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Xenorhabdus spp. are symbiotic bacteria associated with entomopathogenic nematodes to form a model complex that is used for the biological control of insect pests. These bacteria also produce secondary metabolites that have commercial potential in the pharmaceutical and agroforestry industries. Volatile organic compounds (VOCs) produced by the Xenorhabdus indica “strain AB” have been shown to have significant antifungal activity against Fusarium oxysporum f. sp. cucumerinum. Using gas chromatography-mass spectrometry, we identified 61 volatiles in the mixture of VOCs emitted by strain AB compared to a control strain, 6 of which were investigated for their antifungal activities. Of these, methyl anthranilate exhibited the highest mycelial growth suppression toward F. oxysporum, with a minimum inhibitory volume (MIV) of 50 μL/plate. Fluorescence assays, scanning electron microscopy, and measurements of the leakage of intracellular components revealed that the use of methyl anthranilate changed cell wall and cell membrane integrity as well as the permeability of the plasma membrane. Furthermore, methyl anthranilate treatment upregulated the transcription level of target genes related to redox reactions and the cell wall integrity pathway. The results suggest a novel mechanism used by Xenorhabdus spp. to overcome competitors during its life cycle and open up a new approach to using these bacteria in biological control. IMPORTANCE Fungal phytopathogens, particularly Fusarium oxysporum, are a major problem worldwide, especially in the postharvest of vital economic crops. Concerns about negative effects on the environment and human health have led to increasing restrictions on the use of chemical fungicides, and therefore, biological control agents are now being considered alternatives. It is in this context that we investigated the antifungal activity of VOCs produced by X. indica strain AB against F. oxysporum. We found that AB VOCs have a strong effect on the growth of the fungal phytopathogen. In addition, 85% of the identified volatile compounds were determined to be new compounds, opening up new lines of research to discover their properties, effects, and potential for pharmaceutical and agricultural applications. Antifungal assays proved that four of the six compounds with a high concentration in the GC-MS profile had a significant inhibitory effect on pathogen growth. Accordingly, this study opens up a new approach for the use of these bacteria in biocontrol.

High-throughput sequencing (HTS) is a powerful tool that enables the simultaneous detection and potential identification of any organisms present in a sample. The growing interest in the application of HTS technologies for routine diagnostics in plant health laboratories is triggering the development of guidelines on how to prepare laboratories for performing HTS testing. This paper describes general and technical recommendations to guide laboratories through the complex process of preparing a laboratory for HTS tests within existing quality assurance systems. From nucleic acid extractions to data analysis and interpretation, all of the steps are covered to ensure reliable and reproducible results. These guidelines are relevant for the detection and identification of any plant pest (e.g. arthropods, bacteria, fungi, nematodes, invasive plants or weeds, protozoa, viroids, viruses), and from any type of matrix (e.g. pure microbial culture, plant tissue, soil, water), regardless of the HTS technology (e.g. amplicon sequencing, shotgun sequencing) and of the application (e.g. surveillance programme, phytosanitary certification, quarantine, import control). These guidelines are written in general terms to facilitate the adoption of HTS technologies in plant pest routine diagnostics and enable broader application in all plant health fields, including research. A glossary of relevant terms is provided among the Supplementary Material., This article is based upon work from the work package 2 of the project VALITEST (https://www.valitest.eu/), supported by the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 773139.

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.

A new virus was identified in a celery plant showing chlorotic rings, mosaic and strong yellowing symptoms, and its complete genome sequence was determined. The genomic organization of this novel virus is analogous to that of known members of the genus Torradovirus, consisting of two single-stranded RNAs of 6,823 (RNA1) and 4,263 nucleotides (RNA2), excluding the poly(A) tails. BLAST searches against the nucleotide and protein databases showed that this virus is closely related to but different from carrot torradovirus 1 (CaTV1). Comparisons between the two viruses demonstrated relatively low levels of nucleotide and amino acid similarity in different parts of their genomes, as well as considerable differences in the sizes of their two genomic RNAs. However, the protease-polymerase (Pro-Pol) and capsid protein (CP) regions of this virus share >80% amino acid identity with the corresponding regions of CaTV1. Therefore, based on the current ICTV species demarcation criteria for the family Secoviridae, the virus from celery is a divergent strain of CaTV1, named “CaTV1-celery”. Nevertheless, differences between CaTV1 and CaTV1-celery in genome size, as well as in biological and epidemiological features, may warrant their separation into two distinct species in the future.

A novel nepovirus was identified and characterised from caraway, and tentatively named caraway yellows virus (CawYV). Tubular structures with isomeric virus particles typical for nepoviruses were observed in infected tissues by electron microscopy. The whole genome of CawYV was identified by high throughput sequencing (HTS). It consists of two segments with 8026 nt for RNA1 and 6405 nt for RNA2, excluding the poly(A) tails. CawYV-RNA1 shared closest nt identity to peach rosette mosaic virus (PRMV) with 63%, while RNA2 shared 41.5% with blueberry latent spherical virus (BLSV). The amino acid sequences of the CawYV protease-polymerase (Pro-Pol) and capsid protein (CP) regions share the highest identities with those of the subgroup C nepoviruses. The Pro-Pol region shared highest aa identity with PRMV (80.1%), while the CP region shared 39.6% to soybean latent spherical virus. Phylogenetic analysis of the CawYV-Pro-Pol and -CP aa sequences provided additional evidence of their association with nepoviruses subgroup C. Based on particle morphology, genomic organization and phylogenetic analyses, we propose CawYV as a novel species within the genus Nepovirus subgroup C. Electronic supplementary material The online version of this article (10.1186/s12985-019-1181-1) contains supplementary material, which is available to authorized users.

Background Physostegia chlorotic mottle virus (PhCMoV; genus: Alphanucleorhabdovirus, family: Rhabdoviridae) and tomato brown rugose fruit virus (ToBRFV; genus: Tobamovirus, family: Virgaviridae) are newly emerging plant viruses that have a dramatic effect on tomato production. Among various known virus-control strategies, RNAi-mediated defence has shown the potential to protect plants against various pathogens including viral infections. Micro(mi)RNAs play a major role in RNAi-mediated defence. Methods Using in silico analyses, we investigated the possibility of tomato-encoded miRNAs (TomiRNA) to target PhCMoV and ToBRFV genomes using five different algorithms, i.e., miRanda, RNAhybrid, RNA22, Tapirhybrid and psRNATarget. Results The results revealed that 14 loci on PhCMoV and 10 loci on ToBRFV can be targeted by the TomiRNAs based on the prediction of at least three algorithms. Interestingly, one TomiRNA, miR6026, can target open reading frames from both viruses, i.e., the phosphoprotein encoding gene of PhCMoV, and the two replicase components of ToBRFV. There are currently no commercially available PhCMoV- or ToBRFV-resistant tomato varieties, therefore the predicted data provide useful information for the development of PhCMoV- and ToBFRV-resistant tomato plants.

Peas are an important legume for human and animal consumption and are also being used as green manure or intermediate crops to sustain and improve soil condition. Pea production faces constraints from fungal, bacterial, and viral diseases. We investigated the virome of German pea crops over the course of three successive seasons in different regions of pea production to gain an overview of the existing viruses. Pools from 540 plants, randomly selected from symptomatic and asymptomatic peas, and non-crop plants surrounding the pea fields were used for ribosomal RNA-depleted total RNA extraction followed by high-throughput sequencing (HTS) and RT-PCR confirmation. Thirty-five different viruses were detected in addition to nine associated nucleic acids. From these viruses, 25 are classified as either new viruses, novel strains or viruses that have not been reported previously from Germany. Pea enation mosaic virus 1 and 2 were the most prevalent viruses detected in the pea crops, followed by pea necrotic yellow dwarf virus (PNYDV) and turnip yellows virus which was also found also in the surrounding non-legume weeds. Moreover, a new emaravirus was detected in symptomatic peas in one region for two successive seasons. Most of the identified viruses are known to be aphid transmissible. The results revealed a high virodiversity in the German pea fields that poses new challenges to diagnosticians, researchers, risk assessors and policy makers, as the impact of the new findings are currently unknown., Graphical Abstract German pea virome.

In this study, we present the complete genome of a new isolate of soybean dwarf virus (SbDV) (genus Luteovirus, family Luteoviridae) from white clover in Germany. The complete genome of the isolate (JKI ID 23556) consists of 5,858 nucleotides and displays 94.98% nucleotide identity to its most similar SbDV relative (GenBank accession number MN412736).

High-throughput sequencing (HTS) has become increasingly popular as virus diagnostic tool. It has been used to detect and identify plant viruses and viroids in different types of matrices and tissues. A viral sequence enrichment method prior to HTS is required to increase the viral reads in the generated data to ease the bioinformatic analysis of generated sequences. In this study, we compared the sensitivity of three viral enrichment approaches, i.e. double stranded RNA (dsRNA), ribosomal RNA depleted total RNA (ribo-depleted totRNA) and small RNA (sRNA) for plant virus/viroid detection, followed by sequencing on MiSeq and NextSeq Illumina platforms. The three viral enrichment approaches used here enabled the detection of all viruses/viroid used in this study. When the data was normalised, the recovered viral/viroid nucleotides and depths were depending on the viral genome and the enrichment method used. Both dsRNA and ribo-depleted totRNA approaches detected a divergent strain of Wuhan aphid virus 2 that was not expected in this sample. Additionally, Vicia cryptic virus was detected in the data of dsRNA and sRNA approaches only. The results suggest that dsRNA enrichment has the highest potential to detect and identify plant viruses and viroids. The dsRNA approach used here detected all viruses/viroid, consumed less time, was lower in cost, and required less starting material. Therefore, this approach appears to be suitable for diagnostics laboratories.

BGPI : Equipe 2 : Interactions Virus Insecte Plante (VIP); International audience; Vector transmission plays a primary role in the life cycle of viruses, and insects are the most common vectors. An important mode of vector transmission, reported only for plant viruses, is circulative nonpropagative transmission whereby the virus cycles within the body of its insect vector, from gut to salivary glands and saliva, without replicating. This mode of transmission has been extensively studied in the viral families Luteoviridae and Geminiviridae and is also reported for Nanoviridae. The biology of viruses within these three families is different, and whether the viruses have evolved similar molecular/cellular virus-vector interactions is unclear. In particular, nanoviruses have a multipartite genome organization, and how the distinct genome segments encapsidated individually transit through the insect body is unknown. Here, using a combination of fluorescent in situ hybridization and immunofluorescence, we monitor distinct proteins and genome segments of the nanovirus Faba bean necrotic stunt virus (FBNSV) during transcytosis through the gut and salivary gland cells of its aphid vector Acyrthosiphon pisum. FBNSV specifically transits through cells of the anterior midgut and principal salivary gland cells, a route similar to that of geminiviruses but distinct from that of luteoviruses. Our results further demonstrate that a large number of virus particles enter every single susceptible cell so that distinct genome segments always remain together. Finally, we confirm that the success of nanovirus-vector interaction depends on a nonstructural helper component, the viral protein nuclear shuttle protein (NSP), which is shown to be mandatory for viral accumulation within gut cells.IMPORTANCE An intriguing mode of vector transmission described only for plant viruses is circulative nonpropagative transmission, whereby the virus passes through the gut and salivary glands of the insect vector without replicating. Three plant virus families are transmitted this way, but details of the molecular/cellular mechanisms of the virus-vector interaction are missing. This is striking for nanoviruses that are believed to interact with aphid vectors in ways similar to those of luteoviruses or geminiviruses but for which empirical evidence is scarce. We here confirm that nanoviruses follow a within-vector route similar to that of geminiviruses but distinct from that of luteoviruses. We show that they produce a nonstructural protein mandatory for viral entry into gut cells, a unique phenomenon for this mode of transmission. Finally, noting that nanoviruses are multipartite viruses, we demonstrate that a large number of viral particles penetrate susceptible cells of the vector, allowing distinct genome segments to remain together.

A novel virus with a quadruple genome of negative-sense, single-stranded (ss) RNA was identified by high-throughput sequencing (HTS) in a grass sample from Saxony-Anhalt, Germany, and tentatively called Festuca stripe-associated virus (FSaV). The genome of FSaV consists of four segments and a total of 16,535 nucleotides (nt) which encode seven open reading frames. FSaV shares highest nt identity (between 72.84% to 80.74%) to Iranian wheat stripe virus (IWSV) and rice hoja blanca virus (RHBV). Additionally, pairwise comparisons between the amino acid sequences of the open reading frames on the genome of FSaV and the corresponding ones on the genomes of the members of the Tenuvirus genus showed that FSaV shared 83.17% and 90.85% aa identity to IWSV. Moreover, the non-coding intergenic regions (ncIR) shared only between 49.5% to 60.87% nt identity to the corresponding regions on the IWSV genome. Based on the ICTV species demarcation, the results suggest that FSaV may represent a new species of the genus Tenuivirus. Plastid sequence analysis of the HTS data showed that the original host is a member of the genus Festuca most likely the species Festuca pratensis.

The genus Nanovirus consists of plant viruses that predominantly infect legumes leading to devastating crop losses. Nanoviruses are transmitted by various aphid species. The transmission occurs in a circulative nonpropagative manner. It was long suspected that a virus-encoded helper factor would be needed for successful transmission by aphids. Recently, a helper factor was identified as the nanovirus-encoded nuclear shuttle protein (NSP). The mode of action of NSP is currently unknown in contrast to helper factors from other plant viruses that, for example, facilitate binding of virus particles to receptors within the aphids' stylets. In this review, we are summarizing the current knowledge about nanovirus-aphid vector interactions.

Two divergent isolates of turnip yellows virus (TuYV) were identified in pea and rapeseed. The nearly complete genome sequences of the virus isolates share 93.3% nucleotide identity with each other and 89.7% and 92.9% with their closest isolate from South Africa. Additionally, a turnip yellows virus-associated RNA was identified.

A tenuivirus, referred to here as JKI 29327, was isolated from a black medic (Medicago lupulina) plant collected in Austria. The virus was mechanically transmitted to Nicotiana benthamiana, M. lupulina, M. sativa, Pisum sativum and Vicia faba. The complete genome was determined by high throughput sequencing. The genome of JKI 29327 consists of eight RNA segments closely related to those of melon chlorotic spot virus (MeCSV) isolate E11–018 from France. Since segments RNA 7 and 8 of JKI 29327 are shorter, its genome is slightly smaller (by 247 nts) than that of E11–018. Pairwise comparisons between the predicted virus proteins of JKI 29327 and their homologues in E11–018 showed aa identities ranging from 80.6 to 97.2%. Plants infected with E11–081 gave intermediate DAS-ELISA reactions with polyclonal antibodies to JKI 29327. Since JKI 29327 and E11–018 appear to be closely related both serologically and genetically, we propose to regard JKI 29327 as the black medic strain of MeCSV. To our knowledge, JKI 29327 represents the second tenuivirus identified from a dicotyledonous plant. Serological and molecular diagnostic methods were developed for future detection.

During 2019/20, a batch of 6,000 young plants of different Hoya spp. (family: Asclepiadaceae) was grown in a German nursery. The plants had been originally imported from Thailand and a large proportion developed symptoms consistent with …

Southern tomato virus (STV) is a member of the genus Amalgavirus (family: Amalgaviridae). It has been identified in tomatoes (Solanum lycopersicum) in several countries in Asia, Europe, and North and South America (Sabanadzovic et al.,…

Beetroot (Beta vulgaris subsp. vulgaris) is becoming increasingly popular in Germany with an increase in field production from 1,205 ha in 2013 to 1,826 ha in 2018 (Behr, 2019). It is estimated that EU-wide 24,000 ha of beetroot were…

In May 2018, three samples of wild garlic mustard (Alliaria petiolata, Brassicaceae) were collected from a private garden in Koenigslutter, Germany. While sample EPV_18_002 was asymptomatic apart from slight yellowing, samples EPV_18_003…

Pea necrotic yellow dwarf virus (PNYDV), a member of the Nanovirus genus, has been reported from numerous European countries causing yield loss in peas, faba beans, vetches and lentils (Grigoras et al., 2010; Grigoras et al., 2014; Gaafar…

In September 2015, a tomato sample collected in the German state of Hesse was sent to the Julius Kuhn-Institut for analysis. While the fruits showed marbling and discoloration, the leaf samples from this plant did not show any obvious symptoms. Transmission electron microscopy (TEM) revealed the presence of bullet-shaped virus particles indicating the presence of a rhabdovirus. However, immunosorbent electron microscopy using antiserum JKI-1073 for Eggplant mottled dwarf virus (EMDV) could not confirm EMDV infection. The virus was mechanically transmitted to Nicotiana benthamiana, N. clevelandii, and Chenopodium quinoa inducing yellowing and leaf deformation, while mechanical transmission to N. occidentalis (P1 and 37b) failed. Extraction of double stranded-RNA (dsRNA) followed by random-PCR (Froussard 1992), cloning of PCR products, and sequencing failed to reveal any virus sequences. Total RNA was extracted from infected N. benthamiana, followed by ribo-depletion, library preparation and submission for next-generation sequencing (NGS) using an Illumina MiSeq platform as described by Knierim et al. (2017). De novo assembly of the trimmed reads was done with Geneious v 10.1.3 (Biomatters LTD, NZ). Using MEGA BLAST, 13 contigs showed between 95.6 and 98.5% similarity with physostegia chlorotic mottle virus (PhCMoV) isolate PV-1182 (accession no. KX636164). The complete PhCMoV genome (13,321 nt length) was assembled by mapping reads to this reference genome and used to design PhCMoV-specific RT-PCR primers for detection (HZ-343 5′-CGGTGAGTGGGGCAACTAAT-3′/HZ-344 5′-AGCGATGGGGTCTAGTGTCT-3′). RT-PCR confirmed the presence of PhCMoV in the test plants resulting in amplicons of approximately 875 bp. In August 2016, similar symptoms on tomato fruits were observed by a different grower in Hesse. The presence of PhCMoV was confirmed by TEM and RT-PCR. Additionally, the PCR products were sequenced and showed 97% identity to KX636164. Surprisingly, reanalysis of a tomato sample from 2003 that was infected by a hitherto unknown rhabdovirus using NGS also confirmed infection with PhCMoV. This sample also originated from Hesse although the original grower is unknown. The complete genome of the 2003 PhCMoV sample was assembled following the same methods described above. Pairwise comparison between the genomes of 2015 and 2003 isolates resulted in 99.7% nucleotide identity and 96.9% when compared with KX636164. These findings indicate the presence of PhCMoV in tomato in Germany for a long time albeit isolated occurrences in different production areas. PhCMoV was recently identified from Physostegia virginiana plants showing leaf deformation and severe chlorotic and mottle symptoms in Austria (Menzel et al. 2016). However, it is not known if there is a link between PhCMoV isolates infecting P. virginiana and tomato as the routes of transmission and dissemination are currently unknown. The sequences from this report were deposited in GenBank (accession nos. KY706238 and KY859866 [full-length sequences], KY882263 and KY882264 [partial sequences]). To our knowledge, this is the first host record of PhCMoV in tomato and a new country record for Germany.

Pea necrotic yellow dwarf virus (PNYDV) is a nanovirus that was first detected in pea crops ( Pisum sativum ) in Saxony-Anhalt, Germany in 2009 (Grigoras et al ., 2010). In 2016, PNYDV was detected countrywide in both Germany and Austria not…

Pea necrotic yellow dwarf virus (PNYDV) was identified in green peas ( Pisum sativum ) in Germany in 2009 (Grigoras et al ., 2010). In subsequent years, sampling of symptomatic green peas showed that PNYDV was restricted to Saxony and…

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 .