我国黄淮麦区10个短体线虫样品种类的分子鉴定

【目的】短体线虫(Pratylenchus spp.)是植物根系内迁移性寄生线虫,可引起许多作物的根腐线虫病,给世界农业生产造成了极大的危害。为明确我国黄淮麦区与禾谷孢囊线虫复合侵染小麦的短体线虫种类,本研究对采自黄淮4省麦区的10个短体线虫样品进行种类的分子鉴定,分析种群系统进化关系及种内遗传变异,以期为我国小麦根部线虫病害的综合防治提供指导。【方法】对采自江苏、安徽、河南和山东4省小麦孢囊线虫发病田中的10个小麦短体线虫样品进行线虫分离,从各样品中随机挑取5条短体线虫,分别提取单条线虫DNA,扩增r DNA 18S片段并进行测序比对,选取序列有代表性的2个DNA样本进一步扩增其r DNA 28S D2-D3区以及mt DNA-COI基因片段,经序列比对分析后,利用MEGA4.0软件采用邻接法分别构建基于r DNA 18S、28S D2-D3和mt DNA-COI序列的系统进化树,通过聚类关系及相似度分析确定线虫种类,同时利用种特异性引物进行验证。【结果】扩增挑取的50条短体线虫的r DNA 18S区片段,测序得到片段长度在857—935 bp,BLAST比对分析揭示部分样品可能为短体线虫的混合种群;进一步测定的20个代表性DNA样本的r DNA 28S D2-D3区和mt DNA-COI基因的片段长度分别在771—784 bp和415—417 bp;系统进化树以及相似度分析揭示我国黄淮流域4省麦区10个短体线虫样品中有咖啡短体线虫(P.coffeae)、落选短体线虫(P.neglectus)和斯克里布纳短体线虫(P.scribneri),其中,江苏沛县样品JS2和山东潍坊样品SD1是落选短体线虫单一侵染样品,河南永城样品HN2和安徽淮北样品AH3是咖啡短体线虫单一侵染样品,安徽萧县样品AH2、AH5和淮北样品AH4以及河南永城样品HN1和HN3均为落选短体线虫和咖啡短体线虫的混合侵染样品,江苏徐州样品JS1是落选短体线虫和斯克里布纳短体线虫的混合侵染样品。用SCAR特异性引物扩增20个短体线虫单条DNA样本,结果显示,用落选短体线虫特异性引物PNEG-F1/D3B5能够从JS1-2、JS2-1、JS2-2、AH2-2、AH4-1、AH5-1、HN1-2、HN3-2、SD1-1和SD1-2等10个样本中扩增出140 bp的单一条带,用咖啡短体线虫引物PC1/PC2能够从AH2-1、AH3-1、AH3-2、AH4-2、AH5-2、HN1-1、HN2-1、HN2-2和HN3-1等9个样本中扩增出630 bp的单一条带,用斯克里布纳短体线虫引物Ps F7/Ps R7从JS1-1中扩增出130bp的单一条带,种类鉴定结果与上述序列分析结果相一致。【结论】我国黄淮流域4省小麦孢囊线虫发病田中的短体线虫种类有咖啡短体线虫、落选短体线虫和斯克里布纳短体线虫,其中落选短体线虫是优势种,证实了短体线虫不同种群复合侵染小麦的现象较为普遍。基于mt DNA-COI基因构建的系统进化树可以有效区分短体线虫的近缘种,相比r DNA 18S和28S基因更适于作为短体线虫种类鉴定的分子靶标。

Molecular Identification of Pratylenchus Species in 10 Samples Collected from Wheat Field in Huanghuai Region of China

【Objective】The Pratylenchus species are migratory endoparasites of plant roots, causing root lesion of many crops and great damage of agricultural production all over the world. In order to clarify the species of genus Pratylenchus co-infection with Heterodera avenae on wheat from Huanghuai region of China, 10 samples were collected from wheat field in 4 provinces of the region and the Pratylenchus species were molecularly identified. The phylogenetic relationship of Pratylenchus species and genetic variation of intraspecific populations were analyzed. The results will provide valuable information for the integrated control of nematode diseases on wheat root. 【Method】The Pratylenchus nematodes were extracted from 10 samples collected from wheat fields infected with H. avenae in Jiangsu, Anhui, Henan and Shandong provinces. Fivenematodes were randomly picked up from each sample, and the DNA of individual nematode was extracted as the template for PCR amplification. The fragments of rDNA 18S region were amplified and the PCR products were sequenced. The BLAST alignment of 18S sequences revealed the different Pratylenchus species may present in these samples. The DNA templates of two representative specimens from each sample were selected for the further amplification of fragments from rDNA 28S D2-D3 region and mtDNA-COI gene. The fragments were sequenced and the BLAST alignments were performed. The phylogenetic trees were constructed on basis of rDNA 18S, 28S D2-D3 and mtDNA-COI sequences using neighbor-joining method by MEGA4.0 software, respectively. Thespecies identification was supported by the analyses of phylogenetic relationships and sequence similarities. The species-specific primers as the sequence-characterized amplified regions (SCAR) markers were used to validate the identification. 【Result】The fragments amplified from rDNA 18S region of 50 individual nematodes were sequenced and the sizes were 857-935 bp. The BLAST searching revealed that the mixed Pratylenchus populations might present in some samples. The sequence sizes of the rDNA 28S D2-D3 fragments amplified from 20 selected specimens were 771-784 bp, the sizes of mtDNA-COI were 415-417 bp. The phylogenetic analyses as well as the comparisons of sequence similarities both demonstrated that three Pratylenchus species including P. coffeae, P. neglectus and P. scribneri,were found in 10 samples collected from wheat fields in 4 provinces of Huanghuai region. Among 10 samples, the sample JS2 from Peixian of Jiangsu Province and SD1 from Weifang of Shandong Province were infected only with P. neglectus, HN2 from Yongcheng of Henan Province and AH3 from Huaibei of Anhui Province were infected only with P. coffeae. The samples AH2 and AH5 from Xiaoxian and AH4 from Huaibei of Anhui Province as well as HN1 and HN3 from Yongcheng of Henan Province were all co-infected with P. neglectus and P. coffeae, and JS1 from Xuzhou of Jiangsu Province was co-infected with P. neglectus and P. scribneri. The DNA templates from 20 representative specimens were amplified using SCAR primers. The results showed that a single band of 140 bp was amplified from JS1-2, JS2-1, JS2-2, AH2-2, AH4-1, AH5-1, HN1-2, HN3-2, SD1-1 and SD1-2 using specific primer PNEG-F1/D3B5 for P. neglectus, a single band of 630 bp was amplified from AH2-1, AH3-1, AH3-2, AH4-2, AH5-2, HN1-1, HN2-1, HN2-2 and HN3-1 using primer PC1/PC2 for P. coffeae,a single band of 130 bp was amplified from JS1-1 using primer PsF7/PsR7 for P. scribneri. The results of SCAR detection confirmed the species identification mentioned above.【Conclusion】The molecular identification demonstrated that three species, P. coffeae, P. neglectus and P. scribneri, were found in wheat fields infested with H. avenae from four provinces in Huanghuai region, and P. neglectus is the dominant species. The co-infection of different Pratylenchus species occurred quiet common in wheat field from the region. The phylogenetic tree based on mtDNA-COI gene can effectively distinguish the close related Pratylenchus species, therefore, it is more suitable to identify Pratylenchus species than rDNA 18S and 28S markers.