Cyanobacteria are well known producers of bioactive metabolites, including harmful substances. The recently discovered "eagle killer" neurotoxin aetokthonotoxin (AETX) is produced by the epiphytic cyanobacterium Aetokthonos hydrillicola growing on invasive water thyme (Hydrilla verticillata). The biosynthetic gene cluster of AETX was previously identified from an Aetokthonos strain isolated from the J. Strom Thurmond Reservoir, Georgia, USA. Here, a PCR protocol for easy detection of AETX-producers in environmental samples of plant-cyanobacterium consortia was designed and tested. Three different loci of the AETX gene cluster were amplified to confirm the genetic potential for AETX production, along with two variable types of rRNA ITS regions to confirm the homogeneity of the producer´s taxonomic identity. In samples of Hydrilla from three Aetokthonos-positive reservoirs and one Aetokthonos-negative lake, the PCR of all four loci provided results congruent with the Aetokthonos presence/absence detected by light and fluorescence microscopy. The production of AETX in the Aetokthonos-positive samples was confirmed using LC-MS. Intriguingly, in J. Strom Thurmond Reservoir, recently Hydrilla free, an Aetokthonos-like cyanobacterium was found growing on American water-willow (Justicia americana). Those specimens were positive for all three aet markers but contained only minute amounts of AETX. The obtained genetic information (ITS rRNA sequence) and morphology of the novel Aetokthonos distinguished it from all the Hydrilla-hosted A. hydrillicola, likely at the species level. Our results suggest that the toxigenic Aetokthonos spp. can colonize a broader array of aquatic plants, however the level of accumulation of the toxin may be driven by host-specific interactions such as the locally hyper-accumulated bromide in Hydrilla.

Biology Centre of the CAS Institute of Hydrobiology České Budějovice 370 05 Czechia; University of South Bohemia Faculty of Science České Budějovice 370 05 Czechia

Biology Centre of the CAS Institute of Hydrobiology České Budějovice 370 05 Czechia; University of South Bohemia Faculty of Science České Budějovice 370 05 Czechia; Centre Algatech Institute of Microbiology of the CAS Třeboň 379 01 Czechia

Daniel B Warnell School of Forestry and Natural Resources University of Georgia Athens GA 30602 USA

Daniel B Warnell School of Forestry and Natural Resources University of Georgia Athens GA 30602 USA; U S Environmental Protection Agency Office of Research and Development Center for Environmental Measurement and Modeling Athens GA 30605 USA

Institute of Pharmacy Martin Luther University Halle Wittenberg Halle 06120 Germany

U S Environmental Protection Agency Office of Research and Development Center for Environmental Measurement and Modeling Athens GA 30605 USA

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