First Report of Sweet potato leaf curl virus on Blue Morning Glory in Greece.

Blue morning glory (Ipomoea indica, Convolvulaceae) plants are widespread along the Greek coast, where they grow as weeds in addition to being cultivated as ornamentals. Yellow vein symptoms are frequently observed on these plants. These symptoms are similar to those reported for isolates of Sweet potato leaf curl virus (SPLCV) infecting I. indica in Italy and Spain (1,3). SPLCV belongs to the sweepoviruses, a unique group within the genus Begomovirus in the family Geniminiviridae that infects sweet potato (I. batatas) crops around the world. In May 2013, three leaf samples of I. indica showing yellow vein symptoms were collected in Kolymbari (Crete Island), where ~50% of the observed plants were symptomatic, and five asymptomatic leaf samples were collected in Kremasti and Mandriko (Rhodes Island). Total DNA, isolated from all samples, was used as a template in rolling-circle amplification (RCA) using ϕ29 DNA polymerase (TempliPhi kit, GE Healthcare, Little Chalfont, UK) and the product was digested with a set of restriction endonucleases. The samples from Kolymbari and one sample from Kremasti yielded amplification products that were shown to contain a single BamHI site. The DNA fragments of ~2.8 kbp obtained from one sample from each island were cloned into pBluescript II SK(+) (Stratagene, La Jolla, CA). Inserts of two clones from the Kolymbari sample and one clone from the Kremasti sample were completely sequenced (Macrogen, Seoul, South Korea). Sequences were aligned with available sequences of sweepoviruses using MUSCLE and pairwise identity scores were calculated with SDT as described (4). The sequences obtained from Kolymbari (2,830 nt, GenBank Accession Nos. KF697069 and KF697070) were 98.8% similar between them and showed the highest nucleotide identity (97.7%) with a SPLCV isolate obtained from an I. indica plant in Sicily Island (Italy) (AJ586885) (1). The sequence obtained from Kremasti (2,804 nt, KF697071) showed the highest nucleotide identity (92.4%) with a SPLCV isolate (previously named as Ipomoea yellow vein virus, which is currently a synonym of SPLCV [2]) obtained from an I. indica plant from southern Spain (EU839578) (3). Nucleotide sequence identities were above the 91% threshold for begomovirus species demarcation (2), thus confirming that the begomoviruses found infecting I. indica in Greece are isolates of SPLCV. It is worth to note that the infected I. indica plant from Kremasti did not show any conspicuous symptoms, thus highlighting the importance of this species as an alternative host for SPLCV, which could thus affect the sweet potato crop that is grown in Greece in familiar plots. To our knowledge, this is the first report of SPLCV in Greece. References: (1) R. W. Briddon et al. Plant Pathol. 55:286, 2006. (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 , 20 November 2013. (3) G. Lozano et al. J. Gen. Virol. 90:2550, 2009. (4) B. Muhire et al. Arch. Virol. 158:1411, 2013.

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.

Tobacco: A New Natural Host of Tomato chlorosis virus in Spain.

In March 2013, symptoms of mild leaf curling, mosaic, and interveinal yellowing were observed in tobacco (Nicotiana tabacum) plants grown in a row surrounding the exterior of a greenhouse containing a tomato crop in Guía de Isora, Tenerife (Canary Islands, Spain). The tobacco plants were found lightly infested by the whitefly (Hemiptera: Aleyrodidae) Bemisia tabaci. The greenhouses in this area are devoted to the commercial production of tomato. The farmers grow some tobacco plants inside and outside of them as a reservoir of parasitoids and depredators of B. tabaci. This insect is the natural vector of the main viruses severely affecting tomato in the Canary Islands, the begomovirus Tomato yellow leaf curl virus and the crinivirus Tomato chlorosis virus (ToCV). ToCV was detected in Spain in 1997 (2) and has become established in most of the coastal provinces of eastern and southern continental Spain and in the Canary Islands. Approximately 50% of the tomato plants grown inside the greenhouse close to the tobacco plants showed typical ToCV symptoms, and infection by this virus was confirmed in the seven plants tested by reverse transcription (RT)-PCR using specific coat protein gene (CP) primers (see below). Total RNA was extracted with TRIzol Reagent (Invitrogen) from leaves of five tobacco plants showing the symptoms mentioned above and analyzed by dot-blot hybridization using digoxigenin-labeled RNA probes to the CP gene of ToCV. Positive signal was obtained for all five plants. RT-PCR reactions were performed with specific primers 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 size (436 and 763 bp, respectively) were obtained, thus supporting the presence of ToCV in the symptomatic samples. The amplified product of the RdRp gene fragment from one sample was directly sequenced (Macrogen Inc., South Korea) and resulted closely related to ToCV isolates from Sudan (GenBank Accession No. JN411686, 99.6% nt identity) and Spain (DQ983480, 99.4% nt identity), thereby confirming the infection by this virus. Partial sequence of the ToCV isolate from tobacco was deposited in GenBank under accession no. KJ175084. In addition, all five plants resulted positive when analyzed by ELISA for Tomato spotted wilt virus and Potato virus Y and by PCR for Tomato yellow leaf curl virus (data not shown), all three viruses reported to infect naturally tobacco. Although tobacco has been reported as an experimental host of ToCV (4), to our knowledge, this is the first report of this species as a natural host of this virus. The finding of ToCV infecting tobacco raises the question of whether this virus could emerge as a pathogen of this crop and questions the use that farmers make of tobacco as reservoirs of natural enemies for whitefly control in tomato. References: (1) G. Lozano et al. J. Virol. 83:12973, 2009. (2) J. Navas-Castillo et al. Plant Dis. 84:835, 2000. (3) H. P. Trenado et al. Eur. J. Plant Pathol. 118:193, 2007 (4) W. M. Wintermantel and G. C. Wisler. Plant Dis. 90:814, 2006.

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.

First Report of China Rose (Hibiscus rosa-sinensis) as a Host of Alfalfa mosaic virus in Spain.

China rose (Hibiscus rosa-sinensis L.) is an ornamental plant grown throughout the tropics and subtropics. In June 2011, a China rose plant (sample CV-1) showing bright yellow "aucuba"-type mosaic, mainly at the center of the leaves, was found in a public garden in Caleta de Vélez (Málaga Province, southern Spain). Electron microscope examination of negatively stained preparations from the symptomatic plant revealed the presence of semispherical to bacilliform virus-like particles of 30 to 56 × 16 nm. Sap extracts also reacted positively in double-antibody sandwich (DAS)-ELISA to antiserum against Alfalfa mosaic virus (AMV) (Bioreba AG, Reinach, Switzerland). RNA of this sample was extracted with the RNeasy Kit (Qiagen, Valencia, CA) and tested by reverse-transcription (RT)-PCR with AMV specific primers (2), using AMV and GoTaq Master Mixes (Promega, Madison, WI) for cDNA synthesis and amplification, respectively. After cloning and sequencing, the ~750-bp DNA fragment was confirmed as the coat protein (CP) gene of AMV (GenBank Accession No. HE591387) with the highest nucleotide identity of 96% to AMV isolates belonging to subgroup IIA (e.g., GenBank Accession No. AJ130707). Sap from affected leaves of sample CV-1 was mechanically inoculated onto herbaceous indicator plants (Chenopodium amaranticolor, C. quinoa, and Ocimum basilicum). Both Chenopodium species developed chlorotic local lesions followed by mosaic within 3 days after inoculation, and O. basilicum showed bright yellow mosaic of calico type 2 weeks postinoculation. These symptoms are consistent with those reported for AMV in these hosts (1). Virus infection in the inoculated plants was confirmed by DAS-ELISA and RT-PCR. To gain insight on the prevalence and genetic variability of AMV in China rose, a survey was carried out in nearby locations in the provinces of Málaga (14 samples from Torre del Mar and 5 samples from Rincón de la Victoria) and Granada (12 samples from La Herradura). Leaf samples were analyzed by tissue blot hybridization with an AMV-specific digoxigenin-labeled RNA probe obtained from the RNA 1 of the Spanish isolate Tec1 (3), and only two samples from Torre del Mar tested positive. One of these samples (TM-2) was used to amplify by RT-PCR the AMV CP gene that was cloned and sequenced (GenBank Accession No. HE591386). The highest nucleotide identity of the TM-2 CP gene (98%) was with the subgroup IIB Spanish isolate Tec1, whereas identity with the CV-1 isolate was 95%. Nevertheless, phylogenetic analysis (neighbor-joining method) showed that both CV-1 and TM-2 isolates belong to the recently proposed AMV subgroup IIB (3), which includes the Tec1 isolate and two other isolates from ornamental plants, Phlox paniculata from the United States (GenBank Accession No. DQ124429) and Viburnum lucidum from Spain (GenBank Accession No. EF427449). These results show that AMV subgroup IIB is emerging as a complex cluster of virus isolates that currently are reported to infect only ornamentals. To our knowledge, this is the first report of AMV naturally occurring in China rose. References: (1) G. Marchoux et al. Page 163 in: Virus des Solanacées. Quae éditions, Versailles, 2008. (2) G. Parrella et al. Arch. Virol. 145:2659, 2000. (3) G. Parrella et al. Arch. Virol. 156:1049, 2011.

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.

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.

First Report of Sweet potato virus G and Sweet potato virus 2 Infecting Sweet Potato in Spain.

Sweet potato feathery mottle virus (SPFMV), Sweet potato virus G (SPVG), and Sweet potato virus 2 (SPV2) (also known as Ipomoea vein mosaic virus (2) and Sweet potato virus Y) are members of the genus Potyvirus (family Potyviridae), which can synergistically interact with Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae), increasing symptom severity on sweet potato (Ipomoea batatas (L.) Lam.) (1,2,3). During 2002, 2006, and 2007, vine cuttings from sweet potato plants were collected in Malaga (southern Spain), Tenerife, and Lanzarote (Canary Islands, Spain) to be tested for the presence of viruses. Sampled plants ranged from asymptomatic to severely affected by symptoms of sweet potato virus disease (SPVD), caused by dual infection with SPFMV or other potyviruses with SPCSV. Scions collected during 2002 were grafted to the indicator host I. setosa. Foliar samples from I. setosa were used for nitrocellulose membrane (NCM)-ELISA testing with antisera specific to SPVG or SPV2 (provided by C. A. Clark, Louisiana State University) following described procedures (2). NCM-ELISA testing indicated that SPVG was present in samples from Malaga, Tenerife, and Lanzarote, whereas SPV2 was only found in samples from Malaga. Reverse-transcription (RT)-PCR was performed on RNA extracts from sweet potato or I. setosa leaves using primer pairs MA541 (5'-AACAATTCCAGATAGTAGAGGGGTTG-3')/MA542(5'-TGTGGGGACAGCATGATCCAATAG-3') and MA540 (5'-AACCCCAACACCAGCAAAATCAGTTAAG-3')/MA542 corresponding to the capsid protein (CP) genes of SPVG and SPV2, respectively. Thirteen of 47 samples from Malaga and 4 of 30 from the Canary Islands yielded the expected 483-bp DNA fragment with the primers for SPVG. Fifteen of 47 samples from Malaga yielded the expected 627-bp DNA fragment with primers for SPV2. Two RT-PCR amplicons of SPVG, one from Malaga and one from Tenerife, were sequenced. Their nucleotide sequences (GenBank Accession Nos. EF577438 and EF577439, respectively) showed 98% identity to SPVG isolates from Louisiana (2) and China. Sequencing of one RT-PCR amplicon of SPV2 from Malaga resulted in a nucleotide sequence (GenBank Accession No. EF577437) with 99% identity to SPV2 from Lousiana and Australia (3). The presence of SPVG and SPV2 increases the already existing risk of SPVD, since the main viruses involved in the synergism, SPFMV and SPCSV, are present in Spain (4). SPCSV was also detected in some of the plants infected with SPVG or SPV2, in some cases, in coinfection with SPFMV. References: (1) C. D. Kokkinos and C. A. Clark. Plant Dis. 90:1347, 2006. (2) E. R. Souto et al. Plant Dis. 87:1226, 2003. (3) F. Tairo et al. Plant Dis. 90:1120, 2006. (4) R. A. Valverde et al. Plant Dis. 88:428, 2004.

Evidence for a phosphoenolpyruvate dependent sugar-phosphotransferase system in the mollicute Acholeplasma florum.

In order to confirm the presence of a phosphoenolpyruvate (PEP)-dependent sugar-phosphotransferase system (PTS) in the mollicute Acholeplasma florum we studied the ability of cell free extracts of this organism to phosphorylate glucose and/or fructose in the presence of PEP. We also cloned and sequenced a DNA fragment coding for a putative polypeptide showing significant similarity with the enzyme II of the beta-glucoside PTS of Escherichia coli. Taken together, these results show that A florum possesses a fructose-PTS, but not a glucose-PTS, and that the amino acid sequence deduced from the DNA fragment is related to beta-glucoside and sucrose enzymes II of PTS from various bacteria.

Tomato yellow leaf curl virus, an emerging virus complex causing epidemics worldwide.

Tomato yellow leaf curl (TYLC) is one of the most devastating viral diseases of cultivated tomato (Lycopersicon esculentum) in tropical and subtropical regions worldwide, and losses of up to 100% are frequent. In many regions, TYLC is the main limiting factor in tomato production. The causal agents are a group of geminivirus species belonging to the genus Begomovirus of the family Geminiviridae, all of them named Tomato yellow leaf curl virus (TYLCV) (sensu lato). There has been almost 40 years of research on TYLCV epidemics and intensive research programmes have been conducted to find solutions to the severe problem caused by these viruses. This paper provides an overview of the most outstanding achievements in the research on the TYLCV complex that could lead to more effective control strategies.

Detection of double-stranded RNA by ELISA and dot immunobinding assay using an antiserum to synthetic polynucleotides.

An antiserum against polyinosinic-polycytidylic acid (In-Cn) was used to detect double-stranded RNA (dsRNA) by several serological techniques. DsRNA was readily detected by indirect ELISA (ELISA-I) and dot immunobinding assay (DIA). Addition of the antigen to poly-L-lysine-precoated plates and blocking with uncreamed milk powder allowed detection levels of 100 pg.ml-1 In-Cn by ELISA-I. Concentrations as low as 1 ng.ml-1 were detected by DIA using polyvinyliden difluoride (PVDF) membranes. Detection capacity with nitrocellulose membranes was 1000 times lower than with PVDF. ELISA-I and DIA enabled detection of dsRNA in enriched fractions from cucumber mosaic virus (CMV)- and citrus tristeza virus (CTV)-infected plants and from virus-infected Penicillium chrysogenum mycelium. These techniques showed similar or higher sensitivity for detection of dsRNA than separation by polyacrylamide gel electrophoresis and silver staining.

Improvement of the print-capture polymerase chain reaction procedure for efficient amplification of DNA virus genomes from plants and insect vectors.

A rapid and simple procedure is described to amplify efficiently geminivirus DNA genomes by improving the print-capture polymerase chain reaction (PCR) procedure reported recently for RNA viruses. This method, termed print-PCR (P-PCR), allows direct amplification of DNA from infected plant or whitefly tissues printed directly on Whatman 3MM paper, without the need of any grinding, incubation, or washing steps previous to the amplification reaction. P-PCR reduces sample manipulation and avoids previous extraction of nucleic acids, thereby diminishing the possibilities of cross-contamination between samples. P-PCR has been successfully applied to whiteflies and various plant species infected by two different tomato yellow leaf curl viruses, TYLCV-Sr and TYLCV-Is, and for the amplification of the full-length genome of TYLCV-Is from infected plants.

Displacement of Tomato Yellow Leaf Curl Virus (TYLCV)-Sr by TYLCV-Is in Tomato Epidemics in Spain.

ABSTRACT A progressive displacement of tomato yellow leaf curl virus (TYLCV)-Sr by TYLCV-Is was observed in tomato epidemics in southern Spain based on incidence data of both virus species obtained during surveys conducted between 1996 and 1998. Ecological factors that might be involved in such a displacement, such as competition of TYLCV-Sr and TYLCV-Is in tomato, transmission by local biotypes (B and Q) of Bemisia tabaci, and presence in weeds and alternate crops, have been analyzed. No selective advantage is observed for TYLCV-Sr or TYLCV-Is in tomato plants either infected via Agrobacterium tumefaciens or via B. tabaci. However, TYLCV-Is is more efficiently vectored by local biotypes of B. tabaci; and common bean, a bridge crop between tomato crops, is a host for TYLCV-Is but not TYLCV-Sr. Therefore, common bean acts as a reservoir for TYLCV-Is. These two factors are probably responsible for the displacement of TYLCV-Sr by TYLCV-Is as the causative agent of epidemics in tomato in southern Spain.

Molecular variability of the 5'- and 3'-terminal regions of citrus tristeza virus RNA.

ABSTRACT Isolates of citrus tristeza virus (CTV) differ widely in their biological properties. These properties may depend on the structure of viral RNA populations comprising the different isolates. As a first approach to study the molecular basis of the biological variability, we have compared the sequences of multiple cDNA clones of the two terminal regions of the RNA from different CTV isolates. The polymorphism of the 5' untranslated region (UTR) allowed the classification of the sequences into three groups, with intragroup sequence identity higher than 88% and intergroup sequence identity as low as 44%. The variability of an open reading frame (ORF) 1a segment adjacent to the 5' UTR supports the same grouping. Some CTV isolates contained sequences of more than one group. Most sequences from Spanish isolates belonged to group III, whereas a Japanese isolate was composed mostly of sequences of groups I and II. The mildest isolates contained only sequences of group III, whereas the most severe isolates also contained sequences of groups I, II, or both. The most stable secondary structure predicted for the 5' UTR was composed of two stem-loops and remained essentially unchanged as a result of compensatory mutations in the stems and accommodation of most of the variability in the loops. In contrast to the 5'-terminal region, the variability of the 3'-terminal region of CTV RNA was very much restricted, with nucleotide identity values higher than 90%. The presence of a conserved putative "zinc-finger" domain adjacent to a basic region in p23, the predicted product of ORF 11, suggests that this protein might act as a regulatory factor during virus replication.

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.

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.

Evidence of a Naturally Occurring Recombinant Between Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus in Spain.

Tomato yellow leaf curl virus (TYLCV, formerly TYLCV-Is) and Tomato yellow leaf curl Sardinia virus (TYLCSV, formerly TYLCV-Sar) are geminivirus species of the genus Begomovirus that cause the disease known as tomato yellow leaf curl. In Spain, TYLCV and TYLCSV have coexisted in field and greenhouse tomato (Lycopersicon esculentum) crops since 1996 (2). TYLCV is also the causal agent of the leaf crumple disease of common bean (Phaseolus vulgaris) (1), a species that TYLCSV is unable to infect (2). Analysis of field samples from common bean plants affected by leaf crumple disease collected in Almería (southeastern Spain) during 1999 showed that, unexpectedly, several samples hybridized with TYLCV- and TYLCSV-specific probes prepared to the intergenic region (IR) as previously described (1). Polymerase chain reactions (PCR) performed with total nucleic acids extracted from one of these samples (ES421/99) using primer pairs specific to the IR of TYLCV (MA-30/MA-31) or TYLCSV (MA-14/MA-15) (1) gave no amplification product. However, the combination of MA-30 (5' end of TYLCV IR) and MA-15 (3' end of TYLCSV IR) produced a PCR DNA product of the expected size (351 bp). Direct DNA sequencing of this product (GenBank Accession No. AF401478) indicated the presence of a chimeric IR in ES421/99. Comparison of the obtained sequence with those available for isolates reported from Spain showed that the 5' side (149 nt) from the stem-loop structure conserved in the IR of all geminiviruses was 99% identical to the corresponding region of TYLCV (GenBank Accession No. AF071228) and only 62% identical to TYLCSV (GenBank Accession No. Z25751). In contrast, the 3' side (124 nt) from the stem-loop was 98% identical to the corresponding region of TYLCSV and only 57% identical to TYLCV. The 33-nt region involved in the stem-loop was 100% identical to TYLCV and showed one nucleotide change in the loop with respect to TYLCSV. Therefore, this DNA sequence data showed evidence of the occurrence in ES421/99 of a natural recombination between TYLCV and TYLCSV. The biological and epidemiological consequences of the presence of this new interspecific recombinant have yet to be determined. References: (1) J. Navas-Castillo et al. Plant Dis. 83:29, 1999. (2) S. Sánchez-Campos et al. Phytopathology 89:1038, 1999.

Severe Yellowing Outbreaks in Tomato in Spain Associated with Infections of Tomato chlorosis virus.

Since 1997, yellowing disease outbreaks have occurred in tomato (Lycopersicon esculentum) crops in southern Spain. The outbreaks were associated with high populations of the whitefly Bemisia tabaci. Symptoms consisted mainly of interveinal yellowing that developed initially on lower leaves and then progressed to the upper part of the plant. Affected plants were less vigorous and yielded less due to reduced fruit growth and delayed ripening. During 1998 and 1999, the yellowing disease was widespread and occurred at high incidences in the Málaga province. The disease agent was readily transmissible from tomato to tomato by B. tabaci biotype Q. Samples from symptomatic tomato plants were analyzed and shown to be infected with Tomato chlorosis virus (ToCV) (genus Crinivirus, family Closteroviridae). This is the first report of ToCV epidemics in Europe.

First Report of Sweet potato chlorotic stunt virus and Sweet potato feathery mottle virus Infecting Sweet Potato in Spain.

Sweet potato chlorotic stunt virus (SPCSV), family Closteroviridae and Sweet potato feathery mottle virus (SPFMV), family Potyviridae are whitefly and aphid transmitted, respectively, which in double infections cause sweet potato virus disease (SPVD) that is a serious sweet potato (Ipomoea batatas Lam.) disease in Africa (2). During the past decade, sweet potato plants showing symptoms similar to SPVD have been observed in most areas of Spain. Nevertheless, not much information is available about the identity of the viruses infecting this crop in Spain. During the summer of 2002, sweet potato plants with foliar mosaic, stunting, leaf malformation, chlorosis, and ringspot symptoms were observed in several farms in Málaga (southern Spain) and Tenerife and Lanzarote (Canary Islands, Spain). Vine cuttings were collected from 21 symptomatic plants in Málaga and from eight plants on Lanzarote and six on Tenerife. Scions were grafted to the indicator hosts, Brazilian morning glory (I. setosa) and I. nil cv. Scarlett O'Hara. Three weeks after graft inoculations, all plants showed various degrees of mosaic, chlorosis, leaf malformation, and stunting. Four field collections (two from Málaga, one from Tenerife, and one from Lanzarote) with severe symptoms on I. setosa were selected for whitefly (Bemisia tabaci biotype Q) transmission experiments. Acquisition and transmission periods were 48 h. I. setosa was the acquisition host, and I. nil was the transmission host. For each isolate, groups of 10 whiteflies per I. nil plant were used. All I. nil plants used as transmission hosts with the four, field collections showed chlorosis and leaf malformation. Reverse-transcription polymerase chain reaction (RT-PCR) was performed on I. setosa (grafted with the four selected field collections) and I. nil plants (from the whitefly transmission experiments) with primers for the HSP70h gene of SPCSV. A 450-bp DNA fragment was obtained with all I. setosa and I. nil samples. Sequencing of the 450-bp DNA from two samples from Málaga yielded a nucleotide sequence with 98 to 99% similarity to the HSP70h gene of West African SPCSV isolates. Foliar samples from I. setosa, originally grafted with the 21 vine cuttings, were used for nitrocellulose membrane enzyme-linked immunosorbent assay (NCM-ELISA) testing with antiserum specific to SPFMV-RC (provided by J. Moyer, North Carolina State University, Raleigh). Positive control was sap extract from I. setosa that was infected with the common strain of SPFMV. Procedures for NCM-ELISA were as described (4). NCM-ELISA testing suggested that SPFMV was present in all samples. RT-PCR was conducted with degenerate primers POT1/POT2 (1). The nucleotide sequence that was amplified by these two primers spans part of the NIb protein and part of the coat protein gene of potyviruses. All samples yielded the expected 1.3-kb DNA. Sequencing of the RT-PCR products of two isolates from Malaga and sequence comparisons yielded nucleotide sequences with 97% similarity to two East African isolates (Nam 1 and Nam 3) of SPFMV (3). These results confirm the presence of SPCSV and SPFMV in sweet potato in Spain. References: (1) D. Colinet and J. Kummert. J. Virol. Methods 45:149, 1993. (2) R. W. Gibson et al. Plant Pathol. 47:95, 1998. (3) J. F. Kreuze et al. Arch. Virol. 145:567, 2000. (4) E. R. Souto et al. Plant Dis. 87:1226, 2003.

First Report of Tomato Yellow Leaf Curl Virus-Is in Spain: Coexistence of Two Different Geminiviruses in the Same Epidemic Outbreak.

Epidemics of tomato yellow leaf curl have occurred annually in greenhouse- and field-grown tomato (Lycopersicon esculentum Mill.) crops in southern Spain since 1992 (2). The nucleotide sequences of two tomato yellow leaf curl virus (TYLCV) isolates from this region, TYLCV-M (GenBank accession no. Z25751) and TYLCV-Alm (L27708), have been determined and these isolates are closely related to isolates reported from Italy (X61153 and Z28390), suggesting the existence of a geographical cluster of closely related TYLCV isolates in the Western Mediterranean Basin (2). In June 1997, new and unusually severe symptoms of stunting, yellowing, and curling of leaflet margins, with a marked reduction in leaf size, were observed in some plants of a greenhouse-grown tomato crop in Almeria (southeastern Spain). Tomato plants showing milder symptoms similar to those previously described for TYLCV infection in that region (2) were also present in the same greenhouse. Total nucleic acids extracts from plants exhibiting both types of symptoms were analyzed by dot blot hybridization with a probe prepared by random priming on a 1,674-bp SalI fragment of the pSP95 clone of TYLCV-M (3). A strong reaction was obtained with the samples that showed mild symptoms, whereas a weak reaction was observed with the severely affected plants. Specific pairs of primers were prepared to amplify the complete pre-coat (V1) (MA10: 5'-ATGTGGGATCCTTTATTAAATG-3'; MA11: 5'-TCAGGGCTTCTGTACATTC-3') and C2 (MA12: 5'-TAAAGACTCTTAAAAAATGACC-3'; MA13: 5'-AATGCAATCTTCGTCACC-3') genes based on TYLCV-M sequence. With polymerase chain reaction (PCR), the expected fragments were amplified from extracts of both types of plants. The PCR products were submitted to single-strand conformation polymorphism (SSCP) analysis. Clearly distinguishable SSCP patterns were obtained: one for the plants with mild symptoms, identical to that of known TYLCV-M infected plants, and another for the plants with more severe symptoms. Further analyses done on tomato samples collected from the same area showed that both SSCP patterns were present simultaneously in several severely affected plants. The nucleotide sequences of the V1 and C2 PCR products from two samples differing in their SSCP pattern were obtained by direct sequencing, and compared with available TYLCV sequences. The sequences corresponding to the sample with mild symptoms were 100% identical to those previously reported for TYLCV-M. In contrast, the sequences from the sample that showed severe symptoms (GenBank accesion no. AF022219 for V1, and AF022220 for C2) were only 80 and 76% identical to TYLCV-M V1 and C2 genes, respectively, but were 99% identical to the sequence reported for an isolate of TYLCV-Is from Israel (X15656). Epidemics in tomato caused by TYLCV-Is have been recently reported from Portugal (1). Our results demonstrate that the unusually severe symptoms observed are associated with an isolate of TYLCV-Is that coexists in the field with the milder TYLCV previously reported from this area. This is the first report of the occurence of TYLCV-Is in Spain. References: (1) D. Louro et al. Plant Dis. 80:1079, 1996. (2) E. Noris et al. Arch. Virol. 135:165, 1994.