Baseline Sensitivity of Botrytis cinerea from Liaoning Province to Boscalid

Yan LIU,Zhiqiu QI,Baoyan HAN,Bochui ZHAO,Mingshan JI

College of Plant Protection,Shenyang Agricultural University,Shenyang 110866,China

Botrytis,caused byBotrytis cinerea,is a worldwide fungal disease[1-2].In recent years,with the continuous expansion of protected areas,botrytis is becoming severer and severer,and it has become the main disease in facility cultivation of tomato in China,especially in north ern areas[3-4].Currently,benzimidazole fungicide carbendazim,anilino pyrimidine fungicide pyrimethanil and dicarboximide fungicide procymidone are commonly used to prevent and control botrytis in tomato.However,due to long-term use,drug resistance has been generated inB.cinerea.Thus the prevention effect againstB.cinereais greatly reduced,leading to heavy losses to tomato production[5-10].The resistance ofB.cinereato common fungicides has aroused worldwide attention[11-14].

Boscalid is a new nicotinamide fungicidedevelopedbytheBASF Corporation in 1992[15].It is a systemic fungicide,and has broad spectrum,especially against gray mold,powdery mildew,rot,sclerotinia and various rot diseases in economic crops.Boscalid is an inhibitor against coenzyme Q reductase in mitochondrial respiratory chain, affecting celldivision and growth,eventually leading to cell death[16].Ritaet al.[17]found botrytis-resistantB.cinereastrain in tomato.Yuet al.[19]also found botrytis-resistantB.cinereastrain in tomato in Shanxi Province.However,there have been no reports about resistance ofB.cinereato boscalid in tomato in Liaoning Province.In order to avoid and delay the generation of resistance ofB.cinereato boscalid,risk assessment on resistance ofB.cinereato boscalid is urged to be conducted.

Materials and Methods

Materials

Test strainThe samples were collected from ten different urban areas of Liaoning Province,and total165 strains ofB.cinereawere separated.The strains were preserved on PDA plate.They were named in accordance with the principle of"First capital letter of collection site+number".

Test reagentsThe boscalid stock solution(87.4% )was provided by the Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences.The mother liquor of boscalid(10 000 μg/ml)was prepared by diluting the stock solution with acetone.

Test mediumThe prepared PDA medium was cooled to about 45℃and then mixed with different concentrations of boscalid solutions(v∶v,1∶9).Thus the boscalid-containing PDA medium was prepared.

Methods

Determination method of sensitivity of B.cinerea to boscalidThe sensitivity ofB.cinereato boscalid was determined by the mycelial growth rate method[18].Under sterile conditions,the colonies ofB.cinereaon the boscalidcontaining PDA plate were sampled using a punch with a diameter of 5 mm.The sampledB.cinereacolonies were inoculated downward on new PDA plates containing different concentrations (0,0.0625,0.25,1.0,4.0 and 16.0 μg/ml)of boscalid,and cultured at 25℃for 3 d.There were three replicates for each treatment.The diameters of colonies were measured using the crossing method.The mycelial growth inhibition rates ofB.cinereain PDA medium containing different concentrations of boscalid were calculated according to the following formula:

Mycelial growth inhibition rate=(Average colony diameter in the control group-Average colony diameter in the treatmentgroups)/(Average colony diameter in the control group-0.5)×100%.

The data statistics were conducted using the DPS data processing system.The mycelial growth inhibition rates ofB.cinereawere converted into probability values,and the concentrations of boscalid were converted into their logarithms.With the logarithm of boscalid concentration as the horizontal axis,and the probability value as the vertical axis,the regression equation (y=a+bx)was fitted.According to the regression equation of virulence,theEC50values of boscalid against variousB.cinereastrains and the correlation coefficients(r)were calculated.

Establishment of sensitivity baseline of B.cinerea to boscalidAccording to the sensitivity range ofB.cinereato boscalid,theEC50values were divided evenly into several stages.With the final value ofEC50at each stage as the horizontal axis,and the frequency of sensitivity as the vertical axis,the frequency distribution of sensitivities ofB.cinereastrains to boscalid were drawn.If theEC50values were in line with normal distribution,the mean ofEC50values can be treated as the sensitivity baseline ofB.cinereafrom Liaoning Province to boscalid.

Results and Analysis

Establishment of sensitivity baseline of B.cinerea to boscalid

K-S test was conducted for the obtained data by using SPSS software.After removing the 7 strains that did not meet the conditions,theZvalue was 1.335,and thePvalue was 0.057.Since thePvalue was above 0.05,the sensitivity frequency of the 158 testB.cinereastrains to boscalid was in line with normal distribution.Within the sensitivity range ofB.cinereato boscalid,theEC50values were divided into five stages with intercept of 1.000 μg/ml.With theEC50final values as the horizontal axis,and the sensitivity frequency as the vertical axis,the frequency distribution of sensitivity ofB.cinereato boscalid was drawn(Fig.1).

As shown in Fig.1,theEC50values of boscalid against variousB.cinereastrains were mainly concentrated in the range of 1.000-2.000 μg/ml(41.14% ),but the sensitivity frequencies in the other intervals were reduced successively.The averageEC50value of boscalid against the 158B.cinereastrains was 1.9731±1.001 1 μg/ml,and the averageEC50value(1.973 1 μg/ml)was initially treated as the sensitivity baseline ofB.cinereafrom Liaoning Province to boscalid.

Table 1 Sensitivity of B.cinerea to boscalid

Sensitivity comparison of B.cinerea strain to boscalid among different regions

The frequency distribution of sensitivities ofB.cinereastrains to boscalid was determined by the mycelial growth rate method(Fig.2).The sensitivity frequencies were distributed continuously.The differences in sensitivity ofB.cinereato boscalid were also tested among different regions.As shown in Table 1,theEC50values of boscalid against variousB.cinereastrains ranged from 0.080 0 to 7.787 2 μg/ml,and the maximum EC50 value was 97.34 times higher than the minimum.The averageEC50value was highest in Dalian,but was lowest in Dandong.TheEC50values in Dalian were significantly higher than those in the other regions,indicating that theB.cinereastrains in Dalian were less sensitive to boscalid.This might be caused by earlier and more application ofboscalid in Dalian,which still needs further testing and verification.

Cluster analysis of sensitivities of B.cinerea strains from different origins to boscalid

Total 50B.cinereastrains were collected randomly from 10 different regions(5 strains from each region)of Liaoning Province.The cluster analysis(Fig.3)showed that theEC50values of the 50 strains were mainly divided into 7 groups.Total 14 strains were clustered into the first group,including BX-17,FS-09,BX-19,AS-09,FX-11,FS-03,FS-06,CY-14,AS-03,PJ-14,FX-01,SY-08,PJ-08 and FS-10;total 5 strains were clustered into the second group,including DL-12,CY-07,AS-11,HLD-12 and FS-04;total 11 strains clustered into the third group,including DD-01,DD-14,HLD-19,DD-20,SY-07,DD-03,SY-04,FX-05,HLD-15,BX-03 and BX-14;total 13 strains were clustered into the fourth group,including CY-11,AS-01,BX-07,CY-02,CY-20,SY-02,AS-04,SY-01,DD-08,HLD-04,HLD-07,PJ-16 and FX-02;total 4 strains were clustered into the fifth group,including FX-15,PJ-02,DL-01 and PJ-04;there were only two strains clustered into the sixth group,including DL-06 and DL-08;there were only one strain (DL-17)clustered into the seventh group.The strains from different regions were clustered into one group,indicating that the differences in sensitivity ofB.cinereato boscalid have no significant correlation with geographic origin ofB.cinerea.The DL-06 and DL-08 were gathered into one group,and theirEC50values were highest(6.640 9 and 6.872 3 μg/ml).It suggests that these twoB.cinereastrains are least sensitive to boscalid,and drug resistance is easiest to be generated in them.

Conclusions and Discussion

Yuet al.[19]found that theEC50values ofboscalid againstB.cinereastrains from Shanxi Province ranged from 0.22 to 11.67 μg/ml,and the maximumEC50value was 53.05 times higherthantheminimum.Inthis study,theEC50values of boscalid againstB.cinereastrains from Liaoning Province ranged from 0.080 0 to 7.787 2 μg/ml,and the maximumEC50value was 97.34 times higher than the minimum;the averageEC50value of boscalid against the 165 testB.cinereastrains was 2.194 9±1.334 1 μg/ml.Therefore,there is small difference in sensitivity ofB.cinereato boscalid between Shanxi and Liaoning Provinces.In Liaoning Province,although the difference in maximum and minimumEC50values is greater,theB.cinereastrains are still in a sensitive stage to boscalid,indicating that theB.cinereastrains in greenhouse are still sensitive to boscalid.There are great differences inEC50value of boscalid among differentB.cinereastrains,which may be related to physiological differences amongB.cinereastrains in the nature.

There is great genetic variability inB.cinerea,so it is easy to produce drug resistance in production.The establishment of sensitivity baseline ofB.cinereato boscalid is the basis for identifying and detecting resistance inB.cinereaagainst boscalid,and it is of great significance for avoiding and delaying the generation of drug resistance inB.cinerea.Boscalid is an inhibitor against coenzyme Q reductase in mitochondrial respiratory chain,and it has a single site of action,so drug resistance is easy to be generated in pathogenic strains.Therefore,in the application,boscalid,along with other agents with differentmechanisms,should be used alternatively to avoid and delay the generation of drug resistance inB.cinerea,thereby improving the therapeutic effect of boscalid againstB.cinerea.

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