Resistance Management And Synergistic Interaction Of SYP-7017 And Boscalid In Controlling Sclerotinia Stem Rot

The plant-pathogenic fungus Sclerotinia sclerotiorum(Lib.)de Bary has a broad host range and a worldwide distribution.In China,Sclerotinia stem rot seriously reduces the yield and quality of rapeseed.For many years,the control of Sclerotinia stem rot in China has mainly depended on the fungicides carbendazim and dimethachlon.Because of the extensive application of these fungicides,however,fungicide-resistant mutants have developed for S.sclerotiorum and for several other Sclerotinia spp.Strains collected from Sclerotinia-infected stems of oilseed crops in Jiangsu province between 2010 and 2012,were tested for their sensitivity to carbendazim and dimethachlon in order to determine the level of resistance to these fungicides in the population of S.sclerotiorum in that province.Sensitivity tests showed that carbendazim resistant frequency decreased year by year;Among the 3710 isolates,only one isolate in the Lian Yungang region of Jiangsu Province in 2010 showed normal growth at 5?g/ml dimethachlon.The proportion of carbendazim-resistant isolates ranged from 0-44%in different regions of Jiangsu.Moreover,S.sclerotiorum exhibited positive cross resistance between carbendazim and other benzimidazole fungicides;negative cross resistance between carbendazim and diethofencarb.Cross-resistance was also observed between dimethachlon and other dicarboximide fungicides such as iprodione and procymidone.QoIs SYP-7017 and SDHIs boscalid were selected for in vivo and vitro experiments.Results showed that SYP-7017 exhibited better protective than curative activity.However,boscalid showed excellent protective and curative activity.Althoug these two fungicides could be good alternatives to benzimidazoles or dicarboximides fungicides for the control of Sclerotinia stem rot,the work to explore the risk and the mechanism of resistance to SYP-7017 or boscalid in S.sclerotiorum is needed.Baseline sensitivity of S.sclerotiorum to SYP-7017 was determined using 150 isolates.The 50%effective concentration(EC50)values for SYP-7017 inhibiting mycelial growth ranged from 0.006 to 0.047?g/ml.The current study characterized the S.sclerotiorum SYP-7017 or boscalid-resistant(BR)mutants obtained by fungicide induction.However,isolates resistant to SYP-7017 with low resistant frequency is not stable.Moreover,no cross-resistance was detected between SYP-7017 and other QoIs.These results indicate that the risk of resistance to SYP-7017 in S.sclerotiorum is low.However,positive cross-resistance was detected between boscalid and carboxin;and negative cross-resistance was detected between boscalid and kresoxim-methyl.Compared to their parental isolates,BR mutants had slower radial growth,no ability to produce sclerotia,lower virulence and lower oxalic acid content but higher mycelial conductivity and respiration,and succinate dehydrogenase(SDH)activity.Moreover,BR mutants had decreased sensitivity to salicylhydroxamic acid(SHAM)but not to oxidative stress.All the results indicate that the risk of resistance to boscalid in S.sclerotiorum is low to moderate.In many fungal pathogens,point mutations in cyt b or SDH genes are considered for QoIs or SDHIs resistance,respectively.However,no point mutation in cyt b gene was found in SYP-7017 resistant isolates.Interestingly,only in SdhB gene one new point mutation(GCA to GTA)was found in boscalid-resistant mutants,which lead to amino acid mutated from alanine to valine,no other mutation was found.In the current study,replacement of the SdhB gene in a boscalid-sensitive S.sclerotiorum strain with the mutant SdhB gene conferred resistance.Compared to wild-type strains,BR and GSM(SdhB gene in wild-type strain replaced by mutant SdhB gene)mutants lacked the ability to produce sclerotia,and had lower expression of pac1 gene.Importantly,the point mutation was not located in the highly conserved sequence of SDHB subunit.These results suggest different mechanisms might conferred QoIs resistance and resistance based on non-conserved vs.conserved protein domains differs in mechanism.In summary,the risk of resistance to SYP-7017 in S.sclerotiorum is low and the risk of resistance to boscalid in S.sclerotiorum is low to moderate.Different mechanisms might confered with QoIs resistance and point mutation in SDHB subunit(A11V)conferred boscalid resistance in S.sclerotiorum.Mixed fungicides could be not only good alternatives to benzimidazoles or dicarboximides fungicides for the control of Sclerotinia stem rot,but also the best method for delaying fungicide resistance.