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Summary Insect-proof net tunnels can help reduce virus infection of clean virus-tested sweetpotato seed produced by decentralized seed producers. However, optimal management is required to maintain both quality and quantity of seed produced. This study investigated the effect of the ratoon cropping technique on vine production in net tunnels and open fields. Virus-tested planting material of two varieties, Kabode and Mataya, were grown in net tunnels and open fields. Each variety had 80 plants per plot, with 40 following the ratooning technique and 40 a replanting technique. The ratooned crop was harvested six times, comprising the initial harvest and five regrowths. This covered 14 months representing six generations of vine production. The number of vines, number of nodes per vine, and vine length were recorded. The number of plants showing virus symptoms was also recorded. The ratoon cropping technique produced more vines compared with the replanting technique in both net tunnels and open fields. Cv. Kabode produced more vines in open fields compared with net tunnels regardless of cropping technique. On the other hand, cv. Mataya produced relatively equal numbers of vines in net tunnels and open fields. Despite ratooning leading to more vine production compared with replanting, the technique led to higher virus incidences on plants grown in the open. This also varied with variety with the highest virus disease incidences being recorded on cv. Mataya. We recommend the ratoon cropping technique for sweetpotato vine production in net tunnels. Replanting technique should be adopted for vine production in the open fields because it acts as a key control strategy for virus infections even for susceptible varieties.

High-throughput sequencing (HTS) technologies have the potential to become one of the most significant advances in molecular diagnostics. Their use by researchers to detect and characterize plant pathogens and pests has been growing steadily for more than a decade and they are now envisioned as a routine diagnostic test to be deployed by plant pest diagnostics laboratories. Nevertheless, HTS technologies and downstream bioinformatics analysis of the generated datasets represent a complex process including many steps whose reliability must be ensured. The aim of the present guidelines is to provide recommendations for researchers and diagnosticians aiming to reliably use HTS technologies to detect plant pathogens and pests. These guidelines are generic and do not depend on the sequencing technology or platform. They cover all the adoption processes of HTS technologies from test selection to test validation as well as their routine implementation. A special emphasis is given to key elements to be considered: undertaking a risk analysis, designing sample panels for validation, using proper controls, evaluating performance criteria, confirming and interpreting results. These guidelines cover any HTS test used for the detection and identification of any plant pest (viroid, virus, bacteria, phytoplasma, mycetes, nematodes, arthropods, plants) from any type of matrix. Overall, their adoption by diagnosticians and researchers should greatly improve the reliability of pathogens and pest diagnostics and foster the use of HTS technologies in plant health., Scientific publication adapted from the deliverable 2.2 of the Valitest project - www.valitest.eu

Viruses pose a major challenge to sweetpotato production in Tanzania. Use of cleaned-up, virus-tested seed vines distributed through a formal seed system is among the proposed strategies to address this challenge. However, virus-tested seed vines can get infected once in the field and it is not known how they will perform following several seasons of on farm propagation. We assessed the performance of virus-tested seed vines and farmer-sourced seed vines of a susceptible variety, Ejumula, and a relatively tolerant variety, Kabode, over five seasons to understand the trend in root yields, vine yields and virus incidences. The experiments were done in high and low virus pressure areas. The most prevalent viruses were sweet potato chlorotic stunt virus (SPCSV) followed by sweet potato feathery mottle virus (SPFMV) and sweet potato leaf curl virus (SPLCV), respectively. Both farmer-sourced and cleaned-up, virus-tested seed of cv. Ejumula were rapidly infected with SPCSV. The incidence of this virus on Ejumula's farmer-sourced material at the high-virus-pressure area reached 100% by the second season. The incidences for all three viruses remained stable for cv. Kabode across the five seasons. Plants generated from cleaned-up, virus-tested seed had lower incidences for all viruses compared to those from farmer-sourced planting material. Virus-tested seed produced significantly higher root yields for cv. Ejumula in the high-virus-pressure site, with a gradual drop across the seasons. The findings show that regular replenishment of clean, virus-tested seed is more economical in high-virus-pressure areas and for more susceptible varieties like cv. Ejumula. They also indicate that farmers may be reluctant to invest in cleaned-up, virus-tested seed in cases where they have virus-tolerant varieties such as cv. Kabode due to lack of obvious virus effect on yields.

In most developing countries, farmers lack sufficient supply of certified or healthy potato seed tubers. Hence, they often plant their own saved ware potato tubers, a practice that is known to contribute to spread and increase the prevalence of plant viruses. In this study, we proposed options for managing the virus based on the knowledge obtained from surveys of virus prevalence and distribution in potato cultivars grown under such conditions. Potato leaf samples randomly collected from 354 farms in five major potato-growing counties in Kenya were tested for six potato viruses; potato virus Y (PVY), potato leaf roll virus (PLRV), potato virus X (PVX), potato virus M (PVM), potato virus A (PVA) and potato virus S (PVS) through DAS-ELISA. Virus prevalence in the fields was high; 72.9% of the samples were positive for at least one of the six viruses; and 55.9% showed multiple infections. A follow-up survey conducted during three consecutive seasons, in two of the five counties, revealed that virus prevalence fluctuated across seasons. This suggested that updated information on virus prevalence might be of value for designing a virus control strategy. Distribution maps showed the presence of the viruses restricted to specific geographic regions, an indication of where control efforts should be directed. Four cultivars, Sherekea, Shangi, Kenya Karibu and Asante, grown at a high virus-prevalent area, showed low values of average ELISA absorbance (OD), suggest a field resistance to the viruses. This study demonstrated that knowledge of prevalence and distribution may be of value to identify and recommend virus resistant cultivars to replace susceptible ones, especially in the virus hotspot areas.

Potato is an important food security crop in Kenya. High prevalence of potato viruses is one of the major causes of low potato yields. Implementation of effective virus management strategies requires information on farmers’ perceptions and practices on virus epidemiology and control. To better understand farmers’ knowledge and practices in virus control and set possible pathways for intervention, a household survey involving 147 respondents was conducted in two major potato-growing regions in Kenya. Lack of sufficient technical information on potato virus symptoms, vectors and modes of transmission was found to be the major hindrance to farmers’ efforts towards virus control. A spider diagram approach was therefore used to determine the aspect of virus control that required the highest prioritization for immediate intervention by policy makers. Use of virus-resistant cultivars was found to require the most urgent attention compared to other aspects of virus control and was expected to have greater impact in lowering virus prevalence. This study showed that enhancing farmer knowledge needs to be prioritised as this is a key determinant of the success of the virus control strategy to be selected for implementation.

Potato virus Y (PVY) causes major yield losses in potato (Solanum tuberosum L.) crops worldwide. Over the past few decades, two new PVY strains (PVYN−Wi/N:O and PVYNTN) have become prevalent and widely distributed worldwide. In Kenya, there is limited information on the prevalence and distribution of PVY strains in potato cultivars grown in the farmer’s fields. This is a major limiting factor in making decisions for setting up a basis for PVY control. This study was therefore conducted (a) to determine the present prevalence and distribution of the different PVY strains in cultivars grown in different potato-growing counties and (b) to use this knowledge on virus prevalence and distribution in proposing a strategy for PVY control. To this end, a leaf sampling survey was conducted in 354 potato farms in five major potato-growing counties in Kenya with follow-up surveys in two of the five counties for two more seasons. Collected leaf samples were tested using DAS-ELISA, and those found to be infected with PVY were subjected to reverse transcription polymerase chain reactions (RT-PCR) to determine the PVY strains. Overall PVY prevalence was generally low (8.2%). Out of all the PVY-infected plants, PVYN−Wi/N:O was the most prevalent strain (54.9%) followed by PVYO (28.0%) and PVYNTN (17.1%), while PVYN was not detected. The PVYN−Wi/N:O strain occurred across the three seasons and across the counties but only on specific cultivars. Three cultivars were identified as PVY resistant, and the most widely grown cultivar Shangi (60.2%) had only a low PVY prevalence of 0.6%. This is an indication that this cultivar may have already been useful in minimizing virus prevalence in farmer’s fields. This report is the first prevalence survey of PVY strains and resistance; in addition, the results obtained provide a basis for a major focus on PVY resistance breeding in Kenya.

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.

In July 2020, plants with crinkled, chlorotic, occasionally necrotic leaves, typical for Soybean Mosaic Virus (SMV), were observed in eight soybean fields (Glycine max L.) in Flevoland, The Netherlands (Supp. Fig. 1). Disease incidence varied from 5-50% and the plants affected often occurred in small or extensive patches. Leaves from several symptomatic plants were sampled from each of two fields planted with soybean variety Green Shell or Summer Shell. Total RNA was extracted from one plant leaf sample per field using InviTrap Spin Plant RNA Mini Kit (Invitek, Germany). One-tube RT-PCRs employing potyvirus generic primers P9502 and CPUP (Van der Vlugt et al, 1999) and SMV-specific primers SMV-dT (5'-TTTTTTTTTTTTTTTAGGACAAC-3') and SMV-Nib-Fw (5'-CAAGGATGARTTTAAGGAG-3') combined with Sanger sequencing confirmed the presence of SMV in all leaf samples. To exclude the presence of other agents in the samples, total RNA from each cultivar was used in standard Illumina library preparation with ribosomal RNA depletion followed by sequencing on an Illumina NovaSeq6000 (paired-end, 150 bp) which yielded 66,579,158 reads (Summer Shell) and 223,953,206 reads (Green Shell). After quality trimming in CLC Genomics Workbench 20.0.4 (CLC-GWB; Qiagen, Hilden), four million reads were randomly sampled for de novo assembly. Contigs over 500 nucleotides (nts) in length with a minimum of 500 reads were annotated by BLASTn against NCBI GenBank. This identified one contig of 9,883 nts (6,233,397 reads) in Summer Shell and one contig of 9,727 nts (3,139,927 reads) in Green Shell with clear homology to SMV (E-value = 0.0). No other viruses were identified in the datasets. Reference assemblies against the SMV reference sequence (NC_002634) mapped 24,090,763 reads (36.2%) for Summer Shell and 175,459,637 reads (78.3%) for Green Shell. Extracted consensus sequences for SMV in both soybean cultivars were 9,584 nts long (excluding the poly-A tail). Sequence data from the de novo and reference assemblies were combined into consensus sequences which showed over 98% overall nt sequence identity to NC_002634 and 99.6% to each other. Both consensus sequences were deposited in GenBank under accession numbers MW822167 (SMV-Summer Shell) and MW822168 (SMV-Green Shell). In addition, the presence of SMV in the field samples was confirmed with an inoculation assay. Leaf samples from both fields were ground in phosphate buffer (0.1M, pH 7.2) and inoculated on cotyledons and first expanded leaves of soybean plants (unknown cv.) 12 days post-germination. Plants showed veinal chlorosis in systemic leaves from 12 days post-inoculation, which developed into veinal necrosis. SMV infections were confirmed by RT-PCR in systemic, chlorotic leaf samples of all symptomatic plants using the SMV-specific primers described above. To our knowledge, this is the first report of SMV in The Netherlands. As soybean is a relatively new but expanding crop in this country, information about emerging diseases is highly relevant. SMV can be transmitted via seeds and aphids, where seeds can serve as primary source of virus inoculum (Cui et al., 2011; Hartman et al., 2016; Hajimorad et al., 2018). Weeds and non-commercial plants can also serve as origin of SMV, particularly in subsequent growing seasons, although this virus infects a limited host range of six plant families (Cui et al., 2011; Hill & Whitham, 2014). Special monitoring would be advised for the recurrence and possible damage by SMV in Dutch soybean fields.

We present a taxonomic proposal for revision of the family Secoviridae, a taxon of plant viruses in the order Picornavirales. We propose the reorganization of the genus Sadwavirus to create three new subgenera and to update the classification of five existing species. The proposed subgenera are “Satsumavirus” (one species: Satsuma dwarf virus), “Stramovirus” (two species: Strawberry mottle virus and Black raspberry necrosis virus) and “Cholivirus” (two species: Chocolate lily virus A and Dioscorea mosaic associated virus).

Over the past decades, two new strains of potato virus Y (PVYN-Wi (Wilga) and PVYNTN) have been the most widely distributed worldwide in potato crops (Solanum tuberosum L.). Effective resistance genes comprehensive against all the known strains of the virus are available in some commercial cultivars. However, not all resistance genes in cultivars have been challenged by the new PVY strains to confirm effectiveness of the resistances available. This study was set to identify PVY resistant genotypes and estimate yield losses in potato cultivars in Kenya. PVYN-Wi was used for PVY resistance screening because it was the most prevalent strain in Kenya and also was found to produce more severe infection in Kenyan potato genotypes compared with PVYO (the second most prevalent strain). Three cultivars, Kenya Karibu, Sherekea and Unica, were identified as resistant because they did not get PVY infections over three seasons of both greenhouse and field screening experiments. In addition, two early generation breeding clones were identified as resistant at 28 days post-inoculation. In susceptible cultivars Dutch Robyjn, Nyayo and Kenya Mpya, PVYN-Wi infection caused yield losses of 21.6, 39.0 and 53.1%, respectively, after three seasons. This study demonstrated the necessity to screen for PVY resistance based on the most prevalent PVY strains and to make an informed decision on the usefulness of current levels of resistance in potato cultivars.

Potato (Solanum tuberosum L.) is an important crop in Uganda but production is low. There is not a well-functioning official seed system and farmers use potato tubers from a previous harvest as seed. This study investigated how effectively the seed technology positive selection enhanced yield and underlying crop characteristics across multiple seasons, compared to the farmers’ selection method. Positive selection is selecting healthy plants during crop growth for harvesting seed potato tubers to be planted in the next season. Farmers’ selection involves selection of seed tubers from the bulk of the ware potato harvest. Positive selection was compared to farmers’ seed selection for up to three seasons in three field trials in different locations in southwestern Uganda using seed lots from different origins. Across all experiments, seasons and seed lots, yields were higher under positive selection than under farmers’ selection. The average yield increase resulting from positive selection was 12%, but yield increases were variable, ranging from − 5.7% to + 36.9%, and in the individual experiments often not significant. These yield increases were due to higher yields per plant, and mostly higher weights per tuber, whereas the numbers of tubers per plant were not significantly different. Experimentation and yield assessment were hampered by a varying number of plants that could not be harvested because plants had to be rogued from the experimental plots because of bacterial wilt (more frequent under farmers’ selection than under positive selection), plants disappeared from the experimental field and sometimes plants did not emerge. Nevertheless, adoption of positive selection should be encouraged due to a higher production and less virus infection of seed tubers in positive selected plants, resulting in a lower degeneration rate of potato seed tubers.

Smallholder farmers in Uganda commonly use seed potato tubers from the informal sector, especially by seed recycling over several generations. Therefore, seed tubers are highly degenerated with viruses and other pathogens, resulting in poor yield and quality of the produce. Over one cycle of multiplication, degeneration management by positive seed selection was found to be efficient in reducing virus diseases compared with the farmers’ method of selection. The objective of this study was to assess to what extent positive selection over several seasons can reduce six different virus incidences in seed lots of different starting quality in southwestern Uganda. Multi-seasonal trials were carried out in three locations, with five seed lots from four sources and three cultivars. Detection of viruses was based on DAS-ELISA and Luminex xMAP technology. Analysis was carried out with analysis of variance (ANOVA) on angular-transformed percentages of virus incidence. Results showed fluctuations in some viruses over seasons with lower Potato leafroll virus (PLRV) and Potato virus X (PVX) incidences in lots from positive selection compared with lots from farmers’ selection. In contrast, some seed lots were initially highly infected with Potato virus S (PVS) and Potato virus M (PVM) and showed no reduction in virus incidence through positive selection. In general, little infection with Potato virus Y (PVY) and Potato virus A (PVA) was found. Based on these results, it is recommended that smallholder farmers are trained in positive selection to opt for less virus-infected plants and tubers, thus increasing potato production.

The number and diversity of viral sequences that are identified in metagenomic data far exceeds that of experimentally characterized virus isolates. In a recent workshop, a panel of experts discussed the proposal that, with appropriate quality control, viruses that are known only from metagenomic data can, and should be, incorporated into the official classification scheme of the International Committee on Taxonomy of Viruses (ICTV). Although a taxonomy that is based on metagenomic sequence data alone represents a substantial departure from the traditional reliance on phenotypic properties, the development of a robust framework for sequence-based virus taxonomy is indispensable for the comprehensive characterization of the global virome. In this Consensus Statement article, we consider the rationale for why metagenomic sequence data should, and how it can, be incorporated into the ICTV taxonomy, and present proposals that have been endorsed by the Executive Committee of the ICTV.

Despite the seemingly continuous development of newer and ever more elaborate methods for detecting and identifying viruses, very few of these new methods get adopted for routine use in testing laboratories, often despite the many and varied claimed advantages they possess. To understand why the rate of uptake of new technologies is so low, requires a strong understanding of what makes a good routine diagnostic tool to begin. This can be done by looking at the two most successfully established plant virus detection methods: enzyme-linked immunosorbant assay (ELISA) and more recently introduced real-time polymerase chain reaction (PCR). By examining the characteristics of this pair of technologies, it becomes clear that they share many benefits, such as an industry standard format and high levels of repeatability and reproducibility. These combine to make methods that are accessible to testing labs, which are easy to establish and robust in their use, even with new and inexperienced users. Hence, to ensure the establishment of new techniques it is necessary to not only provide benefits not found with ELISA or real-time PCR, but also to provide a platform that is easy to establish and use. In plant virus diagnostics, recent developments can be clustered into three core areas: (1) techniques that can be performed in the field or resource poor locations (e.g., loop-mediated isothermal amplification LAMP); (2) multiplex methods that are able to detect many viruses in a single test (e.g., Luminex bead arrays); and (3) methods suited to virus discovery (e.g., next generation sequencing, NGS). Field based methods are not new, with Lateral Flow Devices (LFDs) for the detection being available for a number of years now. However, the widespread uptake of this technology remains poor. LAMP does offer significant advantages over LFDs, in terms of sensitivity and generic application, but still faces challenges in terms of establishment. It is likely that the main barrier to the uptake of field-based technologies is behavioural influences, rather than specific concerns about the performance of the technologies themselves. To overcome this, a new relationship will need to develop between centralised testing laboratories offering services and those requiring tests; a relationship which is currently in its infancy. Looking further into the future, virus discovery and multiplex methods seem to converge as NGS becomes ever cheaper, easier to perform and can provide high levels of multiplexing without the use of virus specific reagents. So ultimately the key challenge from a routine testing lab perspective will not be one of investment in platforms-which could even be outsourced to commercial sequencing services-but one of having the skills and expertise to analyse the large datasets generated and their subsequent interpretation. In conclusion, only time will tell which of the next-generation of methods currently in development will become the routine diagnostics of the future. This will be determined through a combination of factors. And while the technology itself will have to offer performance advantages over existing methods in order to supplant them, it is likely to be human factors e.g., the behaviours of end users, laboratories and policy makers, the availability of appropriate expertise, that ultimately determine which ones become established. Hence factors cannot be ignored and early engagement with diagnostic stakeholders is essential.

Members of the genus Torradovirus (family Secoviridae, type species Tomato torrado virus, ToTV) are spherical plant viruses transmitted by the whitefly species Trialeurodes vaporariorum and Bemisia tabaci. Knowledge on the mode of vector transmission is lacking for torradoviruses. Here, the mode of transmission was determined for Tomato marchitez virus (ToMarV). A minimal acquisition access period (AAP) and inoculation access period (IAP) of approximately 2h each was required for its transmission by T. vaporariorum, while optimal transmission required an AAP and IAP of at least 16h and 8h, respectively. Whiteflies could retain the virus under non-feeding conditions for at least 8h without loss of transmission efficiency, but upon feeding on a non-host plant in between the AAP and IAP they retained the virus for no more than 8h. Similar conditions supported transmission of isolates of ToTV and Tomato chocolàte virus (ToChV) by T. vaporariorum and B. tabaci. Additionally, similar experiments revealed the banded-winged whitefly (Trialeurodes abutilonea) as a vector for all three virus species. The results are congruent with acquisition and retention periods for semi-persistent virus transmission. RT-PCR detection analysis of ToTV and ToMarV in the vector's body revealed their presence in the stylet, but not in the head where the pharynx of the foregut is located. The results altogether indicate a semi-persistent stylet-borne mode of vector transmission for torradoviruses. Additionally, this is the first group of spherical viruses transmitted by at least three different species of whiteflies.

In this manuscript we provide evidence for the seed transmission of Pepino mosaic virus (PepMV) in tomato. Fruit was harvested from a tomato crop artificially infected with both European and CH2 genotypes of PepMV and more than 100,000 seeds were extracted and cleaned using an enzymatic treatment without disinfection. Infection assays using indicator plants confirmed the presence of viable virus on the seeds. Seeds were distributed to ten different laboratories in three separate batches, where they were germinated and the young plants tested by ELISA. In total over 87,000 plants were tested and 23 positives detected, indicating an overall transmission rate of 0.026%. However, the observed seed transmission rates varied from 0.005% to 0.057%, depending on the seed batch used. Results clearly showed that PepMV can be transmitted from seeds contaminated with virus to seedlings, highlighting the risk of using seeds from PepMV-infected plants and the potential for seed transmission to contribute to the further spread of PepMV.

A new virus was isolated from a tomato plant from Guatemala showing necrotic spots on the bases of the leaves and chocolate-brown patches on the fruits. Structural and molecular analysis showed the virus to be clearly related to but distinct from the recently described Tomato torrado virus (ToTV) and Tomato marchitez virus (ToMarV), both members of the genus Torradovirus. The name tomato chocolàte virus is proposed for this new torradovirus. M. Verbeek and A. Dullemans contributed to this research equally. Nucleotide sequence data reported are available in the GenBank database under accession numbers FJ560489 (RNA1) and FJ560490 (RNA2).

Pepino mosaic virus (PepMV) has recently emerged as a highly infectious viral pathogen in tomato crops. Greenhouse trials were conducted under conditions similar to commercial tomato production. These trials examined whether tomato plants can be protected against PepMV by a preceding infection with an attenuated isolate of this virus. Two potential attenuated isolates that displayed mild leaf symptoms were selected from field isolates. Two PepMV isolates that displayed severe leaf symptoms were also selected from field isolates to challenge the attenuated isolates. The isolates with aggressive symptoms were found to reduce bulk yields by 8 and 24% in single infections, respectively. Yield losses were reduced to a 0–3% loss in plants that were treated with either one of the attenuated isolates, while no effects were observed on the quality of the fruits. After the challenge infection, virus accumulation levels and symptom severity of the isolates with aggressive symptoms were also reduced by cross-protection. Infection with the attenuated isolates alone did neither affect bulk yield, nor quality of the harvested tomato fruits.

International audience; The order Picornavirales includes several plant viruses that are currently classified into the families Comoviridae (genera Comovirus, Fabavirus and Nepovirus) and Sequiviridae (genera Sequivirus and Waikavirus) and into the unassigned genera Cheravirus and Sadwavirus. These viruses share properties in common with other picornavirales (particle structure, positive-strand RNA genome with a polyprotein expression strategy, a common replication block including type III helicase, a 3C-like cysteine proteinase and type I RNA-dependent RNA polymerase). However, they also share unique properties that distinguish them from other picornavirales. They infect plants and use specialized proteins or protein domains to move through their host. In phylogenetic analysis based on their replication proteins, these viruses form a separate distinct lineage within the picornavirales branch. To recognize these common properties at the taxonomic level, we propose to create a new family termed “Secoviridae” to include the genera Comovirus, Fabavirus, Nepovirus, Cheravirus, Sadwavirus, Sequivirus and Waikavirus. Two newly discovered plant viruses share common properties with members of the proposed family Secoviridae but have distinct specific genomic organizations. In phylogenetic reconstructions, they form a separate sub-branch within the Secoviridae lineage. We propose to create a new genus termed Torradovirus (type species, Tomato torrado virus) and to assign this genus to the proposed family Secoviridae.

A new virus was isolated from a lettuce plant grown in an open field in the Netherlands in 2011. This plant was showing conspicuous symptoms that consisted of necrosis and moderate leaf curling. The virus was mechanically transferred to indicator plants, and a total RNA extract of one of these indicator plants was used for next-generation sequencing. Analysis of the sequences that were obtained and further biological studies showed that the virus was related to, but clearly distinct from, viruses belonging to the genus Torradovirus. The name “lettuce necrotic leaf curl virus” (LNLCV) is proposed for this new torradovirus.

Efficient and reliable diagnostic tools for the routine indexing and certification of clean propagating material are essential for the management of pospiviroid diseases in horticultural crops. This study describes the development of a true multiplexed diagnostic method for the detection and identification of all nine currently recognized pospiviroid species in one assay using Luminex bead-based suspension array technology. In addition, a new data-driven, statistical method is presented for establishing thresholds for positivity for individual assays within multiplexed arrays. When applied to the multiplexed array data generated in this study, the new method was shown to have better control of false positives and false negative results than two other commonly used approaches for setting thresholds. The 11-plex Luminex MagPlex-TAG pospiviroid array described here has a unique hierarchical assay design, incorporating a near-universal assay in addition to nine species-specific assays, and a co-amplified plant internal control assay for quality assurance purposes. All assays of the multiplexed array were shown to be 100% specific, sensitive and reproducible. The multiplexed array described herein is robust, easy to use, displays unambiguous results and has strong potential for use in routine pospiviroid indexing to improve disease management strategies.

Flexuous viruses were transmitted from rakkyo (Allium chinense) and wild leek species (especiallyA. commutatum) to plants of crow garlic (A. vineale), by transfer of dry bulb mites. By electron microscope decoration tests using three antisera and by inoculations onto test plants, it was concluded that from each of the two natural host species at least two viruses were isolated. The viruses from wild leeks are both pathogenic onAllium spp. and may be of economic importance. Decoration tests on a virus mixture from shallot obtained earlier, revealed another new mite-borne virus in this species. The mite-borne viruses ofAllium spp. appear to be very common; they are largely diverse and their identification remains difficult.

Tuesday July 6, 2010 we were shocked by the news that that day our former colleague Lute Bos had died unexpectedly at the age of 82. Lute was a world-famous plant virologist. From the start of his career in 1957 until his retirement from the Institute for Plant Protection (IPO-DLO) in Wageningen in 1993, he studied plant viruses and in particular those of legumes and vegetable crops

Tobacco plant lines transformed with the coat protein (CP) gene of the tobacco veinal necrosis strain of potato virus Y (PVYN), and previously shown to be protected against mechanical inoculation with the virus, have now been tested for specificity and protection against virus infection mediated by viruliferous aphids. To determine the specificity of virus protection, two transgenic tobacco lines, A30 and A80, were challenged with several isolates of distinct PVY strains (PVYN, PVYO and PVYC) by mechanical inoculation. Clear levels of protection against the PVYO-isolates tested were maintained in the transgenic plants, although these levels were slightly lower than the protection against the homologous PVYN strain from which the CP gene was derived. Interestingly, no protection against mechanical virus inoculation with the ‘Gladblaadje’ isolate of PVYC could be observed. To assess the levels of protection against aphid-mediated virus infection, two transgenic plant lines, A30 and D25, showing respective levels of protection of 95 and 80% against mechanical virus inoculation, were challenged using PVYN viruliferousMyzus persicae. Virus inoculation using six aphids per plant, resulted in similar levels of protection in both transgenic lines as found previously for mechanical inoculation. Protection was maintained in both lines, even when as many as 60 viruliferous aphids were used per plant in the inoculation experiments.

Multiple susceptibility genes (S), identified in Arabidopsis, have been shown to be functionally conserved in crop plants. Mutations in these S genes result in resistance to different pathogens, opening a new way to achieve plant disease resistance. The aim of this study was to investigate the role of Defense No Death1 (DND1) in susceptibility of tomato and potato to late blight (Phytophthora infestans). In Arabidopsis, the dnd1 mutant has broad-spectrum resistance against several fungal, bacterial, and viral pathogens. However this mutation is also associated with a dwarfed phenotype. Using an RNAi approach, we silenced AtDND1 orthologs in potato and tomato. Our results showed that silencing of the DND1 ortholog in both crops resulted in resistance to the pathogenic oomycete P. infestans and to two powdery mildew species, Oidium neolycopersici and Golovinomyces orontii. The resistance to P. infestans in potato was effective to four different isolates although the level of resistance (complete or partial) was dependent on the aggressiveness of the isolate. In tomato, DND1-silenced plants showed a severe dwarf phenotype and autonecrosis, whereas DND1-silenced potato plants were not dwarfed and showed a less pronounced autonecrosis. Our results indicate that S gene function of DND1 is conserved in tomato and potato. We discuss the possibilities of using RNAi silencing or loss-of-function mutations of DND1 orthologs, as well as additional S gene orthologs from Arabidopsis, to breed for resistance to pathogens in crop plants. Electronic supplementary material The online version of this article (doi:10.1007/s11248-015-9921-5) contains supplementary material, which is available to authorized users.

The complete genomic sequence of an isolate (PRI-509) of the C strain of Potato virus Y (PVYC), which was originally isolated from potato in 1938, was elucidated. The genomic RNA of PRI-509 consists of 9699 nucleotides, with the capacity to encode a polyprotein of 3061 amino acids with a molecular mass of 337 kDa. This is the first full-length sequence of a PVY C isolate from potato that belongs to the C1 phylogenetic subgroup, which was previously thought to exclusively contain non-potato isolates. Electronic supplementary material The online version of this article (doi:10.1007/s00705-010-0853-3) contains supplementary material, which is available to authorized users.

Background Phloem-feeding insects are among the most devastating pests worldwide. They not only cause damage by feeding from the phloem, thereby depleting the plant from photo-assimilates, but also by vectoring viruses. Until now, the main way to prevent such problems is the frequent use of insecticides. Applying resistant varieties would be a more environmental friendly and sustainable solution. For this, resistant sources need to be identified first. Up to now there were no methods suitable for high throughput phenotyping of plant germplasm to identify sources of resistance towards phloem-feeding insects. Results In this paper we present a high throughput screening system to identify plants with an increased resistance against aphids. Its versatility is demonstrated using an Arabidopsis thaliana activation tag mutant line collection. This system consists of the green peach aphid Myzus persicae (Sulzer) and the circulative virus Turnip yellows virus (TuYV). In an initial screening, with one plant representing one mutant line, 13 virus-free mutant lines were identified by ELISA. Using seeds produced from these lines, the putative candidates were re-evaluated and characterized, resulting in nine lines with increased resistance towards the aphid. Conclusions This M. persicae-TuYV screening system is an efficient, reliable and quick procedure to identify among thousands of mutated lines those resistant to aphids. In our study, nine mutant lines with increased resistance against the aphid were selected among 5160 mutant lines in just 5 months by one person. The system can be extended to other phloem-feeding insects and circulative viruses to identify insect resistant sources from several collections, including for example genebanks and artificially prepared mutant collections.