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The genus Diaporthe includes pathogenic species distributed worldwide and affecting a wide variety of hosts. Diaporthe amygdali and Diaporthe eres have been found to cause cankers, dieback, or twig blights on economically important crops such as soybean, almond, grapevine, and blueberry. Despite their importance as plant pathogens, the strategies of species of Diaporthe to infect host plants are poorly explored. To provide a genomic basis of pathogenicity, the genomes of D. amygdali CAA958 and D. eres CBS 160.32 were sequenced and analyzed. Cellular transporters involved in the transport of toxins, ions, sugars, effectors, and genes implicated in pathogenicity were detected in both genomes. Hydrolases and oxidoreductases were the most prevalent carbohydrate-active enzymes (CAZymes). However, analyses of the secreted proteins revealed that the secretome of D. eres CBS 160.32 is represented by 5.4% of CAZymes, whereas the secreted CAZymes repertoire of D. amygdali CAA958 represents 29.1% of all secretomes. Biosynthetic gene clusters (BGCs) encoding compounds related to phytotoxins and mycotoxins were detected in D. eres and D. amygdali genomes. The core gene clusters of the phytotoxin Fusicoccin A in D. amygdali are reported here through a genome-scale assembly. Comparative analyses of the genomes from 11 Diaporthe species revealed an average of 874 CAZymes, 101 secondary metabolite BGCs, 1640 secreted proteins per species, and genome sizes ranging from 51.5 to 63.6 Mbp. This study offers insights into the overall features and characteristics of Diaporthe genomes. Our findings enrich the knowledge about D. eres and D. amygdali, which will facilitate further research into the pathogenicity mechanisms of these species.

Extracellular matrix (ECM)-based bioinks have been steadily gaining interest in the field of bioprinting to develop biologically relevant and functional tissue constructs. Herein, we propose the use of supercritical carbon dioxide (scCO2) technology to extract the ECM components of cell-sheets that have shown promising results in creating accurate 3D microenvironments replicating the cellâ s own ECM, to be used in the preparation of bioinks. The ECM extraction protocol best fitted for cell sheets was defined by considering efficient DNA removal with a minor effect on the ECM. Cell sheets of human dermal fibroblasts (hDFbs) and adipose stem cells (hASCs) were processed using a customised supercritical system by varying the pressure of the reactor, presence, exposure time, and type of co-solvent. A quantification of the amount of DNA, protein, and sulfated glycosaminoglycans (sGAGs) was carried out to determine the efficiency of the extraction in relation to standard decellularization methodologies. The bioinks containing the extracted ECM were fabricated by combining them with alginate as a support polymer. The influence of the alginate (1%, 2% w/vol) and ECM (0.5% and 1.5% w/vol) amounts on the printability of the blends was addressed by analysing the rheological behaviour of the suspensions. Finally, 3D printed constructs were fabricated using an in-house built extrusion-based bioprinter, and the impact of the extrusion process on cell viability was assessed. The optimised scCO2Â protocol allowed efficient removal of DNA while preserving a higher number of proteins and sGAGs than the standard methodologies. The characterization of extractâ s composition also revealed that the ECM produced by hDFbs (fECM) and hASCs (aECM) is distinctively affected by the extraction protocols. Furthermore, rheological analysis indicated an increase in viscosity with increasing ECM composition, an effect even more prominent in samples containing aECM. 3D printing of alginate/ECM constructs demonstrated that cell viability was only marginally affected by the extrusion process, and this effect was also dependent on the ECM source. Overall, this work highlights the benefits of supercritical fluid-based methods for ECM extraction and strengthens the relevance of ECM-derived bioinks in the development of printed tissue-like constructs., ERC -European Research Council(PD/BD/14301/2018)

Functional kleptoplasty is a photosymbiotic relationship, in which photosynthetically active chloroplasts serve as an intracellular symbiont for a heterotrophic host. Among Metazoa, functional kleptoplasty is only found in marine sea slugs belonging to the Sacoglossa and recently described in Rhabdocoela worms. Although functional kleptoplasty has been intensively studied in Sacoglossa, the fundamentals of the specific recognition of the chloroplasts and their subsequent incorporation are unknown. The key to ensure the initiation of any symbiosis is the ability to specifically recognize the symbiont and to differentiate a symbiont from a pathogen. For instance, in photosymbiotic cnidarians, several studies have shown that the host innate immune system, in particular scavenger receptors (SRs) and thrombospondin‐type‐1 repeat (TSR) protein superfamily, is playing a major role in the process of recognizing and differentiating symbionts from pathogens. In the present study, SRs and TSRs of three Sacoglossa sea slugs, Elysia cornigera, Elysia timida, and Elysia chlorotica, were identified by translating available transcriptomes into potential proteins and searching for receptor specific protein and/or transmembrane domains. Both receptors classes are highly diverse in the slugs, and many new domain arrangements for each receptor class were found. The analyses of the gene expression of these three species provided a set of species‐specific candidate genes, that is, SR‐Bs, SR‐Es, C‐type lectins, and TSRs, that are potentially relevant for the recognition of kleptoplasts. The results set the base for future experimental studies to understand if and how these candidate receptors are indeed involved in chloroplast recognition., We investigated the abundance and gene expression of scavenger receptors and thrombospondin‐type‐1 repeat protein superfamily, known to be involved in symbiont recognition, in three plastid‐bearing sea slugs. We could identify candidate receptors, similar to those found in cnidarians for Symbiodinium recognition, that might be relevant in plastid recognition.

Current agricultural methodologies are vulnerable to erratic climate and are dependent on cost-intensive fertilization to ensure high yields. Sustainable practices should be pursued to ensure food security. Phaseolus vulgaris L. is one of the most produced legumes worldwide and may be an alternative to reduce the environmental impact of meat production as a reliable source of high-quality protein. Plant growth-promoting rhizobacteria (PGPR) are emerging as a sustainable option to increase agricultural production. To understand the dynamics between plants and microorganisms, the culturable microbiota of bean roots was isolated and identified at distinct stages of plant development (early and late vegetative growth, flowering, and pod) and root compartments (rhizoplane, endosphere, and nodules). Diversity and abundance of bacteria associated with root compartments differed throughout the plant life cycle. Bacterial plant growth promotion (PGP) and protection abilities (indole-3-acetic acid production, siderophore synthesis, and antifungal activity) were assessed and associated with plant phenology, demonstrating that among the bacteria associated with plant roots, several strains had an active role in the response to plant biological needs at each stage. Several strains stood out for their ability to display one or more PGP traits, being excellent candidates for efficient stage-specific biostimulants for application in precision agriculture.

Fungal communities associated with macroalgae remain largely unexplored. To characterize algicolous fungal communities using culture dependent methods, macroalgae were collected from different sampling sites in the Ria de Aveiro estuary, Portugal. From a collection of 486 isolates that were obtained, 213 representative isolates were selected through microsatellite-primed PCR (MSP-PCR) fingerprinting analysis. The collection yielded 33 different genera, which were identified using the ITS region of the rDNA. The results revealed that the most abundant taxa in all collections were Acremonium-like species: Alternaria, Cladosporium, Leptobacillium and Penicillium. The fungal community composition varied with macroalgae species. Through multilocus phylogenetic analyses based on ITS, tub2, tef1-α and actA sequences, in addition to detailed morphological data, we propose Cladosporium rubrum sp. nov. (type strain=CMG 28=MUM 19.39) and Hypoxylon aveirense sp. nov. (type strain=CMG 29=MUM 19.40) as novel species.

Fungal communities associated with macroalgae remain largely unexplored. To characterize algicolous fungal communities using culture dependent methods, macroalgae were collected from different sampling sites in the Ria de Aveiro estuary, Portugal. From a collection of 486 isolates that were obtained, 213 representative isolates were selected through microsatellite-primed PCR (MSP-PCR) fingerprinting analysis. The collection yielded 33 different genera, which were identified using the ITS region of the rDNA. The results revealed that the most abundant taxa in all collections were

Microbially induced calcification is an ancient, community-driven mineralisation process that produces different types of microbialites. Symbiolites are photosynthesis-induced microbialites, formed by calcifying co-cultures of dinoflagellates from the family Symbiodiniaceae and bacteria. Symbiolites encase the calcifying community as endolithic cells, pointing at an autoendolithic niche of symbiotic dinoflagellates, and provide a rare opportunity to study the role of bacteria in bacterial-algal calcification, as symbiodiniacean cultures display either distinct symbiolite-producing (SP) or non-symbiolite-producing (NP) phenotypes. Using Illumina sequencing, we found that the bacterial communities of SP and NP cultures differed significantly in the relative abundance of 23 genera, 14 families, and 2 phyla. SP cultures were rich in biofilm digesters from the phylum Planctomycetes and their predicted metagenomes were enriched in orthologs related to biofilm formation. In contrast, NP cultures were dominated by biofilm digesters from the Bacteroidetes, and were inferred as enriched in proteases and nucleases. Functional assays confirmed the potential of co-cultures and bacterial isolates to produce biofilms and point at acidic polysaccharides as key stimulators for mineral precipitation. Hence, bacteria appear to influence symbiolite formation primarily through their biofilm-producing and modifying activity and we anticipate that symbiolite formation, as a low-complexity in vitro model, will significantly advance our understanding of photosynthesis-induced microbial calcification processes.

Xenogeneic pericardium-based substitutes are employed for several surgical indications after chemical shielding, limiting their biocompatibility and therapeutic durability. Adverse responses to these replacements might be prevented by tissue decellularization, ideally removing cells and preserving the original extracellular matrix (ECM). The aim of this study was to compare the mostly applied pericardia in clinics, i.e. bovine and porcine tissues, after their decellularization, and obtain new insights for their possible surgical use. Bovine and porcine pericardia were submitted to TRICOL decellularization, based on osmotic shock, detergents and nuclease treatment. TRICOL procedure resulted in being effective in cell removal and preservation of ECM architecture of both species&rsquo, scaffolds. Collagen and elastin were retained but glycosaminoglycans were reduced, significantly for bovine scaffolds. Tissue hydration was varied by decellularization, with a rise for bovine pericardia and a decrease for porcine ones. TRICOL significantly increased porcine pericardial thickness, while a non-significant reduction was observed for the bovine counterpart. The protein secondary structure and thermal denaturation profile of both species&rsquo, scaffolds were unaltered. Both pericardial tissues showed augmented biomechanical compliance after decellularization. The ECM bioactivity of bovine and porcine pericardia was unaffected by decellularization, sustaining viability and proliferation of human mesenchymal stem cells and endothelial cells. In conclusion, decellularized bovine and porcine pericardia demonstrate possessing the characteristics that are suitable for the creation of novel scaffolds for reconstruction or replacement: differences in water content, thickness and glycosaminoglycans might influence some of their biomechanical properties and, hence, their indication for surgical use.

This protocol produces bacteria-depleted (“axenic”) Symbiodiniaceae cultures. The depletion level achieved using the following methods is comparable to what was previously described as an “axenic” culture (Bieri et al., 2016; Xiang et al., 2013). The present protocol was applied to 10 Symbiodiniaceae strains and proved successful in generating bacteria-depleted cultures after its application, for an extended period of time (up to 4 weeks), characterized by both culture-dependent and -independent techniques. Tested cultures showed normal growth and photosynthetic efficiency after recovery from the treatment. Variations of the protocol were developed to increase the success of bacterial depletion for different microalgal strains and to make it applicable to algal strains that do not grow well on solid media. Treatments can also be performed in 24-well plates in order to facilitate the measurement of Fv/Fmthrough the plate bottom using a fiber optic cable, in order to track photophysiology. This protocol was developed in parallel with a recently published method (Costa et al., 2019) to generate bacteria-depleted Aiptasia CC7 polyps, in order to have a toolbox at disposal to disassemble the different compartments of an anthozoan metaorganism for functional testing: the bacterial microbiome, the microalgal symbionts, and the anthozoan host animal. All steps of culture manipulation in this protocol must be done under a sterile environment, using common sterile work practices!

The Symbiodiniaceae are a family of marine dinoflagellates known mostly for their endosymbiotic interactions with invertebrates and protists, but facultatively and exclusively free-living life histories in this family are also evident. A recent systematic revision of the Symbiodiniaceae replaced the clade-based nomenclature of seven divergent lineages of "Symbiodinium" sensu lato with one based on formally described genera. The revised taxonomy was not extended to the whole group because type species to describe a new genus for each of the remaining clades and subclades were lacking. In an effort to characterize benthic habitats of symbiodiniaceans in sediments at Heron Island (Great Barrier Reef, Australia), we isolated >100 monoclonal Symbiodiniaceae cultures. Four of these belonged to Symbiodiniaceae 'subclade' Fr3, and three to Clade H, based on nucleotide sequence similarity (ITS2, LSU, cp23S, and mtCOB), representing the first cultures of these taxa. Based on these isolates, we propose two new genera: Freudenthalidium gen. nov. and Halluxium gen. nov., circumscribing Clades Fr3 and H, respectively. Three new species are described: Freudenthalidium heronense, F. endolithicum, and Halluxium pauxillum. Kofoidian tabulations of motile cells confirm previous observations that amphiesmal vesicle arrangements are generally conserved across the family. These descriptions are an important step toward completing the systematic revision of the Symbiodiniaceae. That this contribution was enabled by isolates from an endopsammic habitat highlights the potential of discovering new symbiodiniacean species in the environment, the study of which will lead to a deeper understanding of free-living versus symbiotic life histories in this ecologically important family of dinoflagellates.

Membranes design for guided tissue engineering have been studied to aid in cell viability and function as tissue barriers. Two asymmetric resorbable membranes for guided bone regeneration (GBR) were produced: chitosan/pectin/poly-caprolactone (PECm) and poly(vinyl alcohol)/polyethylenimine/poly(ethylene glycol) (PVAm). Both membranes were characterized by physical, chemical, mechanical, degradation rate, and in vitro biological assessment. Scanning electron microscopy (SEM) confirmed the membranes asymmetry, in which PECm asymmetry is given by roughness and chemical composition, while PVAm's only by differences in porosity. Fourier transform infrared spectroscopy (FTIR) identified chemical groups and bonds between polymers. Both sides of PVAm revealed to be hydrophobic, whereas the PECm presented one side with higher hydrophobicity than the other. In vitro biological assessment disclosed that PECm presented a higher cell adhesion growth pattern than PVAm, where it seemed to occur a delay in proliferation due to initial low cell adhesion. Both developed membranes are suitable for GBR, since both membranes fulfil the requirements to be used as a tissue barrier. The PECm has an additional role in cell viability that was not observed in the PVAm. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2141-2150, 2018.

Decellularized xenogeneic scaffolds have shown promise to be employed as compatible and functional cardiovascular biomaterials. However, one of the main barriers to their clinical exploitation is the lack of appropriate sterilization procedures. This study investigated the efficiency of a two-step sterilization method, antibiotics/antimycotic (AA) cocktail and peracetic acid (PAA), on porcine and bovine decellularized pericardium. In order to assess the efficiency of the method, a sterilization assessment protocol was specifically designed, comprising: i) controlled contamination with a known amount of bacteria; ii) sterility test; iii) identification of contaminants through MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) mass spectrometry and iv) quantification by the Most Probable Number (MPN) method. This sterilization assessment protocol proved to be a successful tool to monitor and optimize the proposed sterilization method. The treatment with AA + PAA method provided sterile scaffolds while preserving the structural integrity and biocompatibility of the decellularized porcine and bovine tissues. However, surface properties and cellular adhesion resulted slightly impaired on porcine pericardium. This work developed a sterilization method suitable for decellularized pericardial scaffolds that could be adopted for in vivo tissue engineering. Together with the proposed sterilization assessment protocol, this decontamination method will foster the clinical translation of decellularized xenogeneic substitutes. Statement of Significance Clinical application of functional and compatible xenogeneic decellularized scaffolds has been delayed due to the lack of appropriate sterilization methodologies. In this study, it was investigated an effective sterilization method optimized for porcine and bovine decellularized pericardia, based on the use of antibiotics/antimycotics followed by peracetic acid treatment. This treatment effectively sterilizes both species scaffolds, proves to maintain tissue overall structure and components, preserves biocompatibility and biomechanical properties. Furthermore, it was also developed a sterilization assessment protocol used to monitor and validate the previous method, consisting in three main parts: i) controlled contamination; ii) sterility test, and iii) identification and quantification of contaminants. Both methodologies were optimized for the tissues in study but can be applied to other scaffolds and accelerate their clinical translation.

Taxonomical analyses were performed on strain CPA58T, a novel isolate obtained from surface-sterilized aboveground tissues of the halophyte Halimione portulacoides, collected from a salt marsh in Ria de Aveiro, Portugal. The strain was Gram-stain-negative, rod-shaped, oxidase-negative and catalase-positive. Optimal growth was observed at 26 °C, at pH 6–8 and in the presence of 2 to 3 % (w/v) NaCl. Phylogenetic analyses, based on the 16S rRNA gene sequence, showed that strain CPA58T belongs to the genus Zunongwangia , with highest sequence similarities to both Zunongwangia profunda SM-A87T and Zunongwangia mangrovi P2E16T (96.5 %), followed by Zunongwangia atlantica 22II14-10F7T (95.9 %). The principal fatty acids were iso-C15 : 0, summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) and iso-C17 : 0 3-OH. The major respiratory quinone was MK-6 and the DNA G+C content was 35.1 mol%. Phylogenetic and chemotaxonomic analyses clearly placed strain CPA58T in the genus Zunongwangia . However, 16S rRNA gene sequence analysis showed that the threshold for same species relatedness was not surpassed, and biochemical tests revealed diagnostic characteristics that differentiated this strain from other type strains of species of the genus Zunongwangia . Overall, the analyses showed that strain CPA58T represents a novel species within the genus Zunongwangia , for which the name Zunongwangia endophytica sp. nov. is proposed, with the type strain CPA58T (=CECT 9128T=LMG 29517T).

A Gram-stain negative, oxidase- and catalase- positive, motile, aerobic, non-pigmented spirillum, designated CPA1T, was isolated from the surface-sterilized tissues of a halophyte, Halimione portulacoides, collected from a salt marsh in Aveiro, Portugal. The isolate was mesophilic, facultatively alkaliphilic and halophilic, and grew between 18 and 42.5 °C (optimum 30 °C), from pH 5.0 to 11.5 (optimum 7.0–7.5), from 0.5 to 5 % NaCl (w/v, optimum 2 %). Analysis of the 16S rRNA gene sequence showed that this strain belongs to the genus Saccharospirillum , as the highest sequence similarities were observed with Saccharospirillum impatiens EL-105T (96.46 %), Saccharospirillum salsuginis YIM-Y25T (96.32 %) and Saccharospirillum aestuarii IMCC 4453T (95.17 %). The next closest matches were with other genera and below 95.0 %. Phylogenetic analyses revealed that the strain forms a robust clade with other species of the genus Saccharospirillum . The main respiratory quinone was Q-8 and the major fatty acids were C16 : 0 and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). The DNA G+C content was 55.2 mol%. Molecular, physiological and biochemical differences between strain CPA1T and other type strains of species of the genus Saccharospirillum support the addition of this novel species to the genus, and the name Saccharospirillum correiae sp. nov. is proposed, with CPA1T (=CECT 9131T=LMG 29516T) as the type strain.

Three actinobacterial strains were isolated from roots of the salt-marsh plant Halimione portulacoides collected in Ria de Aveiro, Portugal. Molecular typing using enterobacterial repetitive intergenic consensus ERIC-PCR fingerprinting showed the strains to be highly similar. Phylogenetic analyses based on the 16S rRNA gene sequence and multilocus sequence analysis (MLSA) using gyrB, rpoB, recA and ppk and 16S rRNA genes sequences showed that the strains represented a member of the genus Microbacterium, with Microbacterium lacus DSM 18910T as the closest phylogenetic relative. DNA-DNA hybridization between strain RZ63T and its closest relative was below 70 %, supporting the hypothesis that it represented a distinct genomic species. Chemotaxonomic analyses of the novel strains and their DNA G+C contents confirmed their affiliation to the genus Microbacterium, however, the peptidoglycan of RZ63T contained diaminobutyric acid as the diagnostic diamino acid. In addition, physiological and fatty acid analyses revealed differences between these strains and their phylogenetic relatives, reinforcing their status as a distinct species. Based on the physiological, genetic and chemotaxonomic characterisation it is proposed that the strains studied represent a novel species of the genus Microbacterium for which the name Microbacterium diaminobutyricum sp. nov. is proposed (type strain RZ63T=DSM 27101T=CECT 8355T).

Eyewitness memory is widely studied in the forensic context, due to their proneness to make unreliable testimonies. Understanding which factors may impact memory is determinant to avoid wrongful convictions in court. In this exploratory study, the relation between stress and anxiety and memory errors (spontaneous and induced) was analyzed being hypothesized that negative emotions may impair memory performance. Crime and neutral videos were presented to 80 volunteer university students in a between subject-design. They were asked to fill some stress and anxiety scales throughout the experimental task, as well as a free recall task. Also, it was presented several questions about the videos in which spontaneous and induced errors were assessed. Results suggests that stress and anxiety did not influence the quantity of memory errors for both genders. However, overall memory performance was poor for both groups. Our results were discussed in light to existing theories about the relation between stress-anxiety and memory.

Phytoremediation assisted by bacteria is seen as a promising alternative to reduce metal contamination in the environment. The main goal of this study was to characterize endophytic Pseudomonas isolated from Halimione portulacoides, a metal-accumulator plant, in salt marshes contaminated with metal(loid)s. Phylogenetic analysis based on 16S rRNA and gyrB genes showed that isolates affiliated with P. sabulinigri (n = 16), P. koreensis (n = 10), P. simiae (n = 5), P. seleniipraecipitans (n = 2), P. guineae (n = 2), P. migulae (n = 1), P. fragi (n = 1), P. xanthomarina (n = 1), and Pseudomonas sp. (n = 1). Most of these species have never been described as endophytic. The majority of the isolates were resistant to three or more metal(loid)s. Antibiotic resistance was frequent among the isolates but most likely related to species-intrinsic features. Common acquired antibiotic resistance genes and integrons were not detected. Plasmids were detected in 43.6 % of the isolates. Isolates that affiliated with different species shared the same plasmid profile but attempts to transfer metal resistance to receptor strains were not successful. Phosphate solubilization and IAA production were the most prevalent plant growth promoting traits, and 20 % of the isolates showed activity against phytopathogenic bacteria. Most isolates produced four or more extracellular enzymes. Preliminary results showed that two selected isolates promote Arabidopsis thaliana root elongation. Results highlight the diversity of endophytic Pseudomonas in H. portulacoides from contaminated sites and their potential to assist phytoremediation by acting as plant growth promoters and as environmental detoxifiers.

Halimione portulacoides is abundant in salt marshes, accumulates mercury (Hg), and was proposed as useful for phytoremediation and pollution biomonitoring. Endophytic bacteria promote plant growth and provide compounds with industrial applications. Nevertheless, information about endophytic bacteria from H. portulacoides is scarce. Endophytic isolates (n = 665) were obtained from aboveground and belowground plant tissues, from two Hg-contaminated sites (sites E and B) and a noncontaminated site (site C), in the estuary Ria de Aveiro. Representative isolates (n = 467) were identified by 16S rRNA gene sequencing and subjected to functional assays. Isolates affiliated with Proteobacteria (64 %), Actinobacteria (23 %), Firmicutes (10 %), and Bacteroidetes (3 %). Altererythrobacter (7.4 %), Marinilactibacillus (6.4 %), Microbacterium (10.2 %), Salinicola (8.8 %), and Vibrio (7.8 %) were the most abundant genera. Notably, Salinicola (n = 58) were only isolated from site C; Hoeflea (17), Labrenzia (22), and Microbacterium (67) only from belowground tissues. This is the first report of Marinilactibacillus in the endosphere. Principal coordinate analysis showed that community composition changes with the contamination gradient and tissue. Our results suggest that the endosphere of H. portulacoides represents a diverse bacterial hotspot including putative novel species. Many isolates, particularly those affiliated to Altererythrobacter, Marinilactibacillus, Microbacterium, and Vibrio, tested positive for enzymatic activities and plant growth promoters, exposing H. portulacoides as a source of bacteria and compounds with biotechnological applications.

The demand for food with high nutritional value that can sustain the growth of human population while safeguarding sustainability deserves urgent attention. A possible strategy is the inoculation of crops with plant growth promoting (PGP) bacteria. Plants are naturally colonized by bacteria that can exert beneficial effects on growth and stress tolerance. N2 fixation by rhizobia in the root nodules of legumes is a well-known PGP effect. These bacteria can be used as inoculants to boost legumes productivity and can be especially interesting if they are able to survive to abiotic stresses, such as drought. Herein we report the phylogenetic diversity of bacteria colonizing the root nodules of several wild legume species, from four geographic locations in Portugal with different bioclimates. Interestingly, the vast majority of strains belonged to Flavobacterium, Pseudomonas and other genera apart from rhizobia. PGP abilities other than N2 fixation (production of indol acetic acid, siderophores and volatile organic compounds) and osmotolerance were screened. Location and host plant species did not influence PGP abilities and osmotolerance. Taken together, results evidenced that bacterial strains from wild legumes displaying PGP abilities and osmotolerance can be regarded as good candidates for inoculants of a broad range of hosts, including non-legumes.

Two Gram-negative, rod-shaped, motile bacterial strains, named CPA5T and BR75T, were isolated from the halophyte Halimione portulacoides. Both presented optimum growth at 30 °C, pH 7.0–7.5 and 1–2 % NaCl (w/v) for strain CPA5T, and pH 7.5–8.0 and 2 % NaCl (w/v) for strain BR75T. Phylogenetic analyses based on 16S rRNA gene sequences affiliated both strains to the genus Altererythrobacter. CPA5T presented highest 16S rRNA gene sequence similarity with Altererythrobacter aestuarii KYW147T (96.5 %), followed by Altererythrobacter namhicola KYW48T (95.9 %), Novosphingobium indicum H25T (95.6 %) and Altererythrobacter oceanensis Y2T (95.5 %). BR75T displayed highest similarity with Altererythrobacter marensis MSW-14T (96.5 %), followed by Altererythrobacter xinjiangensis S3-63T, Altererythrobacter luteolus SW-109T and Altererythrobacter indicus MSSRF26T (96.1 %). Neither strain contained Bacteriochlorophyll a. The main fatty acids observed for CPA5T were C17 : 1ω6c and summed features 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH) and 8 (C18 : 1 ω7c and/or C18 : 1ω6c). The latter summed feature was the dominant fatty acid observed for strain BR75T as well. The major polar lipids were phosphatidylethanolamine, unidentified phospholipids and unidentified glycolipids for both strains. The predominant ubiquinone was Q-10 for both strains, and the DNA G+C contents were 63.4 mol% and 58.3 mol% for CPA5T and BR75T, respectively. Based on phenotypic and genotypic results, both strains represent novel species belonging to the genus Altererythrobacter for which the names Altererythrobacter halimionae sp. nov. (type strain CPA5T=CECT 9130T=LMG 29519T) and Altererythrobacter endophyticus sp. nov (type strain BR75T=CECT 9129T=LMG 29518T) are proposed.

The goal of this study was to investigate co-selection of antibiotic resistance in gram-negative epiphytic bacteria. Halimione portulacoides samples were collected from metal(loid)-contaminated and non-contaminated salt marshes. Bacterial isolates (n=137) affiliated with Vibrio, Pseudomonas, Shewanella, Comamonas, Aeromonas and with Enterobacteriaceae. Vibrio isolates were more frequent in control site while Pseudomonas was common in contaminated sites. Metal(loid) and antibiotic resistance phenotypes varied significantly according to site contamination, and multiresistance was more frequent in contaminated sites. However, differences among sites were not observed in terms of prevalence or diversity of acquired antibiotic resistance genes, integrons and plasmids. Gene merA, encoding mercury resistance, was only detected in isolates from contaminated sites, most of which were multiresistant to antibiotics. Results indicate that metal(loid) contamination selects for antibiotic resistance in plant surfaces. In salt marshes, antibiotic resistance may be subsequently transferred to other environmental compartments, such as estuarine water or animals, with potential human health risks.

Notwithstanding their wide exploitation, biological prosthetic heart valves are characterized by limited durability (10–15 years). The treatment of biological tissues with chemical crosslinking agents such as glutaraldehyde accounts for the enhanced risk of structural deterioration associated with the early failure of bioprosthetic valves. To overcome the shortcomings of the currently available solutions, adoption of decellularized biological tissues of animal origin has emerged as a promising approach. The present study aims to assess in vitro cardiovascular scaffolds composed of bovine pericardium decellularized with the novel TRITDOC (TRIton-X100 and TauroDeOxyCholic acid) procedure. The effects of the treatment have been assessed by means of histological, biomolecular, cellular, biochemical and biomechanical analyses. The TRITDOC procedure grants the complete decellularization of bovine pericardial scaffolds while preserving the extracellular matrix architecture and the biomechanical properties. With a dedicated ELISA test, the TRITDOC procedure has been proven to ensure the complete removal of the alphaGal antigen, responsible for hyperacute rejection and for long-term deterioration of xenogenic biomaterials. Static seeding of the acellular pericardial patches with human adipose-derived stem cells resulted in an evenly repopulated scaffold without signs of calcification. The in vitro cyto-/immuno-compatibility response of the TRITDOC-bovine pericardium was compared with glutaraldehyde-treated xenogenic pericardium collected from two bioprosthetic devices currently used in clinical practice: PERIMOUNT MAGNA and TRIFECTATM. TRITDOC-bovine pericardium exhibited lower complement activation, lower cytotoxicity and a lower tendency to secrete pro-inflammatory cytokines compared to the tested commercial bioprostheses. Therefore, TRITDOC-decellularized pericardium could be considered as possible candidate material for the production of prosthetic heart valves.

Mycorrhizal fungi form ecologically important connections between plants and soils, and although nitrogen (N) enrichment has been implicated in the decline of ectomycorrhizal fungal diversity, they are rarely considered in studies investigating the effects of increased N availability on plant species diversity. This chapter describes the effects of N enrichment on the soil fungal community and in particular on arbuscular mycorrhizal fungal (AMF) spores, in a Mediterranean ecosystem in a Natura 2000 site in southern Portugal (PTCON0010 Arrabida/Espichel). Soil fungal community structure was affected by the addition of 80 kg N ha−1 year−1 as NH4NO3 within 2 years. The effects of N addition on AMF diversity (richness and evenness) appear to depend on the form of N, since the addition of 40 kg N ha−1 year −1 as ammonium increased AMF spore richness and evenness proportionally more than the addition of 40 kg N ha−1 year−1 as ammonium plus nitrate. The composition of AMF species may serve as a sensitive indicator of N enrichment.