Biological Characterization of Physostegia Chlorotic Mottle Virus, an Emergent Virus Infecting Vegetables in Diversified Production Systems.

In 2014, Physostegia chlorotic mottle virus (PhCMoV) was discovered in Austria in Physostegia virginiana. Subsequent collaborative efforts established a link between the virus and severe fruit symptoms on important crops such as tomato, eggplant, and cucumber across nine European countries. Thereafter, specific knowledge gaps, which are crucial to assess the risks PhCMoV can pose for production and how to manage it, needed to be addressed. In this study, the transmission, prevalence, and disease severity of PhCMoV were examined. This investigation led to the identification of PhCMoV presence in a new country, Switzerland. Furthermore, our research indicates that the virus was already present in Europe 30 years ago. Bioassays demonstrated PhCMoV can result in up to 100% tomato yield losses depending on the phenological stage of the plant at the time of infection. PhCMoV was found to naturally infect 12 new host plant species across eight families, extending its host range to 21 plant species across 15 plant families. The study also identified a polyphagous leafhopper (genus Anaceratagallia) as a natural vector of PhCMoV. Overall, PhCMoV was widespread in small-scale diversified vegetable farms in Belgium where tomato is grown in soil under tunnels, occurring in approximately one-third of such farms. However, outbreaks were sporadic and were associated at least once with the cultivation in tomato tunnels of perennial plants that can serve as a reservoir host for the virus and its vector. To further explore this phenomenon and manage the virus, studying the ecology of the vector would be beneficial.

First report of Melon chlorotic spot virus in cultivated sorrel (Rumex acetosa) in Belgium.

In 2020, symptoms of putative viral origin were observed on 7% of tomatoes in an organic vegetable farm in Belgium (deformed uneven ripened fruits, vein clearing, mosaic and purple leaves, stunted plants). The leaves of twenty symptomatic plants were collected, pooled and screened for viruses using high throughput sequencing technologies (HTS) on Illumina NextSeq500 following a virion-associated nucleic acid (VANA) protocol (Temple et al., 2021, Be_SL1). In total, 3,665,498 reads (PE150) were generated. Bioinformatic analyses (denovo assembly, tblastx search on NCBI and mapping) using Geneious Prime® 2020.1.2 revealed the presence of three viruses known to infect tomatoes: Physostegia chlorotic mottle virus (PhCMoV), 547,142 reads map on NC_055466, potato virus Y (PVY), 4056 reads map on MW595184, and melon chlorotic spot virus (MeCSV), 55 reads mapped to six out of the eight different MeCSV segments (NC_040448-55). Tomato plants have already been artificially inoculated by MeCSV (Lecoq et al., 2019) but this detection (confirmed by independent RT-PCR on the pooled sample) is the first one in natural condition on farm. The high prevalence of symptoms triggered the research of alternative perennial hosts that can serve as a reservoir during inter-cropping season. One plant of Rumex acetosa showing vein clearing (CT-122) was collected in the same greenhouse the year after. Total RNA was extracted, followed by ribodepletion, and Illumina HTS using the protocol described in Temple et al., (2021) for Be_GP1. In total, 4,549,721 PE150 reads were obtained and bioinformatic analyses confirmed the presence of MeCSV (8,816 reads mapped on eight RNA segments NC_040448-55 with an average 96,52% coverage of the reference sequences, supplementary table 1) and suggested the presence of an unclassified partitivirus. Consensus sequences were extracted for each segment of MeCSV (OQ818038-45) and showed between 83% and 87% of nucleotide identity with the reference sequences NC_040448-55. RNA1 segment was used to design MeCSV-specific RT-PCR primers for detection (MeCSV-125F 5'-TTTAAGGCCAGATCCAGAGGTTC-3'/ MeCSV-498R 5'-TGGATGTGACAACCTGGTAGTAC-3'). Thereafter, in July 2022, 42 R. acetosa plants were collected in the same greenhouse. Among them, seven plants showed vein clearing, two showed yellowing with necrosis, two exhibited yellowing and vein clearing (Supplementary figure 1), and one showed mosaic. The 42 plants were subjected to RNA extraction and RT-PCR for MeCSV (Supplementary figure 2) and PhCMoV detection. MeCSV was detected in 13 plants (two asymptomatic plants and all the symptomatic plants except the one exhibiting mosaic where PhCMoV was detected). PhCMoV was also detected in three plants with vein clearing, one with yellowing and one of the two asymptomatic plants infected by MeCSV. Our results report the first detection of MeCSV in R. acetosa and the first detection of MeCSV in Belgium. In addition, according to the hierarchical approach for assessing causal relationships in plant virology (Fox et al., 2020), a preliminary association was observed between symptoms and MeCSV detection [6% prevalence on asymptomatic plants and 92% prevalence on diseased plants (from which seven symptomatic samples were not co-infected by PhCMoV)]. Symptom causality should be further investigated but this results are important for disease management because they suggested that cultivated perennial R. acetosa may serve as a reservoir for two emergent plant viruses (PhCMoV and MeCSV) (Lecoq et al., 2019, Temple et al., 2021).

Generic detection and identification of pospiviroids.

A multiplex one-step RT-PCR aiming at detecting all pospiviroids known to be harmful to cultivated plants has been developed. Specificity, sensitivity, selectivity, repeatability and reproducibility of this test have been assessed in order to fulfill the recommendations of the EPPO standard PM7/98 and provide routine detection laboratories with a cost-effective, easy-to-use and robust pospiviroid detection test. To further understand the epidemiology and ease the management of pospiviroid outbreaks, this RT-PCR diagnostic test can be followed by direct sequencing of the amplicons to identify and characterize the detected pospiviroid isolates.