Cyanobacterial blooms can disrupt aquatic ecosystems and threaten human and animal health through the production of potent toxins. Early detection of toxic cyanobacterial blooms using gene-based methods is predicated on the accurate detection of biosynthetic genes responsible for toxin production. Here, we report the isolation of a nodularin-producing freshwater benthic cyanobacterium from a tychoplanktonic microbial mat potentially linked to donkey deaths at a natural watering hole in Northeast Italy. Morphometric and genomic analyses identified the strain as a member of the genus Planktothrix. The isolated strain produced the demethylated nodularin variant [D-Asp¹]NOD at a high intracellular concentration, with levels reaching up to 556.90 ± 38.34 µg g−1 (dry weight) and extracellular concentrations reaching 97.93 ± 2.95 µg L⁻¹ in spent culture medium. This is the first report of a Planktothrix strain capable of producing nodularin. We identified a complete 47-kb nodularin biosynthetic gene cluster in a complete genome sequence, which shares up to 98–100% sequence identity with microcystin biosynthetic gene clusters from microcystin-producing Planktothrix. A mechanism for how the nodularin biosynthetic gene cluster arose in this strain is presented. Detection of microcystin and nodularin biosynthetic genes over the last two decades has enabled early prediction of toxic blooms. However, especially in benthic communities with co-occurring nodularin- and microcystin-producing strains, such methods cannot discriminate whether one or both toxin classes are produced. These findings underscore the need to refine molecular monitoring strategies and develop more precise genomic or metabolomic tools to accurately predict and manage cyanobacterial toxin risks in freshwater ecosystems worldwide.
