四川省马铃薯孢囊线虫的形态和分子鉴定

Morphological and Molecular Identification of Potato Cyst Nematode from Sichuan

【目的】四川省越西县马铃薯发现疑似线虫侵染症状,为查清病原,对分离获得的线虫进行形态学和分子生物学鉴定。【方法】采集马铃薯根系和根围土壤,对根系内部线虫进行染色观察,用简易漂浮法分离孢囊,并对发现的二龄幼虫、雌虫和孢囊进行显微摄影和形态学测计。提取孢囊和二龄幼虫的DNA,用种特异PCR方法进行鉴定,同时PCR扩增后获得核糖体rDNA部分18S基因、28S基因D2~D3区和ITS区DNA序列,在GenBank中下载已登录的各近似种(球孢囊线虫属)的部分序列,进行序列比对,并使用MrBayes 3.2.3软件进行贝叶斯法系统进化树的构建。【结果】该种线虫能入侵寄主根系内部,二龄幼虫口针基部球近圆形,向后倾斜;口针平均长<23μm;孢囊Granek比值≥3,与马铃薯金线虫高度一致。特异PCR扩增3个孢囊和4条二龄幼虫均得到了430 bp左右的明亮条带,进一步测序后得到的序列长度均为434 bp,与马铃薯金线虫一致。核糖体rDNA 18S和28S进化分析表明,该四川越西马铃薯上发现的孢囊线虫属于球孢囊属线虫,核糖体ITS基因序列进一步分析证实该群体确为马铃薯金线虫。【结论】四川省越西县马铃薯根系分离获得的线虫为马铃薯金线虫,该线虫为我国内检和外检均十分关注的检疫性线虫,一旦传入和定殖扩散,根除十分困难。建议尽快建立马铃薯金线虫疫情监测体系,加强马铃薯种薯的检疫监督管理,增加马铃薯金线虫防控技术的研发投入、研发检测和防控技术,为我国马铃薯主粮化产业健康可持续发展保驾护航。

【Objective】To isolate and identify the pathogen causing cyst nematode infection in Yuexi,Sichuan.【Method】Roots of the diseased potato plants as well as soil around them were collected to find nematodes by staining and examining under a microscope,while cysts separated by means of simple floatation.The second stage juveniles(J2s),female adults,and cysts found were photographed,and their morphological measurements recorded.DNA of the cysts and the J2s were extracted and identified by species-specific PCR.DNA sequences of 18S gene and 28s D2~D3 region,as well as the ITS location in ribosomal DNA,were obtained.Sequences of some cyst nematode species were downloaded from GenBank for alignment.MrBayes 3.2.3 software was used to construct the Bayesian phylogenetic tree.【Result】The isolated nematode could infect the host root system.The J2 stylet knobs rounded to flattened anteriorly;the mean stylet length was less than 23μm;and the cyst Granek’s ratio was higher or equal to 3,which is similar to that for Globodera rostochiensis.The DNA templates of 3cysts and 4 J2s were amplified by species-specific PCR to obtain a band of approximately 430 bp.Further sequencing determined the length to be precisely 434 bp,same as G.rostochiensis.The cyst nematode was shown to be a species of Globodera by an evolutionary analysis on the rDNA 18S and 28S and confirmed to be G.rostochiensis by another evolutionary analysis on ITS gene.【Conclusion】The isolated nematodes from Yuexi,Sichuan was identified as G.rostochiensis,which is of great concern for the domestic disease control and import quarantine,as it can be highly difficult to eradicate once being introduced and colonized in the country.Consequently,a stringent monitoring system must be implemented as soon as possible.Reinforced quarantine,supervision,and management to curtail the possible invasion are urgently needed.Increased efforts on the basic studies as well as developments of detection methodology and prevention and control technology on G.rostochiensis are recommended.