Detection of the sensitivity of Botryosphaeria dothidea to procymidone

Abstract:【Objective】Apple ring rot, caused by Botryosphaeria dothidea, is one of the most important diseases in apples and it is widely distributed in most apple production areas in China. The most effective management strategy for controlling this disease is the application of chemical fungicides. The wide application of chemical fungicides inevitably leads to serious fungicide-resistance problems, and results in more difficulties to control this disease in the fields. Procymidone is a dicarboximides fungicide, which has been widely used to protect apples from fungal infection in both the growing season and postharvest period. The objective of this study was to determine the sensitivity level and baseline of B. dothidea to procymidone using 106 field isolates collected from Shandong, Hebei, Henan, Liaoning, Shanxi and Shaanxi,respectively.【Methods】The sensitivity of each isolate to procymidone was determined by using a mycelial growth assay by PDA containing serial concentrations of procymidone. Techincal grade procymidone(a.i.98.3%) was dissolved in 100% acetone, adjusted to a concentration of 10 000 mg·L^-1 stock solution, and was diluted with 0.1%(ω) polysorbate 80 in water to seven concentrations: 1.25, 2.5, 5.0, 10.0, 20.0, 40.0and 80.0 mg·L^-1, then added to the PDA medium to produce eight standard concentrations: 0, 0.125, 0.25,0.5, 1.0, 2.0, 4.0 and 8.0 mg of procymidone per liter of the medium. A 7 mm mycelial plug, cut from the edge of 4 days old B. dothidea colony, was placed on the center surface of the PDA plate and amended with each concentration of the procymidone. Four replicate plates were used for each concentration. The radial growth was determined by measuring the diameter of the colonies after 4 days of incubation. The effective concentration EC50 (concentration which reduced mycelial growth by 50%) was calculated by regressing the radial growth values (100% control) against the log 10 values of the fungicide concentrations using SPSS16.0 software. The cumulative frequency distribution of EC50 value was produced and calculated by using SPSS16.0 software. The bin width of the histogram was calculated by Scott's rule and the normal distribution of the EC50 values of the 106 isolates was analyzed by using the Shapiro-wilk test. The differences among B. dothidea isolates collected from different provinces in procymidone sensitivity also were statistically analyzed by using a one-way analysis of the variance (ANOVA) with the SPSS 16.0 software, and means were separated using the Student Newman Keuls(SNK) test.【Results】All 106 field isolates of B. dothidea from different provinces of China were relatively sensitive to procymidone, with EC50 values ranging from 0.327 0 to 3.322 8 mg·L^-1. The mean EC50 values of all isolates were(1.289 1±0.060 0)mg·L^-1. The isolate LK3 collected from Shandong province was the most insensitive to procymidone with the highest EC50value(3.322 8 mg·L^-1), compared to the most sensitive isolate SSXDL4 with the lowest EC50value(0.327 0 mg·L^-1) collected from Shaanxi province. The ratio of the highest EC50 value to the lowest EC50 value was 10.16. The Shapiro-wilk test results with a significant level of 95% showed that the skew and kurt values were 0.235 and 0.465, respectively. The normality test was not passed at the level of significance of 0.05 since the p-value is less than this limit. However, the frequency distribution of EC50 values for 106 isolates was continuous, unimodal and positively skewed according to the Shapiro-wilk test results. Therefore, the mean EC50 values of 106 field isolates obtained in this study could be used as the sensitivity baselines of B. dothidea to procymidone. The results of the one-way analysis of variances indicated that there were significant differences in the mean EC50 values among B. dothidea field isolates from the different provinces to procymidone. The B. dothidea isolates collected from Shaanxi province displayed the highest sensitivity to procymidone with the lowest average EC50values(0.551 2±0.134 6 mg·L^-1).The isolates from Liaoning, Hebei and Shanxi provinces showed relatively higher sensitivity to procymidone, the mean EC50 values were(1.277 0±0.151 0) mg·L^-1,(1.070 0±0.085 7) mg·L^-1 and (0.742 2±0.116 1) mg·L^-1, respectively. The isolates from Shandong and Henan displayed the lowest sensitivity to procymidone, the mean EC50 values were (1.451 7±0.083 6) mg·L^-1 and(1.375 8±0.314 0) mg·L-1, respectively.The ratio of the Shandong province to the Shaanxi province was 2.63 using mean EC50 values. Overall,there were significant differences in the mean EC50 values among Shandong, Henan and Shaanxi provinces using the SNK test at the p<0.05 level. However, no significant differences were found in the mean EC50 values between the Shandong and Henan provinces. There were also no differences of sensitivity to procymidone in the tested field isolates collected from Liaoning, Hebei and Shanxi provinces using the SNK test at P<0.05 level【.Conclusion】The results of this study indicate that the procymidone-resistance subisolates of B. dothidea have not been detected in the field. The tested field isolates of B. dothidea have not shifted to reduced sensitivity to procymidone. The mean EC50 values of field isolates obtained in this study could be used as the sensitivity baselines of B. dothidea to procymidone. Moreover, the baseline sensitivity value can be useful for a future procymidone resistance monitoring program for B. dothidea in the China.