The viral content ratio between abdomen and head is informative of the relative efficiency with which Bemisia tabaci populations transmit begomoviruses. [P.12]

Begomoviruses (family Geminiviridae) are transmitted by the whitefly Bemisia tabaci in a circulative non propagative manner. B. tabaci is a species complex composed of at least 24 morphocryptic species which differ in host range, insecticide resistance, endosymbionts and virus transmission. Begomoviruses are supposed to cross the gut barrier at the midgut level and salivary gland barrier in the principal salivary gland (PSG) cells because of the highest virus concentrations in these organs. Thus, the critical steps of the virus circulation in the insect body are (i) the exit of virion from the midgut, (ii) their preservation in the hemocoel and (iii) their entry in the PSG. Thus, we proposed that the efficiency of viral transfer from midgut to PSG may be assessed by measuring the viral content in both compartments and that the deduced viral content ratio may be correlated to viral transmission efficiency by the vector. Our predicition was tested with two invasive B. tabaci species, Middle East-Minor Asia 1 (MEAM1), and Mediterranean (MED), and three begomoviruses: the invasive species Tomato yellow leaf curl virus-Mild (TYLCV-Mld), Tomato leaf curl Comoros virus (ToLCKMV), indigenous from Mayotte and R4, a recombinant between TYLCV and ToLCKMV. In a first approach, PSG and midgut were separated by a cross section through the prothorax and viral loads were estimated in both sections by measuring viral DNA using real time PCR. As the midgut of B. tabaci was reported to be sometimes pushed through the diaphragm separating the abdomen and the thorax, the estimation of the viral content ratio between PSG and midgut may be biased by thorax sectioning. The simple cross sectioning was however validated because the ratio determined with such sections and the ratio determined after a careful gut dissection was similar. Using the simple cross section, the viral content ratio between head and abdomen was higher for MEAM1 than for Med for the three begomoviruses. As predicted, the transmission efficiency was higher with MEAM1 than Med Q2 for the three viruses. These results indicate that viral content ratio may be a reliable predictor of the relative transmission efficiency between different B. tabaci populations. Measuring transmission efficiency is time consuming, involves technically difficult experiments with acquisition and inoculation steps and needs specialized cage and containment equipment. However measuring viral content ratios needs only a few cages for the acquisition step, a binocular lens and an access to the commonly used qPCR machines. This approach might be extended to estimate the relative transmission efficiency of other circulative non propagative viruses. (Résumé d'auteur

Plant resistance-driven emergence of recombinant begomoviruses

The analysis of plant virus genomes reveals that many were shaped by recombination. However, the history of the emergence dynamics of these recombinants is mostly unknown as well as the underlying evolutionary forces that drove their frequency increase. The pivotal role of recombination in geminivirus evolution is supported by the detection of numerous recombination events in sequence data, and by their high propensity to recombine. These typical features were observed with Tomato yellow leaf curl virus (TYLCV), a tomato begomovirus that was extensively studied because of its global economic importance. TYLCV-IS76 is a recombinant TYLCV detected initially in Morocco. It inherited a 76-nt region of tomato yellow leaf curl Sardinia virus (TYLCSV) starting from the origin of replication (OR) towards the V2 gene. Based on field surveys carried out in Morocco and laboratory analysis, a real time emergence of TYLCV-IS76 has been reconstructed from its generation to the displacement of its parental viruses (1). Its emergence coincided with the deployment of Ty-1 resistant tomato cultivars, and a causal link was demonstrated with various competition tests in which positive selection of TYLCV-IS76 was observed in Ty-1 resistant plants (2). TYLCV-IS141 is a TYLCV recombinant detected in Italy (1, 3, 4, 5). It inherited a 141-nt region of TYLCSV between OR and the initiation codon of the V2 gene. TYLCV-IS141 and TYLCV-IS76 exhibit similar recombination profiles and fitness phenotypes in Ty-1 resistant plants. It was inferred from competition tests carried out with various natural and artificially generated TYLCV-IS76 and TYLCV-IS141 clones, that the fitness phenotype of these recombinants was determined by new beneficial intra genomic interaction rather than by a direct effect of specific mutations. Gene silencing is suspected to be involved in the positive selection of these recombinants because Ty-1 is a RNA dependent RNA polymerase gene

Assessing the possible maintenance of TYLCV-satellite association. [O.21]

Viruses of the genus Begomovirus (Family Geminiviridae) are frequently detected with half genome size DNA molecules, either defective DNAs or satellite DNA (? or ?). Whereas some begomoviruses, like Tomato yellow leaf curl virus (TYLCV) were never detected with satellite DNAs, other begomoviruses, like Cotton leaf curl virus (CLCuV), depend upon a betasatellite for their infectivity. Besides the CLCuV-type begomoviruses which may be considered as bipartite begomoviruses, most of the begomoviruses detected with satellites were shown to be infective without their satellites. The alphasatellite was rarely proved to have any impact on the helper virus but the betasatellite was often shown to increase the virulence of its helper virus. Although satellites were never detected with TYLCV in natural conditions, TYLCV was reported as a helper virus for both satellites in artificial conditions and its virulence was dramatically increased when co-inoculated with betasatellites. We have confirmed these results with the Cotton leaf curl Gezira betasatellite (CLCuGB) and two alphasatellites, Cotton leaf curl Gezira alphasatellite (CLCuGA) and Okra leaf curl Burkina Faso alphasatellite (OLCA). If the co-infection of TYLCV and a betasatellite would occur in natural conditions, tomato production may be severely affected. As the probability of such a scenario mainly depends on the maintenance of TYLCV-satellite associations over time, we have studied various factors potentially determining this maintenance: (i) the relative intra-plant accumulation of TYLCV and the satellites, (ii) the cellular co-infection level of TYLCV and satellites, and (iv) the transmission efficiency of satellites by the vector Bemisia tabaci. These various factors were analyzed with CLCuGB, CLCuGA and OLCA. Besides the specific question of the possible maintenance of satellites with TYLCV, the results of our study are expected to provide a new insight on begomoviruses detected in co-infection with satellites in natural conditions, but which were proved to be infectious without satellites. (Résumé d'auteur

Mise au point de deux tests PCR multiplexes pour détecter simultanément des formes recombinantes et non-recombinantes du Tomato yellow leaf curl virus au Maroc

Le Tomato yellow leaf curl virus (TYLCV, genre Begomvirus, famille Geminiviridae) a été isolé pour la première fois au Maroc en 1998 (Peterschmitt et al., 1999) et le Tomato yellow leaf curl Sardinia virus (TYLCSV) en 1999 (Monci et al., 2000). La gravité des attaques de la tomate par ces virus a obligé les agriculteurs à cultiver la tomate sous serre insectproof et à progressivement remplacer les variétés traditionnelles par des variétés tolérantes. Le TYLCV et le TYLCSV étant des virus hautement recombinogènes (Garcia Andres et al., 2007), le risque d'une émergence de recombinants TYLCV/TYLCSV dans une zone infestée par les deux virus était considéré comme très élevé. Pour confirmer ce risque, nous avons mis au point deux tests PCR multiplexes permettant l'identification de tels recombinants. De plus, pour obtenir une première estimation du potentiel d'émergence de ces recombinants ? les recombinants sont-il détectés seuls ou en co-infection avec les parents ? ? les tests ont été conçus pour cibler simultanément les recombinants potentiels et les virus parentaux. Outre la description de la technique, nous présenterons aussi quelques résultats préliminaires qui ont permis de valider la technique. (Texte intégral

Emergence of a recombinant Tomato yellow leaf curl virus and displacement of parental-type viruses in Morocco. [O.01]

Genetic recombination is a major mechanism of virus evolution and it is sometimes associated with serious epidemics especially within the Geminiviridae family. Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) are two geminiviruses of the genus Begomovirus which severely affect tomato crops in the Mediterranean basin. In Morocco, the epidemics caused by these two viruses have been successfully managed by controlling the insect vector, Bemisia tabaci, and by the use of symptomless tomato plants of cultivars bearing the resistant gene Ty1. As virus multiplication is only reduced but not completely abolished in those plants, it was expected that viruses may evolve under the selection pressure exerted by Ty1. In 2010, TYLCV/TYLCSV recombinants were detected in symptomatic Ty1 tolerant cultivars. Further surveys carried out since 2012 evidenced that one of them, IS76, was widespread and had virtually displaced parental-type viruses in the Souss, the region with the highest tomato production of Morocco. As IS76 was not detected in old samples collected before 2003, we hypothesized that its emergence might be associated with the increasing use of tolerant cultivars in the years 2000. Using Bayesian inference with a temporally structured TYLCV and IS76 sequence dataset, the approximate date of occurrence of the recombination event leading to IS76 was determined and found to be consistent with such hypothesis. To further support this hypothesis, the fitness of IS76 was compared with that of potential parental viruses of TYLCV and TYLCSV in controlled conditions. Taking viral DNA accumulation as a proxy for fitness, IS76 and parental viruses were compared in a susceptible and a tolerant Ty1 bearing cultivar. IS76 accumulated significantly more than its parental viruses in co-infected tolerant plants. This advantage did not pay any cost on coinfecting susceptible cultivars in which the accumulation of IS76 was similar to those of the parental viruses. Further studies are needed to understand the genetic determinism of the fitness advantage of IS76. (Résumé d'auteur

Emergence and reemergence of tomato begomoviruses in the North-East of Morocco

In Morocco, Tomato yellow leaf curl disease (Tylc) has emerged in 1997 [1]. Two begomovirus species causing Tylc were identified, Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) [2]. Due to cold winters in North East of Morocco, tomato cannot be grown year round and the populations of the vector Bemisia tabaci are drastically reduced during this season. However, Tylc reemerges every year in the new tomato crops in July and incidences may reach up to 100% in September. The objective of the study was: (i) to assess the relative importance of TYLCV and TYLCSV, (ii) to understand how these viruses are overwintering, and (iii) to estimate the risk of recombinant emergence between TYLCV and TYLCSV. The relative incidence of TYLCV and TYLCSV was estimated with leaf samples collected from 2003 onwards. Among tomato plants exhibiting the typical Tylc symptoms, more than 99% were infected with TYLCV and less than 1% with TYLCSV. Among the weed plants which were detected positive for TYLCV or TYLCSV, 85% were infected with TYLCSV and 15% with TYLCV. The tests of some tomato samples collected in 2001 revealed that shortly after the emergence of Tylc, TYLCSV was detected more frequently, suggesting that TYLCV has partially displaced TYLCSV from tomato. Leaf samples were also collected in winter to detect potential reservoirs of Tylc. TYLCSV was detected on several weeds and on two winter crops, pea and faba bean. However, TYLCV was detected only on the two cultivated hosts and only in mixed infection with TYLCSV. The reemergence of TYLCV in tomato may be due to its overwintering in pea and faba bean. The risk of emergence of TYLCV/TYLCSV recombinants is high, because several plants species were found to be coinfected with these two viruses: tomato, winter crops, and weeds. The risk is increased by the fact that the winter survival of TYLCV, the most damaging virus of tomato, is apparently dependent of its switching to plant species which are the preferred hosts of TYLCSV. We are presently looking for recombinants in field samples of cultivated or weed plants which were detected positive for both TYLCV and TYLCSV. (Résumé d'auteur

TYLCV-IS76 and TYLCV-IS141: fraternal but not identical tomato yellow leaf curl twin recombinant viruses

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

Molecular characterization of Euphorbia caput-medusae stunt virus: Evidence for the existence of a new genus within the family Geminiviridae

Studies focusing on phytoviruses isolated from the wild are rare. Nevertheless, even if those studies remain scarce, it is increasingly accepted that viruses coming from wild plants might play a role on disease emergence and in the functioning of ecosystems. Geminivirus are a major cause of disease on plants of agronomic interest. We hypothesize that strengthening our knowledge of the geminivirus diversity coming from the wild could help reconstructing the evolutionary history of the Geminiviridae family but also could help us understanding and predicting future epidemics. Over the past two decades, rolling circle amplification (RCA) has been more and more employed for the detection of small circular single-stranded DNA viruses, including geminivirus coming from the wild 1. We have used this method for detecting the presence of ssDNA from 236 plants collected in the South African fynbos. We have obtained amplified DNAs from 36% of the plants (85 out of 236 plants). Using classical cloning and sequencing methods, we have obtained ten sequences of which one was identified as a plant virus. This viral sequence corresponds to a new geminivirus, which infects a wild spurge (Euphorbia caput-medusae). This geminivirus is highly divergent from the current known members of the family Geminiviridae and is likely to represent a new previously unknown genus of this agriculturally highly relevant family of viruses. The virus, which we have named Euphorbia caput-medusae stunt virus (EcmSV) is not obviously a recombinant of viruses in the known geminivirus genera, has features most similar to viruses in the genus Mastrevirus (the presence of a repA gene and the production transcripts that are almost certainly spliced), but it also has unique features among geminiviruses (potential product of spliced V2-V3 ORFs). Besides EcmSV providing new information on the evolutionary history of geminiviruses, its discovery stresses the need to better assess viral diversity at the interface between wild and cultivated areas (by in situ sampling) and to study viruses isolated from wild hosts for their potential to infect crop species and vice-versa (by in vitro experimentation). 1. Varsani,A. et al. A highly divergent South African geminivirus species (Texte intégral