Susceptibility of Erwinia amylovora to benziothiazolinone and its resistance risk analysis in Xinjiang

Abstract:【Objective】Fire blight caused by Erwinia amylovora is one of the most destructive diseases in apple, pear and other pome crops worldwide. Application of bactericides is an indispensable strategy for controlling the disease in fruit production. Since the 1950s, the antibiotic streptomycin had often been used for disease management and was the only antibiotic registered to control fire blight. The overuse of streptomycin has resulted in the development of streptomycin resistance in the pathogen in many growing areas. Copper products can be quite effective, but their use was mostly limited to early-season application due to the risk of phytotoxicity to the developing fruit. The research for new chemicals against E. amylovora has resulted in the release of new compounds, which give promising results, commercially available, environmentally safe and non-phytotoxic systemic bactericides, and replacing copper compounds or streptomycin. Benziothiazolinone is a synthetic, non-antibiotic, organic heterocyclic bactericide, and used primarily against different species of the phytopathogenic fungi. It also has been found to be effective against phytopathogenic bacteria. Most recently, benziothiazolinone had been reg-istered to control fire blight in China. The purpose of this study is to assess the susceptibility of E. amylovora strains to benziothiazolinone, establishing the sensitivity baseline to monitor the resistance risk of the novel bactericide, so as to provide reference for the rational use of benziothiazolinone to control fire blight.【Methods】To isolate E. amylovora strains, samples were collected from 2016 to 2020, in ten areas in Xinjing, including Bayinguoleng, Ili, Aksu, Hami, Changji, Hotan, Kashgar, Tacheng and Urumqi. The samples were collected included flower, leaf, fruit and shoot tissues showing typical characteristic fire blight symptoms. Bacterial strains were isolated from the infected plant materials of fragrant pear（Pyrus sinkiangensis), apple (Malus pumila), duli pear (Pyrus betulaefolia), hawthorn (Crataegus pinnatifida) and quince (Cydonia oblonga). The isolates of E. amylovora were verified by colony morphology, physiological and biochemical characteristics, and by polymerase chain reaction (PCR) using primers specific for E. amylovora targeting the ubiquitous plasmid pEA29. A total of eighty-seven strains of E. amylovora were obtained, and fifty representative isolates among them were chosen as tested strains. The sensitivity of the E. amylovora strains to benziothiazolinone were determined in vitro with filter paper disc-inhibition zone method, and baseline sensitivity was established. Single colonies of the strain were grown in nutrient broth shaken at 160 r·min-1 , 28 ℃ for 12-14 h and adjusted to an attenuance (A600) of 1.0. The suspensions of the bacteria (100 µL of approximately 1 × 10⁸ cfu·mL^-1 ) were spread on the nutrient agar media and allowed to dry. The sterilized filter paper discs (diameter 6 mm) were placed on lawns of freshly made pour- plate of E. amylovora strains, then 6 µL dilution was added on the filter paper discs containing 0 to 1600 µg·mL^-1 of benziothiazolinone. Three replicate plate were used for each concentration. The plates were incubated at 28 ℃ for 24-48 h, and the sensitivity was evaluated by observing the presence or absence of a zone of inhibition around the benziothiazolinone discs, measured the diameters of the inhibition zone in each plate and took the average.【Results】The sensitivity and resistance level differed among the strains from different areas, hosts and years in Xinjiang. The half effective concentration value (EC50) of the tested E. amylovora field isolates to benziothiazolinone ranged from 12.40 to 813.13 µg ·mL^-1 , with the mean of (304.61±133.45) µg ·mL-1 . The number of strains with EC50 value ＜ 200 g·mL-1 and EC50 value ＞ 300 g·mL^-1 was thirty-three and seventeen, accounting for 66% and 34% of the tested strains, respectively. The strain Y112 from a hawthorn plant in Urumqi was the most sensitive to the bactericide, with an EC50 value of 12.40 µg ·mL^{- 1} , while the strain Y125 from an apple plant in Luntai County was the lowest in sensitivity, with an EC50 value of 813.31 µg·mL^-1 . The sensititivity frequency distribution of the strains to benziothiazolinone presented a continous, unimodal curve, which was a normal distribution according to the Kolmogorov-Smirnov test results. Therefore, the mean EC50 value of (304.61±133.45) µg·mL-1 could be regarded as the sensitivity baseline of E. amylovora to benziothiazolinone in Xinjiang. The minimum inhibition concentration values (MIC) and resistance factor of the strain to benziothiazolinone varied from 0.5 to 3.75 µg · mL^{- 1} (mean value 2.15 µg · mL^{- 1} ), 0.04 to 2.67 (mean value 1.0), respectively. There were no resistant subgroup strains. The sensitivity of the isolates from pear and apple plants were lower than that of the strains from hawthorn, quince and Duli pear plants. The results indicate that the EC50 and resistance factor of the strains increased along with the bactericide usage.【Conclusion】The sensitivity baseline value of the E. amylovorain isolated strains in Xinjiang to benziothiazolinone could be established according to this study. No resistant subgroup strains had been found, and the resistance risk of E. amylovora to benziothiazolinone was at low level.