Resistance Of Botrytis Cinerea To Pyridoxazole And Boscalid In Shangdong Province And A Novel Management Way

Gray mold is a common fungal disease in protected cultivation,which can infect the flowers,fruits,leaves and stems of crops,thus causing serious yield and quality losses to agricultural production.At present,chemical control is the main management strategy.However,due to its short life cycle,prolific reproduction,and high genetic variability,Botrytis cinerea has developed resistance to a variety of fungicides.In Shandong Province,boscalid,succinate dehydrogenase inhibitor?SDHI?,and pyridoxazole,sterol demethylase inhibitor?DMI?,are commonly used fungicides for controlling gray mold.In recent years,this study systematically determine the sensitivity of B.cinerea to the two fungicides,and examined the relationship between the mutation types of isolates and the resistance.The ability of two new SDHI fungicides,benzovindiflflupyr and pydiflumetofen,to control gray mold was preliminarily evaluated,and explored the feasibility of dipping flower application to control gray mold.The main results are as follows:1.From 2017 to 2019,A total of 283 B.cinerea isolates were used to measure sensitivities to pyridoxazole based on measurements of mycelial growth.In the past three years,there was no significant difference in the sensitivity of B.cinerea to pyrisoxazole.The mean EC₅₀0 values for the past three years were 0.12±0.01 mg L^-1,0.16±0.01 mg L^-1and0.13±0.02 mg L^-1,respectively.This indicated that the pyridoxazole still has a strong inhibitory activity against B.cinerea.However,some insensitive isolates were detected every year.In insensitive isolates,three different point mutations were detected in CYP51 gene,which include alterations from valine acid to isoleucine?V24I?at codon 24,from tyrosine acid to phenylalanine?Y136F?at codon 136 and from arginine to lysine?R464K?at codon464.The CYP51 gene of the sensitive and three mutant isolates were constructed on the pPIC9K vector,and transformed into P.pastoris GS115.The sensitivity of the transformant to pyrisoxazole was determined.The results found that only V24I and Y136F mutations were associated with pyrisoxazole resistance.However,R464K mutation was not associated with pyrisoxazole resistance.This study found that the CYP51,Bcmfs1 and BcatrD can be over-expressed in insensitive isolates,while the expression of the two transporter genes BcatrB and BcmfsM2 related to multiple resistance did not increase significantly.In addition to V24I and Y136F mutation,overexpression of the three genes CYP51,Bcmfs1 and BcatrD may also be involved in the resistance of pyrisoxazole.In addition,insensitive isolates have the ability to survive in the environment.In conclusion,pyrisoxazole can still be used in the field to control gray mold.Considering that resistant isolates have appeared in the field,it is recommended that a strategy should be developed to prevent the rapid development of pyrisoxazole resistance.2.A total of 720 B.cinerea isolates collected in Shandong Province from 2014 to 2019were determined to be sensitive to boscalid.The results showed that the sensitivity of the B.cinerea isolates to boscalid declined gradually over time.The mean EC₅₀0 values of the isolates collected in 2014 and 2019 were 0.3±0.02 mg L^-1(ranging from 0.03 to 1.56 mg L^-1)and 6.39±1.66 mg L^-1(ranging from 0.01 to 85.56 mg L^-1),respectively.The sensitivity of B.cinerea to boscalid in different regions has different degrees of decline.The sensitivity of B.cinerea in Liaocheng and Laiwu areas has decreased the most,and the frequency of resistance has also risen fastest in the two regions.In Shandong Province,the P225F,N230I,H272Y and H272R mutations of SdhB subunit were responsible for the resistance of the B.cinerea isolates to boscalid.The frequency of boscalid-resistant isolates increased gradually over time,changing from 0.81%in 2014 to 28.97%in 2019.Some mutations in the SdhC subunit were also observed in the collected isolates,including the mutants with glycine replaced by alanine at position 85?G85A?,isoleucine by valine at position 93?I93V?,methionine by valine at position 158?M158V?and valine by isoleucine at position 168?V168I?.However,four mutations in the SdhC subunit did not determine boscalid resistance.For the SdhD subunit,no mutations were found in this study.3.In 2019,a total of 107 B.cinerea isolates were used to measure sensitivities to pydiflumetofen based on measurements of mycelial growth and spore germination inhibition.Seventy-four sensitive isolates were used to establish the sensitivity baseline for pydiflumetofen.The EC₅₀0 values ranged from 0.001 to 0.4 mg L^-1with a mean value of 0.07±0.009 mg L^-11 for mycelial growth to 0.001 to 0.06 mg L^-11 with a mean value of 0.02±0.007 mg L^-1for inhibition of spore germination.The frequency distribution of the EC₅₀values of mycelial growth inhibition and spore germination fit a unimodal curve,and he average EC₅₀0 value can be used as sensitivity baseline for detecting the subsequent development of pydiflumetofen resistance.The isolates carrying P225F,N230I,H272Y and H272R mutations in the SdhB subunit were found to be associated with the less sensitive of B.cinerea to pydiflumetofen.The mean EC₅₀0 value of pydiflumetofen for the isolates with P225F,N230I,H272Y and H272R mutation was 0.31±0.09,0.07±0.02,0.29±0.05 and0.08±0.02 mg L^-1,respectively.The P225F and H272Y mutant isolates showed low to moderate levels of resistance to pydiflumetofen,and the N230I and H272R mutant isolates showed low levels of resistance.There was a positive correlation between pydiflumetofen and the other four SDHI fungicides?i.e.,boscalid,fluopyram,isopyrazam and benzovindiflupyr?.The correlation coefficient between pydiflumetofen and isopyrazam was the largest,with a correlation coefficient?r?value of 0.62.In greenhouse experiment,the efficacy of pydiflumetofen at 300 mg L^-11 was much higher than that of boscalid at 400 mg L^-1.The efficacy of pydiflumetofen at 300 mg L^-1for leaves was 80.9%,whereas that of boscalid at400 mg L^-11 was 42.7%.4.In 2017,a total of 103 single-spored B.cinerea isolates were used to test sensitivity to benzovindiflupyr.The EC₅₀0 values ranged from 0.01 to 9.49 mg L^-11 with a mean value of 2.15±0.19 mg L^-1for mycelial growth to 0.01 to 6.49 mg L^-1with a mean value of 0.89±0.14 mg L^-11 for inhibition of spore germination.Benzovindiflupyr has good protective and curative activity against B.cinerea,and the protective activity is superior to its curative activity.However,benzovindiflupyr is poor translocation in cucumber plants.After spraying application,the control efficacy of benzovindiflupyr at 100,200 and 300 mg L^-1on cucumber leaves was 73.51,81.87 and 88.55%,respectively,which were significantly higher than that of iprodione at 500 mg L^-1?52.8%?.However,the efficacy of benzovindiflupyr at 100,200and 300 mg L^-11 on fruits were only 38.75,64.54 and 76.19%,respectively,which were not significantly different from that of the value for the control fungicide iprodione at 500 mg L^-1?44.13%?.In addition,the infection frequency of B.cinerea in cucumber fruits and leaves after spray application was reduced by 7.43 and 12.67%,respectively,but increased by 2.67%in stems.An interesting result is that the lowest concentration of benzovindiflupyr(20 mg L^-1)applied by dipping flower achieved an efficacy of 93.7%,which was much higher than that of iprodione at 200 mg L^-1?59.8%?.The infection frequency in cucumber fruits,leaves and stems after dipping flower application was reduced by 43.33,7.34 and 1.33%,respectively.Therefore,compared with the conventional spray application,dipping flower application is more suitable for controlling gray mold.5.Considering that the labor cost of dipping flower application is too high,the effect and safety of different fungicides and plant growth regulators by dipping flower application to control gray mold were further studied.Both the forchlorfenuron and water controls showed much higher disease indexes than those of the fungicide treatments.Furthermore,compared with other treatments,dipping fruit in fludioxonil at 30 mg L^-11 showed the most satisfactory results,with the proportion of marketable cucumber fruit and the average weight of individual cucumber fruit being 86.7%and 169.99 g,respectively,and an increased yield of 36.7%relative to the forchlorfenuron control.Although dipping fruit with fludioxonil at 35 mg L^-1had a relatively higher residue than did other treatments 8 days after fungicide application,it was still much lower than the maximum residue limit?MRL?of fludioxonil in cucumbers stipulated by the European Union(1.00 mg kg^-1)and Japan(2.00 mg kg^-1).The sensitivity of B.cinerea to boscalid was different in different regions.In Linyi,dipping with 30 mg L^-1?85.03%?could achieve a similar efficacy as spraying with 300 L^-1?82.47%?;the yield of dipping with 40 mg L^-1was significantly higher than that of the control treatments.In Laiwu,boscalid was not effective against cucumber gray mold by either application method,and the efficacy and yield of each treatment were obviously lower than those in Linyi.For example,dipping with 40 mg L^-11 and spraying with 300 mg L^-11 in Laiwu only showed efficacy of67.38%and 43.11%,respectively.