that of the resistant isolates,indicating that the affinity of the sensitive strain between MDL and pyrimethanil was higher than that of the resistance isolates.MIC(mininum inhibitive concentration)is recommened to determine the in-vitro sensitivity of S.sclerotiorum against pyrimethanil.S.sclerotiorum is easy to produce fastgrowing sectors on L-asp medium,which causes abnormal colony,interfering with accurate determination of the in-vitro sensitivity against pyrimethnil.Therefore,fast-growing sectors of pyrimethanil-resistant and-sensitive isolates from S.sclerotiorum were obtained on L-asp medium to compare with the biology of the fast-growing sectors.The result was exhibited that the mycelial growth of the fast-growing sectors was decreased compared to its parental strain.The sclerotia-producing ability of the resistant strain CS18 fast-growing sectors was higher than that of the parent strain,while the sclerotia product ability of resistant strain CS52 fast-growing sectors lower than that of the parent strain.The membrane permeability of fastgrowing sectors of resistant isolates was enhanced,while the membrane permeability of fastgrowing sectors of sensitive isolates was decreased.After treatment with the stress agents,the mycelial growth of fast-growing sectors of resistant isolates varied with the isolates.The intracellular glycerol contents of fast-growing sectors of resistant isolates decreased when treated with pyrimethanil.No significant difference was found in the oxalic acid content of resistant fast-growing sectors treated with pyrimethanil.When transferred to PDA plates 3 times,the sensitivity of fast-growing sectors to pyrimethanil return to their respective original levels.