M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Miklukho-Maklaya st., 16/10, Moscow 117997, Russia.
M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Miklukho-Maklaya st., 16/10, Moscow 117997, Russia.
Int J Food Microbiol. 2016 May 16;225:27-37. doi: 10.1016/j.ijfoodmicro.2016.02.012. Epub 2016 Feb 22.
The fungus Fusarium avenaceum and its closest relatives are responsible for contamination of agricultural plants and their products by mycotoxins such as enniatins and moniliformin. Precise identification of mycotoxin producers is necessary for estimation of the accumulation risk of those compounds and for preventing the consumption of highly contaminated products. Nucleic acids amplification-based techniques proved to be the most rapid and reliable approach for pathogen diagnostics and identification. In this study partial phosphate permease gene (PHO) sequences were determined for Fusarium avenaceum (including one isolate identified as F. arthrosporioides), F. tricinctum, F. acuminatum and F. torulosum. Phylogenetic analysis of 40 isolates of those species from different climates and geographical regions of Russia and some neighboring countries based on sequences of PHO, translation elongation factor 1 alpha (TEF1α), beta-tubulin (β-TUB), enniatin synthetase (Esyn1) genes and combined data set demonstrated that the PHO gene possesses the highest rate of variability among them and can be considered as an informative marker for phylogenetic studies of these species. According to the combined data set phylogeny, the isolates of each species formed clusters with a high bootstrap support. Analysis of PHO sequences revealed a high intraspecific variability of F. avenaceum: there were 5 independent clusters on the dendrogram, including one cluster which was closer to F. torulosum than to other F. avenaceum isolates. Variable sites in PHO sequences have been used for the design of species-specific primers and a fluorescent hydrolysis probe. The specificity of the assay was shown for DNA samples extracted from 68 isolates of 23 Fusarium species. Quantitative PCR approach was applied to estimate the contamination rate of 17 naturally infected oat and barley samples, previously characterized by microbiological procedures.
镰刀菌属 Fusarium avenaceum 及其近亲是造成农作物及其产品受真菌毒素（如恩镰孢菌素和单端孢霉烯族化合物）污染的原因。准确鉴定真菌毒素的产生者对于评估这些化合物的积累风险以及防止食用高度污染的产品是必要的。基于核酸扩增的技术已被证明是病原体诊断和鉴定最快速和可靠的方法。在本研究中，测定了镰刀菌属 Fusarium avenaceum（包括一个被鉴定为 F. arthrosporioides 的分离株）、F. tricinctum、F. acuminatum 和 F. torulosum 的部分磷酸载体基因（PHO）序列。基于 PHO、延伸因子 1α（TEF1α）、β-微管蛋白（β-TUB）、恩镰孢菌素合成酶（Esyn1）基因序列和组合数据集，对来自俄罗斯不同气候和地理区域以及一些邻国的 40 个这些物种的分离株进行了系统发育分析，结果表明 PHO 基因在这些物种中具有最高的变异性，可作为这些物种系统发育研究的有用标记。根据组合数据集的系统发育，每个种的分离株形成了具有高自举支持的聚类。PHO 序列分析揭示了 F. avenaceum 的种内高度变异性：在系统发育树上有 5 个独立的聚类，其中一个聚类与 F. torulosum 比与其他 F. avenaceum 分离株更接近。PHO 序列中的变异位点已被用于设计种特异性引物和荧光水解探针。该测定的特异性已通过从 23 种镰刀菌属的 68 个分离株提取的 DNA 样本得到证明。已应用定量 PCR 方法来估计先前通过微生物学程序进行了特征描述的 17 个自然感染燕麦和大麦样本的污染率。
