浙贝母黑斑病致病菌的分离鉴定及分子检测

目的]分离、纯化和鉴定浙贝母黑斑病的病原真菌,为浙贝母黑斑病的监测及防治提供实验基础。方法]采用患病组织分离法在马铃薯葡萄糖琼脂(potato dextrose agar,PDA)培养基上分离纯化病原真菌,PCR扩增基因组DNA的核糖体内转录间隔区(internal transcribed spacer,ITS)、翻译延伸因子-1α(translation elongation factor-1α,TEF-1α)、甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,G3PDH)和RNA聚合酶Ⅱ亚基(the second largest subunit of the nuclear RNA polymerase enzymeⅡ,RPB2)序列并双向测序、拼接,与GenBank中序列同源性比对后构建系统发育树,确定真菌种类,以摩擦接种法检测致病性。结果]从浙贝母黑斑病病株中分离纯化获得2种链格孢属真菌。分离物1的ITS、TEF-1α、G3PDH、RPB2扩增片段与GenBank中链格孢菌(登录号:MG182428、KY175227、XM018523704、MG250634)的同源性均为100%;分离物2则与细极链格孢菌(登录号:MK967997、LC136862、KY290574、LT707523)的同源性均为100%。经摩擦接种确定两种链格孢属真菌均为浙贝母黑斑病致病菌。结论]浙贝母黑斑病的病原真菌有链格孢菌和细极链格孢菌,其中细极链格孢菌首次被证明可引起浙贝母黑斑病。

Isolation and Molecular Identification of the Pathogenic Fungi of Black Spot Disease of Fritillaria thunbergii Miq

Objective]To isolate,purify and identify the pathogenic fungi of black spot disease of Fritillaria thunbergii(F.thunbergii),and provide experimental basis for the monitoring and control of black spot disease of F.thunbergii.Methods]Pathogenic tissue isolation method was used to isolate and purify pathogenic fungi from F.thunbergii.Partial fragments of ribosomal internal transcribed spacer(ITS),translation elongation factor-1α(TEF-1α)glyceraldehyde-3-phosphate dehydrogenase(G3PDH)and the second largest subunit of the nuclear RNA polymerase enzymeⅡ(RPB2)of the fungi were obtained by PCR amplification with specific primers.After two-way sequencing and splicing,the amplified fragments were homologous with the corresponding sequences in GenBank,and the phylogenetic tree was constructed to identify fungal species.The pathogenicity of fungi of F.thunbergii was detected by friction inoculation method.Results]Two fungi belonging to Alternaria genus were isolated and purified from F.thunbergii plants infected with black spot disease.The ITS,TEF-1α,RPB2,and G3 PDH amplified fragments of the isolate 1 were 100%homologous to Alternaria alternata(A alternata,accession number:MG182428,KY175227,XM018523704,MG250634,respectively)in Gen Bank,while those of the isolate 2 was 100%homologous to Alternaria tenuissima(A.tenuissima,accession numbers:MK967997,LC136862,KY290574,LT707523,respectively).Both of the fungi were identified as pathogenic fungi of F.thunbergii black spot disease by friction inoculation.Conclusion]A.alternata and A.tenuissima are the pathogenic fungi of black spot disease of F.thunbergii.A.tenuissima is first reported to cause black spot disease of F.thunbergii.