浙贝母叶斑病病原真菌的分子鉴定及杀菌剂筛选

Molecular Identification and Indoor Fungicide Screening of Pathogenic Fungi of Leaf Spot Disease of Fritillaria Thunbergii Miq

[目的]明确浙贝母叶斑病的致病菌种类,并筛选有效杀菌剂。[方法]采用病组织分离法,从浙贝母叶斑病病株中分离纯化真菌,以聚合酶链式反应(polymerase chain reaction,PCR)扩增基因组DNA的内源转录间隔区(internal transcribed spacer,ITS)、翻译延伸因子-1a(translation elongation factor-1a,TEF-1a)和微管蛋白基因(tubulin gene,TUB)并双向测序,经DNAMAN 9.0软件拼接后利用基本局部对齐搜索工具(basic local alignment search tool,BLAST)进行分析。采用邻接法构建系统发育树以鉴定真菌种类,采用菌饼接种寄主植物和组培苗的柯赫氏法检测致病性,以生长速率法和抑菌圈法室内筛选病原真菌的有效杀菌剂。[结果]从浙贝母叶斑病病株中分离纯化获得1种间座壳属(Diaporthe)真菌。分离物的ITS、TEF-1a和TUB扩增片段与GenBank中Diaporthe eres(无性型为Phomopsis oblonga,P.oblonga)相应片段的同源性分别为100%、99.73%和96.95%(登录号:LC342971、MW804672、MT561360)。在间座壳属中,分离物与P.oblonga的亲缘关系最近。该菌株接种浙贝母植株和组培苗后诱发浙贝母叶斑病。吡唑醚菌酯、咯菌腈、异菌脲、苯醚甲环唑、腈菌唑、咪鲜胺对该真菌的半最大效应浓度(concentration for 50%of maximal effect,EC_(50))分别为0.174、0.084、1.092、0.041、0.619和0.004μg·mL^(-1);6种杀菌剂对P.oblonga孢子萌发的抑制效果为咪鲜胺>吡唑醚菌酯>苯醚甲环唑>腈菌唑>咯菌腈和异菌脲。[结论]本研究证实,P.oblonga是浙贝母叶斑病病原真菌,咪鲜胺和苯醚甲环唑是防治浙贝母叶斑病的有效杀菌剂。

[Objective]To clarify the pathogenic fungi species of the leaf spot disease of Fritillaria thunbergii Miq(F.thunbergii)and screen the effective fungicides.[Methods]The fungi were isolated and purified from F.thunbergii infected with leaf spot disease by tissue separation method.The internal transcribed spacer(ITS),translation elongation factor-1a(TEF-1a)and tubulin gene(TUB)of genomic DNA were amplified by polymerase chain reaction(PCR)and sequenced bidirectionally.After being spliced by DNAMAN 9.0 software,the sequences were analyzed by basic local alignment search tool(BLAST),and the phylogenetic tree was constructed by adjacency method to identify fungal species.The pathogenicity was detected by the Cochrane method of inoculating host plants and tissue culture seedling with fungus cake.The growth rate method and the inhibition zone method were used to screen the effective fungicides of pathogenic fungi in laboratory.[Results]A fungus of the genus Diaporthe was isolated and purified from F.thunbergii infected with the leaf spot disease by the method of diseased tissue isolation.The homology of ITS,TEF-1a and TUB amplified fragments of the isolates with the corresponding fragments of Diaporthe eres(Phomopsis oblonga)in GenBank was 100%,99.73%and 96.95%respectively(accession number:LC342971,MW804672,MT561360).The isolates were most closely related to P.oblonga in the genus oblonga.Leaf spot disease of F.thunbergii was induced by inoculating plants and tissue culture seedlings with the fungus.The concentrations for 50%of maximal effect(EC_(50))of pyraclostrobin,fludioxonil,iprodione,difenoconazole,myclobutanil and prochloraz were 0.174,0.084,1.092,0.041,0.619 and 0.004 mg·mL^(-1)respectively.The inhibition effect of the six fungicides on conidial germination of P.oblonga was prochloraz>pyraclostrobin>difenoconazole>myclobutanil>fludioxonil and iprodione.[Conclusion]This study confirmed that P.oblonga was the pathogen of F.thunbergii leaf spot disease.Prochloraz and difenoconazole were effective fungicides against leaf spot disease of F.thunbergii.