我国黄淮麦区10个短体线虫样品种类的分子鉴定

doi:10.3864/j.issn.0578-1752.2018.15.006

Abstract

Abstract: 【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.

Key words: Pratylenchus spp., species identification, rDNA, mtDNA, phylogeny, SCAR

LIU HaiLu, WANG Xuan, LI HongMei, LI YanXia, XUE BoWen, MA JuKui. Molecular Identification of Pratylenchus Species in 10 Samples Collected from Wheat Field in Huanghuai Region of China[J].Scientia Agricultura Sinica, 2018, 51(15): 2898-2912.

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