Evidence that an Unnamed Isometric Virus Associated with Potato Rugose Disease in Peru Is a New Species of Genus Torradovirus.

A previously uncharacterized torradovirus species infecting potatoes was detected by high-throughput sequencing from field samples from Peru and in customs intercepts in potato tubers that originated from South America in the United States of America and the Netherlands. This new potato torradovirus showed high nucleotide sequence identity to an unidentified isometric virus (SB26/29), which was associated with a disease named potato rugose stunting in southern Peru characterized over two decades ago. Thus, this virus is tentatively named potato rugose stunting virus (PotRSV). The genome of PotRSV isolates sequenced in this study were composed of two polyadenylated RNA segments. RNA1 ranges from 7,086 to 7,089 nt and RNA2 from 5,228 to 5,230 nt. RNA1 encodes a polyprotein containing the replication block (helicase-protease-polymerase), whereas RNA2 encodes a polyprotein cleaved into a movement protein and the three capsid proteins (CPs). Pairwise comparison among PotRSV isolates revealed amino acid identity values greater than 86% in the protease-polymerase (Pro-Pol) region and greater than 82% for the combined CPs. The closest torradovirus species, squash chlorotic leaf spot virus, shares amino acid identities of ∼58 and ∼41% in the Pro-Pol and the combined CPs, respectively. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

First report of lettuce ring necrosis virus in chili pepper and tomato in Belgium and The Netherlands.

Lettuce ring necrosis virus (LRNV), genus Ophiovirus, was detected by the Netherlands Institute for Vectors, Invasive plants and Plant health (NIVIP) in June and November of 2021 in two samples of chili pepper fruits (Capsicum spp.), both in mixed infection with other viruses. The first sample originated from a production site in Belgium (Sample ID: 40009704) and the second from a production site in the Netherlands (Sample ID: 41115269). One of the fruits of 40009704 showed a light purple circular pattern, while fruits from 41115269 showed colored (ring)spots. The samples were analyzed using Illumina sequencing on a NovaSeq 6000 platform (PE 150) as described previously (Hammond et al., 2021), obtaining 39.9M and 22.8M total reads for 40009704 and 41115269. The corresponding sequence read archives (SRA) were deposited in the NCBI SRA database under BioProject accession number PRJNA917231. From both samples, the nearly complete genome of LRNV (RNA1-4) was obtained and deposited in GenBank (40009704, OQ160823- OQ160826 (7616, 1799, 1502, 1382 nt, mapped reads: 40K, 12K, 114K, 12K , average read coverage (ARC): 0.8K, 0.9K, 11.3K and 1.1K); 41115269, OQ160827- OQ160830 (7616, 1801, 1518, 1389 nt, mapped reads: 112K, 7K, 357K, 55K reads, ARC: 2.2K, 0.6K, 34K and 5.8K)). The shared sequence identities with the Genbank reference sequence of LRNV (NC_006051-NC_006051) were 99.2 and 99.2% (RNA1), 99.1 and 99.1% (RNA2), 98.3 and 98.8% (RNA3), 99.0 and 98.9% (RNA4) for 40009704 and 41115269 respectively. The shared sequence identities between 40009704 and 41115269 were 99.9 (RNA1), 99.0 (RNA2), 99.1 (RNA3) and 99.5% (RNA4). In addition to LRNV, the ophiovirus ranunculus white mottle virus (RWMV) was detected in both samples (OQ160831-OQ160834; OQ160835-OQ160838), while the tobamovirus pepper mild mottle virus (PMMoV) was present in the fruits of 41115269 (OQ160839). Since RWMV has been associated with leaf symptoms in pepper (Gambley et al., 2019; Rivarez et al., 2022) and the colored (ring)spots of 41115269 were very similar to reported symptoms of PMMoV-infected pepper fruits (Martínez-Ochoa et al., 2003), it remains unclear whether LRNV contributed to the observed symptoms. Additionally, LRNV was detected in tomato (Solanum lycopersicum) in Belgium in 2020. In the frame of a metagenomic survey using Virion-Associated Nucleic Acids (VANA)-based protocol (Maclot et al., 2021) on a Nextseq 500 platform (PE 150), partial genome sequences of LRNV were detected in two pools of tomato plants. One pool was made of 44 asymptomatic cultivars from a non-commercial grower (one sample per cultivar) yielding 118K total reads of which 84, 59, 335, and 18 reads mapped on RNA1, 2, 3, and 4, covering 35%, 69%, 100% and 55% of the genome, respectively. The other pool consisted of 15 plants from one cultivar from a production site yielding 3.1M total reads of which 6 and 5 reads mapped on RNA3 and 4, respectively. The detection of LRNV was confirmed for both pooled samples using the real-time RT-PCR method, targeting the CP gene, as described by Maachi et al. (2021). To our knowledge this is the first report of LRNV in pepper anywhere in the world. Additionally, although the disease lettuce ring necrosis in lettuce (Lactuca sativa) has been described in Belgium and the Netherlands before the causal agent was identified (Bos & Huijberts, 1996), this is the first official report of this virus in Belgium and the Netherlands. This publication resulted from pre-publication data sharing of sequences and biological data among plant virologists to provide more context to two independent findings (Hammond et al., 2021).

The large Gunnera's (G. tinctoria and G. manicata) in Europe in relation to EU regulation 1143/2014.

Incorrect labelling of plants in the horticultural trade and misidentification is widespread. For the inspection services of the EU member states, correct identification of G. tinctoria has become important since the species was added to the List of Union concern in accordance with EU regulation 1143/2014 in August 2017. In the horticultural trade Gunnera plants are generally of modest dimensions and rarely flowering, so that the major distinguishing morphological characters for the identification of the two large species, G. tinctoria and G. manicata, are missing. As G. tinctoria is included in the EU regulation, its trade is prohibited, although the closely related species, G. manicata is not included on the list. Given that it is often difficult to distinguish between these two large herbaceous species using morphological attributes we used standard chloroplast DNA barcode markers, supplemented at a later stage by ITS markers. Plant material of putative G. tinctoria or G. manicata was obtained from the native and introduced range, both from "wild" sources, botanical gardens, and the horticultural trade. In western Europe plants circulating in the horticultural trade turned out to be predominantly G. tinctoria, with only one plant in cultivation identified as true G. manicata and the G. manicata found in botanical gardens was a hybrid recently described as G. x cryptica.

African eggplant-associated virus: Characterization of a novel tobamovirus identified from Solanum macrocarpon and assessment of its potential impact on tomato and pepper crops.

A novel tobamovirus was identified in a fruit of Solanum macrocarpon imported into the Netherlands in 2018. This virus was further characterized in terms of host range, pathotype and genomic properties, because many tobamoviruses have the potential to cause severe damage in important crops. In the original fruit, two different genotypes of the novel virus were present. The virus was able to infect multiple plant species from the Solanaceae family after mechanical inoculation, as well as a member of the Apiaceae family. These species included economically important crops such as tomato and pepper, as well as eggplant and petunia. Both tomato and pepper germplasm were shown to harbor resistance against the novel virus. Since most commercial tomato and pepper varieties grown in European greenhouses harbor these relevant resistances, the risk of infection and subsequent impact on these crops is likely to be low in Europe. Assessment of the potential threat to eggplant, petunia, and other susceptible species needs further work. In conclusion, this study provides a first assessment of the potential phytosanitary risks of a newly discovered tobamovirus, which was tentatively named African eggplant-associated virus.

Biological and Genetic Characterization of Physostegia Chlorotic Mottle Virus in Europe Based on Host Range, Location, and Time.

Application of high throughput sequencing (HTS) technologies enabled the first identification of Physostegia chlorotic mottle virus (PhCMoV) in 2018 in Austria. Subsequently, PhCMoV was detected in Germany and Serbia on tomatoes showing severe fruit mottling and ripening anomalies. We report here how prepublication data-sharing resulted in an international collaboration across eight laboratories in five countries, enabling an in-depth characterization of PhCMoV. The independent studies converged toward its recent identification in eight additional European countries and confirmed its presence in samples collected 20 years ago (2002). The natural plant host range was expanded from two to nine species across seven families, and we confirmed the association of PhCMoV presence with severe fruit symptoms on economically important crops such as tomato, eggplant, and cucumber. Mechanical inoculations of selected isolates in the greenhouse established the causality of the symptoms on a new indexing host range. In addition, phylogenetic analysis showed a low genomic variation across the 29 near-complete genome sequences available. Furthermore, a strong selection pressure within a specific ecosystem was suggested by nearly identical sequences recovered from different host plants through time. Overall, this study describes the European distribution of PhCMoV on multiple plant hosts, including economically important crops on which the virus can cause severe fruit symptoms. This work demonstrates how to efficiently improve knowledge on an emergent pathogen by sharing HTS data and provides a solid knowledge foundation for further studies on plant rhabdoviruses.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Interlaboratory Comparison Study on Ribodepleted Total RNA High-Throughput Sequencing for Plant Virus Diagnostics and Bioinformatic Competence.

High-throughput sequencing (HTS) technologies and bioinformatic analyses are of growing interest to be used as a routine diagnostic tool in the field of plant viruses. The reliability of HTS workflows from sample preparation to data analysis and results interpretation for plant virus detection and identification must be evaluated (verified and validated) to approve this tool for diagnostics. Many different extraction methods, library preparation protocols, and sequence and bioinformatic pipelines are available for virus sequence detection. To assess the performance of plant virology diagnostic laboratories in using the HTS of ribosomal RNA depleted total RNA (ribodepleted totRNA) as a diagnostic tool, we carried out an interlaboratory comparison study in which eight participants were required to use the same samples, (RNA) extraction kit, ribosomal RNA depletion kit, and commercial sequencing provider, but also their own bioinformatics pipeline, for analysis. The accuracy of virus detection ranged from 65% to 100%. The false-positive detection rate was very low and was related to the misinterpretation of results as well as to possible cross-contaminations in the lab or sequencing provider. The bioinformatic pipeline used by each laboratory influenced the correct detection of the viruses of this study. The main difficulty was the detection of a novel virus as its sequence was not available in a publicly accessible database at the time. The raw data were reanalysed using Virtool to assess its ability for virus detection. All virus sequences were detected using Virtool in the different pools. This study revealed that the ribodepletion target enrichment for sample preparation is a reliable approach for the detection of plant viruses with different genomes. A significant level of virology expertise is needed to correctly interpret the results. It is also important to improve and complete the reference data.

Pennisetum setaceum or Pennisetum advena cultivars, what ornamental do we have in our garden.

Pennisetum Rich. or following recent taxonomic insights Cenchrus L. is a genus with some 120 species worldwide, especially in warm areas. The genus includes some crops, some ornamentals but mostly species that are considered weedy. The name of one of the weedy species Pennisetum setaceum (Forssk.) Chiov. is also found on labels of ornamental grasses as P. setaceum "Rubrum." It has been debated to belong to a species on its own Pennisetum advena Wipff & Veldkamp or Cenchrus advena (Wipff & Veldkamp) Morrone, only known from cultivation, whereas others still adhere to a broader species concept of P. setaceum. The recent inclusion of P. setaceum on the EU List of Union concern has revitalized the discussion on this issue for commercial reasons. Based on a morphological and molecular comparison (ITS, rbcL, and the trnh-psbA intergenic spacer sequences) of the type specimen of P. advena, five of its "cultivars" in trade and collections of P. setaceum from different regions of the world we conclude that plants currently in trade in Western Europe belong to a separate species P. advena. A drooping inflorescence is consistent as is the difference in width of the leaf blade, the leaf blade being flat or involute, the central vein being swollen or not, and the length of the stipe being 0.3-1.1 mm in P. advena and 1.1-3.1 mm in P. setaceum. On the chloroplast markers rbcL and trnH-psbA, the species consistently differ in 2 and 4 base pairs, respectively. On the nuclear ITS sequence, there is only 90% overlap between the two species. This justifies these ornamentals to be excluded from the List of Union concern of EU regulation 1143/2014.

Competition between Usutu virus and West Nile virus during simultaneous and sequential infection of Culex pipiens mosquitoes.

Usutu virus (USUV) and West Nile virus (WNV) are closely related mosquito-borne flaviviruses that are mainly transmitted between bird hosts by vector mosquitoes. Infections in humans are incidental but can cause severe disease. USUV is endemic in large parts of Europe, while WNV mainly circulates in Southern Europe. In recent years, WNV is also frequently detected in Northern Europe, thereby expanding the area where both viruses co-circulate. However, it remains unclear how USUV may affect the future spread of WNV and the likelihood of human co-infection. Here we investigated whether co-infections with both viruses in cell lines and their primary mosquito vector, Culex pipiens, affect virus replication and transmission dynamics. We show that USUV is outcompeted by WNV in mammalian, avian and mosquito cells during co-infection. Mosquitoes that were exposed to both viruses simultaneously via infectious blood meal displayed significantly reduced USUV transmission compared to mosquitoes that were only exposed to USUV (from 15% to 3%), while the infection and transmission of WNV was unaffected. In contrast, when mosquitoes were pre-infected with USUV via infectious blood meal, WNV transmission was significantly reduced (from 44% to 17%). Injection experiments established the involvement of the midgut in the observed USUV-mediated WNV inhibition. The competition between USUV and WNV during co-infection clearly indicates that the chance of concurrent USUV and WNV transmission via a single mosquito bite is low. The competitive relation between USUV and WNV may impact virus transmission dynamics in the field and affect the epidemiology of WNV in Europe.

Real-time tracking of Tomato brown rugose fruit virus (ToBRFV) outbreaks in the Netherlands using Nextstrain.

Tomato brown rugose fruit virus (ToBRFV) is a Tobamovirus that was first observed in 2014 and 2015 on tomato plants in Israel and Jordan respectively. Since the first description, the virus has been reported from all continents except Oceania and Antarctica, and has been found infecting both tomato and pepper crops. In October 2019, the Dutch National Plant Protection Organization received a ToBRFV infected tomato sample as part of a generic survey targeting tomato pests. Presence of the virus was verified using Illumina sequencing. A follow-up survey was initiated to determine the extent of ToBRFV presence in the Dutch tomato horticulture and identify possible linkages between ToBRFV genotypes, companies and epidemiological traits. Nextstrain was used to visualize these potential connections. By November 2019, 68 companies had been visited of which 17 companies were found to be infected. The 50 ToBRFV genomes from these outbreak locations group in three main clusters, which are hypothesized to represent three original sources. No correlation was found between genotypes, companies and epidemiological traits, and the source(s) of the Dutch ToBRFV outbreak remain unknown. This paper describes a Nextstrain build containing ToBRFV genomes up to and including November 2019. Sharing data with this interactive online tool will enable the plant virology field to better understand and communicate the diversity and spread of this new virus. Organizations are invited to share data or materials for inclusion in the Nextstrain build, which can be accessed at https://nextstrain.nrcnvwa.nl/ToBRFV/20191231.

Development and validation of a real-time RT-PCR test for screening pepper and tomato seed lots for the presence of pospiviroids.

Potato spindle tuber viroid and other pospiviroids can cause serious diseases in potato and tomato crops. Consequently, pospiviroids are regulated in several countries. Since seed transmission is considered as a pathway for the introduction and spread of pospiviroids, some countries demand for the testing of seed lots of solanaceous crops for the presence of pospiviroids. A real-time RT-PCR test, named PospiSense, was developed for testing pepper (Capsicum annuum) and tomato (Solanum lycopersicum) seeds for seven pospiviroid species known to occur naturally in these crops. The test consists of two multiplex reactions running in parallel, PospiSense 1 and PospiSense 2, that target Citrus exocortis viroid (CEVd), Columnea latent viroid (CLVd), pepper chat fruit viroid (PCFVd), potato spindle tuber viroid (PSTVd), tomato apical stunt viroid (TASVd), tomato chlorotic dwarf viroid (TCDVd) and tomato planta macho viroid (TPMVd, including the former Mexican papita viroid). Dahlia latent viroid (DLVd) is used as an internal isolation control. Validation of the test showed that for both pepper and tomato seeds the current requirements of a routine screening test are fulfilled, i.e. the ability to detect one infested seed in a sample of c.1000 seeds for each of these seven pospiviroids. Additionally, the PospiSense test performed well in an inter-laboratory comparison, which included two routine seed-testing laboratories, and as such provides a relatively easy alternative to the currently used tests.

Patterns of inter- and intrasubspecific homologous recombination inform eco-evolutionary dynamics of Xylella fastidiosa.

High rates of homologous recombination (HR) in the bacterial plant pathogen Xylella fastidiosa have been previously detected. This study aimed to determine the extent and explore the ecological significance of HR in the genomes of recombinants experimentally generated by natural transformation and wild-type isolates. Both sets of strains displayed widespread HR and similar average size of recombined fragments consisting of random events (2-10 kb) of inter- and intrasubspecific recombination. A significantly higher proportion and greater lengths (>10 kb, maximum 31.5 kb) of recombined fragments were observed in subsp. morus and in strains isolated in Europe from intercepted coffee plants shipped from the Americas. Such highly recombinant strains pose a serious risk of emergence of novel variants, as genetically distinct and formerly geographically isolated genotypes are brought in close proximity by global trade. Recently recombined regions in wild-type strains included genes involved in regulation and signaling, host colonization, nutrient acquisition, and host evasion, all fundamental traits for X. fastidiosa ecology. Identification of four recombinant loci shared between wild-type and experimentally generated recombinants suggests potential hotspots of recombination in this naturally competent pathogen. These findings provide insights into evolutionary forces possibly affecting the adaptive potential to colonize the host environments of X. fastidiosa.

Virus Detection by High-Throughput Sequencing of Small RNAs: Large-Scale Performance Testing of Sequence Analysis Strategies.

Recent developments in high-throughput sequencing (HTS), also called next-generation sequencing (NGS), technologies and bioinformatics have drastically changed research on viral pathogens and spurred growing interest in the field of virus diagnostics. However, the reliability of HTS-based virus detection protocols must be evaluated before adopting them for diagnostics. Many different bioinformatics algorithms aimed at detecting viruses in HTS data have been reported but little attention has been paid thus far to their sensitivity and reliability for diagnostic purposes. Therefore, we compared the ability of 21 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 12 plant viruses through a double-blind large-scale performance test using 10 datasets of 21- to 24-nucleotide small RNA (sRNA) sequences from three different infected plants. The sensitivity of virus detection ranged between 35 and 100% among participants, with a marked negative effect when sequence depth decreased. The false-positive detection rate was very low and mainly related to the identification of host genome-integrated viral sequences or misinterpretation of the results. Reproducibility was high (91.6%). This work revealed the key influence of bioinformatics strategies for the sensitive detection of viruses in HTS sRNA datasets and, more specifically (i) the difficulty in detecting viral agents when they are novel or their sRNA abundance is low, (ii) the influence of key parameters at both assembly and annotation steps, (iii) the importance of completeness of reference sequence databases, and (iv) the significant level of scientific expertise needed when interpreting pipeline results. Overall, this work underlines key parameters and proposes recommendations for reliable sRNA-based detection of known and unknown viruses.

Euphresco Sendo: An international laboratory comparison study of molecular tests for Synchytrium endobioticum detection and identification.

An international test performance study (TPS) was organised to generate validation data for three molecular Synchytrium endobioticum tests: van den Boogert et al. (European Journal of Plant Pathology 113, 47-57, 2005), and van Gent-Pelzer et al. (European Journal of Plant Pathology, 126, 129-133, 2010) for the detection of S. endobioticum, and the pathotype 1(D1) identification test described by Bonants et al. (European Journal of Plant Pathology, 143, 495-506, 2015). Two TPS rounds were organised focussing on different test matrices, i.e. round 1: warted potato tissue, and round 2: resting spore suspensions. When using the tests for detection and identification of S. endobioticum in warted potato tissue, no significant differences were observed for diagnostic sensitivity, diagnostic specificity, overall accuracy, analytical sensitivity and robustness. When using the tests for detection and identification of S. endobioticum in resting spore suspensions, the van den Boogert and van Gent-Pelzer tests significantly outperform the Bonants test for diagnostic sensitivity and diagnostic specificity. For overall accuracy and analytical sensitivity, the van Gent-Pelzer significantly outperforms the van den Boogert and Bonants tests and is regarded as the test of choice when identifying S. endobioticum from resting spores. Tests regarded fit for purpose for routine testing of wart material and resting spore suspensions are proposed for the update of EPPO standard PM7/28(1) Synchytrium endobioticum.

Assessing the Pathogenic Ability of Ralstonia pseudosolanacearum (Ralstonia solanacearum Phylotype I) from Ornamental Rosa spp. Plants.

Ralstonia pseudosolanacearum (Ralstonia solanacearum phylotype I) isolates found in stunted, yellowing, and wilted ornamental rose (Rosa spp.) were assessed for their pathogenic ability in two rose cultivars (cv. "Armando" and cv. "Red Naomi") and in four solanaceous crops: tomato (Solanum lycopersicum cv. "Money Maker"), tobacco (Nicotiana tabacum cv. "White Burley"), eggplant (Solanum melongena cv. "Black Beauty") and sweet pepper (Capsicum annum cv. "Yolo Wonder"). Significant differences were observed in susceptibility between the two rose cultivars as well as between the two modes of inoculation performed. The cultivar "Armando" was significantly more susceptible than cultivar "Red Naomi," exhibiting higher disease severity and incidence. Similarly, stem inoculation after wounding was found to be significantly more effective than soil drenching, resulting in higher disease severity. Additionally, a temperature dependency in susceptibility was observed for both cultivars irrespective of the mode of inoculation, however, this was significantly more pronounced upon soil drenching. The solanaceous crops all showed to be susceptible to the R. pseudosolanacearum isolates originated from the Rosa spp. plants. Furthermore, both rose cultivars were able to harbor symptomless infections with other R. pseudosolanacearum and R. solanacearum isolates than those isolated from rose. Our results clearly demonstrated that latent infections in a rose cultivar such as cv. "Red Naomi" do occur even at temperatures as low as 20°C. This latency poses high risks for the entire floricultural industry as latently infected Rosa spp. plants are propagated and distributed over various continents, including areas where climatic conditions are optimal for the pathogen.

C. elegans flavin-containing monooxygenase-4 is essential for osmoregulation in hypotonic stress.

Studies in Caenorhabditis elegans have revealed osmoregulatory systems engaged when worms experience hypertonic conditions, but less is known about measures employed when faced with hypotonic stress. Inactivation of fmo-4, which encodes flavin-containing monooxygenase-4, results in dramatic hypoosmotic hypersensitivity; worms are unable to prevent overwhelming water influx and swell rapidly, finally rupturing due to high internal hydrostatic pressure. fmo-4 is expressed prominently in hypodermis, duct and pore cells but is excluded from the excretory cell. Thus, FMO-4 plays a crucial osmoregulatory role by promoting clearance of excess water that enters during hypotonicity, perhaps by synthesizing an osmolyte that acts to establish an osmotic gradient from excretory cell to duct and pore cells. C. elegans FMO-4 contains a C-terminal extension conserved in all nematode FMO-4s. The coincidently numbered human FMO4 also contains an extended C-terminus with features similar to those of FMO-4. Although these shared sequence characteristics suggest potential orthology, human FMO4 was unable to rescue the fmo-4 osmoregulatory defect. Intriguingly, however, mammalian FMO4 is expressed predominantly in the kidney - an appropriate site if it too is, or once was, involved in osmoregulation.

Winter Activity and Aboveground Hybridization Between the Two Biotypes of the West Nile Virus Vector Culex pipiens.

Culex (Cx.) pipiens mosquitoes are important vectors of West Nile virus (WNV). In Europe, the species Cx. pipiens consists of two biotypes, pipiens and molestus, which are morphologically identical, but differ in behavior. Typical behavior of the molestus biotype is the ability to remain active during winter, whereas the pipiens biotype enters diapause. The current paradigm is that the two biotypes occur sympatrically in southern Europe, but occur in isolated above- and belowground populations in northern Europe. In northern Europe, hybridization between biotypes is considered to be low because of the barrier that exists between typical habitats. Data on the occurrence of the biotypes and hybrids in northern Europe, however, are scarce, because identification to the level of biotype is often not performed. Our objective was to clarify the distribution of the Cx. pipiens biotypes and to determine hybridization rates in The Netherlands. Cx. pipiens mosquitoes were collected using three different approaches. First, traps were deployed randomly throughout The Netherlands during the summers of 2011 and 2012 (active surveillance). Second, using a web-based reporting platform and media campaign, Dutch citizens were asked to send dead mosquitoes to our laboratory during the winter and summer of 2014 (passive surveillance). Third, larvae and adults were collected during the summer of 2014 from aboveground locations in Amsterdam to identify molestus larval habitats. Real-time PCR was used for identification to the level of biotype. We found that biotype molestus and hybrids were feeding indoors during winter and summer in The Netherlands and that hybridization rates ranged between 6% and 15%. Larval habitats of biotype molestus were found to occur aboveground. The high percentage of hybridization has implications for assessing the risk of WNV transmission, because hybrids are thought to have ideal characteristics for bridging WNV between birds and humans.

Phytophthora terminalis sp. nov. and Phytophthora occultans sp. nov., two invasive pathogens of ornamental plants in Europe.

In the past decade several Phytophthora strains were isolated from diseased Pachysandra terminalis plants suffering stem base and root rot, originating from the Netherlands and Belgium. All isolates were homothallic and had a felt-like colony pattern, produced semi-papillate sporangia, globose oogonia and had a maximum growth at ~ 27 C. Several additional Phytophthora strains were isolated from diseased Buxus sempervirens plants, originating from the Netherlands and Belgium, which had sustained stem base and root rot; similar strains also were isolated from Acer palmatum, Choisya ternata and Taxus in the United Kingdom. All isolates were homothallic and had a stellate colony pattern, produced larger semi-papillate sporangia and smaller globose oogonia than the isolates from Pa. terminalis and had a maximum growth temperature of ~ 30 C. Phylogenetic analyses of both species using the internal transcribed spacer region of the nuc rDNA (ITS), mt cytochrome oxidases subunit I gene (CoxI) and nuc translation elongation factor 1-α gene (TEF1α) revealed that all sequences of each species were identical at each locus and unique to that species, forming two distinct clusters in subclade 2a. Sequence analysis of partial ß-tubulin genes showed that both taxa share an identical sequence that is identical to that of Ph. himalsilva, a species originating from Asia, suggesting a common Asian origin. Pathogenicity trials demonstrated disease symptoms on their respective hosts, and re-isolation and re-identification of the inoculated pathogens confirmed Koch's postulates.

A simplified counter-selection recombineering protocol for creating fluorescent protein reporter constructs directly from C. elegans fosmid genomic clones.

BACKGROUND: Recombineering is a genetic engineering tool that enables facile modification of large episomal clones, e.g. BACs, fosmids. We have previously adapted this technology to generate, directly from fosmid-based genomic clones, fusion gene reporter constructs designed to investigate gene expression patterns in C. elegans. In our adaptation a rpsL-tet(A) positive/negative-selection cassette (RT-cassette) is first inserted and then, under negative selection, seamlessly replaced with the desired sequence. We report here on the generation and application of a resource comprising two sets of constructs designed to facilitate this particular recombineering approach. RESULTS: Two complementary sets of constructs were generated. The first contains different fluorescent protein reporter coding sequences and derivatives while the second set of constructs, based in the copy-number inducible vector pCC1Fos, provide a resource designed to simplify RT-cassette-based recombineering. These latter constructs are used in pairs the first member of which provides a template for PCR-amplification of an RT-cassette while the second provides, as an excised restriction fragment, the desired fluorescent protein reporter sequence. As the RT-cassette is flanked by approximately 200 bp from the ends of the reporter sequence the subsequent negative selection replacement step is highly efficient. Furthermore, use of a restriction fragment minimizes artefacts negating the need for final clone sequencing. Utilizing this resource we generated single-, double- and triple-tagged fosmid-based reporters to investigate expression patterns of three C. elegans genes located on a single genomic clone. CONCLUSIONS: We describe the generation and application of a resource designed to facilitate counter-selection recombineering of fosmid-based C. elegans genomic clones. By choosing the appropriate pair of 'insertion' and 'replacement' constructs recombineered products, devoid of artefacts, are generated at high efficiency. Gene expression patterns for three genes located on the same genomic clone were investigated via a set of fosmid-based reporter constructs generated with the modified protocol.

Seamless replacement of Autographa californica multiple nucleopolyhedrovirus gp64 with each of five novel type II alphabaculovirus fusion sequences generates pseudotyped virus that fails to transduce mammalian cells.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV), a member of the type I alphabaculoviruses, is able to transduce and deliver a functional gene to a range of non-host cells, including many mammalian lines and primary cells, a property mediated by the envelope fusion protein GP64. AcMNPV is non-cytopathic and inherently replication deficient in non-host cells. As such, AcMNPV represents a possible new class of gene therapy vector with potential future clinical utility. Whilst not a problem for in vitro gene delivery, the broad tropism displayed for non-host cells is less desirable in a gene therapy vector. The fusion protein F of type II alphabaculoviruses can substitute functionally for GP64, and such pseudotyped viruses display a severely impaired capacity for non-host-cell transduction. Thus, surface decoration of such an F-pseudotyped AcMNPV with cell-binding ligands may restore transduction competence and generate vectors with desirable cell-targeting characteristics. By seamlessly swapping the native gp64 coding sequence with each of five sequences encoding different F proteins, a set of F-pseudotyped AcMNPV was generated. This report details their relative abilities both to functionally replace GP64 in viral growth and to transduce human Saos-2 and HeLa cells. All five supported viable infections in insect cell cultures and one, the Mamestra configurata NPV (MacoNPV) F pseudotype, could be amplified to titres close to those of native AcMNPV. In contrast, none was able to transduce the Saos-2 and HeLa cell lines. The robust support provided by MacoNPV F in virus production makes the corresponding pseudotype a viable scaffold to display surface ligands to direct selective mammalian cell targeting.