Specificity of Resistance and Tolerance to Cucumber Vein Yellowing Virus in Melon Accessions and Resistance Breaking with a Single Mutation in VPg.

Cucumber vein yellowing virus (CVYV) is an emerging virus on cucurbits in the Mediterranean Basin, against which few resistance sources are available, particularly in melon. The melon accession PI 164323 displays complete resistance to isolate CVYV-Esp, and accession HSD 2458 presents a tolerance, i.e., very mild symptoms despite virus accumulation in inoculated plants. The resistance is controlled by a dominant allele Cvy-11, while the tolerance is controlled by a recessive allele cvy-2, independent from Cvy-11. Before introducing the resistance or tolerance in commercial cultivars through a long breeding process, it is important to estimate their specificity and durability. Upon inoculation with eight molecularly diverse CVYV isolates, the resistance was found to be isolate-specific because many CVYV isolates induced necrosis on PI 164323, whereas the tolerance presented a broader range. A resistance-breaking isolate inducing severe mosaic on PI 164323 was obtained. This isolate differed from the parental strain by a single amino acid change in the VPg coding region. An infectious CVYV cDNA clone was obtained, and the effect of the mutation in the VPg cistron on resistance to PI 164323 was confirmed by reverse genetics. This represents the first determinant for resistance-breaking in an ipomovirus. Our results indicate that the use of the Cvy-11 allele alone will not provide durable resistance to CVYV and that, if used in the field, it should be combined with other control methods such as cultural practices and pyramiding of resistance genes to achieve long-lasting resistance against CVYV.

Can Winged Aphid Abundance Be a Predictor of Cucurbit Aphid-Borne Yellows Virus Epidemics in Melon Crop?

Aphid-borne viruses are frequent yield-limiting pathogens in open field vegetable crops. In the absence of curative methods, virus control relies exclusively on measures limiting virus introduction and spread. The efficiency of control measures may greatly benefit from an accurate knowledge of epidemic drivers, in particular those linked with aphid vectors. Field experiments were conducted in southeastern France between 2010 and 2019 to investigate the relationship between the epidemics of cucurbit aphid-borne yellows virus (CABYV) and aphid vector abundance. Winged aphids visiting melon crops were sampled daily to assess the abundance of CABYV vectors (Aphis gossypii, Macrosiphum euphorbiae and Myzus persicae) and CABYV was monitored weekly by DAS-ELISA. Epidemic temporal progress curves were successfully described by logistic models. A systematic search for correlations was undertaken between virus variables including parameters µ (inflection point of the logistic curve) and γ (maximum incidence) and aphid variables computed by aggregating abundances on periods relative either to the planting date, or to the epidemic peak. The abundance of A. gossypii during the first two weeks after planting was found to be a good predictor of CABYV dynamics, suggesting that an early control of this aphid species could mitigate the onset and progress of CABYV epidemics in melon crops.

Sixty Years After the First Description: Genome Sequence and Biological Characterization of European Wheat Striate Mosaic Virus Infecting Cereal Crops.

High-throughput sequencing technologies were used to identify plant viruses in cereal samples surveyed from 2012 to 2017. Fifteen genome sequences of a tenuivirus infecting wheat, oats, and spelt in Estonia, Norway, and Sweden were identified and characterized by their distances to other tenuivirus sequences. Like most tenuiviruses, the genome of this tenuivirus contains four genomic segments. The isolates found from different countries shared at least 92% nucleotide sequence identity at the genome level. The planthopper Javesella pellucida was identified as a vector of the virus. Laboratory transmission tests using this vector indicated that wheat, oats, barley, rye, and triticale, but none of the tested pasture grass species (Alopecurus pratensis, Dactylis glomerata, Festuca rubra, Lolium multiflorum, Phleum pratense, and Poa pratensis), are susceptible. Taking into account the vector and host range data, the tenuivirus we have found most probably represents European wheat striate mosaic virus first identified about 60 years ago. Interestingly, whereas we were not able to infect any of the tested cereal species mechanically, Nicotiana benthamiana was infected via mechanical inoculation in laboratory conditions, displaying symptoms of yellow spots and vein clearing evolving into necrosis, eventually leading to plant death. Surprisingly, one of the virus genome segments (RNA2) encoding both a putative host systemic movement enhancer protein and a putative vector transmission factor was not detected in N. benthamiana after several passages even though systemic infection was observed, raising fundamental questions about the role of this segment in the systemic spread in several hosts.

Resistance Against Melon Chlorotic Mosaic Virus and Tomato Leaf Curl New Delhi Virus in Melon.

Thirty-one melon accessions were screened for resistance to the begomoviruses Melon chlorotic mosaic virus (MeCMV) and Tomato leaf curl New Delhi virus (ToLCNDV). Five accessions presented nearly complete resistance to both viruses. Accession IC-274014, showing the highest level of resistance to both viruses, was crossed with the susceptible cultivar Védrantais. The F1, F2, F3/F4, and both backcross progenies were mechanically inoculated with MeCMV. Plants without symptoms or virus detection by enzyme-linked immunosorbent assay and/or PCR were considered as resistant. The segregations were compatible with two recessive and one dominant independent genes simultaneously required for resistance. Inheritance of resistance to ToLCNDV in the F2 was best explained by one recessive gene and two independent dominant genes simultaneously required. Some F3 and F4 families selected for resistance to MeCMV also were resistant to ToLCNDV, suggesting that common or tightly linked genes were involved in resistance to both viruses. We propose the names begomovirus resistance-1 and Begomovirus resistance-2 for these genes (symbols bgm-1 and Bgm-2). Resistance to MeCMV in IC-274014 was controlled by bgm-1, Bgm-2, and the recessive gene melon chlorotic mosaic virus resistance (mecmv); resistance to ToLCNDV was controlled by bgm-1, Bgm-2, and the dominant gene Tomato leaf curl New Delhi virus resistance (Tolcndv).

Impact of Vat resistance in melon on viral epidemics and genetic structure of virus populations.

Cultivar choice is at the heart of cropping systems and resistant cultivars should be at the heart of disease management strategies whenever available. They are the easiest, most efficient and environmentally friendly way of combating viral diseases at the farm level. Among the melon genetic resources, Vat is a unique gene conferring resistance to both the melon aphid Aphis gossypii and the viruses it carries. The 'virus side' of this pleiotropic phenotype is seldom regarded as an asset for virus control. Indeed, the effect of Vat on virus epidemics in the field is expected to vary according to the composition of aphid populations in the environment and long-term studies are needed to draw a correct trend. Therefore, the first objective of the study was to re-evaluate the potential of Vat to reduce viral diseases in melon crops. The second objective was to investigate the potential of Vat to exert a selection pressure on virus populations. We monitored the epidemics of Cucurbit aphid-borne yellows virus (CABYV), Cucumber mosaic virus (CMV), Watermelon mosaic virus (WMV) and Zucchini yellow mosaic virus (ZYMV) in two melon lines having a common genetic background, a resistant line (R) and a susceptible line (S), in eight field trials conducted in southeastern France between 2011 and 2015. Vat had limited impact if any on WMV epidemics probably because A. gossypii is not the main vector of WMV in the field, but a favorable impact on CMV, yet of variable intensity probably related to the importance of A. gossypii in the total aphid population. Vat had a significant impact on CABYV epidemics with mean incidence reduction exceeding 50% in some trials. There was no effect of Vat on the structure of virus populations, both for the non-persistent WMV transmitted by numerous aphid species and for the persistent CABYV transmitted predominantly by A. gossypii.

Characterization of a new cucurbit-infecting ipomovirus from Sudan.

Two members of the genus Ipomovirus (family Potyviridae) are known to infect cucurbits: cucumber vein yellowing virus (CVYV), which is emerging throughout the Mediterranean Basin, and squash vein yellowing virus (SqVYV), which has been described in America and the Caribbean Basin, and more recently in Israel. In this work, an ipomovirus different from CVYV and SqVYV, tentatively named coccinia mottle virus (CocMoV), was detected in a sample of the cucurbit Coccinia grandis collected in central Sudan in 2012. Sequence identity in nt was 68 % with CVYV, 59-60 % with SqVYV, cassava brown streak virus and Ugandan cassava brown streak virus, and less than 50 % with other members of the family Potyviridae. Preliminary biological and epidemiological studies indicate that CocMoV has a narrow natural host range and a low prevalence.

NBS-LRR-mediated resistance triggered by aphids: viruses do not adapt; aphids adapt via different mechanisms.

BACKGROUND: Aphids are serious pest on crops. By probing with their stylets, they interact with the plant, they vector viruses and when they reach the phloem they start a continuous ingestion. Many plant resistances to aphids have been identified, several have been deployed. However, some resistances breaking down have been observed. In the melon, a gene that confers resistance to aphids has been deployed in some melon-producing areas, and aphid colony development on Vat-carrying plants has been observed in certain agrosystems. The Vat gene is a NBS-LRR gene that confers resistance to the aphid species Aphis gossypii and exhibits the unusual characteristic of also conferring resistance to non-persistently transmitted viruses when they are inoculated by the aphid. Thus, we characterized patterns of resistance to aphid and virus using the aphid diversity and we investigated the mechanisms by which aphids and viruses may adapt to the Vat gene. RESULTS: Using a Vat-transgenic line built in a susceptible background, we described the Vat- spectrum of resistance to aphids, and resistance to viruses triggered by aphids using a set of six A. gossypii biotypes. Discrepancies between both resistance phenotypes revealed that aphid adaptation to Vat-mediated resistance does not occur only via avirulence factor alterations but also via adaptation to elicited defenses. In experiments conducted with three virus species serially inoculated by aphids from and to Vat plants, the viruses did not evolve to circumvent Vat-mediated resistance. We confirmed discrepancies between both resistance phenotypes by testing each aphid biotype with a set of thirteen melon accessions chosen to reflect the natural diversity of the melon. Inheritance studies revealed that patterns of resistance to virus triggered by aphids are controlled by different alleles at the Vat locus and at least another locus located at a short genetic distance. Therefore, resistance to viruses triggered by aphids is controlled by a gene cluster. CONCLUSIONS: Under the Flor model, changes in the avirulence gene determine the ability of the pathogen to overcome the resistance conferred by a plant gene. The Vat gene belongs to a resistance gene family that fits this pest/pathogen-plant interaction, and we revealed an additional mechanism of aphid adaptation that potentially exists in other interactions between plants and pests or pathogens.

Can plant viruses cross the kingdom border and be pathogenic to humans?

Phytoviruses are highly prevalent in plants worldwide, including vegetables and fruits. Humans, and more generally animals, are exposed daily to these viruses, among which several are extremely stable. It is currently accepted that a strict separation exists between plant and vertebrate viruses regarding their host range and pathogenicity, and plant viruses are believed to infect only plants. Accordingly, plant viruses are not considered to present potential pathogenicity to humans and other vertebrates. Notwithstanding these beliefs, there are many examples where phytoviruses circulate and propagate in insect vectors. Several issues are raised here that question if plant viruses might further cross the kingdom barrier to cause diseases in humans. Indeed, there is close relatedness between some plant and animal viruses, and almost identical gene repertoires. Moreover, plant viruses can be detected in non-human mammals and humans samples, and there are evidence of immune responses to plant viruses in invertebrates, non-human vertebrates and humans, and of the entry of plant viruses or their genomes into non-human mammal cells and bodies after experimental exposure. Overall, the question raised here is unresolved, and several data prompt the additional extensive study of the interactions between phytoviruses and non-human mammals and humans, and the potential of these viruses to cause diseases in humans.

Control of cucurbit viruses.

More than 70 well-characterized virus species transmitted by a diversity of vectors may infect cucurbit crops worldwide. Twenty of those cause severe epidemics in major production areas, occasionally leading to complete crop failures. Cucurbit viruses' control is based on three major axes: (i) planting healthy seeds or seedlings in a clean environment, (ii) interfering with vectors activity, and (iii) using resistant cultivars. Seed disinfection and seed or seedling quality controls guarantee growers on the sanitary status of their planting material. Removal of virus or vector sources in the crop environment can significantly delay the onset of viral epidemics. Insecticide or oil application may reduce virus spread in some situations. Diverse cultural practices interfere with or prevent vector reaching the crop. Resistance can be obtained by grafting for soil-borne viruses, by cross-protection, or generally by conventional breeding or genetic engineering. The diversity of the actions that may be taken to limit virus spread in cucurbit crops and their limits will be discussed. The ultimate goal is to provide farmers with technical packages that combine these methods within an integrated disease management program and are adapted to different countries and cropping systems.

A short motif in the N-terminal part of the coat protein is a host-specific determinant of systemic infectivity for two potyviruses.

Although the biological variability of Watermelon mosaic virus is limited, isolates from the three main molecular groups differ in their ability to infect systemically Chenopodium quinoa. Mutations were introduced in a motif of three or five amino acids located in the N-terminal part of the coat protein, and differing in isolates from group 1 (motif: lysine-glutamic acid-alanine (Lys-Glu-Ala) or KEA, systemic on C. quinoa), group 2 (Lys-Glu-Thr or KET, not systemic on C. quinoa) and group 3 (KEKET, not systemic on C. quinoa). Mutagenesis of KEKET in an isolate from group 3 to KEA or KEKEA was sufficient to make the virus systemic on C. quinoa, whereas mutagenesis to KET had no effect. Introduction of a KEA motif in Zucchini yellow mosaic virus coat protein also resulted in systemic infection on C. quinoa. These mutations had no obvious effect on the disorder profile or potential post-translational modifications of the coat protein as determined in silico.

General strategy for ordered noncovalent protein assembly on well-defined nanoscaffolds.

Here we develop a novel approach allowing the noncovalent assembly of proteins on well-defined nanoscaffolds such as virus particles. The antibody-binding peptide Z33 was genetically fused to the monomeric yellow fluorescent protein and 4-coumarate:CoA-ligase 2. This Z33 "tag" allowed their patterning on the surface of zucchini yellow mosaic virus by means of specific antibodies directed against the coat protein of the virus. The approach was validated by affinity assays and correlative microscopy. The coverage efficiency was ≈ 87%. Fluorescence and enzymatic activity were fully retained after assembly. The principle of using the combination of a scaffold-specific antibody and Z33-fusion proteins can be extended to a wide variety of proteins/enzymes and antigenic scaffolds to support coupling for creating functional "biochips" with optical or catalytic properties.

Tobacco mosaic virus in the lungs of mice following intra-tracheal inoculation.

Plant viruses are generally considered incapable of infecting vertebrates. Accordingly, they are not considered harmful for humans. However, a few studies questioned the certainty of this paradigm. Tobacco mosaic virus (TMV) RNA has been detected in human samples and TMV RNA translation has been described in animal cells. We sought to determine if TMV is detectable, persists, and remains viable in the lung tissues of mice following intratracheal inoculation, and we attempted to inoculate mouse macrophages with TMV. In the animal model, mice were intratracheally inoculated with 10(11) viral particles and were sacrificed at different time points. The virus was detected in the mouse lungs using immunohistochemistry, electron microscopy, real-time RT-PCR and sequencing, and its viability was studied with an infectivity assay on plants. In the cellular model, the culture medium of murine bone marrow derived macrophages (BMDM) was inoculated with different concentrations of TMV, and the virus was detected with real-time RT-PCR and immunofluorescence. In addition, anti-TMV antibodies were detected in mouse sera with ELISA. We showed that infectious TMV could enter and persist in mouse lungs via the intratracheal route. Over 14 days, the TMV RNA level decreased by 5 log(10) copies/ml in the mouse lungs and by 3.5 log(10) in macrophages recovered from bronchoalveolar lavage. TMV was localized to lung tissue, and its infectivity was observed on plants until 3 days after inoculation. In addition, anti-TMV antibody seroconversions were observed in the sera from mice 7 days after inoculation. In the cellular model, we observed that TMV persisted over 15 days after inoculation and it was visualized in the cytoplasm of the BMDM. This work shows that a plant virus, Tobacco mosaic virus, could persist and enter in cells in mammals, which raises questions about the potential interactions between TMV and human hosts.

Viruses of cucurbit crops in the Mediterranean region: an ever-changing picture.

Cucurbit crops may be affected by at least 28 different viruses in the Mediterranean basin. Some of these viruses are widely distributed and cause severe yield losses while others are restricted to limited areas or specific crops, and have only a negligible economic impact. A striking feature of cucurbit viruses in the Mediterranean basin is their always increasing diversity. Indeed, new viruses are regularly isolated and over the past 35 years one "new" cucurbit virus has been reported on average every 2 years. Among these "new" viruses some were already reported in other parts of the world, but others such as Zucchini yellow mosaic virus (ZYMV), one of the most severe cucurbit viruses and Cucurbit aphid-borne yellows virus (CABYV), one of the most prevalent cucurbit viruses, were first described in the Mediterranean area. Why this region may be a potential "hot-spot" for cucurbit virus diversity is not fully known. This could be related to the diversity of cropping practices, of cultivar types but also to the important commercial exchanges that always prevailed in this part of the world. This chapter describes the major cucurbit viruses occurring in the Mediterranean basin, discusses factors involved in their emergence and presents options for developing sustainable control strategies.

Snake melon asteroid mosaic virus, a Tentative New Member of the Genus Sobemovirus Infecting Cucurbits.

A virus isolate (Su-95-67) was obtained from a snake melon (Cucumis melo var. flexuosus) plant presenting severe chlorotic spots, mosaic, stunting, and leaf deformations collected in Eastern Sudan in 1995. Su-95-67 was easily mechanically transmissible and had a host range limited to a few cucurbit species. Isometric virus particles approximately 30 nm in diameter were observed in leaf dip preparations. A cytopathological study did not reveal alterations specific for a virus genus or family. A polyclonal antiserum was obtained and used in double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Su-95-67 was transmitted by seed at a low rate, by the red melon beetle (Aulacophora foveicollis), but not by the melon aphid (Aphis gossypii). Because Su-95-67 shared several properties with sobemoviruses, generic Sobemovirus reverse-transcription polymerase chain reaction primers were developed. They allowed amplification of a 384-bp fragment from extracts of plants infected by two sobemoviruses or by Su-95-67 but not from healthy plants extracts. Sequence comparison confirmed that Su-95-67 belongs to a new tentative Sobemovirus species for which we propose the name Snake melon asteroid mosaic virus (SMAMV). DAS-ELISA tests conducted on extracts of virus-infected cucurbit plants collected from 1992 to 2003 revealed the presence of SMAMV in 10.2% of 600 samples originating from different regions of Sudan.

Asymmetrical over-infection as a process of plant virus emergence.

Disentangling the role of epidemiological factors in plant pathogen emergences is a prerequisite to identify the most likely future invaders. An example of emergence was recently observed in France: in 10 years, "classic" (CL) strains of Watermelon mosaic virus (WMV) were displaced at a regional scale by newly introduced "emerging" (EM) strains. Here we analyse a 3 years dataset describing the co-dynamics of CL and EM strains at field scale using state-space models estimating jointly: (i) probabilities of primary and secondary infection and (ii) probabilities of over-infecting with a CL [EM] strain a plant already infected with an EM [CL] strain. Results especially indicate that it is more than 3 times less probable for a CL strain to over-infect an EM infected plant than for an EM strain to over-infect a CL infected plant. To investigate if these asymmetric interactions can explain the CL/EM shift observed at regional scale, an exploratory model describing WMV epidemiology over several years in a landscape composed of a reservoir and a cultivated compartment is introduced. In most simulations a shift is observed and both strains do coexist in the landscape, reaching an equilibrium that depends on the probabilities of over-infection.

Pepper mild mottle virus, a plant virus associated with specific immune responses, Fever, abdominal pains, and pruritus in humans.

BACKGROUND: Recently, metagenomic studies have identified viable Pepper mild mottle virus (PMMoV), a plant virus, in the stool of healthy subjects. However, its source and role as pathogen have not been determined. METHODS AND FINDINGS: 21 commercialized food products containing peppers, 357 stool samples from 304 adults and 208 stool samples from 137 children were tested for PMMoV using real-time PCR, sequencing, and electron microscopy. Anti-PMMoV IgM antibody testing was concurrently performed. A case-control study tested the association of biological and clinical symptoms with the presence of PMMoV in the stool. Twelve (57%) food products were positive for PMMoV RNA sequencing. Stool samples from twenty-two (7.2%) adults and one child (0.7%) were positive for PMMoV by real-time PCR. Positive cases were significantly more likely to have been sampled in Dermatology Units (p<10(-6)), to be seropositive for anti-PMMoV IgM antibodies (p = 0.026) and to be patients who exhibited fever, abdominal pains, and pruritus (p = 0.045, 0.038 and 0.046, respectively). CONCLUSIONS: Our study identified a local source of PMMoV and linked the presence of PMMoV RNA in stool with a specific immune response and clinical symptoms. Although clinical symptoms may be imputable to another cofactor, including spicy food, our data suggest the possibility of a direct or indirect pathogenic role of plant viruses in humans.

Algerian watermelon mosaic virus (AWMV): a new potyvirus species in the PRSV cluster.

A potyvirus was isolated from a naturally infected squash plant in Algeria in 1986. Biological and serological data have revealed that the virus, initially described as H4, is related to other cucurbit-infecting potyviruses, particularly Moroccan watermelon mosaic virus (MWMV) and Papaya ringspot virus (PRSV). To establish unequivocally the taxonomic status of H4, its full-length genome sequence was established. H4 shared identities of 70% and 65% at the amino acid level with MWMV and PRSV, respectively, indicating that H4 is a distinct species of the PRSV cluster. The name Algerian watermelon mosaic virus (AWMV) is proposed for this new potyvirus species.

Potential Involvement of Melon Fruit in the Long Distance Dissemination of Cucurbit Potyviruses.

Papaya ringspot virus (PRSV) and Zucchini yellow mosaic virus(ZYMV) are potyviruses frequently reported in cucurbits in Mediterranean, subtropical, and tropical regions. Occasionally, epidemics are also observed in more temperate regions, but the ways these viruses are introduced into new areas are not yet fully determined. We investigated the possibility that infected imported melon fruit could be a route for the introduction of PRSV and ZYMV. Imported melon fruits of the yellow canary type infected by ZYMV and PRSV were exposed in the fields next to healthy melon or squash bait plants. During this period, aphids were observed landing and probing on the fruits. In four independent experiments using different fruits, 3.1 to 25% of bait plants were infected by ZYMV and/or PRSV. PRSV was more frequently transmitted to bait plants than ZYMV. Comparison of partial sequences of the isolates from fruits and from bait plants showed a very high, if not complete, identity within each experiment, confirming that a natural transmission did occur from the fruit to the bait plants. These results suggest that globalization of melon production and international trade may be a factor in the spread of cucurbit potyviruses between countries or continents.

Biological and Molecular Characterization of Moroccan watermelon mosaic virus and a Potyvirus Isolate from Eastern Sudan.

A potyvirus (Su-94-54) was isolated from a naturally infected snake cucumber (Cucumis melo var. flexuosus) plant with severe mosaic and leaf deformation symptoms collected in Eastern Sudan. This isolate has a host range limited to cucurbits and is serologically distantly related to Moroccan watermelon mosaic virus (MWMV) and to Papaya ringspot virus (PRSV). Coat protein sequence analysis of Su-94-54 and MWMV and comparison with other potyviruses indicate that Su-94-54 is more closely related to MWMV than to any other potyvirus. Based on the amino acid sequence identity in the core part of the coat protein with MWMV (86%), this isolate could be regarded as a distinct species. However, because of biological, cytological, and serological affinities with MWMV, we propose that this isolate be considered as a strain of MWMV, possibly an evolutionary intermediate between MWMV and PRSV, until more is known on the structure of the PRSV subgroup within the genus Potyvirus.

Melon Rugose Mosaic Virus: Characterization of an Isolate from Sudan and Seed Transmission in Melon.

Melon rugose mosaic virus (MRMV) was isolated from snake cucumber (Cucumis melo var. flexuosus) in the Kassala region of Sudan in 1993. The host range of the virus was mostly limited to cucurbits, where it induced severe mosaic and leaf deformations. Cytopathological studies revealed severe chloroplast alterations, including vesicles at their periphery and the tendency to aggregate, which are typical of tymovirus infections, providing further evidence that MRMV is a tentative member of the genus Tymovirus. In melon and snake cucumber, MRMV was found to be seed transmitted at rates of 0.9 and 3.8%, respectively. Seed dissection experiments revealed that the virus could be detected in the seed coat, papery layer, and embryo. Seed disinfection treatments did not reduce seed transmission rates, which suggests an internal transmission. A preliminary screening for resistance in melon revealed some resistance in two out of 367 accessions tested.