First Report of Tomato yellow leaf curl virus in Tomato in Costa Rica.

One of the most important invasive and harmful members of the genus Begomovirus (family Geminiviridae) is the monopartite Tomato yellow leaf curl virus (TYLCV), which is widespread over the world associated with tomato yellow leaf curl disease (TYLCD). Tomato (Solanum lycopersicum) plants infected with TYLCV show upward leaf curling and yellowing. In Latin America, isolates of TYLCV have been reported from Cuba, the Dominican Republic, Mexico and Puerto Rico (1), Guatemala (GenBank Accession No. GU355941), and Venezuela (partial genome sequence DQ302033). In Costa Rica, only isolates of the bipartite begomoviruses Tomato leaf curl Sinaloa virus (TLCSiV) (3) and Tomato yellow mottle virus (KC176780, KC176781) have been reported infecting tomatoes. During a survey conducted in 2012, similar begomovirus-like symptoms (leaf yellowing and upward leaf curling) were observed in tomato plants of five commercial growing areas in the Central Valley (Grecia region) of Costa Rica. In total, 65 tomato samples were randomly collected, 14 from greenhouses and 41 from open fields. Symptoms of upward leaf curling and yellowing were observed in three samples. Total DNA was extracted from collected samples and tested by dot blot hybridization using a probe to the coat protein (CP) gene of a Guatemalan isolate of Bean golden yellow mosaic virus (3). Only the three symptomatic samples tested positive, which represents an incidence of 14% (2 samples) in greenhouses and 2.4% (1 sample) in open field crops. These samples were subjected to rolling circle amplification (RCA) for viral circular genome amplification (2). The amplified products were then digested with MspI restriction endonuclease, which resulted into DNA fragments of 2,320 and 458 bp for all three samples. This suggested infection with a monopartite begomovirus. In order to obtain the full-length clone, the RCA product of two samples (5240 and 5241) was digested with BamHI, and the ~2.8 kb DNA fragment was cloned into pBluescript II SK(+) (Stratagene, La Jolla, CA) vector. After transformation of Escherichia coli DH5α, recombinant plasmids with inserts of expected size were selected and the insert was sequenced by primer walking (Macrogen Inc., Korea). The inserts of three clones from the two samples (CR:5240-16:2012, CR:5240-17:2012, and CR:5241-14:2012) were sequenced (deposited in GenBank as KF533855, KF533856, and KF533857, respectively). Sequences were all 2,781 nt long and shared 100% identity between themselves (1-nt mismatch between CR:5240-16:2012 and CR:5240-17:2012, and CR:5240-16:2012 and CR:5241-14:2012; and 2-nt mismatches between CR:5240-17:2012 and CR:5241-14:2012) and 99% with the sequence of Tomato yellow leaf curl virus-Israel[Japan:Haruno:2005] (TYLCV-IL[JR:Har:05]) (AB192966). These sequences represented full length genomes of isolates of the monopartite begomovirus TYLCV-IL and grouped in a phylogenetic clade (4) that comprised TYLCV-IL isolates reported from Asia (China and Japan) and from Mexico, while more distantly related to the clade comprising TYLCV-IL isolates reported from Central America (Cuba, Guatemala, Puerto Rico) and the United States, suggesting a distinct introduction event in Costa Rica. This is the first report of the presence of TYLCV in Costa Rica, therefore it is imperative to study the incidence and geographical spread of this virus in the country as well as its genetic diversity, since TYLCV infections might lead to significant yield losses, as reported in other countries. References: (1) A. M. Idris et al. Plant Dis. 83:303, 1999. (2) A. K. Inoue-Nagata et al. J. Virol. Methods 116:209, 2004. (3) M. K. Nakla et al. Acta Hortic. 695:277, 2005. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

First Detection of Tomato leaf curl New Delhi virus Infecting Zucchini in Spain.

In September 2012, a novel disease syndrome was observed in zucchini (Cucurbita pepo L.) crops in Murcia Province (southeastern Spain). Symptoms included curling, vein swelling, and severe mosaic in young leaves, short internodes, and fruit skin roughness, resembling begomovirus infection. Similar symptoms were observed in May 2013 in Almería Province (southern Spain). DNA was isolated from 8 and 7 symptomatic leaf samples collected in Murcia and Almería, respectively, and analyzed by PCR with primers GemCP-V-5' and GemCP-C-3' designed to detect begomoviruses by amplifying the core of coat protein gene (CP) (3). DNA fragments of the expected size (~600 bp) were amplified supporting a begomovirus infection. The DNA sequences obtained from four samples were identical. BLAST analysis showed the highest nucleotide identity (98%) with partial CP gene sequences from isolates of Tomato leaf curl New Delhi virus (ToLCNDV) infecting cucumber in India (GenBank Accession No. KC846817). ToLCNDV, a bipartite begomovirus first reported from tomato, also infects other solanaceous and cucurbitaceous crops in India and neighboring countries (1). DNA from two samples from Murcia and three from Almería was used for rolling-circle amplification using ϕ29 DNA polymerase (TempliPhi kit, GE Healthcare, Little Chalfont, UK) and digested with a set of restriction endonucleases. All five samples yielded amplification products with identical restriction patterns. Two samples from Murcia (MU-8.1 and MU-11.1) and one from Almería (AL-661) were selected to clone the putative DNA-A and DNA-B begomovirus genome components by using single BamHI or NcoI sites. Inserts of two clones from each sample, one corresponding to DNA-A and one to DNA-B, were completely sequenced. The cloned genomes exhibited the typical organization of Old World bipartite begomoviruses (1). Sequences were aligned with begomovirus sequences available in databases using MUSCLE and pairwise identity scores were calculated with SDT (species demarcation tool [4]). DNA-A sequences obtained from Murcia (2,738 nt, KF749224 and KF749225) and Almería (2,738 nt, KF749223) shared >99% nucleotide identity, with the highest nucleotide identity (91.3 to 91.5%) with that of an Indian ToLCNDV isolate from chilli (HM007120). DNA-B sequences (2,684 nt, KF749226, KF749227, and KF749228) shared >99% nucleotide identity, and showed the highest nucleotide identity (83.1 to 83.3%) with that of a Pakistani ToLCNDV isolate from Solanum nigrum (AJ620188). Nucleotide sequence identity of DNA-A with the most closely related begomoviruses was above the 91% threshold for species demarcation (2), thus confirming that the begomoviruses found infecting zucchini in Spain are isolates of ToLCNDV. In fall 2013, the disease was widespread in zucchini both in Murcia and Almería, and ToLCNDV has also been found infecting melon and cucumber crops. To our knowledge, this is the first report of a bipartite begomovirus in Spain and Europe. References: (1) J. K. Brown et al. Page 351 in: Virus Taxonomy. Ninth Report of the ICTV. A. M. Q. King et al., eds. Elsevier/Academic Press, London, 2012. (2) ICTV Geminiviridae Study Group. New species and revised taxonomy proposal for the genus Begomovirus (Geminiviridae). ICTV. Retrieved from http://talk.ictvonline.org/files/proposals/ taxonomy_proposals_plant1/m/plant04/4720.aspx , 10 October 2013. (3) H. Lecoq and C. Desbiez. Adv. Virus Res. 84:67, 2012. (4) B. Muhire et al. Arch. Virol. 158:1411, 2013.

Fulfilling Koch's postulates confirms the monopartite nature of tomato leaf deformation virus: a begomovirus native to the New World.

The monopartite nature of the begomovirus tomato leaf deformation virus (ToLDeV) reported in Peru is demonstrated here. The DNA molecule cloned from an infected plant was shown to be fully infectious in tomatoes inducing leaf curling and stunted growth similar to that observed in field-infected plants. The viral DNA was reisolated from systemically infected tissues of inoculated plants, thus fulfilling Koch's postulates. ToLDeV was demonstrated, therefore, as the causal agent of the disease syndrome widespread in tomato crops in Peru. This virus was shown to be present throughout the major tomato-growing regions of this country, both in tomatoes and wild plants. Analyses of the sequences of 51 ToLDeV isolates revealed a significant genetic diversity with three major genetic types co-circulating in the population. A geographical segregation was observed which should be taken into account for virus control. Constraints to genetic divergence found for the C4 gene of ToLDeV isolates suggest a relevant function for this protein. The results obtained confirm ToLDeV as a monopartite begomovirus native to the New World, which is a significant finding for this region.

First Report of Pepper vein yellows virus Infecting Sweet Pepper in Spain.

In November 2012, unusual symptoms were observed in plants of sweet pepper (Capsicum annuum L.) grown in commercial greenhouses of Almería Province, southeastern Spain. Symptoms included interveinal yellowing, upward leaf curling, and internode shortening, and were more evident in the upper part of the plant. Abnormal ripening of fruits was observed in symptomatic plants, with fruits remaining orange in the red varieties and yellow in the orange varieties, thus reducing their marketability. During December 2012 and January 2013, severe outbreaks of this disease syndrome occurred, with many greenhouses exhibiting almost 100% incidence. The symptoms observed were similar to those reported for isolates of Pepper vein yellows virus (PeVYV, genus Polerovirus, family Luteoviridae) (previously also named Pepper yellow leaf curl virus [PYLCV] and Pepper yellows virus [PYV]) (2,4). Twenty five symptomatic leaf and/or fruit samples (some of them supplied by Zeraim Ibérica, S.A.), each from a different greenhouse, were analyzed and all reacted positively in double-antibody sandwich-ELISA with an antiserum against the polerovirus Cucurbit aphid-borne yellows virus (CABYV) (Sediag, Longvic, France), known to cross-react with PeVYV (2). Total RNA was extracted by TRIsure reagent (Bioline, London, United Kingdom) from symptomatic leaves and analyzed by reverse transcription (RT)-PCR with primers Pol-G-F (5'-GAYTGCTCYGGYTTYGACTGGAG-3') and Pol-G-R (5'-GATYTTATAYTCATGGTAGGCCTTGAG-3') designed for universal detection of poleroviruses by amplifying the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) partial genes (3). DNA fragments of the expected size (1.1 kbp) were amplified supporting a polerovirus infection in all the analyzed samples. The PCR product obtained from one sample (Almería-1) was extracted from agarose gel with a QIAquick gel extraction kit (Qiagen, Hilden, Germany), cloned in pGEM-T Easy vector (Promega, Madison, WI), and one clone was sequenced (Macrogen Inc., Seoul, South Korea). The PCR products amplified from three other samples (2-13, 7-13, and 8-13) were directly sequenced. The nucleotide identity between the amplified fragments (GenBank Accession Nos. KC769487, KC839992 to 94), calculated after alignment with ClustalW, was 99.7 to 100%. The highest nucleotide identity of the Spanish sequences was with a PeVYV isolate from Turkey (FN600344, named as PYV) (98.5 to 98.7%). The spread of PeVYV in Spain is additional evidence of the emergence of this virus as a global threat for pepper crops after its first detection in Japan in 1995 and recent reports from the Mediterranean Basin (1,2). References: (1) N. Buzkan et al. Arch. Virol. 158:881, 2013. (2) A. Dombrovsky et al. Phytoparasitica 38:477, 2010. (3) D. Knierim et al. Plant Pathol. 59:991, 2010. (4) R. Murakami et al. Arch. Virol. 156:921, 2011.

Whitefly resistance traits derived from the wild tomato Solanum pimpinellifolium affect the preference and feeding behavior of Bemisia tabaci and reduce the spread of Tomato yellow leaf curl virus.

Breeding of tomato genotypes that limit whitefly (Bemisia tabaci) access and feeding might reduce the spread of Tomato yellow leaf curl virus (TYLCV), a begomovirus (genus Begomovirus, family Geminiviridae) that is the causal agent of tomato yellow leaf curl disease. TYLCV is restricted to the phloem and is transmitted in a persistent manner by B. tabaci. The tomato breeding line ABL 14-8 was developed by introgressing type IV leaf glandular trichomes and secretion of acylsucroses from the wild tomato Solanum pimpinellifolium accession TO-937 into the genetic background of the whitefly- and virus-susceptible tomato cultivar Moneymaker. Results of preference bioassays with ABL 14-8 versus Moneymaker indicated that presence of type IV glandular trichomes and the production of acylsucrose deterred the landing and settling of B. tabaci on ABL 14-8. Moreover, electrical penetration graph studies indicated that B. tabaci adults spent more time in nonprobing activities and showed a reduced ability to start probing. Such behavior resulted in a reduced ability to reach the phloem. The superficial type of resistance observed in ABL 14-8 against B. tabaci probing significantly reduced primary and secondary spread of TYLCV.

First Report of Tomato chlorosis virus Infecting Tomato in Sudan.

In March 2011, interveinal yellowing and necrosis symptoms on middle and lower leaves were observed in tomato (Solanum lycopersicum L., cv. Castle Rock) plants grown in three adjacent greenhouses of the Agricultural Research Corporation at Wad Medani (Gezira State, Sudan). These symptoms resembled those caused by Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV) (4) (genus Crinivirus, family Closteroviridae). Whitefly (Bemisia tabaci) infestation was also observed in these greenhouses. Total RNA was extracted by TRIzol Reagent (Invitrogen, Carlsbad, CA) from symptomatic leaves and analyzed by dot-blot hybridization with digoxigenin-labelled RNA probes to the coat protein (CP) gene of ToCV and to the minor coat protein (CPm) gene of TICV. Positive signal was obtained only with the ToCV probe. Reverse transcription (RT)-PCR reactions were performed with two pairs of primers specific for the detection of ToCV, MA380(+) (5'-GTGAGACCCCGATGACAGAT-3') and MA381(-) (5'-TACAGTTCCTTGCCCTCGTT-3'), specific to the CP gene (ToCV RNA 2) (3), and MA396(+) (5'-TGGTCGAACAGTTTGAGAGC-3') and MA397(-) (5'-TGAACTCGAATTGGGACAGA-3'), specific to the RNA-dependent RNA polymerase (RdRp) gene (ToCV RNA 1) (1). DNA fragments of the expected sizes (436 and 763 bp, respectively) were obtained, thus supporting the presence of ToCV in the symptomatic samples. Amplified DNA fragments were cloned in pGEM-T Easy vector (Promega, Madison, WI) and one clone per amplicon was sequenced (Macrogen Inc., Seoul, South Korea). The highest nucleotide sequence identity of the CP gene fragment obtained (GenBank Accession No. JN411685) was 99.2% related with North American ToCV isolates from Florida (DQ234674), Colorado (DQ234675), and Georgia (HQ879842), while the RdRp gene fragment (JN411686) was more closely related (99.0%) to the Spanish AT80/99 isolate (DQ983480). Although yellowing symptoms similar to those reported here have been observed sporadically during the last few years in open-field tomato crops in the state of Gezira, additional studies are needed to determine the prevalence and economic impact of ToCV infections in tomato cultivation in Sudan. To our knowledge, ToCV has been found in continental Africa only in Morocco and South Africa, in the Mediterranean climate areas in the northern and southern edges of the continent, respectively (2). The finding of ToCV infecting tomato in Sudan raises the question of whether this virus is emerging also in other tropical areas of the continent and illustrates the need to monitor whitefly-infested areas within Africa for the presence of ToCV. References: (1) G. Lozano et al. J. Virol. 83:12973, 2009. (2) J. Navas-Castillo et al. Annu. Rev. Phytopathol. 49:219, 2011. (3) H. P. Trenado et al. Eur. J. Plant Pathol. 118:193, 2007. (4) G. C. Wisler et al. Plant Dis. 82:270, 1998.

Multiple Resistance to Meloidogyne spp. and to Bipartite and Monopartite Begomovirus spp. in Wild Solanum (Lycopersicon) Accessions.

The Ty-1 locus confers tolerance to monopartite and bipartite Begomovirus spp. (genus Begomovirus, family Geminiviridae) and this phenotype is improved in homozygous tomato lines. However, the gene Mi (Meloidogyne spp. resistance) is in repulsion phase linkage with Ty-1, which hampers the large-scale development of multiresistant inbred lines. Seventy-one Solanum (section Lycopersicon) accessions were whitefly inoculated with the bipartite Begomovirus sp. Tomato rugose mosaic virus (ToRMV) and simultaneously infested with a mixture of Meloidogyne incognita and M. javanica under greenhouse conditions in Brazil. Accessions were then transplanted into a nematode-infested field with natural ToRMV infection. A severity index was used to evaluate ToRMV reaction. Nematode evaluation was done by counting the number of galls per root system. Seventeen accessions with Meloidogyne spp. and ToRMV resistance were selected and evaluated in Spain against three monopartite Begomovirus spp. associated with the tomato yellow leaf curl virus disease, using infectious clones. Systemic infection was monitored by DNA hybridization. Five S. peruvianum accessions (PI-306811, PI-365951, LA-1609, LA-2553, and CNPH-1194) displayed nematode and broad-spectrum resistance to all Begomovirus spp. tested in both continents. From the breeding standpoint, accessions combining resistance to Meloidogyne spp. and to bipartite and monopartite Begomovirus spp. would be useful for the development of elite lines expressing all traits in homozygous condition.

Resistance-driven selection of begomoviruses associated with the tomato yellow leaf curl disease.

Tomato yellow leaf curl disease (TYLCD) causes severe damage to tomato crops worldwide. The deployment of host-plant resistance is the most desirable mean to control this disease. However, some concerns exist because it may place a selection pressure on the virus. Field and experimental data are provided which suggest that the use of TYLCD resistance in tomato crops might have contributed to the emergence of tomato yellow leaf curl virus in the TYLCD-associated virus population, a virus species that fits better in the resistant genotypes. Emergence of recombinant variants was observed during mixed infections of TYLCD-associated viruses in Ty-1 resistant plants, as already observed for susceptible tomatoes. Therefore, selection may be occurring for virus variants with novel genome combination to infect the resistant genotypes with this resistance gene.

Phenotypic expression, stability, and inheritance of a recessive resistance to monopartite begomoviruses associated with tomato yellow leaf curl disease in tomato.

Tomato-infecting begomoviruses comprise a complex of monopartite and bipartite virus species that cause severe yield and quality losses worldwide. Therefore, the availability of wide spectrum resistance for begomovirus control is desirable. However, limited sources of resistance are available. In this study, three tomato inbred lines with resistance to bipartite begomoviruses of Brazil were tested for resistance to monopartite begomoviruses associated with the tomato yellow leaf curl disease (TYLCD). Stable resistance to Tomato yellow leaf curl virus was observed either by inoculation with Bemisia tabaci or with Agrobacterium tumefaciens using an infectious clone. The resistance resulted in a complete absence of TYLCD symptoms and restricted virus accumulation. Further studies performed with the line '468-1-1-12' indicated that the resistance was also effective against three other virus species associated with TYLCD, indicating wide spectrum resistance of this source. Quantitative genetics analyses suggested that a major recessive locus with epistatic interactions is controlling the resistance to TYLCD in '468-1-1-12', which could facilitate introgression of this trait into elite tomato lines. The resistance was stable under field conditions with high TYLCD pressure. Mild symptoms could be observed in these conditions, and recovery from disease and from virus infection suggested an active host antiviral defense mechanism. The differential reaction of '468-1-1-12' against a number of TYLCD-associated viruses and artificial chimeras between them allowed to identify a region of the virus genome that presumably contains a virus determinant for breaking the resistance to infection observed in '468-1-1-12'.

Recommendations for the classification and nomenclature of the DNA-beta satellites of begomoviruses.

The symptom-modulating, single-stranded DNA satellites (known as DNA-beta) associated with begomoviruses (family Geminiviridae) have proven to be widespread and important components of a large number of plant diseases across the Old World. Since they were first identified in 2000, over 260 full-length sequences (approximately 1,360 nucleotides) have been deposited with databases, and this number increases daily. This has highlighted the need for a standardised, concise and unambiguous nomenclature for these components, as well as a meaningful and robust classification system. Pairwise comparisons of all available full-length DNA-beta sequences indicate that the minimum numbers of pairs occur at a sequence identity of 78%, which we propose as the species demarcation threshold for a distinct DNA-beta. This threshold value divides the presently known DNA-beta sequences into 51 distinct satellite species. In addition, we propose a naming convention for the satellites that is based upon the system already in use for geminiviruses. This maintains, whenever possible, the association with the helper begomovirus, the disease symptoms and the host plant and provides a logical and consistent system for referring to already recognised and newly identified satellites.

Geminivirus strain demarcation and nomenclature.

Geminivirus taxonomy and nomenclature is growing in complexity with the number of genomic sequences deposited in sequence databases. Taxonomic and nomenclatural updates are published at regular intervals (Fauquet et al. in Arch Virol 145:1743-1761, 2000, Arch Virol 148:405-421, 2003). A system to standardize virus names, and corresponding guidelines, has been proposed (Fauquet et al. in Arch Virol 145:1743-1761, 2000). This system is now followed by a large number of geminivirologists in the world, making geminivirus nomenclature more transparent and useful. In 2003, due to difficulties inherent in species identification, the ICTV Geminiviridae Study Group proposed new species demarcation criteria, the most important of which being an 89% nucleotide (nt) identity threshold between full-length DNA-A component nucleotide sequences for begomovirus species. This threshold has been utilised since with general satisfaction. More recently, an article has been published to clarify the terminology used to describe virus entities below the species level [5]. The present publication is proposing demarcation criteria and guidelines to classify and name geminiviruses below the species level. Using the Clustal V algorithm (DNAStar MegAlign software), the distribution of pairwise sequence comparisons, for pairs of sequences below the species taxonomic level, identified two peaks: one at 85-94% nt identity that is proposed to correspond to "strain" comparisons and one at 92-100% identity that corresponds to "variant" comparisons. Guidelines for descriptors for each of these levels are proposed to standardize nomenclature under the species level. In this publication we review the status of geminivirus species and strain demarcation as well as providing updated isolate descriptors for a total of 672 begomovirus isolates. As a consequence, we have revised the status of some virus isolates to classify them as "strains", whereas several others previously classified as "strains" have been upgraded to "species". In all other respects, the classification system has remained robust, and we therefore propose to continue using it. An updated list of all geminivirus isolates and a phylogenetic tree with one representative isolate per species are provided.

Tomato torrado virus is Transmitted by Bemisia tabaci and Infects Pepper and Eggplant in Addition to Tomato.

Torrao or torrado is an emerging disease that is causing serious economic losses in tomato crops of southeastern Spain. The causal agent has been shown to be a new picorna-like plant virus, tentatively named Tomato torrado virus (ToTV) (4). By using trap tomato plants in a greenhouse affected by torrado located in the Murcia Region of Spain, we obtained a ToTV isolate (ToTV-CE) that we have biologically and molecularly characterized. Subtracted cDNA libraries (1) and expressed sequence tags sequencing were used to determine the partial nucleotide sequence of ToTV-CE. We covered ≈53% of the virus genome (GenBank Accession Nos. EU476181 and EU476182) and found that ToTV-CE RNAs 1 and 2 had a high nucleotide similarity (98 and 99%, respectively) with the ToTV published sequences (2,4). ToTV-CE sequences also showed a 70% nt similarity with those of Tomato apex necrosis virus, a newly identified virus in tomato crops of the Culiacan area (Sinaloa, Mexico) (3). To characterize the host range of ToTV-CE, 6 to 10 plants belonging to 14 species were mechanically inoculated with extracts from ToTV-CE-infected Nicotiana benthamiana plants. The presence of ToTV in these plants was analyzed at 3 and 6 weeks postinoculation (PI) by molecular hybridization in dot-blots. The determined host range was in agreement with that described earlier (2,4), but additional hosts and nonhosts were identified. Thus, the virus did not infect melon (Cucumis melo var. cantaloupe), cucumber (C. sativus cv. Marketmore), squash (Cucurbita pepo cv. Negro Belleza), Chenopodium album ssp. Amaranticolor, or Chenopodium quinoa. The virus infected systemically N. benthamiana, N. glutinosa, N. rustica, tobacco (N. tabacum cvs. Xanthi nc and Samsun), Physalis floridana, pepper (Capsicum annuum cv. Italian Long Sweet), tomato (Solanum lycopersicum cv. Boludo), and eggplant (S. melongena cv. Black Beauty). Pepper plants displayed severe symptoms of infection consisting of marked mosaics and stunting (but no necrosis), but eggplant remained asymptomatic for up to 6 weeks PI. A simple assay was devised to analyze whether ToTV can be transmitted by whiteflies. ToTV-CE-infected tomato plants were placed together with three to eight healthy tomato seedlings inside insect-proof glass boxes. Adult Bemisia tabaci (100 to 800 individuals in three replicates) or Trialeurodes vaporariorum (100 individuals in one replicate) were released into each box. For both treatments, symptoms typically induced by ToTV appeared in one to seven tomato seedlings by 1 week after the release of the whiteflies. ToTV infection was confirmed by molecular hybridization in tissue prints of petiole cross sections at 10 days PI. These data are in agreement with those reported by Pospieszny et al. (2) and strongly suggest that both B. tabaci and T. vaporariorum can transmit ToTV. References: (1) L. Diachenko et al. Proc. Natl. Acad. Sci. USA 93:6025, 1996. (2) H. Pospieszny et al. Plant Dis. 91:1364, 2007 (3) M. Turina et al. Plant Dis. 91:932, 2007. (4) M. Verbeek et al. Arch. Virol. 152:881, 2007.

Complete nucleotide sequence of the RNA2 of the crinivirus tomato chlorosis virus.

The complete sequence of genomic RNA2 of Tomato chlorosis virus (ToCV; genus Crinivirus, family Closteroviridae), isolate AT80/99 from Spain, was determined and compared with those from the other members of the genus sequenced to date. RNA2 is 8244 nucleotides (nt) long and putatively encodes nine ORFs that encompass the hallmark gene array of the family Closteroviridae, which includes a heat shock protein 70 family homologue, a 59 kDa protein, the coat protein, and a diverged coat protein. Phylogenetic analysis confirmed assignment of ToCV in the genus Crinivirus, being most similar to sweet potato chlorotic stunt virus and cucurbit yellow stunting disorder virus.

Pepper (Capsicum annuum) Is a Dead-End Host for Tomato yellow leaf curl virus.

ABSTRACT Tomato yellow leaf curl (TYLC) is one of the most devastating pathogens affecting tomato (Lycopersicon esculentum) worldwide. The disease is caused by a complex of begomovirus species, two of which, Tomato yellow leaf curl Sardinia virus (TYLCSV) and Tomato yellow leaf curl virus (TYLCV), are responsible for epidemics in Southern Spain. TYLCV also has been reported to cause severe damage to common bean (Phaseolus vulgaris) crops. Pepper (Capsicum annuum) plants collected from commercial crops were found to be infected by isolates of two TYLCV strains: TYLCV-Mld[ES01/99], an isolate of the mild strain similar to other TYLCVs isolated from tomato crops in Spain, and TYLCV-[Alm], an isolate of the more virulent TYLCV type strain, not previously reported in the Iberian Peninsula. In this work, pepper, Nicotiana benthamiana, common bean, and tomato were tested for susceptibility to TYLCV-Mld[ES01/99]and TYLCV-[Alm] by Agrobacterium tumefaciens infiltration, biolistic bombardment, or Bemisia tabaci inoculation. Results indicate that both strains are able to infect plants of these species, including pepper. This is the first time that infection of pepper plants with TYLCV clones has been shown. Implications of pepper infection for the epidemiology of TYLCV are discussed.

Variability and genetic structure of the population of watermelon mosaic virus infecting melon in Spain.

The genetic structure of the population of Watermelon mosaic virus (WMV) in Spain was analysed by the biological and molecular characterisation of isolates sampled from its main host plant, melon. The population was a highly homogeneous one, built of a single pathotype, and comprising isolates closely related genetically. There was indication of temporal replacement of genotypes, but not of spatial structure of the population. Analyses of nucleotide sequences in three genomic regions, that is, in the cistrons for the P1, cylindrical inclusion (CI) and capsid (CP) proteins, showed lower similar values of nucleotide diversity for the P1 than for the CI or CP cistrons. The CI protein and the CP were under tighter evolutionary constraints than the P1 protein. Also, for the CI and CP cistrons, but not for the P1 cistron, two groups of sequences, defining two genetic strains, were apparent. Thus, different genomic regions of WMV show different evolutionary dynamics. Interestingly, for the CI and CP cistrons, sequences were clustered into two regions of the sequence space, defining the two strains above, and no intermediary sequences were identified. Recombinant isolates were found, accounting for at least 7% of the population. These recombinants presented two interesting features: (i) crossover points were detected between the analysed regions in the CI and CP cistrons, but not between those in the P1 and CI cistrons, (ii) crossover points were not observed within the analysed coding regions for the P1, CI or CP proteins. This indicates strong selection against isolates with recombinant proteins, even when originated from closely related strains. Hence, data indicate that genotypes of WMV, generated by mutation or recombination, outside of acceptable, discrete, regions in the evolutionary space, are eliminated from the virus population by negative selection.

First Report of Sweet Pepper (Capsicum annuum) as a Natural Host Plant for Tomato chlorosis virus.

Since 1997, epidemics of a tomato yellowing disease have occurred in the Málaga and Almería provinces of southern Spain. These epidemics have been associated with infections of Tomato chlorosis virus (ToCV) (genus Crinivirus, family Closteroviridae) (2). During the past few years, an increasing incidence of the disease was observed and it spread to new areas including eastern Spain and the Balearic and Canary Islands (G. Lozano, E. Moriones, and J. Navas-Castillo, unpublished results and [1]). In 1999, plants of sweet pepper (Capsicum annuum L.) exhibiting symptoms of interveinal yellowing, mild upward leaf curling, and stunting were observed in greenhouses of Almería that were heavily infested with the whitefly, Bemisia tabaci. Symptomatic plants were tested for the presence of the begomovirus, Tomato yellow leaf curl virus, a virus previously reported in sweet pepper (3) by molecular hybridization or polymerase chain reaction (PCR). Some of these plants tested positive. Total RNA extracts from the symptomatic plants were also analyzed for the presence of tomato criniviruses using reverse transcription (RT)-PCR with primers MA59 and MA60 for the HSP70h gene (2). A PCR DNA product of the expected size (587 bp) was obtained from several samples. The cloning and sequencing of the PCR product obtained from one of these samples confirmed the presence of ToCV, with a sequence 100% identical to the equivalent region of the first ToCV isolated from tomato in Málaga (2). Total RNA extracts from plants that tested positive using RT-PCR were also positive with molecular hybridization using a probe for the HSP70h gene of ToCV. To our knowledge, this is the first report of sweet pepper as a natural host of a tomato crinivirus, which may have important epidemiological consequences in regions where both crops are grown. Association between ToCV infection and specific symptoms observed in sweet pepper plants is under study. References: (1) M. I. Font et al. Bol. San. Veg. Plagas 29:109, 2003. (2) J. Navas-Castillo et al. Plant Dis. 84:835, 2000. (3) J. Reina et al. Plant Dis. 83:1176, 1999.

Nucleotide sequence and infectious transcripts from a full-length cDNA clone of the carmovirus Melon necrotic spot virus.

We have studied the biological and molecular characteristics of a MNSV isolate collected in Spain (MNSV-Malpha5) and generated a full-length cDNA clone from which infectious RNA transcripts can be produced. The host range of MNSV-Malpha5 appeared to be limited to cucurbits and did not differ from that of MNSV-Dutch [4, 21]. However, differences were observed in the type of symptoms that both isolates could induce. A full-length cDNA of MNSV-Malpha5 was directly amplified by reverse-transcription polymerase chain reaction (RT-PCR) using a 5'-end primer anchoring a T7 RNA promoter sequence and a 3'-end primer, and cloned. Uncapped RNAs transcribed from this cDNA clone were infectious and caused symptoms indistinguishable from those caused by viral RNA when mechanically inoculated onto melon, cucumber or watermelon plants. The complete genome sequence of MNSV-Malpha5 was deduced from the full length cDNA clone. It is 4271 nt long and, similarly to MNSV-Dutch, consists of 5' and 3' untranslated regions (UTRs) and five open reading frames (ORFs) coding for 29, 89, 42 and two small 7 kDa proteins. One notable difference between MNSV-Malpha5 and other sequenced MNSV isolates was found, as for MNSV-Malpha5 the first of the two small ORFs, which are contiguous in the genome, terminates with a genuine stop codon, whereas for MNSV-Dutch and other sequenced MNSV isolates it terminates with an amber codon. This suggested that the putative p14 readthrough protein that could be expressed from the MNSV-Dutch and other MNSV genomes could not be expressed from the MNSV-Malpha5 genome. Also, the nucleotide and amino acid sequences comparisons showed a distant relationship of MNSV-Malpha5 with other known MNSV isolates.

Spanish Melon necrotic spot virus Isolate Overcomes the Resistance Conferred by the Recessive nsv Gene of Melon.

Melon necrotic spot virus (MNSV, genus Carmovirus, family Tombusviridae) is a worldwide natural pathogen that can cause significant economic losses in cucurbit crops (2). Use of commercial cultivars that carry the recessive nsv gene, the only resistance to MNSV described until now (1), is an effective means of controlling MNSV on melon crops. We report here the appearance of MNSV isolates able to overcome nsv resistance. Typical MNSV symptoms were observed in plants with an nsv/nsv genotype during a survey of melon in Almería (southeastern Spain) in 1999. The presence of MNSV in symptomatic plants was confirmed by standard double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using commercial antisera (Loewe Biochemica GmbH, Sauerlach, Germany). MNSV was isolated from these samples by three serial single-lesion inoculation passages in melon (cv. Bola de Oro). The isolate obtained (MNSV-264) was characterized by sequencing a fragment of its RNA genome corresponding to most of the capsid protein (CP) gene. Briefly, total RNA extracts (TRI reagent, Sigma Chemical, St. Louis, MO) of MNSV-264-infected plants were used in a reverse transcription polymerase chain reaction (RT-PCR) in which the oligonucleotide for the reverse transcription was 5'-TTAGGCGAGGTAAGCAGTTTC-3' (MA34), and the oligonucleotides for the PCR were MA34 and 5'-ATGGCGATGGTTAAACGC-3' (MA32). MA32 and MA34 were designed based on an alignment of nucleotide sequences of CP genes of diverse MNSV isolates. A DNA product of approximately 1.1 kbp was obtained, purified, and sequenced (GenBank Accession No. AF488692). Sequence comparisons (GCG Software Package, Madison, WI) of MNSV-264 and MNSV-Dutch (2) showed a 93% nucleotide sequence identity and a 96% similarity of the deduced amino acid sequence. Therefore, both isolates appear to be closely related. MNSV-264 was inoculated on melon accessions known to be susceptible (cvs. Panal and Bola de Oro) or resistant (cvs. Primal F1, Planters Jumbo, and PI161375) to common MNSV strains. Inoculations with MNSV-Dutch were used as a control for this experiment. The results showed that MNSV-264 was able to infect plants of all genotypes, whereas MNSV-Dutch infected only plants of the susceptible genotypes. Therefore, MNSV-264 is an isolate able to overcome the resistance conferred by nsv. The genetic determinant of the MNSV-264 virulence on resistant genotypes is currently under investigation. From 55 MNSV isolates obtained from fields in southeastern Spain during the last 3 years, only one could overcome nsv, like MNSV-264. Therefore, resistance-breaking isolates are not frequent in the MNSV populations in Spain. It is plausible that the situation may change as a consequence of the widespread use of commercial melon cultivars with the nsv genotype. A search for new sources of genetic resistance to MNSV isolates like MNSV-264 is in progress in our laboratory. References: (1) D. L. Coudriet et al. J. Am. Soc. Hortic. Sci. 106:789, 1981. (2) C. J. Riviere et al. J. Gen. Virol. 70:3033, 1989.

High Genetic Stability of the Begomovirus Tomato yellow leaf curl Sardinia virus in Southern Spain Over an 8-Year Period.

ABSTRACT The evolution of the plant single-stranded DNA virus Tomato yellow leaf curl Sardinia virus (TYLCSV) (genus Begomovirus, family Geminiviridae) has been monitored for 8 years after its appearance in southern Spain. Variation within three genomic regions of 166 TYLCSV isolates collected from three locations was assessed by single-strand conformation polymorphism (SSCP) analysis. According to SSCP, the intergenic region (IR) was the most variable. Low genetic diversity was found within the population and geographical or temporal differences were not evident. Nucleotide sequences of specific genomic regions of haplotypes identified by SSCP indicated close relationships among them. Therefore, the Spanish TYLCSV population appears to represent a single, undifferentiated population. The analysis of IR sequences for a subsample of 76 randomly chosen isolates confirmed the limited genetic diversity revealed by the SSCP analysis. A tendency to a lineal increase in diversity over time was observed in Málaga and Almería subpopulations; however, no accumulation of mutations in single isolates was evident. Negative selection to variation seems to operate to conserve certain regions of the genome. Thus, the low genetic diversity found in the studied TYLCSV population might be the result of a founder effect with subsequent selection against less fit variants arising by mutation.