Establishment of sensitivity baseline and evaluation of resistance to tetramycin of Erwinia amylovora

Abstract: 【Objective】Pear fire blight caused by Erwinia amylovora is an international quarantine bacterial disease. It is one of the top ten plant pathogenic bacteria in the world with fast transmission speed, multiple transmission routes and wide host range. Biopesticide tetramycin is a pure green biopesticide developed by Liaoning Academy of Microbial Sciences in the 1970s and was productized in the early 1990s. Tetramycin can inhibit both bacterial and fungal plant diseases. At present, there are no reports on the establishment of sensitivity baseline and the evaluation of resistance to tetramycin of E. amylovora in China.The study aimed to establish a sensitivity baseline for E. amylovora to tetramycin in Korla and Aksu regions of Xinjiang, and monitor the resistance level of pear fire blight to tetramycin in the region and provide a scientific basis for the field administration of tetramycin against pear fire blight, antibiotic resistance monitoring and antibiotic resistance management.【Methods】The samples were collected, in ten areas in Xinjing, including Bayinguoleng and Aksu in 2021 and 2023. The bacterial strains were isolated from the infected plant materials of Korla fragrant pear. The sensitivity of 100 strains of E. amylovora isolated to tetramycin was determined by the inhibition zone method. The drug-resistant mutants were acclimated by streak plate method and drug containing medium method, followed by drugresistance evaluation. The preserved strains were separated in the NA medium, incubated at 28 ℃ for 48 h, and single colonies were obtained by secondary activation. The single colony was transferred to the NB culture medium at 28 ℃, 180 r· min-1 for 12 h, and the test bacterial suspension was obtained when OD600 was determined to be 1.0. After the bacterial suspension was diluted to 1×104 cfu · mL- 1 , 100 µL was coated in the NA medium until dry. Three pieces of sterilized filter paper with a diameter of 6 mm were placed on the NA medium in the petri dish. 6 µL of different concentrations of drug drops were absorbed on the filter paper sheet, with 5 treatment concentrations for each drug solution and 2 dishes for each concentration, for a total of 6 repetitions, with sterile water as the control. After being incubated at 28 ℃ for 36 h, the diameter of the antibacterial zone was measured by the cross method, and the antibacterial rate was calculated according to the formula. The correlation coefficient and EC50 value were calculated according to the bacteriostasis rate. Based on the average EC50 values of the all tested strains, the sensitivity baseline of tetramycin in Xinjiang was established.【Results】The results showed that from the all tested strains, 74 sensitive strains were detected, accounting for 74%, and 26 resistant strains were detected, accounting for 26%. The distribution of EC50 values of 100 strains of E. amylovora ranged from 0.199 to 4.84 µg·mL-1 , with the maximum EC50 was 24.32 times as high as the minimum EC50, the average EC50 was (1.59±1.029) µg ·mL-1 , and the 95% confidence interval was 1.389 2- 1.797 4 µg·mL-1 The distribution of tetramycin to E. amylovora strains showed a unimodal curve, which was similar to the normal distribution, and no E. amylovora strains with significantly decreased sensitivity were found. Therefore, the mean EC50 value of (1.59±1.029) µg ·mL-1 could be used as the baseline for the sensitivity of E. amylovora strains to tetramycin in southern Xinjiang. There were some differences in the sensitivity of the 100 strains of E. amylovora between 4 regions, the strains from Aksu were more sensitive, with mean EC50 value of 0.780±0.436 µg ·mL- 1 , which was significantly different from the strains from Awati Township, Halayugong Township and Tiemengguan. After the four resistance mutants of E. amylovora were transferred to the NA medium for 10 successive generations, the resistance levels of the 11th generation resistance mutants were 0.275 µg·mL-1 , 0.43 µg·mL-1 , 0.145 µg·mL-1 and 0.564 µg · mL- 1 , respectively, which all decreased from the resistance level of low antibacterial strains to the resistance level of sensitive strains. The results indicated that the 4 resistant mutants was not genetically stabile. The EC50 values of the E. amylovora resistance mutant against tetramycin were highly correlated with the EC50 values of 84% benziothiazolinone and zhongshengmycin, and the correlation coefficients were 0.765 8 and 0.900 8, respectively, and the absolute values of all correlation coefficients were higher than 0.75. It can be seen that there is a cross-resistance problem between tetramycin and 84% benziothiazolinone and zhongshengmycin, which are commonly used in field to control E. amylovora, so the three fungicides should be avoided as alternate standby pesticides in the field to avoid cross- resistance problems. Thus, it was confirmed that tetramycin had cross- resistance with the 84% benziothiazolinone and zhongshengmycin.【Conclusion】The basic resistance of the strains of E. amylovora from different regions to fungicides was determined by both the agent and the pathogen itself. The sensitivity baseline value of the E. amylovora isolated strains in Xinjiang to benziothiazolinone could be established according to this study. Only a few low resistant subgroup strains were detected, and the risk of resistance to tetramycin was at low level.