De Novo Assembly of the Dirofilaria immitis Genome by Long-Read Nanopore-Based Sequencing Technology on an Adult Worm from a Canine Cardiopulmonary Dirofilariosis Case.

Dirofilaria immitis is a zoonotic parasitic nematode that infects domestic and wild canids, among its vertebrate hosts. The genetic analysis of D. immitis nowadays transcends the need for genetic taxonomy of nematodes, such as the study of resistance to macrocyclic lactone. We expanded the use of long-read nanopore-based sequencing technology on nematodes by performing genomic de novo assembly of a D. immitis specimen retrieved from a canine cardiopulmonary dirofilariasis case using the ONT MinION platform, followed by the study of macrocyclic lactone resistance. The assembled genome of D. immitis consists of 110 contigs with an N50 of 3687191. The genome size is 87899012 and contains a total of 9741 proteins; 6 ribosomal RNAs, with three belonging to the small subunit (18S) and three to the large subunit (28S); and 73 tRNAs. Subsequent analysis of six loci previously characterized as being associated to macrocyclic lactone resistance selection pressure showed that four have a genotype associated with either some loss of efficacy or the resistance phenotype. Considering the zoonotic potential of D. immitis, the identification of a resistant parasite alerts for the overuse of macrocyclic lactone in the region, which poses a potential risk to both veterinary and human public health.

Occurrence and molecular characterization of human pegivirus-1 (HPgV-1) viremia in healthy volunteer blood donors from Northern Portugal.

Human pegivirus-1 (HPgV-1) is a member of the Flaviviridae family and the Pegivirus genus. Despite having been discovered 25 years ago, there is still much to know regarding HPgV-1 clinical impact, as this virus is currently not associated with any pathology. Yet, HPgV-1 prevalence and molecular characterization are still unknown in many countries, including Portugal. To fill in this knowledge gap, this study aimed to determine the occurrence and molecular characterization of HPgV-1 in a group of healthy blood donors from the north of Portugal. Blood samples from 465 Portuguese blood donors were collected from a major Hospital Center in the north of Portugal. RNA was extracted and an initial nested RT-PCR was performed targeting the conserved 5'-untranslated region  region of the HPgV-1 genome. A second nested RT-PCR targeting the E2 region was performed for genotyping. Only one sample tested positive for HPgV-1 RNA, resulting in a prevalence of approximately 0.22%. Phylogenetic analyses confirmed the characterization as genotype 2, the most prevalent in Europe.

High-Quality Draft Genome Sequences of Marine Fish Gut Bacillus sp. Strains ABP1 and ABP2 with Nonstarch Polysaccharide Hydrolytic Potential.

Here, we present the genome sequences of two environmental Bacillus strains with broad hydrolytic capacity toward different nonstarch polysaccharides (NSPs) that were isolated from the gut of marine fish fed NSP-rich diets. Several genes that may contribute to the NSP-degrading behavior were identified through in silico analysis.

Quantification of dose-mortality responses in adult Diptera: Validation using Ceratitis capitata and Drosophila suzukii responses to spinosad.

Quantitative laboratory bioassay methods are required to evaluate the toxicity of novel insecticidal compounds for pest control and to determine the presence of resistance traits. We used a radioactive tracer based on 32P-ATP to estimate the volume of a droplet ingested by two dipteran pests: Ceratitis capitata (Tephritidae) and Drosophila suzukii (Drosophilidae). Using blue food dye it was possible to distinguish between individuals that ingested the solution from those that did not. The average volume ingested by C. capitata adults was 1.968 µl. Females ingested a ~20% greater volume of solution than males. Adults of D. suzukii ingested an average of 0.879 µl and females ingested ~30% greater volume than males. The droplet feeding method was validated using the naturally-derived insecticide spinosad as the active ingredient (a.i.). For C. capitata, the concentration-mortality response did not differ between the sexes or among three different batches of insects. Lethal dose values were calculated based on mean ingested volumes. For C. capitata LD50 values were 1.462 and 1.502 ng a.i./insect for males and females, respectively, equivalent to 0.274 and 0.271 ng a.i./mg for males and females, respectively, when sex-specific variation in body weight was considered. Using the same process for D. suzukii, the LD50 value was estimated at 2.927 ng a.i./insect, or 1.994 ng a.i./mg based on a mean body weight of 1.67 mg for both sexes together. We conclude that this technique could be readily employed for determination of the resistance status and dose-mortality responses of insecticidal compounds in many species of pestiferous Diptera.

The use of stable and unstable green fluorescent proteins for studies in two bacterial models: Agrobacterium tumefaciens and Xanthomonas campestris pv. campestris.

Fluorescent proteins have been used to track plant pathogens to understand their host interactions. To be useful, the transgenic pathogens must present similar behaviour than the wild-type isolates. Herein, a GFP marker was used to transform two plant pathogenic bacteria, Agrobacterium and Xanthomonas, to localize and track the bacteria during infection. The transgenic bacteria were evaluated to determine whether they showed the same fitness than the wild-type strains or whether the expression of the GFP protein interfered in the bacterial activity. In Agrobacterium, the plasmid used for transformation was stable in the bacteria and the strain kept the virulence, while Xanthomonas was not able to conserve the plasmid and transformed strains showed virulence variations compared to wild-type strains. Although marking bacteria with GFP to track infection in plants is a common issue, works to validate the transgenic strains and corroborate their fitness are not usual. Results, presented here, confirm the importance of proper fitness tests on the marked strains before performing localization assays, to avoid underestimation of the microbe population or possible artificial effects in its interaction with the plant.

Contribution of the non-effector members of the HrpL regulon, iaaL and matE, to the virulence of Pseudomonas syringae pv. tomato DC3000 in tomato plants.

BACKGROUND: The phytohormone indole-3-acetic acid (IAA) is widely distributed among plant-associated bacteria. Certain strains of the Pseudomonas syringae complex can further metabolize IAA into a less biologically active amino acid conjugate, 3-indole-acetyl-ε-L-lysine, through the action of the iaaL gene. In P. syringae and Pseudomonas savastanoi strains, the iaaL gene is found in synteny with an upstream gene, here called matE, encoding a putative MATE family transporter. In P. syringae pv. tomato (Pto) DC3000, a pathogen of tomato and Arabidopsis plants, the HrpL sigma factor controls the expression of a suite of virulence-associated genes via binding to hrp box promoters, including that of the iaaL gene. However, the significance of HrpL activation of the iaaL gene in the virulence of Pto DC3000 is still unclear. RESULTS: A conserved hrp box motif is found upstream of the iaaL gene in the genomes of P. syringae strains. However, although the promoter region of matE is only conserved in genomospecies 3 of this bacterial group, we showed that this gene also belongs to the Pto DC3000 HrpL regulon. We also demonstrated that the iaaL gene is transcribed both independently and as part of an operon with matE in this pathogen. Deletion of either the iaaL or the matE gene resulted in reduced fitness and virulence of Pto DC3000 in tomato plants. In addition, we used multicolor fluorescence imaging to visualize the responses of tomato plants to wild-type Pto DC3000 and to its ΔmatE and ΔiaaL mutants. Activation of secondary metabolism prior to the development of visual symptoms was observed in tomato leaves after bacterial challenges with all strains. However, the observed changes were strongest in plants challenged by the wild-type strain, indicating lower activation of secondary metabolism in plants infected with the ΔmatE or ΔiaaL mutants. CONCLUSIONS: Our results provide new evidence for the roles of non-type III effector genes belonging to the Pto DC3000 HrpL regulon in virulence.

Translocation and functional analysis of Pseudomonas savastanoi pv. savastanoi NCPPB 3335 type III secretion system effectors reveals two novel effector families of the Pseudomonas syringae complex.

Pseudomonas savastanoi pv. savastanoi NCPPB 3335 causes olive knot disease and is a model pathogen for exploring bacterial infection of woody hosts. The type III secretion system (T3SS) effector repertoire of this strain includes 31 effector candidates plus two novel candidates identified in this study which have not been reported to translocate into plant cells. In this work, we demonstrate the delivery of seven NCPPB 3335 effectors into Nicotiana tabacum leaves, including three proteins from two novel families of the P. syringae complex effector super-repertoire (HopBK and HopBL), one of which comprises two proteins (HopBL1 and HopBL2) that harbor a SUMO protease domain. When delivered by P. fluorescens heterologously expressing a P. syringae T3SS, all seven effectors were found to suppress the production of defense-associated reactive oxygen species. Moreover, six of these effectors, including the truncated versions of HopAA1 and HopAZ1 encoded by NCPPB 3335, suppressed callose deposition. The expression of HopAZ1 and HopBL1 by functionally effectorless P. syringae pv. tomato DC3000D28E inhibited the hypersensitive response in tobacco and, additionally, expression of HopBL2 by this strain significantly increased its competitiveness in N. benthamiana. DNA sequences encoding HopBL1 and HopBL2 were uniquely detected in a collection of 31 P. savastanoi pv. savastanoi strains and other P. syringae strains isolated from woody hosts, suggesting a relevant role of these two effectors in bacterial interactions with olive and other woody plants.

Identification of novel virulence genes and metabolic pathways required for full fitness of Pseudomonas savastanoi pv. savastanoi in olive (Olea europaea) knots.

Comparative genomics and functional analysis of Pseudomonas syringae and related pathogens have mainly focused on diseases of herbaceous plants; however, there is a general lack of knowledge about the virulence and pathogenicity determinants required for infection of woody plants. Here, we applied signature-tagged mutagenesis (STM) to Pseudomonas savastanoi pv. savastanoi during colonization of olive (Olea europaea) knots, with the goal of identifying the range of genes linked to growth and symptom production in its plant host. A total of 58 different genes were identified, and most mutations resulted in hypovirulence in woody olive plants. Sequence analysis of STM mutations allowed us to identify metabolic pathways required for full fitness of P. savastanoi in olive and revealed novel mechanisms involved in the virulence of this pathogen, some of which are essential for full colonization of olive knots by the pathogen and for the lysis of host cells. This first application of STM to a P. syringae-like pathogen provides confirmation of functional capabilities long believed to play a role in the survival and virulence of this group of pathogens but not adequately tested before, and unravels novel factors not correlated previously with the virulence of other plant or animal bacterial pathogens.

Pseudomonas savastanoi pv. savastanoi: some like it knot.

Pseudomonas savastanoi pv. savastanoi is the causal agent of olive (Olea europaea) knot disease and an unorthodox member of the P. syringae complex, causing aerial tumours instead of the foliar necroses and cankers characteristic of most members of this complex. Olive knot is present wherever olive is grown; although losses are difficult to assess, it is assumed that olive knot is one of the most important diseases of the olive crop. The last century witnessed a large number of scientific articles describing the biology, epidemiology and control of this pathogen. However, most P. savastanoi pv. savastanoi strains are highly recalcitrant to genetic manipulation, which has effectively prevented the pathogen from benefitting from the scientific progress in molecular biology that has elevated the foliar pathogens of the P. syringae complex to supermodels. A number of studies in recent years have made significant advances in the biology, ecology and genetics of P. savastanoi pv. savastanoi, paving the way for the molecular dissection of its interaction with other nonpathogenic bacteria and their woody hosts. The selection of a genetically pliable model strain was soon followed by the development of rapid methods for virulence assessment with micropropagated olive plants and the analysis of cellular interactions with the plant host. The generation of a draft genome of strain NCPPB 3335 and the closed sequence of its three native plasmids has allowed for functional and comparative genomic analyses for the identification of its pathogenicity gene complement. This includes 34 putative type III effector genes and genomic regions, shared with other pathogens of woody hosts, which encode metabolic pathways associated with the degradation of lignin-derived compounds. Now, the time is right to explore the molecular basis of the P. savastanoi pv. savastanoi-olive interaction and to obtain insights into why some pathovars like it necrotic and why some like it knot. SYNONYMS: Pseudomonas syringae pv. savastanoi. TAXONOMY: Kingdom Bacteria; Phylum Proteobacteria; Class Gammaproteobacteria; Family Pseudomonadaceae; Genus Pseudomonas; included in genomospecies 2 together with at least P. amygdali, P. ficuserectae, P. meliae and 16 other pathovars from the P. syringae complex (aesculi, ciccaronei, dendropanacis, eriobotryae, glycinea, hibisci, mellea, mori, myricae, phaseolicola, photiniae, sesami, tabaci, ulmi and certain strains of lachrymans and morsprunorum); when a formal proposal is made for the unification of these bacteria, the species name P. amygdali would take priority over P. savastanoi. MICROBIOLOGICAL PROPERTIES: Gram-negative rods, 0.4-0.8 × 1.0-3.0 µm, aerobic. Motile by one to four polar flagella, rather slow growing, optimal temperatures for growth of 25-30 °C; oxidase negative, arginine dihydrolase negative; elicits the hypersensitive response on tobacco; most isolates are fluorescent and levan negative, although some isolates are nonfluorescent and levan positive. HOST RANGE: P. savastanoi pv. savastanoi causes tumours in cultivated and wild olive and ash (Fraxinus excelsior). Although strains from olive have been reported to infect oleander (Nerium oleander), this is generally not the case; however, strains of P. savastanoi pv. nerii can infect olive. Pathovars fraxini and nerii are differentiated from pathovar savastanoi mostly in their host range, and were not formally recognized until 1996. Literature before about 1996 generally names strains of the three pathovars as P. syringae ssp. savastanoi or P. savastanoi ssp. savastanoi, contributing to confusion on the host range and biological properties. DISEASE SYMPTOMS: Symptoms of infected trees include hyperplastic growths (tumorous galls or knots) on the stems and branches of the host plant and, occasionally, on leaves and fruits. EPIDEMIOLOGY: The pathogen can survive and multiply on aerial plant surfaces, as well as in knots, from where it can be dispersed by rain, wind, insects and human activities, entering the plant through wounds. Populations are very unevenly distributed in the plant, and suffer drastic fluctuations throughout the year, with maximum numbers of bacteria occurring during rainy and warm months. Populations of P. savastanoi pv. savastanoi are normally associated with nonpathogenic bacteria, both epiphytically and endophytically, and have been demonstrated to form mutualistic consortia with Erwinia toletana and Pantoea agglomerans, which could result in increased bacterial populations and disease symptoms. DISEASE CONTROL: Based on preventive measures, mostly sanitary and cultural practices. Integrated control programmes benefit from regular applications of copper formulations, which should be maintained for at least a few years for maximum benefit. Olive cultivars vary in their susceptibility to olive knot, but there are no known cultivars with full resistance to the pathogen. USEFUL WEBSITES: http://www.pseudomonas-syringae.org/; http://genome.ppws.vt.edu/cgi-bin/MLST/home.pl; ASAP access to the P. savastanoi pv. savastanoi NCPPB 3335 genome sequence https://asap.ahabs.wisc.edu/asap/logon.php.

Fate of a Pseudomonas savastanoi pv. savastanoi type III secretion system mutant in olive plants (Olea europaea L.).

Pseudomonas savastanoi pv. savastanoi strain NCPPB 3335 is a model bacterial pathogen for studying the molecular basis of disease production in woody hosts. We report the sequencing of the hrpS-to-hrpZ region of NCPPB 3335, which has allowed us to determine the phylogenetic position of this pathogen with respect to previously sequenced Pseudomonas syringae hrp clusters. In addition, we constructed a mutant of NCPPB 3335, termed T3, which carries a deletion from the 3' end of the hrpS gene to the 5' end of the hrpZ operon. Despite its inability to multiply in olive tissues and to induce tumor formation in woody olive plants, P. savastanoi pv. savastanoi T3 can induce knot formation on young micropropagated olive plants. However, the necrosis and formation of internal open cavities previously reported in knots induced by the wild-type strain were not observed in those induced by P. savastanoi pv. savastanoi T3. Tagging of P. savastanoi pv. savastanoi T3 with green fluorescent protein (GFP) allowed real-time monitoring of its behavior on olive plants. In olive plant tissues, the wild-type strain formed aggregates that colonized the intercellular spaces and internal cavities of the hypertrophic knots, while the mutant T3 strain showed a disorganized distribution within the parenchyma of the knot. Ultrastructural analysis of knot sections revealed the release of extensive outer membrane vesicles from the bacterial cell surface of the P. savastanoi pv. savastanoi T3 mutant, while the wild-type strain exhibited very few vesicles. This phenomenon has not been described before for any other bacterial phytopathogen during host infection.

Annotation and overview of the Pseudomonas savastanoi pv. savastanoi NCPPB 3335 draft genome reveals the virulence gene complement of a tumour-inducing pathogen of woody hosts.

Pseudomonas savastanoi pv. savastanoi is a tumour-inducing pathogen of Olea europaea L. causing olive knot disease. Bioinformatic analysis of the draft genome sequence of strain NCPPB 3335, which encodes 5232 predicted coding genes on a total length of 5856 998 bp and a 57.12% G + C, revealed a large degree of conservation with Pseudomonas syringae pv. phaseolicola 1448A and P. syringae pv. tabaci 11528. However, NCPPB 3335 contains twelve variable genomic regions, which are absent in all previously sequenced P. syringae strains. Various features that could contribute to the ability of this strain to survive in a woody host were identified, including broad catabolic and transport capabilities for degrading plant-derived aromatic compounds, the duplication of sequences related to the biosynthesis of the phytohormone indoleacetic acid (iaaM, iaaH) and its amino acid conjugate indoleacetic acid-lysine (iaaL gene), and the repertoire of strain-specific putative type III secretion system effectors. Access to this seventh genome sequence belonging to the 'P. syringae complex' allowed us to identify 73 predicted coding genes that are NCPPB 3335-specific. Results shown here provide the basis for detailed functional analysis of a tumour-inducing pathogen of woody hosts and for the study of specific adaptations of a P. savastanoi pathovar.

A simple PCR-based genotyping method for M105I mutation of alpha-SNAP enhances the study of early pathological changes in hyh phenotype.

alpha-SNAP is an essential component of the protein machinery responsible for membrane fusion events in different cell types. The hyh (hydrocephalus with hop gait) mouse carries a missense mutation in Napa gene that results in a point mutation (M105I) in alpha-SNAP protein. Homozygous animals for the mutant allele have been identified by the clinical and/or neuropathological phenotype, or by direct sequencing of PCR products. The aims of the present study were (i) to develop a high-throughput technique to genotype hyh mice, (ii) to correlate genotype-phenotype, and (iii) to analyze the earliest pathological changes of hyh mutant mice. As no restriction sites are affected by the hyh mutation, we resolved this problem by creating a BspHI restriction site with a modified (mismatch) polymerase chain reaction (PCR) primer in wild-type allele. This artificially created restriction site (ACRS)-PCR technique is a simple, rapid and reliable method to genotype hyh mice in a day-work procedure. Biochemical and histological analysis of genotyped hyh embryos at different developmental stages allowed us to identify and characterize the earliest brain pathological changes of the hyh phenotype, including the first signs of neuroepithelial disruption and neuronal ectopia. In addition, genotype-phenotype analysis of 327 animals confirmed that (i) hyh is a single-gene autosomal recessive disorder, and (ii) the disorder has 100% penetrance (i.e., the mutation was only present in affected mice). The genotyping method described here enhances the potentiality of hyh mouse as a unique in vivo model to study the role of membrane trafficking in different developmental and physiological processes.

Pseudomonas savastanoi pv. savastanoi contains two iaaL paralogs, one of which exhibits a variable number of a trinucleotide (TAC) tandem repeat.

In this study, Pseudomonas savastanoi pv. savastanoi isolates were demonstrated to contain two iaaL paralogs, which are both chromosomally located in most strains. Comparative analysis of iaaL nucleotide sequences amplified from these two paralogs revealed that one paralog, iaaL(Psn), is 100% identical to iaaL from P. savastanoi pv. nerii, while the other paralog, iaaL(Psv), exhibited 93% identity to iaaL from Pseudomonas syringae pv. tomato (iaaL(Pto)). A 3-nucleotide motif (TAC) comprised of 3 to 15 repeats, which remained stable after propagation of the strains in olive plants, was found in iaaL(Psv). Based on the observed nucleotide sequence variations, a restriction fragment length polymorphism assay was developed that allowed differentiation among iaaL(Psn), iaaL(Psv), and iaaL(Pto)(.) In addition, reverse transcriptase PCR on total RNA from P. savastanoi pv. savastanoi strains demonstrated that both iaaL(Psv) and iaaL(Psn) containing 14 or fewer TAC repeats are transcribed. Capillary electrophoresis analysis of PCR-amplified DNA fragments containing the TAC repeats from iaaL(Psv) allowed the differentiation of P. savastanoi pv. savastanoi isolates.