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 pre-publication 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 ago (2002). The natural plant host range was expanded from two species 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 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 genomes 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 which the virus can cause severe fruit symptoms for. 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.

Several outbreaks of pospiviroids have been reported in pepper and tomato crops worldwide. Tracing back the origin of the infections has led to different sources. In some cases, the infections were considered to result from seed transmission. Other outbreaks were related to transmission from ornamental crops and weeds. Pospiviroids, in particular potato spindle tuber viroid, are regulated by many countries because they can be harmful to potatoes and tomatoes. Seed transmission has been considered an important pathway of introduction and spread. However, the importance of this pathway can be questioned. This paper presents data on seed transmission from large-scale grow-out trials of infested pepper and tomato seed lots produced under standard seed-industry conditions. In addition, it presents the results of a systematic review of published data on seed transmission and outbreaks in commercial pepper and tomato crops. Based on the results of the grow-out trials and review of the literature, it was concluded that the role of seed transmission in the spread of pospiviroids in practice is possibly overestimated.

A cell-associated fructosyltransferase produced by Streptococcus salivarius was irreversibly inactivated in a time-dependent manner when resting or permeabilized cell suspensions were incubated with low concentrations (less than 1.0 microM) of copper. In addition to copper, the inactivation was dependent on oxygen and on a fermentable carbon source (endogenous intracellular polysaccharide or glucose). In starved, permeabilized cell suspensions, the fermentable carbon source could be replaced by NADH but not by NADPH or ATP. Of several other S. salivarius enzymes tested, only fructosyltransferase was inactivated under these conditions. The available evidence indicated that NADH oxidase is the enzyme responsible for fructosyltransferase inactivation. Results from oxygen radical scavenger studies implicated one or more species of oxygen radicals and hydrogen peroxide in the inactivation reaction.

Tomato mottle mosaic virus (ToMMV) is a tobamovirus found in a Solanum lycopersicum sample collected in Mexico in 2009. To assess the possible presence of ToMMV in Europe, accessions from a historic seed collection were tested by real-time RT-PCR and Illumina sequencing. ToMMV was identified in historical seed accessions produced in France, the Netherlands and Spain. Three different near complete genome sequences were obtained, each corresponding to the country in which the seeds had been produced. Positive samples from France and Spain could be related to the same production location and year, respectively, while the identical genome sequences from the Netherlands were obtained from samples produced in different locations and years between 1981 and 2007. The latter could be due to the fact that the Dutch seed accessions had been repacked in the past at the same location and time as accessions with a relatively high virus load from 2007. This indicates that possibly only the seeds from 2007 originated from ToMMV-infected plants, while the detection of ToMMV in the older seed accessions resulted from cross contamination. This data shows that ToMMV has been around in Europe before its first description and is possibly more widespread than currently known.

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). Bos, L., & Huijberts, N. (1996). Lettuce ring necrosis, caused by a chytrid-borne agent distinct from lettuce big-vein ‘virus’. European Journal of Plant Pathology, 102(9), 867-873. https://doi.org/10.1007/bf01877057 Gambley, C., Persley, D., & Thomas, J. E. (2019). First record of Ranunculus white mottle virus from Australia. New Disease Reports, 40(1), 13-13. https://doi.org/10.5197/j.2044-0588.2019.040.013 Hammond, J., Adams, I. P., Fowkes, A. R., McGreig, S., Botermans, M., Oorspronk, J. J. A., Westenberg, M., Verbeek, M., Dullemans, A. M., Stijger, C. C. M. M., Blouin, A. G., Massart, S., De Jonghe, K., Heyneman, M., Walsh, J. A., & Fox, A. (2021). Sequence analysis of 43‐year old samples of Plantago lanceolata show that Plantain virus X is synonymous with Actinidia virus X and is widely distributed. Plant Pathology, 70, 249-258. https://doi.org/10.1111/ppa.13310 Maachi, A., Torre, C., Sempere, R. N., Hernando, Y., Aranda, M. A., & Donaire, L. (2021). Use of High-Throughput Sequencing and Two RNA Input Methods to Identify Viruses Infecting Tomato Crops. Microorganisms, 9(5). https://doi.org/10.3390/microorganisms9051043 Maclot, F. J., Debue, V., Blouin, A. G., Fontdevila Pareta, N., Tamisier, L., Filloux, D., & Massart, S. (2021). Identification, molecular and biological characterization of two novel secovirids in wild grass species in Belgium. Virus Res, 298, 198397. https://doi.org/10.1016/j.virusres.2021.198397 Martínez-Ochoa, N., Langston, D. B., Mullis, S. W., & Flanders, J. T. (2003). First Report of Pepper mild mottle virus in Jalapeno Pepper in Georgia. Plant Health Progress, 4(1). https://doi.org/10.1094/php-2003-1223-01-hn Rivarez, M. P. S., Kogej, Z., Jakos, N., Pecman, A., Seljak, G., Vucurovic, A., Ravnikar, M., Mehle, N., & Kutnjak, D. (2022). First Report of Ranunculus White Mottle Ophiovirus in Slovenia in Pepper with Yellow Leaf Curling Symptom and in Tomato. Plant Dis, PDIS08211624PDN. https://doi.org/10.1094/PDIS-08-21-1624-PDN