Azolla as a Safe Food: Suppression of Cyanotoxin-Related Genes and Cyanotoxin Production in Its Symbiont, Nostoc azollae

The floating freshwater fern <i>Azolla</i> is the only plant that retains an endocyanobiont, <i>Nostoc azollae</i> (aka <i>Anabaena azollae</i>), during its sexual and asexual reproduction. The increased interest in <i>Azolla</i> as a potential source of food and its unique evolutionary history have raised questions about its cyanotoxin content and genome. Cyanotoxins are potent toxins synthesized by cyanobacteria which have an anti-herbivore effect but have also been linked to neurodegenerative disorders including Alzheimer's and Parkinson's diseases, liver and kidney failure, muscle paralysis, and other severe health issues. In this study, we investigated 48 accessions of <i>Azolla-Nostoc</i> symbiosis for the presence of genes coding microcystin, nodularin, cylindrospermopsin and saxitoxin, and BLAST analysis for anatoxin-a. We also investigated the presence of the neurotoxin β-N-methylamino-L-alanine (BMAA) in <i>Azolla</i> and <i>N. azollae</i> through LC-MS/MS. The PCR amplification of saxitoxin, cylindrospermospin, microcystin, and nodularin genes showed that <i>Azolla</i> and its cyanobiont <i>N. azollae</i> do not have the genes to synthesize these cyanotoxins. Additionally, the matching of the anatoxin-a gene to the sequenced <i>N. azollae</i> genome does not indicate the presence of the anatoxin-a gene. The LC-MS/MS analysis showed that BMAA and its isomers AEG and DAB are absent from <i>Azolla</i> and <i>Nostoc azollae</i>. <i>Azolla</i> therefore has the potential to safely feed millions of people due to its rapid growth while free-floating on shallow fresh water without the need for nitrogen fertilizers.