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The hemolytic ability, the presence of cyl genes, and the diagnostic accuracy of cytolysin molecular detection were investigated in the genus Enterococcus by using 164 strains from 20 different species (26 reference strains, 42 clinical isolates from human and veterinary origin, and 96 isolates from ewe cheese and milk). Hemolysis was assayed with sheep and horse erythrocytes and under aerobic or anaerobic conditions. Screening of cytolysin genes (cylL L , cylL S , cylM, cylB, and cylA) was performed with new specific primers and the anaerobic assay of beta-hemolysis was used as the "gold standard" for the evaluation of cyl gene-based PCRs. Since beta-hemolysis and cyl genes were found in 10 and 14 species, respectively, the hemolytic ability seems to be spread throughout the genus Enterococcus. Beta-hemolysis was observed in 6 of 26 (23%) reference strains, 14 of 42 (33%) clinical isolates, and 6 of 96 (6%) food isolates. The presence of cyl genes was detected in 15 of 26 (58%) reference strains, 37 of 42 (88%) clinical isolates, and 67 of 96 (70%) food isolates. These data indicate a virulence potential in food isolates, reinforcing the need of their safety assessment. Analysis of phenotypicgenotypic congruence suggests a divergent sequence evolution of cyl genes and the effect of environmental factors in the regulation of cytolysin expression. Evaluation of the diagnostic accuracy of cytolysin molecular detection points to cylL L -based PCR and cylL L L S MBA-based PCR as the most reliable approaches. Nevertheless, the low sensitivity (46%) and gene variability indicated by our study strongly recommend the phenotypic assay for the assessment of hemolytic ability in enterococci, followed by the molecular screening of cyl genes in nonhemolytic strains to evaluate their virulence potential.

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Summary Cryphonectria parasitica, the causal agent of chestnut blight, has been responsible for the decline in chestnut in Portugal for the last two decades. In order to improve understanding of C. parasitica diversity, a total of 617 isolates from all affected chestnut‐growing areas in continental Portugal, Madeira and the Azores archipelagos were studied. Nine vegetative compatibility (vc) types were identified among the isolates. EU‐11 was the most widespread vc type comprising 80.2% of the isolates, followed by EU‐12 (7.1%) and EU‐66 (6.6%). Two of the Portuguese vc types could not be assigned to a known European vc type. The diversity of vc types was low in the Portuguese populations of C. parasitica, but comparable with other areas where C. parasitica was introduced recently. The frequent occurrence of perithecia and both mating types of C. parasitica indicates that sexual reproduction of the chestnut blight fungus is common in Portugal. One C. parasitica isolate from Trás‐os‐Montes showed a white culture morphology and contained dsRNA, indicating the presence of hypovirulence in this area.

Microorganisms are ubiquitous in all habitats and are recognized by their metabolic versatility and ability to produce many bioactive compounds, including toxins. Some of the most common toxins present in water are produced by several cyanobacterial species. As a result, their blooms create major threats to animal and human health, tourism, recreation and aquaculture. Quite a few cyanobacterial toxins have been described, including hepatotoxins, neurotoxins, cytotoxins and dermatotoxins. These toxins are secondary metabolites, presenting a vast diversity of structures and variants. Most of cyanobacterial secondary metabolites are peptides or have peptidic substructures and are assumed to be synthesized by non-ribosomal peptide synthesis (NRPS), involving peptide synthetases, or NRPS/PKS, involving peptide synthetases and polyketide synthases hybrid pathways. Besides cyanobacteria, other bacteria associated with aquatic environments are recognized as significant toxin producers, representing important issues in food safety, public health, and human and animal well being. Vibrio species are one of the most representative groups of aquatic toxin producers, commonly associated with seafood-born infections. Some enterotoxins and hemolysins have been identified as fundamental for V. cholerae and V. vulnificus pathogenesis, but there is evidence for the existence of other potential toxins. Campylobacter spp. and Escherichia coli are also water contaminants and are able to produce important toxins after infecting their hosts. Other bacteria associated with aquatic environments are emerging as toxin producers, namely Legionella pneumophila and Aeromonas hydrophila, described as responsible for the synthesis of several exotoxins, enterotoxins and cytotoxins. Furthermore, several Clostridium species can produce potent neurotoxins. Although not considered aquatic microorganisms, they are ubiquitous in the environment and can easily contaminate drinking and irrigation water. Clostridium members are also spore-forming bacteria and can persist in hostile environmental conditions for long periods of time, contributing to their hazard grade. Similarly, Pseudomonas species are widespread in the environment. Since P. aeruginosa is an emergent opportunistic pathogen, its toxins may represent new hazards for humans and animals. This review presents an overview of the diversity of toxins produced by prokaryotic microorganisms associated with aquatic habitats and their impact on environment, life and health of humans and other animals. Moreover, important issues like the availability of these toxins in the environment, contamination sources and pathways, genes involved in their biosynthesis and molecular mechanisms of some representative toxins are also discussed.

In order to assess the potential of several molecular targets for the identification, typing and traceability of cyanobacteria in freshwater reservoirs, molecular techniques were applied to 118 cyanobacterial isolates mostly sourced from Portuguese freshwater reservoirs and representative of three orders of cyanobacteria: Chroococcales (54), Oscillatoriales (15) and Nostocales (49). The isolates were previously identified by morphological methods and subsequently characterized by composite hierarchical cluster analysis of STRR and LTRR (short and long tandemly repeated repetitive sequences) PCR fingerprinting profiles. Representative isolates were selected from each cluster and their molecular identification, at the species level, was obtained or confirmed by phylogenetic positioning using 16S rRNA gene and rpoC1 phylogenies. A highly congruent association was observed between STTR-and LTRR-based clusters and taxonomic affiliation, revealing the usefulness of such PCR fingerprinting profiles for the identification of cyanobacteria. Composite analysis of hierarchical clustering of M13 and ERIC PCR fingerprints also appeared suitable for strain typing and traceability within a reservoir, indicating its potential for use in cyanobacterial monitoring, as a quality management control. Based on Simpson (D) and Shannon-Wiener (J9) indices a high diversity was observed within all species, with Planktothrix agardhii showing the lowest diversity values (D50.83; J950.88) and Aphanizomenon flos-aquae the highest ones (D5J950.99). A diagnostic key based on 16S-ARDRA, ITS amplification and ITS-ARDRA for identification purposes is also presented.

Genetic species identification of non-invasively collected samples has become an important tool in ecological research, management and conservation and wildlife forensics. This is especially true for carnivores, due to their elusive nature, and is crucial when several ecologically and phylogenetically close species, with similar faeces, hairs, bones and/or pelts, occur in sympatry. This is the case of the Iberian Peninsula, a region with a carnivore community of 16 species-about two-thirds of the European carnivore fauna. Here we present a simple, efficient and reliable PCR-based protocol, using a novel set of species-specific primers, for the unambiguous identification to species of non-invasively collected samples or forensic materials from Iberian carnivores. For each species, from the consensus of all cytochrome b haplotypes, found here and previously reported, we designed speciesspecific primer pairs for short fragments, the most likely to persist in low-quantity and degraded DNA samples. The predicted specificity of each primer pair was assessed through PCR of positive DNA extracts from the carnivore species, from an exhaustive array of potential prey and from humans. The robustness of PCR amplification for non-invasively sampled DNA was tested with scat samples. The primers did not produce false positives and correctly identified all carnivore samples to the species level. In comparison with sequencing and PCR-RFLP assays, our method is, respectively, cost-and time-effective, and is especially suited for monitoring surveys targeting multiple populations/species. It also introduces an approach that works for a whole community of carnivores living sympatrically over a large geographic area.

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