Xylella fastidiosa is a Gram-negative bacterium native to the Americas and classified as a priority pest under EU regulations. This xylem-limited plant pathogenic bacterium has a wide host range and is transmitted by insect vectors. Since 2013, X. fastidiosa has been identified in several European countries including Italy, France, Spain and Portugal, with different subspecies and sequence types (ST) detected. Since 2015, most strains identified in France are of the subspecies multiplex, specifically ST6 and ST7. Two new STs of X. fastidiosa subsp. multiplex, ST88 and ST89, were recently detected in the region Provence-Alpes-Côte d’Azur (PACA), and one strain of each ST has been isolated from infected plants. To investigate the phylogenetic relationships between the four STs present in France, a complete circular genome and a single-contig genome were assembled for the ST89 and ST88 strains, respectively, by combining PacBio and Illumina sequencing data. A phylogenomic analysis was performed to investigate the phylogenetic position and potential origin of these new strains. This data article contributes to improve our knowledge of the diversity and origin of X. fastidiosa subsp. multiplex in France and Europe.

There is limited information on the compared performances of biological, serological. and molecular assays with high-throughput sequencing (HTS) for viral indexing in temperate fruit crops. Here, using a range of samples of predetermined virological status, we compared two performance criteria (inclusivity and analytical sensitivity) of enzyme-linked immunosorbent assay (ELISA), molecular hybridization, reverse transcription (RT)-PCR, and double-stranded RNA (dsRNA) HTS for the detection of a total of 14 viruses (10 genera) and four viroids (three genera). When undiluted samples from individual plants were used, ELISA had the lowest performance, with an overall detection rate of 68.7%, followed by RT-PCR (82.5%) and HTS (90.7%; 100% if considering only viruses). The lower performance of RT-PCR reflected the inability to amplify some isolates as a consequence of point mutations affecting primer-binding sites. In addition, HTS identified viruses that had not been identified by other assays in nearly two-thirds of the samples. Analysis of serial dilutions of fruit tree samples allowed comparison of analytical sensitivities for various viruses. ELISA showed the lowest analytical sensitivity, but RT-PCR showed higher analytical sensitivity than HTS for most of the samples. Overall, these results confirm the superiority of HTS over biological indexing in terms of speed and inclusivity and show that while the absolute analytical sensitivity of RT-PCR tends to be higher than that of HTS, PCR inclusivity is affected by viral genetic diversity. Taken together, these results make a strong case for the implementation of HTS-based approaches in fruit tree viral testing protocols supporting quarantine and certification programs.

High-throughput sequencing of two lettuces showing virus-like symptoms in France provided evidence of infection by members of the family Secoviridae. One plant (JG1) had a complex mixed infection that involved, among others, a novel waikavirus (lettuce waikavirus 1) and two isolates of a sequivirus related to lettuce mottle virus (LeMoV). The second lettuce plant (JG2) was singly infected by LeMoV. Complete genomic sequences were obtained for all four isolates and, in addition, near complete genome sequences were obtained for other LeMoV or LeMoV-related isolates (from French cultivated and wild lettuces and from a Brazilian cultivated lettuce) and for two isolates of another family Asteraceae-infecting sequivirus, dandelion yellow mosaic virus (DaYMV). Analysis of these genomic sequences allows the proposal of tentative genome organization for the various viruses and clarification of their phylogenetic relationships. Sequence and host range comparisons point to significant differences between the two sequivirus isolates identified in the JG1 plant and LeMoV isolates from France and Brazil, suggesting they belong to a novel species for which the name lettuce star mosaic virus is proposed.

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, fungi and fungus-like protists, 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.

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.

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.

Viruses constitute important problems for cucurbit production. In France, aphid-transmitted viruses remain the most important (Desbiez et al., 2020), but whitefly-transmitted viruses, particularly begomoviruses, now emerging in the Mediterranean Basin (Lecoq & Desbiez, 2012) constitute major threats. A few plants showing leaf crumpling, yellowing and downward curling were observed in a courgette (Cucurbita pepo) plot in Bouches-du-Rhône department (southeastern France) in September 2020. The symptoms were similar to those of Tomato leaf curl New Delhi virus (ToLCNDV), an emerging bipartite begomovirus affecting cucurbits in the Mediterranean Basin (Fortes et al., 2016). After DNA extraction, PCR was performed on a symptomatic sample (CD20001) with specific primers TLCNDV-CP-5 and TLCNDV-CP-3, and ToLCNDV-B-900-5 and ToLCNDV-B-1940-3 (Romay et al., 2019) targeting ToLCNDV DNA-A and DNA-B respectively. The amplified fragments (760 bp and 1072 bp) displayed 99.1% and 99.6% identity respectively to ToLCNDV isolate MU.12.ZU/1/2 (MH577761) from Spain. To complete the sequence of DNA-A, PCR amplification was performed with primers TLCNDV-fCP-F (5′-GTGACGGGAGGAACRTATGC-3′) and TLCNDV-dCP-R (5′-CTAACACACATGACTTTGCC-3′) before sequencing. For DNA-B, rolling circle amplification (RCA) was performed on the extracted DNA with the TempliPhi kit (GE Healthcare). A 2.7 Kb fragment was obtained after digestion with BamHI and cloned in linearised pBlueScript(KS)+ before sequencing. Two clones were sequenced and were identical. The complete sequences of DNA-A and DNA-B (GenBank Accession Nos. MW310624 and MW310625. respectively) were 98–99% identical to isolates from the “Mediterranean” ToLCNDV clade (Fortes et al., 2016). Five other symptomatic courgette plants were collected in the same plot and 12 plants in two other plots in Bouches-du-Rhône, at a distance of 10 km (8 plants) and 6 km (4 plants) respectively from the first plot (Figure 1). Four courgette samples with similar symptoms were also collected in Gard department, 30 km away from the first plot. The samples were tested by PCR with primers Beg-CP-F and Beg-580-R (Saison et al., 2015). A 580 bp product was obtained for 17 of the 21 tested samples (3/5 in the first plot, 8/8 and 4/4 respectively in the two other plots in Bouches-du-Rhône and 2/4 in Gard) and determined as ToLCNDV by sequencing, confirming the presence of the virus in all four areas. The sequences displayed 99.3-100% identity with isolate CD20001. This is the first report of ToLCNDV in France. Since ToLCNDV is highly damaging in cucurbits and is a quarantine pest in Europe, it is important to prevent its emergence in France, particularly in the southeast which constitutes France's most important cucurbit production area. The presence of ToLCNDV in four locations within a 30 km range suggests that it has begun to spread efficiently or that multiple introductions have occurred. It is now important to prevent virus overwintering in weeds (Juárez et al., 2019) and be careful to detect and eradicate potential infections in the next growing season.

Understanding how viruses and subviral agents initiate disease is central to plant pathology. Whether RNA silencing mediates the primary lesion triggered by viroids (small non-protein-coding RNAs), or just intermediate-late steps of a signaling cascade, remains unsolved. While most variants of the plastid-replicating peach latent mosaic viroid (PLMVd) are asymptomatic, some incite peach mosaics or albinism (peach calico, PC). We have previously shown that two 21-nt small RNAs (PLMVd-sRNAs) containing a 12-13-nt PC-associated insertion guide cleavage, via RNA silencing, of the mRNA encoding a heat-shock protein involved in chloroplast biogenesis. To gain evidence supporting that such event is the initial lesion, and more specifically, that different chloroses have different primary causes, here we focused on a PLMVd-induced peach yellow mosaic (PYM) expressed in leaf sectors interspersed with others green. First, sequencing PLMVd-cDNAs from both sectors and bioassays mapped the PYM determinant at one nucleotide, a notion further sustained by the phenotype incited by other natural and artificial PLMVd variants. And second, sRNA deep-sequencing and RNA ligase-mediated RACE identified one PLMVd-sRNA with the PYM-associated change that guides cleavage, as predicted by RNA silencing, of the mRNA encoding a thylakoid translocase subunit required for chloroplast development. RT-qPCR showed lower accumulation of this mRNA in PYM-expressing tissues. Remarkably, PLMVd-sRNAs triggering PYM and PC have 5MODIFIER LETTER PRIME-terminal Us, involving Argonaute 1 in what likely are the initial alterations eliciting distinct chloroses.

‘ Candidatus Liberibacter solanacearum’ (Lso) has emerged as a serious threat on solanaceous and apiaceous crops worldwide. Five Lso haplotypes (LsoA, LsoB, LsoC, LsoD and LsoE) have been identified so far. To decipher genetic relationships between Lso strains, a MLSA study of seven housekeeping genes ( acnA, atpD, ftsZ, glnA, glyA, gnd and groEL ) was performed on a representative bacterial collection of 49 Lso strains. In all, 5415 bp spanning the seven loci were obtained from each of the 49 strains of our bacterial collection. Analysis of sequence data was consistent with a clonal population structure with no evidence of recombination. Phylogenies reconstructed from individual genes, and with concatenated data, were globally congruent with each other. In addition to the five highly supported and distinct genetic clusters, which correspond to the five established haplotypes, our phylogenetic data revealed the presence of a sixth haplotype, designated ‘LsoG’. This new haplotype is currently represented by two strains from France which had distinct sequences in four out of the seven tested housekeeping genes. Altogether, the data presented here provide new information regarding the genetic structure of Lso and the evolutionary history of the haplotypes defined within this bacterial species.

High‐throughput sequencing (HTS) technologies have revolutionized plant pest research and are now raising interest for plant pest diagnostics, with plant virus diagnostics at the forefront of development. However, the application of HTS in plant pest diagnostics raises important challenges that plant health regulators will have to address. Adapted infrastructures, technical guidelines and training are pivotal for further use and adoption of the HTS technologies in the phytosanitary framework.

Begomoviruses (family Geminiviridae) are frequently associated with alphasatellites and betasatellites in the Old World. Tomato yellow leaf curl virus, one of the most damaging begomovirus species worldwide, was recently found associated with betasatellites in the eastern coast of the Mediterranean Sea, and in the Middle East region. Tomato yellow leaf curl virus (TYLCV)/betasatellite associations were shown to increase TYLCV virulence in experimental conditions. The sustainability of TYLCV/satellite associations in tomato was assessed here by estimating accumulation levels of satellites in comparison to TYLCV, vector transmission efficiency, and by testing how far the popular Ty-1 resistance gene used in most TYLCV-resistant tomato cultivars in the Mediterranean Basin is effective against betasatellites. Three satellites previously isolated from okra in Burkina Faso-of the species Cotton leaf curl Gezira betasatellite, Cotton leaf curl Gezira alphasatellite and Okra leaf curl Burkina Faso alphasatellite-were shown to accumulate at levels similar to, or higher than, the helper virus TYLCV-Mld in tomato plants from 32 to 150 days post inoculation (dpi). Cotton leaf curl Gezira betasatellite (CLCuGB) reduced TYLCV-Mld accumulation whereas alphasatellites did not. Transmission tests were performed with B. tabaci from plants infected with TYLCV-Mld/CLCuGB- or TYLCV-Mld/Okra leaf curl Burkina Faso alphasatellite. At 32 dpi, both satellites were transmitted to more than 50% of TYLCV-infected test plants. Betasatellite transmission, tested further with 150 dpi source plants was successful in more than 30% of TYLCV-infected test plants. Ty-1 resistant tomato plants co-infected with TYLCV (-Mld or -IL) and CLCuGB exhibited mild leaf curling and mosaic symptoms at the early stage of infection associated with a positive effect on TYLCV-IL accumulation, while resistant plants infected with TYLCV only, were asymptomatic. Together with previous experimental studies, these results further emphasize the potential risk of betasatellites to tomato cultivation, including with Ty-1 resistant cultivars.

Following the recent detections of 'Candidatus Liberibacter solanacearum' on carrots in Europe and particularly in fields used for seed production in France in 2012, experiments to study the transmission of this pathogen by seeds were conducted. Three batches of carrot seeds harvested in three infected carrot fields in France and one batch of commercial carrot seeds, as negative control, were used. Except for the commercial seed lot, 'Ca. L. solanacearum' was detected in each batch of seeds at a level close to 100%. The first three months, the experiments were conducted in greenhouse. Then, seedlings were placed in overwintering conditions for 4 months. Each month, during 9 months and for each batch, 100 seedlings were analyzed individually by real-time PCR for the presence of the bacterium. At the end of the first three months, no positive result was obtained. After 9 months, no seedling was found positive. These results are not consistent with those obtained in a Spanish laboratory and suggesting that 'Ca. L. solanacearum' should be considered as a seed-borne pathogen. Indeed, the bacterium was detected in seedlings after 3 months. After 5 months, between 15 and 42% of the seedlings from positive seed lots were tested positive. The overwintering conditions applied on the French carrot seedlings may have an impact on the transmissibility of the bacterium. Anyway, these results indicate the need to duplicate tests to conclude on seed transmission of 'Ca. L. solanacearum' by carrot seeds.

‘Candidatus Liberibacter solanacearum’ is a bacterium associated with several vegetative disorders on solanaceous and apiaceous crops. Following the recent detection of the bacterium in carrots in Europe, and particularly carrot plants used for seed production in France, two independent laboratories conducted experiments on the transmission of this pathogen by seed and had discordant results: one study showed no bacterial transmission to plants, and the other showed transmission to carrot seedlings starting from the fourth month of culture. To test the hypothesis that growing conditions affect seed transmission efficiencies, trials were renewed in 2015 on four lots of 500 carrot seeds naturally contaminated with ‘Ca. L. solanacearum’ and two lots of 100 healthy seeds. The plants were grown for 6 months in an insect-proof NS2 greenhouse. Sets of 108 plants from the contaminated lots and 24 plants from the healthy lots were individually analyzed each month using real-time PCR to detect the bacterium. The detection tests on seeds and plants from healthy lots were always negative. During the 6 months of the trial, no plants from the contaminated seed lots tested positive for the bacterium or showed any infection symptoms. These results indicate that transmission of ‘Ca. L. solanacearum’ by carrot seed is rare and difficult to reproduce.