烟草赤星病菌对嘧菌酯抗性突变体的诱导及其生物学特性

Induction and biological characterization of mutants of Alternaria alternata resistant to azoxystrobin

为评价烟草赤星病致病菌链格孢Alternaria alternata对嘧菌酯的抗性风险,以敏感菌株J6为试材,通过菌丝药剂驯化和分生孢子紫外诱变诱导抗性突变体,并对抗性突变体的生物学特性进行了研究,同时对抗性突变体与敏感菌株线粒体的细胞色素b基因(cyt b) cDNA序列全长进行了测序分析。结果表明:经药剂驯化未获得抗性突变体,而紫外诱变共获得7株抗性突变体,突变频率约为0.007%,抗性水平分别为5.27、8.28、25.28、12.82、6.14、9.28和52.91倍。适合度研究表明,抗性突变体与敏感菌株的分生孢子萌发能力及致病力相当,但分生孢子产生量均高于敏感菌株,菌丝生长速率除突变体6-1外均快于敏感菌株。cyt b基因cDNA序列分析表明:有4株抗性突变体在不同位点上发生了核苷酸突变,其中突变体6-7 cyt b的249位和871位碱基由T突变为C,但其编码的氨基酸未发生突变;突变体6-8 cyt b的734位碱基由T突变为C,引起所编码的245位丙氨酸突变为缬氨酸(V245A);突变体6-9 cyt b的510位碱基由T突变为A,所编码的170位由精氨酸替代了丝氨酸(S170R);突变体6-11cyt b的732位碱基由T突变为A,所编码的244位由苯丙氨酸替代了亮氨酸(L244F),其776位碱基由T突变为C,所编码的259位由丙氨酸替代了缬氨酸(V259A),其1 156位碱基由A突变为G,所编码的氨基酸未发生变化。研究结果初步表明,烟草赤星病菌对嘧菌酯存在潜在的抗药性风险,其cyt b基因的点突变与其对嘧菌酯的抗药性有关。

The resistance risk of Alternaria alternata to azoxystrobin was evaluated.Mycelial plugs of the sensitive parent isolate J6 was inoculated onto agar plates amended with azoxystrobin and the conidia of J6 was induced with UV irradiation.The biological characterization of resistant mutants was studied,and the resistance mechanism of mutants was also investigated by the full-length cDNA of target gene cyt b sequence analysis.Seven mutants of A.alternata resistant to azoxystrobin were acquired by UV-induced mutation of conidia,which exhibited a mutation frequency of around 0.007%.The resistance ratios for seven mutants were 5.27,8.28,25.28,12.82,6.14,9.28 and 52.91,respectively.However,no mutants were obtained by mycelial plug transferring on the agar plates amended with azoxystrobin.Studies on biological characters of the wild-type and mutant strains of A.alternata showed that all resistant mutants and the parent isolate J6 had equal abilities in the conidia germination and pathogenicity,while resistant mutants had higher conidia production ability and stronger mycelial growth ability(except mutant 6-1)than those of J6.Sequence analysis of the full-length cDNA sequence of cyt b gene showed that there were different point mutations on four mutations of A.alternata.Mutant 6-7 showed mutations at 249 and 871 positions(T mutated to C),while no amino acids mutated.Mutant 6-8 had a point mutation at 734 position(T mutated to C),which resulted in alanine mutated to valine(V245 A).Mutant 6-9 had a point mutation at 510 position(T mutated to A),which resulted in arginine mutated to serine(S170 R).And mutant 6-11 had a mutation at 732 position(T mutated to A),which resulted in leucine mutated to phenylalanine(L244 F);had a mutation at 776 position(T mutated to C),which resulted in valine mutated to alanine(V259 A);and had a mutation at 1 156 position(A mutated to G),while no amino acids mutated.These studies suggested that A.alternata has the potential resistant risk to azoxystrobin,and the mutation of cyt b gene in A.alternata was related to the resistance to azoxystrobin.