Impact of PET micro/nanoplastics on the symbiotic system Azolla filiculoides-Trichormus azollae

The symbiotic system Azolla filiculoides-Trichormus azollae was exposed for ten days to environmentally relevant concentrations (i.e. 0.05 and 0.1 g L^-1) of polyethylene terephthalate micro-nanoplastics (PET-MNPs). Plastic particles did not induce any visible toxicity symptoms or growth disorders to the fern, as well as any effects on leaf anatomy and chlorophyll fluorescence parameters. Nonetheless, in treated plants a decrease of chlorophyll content occurred and was coupled to reduction of Nitrogen Balance Index (NBI), an informative parameter of the plant nitrogen status. In the presence of MNPs, plants exhibited a substantial decline in the absorption of essential elements, as evidenced by decreased tissue concentration of Ca, Mg, Co and Mn. The exposure to the pollutants compromised root integrity and possibly its functioning in nutrient accumulation, with evident physical damages not only in the rhizodermis and cortex, but also in the vascular system. In addition, a DNA-based estimation of T. azollae revealed a decreasing trend in the relative abundance of the N₂-fixing cyanobacteria for PET-treated samples. This was coupled with an alteration of the symbiont's phenotype highlighted by microscopy analysis, showing a reduction in number of vegetative cells between two consecutive heterocysts and in heterocyst size. This work is the first evidence of MNPs disturbing a strict symbiosis, with possible implications on nitrogen cycling in ecosystems, bio fertilization of agricultural lands and evolutionary pathways.