The Azolla Metagenome: Foul Play In the Pocket?

Ferns from the genus Azolla are floating invasive weeds in waterways worldwide, yet industrialized biomass production with Azolla may be advantageous not the least because of their N₂-fixing symbionts, Nostoc azollae. Experimental production with A. filiculoides in the absence of N-fertilizer yielded over 1200 kg N ha^-1yr^-1 fixed in the biomass harvested implying that Azolla should be considered as forage to substitute soybean imports in European latitudes where water is available. The genome of A. filiculoides was sequenced, assembled and annotated towards domestication and breeding, yet we wondered about biosafety of the biomass and therefore began to characterize the metagenome of A. filiculoides from a Dutch ditch and the corresponding sterilized strain grown in the laboratory. The approach included metagenome sequencing, taxonomic assignments using rRNA gene sequences, assembly of genome sequences and recruitment analyses. In addition, nitrogen cycle enzymes in the assembled persistent Rhizobium endophyte were annotated, and ¹⁵N₂ fixation and biomass δ¹⁵N measured. We conclude 1) the immune system of A. filiculoides is very selective, 2) Azolla's main symbiont in the leaf pockets is N. azollae, 3) denitrifying Rhizobia are generally associated with Azolla species in culture or in A. filiculoides from the ditch 4) Rhizobia are enriched in leaf pockets and may detoxify excess NH₄⁺ produced by N. azollae or present in waterlogged anoxic sediments where nitrification is inhibited and NH₄⁺becomes the primary N-source.