The sensitivity of Botryosphaeria dothidea to thiophanate-methyl, captanand metiram

Abstract:【Objective】Botryosphaeria dothidea is widely distributed in most apple growing regions in China, causing ring rot disease of apple. For many years, use of chemical fungicides has been effective forcontrolling the disease in the areas of apple production. However, the effectiveness of these fungicides hasbeen threatened by the emergence of resistant pathogens in the field. Thiophanate-methyl, captan and me⁃tiram have been widely used for controlling the disease in China for many years. So far there has been noreport on the sensitivity of B. dothidea to these fungicides in China. The objective of this study was to de⁃tect the sensitivity level of B. dothidea to thiophanate-methyl, captan and metiram using 106 field isolatescollected from Shandong, Hebei, Henan, Liaoning, Shanxi and Shaanxi, respectively.【Methods】The sen⁃sitivity of B. dothidea to fungicides was assessed based on inhibition of mycelial growth by PDA contain⁃ing serial concentrations of fungicides. Technical grade thiophanate- methyl (a.i. 95.0% ), captan (a.i.95.0%), and metiram (a.i. 87.0%) were dissolved in 100% acetone, respectively, being adjusted to a concentration of 10 000 mg·L^-1 stock solution. The stock solution was diluted with 0.1% (ω) polysorbate 80 inwater to eight concentrations: 5.0, 10.0, 20.0, 40.0, 80.0, 160.0, 320.0 and 640.0 mg·L^-1, then added tothe PDA medium to produce eight standard concentrations: 0, 0.5, 1.0, 2.0, 4.0, 8.0, 16.0, 32.0 and 64.0mg of each fungicide per liter of the medium. A 7-mm-diameter agar disk taken from the margin of a 4-day-old B. dothidea colony were placed at the center surface of a series of petri dishes (9 cm diameter)containing PDA amended with various concentrations of fungicides. Four replicate plates were used foreach concentration. Mycelial growth was quantified after 4 days of incubation at 26 ℃ in darkness by mea⁃suring two perpendicular diameters of the growth ring in each plate, and taking the average (the diameterof the original plug subtracted). The effective concentration EC50 (concentration which reduced mycelialgrowth by 50%) was calculated by regressing the radial growth values (100% control) against the log10 val⁃ues of the fungicide concentrations using SPSS 16.0 software. The cumulative frequency distribution ofEC50 value was produced and calculated by using SPSS 16.0 software. The bin width of the histogram wascalculated by Scott’s rule and the normal distribution of the EC50 values of the 106 isolates was analyzedusing the Shapiro-wilk test. Cross-resistance patterns were analyzed by lgEC50 values using the Spearmanrank correlation and a line regression analysis. The differences of the sensitivities among B. dothidea iso⁃lates collected from different provinces were statistically analyzed 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】The EC50 values of 106 B. dothidea field isolates collected from different provinces ofChina to thiophanate-methyl varied from 0.814 5 to 10.700 8 mg·L^-1, with an average of (3.357 9±0.160 7) mg·L^-1. The frequency distribution of EC₅₀ values of 106 isolates to thiophanate-methyl was continuous,unimodal and positively skewed according to the Shapiro-wilk test results. The sensitivities of B. dothideafield isolates to thiophanate-methyl were still in high levels. The EC50 values to captan were from 1.672 2to 38.487 3 mg·L^-1, with discontinuous frequency distribution curve. Isolate XY7 (Henan province) hadthe highest EC50 values to captan (38.487 3 mg·L^-1) among the 106 isolates. However, the frequency distribution of EC50 values of the other 105 isolates to captan was continuous, unimodal and positively skewedaccording to the Shapiro-wilk test results. The mean EC50 values of the other 105 isolates were (10.268 1±0.460 0) mg·L^-1, which could be used as the sensitivity baselines of B. dothidea to captan. The EC₅₀ values of 106 B. dothidea field isolates to metiram were from 1.276 3 to 32.132 4 mg·L^-1, with an average of (15.511 9±0.710 7) mg·L^-1. The frequency distribution of EC50 values of the 106 isolates to metiram wascontinuous, unimodal and positively skewed according to the Shapiro-wilk test results. Therefore，the sen⁃sitivity baseline of B. dothidea to metiram could be established in this study. Spearman rank correlationanalysis by lgEC50 values indicated that there were positively significant correlations between sensitivitiesof B. dothidea isolates to thiophanate-methyl and captan (r=0.281 3, P=0.003 5), but there were no significant correlations between sensitivities of B. dothidea isolates to thiophanate-methyl and metiram (r=0.179 7,P=0.065 3), captan and metiram (r=0.081 1, P=0.408 6), respectively. There were no significant differences among the sensitivities of 106 B. dothidea field isolates to each fungicide using the SNK test at P=0.05level (P＞0.05).【Conclusion】The 106 B. dothidea field isolates showed a relatively high sensitive level tothiophanate-methyl, captan, and metiram, respectively. The sensitivity baselines of B. dothidea to captanand metiram could be established according to this study.