Monitoring And Moleculer Mechanisms Of Resistance To Boscalid By Didlymella Bryoniae In Southwest China

As an important commercial crop,watermelon has a huge planting area in China.Watermelon stem blight(caused by Didymella bryoniae)is one of the serious diseases,which has great destruction to yield and quality of watermelon.At present,the main controlling measure in China is chemical application,such as carbendazim and difenoconazole.Inevitably,long-term usage of fungicids could cause resistance to watermelon stem blight,which not only reduce the control effects but also increase the control cost.Boscalid is the most popular fungicide in SDHIs,which has been used in many crops to prevent different disease.However,the report of boscalid to prevent and control stem blight is still less.Therefore,this paper carried out the research on sensitivity baseline,resistance monitoring,biological characteristics,cross resistance and molecular mechanism of mutant isolates of D.bryoniae collected from southwest China.These results will provide a theoretical basis to prevention strategies for fungicide application and resistance risk in watermelon stem blight.1.Sensitivity baseline and resistance monitoring of D.bryoniae to boscalidA hundred isolates of D.bryoniae collected in Chongqing Municipality,Guizhou Province,and Sichuan Province were selected randomly and their sensitivity to boscalid was measured by using the mycelial growth diameter method.The results showed that the EC50 value were ranged from 0.033 ?g/m L to 0.180 ?g/m L;the average EC50 value was 0.085 ± 0.037 ?g/m L;the maximum EC50 value was 5.4 times compared to the minimum EC50 value;and these isolates were all collected from Hechuan Distract,Chongqing.The frequency of those 100 isolates to boscalid sensitivity was successive distribution,and the frequency distribution approximate normal monopeak curve.Therefore,the average EC50 value can be used as the sensitivity baseline to evaluate the sensitivity of D.bryoniae to boscalid.The MIC values of those 100 isolates to boscalid were range from 4 ?g/m L to 10 ?g/mL.Based on the MIC value and sensitivity baseline,10 ?g/mL was assigned as the differential dose to monitor the boscalid-resistance and boscalid-sensitive isolates of D.bryoniae.Subsequently,615 isolates collected in 2014 to 2016 were monitored,and the result showed that no resistance isolate to boscalid in the felid,and the resistance frequency was 0,indicating that isolates in southwest China were sensitive to boscalid,and boscalid could be used as carbendazim alternative fungicide to control watermelon stem blight in these areas.2.Obtain and genetic stability of D.bryoniae boscalid-resistant mutantsFour suspected resistant mutant isolates were obtained by chemical taming and UV irradiation.Of the four isolates,two of them belonged to intermediate boscalid-resistant mutants and other two isolates belonged to high boscalid-resistant mutants of D.bryoniae.EC50 values of two intermediate boscalid-resistant mutants,XF98-1 and XF21-1,were 1.489 ?g/mL and 1.182 ?g/mL,respectively(resistance factors of 15 and 14,respectively).EC50 values of two high boscalid-resistant mutants,XF21-3 and YC60-1,were 108.073 ?g/m L and 124.650 ?g/m L,respectively(resistance factors of 1,010 and 1,340,respectively).After continuously culture on PDA plates without the fungicide for 8 generations,their sensitivities to boscalid were measured.The final results showed that two intermediate boscalid-resistant isolates became sensitive to boscalid,suggesting that their resistance cannot heredity stable.In contrast,EC50 values of two high boscalid-resistant mutants,XF21-3 and YC60-1,were 73.046 ?g/m L and 91.971 ?g/mL,respectively(resistance factors of 683 and 989,respectively),indicating these two mutants still keep their high resistance,and their resistance can heredity stably.Therefore,XF21-3 and YC60-1 were used to study biological characteristics and molecular mechanisms.3.Biological characteristics of boscalid-resistance mutantsThe growth,breeding,pathogenicity and other biological characteristics of two high resistant mutants were compared to parental strain.The results showed that the mycelial growth rate of one high boscalid-resistance mutant was faster than parental strain.However,another high boscalid-resistance mutant showed no significant difference with parental strain.There has no significant difference on optimum growth temperature for mycelium growth between mutants and parental strain,and the optimum growth temperature remains 20?-25?.The sporulation of one high boscalid-resistance mutant was higher than parental strain,but another high boscalid-resistance mutant still showed no significant difference with parental strain.Compared to their parental strains,two resistant mutants showed lower sensitivity to the high osmotic pressure.Pathogenicity test results suggested that no significant difference between mutants and parental strains.Above results revealed that there was no obvious difference on comprehensive fitness of boscalid-resistance between mutants and parental strains.Cross-resistance of boscalid to carboxin,thifluzamid,diethofencrab,tebuconazole,and kresoxoim-methyl were tested.The results showed that mutants and parental strains were all sensitive to carboxin(SDHI),diethofencrab(Carbamates),tebuconazole(DMI),and kresoxoim-methyl(QoI),suggesting there was no cross-resistance between them.However,boscalid-resistant mutants resistant to thifluzamid(SDHI),indicating that there was a cross-resistance between boscalid and thifluzamid.Therefore,boscalid and thifluzamid should not application interchangeably,whereas choose other types of fungicide as tebuconazole.4.Molecular mechanisms of boscalid-resistance mutantsSpecific primers WSQ1(5'-CTTGTCCCTGACATGACACTT-3')and WSQ2(5'-TCCTTGAGCAGTTGAGAATGG-3')were designed and used to amplification of Sdh B gene of XF21-3,YC60-1 and XF21,YC60,and a 337 bp sequence of genomic DNA fragment was obtained.Sequencing and alignment results show that the coding sequences at site 277 in Sdh B gene was mutated from CAC to TAC(amino acid was mutated from His to Tyr),and other amino acid sites had no change.Therefore,we initially estimated the reason for boscalid-mutant was that the amino acid at site 277 in Sdh B gene was mutated from His to Tyr.