Mediterranean ecosystems have not been consistently investigated as natural habitats for microbes in general, and fungi in particular. Here we present the results of a survey of epiphytic mycobiota (filamentous fungi and yeasts) on the phylloplane of selected plants in the Arrábida Natural Park, an ecosystem of Mediterranean characteristics in Portugal, using conventional culture-dependent isolation methods. Leaves from the species Acer monspessulanum and Quercus faginea (deciduous trees) and Cistus albidus, Pistacia lentiscus, and Osyris quadripartita (evergreen shrubs) were collected twice a year for two consecutive years, at two distinct locations of Serra da Arrábida: the more humid northern slope and the drier southern slope. A total of 1029 strains of filamentous fungi and 540 strains of yeasts were isolated, which represented at least 36 and 46 distinct species, respectively. Total counts were higher on the plants from the northern slope and there was a general increase from spring to autumn, notably on the deciduous trees for the yeasts. Plant species that had higher numbers of leaf colonists (A. monspessulanum, C. albidus, and Q. faginea) also yielded a wider range of species. Among the filamentous fungi there was a predominance of species of ascomycetous affinity, whereas basidiomycetous species dominated among yeast isolates. Some of the taxa recovered were common to other phylloplane studies (e.g., ubiquitous molds and yeasts such as Cladosporium spp. and Cryptococcus spp., respectively), but less common species were also found, some of which appeared to represent undescribed taxa. Interestingly, a few species seemed to be associated with a particular plant, notably in the case of the evergreen shrub C. albidus. However, for a considerable number of fungi and yeasts the same taxon was recovered throughout the year from more than one plant and at both sites, suggesting that such species might be genuine phylloplane inhabitants (or at least of aerial plant surfaces) even though they appeared not to display host specificity.

A collaborative study was conducted for the determination of paralytic shellfish poisoning (PSP) toxins in shellfish. The method used liquid chromatography with fluorescence detection after prechromatographic oxidation of the toxins with hydrogen peroxide and periodate. The PSP toxins studied were saxitoxin (STX), neosaxitoxin (NEO), gonyautoxins 2 and 3 (GTX2,3; together), gonyautoxins 1 and 4 (GTX1,4; together), decarbamoyl saxitoxin (dcSTX), B-1 (GTX5), C-1 and C-2 (C1,2; together), and C-3 and C-4 (C3,4; together). B-2 (GTX6) toxin was also included, but for qualitative identification only. Mussels, both blank and naturally contaminated, were mixed and homogenized to provide a variety of PSP toxin mixtures and concentration levels. The same procedure was followed with clams, oysters, and scallops. Twenty-one test samples in total were sent to 21 collaborators who agreed to participate in the study. Results were obtained from 18 laboratories representing 14 different countries. It is recommended that the method be adopted First Action by AOAC INTERNATIONAL.

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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.