Sensitivity analysis of pitaya canker disease Neoscytalidium dimidiatum to pyraclostrobin in Hainan province, China

Abstract: 【Objective】Pitaya canker disease caused by Neoscytalidium dimidiatum is the main disease of pitaya in Hainan province. With the expansion of pitaya planting area and the increase of planting years, the occurrence of N. dimidiatum in the planting area has been increasing. The pathogen mainly damages the stem and the fruit of pitaya and causes great loss in production. According to investigation, pyraclostrobin is used to control the disease in Hainan province all year round, but the sensitivity of N. dimidiatum to pyraclostrobin has not been reported yet. Therefore, the purpose of this study was to de-termine the sensitivity baseline and sensitivity level of N. dimidiatum to pyrazolesterim, so as to provide a reference for scientific application of N. dimidiatum in Hainan province.【Methods】N. dimidia-tum collected from eight regions in Hainan province were isolated from the infected tissues. First, some diseased tissue pieces (5 mm×5 mm) were cut off and sterilized with 75% ethanol for 35 s and then with 5% NaClO for 40 s, and washed with sterile water for 3 times. The diseased tissue pieces were then pasted on PDA plates and cultured in an incubator at 28 ℃ for 2-3 days. After mycelia grew on the PDA plate, a single mycelium was transferred to a new PDA plate and cultured for 5 days. The patho-gen was preliminarily confirmed by microscopy after sporulating. Next, single spores isolated was used to obtain pure colonies. Finally, a total of 59 purified isolates were obtained and their sensitivities to pyraclostrobin were evaluated through mycelial growth rate method. The medium with final concentrations of 6, 4, 2, 0.5 and 0.2 μg·mL^-1 were prepared for each isolate. The pyraclostrobin-free PDA medi-um was added with 1 mL acetone as the solvent control, and the PDA medium with 1 mL sterile water as the blank control. In addition, salicylhydroxamic acid with a final concentration of 50 mg·L^-1 was added to all the tested mediums to reduce the effect of other respiratory pathway on the fungicide. The agar disks containing mycelium of N. dimidiatum were inoculated facing up on the pyraclostrobin-con-taining mediums and the control medium. Each concentration was set up with 3 replicates and cultured in an incubator at 28 ℃. After 48 h, the criss-cross method was used to measure diameters of these colo-nies. Microsoft Excel 2016 software was used to calculate the regression equation and correlation coefficient for each isolate, and the effective inhibiting medium concentrations (EC50) was calculated accord-ing to the regression equation. The EC50 values of 59 isolates, were tested for normality and variance ho-mogeneity by R language, and the sampling site was used as single factor for one-way analysis of vari-ance. The resistance level was determined based on the sensitivity baseline of N. dimidiatum to pyra-clostrobin, and the resistance level (RF) was calculated as EC50 (isolates)/sensitivity baseline. When EC50 was less than 5 folds of the sensitive baseline, the strain was defined as sensitive strain. When EC50 was 5-10 folds of the sensitive baseline, the strain was defined as low resistance. When the EC50 value was 10-40 folds of the sensitive baseline, the strain was defined as medium resistant strain. When the EC50 value was higher than 40 folds of the sensitivity baseline, the strain was defined as highly resistant strain.【Results】A total of 59 isolates of N. dimidiatum were obtained by the tissue isolation method.The EC50 values of these isolates to pyraclostrobin were measured and used for the test and analysis.Our results showed that the EC50 values of 58 isolates followed the normal distribution pattern, and the mean value of EC50 values was 0.962 7 μg·mL^-1, which was ultimately used as the sensitivity baseline of N. dimidiatum to pyraclostrobin. The results of regional sensitivity analysis showed that the sensitivity of the 59 isolates to pyraclostrobin were hardly different in Hainan province, with EC50 ranging from 0.254 0 to 2.223 3 μg·mL^-1. There was no significant difference in sensitivity among different regions except for Ledong and Baisha. Isolates collected from Ledong county were very sensitive to pyraclos-trobin, while isolates from Baisha county were relatively less sensitive. The difference of EC50 between Baisha and Ledong county was the most significant. The mean value of EC50 in Baisha county was 2.69 times that of Ledong county, and the average resistance was 2.71 times, which was consistent with our field survey results of pyraclostrobin applications.【Conclusion】The sensitivity of N. dimidiatum to pyraclostrobin was characterized by the EC50 values of the tested isolates. All the EC50 values were in the range of 0.254 0-2.223 3 μg·mL^-1, and the average EC50 value was 0.984 1 μg·mL^-1. The sensitive baseline of N. dimidiatum to pyraclostrobin in Hainan province was 0.9627 μg·mL^-1. In general, the N.dimidiatum isolates collected from different regions in Hainan province had minor differences in sensi-tivity to pyraclostrobin, and the isolates were still sensitive to pyraclostrobin. However, with the in-crease of planting years and the constant accumulation of the fungicide, the risk of resistance cannot be completely excluded.