摘要
【目的】阐明江苏丘陵地区葡萄炭疽病菌(Colletotrichum spp.)种群(GG)对多菌灵的抗药性流行动态及抗性分子机制。【方法】采用区分剂量法和菌丝生长速率法分别测定GG的抗药性流行动态和敏感性;田间人工接种GG后再检测回分离菌株对药剂敏感性的方法评价GG的抗药性流行演化;药剂作用标靶基因序列分析阐明抗性分子机制。【结果】2013年和2017年GG对多菌灵的EC50均值(ρ)分别为0.528 5 mg·L-1和7.787 8 mg·L^-1。GG对多菌灵的抗性菌株比率从2013年的2.34%上升至2017年的32.21%。2017年和2018年多菌灵接种抗性菌株处理的防效分别为0.64%和4.18%。回分离GG中抗性菌株/敏感菌株的比率2017年和2018年分别为65.00%和68.18%,均高于接种比率50%。抗性菌株靶标基因(TUB2)第198位的谷氨酸突变成丙氨酸(E198A),或第200位的苯丙氨酸突变成络氨酸(F200Y)。【结论】江苏丘陵地区GG对多菌灵已形成抗药性流行。多菌灵持续施用是GG田间抗药性流行的驱动因子。GG抗性菌株标靶基因突变类型为E198A或F200Y。
【Objective】Grapevine anthracnose caused by Colletotrichum spp.species complex population(GG)has been the main disease during the grapevine spike stage in the hilly area of Jiangsu province.In recent years,the controlling failure of common chemicals to the disease has caused serious yield loss.Therefore,it has become an important research target to clarify the evolution,epidemic dynamics and molecular mechanism of resistance of GG to benzimidazole fungicide,and to provide decision-making basis for resistance control.【Methods】Employing the methods of discriminative dose(a concentration that fully inhibits mycelial growth of the sensitive isolates)and effective inhibition medium concentration(inhibits mycelia growth by 50%relative to the control,EC50),the EC50 values were identified to distinguish sensitivity and sensitive baseline migration to carbendazim.According to previous studies,the discriminatory concentration of carbendazim was 10 mg·L^-1.Then EC50 values of carbendazim were determined from 98 isolates and 34 isolates in 2013 and 2017,respectively.Carbendazim solution was added to PDA to produce final active ingredient concentrations of 0,0.04,0.12,0.37,1.11,3.33,10.00 and 30.00 mg·L^-1.For each isolate,three replicates per concentration were used.The conidia suspension of sensitive,resistant and mixed isolates(the resistant isolate and sensitive isolate with the same conidia concentration were mixed in equal volume)was inoculated during young berry growing stage after spray with carbendazim.The concentration of conidia suspension was 1×10^4 spores per mL.The concentration of carbendazim was 1000 mg·L^-1.The controlling efficacy was investigated during berry ripening stage,and the sensitivity of the back separative isolates to the carbendazim was detected to evaluate the resistant evolution of GG.This field control test was repeated twice in 2017 and2018,respectively.Furthermore,the molecular mechanisms of carbendazim were determined by the sequence analysis of target gene(TUB2).First,DNA from fungal mycelia was extracted using a DNA kit.One primer pairs,TubF1(5’-ACTTCGTCTTCGGCCAGTCTG-3’)and TubR1(5’-TTCTGGACGTTGCGCATCTG-3’)was used.PCR products were examined by electrophoresis in a 1.2%agarose gel in1×TAE buffer.DNASTAR software was used to assemble and align the nucleotide and amino acid sequences.All nucleotide sequences were compared to previously reported sequences using BLAST.【Results】The mean EC50 values of GG to carbendazim were 0.5285 mg·L^-1 and 7.7878 mg·L^-1 in 2013 and 2017,respectively.The sensitivity of GG shifted from the baseline significantly.The resistant frequencies of GG to carbendazim in different sampling areas in the same year and in different sampling years in the same region were different.However,when the whole city was taken as a sampling unit,the resistant frequencies of GG to carbendazim within five years(2013-2017)increased year by year.The resistant frequencies of GG to carbendazim increased from 2.34%in 2013 to 32.21%in 2017.Field test of carbendazim at twice the recommended field dose(a.i.1000 mg·L^-1)against resistant isolates showed that the controlling efficacy of carbendazim was very low,only 0.64%and 4.18%in 2017 and2018,respectively.The resistance frequencies of back separative isolates were 65.00%and 68.18%in2017 and 2018,respectively,which was significantly higher than the initial ratio of inoculated resistant isolates(50%).All the resistant isolates harbored the E198 A(glutamate substituted by alanine)or F200 Y(phenylalanine substituted by arginine)point mutation in TUB2.【Conclusion】The resistance frequencies of GG to carbendazim in the hilly area of Jiangsu province fluctuated from year to year,but the resistance frequencies of the population showed an overall upward trend within five years,and resistant epidemic formed.The results also showed that the current resistance control measures had poor effect on the resistance management of GG.We suggested stopping carbendazim application completely.Carbendazim should be replaced by fungicides with different mechanisms.The selection pressure caused by the continuous application of carbendazim was the main driving factor for the emergence and prevalence of resistance to GG in the field.The genotype of resistance to carbendazim of GG belonged to the point mutation of single base of target gene,which resulted in the substitution of amino acids,i.e.E198 A or F200 Y,and no other genotypes were found in this research.
作者
杨敬辉
许媛
肖婷
褚姝频
芮东明
姚克兵
YANG Jinghui;XU Yuan;XIAO Ting;CHU Shupin;RUI Dongming;YAO Kebing(Zhenjiang Institute of Agricultural Sciences of Jiangsu,Jurong 212400,Jiangsu,China;Jiangsu Plant Protection and Quarantine Station,Nanjing 200000,Jiangsu,China)
出处
《果树学报》
CAS
CSCD
北大核心
2021年第2期242-249,共8页
Journal of Fruit Science
基金
江苏现代农业(葡萄)产业技术体系(JATS[2020]305)
江苏省“333工程”资助项目(BRA2020167)。
关键词
葡萄
炭疽病菌
多菌灵
抗药性监测
分子机制
Grape
Colletotrichum spp.
Carbendazim
Resistance monitoring
Molecular mechanism
