新疆梨火疫病菌对噻霉酮的敏感性及抗药性分析

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

【目的】明确梨火疫病菌对噻霉酮的敏感性,建立梨火疫病菌对噻霉酮的敏感基线,监测梨火疫病菌对噻霉酮的抗药性水平,为噻霉酮的合理使用和梨火疫病的高效防控提供科学依据。【方法】选取2016-2020年从新疆10个地州(区/市)的香梨、苹果、山楂、榅桲和杜梨病样中分离获得的50个梨火疫病菌(Erwinia amylovora)菌株,采用滤纸片-抑菌圈法测定供试梨火疫病菌菌株对噻霉酮的室内毒力。【结果】来自新疆不同地区、不同寄主和年份的梨火疫病菌分离菌株对噻霉酮敏感性、抗性水平都存在一定差异。噻霉酮对供试菌株的EC_(50)值范围为12.40~813.13μg·mL^(-1),其中Y112(乌鲁木齐市的山楂分离物)为最敏感的菌株,EC_(50)为12.40μg·mL^(-1),Y125菌株(轮台县的苹果分离物)的敏感性最低,EC_(50)813.31μg·mL^(-1)是最敏感菌株的65.59倍。不同菌株的敏感性分布频率呈连续性单峰曲线,EC50值符合正态分布,确定EC50平均值(304.61±133.45μg·mL^(-1))作为新疆梨火疫病菌对噻霉酮的敏感基线值。噻霉酮对菌株的最小抑制浓度(ρ)(MIC)在0.5~3.75μg·mL^(-1)之间(均值为2.15μg·mL^(-1)),抗性水平为0.04~2.67倍之间(均值为1.0倍),未发现抗药性菌株。苹果和香梨寄主上分离菌株的敏感性低于山楂、榲桲和杜梨寄主上的分离菌株;菌株的EC50和抗性倍数有随药剂使用年份延长而增加的趋势。【结论】建立了新疆梨火疫病菌分离菌株对噻霉酮药剂的敏感基线,未发现抗药性菌株,梨火疫病菌对噻霉酮的抗性风险低。

【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 over use 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,com-mercially available,environmentally safe and non-phytotoxic systemic bactericides,and replacing cop-per 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 registered to control fire blight in China.The purpose of this study is to assess the susceptibility of E.amy-lovora 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 char-acteristic fire blight symptoms.Bacterial strains were isolated from the infected plant materials of fra-grant pear(Pyrus sinkiangensis),apple(Malus pumila),duli pear(Pyrus betulaefolia),hawthorn(Cra-taegus 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)us-ing 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 test-ed 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 at-tenuance(A600)of 1.0.The suspensions of the bacteria(100μL of approximately 1×10^(8) 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 6μL dilution was added on the filter paper discs containing 0 to 1600μg·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 Xinji-ang.The half effective concentration value(EC50)of the tested E.amylovora field isolates to benziothia-zolinone ranged from 12.40 to 813.13μg·mL^(-1),with the mean of(304.61±133.45)μg·mL^(-1).The num-ber of strains with EC50 value<200 g·mL^(-1) and EC50 value>300 g·mL^(-1) was thirty-three and seven-teen,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 12.40μg·mL^(-1),while the strain Y125 from an apple plant in Luntai County was the lowest in sensitivity,with an EC50 value of 813.31μg·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 val-ues(MIC)and resistance factor of the strain to benziothiazolinone varied from 0.5 to 3.75μg·mL^(-1)(mean value 2.15μg·mL^(-1)),0.04 to 2.67(mean value 1.0),respectively.There were no resistant sub-group 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 fac-tor of the strains increased along with the bactericide usage.【Conclusion】The sensitivity baseline val-ue of the E.amylovorain isolated strains in Xinjiang to benziothiazolinone could be established accord-ing to this study.No resistant subgroup strains had been found,and the resistance risk of E.amylovora to benziothiazolinone was at low level.