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TYLCV-IS76 is a recombinant begomovirus (family Geminiviridae) between Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus. It was detected in 2010 in Southern Morocco, and subsequent large field surveys showed that it had replaced its parental viruses. The fact that TYLCV-IS76 was positively selected in tomato plants carrying the begomovirus resistance gene Ty-1, and the co-occurrence of its emergence with the replacement of susceptible tomato cultivars with Ty-1 resistant cultivars, both support a Ty-1 resistancedriven emergence of TYLCV-IS76. The virus-plant interactions that determine positive selection of begomovirus recombinants are thought to involve gene silencing, because the Ty-1 gene encodes a gamma-clade RNAdependent RNA polymerase (RDRγ) implicated in biogenesis of viral small interfering RNAs (siRNAs) and methylation of viral DNA. To further investigate the mechanisms of RDRγ-mediated antiviral defence and defence evasion by TYLCV-IS76, we performed a comparative small RNA-ome and transcriptome profiling of Ty- 1 resistant and control susceptible plants infected with TYLCV or TYLCV-IS76. We found that RDRγ strongly enhances production of 22 and 24 nt viral siRNAs from both TYLCV and IS76 recombinant. The TYLCV-IS76 recombination event enhances transcription of viral silencing suppressor and coat protein genes and triggers more potent antiviral silencing responses. Compared to TYLCV, TYLCV-IS76 better evades transcriptional silencing of viral genes promoted by RDRγ, which may explain why the recombinant virus outcompetes its parents in Ty-1 plants.

Begomovirus-induced tomato yellow leaf curl disease challenges tomato production worldwide. Following geographic spread, the distribution areas of the Middle Eastern tomato yellow leaf curl virus (TYLCV) and the Western Mediterranean tomato yellow leaf curl Sardinia virus (TYLCSV) overlapped. Recombinants between TYLCV and TYLCSV were detected in overlapping areas and some of them, exhibiting unusual recombination patterns, were found to be invasive in Morocco (TYLCV-IS76) and presumably in Italy (TYLCV-IS141). Field surveys and laboratory experiments revealed a strong positive selection of these unusual recombinants in tomato plants encoding the Ty-1-resistance gene. Experiments conducted in controlled conditions showed that their fitness advantage was induced by beneficial intragenomic interactions rather than by specific mutations. The Ty-1-resistance gene is coding for an RNA-dependent RNA polymerase (RDR) of a distinct γ-clade, related to α-clade RDRs involved in gene silencing-based antiviral defense. Hence, the positive selection of recombinants by Ty-1-resistant plants is thought to be associated with gene silencing. Several hypotheses were proposed to identify viral determinants of potentially new molecular interactions triggered by the recombination event and some of them were tested. As the emergence of TYLCV-IS76 was driven by the deployment of Ty-1-resistant plants, new virus–plant interactions are expected. In the absence of any strong hypothesis to explain the selective advantage of the invasive recombinants and particularly their strong negative impact on TYLCV accumulation, an RNAomics approach is recommended. The selective advantage of TYLCV-IS76 in Ty-1-resistant plants was detected, irrespective of the three allelic combinations tested. To control these new invading TYLCV recombinants, new resistance genes need to be identified and preferably genes that block virus replication to limit the emergence of resistance-breaking viruses.

The rising global population puts constant pressure on food production and intensive agricultural practices. Alongside the injudicious use of insecticides, this has modified agrarian ecosystems, causing more plant vectorborne viral diseases than ever before. The EU-funded GuardSym project will focus on the whitefly Bemisia tabaci, one of the worst pest insects, as a vector to investigate its multitrophic interactions with its bacterial symbionts, vectored virus, and host plants. The project will form the basis for the development of population modification strategies where natural populations are replaced with virus-resistant ones that can no longer cause plant disease.

It was only with the advent of viral metagenomics in the 2010s that aphid-transmitted geminiviruses were discovered. The genus Capulavirus was established to accommodate them within the family Geminiviridae. Euphorbia caput-medusae latent virus (EcmLV) is the type member of the new genus, which includes Alfalfa leaf curl virus (ALCV), French bean severe leaf curl virus (FbSLCV), and Plantago lanceolata latent virus (PlLV). Several reasons can explain why they went unnoticed. EcmLV and PlLV are latent and were detected on noncultivated hosts. The symptoms induced by FbSLCV in French beans resemble those induced by some whitefly-transmitted geminiviruses and FbSLCV incidence is generally around 2%. The symptoms associated with ALCV were previously associated with a rhabdovirus. A detailed analysis of the transmission cycle of an agroinfectious clone of ALCV revealed that it is efficiently acquired by its aphid vector Aphis craccivora but its excretion rate is dramatically low compared to that of the nanovirus Faba bean necrotic stunt virus tested in parallel with the same aphid population. Consistent with its low excretion, the transmission rate of ALCV is much lower than that of nonaphid-transmitted geminiviruses, irrespective of the three A. craccivora populations tested. The transmission rate of a field-collected isolate of ALCV was similarly low. According to preliminary results and the literature, transmission rates of other capulaviruses seem to be low as well suggesting that low transmission rates are a typical feature of capulaviruses inherent to geminivirus–aphid combinations. Inefficient virus transmission may explain why capulaviruses were detected mostly in hardy perennial plants where high transmission rates are not critical. The exception of FbSLCV on French beans may be explained by a spillover infection from a potential perennial host where it may be maintained.

One of the most harmful viruses to vegetable crops, especially in the Mediterranean Basin, is the family Geminiviridae known as the "geminivirus." A project on the prevention and control of new and invasive geminiviruses infecting vegetables in the Mediterranean was launched through an international 3-year collaboration federated by a PRIMA call (Partnership for research and innovation in the Mediterranean area). This project involved public and private institutions from Italy, Jordan, Tunisia, Morocco, and France. Virusspecific PCR tests on DNA extracted from symptomatic tomato and cucurbit plants have been used to monitor the prevalence of geminiviruses previously described in this region, including tomato yellow leaf curl associated viruses, tomato leaf curl New Delhi virus, chickpea chlorotic dwarf virus, squash leaf curl virus, and watermelon chlorotic stunt virus. Meanwhile, deep sequencing of DNA extracted from insects collected in the surveyed areas (mainly whiteflies, aphids, and leafhoppers) is being used to look into the possibility of novel geminiviruses circulating in the region. To compare results of viral incidence between countries, standardized procedures for collecting, preserving, and analyzing leaf and insect samples have been established. In different agroecosystems in the following countries in 2020-2021, more than a thousand leaf samples and about 200 insect samples were collected: Morocco, in the Berkane area in the North-East and Souss region in the South; Tunisia, in the Center, North, and Sahel regions; Italy, in Campania and Sicily; and the Northern area of Jordan. These surveys are being used to update data on geminivirus prevalence across the Mediterranean Basin.

Begomoviruses are circular single stranded DNA (css) plant viruses with bipartite (A and B) or monopartite (A-like component) genomes. They are sometimes associated with satellites, cssDNA molecules, namely alphasatellites and betasatellites. Like the B component of bipartite begomoviruses, satellites depend on the A or A-like component for their replication (betasatellite) and encapsidation (alphasatellites and betasatellites). Although Tomato yellow leaf curl virus (TYLCV) was only rarely reported with satellites, alphasatellites and betasatellites of various geographic origins are readily assisted by TYLCV in experimental conditions. This result was consistent with the observation that satellite DNA contents were mostly higher than that of TYLCV (Conflon et al., 2018). The ease with which satellites can be assisted with TYLCV was supported further by FISH observations, which showed that the frequency of TYLCV-infected cells that were co-infected with a satellite exceeded 85% for an alphasatellite, and 95% for a betasatellite. Interestingly, a substantial number of cell nuclei were positive only for the satellite, suggesting that the assistance seems to be possible, even with a low amount of TYLCV DNA, and possibly no TYLCV DNA. This later possibility that need to be confirmed with further tests, is according to the “multicellular way of life” theory proposed recently for Faba bean necrotic stunt virus, a multipartite nanovirus with eight separately encapsidated components (Sicard et al.,2019).

Tomato yellow leaf curl virus (TYLCV), tomato yellow leaf curl Sardinia virus (TYLCSV) and TYLCV/TYLCSV recombinants (Begomovirus, Geminiviridae) are responsible of the damaging tomato leaf curl disease (TYLCD) in the Mediterranean. Tomato cultivars having the Ty-1 resistance gene were deployed in the Mediterranean Basin in the 2000s. Plants of those cultivars prevent symptom expression and reduce virus accumulation. The shift from susceptible to Ty-1 resistant cultivars in Morocco coincided with the invasion of a peculiar TYLCV/TYLCSV recombinant virus (TYLCV-IS76) discovered in 2010 in symptomatic resistant plants (1). It replaced the parental-type viruses in the South of Morocco and spread to the whole country. The selective advantage of TYLCV-IS76 assessed experimentally in comparison with parental type viruses is consistent with a selection driven emergence (2). According to phylogenetic analysis, and the recombination profiles of recombinants generated in tomato plants co-inoculated with TYLCV and TYLCSV, TYLCV-IS76 has emerged through a rare process (3). TYLCV-IS141, another peculiar TYLCV/TYLCSV exhibit some but not all the features of TYLCV-IS76. Like TYLCV-IS76 it inherited a very short TYLCSV fragment of about 120nts, it was detected on symptomatic Ty-1 resistant plants and exhibit a selective advantage over parental viruses in experimental conditions. However, unlike TYLCV-IS76, TYLCV-IS141 was detected in Italy (1, 4, 5) and was found to be generated and positively selected in experimental conditions (3). The sampling effort is presently too low to know if TYLCV-IS141 has the same invasion potential as TYLCV-IS76. Both recombinants will be useful to identify the determinants of their similar competitiveness.

The genus Capulavirus is a new genus in the family Geminiviridae that contains so far four species (Alfalfa leaf curl virus, Euphorbia caput-medusae latent virus, French bean severe leaf curl virus and Plantago lanceolata latent virus) that infect both cultivated and non-cultivated plants. Among them, plantago lanceolata latent virus (PlLV) infects ribwort plantain (Plantago lanceolata L.), a perennial herb that is native from Eurasia and is now widespread all over the world. PlLV is transmitted by the aphid Dysaphis plantaginea, which has a holocyclic lifestyle with two successive host plants: apple and plantain. Mechanical inoculation of sap using carborundum powder or syringe have also proved effective to infect various plants including P. lanceolata. Although plants naturally infected with PlLV displayed yellowing symptoms, agroinoculation in controlled conditions of an agroinfectious clone of PlLV in plantain did not reveal evidence of symptoms. In order to better decipher the prevalence and the diversity of PlLV, P. lanceolata plant samples were collected from four European countries (Finland, France, Italy and Spain), as well as from Iran and South Africa, and were tested by PCR for the presence of PlLV. While PlLV was initially reported from the Åland Islands (South-West Finland), we show that PlLV is also found from all sampled countries but South Africa. These results suggest that the geographic distribution of PlLV is larger than initially thought and that PlLV prevalence is probably relatively high in the Old World. Six complete genomes of PlLV from four distinct plant populations [Finland (2 genomes), France (1), Italy (1), Spain (1)] were amplified by RCA and/or PCR using a pair of abutting primers from infected samples, cloned and sequenced (Sanger). Partial nucleotidic sequences of the coat protein (cp) and replication-associated protein (rep) genes of PlLV were also obtained by PCR from samples collected in France (1) and Iran (2). The six PlLV complete genome sequences ranged in size from 2832 to 2834 nt in length and shared 94.1% genome-wide pairwise identity. The characterization of capulaviruses from wild asymptomatic plants such as PlLV is essential to better understanding the ecology and the evolution of geminiviruses.

Nanoviruses and geminiviruses are important ssDNA plant viruses with a dramatic impact on vegetable crops. They share ecological and molecular features. Both groups of viruses are restricted to the plant vascular system where they are acquired and then transmitted plant-to-plant by insect vectors through the circulative non-propagative mode. Investigating putative interactions between nanoviruses and geminiviruses is essential to determine the differences or similarities in their transmission process. The Faba Bean Necrotic Stunt Virus (FBNSV) is a legume nanovirus vectored by different aphid species including Aphis craccivora. The Alfalfa Leaf Curl Virus (ALCV) also infects legumes and is so far the only reported geminivirus vectored by an aphid: Aphis craccivora. After ingestion with the plant sap, FBNSV and ALCV virions massively accumulate in cells of the aphid anterior midgut (AMG) before passage in the hemolymph and to the salivary glands. Though the precise gut-crossing mechanisms remain elusive, previous experiments have definitely established that FBNSV needs the presence of the virus-encoded Nuclear Shuttle Protein (NSP) for successful aphid-transmission while ALCV uses the capsid strategy and does not require any transmission factor. In co-agroinoculated faba bean, we observed co-infection in a small proportion (approximately 10 %) of the test plants. Transmission tests using these co-infected plants and study of the accumulation and localization of the two viruses in aphid guts indicated that they follow a distinct intracellular route and do not interfere, suggesting for the first time that nanoviruses and capulaviruses may interact differently with their common aphid vector.

Tomato yellow leaf curl disease (TYLCD) is caused by two begomovirus species (Geminiviridae, ss circular DNA), Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) in the Mediterranean region. Interspecies recombinants were frequently reported in both naturally infected tomato plants and in plants co-inoculated with TYLCV and TYLCSV. The TYLCV/TYLCSV recombination profiles were relatively homogenous with a recombination breakpoint (RB) always located at the origin of replication (OR), and other RBs distant from the OR. None of these recombinants has been reported to be associated with breaking of the resistance induced by the Ty-1 resistance gene which is virtually present in all TYLCD-resistant tomato cultivars used in the Mediterranean. Unusual recombinants for which the non-OR RB was very close to the OR were detected in Ty-1 tomato plants in Morocco (TYLCV-IS76) and Italy (TYLCV-IS141) and exhibited invasive and Ty-1 resistance breaking features. Unlike TYLCV-IS141, TYLCV-IS76 was not positively selected in tomato plants co-inoculated with TYLCV and TYLCSV, which suggest that their generation and pre-emergence in natural conditions was probably different. However, both recombinants were shown to have a selective advantage in Ty-1 plants with a tremendous negative impact on the accumulation of TYLCV, the most competitive parental virus. This advantage was shown to be associated to the short 76-nt TYLCSV inherited fragment. The molecular determinism of the competitiveness of these recombinants was investigated according to three hypotheses: (i) the replication of the recombinants may be more efficient due to their 8 nts shorter genome in the recombinant region, (ii) a lower GC content which may reduce the transcriptional gene silencing which is expected to be involved due to the RNA dependent RNA polymerase encoded by Ty-1, and (iii) the TYLCV region that was switched in the recombinants has relieved the genome from a fitness load. Three variants were generated and their competitiveness with TYLCV was compared to that of the wild type TYLCV-IS76.

For decades, whiteflies, leafhoppers and treehoppers were the only known vectors of geminiviruses (family Geminiviridae). Metagenomic analysis revealed four divergent members of this family that are now classified in a new genus named Capulavirus according to their type member Euphorbia Caput medusae latent virus (EcmLV). Among them, Alfalfa leaf curl virus (ALCV) infects mostly Fabaceae and has been shown in our lab to be transmitted by Aphis craccivora. As it was the first report of an aphid-transmitted geminivirus, the mode of transmission was characterized. The transmission journey of ALCV starts in the phloem (Vicia faba) where it was shown to be restricted using FISH and EPG. To reach a 50% transmission success it needs at least 10 insects per inoculated plant following an acquisition access period of at least 24 hours. Once the aphid has acquired virus, the viral load remains constant in its body and transmission was still possible up to 11 days post-acquisition. Viral DNA was detected in the midgut and salivary glands which altogether support a circulative persistent mode of transmission involving internalization into insect epithelia mediated by potential receptors. The relatively low transmission efficiency of ALCV may be explained by the non-uniform distribution of ALCV (FISH and EPG) which may hinder its acquisition, and a non-efficient release from the viruliferous aphid into the inoculated tissue as revealed by qPCR. The aphid transmission of ALCV is highly specific because Aphis tirucallis, a close relative of A. craccivora, undistinguishable with CO1 phylogeny (Coeur d'Acier et. al., 2014), did no transmit ALCV although we identified it as the vector of EcmLV. Thus, we compared the pathways of ALCV between the two aphids to enlight potential transmission barriers in A. tirucallis body (qPCR, FISH), and initiate the identification of potential receptors in A. craccivora with a Y2H approach.

Disease emergence events regularly result from human activities such as agriculture, which frequently brings large populations of genetically uniform hosts into contact with potential pathogens. Although viruses cause nearly 50% of emerging plant diseases, there is little systematic information about virus distribution across agro‐ecological interfaces and large gaps in understanding of virus diversity in nature. Here we applied a novel landscape‐scale geometagenomics approach to examine relationships between agricultural land use and distributions of plant‐associated viruses in two Mediterranean‐climate biodiversity hotspots (Western Cape region of South Africa and Rhône river delta region of France). In total, we analysed 1725 geo‐referenced plant samples collected over two years from 4.5 km x 4.5 km grids spanning farmlands and adjacent uncultivated vegetation. We found substantial virus prevalence (25.8–35.9%) in all ecosystems, but prevalence and identified family‐level virus diversity were greatest in cultivated areas, with some virus families displaying strong agricultural associations. Our survey revealed 94 previously unknown virus species, primarily from uncultivated plants. This is the first effort to systematically evaluate plant‐associated viromes across broad agro‐ecological interfaces. Our findings indicate that agriculture substantially influences plant virus distributions and highlight the extent of current ignorance about the diversity and roles of viruses in nature.

Tomato yellow leaf curl virus is one of the most damaging plant viruses. Following its progression from the Middle East to the Mediterranean Basin, TYLCV has spread globally during the last 30 years. TYLCV induce dramatic losses in tomato and its control requires important investments, for insecticides, the building of insectproof shelters and the selection of resistant cultivars. All resistant commercial varieties deployed in the Mediterranean Basin in the 2000s, have the Ty-1 gene with which the symptom expression is blocked. As the Ty-1 gene reduce but does not block virus multiplication, the infected plants are healthy carrier in which TYLCV was able to efficiently evolve through its propensity to recombine. Thus, in 2010, a recombinant, TYLCV-IS76, has been detected in Morocco in a few plants which exhibited TYLCV symptoms. IS76 is a recombinant between TYLCV and tomato yellow leaf curl Sardinia virus (TYLCSV), a virus endemic to the Mediterranean Basin. Surveys have shown that IS76 has replaced parental viruses in the South of Morocco and that it has spread to the whole country. IS76 was experimentally shown to be more competitive than parental viruses and particularly in Ty-1 resistant plants. As IS76 could not be detected on plants sampled before the deployment of Ty-1 varieties, its invasion has been associated with positive selection by these varieties. Similar results are presently obtained with a TYLCV/TYLCSV recombinant detected in Sicily on symptomatic resistant plants.