The Sensitivity Detection Of Botryosphaeria Dothidea To Eight Fungicides And The Effective Fungicide Screening

Apple ring rot disease, caused by Botryosphaeria dothidea, is widely distributed in most apple planting areas, and has become one of the most serious diseases in the apple production in China. This disease can occur at the growing stage before harvest or anytime during the storage. Currently, the most popular and effective management strategy for controlling ring rot disease in China is the application of synthetic fungicides. However, wide application of chemical fungicides inevitably leads to serious fungicide-resistance problems, and result in more difficulties to control apple ring rot. The objective of the present study was to evaluate the sensitivity level and baseline of B. dothidea to eight fungicides using 106 field isolates collected from Shandong, Hebei, Henan, Liaoning, Shanxi and Shaanxi, respectively. To select the synergistic mixture of difenoconazole with other seven fungicides against apple ring rot disease, the toxicity of these mixtures with different ratios against the B. dothidea were also tested in the laboratory by using mycelial growth assays, and the optimal combination and mixture ratio of difenoconazole with other seven fungicides were determined by the co-toxicity cofficient method. The results of our study may provide a basis for the detection and management of fungicide resistance to B. dothidea, also for using fungicides efficiently to prevent and control the apple ring rot disease in the field.The sensitivity level of each field isolate of B. dothidea to difenoconazole, propiconazole, epoxiconazole, procymidone, thiophanate-methyl, LH-2010 e, captan and metiram, respectively, was determined by using mycelial growth assay on PDA containing serial concentrations of each fungicide. The sensitivity baseline of B. dothidea to each fungicide also was established based on the mean EC50 values of 106 filed isolates. The sensitivity results showed that, the EC50 values in the same fungicide were similar for all 106 field isolates for B. dothidea. However, there were significant differences of mean EC50 values of 106 field isolates in the eight tested fungicides. The mean EC50 values of three triazole fungicides(difenoconazole, propiconazole and epoxiconazole) was the lowest and no more than 0.25 mg·L-1. The EC50 values for difenoconazole, propiconazole and epoxiconazole ranged from 0.0063-0.6782, from 0.0160-0.9310 and from 0.0241-0.7828 mg·L-1, with the mean EC50 value and standard deviation of 0.1346±0.0139, 0.1880± 0.0184 and 0.2134± 0.0165 mg·L-1, respectively. The EC50 values for procymidone(dicarboximides fungicide) and thiophanate-methyl(substitutive benzene fungicide) ranged from 0.3270-3.3228 and from 0.8145-10.7008 mg·L-1, with the mean EC50 value and standard deviation of 1.2891± 0.0600 and 3.3579± 0.1607 mg·L-1, respectively. The LH-2010e(phenylamide fungicides) had the relatively lower toxicity to B. dothidea, the EC50 values ranged from 0.9913-16.9868 mg·L-1, with the mean EC50 value and standard deviation of 5.9254±0.2881 mg·L-1. The traditional fungicides, captan(dicarboximides fungicide) and metiram(dithiocarbamates fungicide) had the lowest toxicity to B. dothidea with the highest mean mean EC50 value compared with other six tested fungicides. The EC50 values for aptan and metiram ranged from 1.6722-27.0095 and from 1.2763-32.1324 mg·L-1, and the mean EC50 value and standard deviation was 10.2681± 4.6002 and 15.5119± 0.7107 mg·L-1, respectively. Since the frequency distribution of EC50 values for each fungicide in 106 isolates was continuous, unimodal and positively skewed, except metiram with a normal distribution, the mean EC50 values of each fungicide could be used as the sensitivity baselines.The co-toxicity of difenoconazole mixture with each of other seven fungicides at different ratios against B. dothidea also was determined by using mycelial growth assay on PDA containing serial concentrations of each fungicide mixture at different ratios. The results showed that, the significant synergism was observed in all tested mixtures combined difenoconazole with propiconazole, epoxiconazole, procymidone, thiophanate-methyl, LH-2010 e, captan and metiram, respectively. The mixture of difenoconazole and propiconazole showed the synergism in the ratios ranged from 1:1 to 1:3. The mixture of difenoconazole and epoxiconazole showed the synergism in the ratios of 1:4. The mixture of difenoconazole and procymidone showed the synergism in the ratios of 1:7. The mixture of difenoconazole and thiophanate-methyl showed the synergism in the ratios ranged from 1:9 to 1:10. The mixture of difenoconazole and LH-2010 e showed the synergism in the ratios ranged from 1:5 to 1:6 and 1:9-1:10, respectively. The mixture of difenoconazole and captan showed the synergism in the ratios ranged from 1:2to 1:7. The mixture of difenoconazole and metiram showed the synergism in the ratios ranged from 1:1 to 1:3 or of 1:5 and 1:7, respectively. The co-toxicity results of our study may provide a basis for the next research and development of mixture fungicide to prevent and control the apple ring rot disease in the field.