C. Wipf-Scheibel

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.

Serological and molecular variability of watermelon mosaic virus (genus Potyvirus)

SummaryWatermelon mosaic virus (WMV, genus Potyvirus) is very common in cucurbits worldwide, but its variability has been little studied. In France, where WMV has been known since 1974, unusually severe symptoms on zucchini squash have been found to be associated with WMV since 1999. We have developed serological and molecular tools to study WMV variability and the origin of severe strains. Eight monoclonal antibodies were obtained, characterized by epitope mapping, and used to assess the serological variability of 42 isolates from different countries. Sequence analysis based on the NIb-CP region revealed an important variability, with three distinct molecular groups. These analyses also suggested frequent intraspecific recombination in WMV.

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.

Molecular epidemiology of Zucchini yellow mosaic virus in France: an historical overview.

Cucurbit viruses are involved in complex and changing pathosystems in France, with new virus strains or species regularly reported. Zucchini yellow mosaic virus (ZYMV) is an archetypal emerging virus that was reported in France in 1979. It has since caused sporadic but occasionally very severe economic losses and its epidemiology still remains poorly understood. Partial sequencing of the viral genome has been used to characterize ZYMV isolates that occurred over a 29-year period in experimental plots at Montfavet, France (n=227), or that were received through a national survey for cucurbit viruses conducted in France from 2004 to 2007 (n=198). A total of 34 haplotypes were differentiated belonging to five molecular groups, three including isolates already described in France and two corresponding to isolates that emerged in France within the last 5 years. Comparison of haplotypes found at one location during successive years revealed contrasting situations. When they were either the same or closely related haplotypes, this suggested the availability of overwintering hosts, whereas when they belonged to different molecular groups this indicated shifts in viral populations with possible new introductions. The contribution of molecular epidemiology in tracing the origin of ZYMV in the French West Indies is also reviewed.

Transmission of cucumber leaf spot virus by Olpidium radicale

The ability of zoospores of four cultures of Olpidium radicale and one of O. brassicae to transmit viruses acquired in vitro from dilute virus solutions was compared. Transmission was demonstrated by infectivity and serological assays of the roots of cucumber seedlings 6 days after inoculation. A bulk culture of O. radicale, from cucumber plant roots collected near Nantes, France, a single-sporangial culture derived from it, and a single-sporangial culture from melon plant roots collected near Woodland, California, U.S.A., transmitted cucumber leaf spot virus (CLSV) and the cucumber fruit streak strain of CLSV (CLSV-FS). A bulk culture of O. radicale from melon plant roots collected at Montfavet, France, did not transmit CLSV or CLSV-FS. All four cultures transmitted cucumber necrosis and melon necrotic spot viruses, used as positive controls, but they did not transmit cucumber soil-borne, squash necrosis, petunia asteroid mosaic or tobacco necrosis viruses. In each of the trials a single-sporangial culture of O. brassicae from lettuce plant grown in California transmitted only tobacco necrosis virus.

Biological characterization and complete nucleotide sequence of a Tunisian isolate of Moroccan watermelon mosaic virus

SummaryDuring a survey conducted in October 2005, cucurbit leaf samples showing virus-like symptoms were collected from the major cucurbit-growing areas in Tunisia. DAS-ELISA showed the presence of Moroccan watermelon mosaic virus (MWMV, Potyvirus), detected for the first time in Tunisia, in samples from the region of Cap Bon (Northern Tunisia). MWMV isolate TN05-76 (MWMV-Tn) was characterized biologically and its full-length genome sequence was established. MWMV-Tn was found to have biological properties similar to those reported for the MWMV type strain from Morocco. Phylogenetic analysis including the comparison of complete amino-acid sequences of 42 potyviruses confirmed that MWMV-Tn is related (65% amino-acid sequence identity) to Papaya ringspot virus (PRSV) isolates but is a member of a distinct virus species. Sequence analysis on parts of the CP gene of MWMV isolates from different geographical origins revealed some geographic structure of MWMV variability, with three different clusters: one cluster including isolates from the Mediterranean region, a second including isolates from western and central Africa, and a third one including isolates from the southern part of Africa. A significant correlation was observed between geographic and genetic distances between isolates. Isolates from countries in the Mediterranean region where MWMV has recently emerged (France, Spain, Portugal) have highly conserved sequences, suggesting that they may have a common and recent origin. MWMV from Sudan, a highly divergent variant, may be considered an evolutionary intermediate between MWMV and PRSV.

First Report of Tomato torrado virus in Tomato Crops in France

In June 2008, tomato (Solanum lycopersicum L.) plants cv. Fer De Lance (De Ruiter Seeds, Bergschenhoek, the Netherlands) grown in greenhouses near Perpignan (southern France) showed growth reduction and necrotic lesions on fruits, stems, and basal parts of the leaves. Tomato torrado virus (ToTV) was suspected on the basis of symptoms and its recent description in Spain (4). Primer set A (3), designed to ToTV RNA-2, was used for reverse transcription (RT)-PCR experiments on RNA extracted from four infected plants and allowed the amplification of a 493-bp fragment. No amplification was observed from healthy plant extracts. The RT-PCR product was directly sequenced (GQ303330) and a BLAST search in GenBank revealed 99.8- and 99.5%-nt identity with Polish (EU563947) and Spanish type strain (DQ388880) isolates of ToTV, respectively. Double-antibody sandwich-ELISA tests were conducted on these four samples to check for the presence of other viruses commonly found in tomato crops in France. Tomato spotted wilt virus, Parietaria mottle virus, Cucumber mosaic virus, Tomato mosaic virus, and Potato virus Y were not detected but Pepino mosaic virus (PepMV) was detected in all samples. ToTV was mechanically transmitted to Physalis floridana but PepMV was not. This plant was used to inoculate healthy tomatoes that served as a ToTV source for further experiments. Mechanical inoculation to test plants showed that Nicotiana benthamiana, N. clevelandii, N. debneyi, N. glutinosa, Capsicum annuum, Solanum melongena, and some tomato cultivars (including Fer De Lance), in which typical necrotic symptoms were observed, were systemically infected by the virus. Isometric particles ~28 nm in diameter were observed by electron microscopy in crude extracts of infected plants negatively stained with 1% ammonium molybdate, pH 7. To confirm ToTV identification, whitefly transmission experiments were performed with Trialeurodes vaporariorum and Bemisia tabaci. Adult whiteflies were placed in cages with infected tomato plants for 1-, 24-, or 48-h acquisition access periods (AAP) before transferring them by groups of ~50 on susceptible tomato plantlets placed under small containers (six plants per AAP). Forty-eight hours later, plants were treated with an insecticide and transferred to an insect-proof containment growth room. Ten days later, RNA preparation from all plants was tested by RT-PCR for the presence of ToTV. No transmission was observed with a 1-h AAP. With a 24-h AAP, transmission to four of six test plants was observed with both whitefly species, while at 48 h, AAP transmission to three and four plants of six was observed with T. vaporariorum and B. tabaci, respectively. Noninoculated control plants were all negative by RT-PCR. These experiments confirm T. vaporariorum and B. tabaci as natural vectors of ToTV as previously described (1,2). ToTV has been already reported in Spain, Poland, Hungary, and Australia, but to our knowledge, this is the first report of ToTV in France. Our detection of ToTV in April 2009 from the same area revealed 7 positive tomato plants of 17 tested. This observation suggests the persistence of the disease in the Perpignan Region. References: (1) K. Amari et al. Plant Dis. 92:1139, 2008. (2) H. Pospieszny et al. Plant Dis. 91:1364, 2007 (3) J. Van der Heuvel et al. Plant Virus Designated Tomato Torrado Virus. Online publication. World Intellectual Property Organization WO/2006/085749, 2006. (4) M. Verbeek et al. Arch. Virol. 152:881, 2007.

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.

Characterization and occurrence of squash chlorotic leaf spot virus, a tentative new torradovirus infecting cucurbits in Sudan

During a survey conducted in Sudan in 2012, a virus with spherical particles was isolated from a squash plant showing chlorotic leaf spots. The virus was transmitted mechanically and by two whitefly species, but not by aphids. RT-PCR with generic torradovirus primers yielded a band of expected size from total RNA of a symptomatic plant. Next-generation sequencing confirmed that this is tentatively a new torradovirus, for which we propose the name ‘squash chlorotic leaf spot virus’. Using specific RT-PCR primers, the virus was detected in cucurbit samples collected since 1992 at different locations in Sudan.

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.