# 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.

## Key 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.