Novel DNA Barcoding and Multiplex PCR Strategy for the Molecular Identification and Mycotoxin Gene Detection of Fusarium spp. in Maize from Bulgaria

TL;DR

This study presents a new DNA barcoding and PCR method to identify Fusarium species in Bulgarian maize and detect their mycotoxin genes.

Contribution

A refined molecular protocol combining four genomic regions and a novel multiplex PCR strategy for improved Fusarium spp. identification and toxin gene detection.

Findings

F. proliferatum was the most prevalent Fusarium species in the tested maize samples.

Eighteen percent of isolates co-harbored genes for both fumonisins and zearalenone.

The dual-protocol approach improves diagnostic accuracy for mycotoxin risk management.

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.

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