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).

Correction: The genetic identity of neighboring plants in intraspecific mixtures modulates disease susceptibility of both wheat and rice.

[This corrects the article DOI: 10.1371/journal.pbio.3002287.].

The genetic identity of neighboring plants in intraspecific mixtures modulates disease susceptibility of both wheat and rice.

Mixing crop cultivars has long been considered as a way to control epidemics at the field level and is experiencing a revival of interest in agriculture. Yet, the ability of mixing to control pests is highly variable and often unpredictable in the field. Beyond classical diversity effects such as dispersal barrier generated by genotypic diversity, several understudied processes are involved. Among them is the recently discovered neighbor-modulated susceptibility (NMS), which depicts the phenomenon that susceptibility in a given plant is affected by the presence of another healthy neighboring plant. Despite the putative tremendous importance of NMS for crop science, its occurrence and quantitative contribution to modulating susceptibility in cultivated species remains unknown. Here, in both rice and wheat inoculated in greenhouse conditions with foliar fungal pathogens considered as major threats, using more than 200 pairs of intraspecific genotype mixtures, we experimentally demonstrate the occurrence of NMS in 11% of the mixtures grown in experimental conditions that precluded any epidemics. Thus, the susceptibility of these 2 major crops results from indirect effects originating from neighboring plants. Quite remarkably, the levels of susceptibility modulated by plant-plant interactions can reach those conferred by intrinsic basal immunity. These findings open new avenues to develop more sustainable agricultural practices by engineering less susceptible crop mixtures thanks to emergent but now predictable properties of mixtures.

Managing the deluge of newly discovered plant viruses and viroids: an optimized scientific and regulatory framework for their characterization and risk analysis.

The advances in high-throughput sequencing (HTS) technologies and bioinformatic tools have provided new opportunities for virus and viroid discovery and diagnostics. Hence, new sequences of viral origin are being discovered and published at a previously unseen rate. Therefore, a collective effort was undertaken to write and propose a framework for prioritizing the biological characterization steps needed after discovering a new plant virus to evaluate its impact at different levels. Even though the proposed approach was widely used, a revision of these guidelines was prepared to consider virus discovery and characterization trends and integrate novel approaches and tools recently published or under development. This updated framework is more adapted to the current rate of virus discovery and provides an improved prioritization for filling knowledge and data gaps. It consists of four distinct steps adapted to include a multi-stakeholder feedback loop. Key improvements include better prioritization and organization of the various steps, earlier data sharing among researchers and involved stakeholders, public database screening, and exploitation of genomic information to predict biological properties.

First report of lettuce ring necrosis virus in chili pepper and tomato in Belgium and The Netherlands.

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).

Virion-Associated Nucleic Acid-Based Metagenomics: A Decade of Advances in Molecular Characterization of Plant Viruses.

Over the last decade, viral metagenomic studies have resulted in the discovery of thousands of previously unknown viruses. These studies are likely to play a pivotal role in obtaining an accurate and robust understanding of how viruses affect the stability and productivity of ecosystems. Among the metagenomics-based approaches that have been developed since the beginning of the 21st century, shotgun metagenomics applied specifically to virion-associated nucleic acids (VANA) has been used to disentangle the diversity of the viral world. We summarize herein the results of 24 VANA-based studies, focusing on plant and insect samples conducted over the last decade (2010 to 2020). Collectively, viruses from 85 different families were reliably detected in these studies, including capsidless RNA viruses that replicate in fungi, oomycetes, and plants. Finally, strengths and weaknesses of the VANA approach are summarized and perspectives of applications in detection, epidemiological surveillance, environmental monitoring, and ecology of plant viruses are provided. [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.

Biological and Genetic Characterization of Physostegia Chlorotic Mottle Virus in Europe Based on Host Range, Location, and Time.

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.

Plant neighbour-modulated susceptibility to pathogens in intraspecific mixtures.

As part of a trend towards diversifying cultivated areas, varietal mixtures are subject to renewed interest as a means to manage diseases. Besides the epidemiological effects of varietal mixtures on pathogen propagation, little is known about the effect of intraspecific plant-plant interactions and their impact on responses to disease. In this study, genotypes of rice (Oryza sativa) or durum wheat (Triticum turgidum) were grown with different conspecific neighbours and manually inoculated under conditions preventing pathogen propagation. Disease susceptibility was measured together with the expression of basal immunity genes as part of the response to intra-specific neighbours. The results showed that in many cases for both rice and wheat susceptibility to pathogens and immunity was modified by the presence of intraspecific neighbours. This phenomenon, which we term 'neighbour-modulated susceptibility' (NMS), could be caused by the production of below-ground signals and does not require the neighbours to be infected. Our results suggest that the mechanisms responsible for reducing disease in varietal mixtures in the field need to be re-examined.