葡萄胶孢炭疽病菌复合种(Colletotrichum gloeosporioides species complexes)对嘧菌酯的抗药性研究

Resistance of Colletotrichum gloeosporioides Species Complexes from Grape to Azoxystrobin

研究了江苏丘陵地区葡萄胶孢炭疽病菌复合种(Colletotrichum gloeosporioides species complexes,CGSC)对嘧菌酯的敏感性及抗性分子机理。利用孢子萌发法和区分剂量法确定3个种(C.aenigma、C.viniferum和C.fructicola)51个单孢分离株对嘧菌酯的抑制中浓度(EC_(50))和敏感性表型,采用田间人工接种防治方法评价其抗药性,通过嘧菌酯作用标靶基因(CYTB)序列分析确定抗性的分子机制。27个C.aenigma和16个C.viniferum中对嘧菌酯敏感的菌株分别有20和14个,EC_(50)分别为0.0388(0.0150~0.0770)和0.0614(0.0213~0.0906)mg·L^(-1),而抗性菌株的EC_(50)均大于100 mg·L^(-1);C.fructicola菌株均为抗性菌株,其EC_(50)大于100 mg·L^(-1)。田间接种防治试验证明嘧菌酯2倍推荐剂量(250 mg·L^(-1))对抗性菌株C.fructicola MS206失去防效(防治效果7.11%)。所有抗性菌株的CYTB基因上都只含有G143A点突变;CYTB基因G143位氨基酸两侧内元呈现多样性。江苏丘陵地区葡萄炭疽病种群中的不同种对嘧菌酯的敏感性不同,抗药种群的流行是造成田间防效下降的主因子,所有抗性菌株的抗性分子机制均为CYTB基因G143A点突变,未发现其他点突变类型。

The sensitivity and resistance molecular mechanism of Colletotrichum gloeosporioides species complexes(CGSC)derived from the hilly area of Jiangsu Province to azoxystrobin were studied.Employing the methods of spore germination and discriminative dose(a concentration that fully inhibits mycelial growth of the sensitive strains),the effective inhibition medium concentration values(inhibits mycelia growth by 50%relative to the control,EC_(50))to azoxystrobin and sensitivity types of 51 single spore isolates of CGSC(C.aenigma,C.viniferum and C.fructicola)were identified.The field control efficacy test inoculated with conidia suspension of sensitive,resistant and mixed strains(the resistant strain and sensitive strain with the same conidia concentration were mixed in equal volume)was used to evaluate the epidemic of fungicides resistance of CGSC.Furthermore,the molecular mechanisms of azoxystrobin were determined by the sequence analysis of the target gene(mitochondrial cytochrome b,CYTB).Among the 27 strains of C.aenigma and 16 strains of C.viniferum,20 and 14 strains were sensitive to axoxystrobin,with EC_(50)values of 0.0388(0.0150–0.0770)and 0.0614(0.0213–0.0906)mg·L^(-1),respectively,while the EC_(50)values of resistant strains were greater than 100 mg·L^(-1).All the C.fructicola strains were resistant and their EC_(50)values were greater than 100 mg·L^(-1).Field inoculated test of azoxystrobin at twice recommended field dose(250 mg·L^(-1))against resistant strains have lost the control efficacy(control efficacy 7.11%).Sequence analysis of target gene(CYTB)showed that all resistant strains harbored G143A(Glycine substituted by Alanine)point mutation in CYTB.The CYTB can be divided into two types based on whether there are partial deletions in the sequence of the intro adjoining after the G143 amino acid position.The sensitivity of different species in CGSC to azoxystrobin exhibits heterogeneity.The prevalence of resistant populations was the main factor leading to a decrease in field control effectiveness.The genotype of resistance to azoxystrobin of CGSC belonged to the point mutation of single base of target gene,which resulted in the substitution of amino acids,i.e.G143A,and no other genotypes of point mutations were found in this research.