山西省植物病原镰孢菌种群分布及遗传变异分析

Analysis of Population Distribution and Genetic Variation of Plant Pathogenic Fusarium in Shanxi Province

【目的】通过对镰孢菌(Fusarium)ITS、EF-1α和β-tubulin 3个基因序列比较分析,筛选适合于镰孢菌种类鉴定的基因序列,并以此序列分析山西省植物病原镰孢菌种群分布及遗传变异情况。【方法】从2013—2015年在山西省11市28县(区)采集分离的625株镰孢菌菌株中选取形态学清晰的菌株进行ITS、EF-1α和β-tubulin基因序列联合分析,运用Sequencher软件对序列进行拼接和校对,将测序结果与NCBI及FUSARIUM-ID数据库中所有已公布的序列进行BLAST分析,结合下载概念清晰或标准种序列,运用Clustal X和Gel Doc软件进行序列对齐和编辑,运用MEGA、Excel、DNAstar和Taxon Gap软件分析镰孢菌种内种间遗传变异情况,从ITS、EF-1α和β-tubulin中筛选适合镰孢菌种类鉴定的基因序列,并以此序列分析山西省镰孢菌的种群分布特点等。【结果】在3个候选基因序列中,EF-1α基因序列是最适用于镰孢菌种类鉴定的基因序列,其种间平均遗传距离分别是种内平均遗传距离的24倍,种内差异小于种间差异的种的数量最多,达到供试种的73%,物种鉴定准确率最强,达到87%。基于EF-1α基因片段的系统发育分析结果表明,在供试的27种镰孢菌中,有22种表现出单系性,相同种的不同菌株以较高支持率聚成独立支。在27种镰孢菌中,F.oxysporum是山西省镰孢菌的优势种,分离频率最高(22.1%),且分布最广,在23个县(区)均有分布;其次是F.solani(13.8%),在14个县(区)有分布。从不同地区镰孢菌的种群结构及分布来看,运城、临汾、忻州、长治、吕梁、晋中和太原均以F.oxysporum为优势种,朔州以F.lateritium为优势种,大同以F.solani为优势种,晋城以F.verticillioides为优势种,阳泉以F.incarnatum为优势种;其中晋中和忻州的镰孢菌种类最丰富,临汾次之,朔州最少。从不同寄主镰孢菌的种群结构及分布来看,番茄上镰孢菌的种类最多(15种),其次是马铃薯(13种)和大豆(12种);番茄、黄瓜、西瓜、马铃薯、茄子、西葫芦和甘蓝,均以F.oxysporum为优势种,大豆和玉米以F.verticillioides为优势种,小麦以F.avenaceum和F.graminearum为优势种。【结论】镰孢菌种内种间存在丰富的遗传变异,其中EF-1α基因序列的遗传变异最适用于镰孢菌种类的鉴定,但形态学种和系统发育学种不完全吻合。F.oxysporum是山西省镰孢菌的优势种,不同地区、不同寄主上镰孢菌种群存在明显的遗传分化;研究结果可为镰孢菌的分类鉴定、DNA条形码筛选、检验检疫及其综合防治提供理论依据。

[Objective] The objectives of this study are to obtain the sequences of ITS, EF-la and β-tubulin gene regions from plant pathogen, Fusarium spp., compare the suitable gene sequences for species identification, and to analyze the population distribution and genetic variation of Fusarium spp. in Shanxi Province by using the suitable gene sequences. [ Method] A total of 625 Fusarium strains were collected from 28 counties and 11 cities in Shanxi Province from 2013 to 2015. The ITS, EF-la and β-tubulin gene fragments of the morphology of clear strains from 625 strains were sequenced and analyzed. The sequences were assembled and edited using Sequencher software, then blasted in GenBank in NCBI and FUSARIUM-ID databases. The sequences from the clearly documented and reference species were downloaded. All the sequences were aligned and edited manually using ClustalX and GelDoc. The inter- and intra-specific variations of Fusarium spp. were analyzed with MEGA, Excel, DNAstar and TaxonGap. All the three gene sequences consisting of ITS, EF-la and β-tubulin were selected for the taxonomy of the Fusarium species and then the best optimal fragment genes were used in the analysis of the population distributions ofFusarium spp. in Shanxi Province. [Result] EF-1α was the best gene fragment for identifying Fusarium species among the three candidate gene fragments. Intra-specific pairwise distances was 24 times higher than that of the inter-specific pairwise distances. The intra-specific variation was smaller than those of the inter-specific variation by 73% of the tested species. The accuracy rate for identification was the highest, reached 87%. The phylogenetic relationships derived from the EF-1α sequences showed that different strains in 22 of 27 Fusarium species were monophyly, clustering in a same clade with high supporting values. Among the 27 species of Fusarium, F. oxysporum was the dominant species with 22.1% frequency and covered the largest geographical distribution in the 23 counties or regions in Shanxi Province. Followed by F. solani with 13.8% in 14 counties or regions. The results of population distribution of Fusarium in different geographical regions in Shanxi Province showed that F. oxysporum was the dominant species in Yuncheng, Linfen, Xinzhou, Changzhi, Luliang, Jinzhong and Taiyuan city; F. lateritium was the dominant species in Shuozhou, F. solani in Datong, F. verticillioides in Jincheng and F. incarnatum in Yangquan. Among the different regions in Shanxi Province, the most abundant Fusarium species was in Jinzhong and Xinzhou, followed by Linfen, and the least distribution was in Shuozhou. The results of the population distribution of Fusarium from different hosts showed that the 15 Fusarium species extracted from Lycopersicon esculentum, 13 species from Solanum tuberosum and 12 from Glycine max. In these hosts, F. oxysporum was the dominant species in Lycopersicon esculentum, Cucumis sativus, Citrullus lanatus, Solanum tuberosum, Solanum melongena, Cucurbita pepo and Brassica oleracea. F. verticillioides was the dominant species in Glycine max and Zea mays. F. avenaceum and F. graminearum were the dominant species in Triticum aestivum. [Conclusion] There are abundant genetic variation between inter- and intra-specific of Fusarium. Of the three gene regions, EF-1α is the most suitable region for Fusarium species identification. Fusarium morphological taxonomy results are not fully agreed with the molecular phylogenetic results using EF-1α sequences. F. oxysporum is the dominant Fusarium species in Shanxi Province. There are obvious genetic variations of Fusarium populations in different districts and hosts. The research results will provide a theoretic and scientific basis for Fusarium taxonomy, DNA barcode screening, quarantine and integrated control.