Studies of cyanobacterial nostocacean taxa are important to the global scientific community, mainly because a significant number of beneficial strains that belong to the order Nostocales fix atmospheric nitrogen, thus contributing to the fertility of agricultural soils worldwide, while others behave as nuisance microorganisms in aquatic ecosystems due to their involvement in toxic bloom events. However, in spite of their ecological importance and environmental concerns, their identification and taxonomy are still problematic and doubtful, often being based on current morphological and physiological studies, which generate confusing classification systems and usually vary under different conditions. Therefore, the present research aimed to investigate through a polyphasic approach differences in morphological, biochemical and genotypic features of three nostocacean cyanobacterial strains isolated from central-western Portuguese shallow freshwater bodies. Morphometric, genetic (16S rRNA, nifH and hetR fragments) and biochemical (fatty acid methyl ester; FAME profiles) data were used to characterize the strains. Morphological analysis and sequencing of 16S rRNA fragments showed that the strains belonged to Anabaena cylindrica (UTAD_A212), Aphanizomenon gracile (UADFA16) and Nostoc muscorum (UTAD_N213) species. These strains showed clear distinct morphological and genetic features, allowing easy allocation to their respective genera. The same happened by using partial sequences of hetR and nifH genes, in spite of the scarcity of deposited sequences. Biochemical characterization showed that the FAME profiles obtained were consistent with both morphological and molecular analyses. It was suggested that the ratio of monounsaturated to polyunsaturated FAMEs, together with the unsaturation index, could be used as genus-specific chemotaxonomic biomarkers.

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Rice fields are frequently exposed to environmental contamination by herbicides and cyanobacteria, as primary producers of these aquatic ecosystems, are adversely affected. Anabaena cylindrica is a cyanobacterium with a significantly widespread occurrence in Portuguese rice fields. This strain was studied throughout 72 h in laboratory conditions for its stress responses to sublethal concentrations (0.75-2 mM) of bentazon, a selective postemergence herbicide recommended for integrated weed management in rice, with special reference to oxidative stress, role of proline and intracellular antioxidant enzymes in herbicide-induced free radicals detoxification. Activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione S-transferase (GST) increased in a time- and herbicide dose-response manner and were higher than those in the control samples after 72 h. A time- and concentration-dependent increase of malondialdehyde (MDA) levels and the enhanced cell membrane leakage following bentazon exposure are indicative of lipid peroxidation, free radicals formation, and oxidative damage, while increased amounts of SOD, CAT, APX, GST, and proline indicated their involvement in free radical scavenging mechanisms. The appreciable decline in the reduced glutathione (GSH) pool after 72 h at higher bentazon concentrations could be explained by the reduction of the NADPH-dependent glutathione reductase (GR) activity. The obtained results suggested that the alterations of antioxidant systems in A. cylindrica might be useful biomarkers of bentazon exposure. As the toxic mechanism of bentazon is a complex phenomenon, this study also adds relevant findings to explain the oxidative stress pathways of bentazon promoting oxidative stress in cyanobacteria.

The effects of bentazon and molinate, two selective herbicides recommended for integrated weed management in rice, were studied in Anabaena cylindrica, an abundant cyanobacterium isolated from a Portuguese rice field agro-ecosystem. Comparative effects of both herbicides on A. cylindrica were estimated under laboratory conditions by measuring its dry weight yield, photopigments, and carbohydrate and protein contents in a time-and dose-dependent exposure throughout 72 h. Photosynthesis and respiration were also monitored. The results revealed that both herbicides exerted a pleiotropic effect on the cyanobacterium at the range of concentrations tested (0.75-2 mM). Growth, chlorophyll a, carotenoids and phycobiliproteins were more adversely affected by molinate than by bentazon.Cyanobacterial growth inhibitions of over 50% were observed after 48 h when 1.5-2 mM of molinate were applied. Bentazon concentrations ranging from 0.75 to 2 mM did not significantly modified chlorophyll a content with time, however, considerable reductions in chlorophyll a, carotenoids and specially phycobiliproteins were observed with molinate. Protein content increased with both herbicides although the effect was particularly noticeable with the highest concentration of molinate. Herbicide effects on carbohydrate content were contrasting: molinate increased this organic fraction whereas bentazon decrease it. Photosynthesis and respiration were inhibited by both herbicides and higher molinate concentrations (1.5-2 mM) completely ceased O 2 evolution after 48 h. Since A. cylindrica is abundant in Portuguese rice fields and could be used as an inoculum source in rice biofertilization programs, their protection from potential residual effects of herbicides is fundamental for a correct management of local soil fertility.