Spray irrigation with microcystins-rich water affects plant performance from the microscopic to the functional level and food safety of spinach (Spinacia oleracea L.).

Irrigation water coming from freshwater bodies that suffer toxic cyanobacterial blooms causes adverse effects on crop productivity and quality and raises concerns regarding food contamination and human exposure to toxins. The common agricultural practice of spray irrigation is an important exposure route to cyanotoxins, yet its impact on crops has received little attention. In the present study we attempted an integrated approach at the macro- and microscopic level to investigate whether spray or drip irrigation with microcystins (MCs)-rich water differently affect spinach performance. Growth and functional features, structural characteristics of stomata, and toxin bioaccumulation were determined. Additionally, the impact of irrigation method and water type on the abundance of leaf-attached microorganisms was assessed. Drip irrigation with MCs-rich water had detrimental effects on growth and photosynthetic characteristics of spinach, while spray irrigation ameliorated to various extents the observed impairments. The stomatal characteristics were differently affected by the irrigation method. Drip-irrigated spinach leaves showed significantly lower stomatal density in the abaxial epidermis and smaller stomatal size in the adaxial side compared to spray-irrigation treatment. Nevertheless, the latter deteriorated traits related to fresh produce quality and safety for human consumption; both the abundance of leaf-attached microorganisms and the MCs bioaccumulation in edible tissues well exceeded the corresponding values of drip-irrigated spinach with MC-rich water. The results highlight the significance of both the use of MCs-contaminated water in vegetable production and the irrigation method in shaping plant responses as well as health risk due to human and livestock exposure to MCs.

Time series metagenomic sampling of the Thermopyles, Greece, geothermal springs reveals stable microbial communities dominated by novel sulfur-oxidizing chemoautotrophs.

Geothermal springs are essentially unaffected by environmental conditions aboveground as they are continuously supplied with subsurface water with little variability in chemistry. Therefore, changes in their microbial community composition and function, especially over a long period, are expected to be limited but this assumption has not yet been rigorously tested. Toward closing this knowledge gap, we applied whole metagenome sequencing to 17 water samples collected between 2010 and 2016 from the Thermopyles sulfur-rich geothermal springs in central Greece. As revealed by 16S rRNA gene fragments recovered in the metagenomes, Epsilonproteobacteria-related operational taxonomic units (OTUs) dominated most samples and grouping of samples based on OTU abundances exhibited no apparent seasonal pattern. Similarities between samples regarding functional gene content were high, with all samples sharing >70% similarity in functional pathways. These community-wide patterns were further confirmed by analysis of metagenome-assembled genomes (MAGs), which showed that novel species and genera of the chemoautotrophic Campylobacterales order dominated the springs. These MAGs carried different pathways for thiosulfate or sulfide oxidation coupled to carbon fixation pathways. Overall, our study showed that even in the long term, functions of microbial communities in a moderately hot terrestrial spring remain stable, presumably driving the corresponding stability in community structure.

Irrigation of radish (Raphanus sativus L.) with microcystin-enriched water holds low risk for plants and their associated rhizopheric and epiphytic microbiome.

Microcystins (MCs) are toxins produced during cyanobacterial blooms. They reach soil and translocated to plants through irrigation of agricultural land with water from MC-impacted freshwater systems. To date we have good understanding of MC effects on plants, but not for their effects on plant-associated microbiota. We tested the hypothesis that MC-LR, either alone or with other stressors present in the water of the Karla reservoir (a low ecological quality and MC-impacted freshwater system), would affect radish plants and their rhizospheric and phyllospheric microbiome. In this context a pot experiment was employed where radish plants were irrigated with tap water without MC-LR (control) or with 2 or 12 µg L-1 of pure MC-LR (MC2 and MC12), or water from the Karla reservoir amended (12 µg L-1) or not with MC-LR. We measured MC levels in plants and rhizospheric soil and we determined effects on (i) plant growth and physiology (ii) the nitrifying microorganisms via q-PCR, (ii) the diversity of bacterial and fungal rhizospheric and epiphytic communities via amplicon sequencing. MC-LR and/or Karla water treatments resulted in the accumulation of MC in taproot at levels (480-700 ng g-1) entailing possible health risks. MC did not affect plant growth or physiology and it did not impose a consistent inhibitory effect on soil nitrifiers. Karla water rather than MC-LR was the stronger determinant of the rhizospheric and epiphytic microbial communities, suggesting the presence of biotic or abiotic stressors, other than MC-LR, in the water of the Karla reservoir which affect microorganisms with a potential role (i.e. pathogens inhibition, methylotrophy) in the homeostasis of the plant-soil system. Overall, our findings suggest that MC-LR, when applied at environmentally relevant concentrations, is not expected to adversely affect the radish-microbiota system but might still pose risk for consumers' health.

Cyanotoxins as the "common suspects" for the Dalmatian pelican (Pelecanus crispus) deaths in a Mediterranean reconstructed reservoir.

Toxic cyanobacterial blooms have been implicated for their negative consequences on many terrestrial and aquatic organisms. Water birds belong to the most common members of the freshwater food chains and are most likely to be affected by the consumption of toxic cyanobacteria as food. However, the contribution of cyanotoxins in bird mortalities is under-studied. The aim of the study was to investigate the likely role of cyanotoxins in a mass mortality event of the Dalmatian pelican (Pelecanus crispus) in the Karla Reservoir, in Greece. Water, scum, tissues and stomach content of dead birds were examined for the presence of microcystins, cylindrospermopsins and saxitoxins by an enzyme-linked immunosorbent assay. High abundances of potential toxic cyanobacterial species and significant concentrations of cyanotoxins were recorded in the reservoir water. All examined tissues and stomach content of the Dalmatian pelicans contained significant concentrations of microcystins and saxitoxins. Cylindrospermopsin concentrations were detected in all tissues except from the brain. Our results suggest that cyanotoxins are a plausible cause for this bird mass mortality episode in the Karla Reservoir.

Artificially-born "killer" lake: phytoplankton based water quality and microcystin affected fish in a reconstructed lake.

Lake Karla (Greece) is an example of a lake ecosystem which was dried in 1960's and now is restored, facing various anthropogenic pressures, whereas it is also listed in the network of Greek protected areas in terms of its conservation value. The objective of the present study was to determine the presence of microcystins (MCYST) in the lake water and their accumulation in tissues of the commercial fish species Cyprinus carpio, along with the highlighting of phytoplankton community and general limnological features of Lake Karla, a newly reconstructed lake, the first year of its refilling. MCYST concentrations in water and fish tissues were determined by an enzyme-linked immunosorbent assay (ELISA). Results suggest that Lake Karla has undergone a progressive cultural eutrophication with frequent cyanobacterial blooms. The most dominant species in lake's phytoplankton were Anabaenopsis elenkinii, Sphaerospermopsis and Planktothrix agardhii. MCYST concentrations were detected in water samples comparable to those reported for other eutrophicated Mediterranean lakes while considerable amounts of MCYST were detected in the tissues of the species C. carpio in the following order: liver>kidney>brain>intestine>muscles. The presence of prominent cyanobacterial blooms dominated by toxic species highlights the need to undertake eutrophication control measures so as to avoid further toxicological problems.

Indigenous and spoilage microbiota of farmed sea bream stored in ice identified by phenotypic and 16S rRNA gene analysis.

Investigation of the initial and spoilage microbial diversity of iced stored sea bream was carried out. Culture dependent methods were used for bacterial enumeration and phenotypic identification of bacterial isolates, while culture independent methods, using bacterial 16S rRNA gene amplification, cloning and sequencing of DNA extracted directly from the flesh were also employed. The culture dependent approach revealed that the initial microbiota was dominated by Acinetobacter, Shewanella, Pseudomonas and Flavobacterium, while at the end of shelf-life determined by sensory analysis (16 days), the predominant microbiota was Pseudomonas and Shewanella. Culture independent approach showed that initially the sea bream flesh was strongly dominated by Acinetobacter, while Pseudomonas, Aeromonas salmonicida and Shewanella were the predominant phylotypes at the end of shelf-life. Initial and spoilage microbiota comprised of phylotypes previously identified by others using traditional or molecular techniques. However, Aeromonas has not been reported as part of the dominant microbiota of sea bream at the time of spoilage. Combination of classical and molecular methodologies better reveals the microbiota during storage by revealing bacteria that escape standard approaches and, thus, provides valuable complementary information regarding microbiological spoilage.

Morphology and molecular evaluation of Iphinoe spelaeobios gen. nov., sp. nov. and Loriellopsis cavernicola gen. nov., sp. nov., two stigonematalean cyanobacteria from Greek and Spanish caves.

Caves have generally been found to host phototrophic micro-organisms from various taxonomic groups, with cyanobacteria comprising an important group that have adapted to these stable and highly specific environments. A polyphasic study based on aspects of classical morphology and molecular data revealed two new monospecific genera from fresh material of Greek and Spanish caves. Both taxa are characterized by obligatory true branching (T-type, V-type and false branching), the presence of heterocysts, and reproduction by hormocysts and akinetes. They shared some similarities in their morphological characteristics as revealed by light, scanning electron and transmission electron microscopy, but phylogenetic analysis based on 16S rRNA gene sequences showed that the two phylotypes were different (89.8% similarity); this represents an example of shared morphology in genetically different strains of cave-adapted species. Phenotypic and genetic traits strongly support classification of the phylotypes as independent taxa in the order Stigonematales (the most differentiated and complicated group of cyanobacteria), family Loriellaceae Geitl 1925. Hence, the names Iphinoe spelaeobios Lamprinou and Pantazidou gen. nov., sp. nov. and Loriellopsis cavernicola Hernández-Mariné and Canals gen. nov., sp. nov. are proposed.

Multiple biomarkers of pollution effects in caged mussels on the Greek coastline.

A suite of biomarkers was measured in caged mussels at areas impacted by different anthropogenic activities along the Greek coastline to assess biological effects of environmental pollution. Mussels were caged at coastal sites in the vicinity of major cities, in areas influenced by major industries, agricultural practices and in islands away from known sources of pollution. Biomarkers indicative of neurotoxicity (acetylcholinesterase, AchE), oxidative stress (catalase, CAT), phase II biotransformation of xenobiotics (glutathione S-transferase, GST), metal exposure (metallothioneins, MTs) and protein synthesis (RNA:DNA ratio) were measured to assess effects of various types of pollutants. AchE activity proved to be the most responsive biomarker with decreased values at sites influenced by agricultural, urban and industrial activities. Decreased CAT and GST activities and increased MTs levels were recorded at a number of anthropogenic-impacted sites. RNA:DNA ratio showed a biphasic response as both high and low values were found at impacted sites. Principal component analysis clearly distinguished sites receiving pollution inputs from non-polluted sites. The combination of the selected biomarkers used in caged mussels resulted useful in the assessment of the effects of environmental pollution.

Characterization of methanogenic and prokaryotic assemblages based on mcrA and 16S rRNA gene diversity in sediments of the Kazan mud volcano (Mediterranean Sea).

The diversity of the methyl-coenzyme reductase A (mcrA) and 16S rRNA genes was investigated in gas hydrate containing sediment from the Kazan mud volcano, eastern Mediterranean Sea. mcrA was detected only at 15 and 20 cm below seafloor (cmbsf) from a 40-cm long push core, while based on chemical profiles of methane, sulfate, and sulfide, possible anaerobic oxidation of methane (AOM) depth was inferred at 12-15 cmbsf. The phylogenetic relationships of the obtained mcrA, archaeal and bacterial 16S rRNA genes, showed that all the found sequences were found in both depths and at similar relative abundances. mcrA diversity was low. All sequences were related to the Methanosarcinales, with the most dominant (77.2%) sequences falling in group mcrA-e. The 16S rRNA-based archaeal diversity also revealed low diversity and clear dominance (72.8% of all archaeal phylotypes) of the Methanosarcinales and, in particular, ANME-2c. Bacteria showed higher diversity but 83.2% of the retrieved phylotypes from both sediment layers belonged to the delta-Proteobacteria. These phylotypes fell in the SEEP-SRB1 putative AOM group. In addition, the rest of the less abundant phylotypes were related to yet-uncultivated representatives of the Actinobacteria, Spirochaetales, and candidate divisions OP11 and WS3 from gas hydrate-bearing habitats. These phylotype patterns indicate that AOM is occurring in the 15 and 20 cmbsf sediment layers.

Application of rpoB sequence similarity analysis, REP-PCR and BOX-PCR for the differentiation of species within the genus Geobacillus.

AIM: To investigate the applicability of rpoB gene, which encodes the beta subunit of RNA polymerase, to be used as an alternative to 16S rRNA for sequence similarity analysis in the thermophilic genus Geobacillus. Rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (REP- and BOX-polymerase chain reaction) were also used. METHODS AND RESULTS: rpoB DNA (458 bp) were amplified from 21 Geobacillus- and Bacillus type strains, producing different BOX- and REP-PCR profiles, in addition to 11 thermophilic isolates of Geobacillus and Bacillus species from a Santorini volcano habitat. The sequences and the phylogenetic tree of rpoB were compared with those obtained from 16S rRNA gene analysis. The results demonstrated between 90-100% (16S rRNA) and 74-100% (rpoB) similarity among examined bacteria. CONCLUSION: BOX- and REP-PCR can be applied for molecular typing within Geobacillus genus. rpoB sequence similarity analysis permits a more accurate discrimination of the species within the Geobacillus genus than the more commonly used 16S rRNA. SIGNIFICANCE AND IMPACT OF THE STUDY: The obtained results suggested that rpoB sequence similarity analysis is a powerful tool for discrimination between species within the ecologically and industrially important strains of Geobacillus genus.

Dynamic characteristics of Prochlorococcus and Synechococcus consumption by bacterivorous nanoflagellates.

We compared the characteristics of ingestion of Prochlorococcus and Synechococcus by the marine heterotrophic nanoflagellate Pseudobodo sp. and by a mixed nanoflagellate culture (around 3 microm in size) obtained from an open sea oligotrophic area. Maximum ingestion rate on Synechococcus (2.7 Syn flagellate(-1) h(-1)) was reached at concentrations of 5 x 10(5) Syn mL(-1) and decreased between 6 x 10(5) and 1.5 x 10(6) Syn mL(-1). In order to validate laboratory data, one set of data on Synechococcus grazing was obtained during a field study in the oligotrophic northeastern Mediterranean Sea. Ingestion rates by heterotrophic nanoflagellates were related to Synechococcus abundance in the water, and the feeding rate showed a clear diel rhythm with consumption being highest during the night, declining during the day hours, and being lowest at dusk. Ingestion rates on Prochlorococcus increased linearly for the whole range of prey density used (i.e., from 1 x 10(3) to 3 x 10(6) Proc mL(-1)), with maximum ingestion of 6.7 Proc flagellate(-1) h(-1). However, for prey concentrations in the range of 10(3)-10(5), which are usually encountered in aquatic systems, ingestion rates were significantly less than on Synechococcus. In our experiments, both Prochlorococcus and Synechococcus proved to be poor food items for support of nanoflagellate growth.