Species of Bursaphelenchus Fuchs, 1937 (Nematoda: Parasitaphelenchidae) associated with maritime pine in Portugal

Summary – Species of Bursaphelenchus associated with maritime pine, Pinus pinaster, from Portugal – within and outside the quarantine restricted demarcated zone of B. xylophilus – are described and characterised both morphologically (LM and SEM) and with the use of molecular biology (ITS-RFLP).A new staining method for spicules is proposed. Species include B. hellenicus, B. hylobianum, B. leoni, B. pinophilus, B. sexdentati, B. tusciae, B. teratospicularis, B. xylophilus and Bursaphelenchus sp. 1. Bursaphelenchus hylobianum was collected from the insect Hylobius sp. The most frequent species in the demarcated zone, besides B. xylophilus, was Bursaphelenchus sp. 1. Morphological characterisation is compared with the original descriptions and discussed. The differentiation between B. pinophilus and B. sexdentati is not clear in the literature and is discussed. Since differentiation of B. xylophilus (mucronate form) from B. mucronatus, and B. pinophilus from B. sexdentati, as well as their juvenile forms, is almost im possible on the basis of morphological features, a molecular approach based on ITS-RFLPs was used. Ribosomal DNA containing the 5.8S gene, the internal transcribed spacer region 1 and 2, and partial regions of 18S and 28S gene were amplified by PCR. Restriction profiles of the amplified products generated species-specific differences, leading to the unambiguous identification of isolates belonging to B. xylophilus, B. mucronatus, B. sexdentati, B. tusciae and B. hylobianum

Genome diversity in the genera Fructobacillus, Leuconostoc and Weissella determined by physical and genetic mapping

Pulsed-field gel electrophoresis analysis of chromosomal single and double restriction profiles of 17 strains belonging to three genera of ‘Leuconostocaceae’ was done, resulting in physical and genetic maps for three Fructobacillus, six Leuconostoc and four Weissella strains. AscI, I-CeuI, NotI and SfiI restriction enzymes were used together with Southern hybridization of selected probes to provide an assessment of genomic organization in different species. Estimated genome sizes varied from 1408 kb to 1547 kb in Fructobacillus, from 1644 kb to 2133 kb in Leuconostoc and from 1371 kb to 2197 kb in Weissella. Other genomic characteristics of interest were analysed, such as oriC and terC localization and rrn operon organization. The latter seems markedly different in Weissella, in both number and disposition in the chromosome. Comparisons of intra- and intergeneric features are discussed in the light of chromosome rearrangements and genomic evolution

Microbial Characterization of Yellow Curing Process of Codfish

Research articleYellow cured codfish has a typical yellow colour, distinctive taste, and low salt content due to its special curing process of the raw salted codfish involving several soaks in water of the raw salted codfish, alternated with drying steps. The purpose of this study was to assess the main functional groups of bacteria involved in this process and relate them with physicochemical properties of the product. A total of 28 codfish from Iceland were supplied by two local companies. Seven stages of the curing process were analyzed. From each of these seven stages, four fish samples were collected to carry out the microbial and physicochemical analyses (moisture, salt content, pH, total volatile basic nitrogen (TVB-N), and trimethylamine nitrogen (TMA-N)). Bacteria counts were performed using the MPN method and adequate culture media for aerobic, proteolytic, sulphite-reducing, biogenic amine, and trimethylamine-producing and ammonifying bacteria. Strains isolated from the highest dilutions with microbial growth were used to characterize the predominant bacteria. The results showed that total aerobic counts increased from 3.9 log MPN/g in raw salted codfish to 5.9 log MPN/g in the final. Proteolytic, ammonifying, and trimethylamine bacteria producers also increased to 8, 7.5, and 6.5 log MPN/g, respectively. The salt content decreases (from 17% until 8%) and moisture increases (53% until 67%) during the salted-raw-codfish soaking, favoring sulphite-reducing and biogenic amine-producing species, confirming that desalting enhances potential spoilers. The subsequent drying step benefits proteolytic, ammonifying, and trimethylamine-producing bacteria, with a corresponding non-protein-nitrogen content (TVB-N and TMA-N) increase. The dominant bacteria during yellow curing belong to the genera Staphylococcus, Psychrobacter, Pseudomonas, and Alcaligenes with a clear positive correlation between the content of Staphylococcus and Psychrobacter and TVB-N and TMA-N concentration. Staphylococcus spp. are the dominant bacteria in the steps where the product has a higher salt concentration; thus, it could be particularly useful as an indicator to control the industrially yellow curing process and could have an important role in the development of the final characteristics of this productinfo:eu-repo/semantics/publishedVersio

An Optimized in situ Quantification Method of Leaf H2O2 Unveils Interaction Dynamics of Pathogenic and Beneficial Bacteria in Wheat

Hydrogen peroxide (H2O2) functions as an important signaling molecule in plants during biotic interactions. However, the extent to which H2O2 accumulates during these interactions and its implications in the development of disease symptoms is unclear. In this work, we provide a step-by-step optimized protocol for in situ quantification of relative H2O2 concentrations in wheat leaves infected with the pathogenic bacterium Pseudomonas syringae pv. atrofaciens (Psa), either alone or in the presence of the beneficial bacterium Herbaspirillum seropedicae (RAM10). This protocol involved the use of 3-3'diaminobenzidine (DAB) staining method combined with image processing to conduct deconvolution and downstream analysis of the digitalized leaf image. The application of a linear regression model allowed to relate the intensity of the pixels resulting from DAB staining with a given concentration of H2O2. Decreasing H2O2 accumulation patterns were detected at increasing distances from the site of pathogen infection, and H2O2 concentrations were different depending on the bacterial combinations tested. Notably, Psa-challenged plants in presence of RAM10 accumulated less H2O2 in the leaf and showed reduced necrotic symptoms, pointing to a potential role of RAM10 in reducing pathogen-triggered H2O2 levels in young wheat plants.info:eu-repo/semantics/publishedVersio

Identification of Sinorhizobium (Ensifer) medicae based on a specific genomic sequence unveiled by M13-PCR fingerprinting

A collection of nodule isolates from Medicago polymorpha obtained from southern and central Portugal was evaluated by M13-PCR fingerprinting and hierarchical cluster analysis. Several genomic clusters were obtained which, by 16S rRNA gene sequencing of selected representatives, were shown to be associated with particular taxonomic groups of rhizobia and other soil bacteria. The method provided a clear separation between rhizobia and co-isolated non-symbiotic soil contaminants. Ten M13-PCR groups were assigned to Sinorhizobium (Ensifer) medicae and included all isolates responsible for the formation of nitrogen-fixing nodules upon re-inoculation of M. polymorpha test-plants. In addition, enterobacterial repetitive intergenic consensus (ERIC)-PCR fingerprinting indicated a high genomic heterogeneity within the major M13-PCR clusters of S. medicae isolates. Based on nucleotide sequence data of an M13-PCR amplicon of ca. 1500 bp, observed only in S. medicae isolates and spanning locus Smed_3707 to Smed_3709 from the pSMED01 plasmid sequence of S. medicae WSM419 genome’s sequence, a pair of PCR primers was designed and used for direct PCR amplification of a 1399-bp sequence within this fragment. Additional in silico and in vitro experiments, as well as phylogenetic analysis, confirmed the specificity of this primer combination and therefore the reliability of this approach in the prompt identification of S. medicae isolates and their distinction from other soil bacteria. [Int Microbiol 2009; 12(4):215-225

Modulation of the Wheat Seed-Borne Bacterial Community by Herbaspirillum seropedicae RAM10 and Its Potential Effects for Tryptophan Metabolism in the Root Endosphere

Plants and their associated microbiota share ecological and evolutionary traits that are considered to be inseparably woven. Their coexistence foresees the use of similar metabolic pathways, leading to the generation of molecules that can cross-regulate each other’s metabolism and ultimately influence plant phenotype. However, the extent to which the microbiota contributes to the overall plant metabolic landscape remains largely unexplored. Due to their early presence in the seed, seed-borne endophytic bacteria can intimately colonize the plant’s endosphere while conferring a series of phytobeneficial services to their host. Understanding the dynamics of these endophytic communities is a crucial step toward the formulation of microbial inoculants that can modulate the functionality of the plant-associated microbiota for improved plant fitness. In this work, wheat (Triticum aestivum) roots non-inoculated and inoculated with the bacterium Herbaspirillum seropedicae strain RAM10 were analyzed to explore the impact of inoculant–endophyte–wheat interrelationships on the regulation of tryptophan (Trp) metabolism in the endosphere environment. Root inoculation with H. seropedicae led to phylum-specific changes in the cultivable seed-borne endophytic community. This modulation shifted the metabolic potential of the community in light of its capacity to modulate the levels of key Trp-related metabolites involved in both indole-3-acetic acid (IAA) biosynthesis and in the kynurenine pathway. Our results support a mode of action of H. seropedicae relying on a shift in both the composition and functionality of the seed-borne endophytic community, which may govern important processes such as root growth. We finally provide a conceptual framework illustrating that interactions among roots, inoculants, and seed-borne endophytes are critical to fine-tuning the levels of IAA in the endosphere. Understanding the outcomes of these interactions is a crucial step toward the formulation of microbial inoculants based on their joint action with seed-borne endophytic communities to promote crop growth and health in a sustainable manner.info:eu-repo/semantics/publishedVersio

Genomic diversity of Oenococcus oeni from different winemaking regions of Portugal

Oenococcus oeni is an alcohol-tolerant, acidophilic lactic acid bacterium that plays an important role in the elaboration of wine. It is often added as a starter culture to carry out malolactic conversion. Given the economic importance of this reaction, the taxonomic structure of this species has been studied in detail. In the present work, phenotypic and molecular approaches were used to identify 121 lactic acid bacteria strains isolated from the wines of three winemaking regions of Portugal. The strains were differentiated at the genomic level by M13-PCR fingerprinting. Twenty-seven genomic clusters represented by two or more isolates and 21 single-member clusters, based on an 85% similarity level, were recognized by hierarchic numerical analysis. M13-PCR fingerprinting patterns revealed a high level of intraspecific genomic diversity in O. oeni. Moreover, this diversity could be partitioned according to the geographical origin of the isolates. Thus, M13-PCR fingerprint analysis may be an appropriate methodology to study the O. oeni ecology of wine during malolactic fermentation as well as to trace new malolactic starter cultures and evaluate their dominance over the native microbiota

Whole Genome Sequencing Refines Knowledge on the Population Structure of Mycobacterium bovis from a Multi-Host Tuberculosis System

Classical molecular analyses of Mycobacterium bovis based on spoligotyping and Variable Number Tandem Repeat (MIRU-VNTR) brought the first insights into the epidemiology of animal tuberculosis (TB) in Portugal, showing high genotypic diversity of circulating strains that mostly cluster within the European 2 clonal complex. Previous surveillance provided valuable information on the prevalence and spatial occurrence of TB and highlighted prevalent genotypes in areas where livestock and wild ungulates are sympatric. However, links at the wildlife–livestock interfaces were established mainly via classical genotype associations. Here, we apply whole genome sequencing (WGS) to cattle, red deer and wild boar isolates to reconstruct the M. bovis population structure in a multi-host, multi-region disease system and to explore links at a fine genomic scale between M. bovis from wildlife hosts and cattle. Whole genome sequences of 44 representative M. bovis isolates, obtained between 2003 and 2015 from three TB hotspots, were compared through single nucleotide polymorphism (SNP) variant calling analyses. Consistent with previous results combining classical genotyping with Bayesian population admixture modelling, SNP-based phylogenies support the branching of this M. bovis population into five genetic clades, three with apparent geographic specificities, as well as the establishment of an SNP catalogue specific to each clade, which may be explored in the future as phylogenetic markers. The core genome alignment of SNPs was integrated within a spatiotemporal metadata framework to further structure this M. bovis population by host species and TB hotspots, providing a baseline for network analyses in different epidemiological and disease control contexts. WGS of M. bovis isolates from Portugal is reported for the first time in this pilot study, refining the spatiotemporal context of TB at the wildlife–livestock interface and providing further support to the key role of red deer and wild boar on disease maintenance. The SNP diversity observed within this dataset supports the natural circulation of M. bovis for a long time period, as well as multiple introduction events of the pathogen in this Iberian multi-host system.info:eu-repo/semantics/publishedVersio

New Insights on Streptococcus dysgalactiae subsp. dysgalactiae Isolates

Funding Information: This work was supported by the Unidade de Ciências Biomoleculares Aplicadas-UCIBIO, which is financed by Funding Information: Funding. This work was supported by the Unidade de Ci?ncias Biomoleculares Aplicadas-UCIBIO, which is financed by national funds from FCT/MEC (UIDP/04378/2020 and UIDB/04378/2020) and also by projects PTDC/CVT-EPI/4651/2012 and PTDC/CVT-EPI/6685/2014. FCT-MEC is also acknowledged for grant SFRH/BD/118350/2016 to CA-B. Publisher Copyright: © Copyright © 2021 Alves-Barroco, Caço, Roma-Rodrigues, Fernandes, Bexiga, Oliveira, Chambel, Tenreiro, Mato and Santos-Sanches.Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) has been considered a strict animal pathogen. Nevertheless, the recent reports of human infections suggest a niche expansion for this subspecies, which may be a consequence of the virulence gene acquisition that increases its pathogenicity. Previous studies reported the presence of virulence genes of Streptococcus pyogenes phages among bovine SDSD (collected in 2002–2003); however, the identity of these mobile genetic elements remains to be clarified. Thus, this study aimed to characterize the SDSD isolates collected in 2011–2013 and compare them with SDSD isolates collected in 2002–2003 and pyogenic streptococcus genomes available at the National Center for Biotechnology Information (NCBI) database, including human SDSD and S. dysgalactiae subsp. equisimilis (SDSE) strains to track temporal shifts on bovine SDSD genotypes. The very close genetic relationships between humans SDSD and SDSE were evident from the analysis of housekeeping genes, while bovine SDSD isolates seem more divergent. The results showed that all bovine SDSD harbor Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas IIA system. The widespread presence of this system among bovine SDSD isolates, high conservation of repeat sequences, and the polymorphism observed in spacer can be considered indicators of the system activity. Overall, comparative analysis shows that bovine SDSD isolates carry speK, speC, speL, speM, spd1, and sdn virulence genes of S. pyogenes prophages. Our data suggest that these genes are maintained over time and seem to be exclusively a property of bovine SDSD strains. Although the bovine SDSD genomes characterized in the present study were not sequenced, the data set, including the high homology of superantigens (SAgs) genes between bovine SDSD and S. pyogenes strains, may indicate that events of horizontal genetic transfer occurred before habitat separation. All bovine SDSD isolates were negative for genes of operon encoding streptolysin S, except for sagA gene, while the presence of this operon was detected in all SDSE and human SDSD strains. The data set of this study suggests that the separation between the subspecies “dysgalactiae” and “equisimilis” should be reconsidered. However, a study including the most comprehensive collection of strains from different environments would be required for definitive conclusions regarding the two taxa.publishersversionpublishe