Baseline sensitivity and resistance risk assessment of Peronophythora litchii to dimethomorph

Abstract: 【Objective】Litchi downy blight caused by peronophythora litchii is a devastating disease, which seriously affects the quality and yield of litchi. With the continuous use of fungicides, the effectiveness of fungicides has been threatened by the emergence of resistant pathogens. Dimethomorph is a CAAs fungicide, which has been widely used for controlling litchi downy blight for many years. Up to now, few studies on the sensitivity of P. litchii to dimethomorph have been reported. The resistance risk of P. litchii to dimethomorph is still largely unknown. The objective of this study is to (i) establish the baseline sensitivity of P. litchii isolates collected from five provinces or autonomous region (Guangdong, Guangxi, Fujian, Yunnan and Sichuan) to dimethomorph. (ii) obtain the dimethomorph- resistant mutants by fungicide adaption, and investigate the biological fitness of resistant mutants. The results might provide scientific guidance for highly effective dimethomorph use in controlling litchi downy blight.【Methods】A total of 320 P. litchii isolates were obtained from diseased fruits from Guangdong, Guangxi, Fujian, Yunnan and Sichuan provinces or autonomous region of China during 2019 to 2020. Growing mycelia assay in vitro was used to determine the median effective concentration (EC50) values of P. litchii isolates to dimethomorph. Technical grade dimethomorph (a.i. 99.0%) was dissolved in dimethyl sulfoxide. The solution was diluted to different concentrations. Mycellium plugs (5-mm-diameter) were cut from the fresh growing edge of a 4-day-old P. litchii colony, and placed on carrot agar medium plates (90-mm- diameter) amended with 0.05, 0.1, 0.15, 0.2, 0.25, 0.3 mg · mL^{- 1} dimethomorph.Three replicates were determined for each concentration. Mycelial growth was measured after 5 days incubation at 25 ℃ in darkness. The EC50 value for each isolate was calculated by regression analysis using the percentage of growth inhibition against the logarithmic value of dimethomorph concentration. Kolmogorov- smirnov test was used to check the normal distribution. All statistical analysis was performed using SPSS 23.0. Differences between the means were determined using Duncan’s multiple range test (DMRT) and independent sample T- test. The resistant mutants were obtained by dimethomorph adaptation test. Resistance stability of the mutants was assayed by placing the mutants on the fungicide-free carrot agar medium plate, after nine successive transfers to fresh media, and the sensitivities of resistant mutants to dimethomorph in the 1st, 3rd, 5th, 7th and 9th generations were determined. The resistance factor (RF) was the ratio of the EC50 value of a resistant mutant to the EC50 value of its parental isolate. The biological fitness of resistant mutants was measured in terms of sporangial size, sporangial germination, resting spore germination and oospore production. The virulence of dimethomorphresistant mutants and their parental isolates were carried out by inoculating six fresh leaves (Feizixiao) with the suspension of sporangia (1×104 mL^{- 1} ). All inoculated leaves were kept in a dark, and 25 ℃ chamber with 80% relative humidity. After 48 hours, the disease incidence and proportional area of lesion were determined using the Image J software. Each assay contained three biological replicates and all experiments were conducted twice.【Results】The mean median effective concentration (EC50) values of 317 P. litchii isolates obtained from five provinces or autonomous region to dimethomorpth ranged from 0.086 3 to 0.173 3 µg·mL^-1 , with the average of（0.122 2±0.000 9）µg·mL^-1 , and the ratio of the maximum to the minimum of EC50 values was 2.01. After three outliers were excluded, the frequency distribution of the EC50 values of P. litchii isolates showed a unimodal curve with a positive skew. The sensitivities of P. litchii isolates to dimethomorpth were still at high levels, and no dimethomorpth-resistant isolate was observed in the field. The mean EC50 values of P. litchii isolates collected in 2020 declined compared to those in 2019. The sensitivities of P. litchii isolates collected from different geographic regions to dimethomorpth were different. The mean EC50 values of P. litchii isolates from Guangdong were the highest (0.128 9±0.001 5 µg · mL^{- 1} ), while those from Sichuan were the lowest (0.115 4±0.001 7 µg · mL^{- 1} ). Three dimethomorph-resistant mutants of P. litchii (GZ3-3R , DH1-9R and LZ4- 10R ) were obtained by fungicide adaption, with RF ranging from 77.17 to 360.11. The mutants showed a stable resistance to dimethomorph after 9 transfers on dimethomorph-free medium. Comparing with their parental isolates, sporangial germination rate and oospore production of mutants were reduced. The disease incidence and proportional area of lesion on leaves inoculated by mutants were similar to those by their parental isolates (p＜0.05).【Conclusion】In this study, baseline sensitivity of P. litchii to dimethomorph was established. P. litchii isolates collected from Guangdong, Guangxi, Fujian, Yunnan and Sichuan provinces or autonomous region of China during 2019 to 2020 were sensitive to dimethomorph, which could be still used as an effective fungicide in controlling litchi downy mildew. Considering the fitness cost of the dimethomorph- resistant mutants (lower sporangial germination rate and oospore production, slightly lower virulence and high RF), it is necessary to strengthen monitoring the resistance to dimethomorph regularly and utilize fungicide rationally to prevent and control the occurrence and development of litchi downy blight in the field.