Daniela Stoeva, Deyana Gencheva, Georgi Radoslavov, Peter Hristov, Rozalina Yordanova, Georgi Beev
{"title":"Novel DNA Barcoding and Multiplex PCR Strategy for the Molecular Identification and Mycotoxin Gene Detection of <i>Fusarium</i> spp. in Maize from Bulgaria.","authors":"Daniela Stoeva, Deyana Gencheva, Georgi Radoslavov, Peter Hristov, Rozalina Yordanova, Georgi Beev","doi":"10.3390/mps8040078","DOIUrl":null,"url":null,"abstract":"<p><p><i>Fusarium</i> spp. represent a critical threat to maize production and food safety due to their mycotoxin production. This study introduces a refined molecular identification protocol integrating four genomic regions-ITS1, IGS, <i>TEF-1α</i>, and <i>β-TUB</i>-for robust species differentiation of <i>Fusarium</i> spp. isolates from post-harvest maize in Bulgaria. The protocol enhances species resolution, especially for closely related taxa within the <i>Fusarium fujikuroi</i> species complex (FFSC). A newly optimized multiplex PCR strategy was developed using three primer sets, each designed to co-amplify a specific pair of toxigenic genes: <i>fum6/fum8</i>, <i>tri5/tri6</i>, and <i>tri5/zea2</i>. Although all five genes were analyzed, they were detected through separate two-target reactions, not in a single multiplex tube. Among 17 identified isolates, <i>F. proliferatum</i> (52.9%) dominated, followed by <i>F. verticillioides</i>, <i>F. oxysporum</i>, <i>F. fujikuroi</i>, and <i>F. subglutinans</i>. All isolates harbored at least one toxin biosynthesis gene, with 18% co-harboring genes for both fumonisins and zearalenone. This dual-protocol approach enhances diagnostic precision and supports targeted mycotoxin risk management strategies.</p>","PeriodicalId":18715,"journal":{"name":"Methods and Protocols","volume":"8 4","pages":""},"PeriodicalIF":2.0000,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12286001/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Methods and Protocols","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.3390/mps8040078","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
Abstract
Fusarium spp. represent a critical threat to maize production and food safety due to their mycotoxin production. This study introduces a refined molecular identification protocol integrating four genomic regions-ITS1, IGS, TEF-1α, and β-TUB-for robust species differentiation of Fusarium spp. isolates from post-harvest maize in Bulgaria. The protocol enhances species resolution, especially for closely related taxa within the Fusarium fujikuroi species complex (FFSC). A newly optimized multiplex PCR strategy was developed using three primer sets, each designed to co-amplify a specific pair of toxigenic genes: fum6/fum8, tri5/tri6, and tri5/zea2. Although all five genes were analyzed, they were detected through separate two-target reactions, not in a single multiplex tube. Among 17 identified isolates, F. proliferatum (52.9%) dominated, followed by F. verticillioides, F. oxysporum, F. fujikuroi, and F. subglutinans. All isolates harbored at least one toxin biosynthesis gene, with 18% co-harboring genes for both fumonisins and zearalenone. This dual-protocol approach enhances diagnostic precision and supports targeted mycotoxin risk management strategies.