RESUMEN

Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV) are two whitefly transmitted viruses which are classified in the genus Crinivirus of the family Closteroviridae. Both induce similar yellowing symptoms in tomato and are responsible for severe economic losses. ToCV is transmitted by Bemisia tabaci Gennadious, Trialeurodes vaporariorum Westwood and Trialeurodes abutilonea Haldeman, whereas TICV is transmitted only by T. vaporariorum. An extensive study was conducted during 2009-2012 in order to identify the virus species involved in tomato yellowing disease in Greece. Samples from tomato, other crops and weeds belonging to 44 species from 26 families were collected and analyzed using molecular methods. In addition, adult whiteflies were collected and analyzed using morphological characters and DNA markers. Results showed that TICV prevailed in tomato crops (62.5%), while ToCV incidence was lower (20.5%) and confined in southern Greece. ToCV was also detected in lettuce plants showing mild yellowing symptoms for the first time in Greece. Approximately 13% of the tested weeds were found to be infected, with TICV being the predominant virus with an incidence of 10.8%, whereas ToCV was detected only in 2.2% of the analyzed samples. These results indicate that the host range of TICV and ToCV in Greece is far more extensive than previously believed. T. vaporariorum was the most widespread whitefly species in Greece (80%), followed by B. tabaci (biotypes B and Q) (20%). Sequence analysis of the CP and CPm genes from Greek tomato and weed isolates of ToCV and TICV showed that even though both viruses have very wide host ranges their populations show very low molecular divergence.

Asunto(s)

Crinivirus/genética , ADN Viral/genética , Hemípteros/genética , Insectos Vectores/genética , Filogenia , Enfermedades de las Plantas/virología , Solanum lycopersicum/virología , Animales , Conducta Animal , Evolución Biológica , Crinivirus/clasificación , Crinivirus/aislamiento & purificación , Conducta Alimentaria , Variación Genética , Grecia , Hemípteros/clasificación , Hemípteros/virología , Especificidad del Huésped , Interacciones Huésped-Parásitos , Insectos Vectores/clasificación , Insectos Vectores/virología , Lactuca/parasitología , Lactuca/virología , Solanum lycopersicum/parasitología , Filogeografía , Enfermedades de las Plantas/parasitología , Malezas/parasitología , Malezas/virología

RESUMEN

An extensive study was conducted during 2007 and 2008 in three major tomato production areas of Cyprus, where Tomato yellow leaf curl virus (TYLCV) is commonly found, to assess the incidence and prevalence of naturally infected weed species that could serve as TYLCV reservoirs. Approximately 4,000 of the most common dicotyledonous plants belonging to 122 species from 25 families were collected, identified, and tested for TYLCV presence using serological and molecular methods. The tests included a previously reported conventional polymerase chain reaction (PCR) assay and a real-time TaqMan PCR assay developed and optimized in this study. Real-time PCR was found to be the most sensitive technique, and enabled the detection of TYLCV in 461 samples of 49 different species belonging to the families Amaranthaceae, Chenopodiaceae, Compositae, Convolvulaceae, Cruciferae, Euphorbiaceae, Geraniaceae, Leguminosae, Malvaceae, Orobanchaceae, Plantaginaceae, Primulaceae, Solanaceae, Umbelliferae, and Urticaceae. The results further indicated that the host range of TYLCV in Cyprus is far more extensive than previously documented and, therefore, new management strategies are required. These should focus on the control of alternative virus hosts during the growing season and in crop-free periods.

RESUMEN

Pepino mosaic virus (PepMV) (genus Potexvirus, family Flexiviridae) is a mechanically transmitted virus that has emerged as a significant problem of greenhouse tomato crops in Europe and around the world during the past 10 years (1). In spring of 2010, mosaic symptoms were observed on leaves of cherry tomato (Lycopersicon esculentum var. cerasiforme) greenhouse crops (hybrids Shiren, Tomito, and Rubino top) in the areas of Drymos and Vonitsa, located at Aitoloakarnania Prefecture, in Greece. A total of 63 tomato samples (55 from symptomatic and 8 from asymptomatic plants) were collected from 11 greenhouses where disease incidence ranged from 10 to 20%. All samples were tested by double-antibody sandwich (DAS)-ELISA using polyclonal antibodies from BIOREBA, AG (Reinach, Switzerland) for the presence of PepMV, Cucumber mosaic virus (CMV), and Tomato mosaic virus (ToMV). Leaf tissue from PepMV-, CMV-, and ToMV-infected samples and virus-free tomato plants were included in all tests as positive and negative controls, respectively. Results showed that 53 symptomatic samples collected from all greenhouses were infected with PepMV and two were co-infected with PepMV and CMV. Total RNA was extracted from all infected plants with a commercially available kit (Qiagen, Hilden, Germany) and amplified by conventional and real-time reverse transcription (RT)-PCR, using previously reported protocols (2). Positive and negative controls were also included in each assay. The 200-bp amplified PCR fragments of Triple Gene Block 3 (TGB3) obtained from five infected samples were purified and both strands were sequenced. Sequencing data were analyzed, deposited in the GenBank, and compared with other reported sequences. In addition, leaf tissue from five samples infected with only PepMV was used for mechanical inoculation of four plants of Nicotiana glutinosa, N. benthamiana, and tomato (L. esculentum FA 179 hybrid) plants. As negative controls, two plants from each species were used. Sequencing analysis showed that all five PepMV sequences were identical (GenBank Accession Nos. FR686904 to FR686908) and possessed 100% identity PepMVstrain CH2 (DQ000985). Inoculation results showed that the virus was successfully transmitted to N. benthamiana and tomato plants which developed mosaic symptoms, and tested positive by DAS-ELISA and RT-PCR. N. glutinosa plants did not develop any symptoms and were found to be free of PepMV when tested by DAS-ELISA and RT-PCR. To our knowledge, this is the first report of PepMV in Greece. Further studies on the disease prevalence and incidence and its economic impact on tomato production are required. PepMV is currently under quarantine status in the EU and therefore new protective measures should be recommended to prevent the spread of PepMV to other regions of Greece. References: (1) I. M. Hanssen and B. P. H. J. Thomma. Mol. Plant Pathol. 11:179, 2010. (2) K. S. Ling et al. J. Virol. Methods 144:65, 2007.

RESUMEN

During the past four decades, Tomato yellow leaf curl disease has become one of the major constraints in tomato production worldwide. In the Mediterranean basin, several isolates from two major Begomovirus species are involved in outbreaks and persistent epidemics. A real-time TaqMan PCR assay was developed and evaluated for the rapid and multiplex detection and differentiation of two begomoviruses often found in mixed infections in the region, Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV). This assay was 1000-fold more sensitive than conventional PCR assays described previously, allowing the use of simple template preparation methods and eliminating the need for total nucleic acid purification. The viral DNA template was obtained by spotting sap extract derived from TYLCV or TYLCSV infected tissues on a nylon membrane or by directly using crude plant extracts in the real-time reaction cocktail. Preliminary results showed that this method can successfully detect and discriminate virus species from infected tomato, bean, pepper and different weed species obtained from the Mediterranean basin, the USA and Japan, allowing the simple, fast and cost-effective testing of a large number of samples in certification schemes. The assay can also be used for the detection of these two begomovirus species in their whitefly vector biotypes of the Bemisia tabaci (Gennadius) species group.

Asunto(s)

Begomovirus/clasificación , Begomovirus/aislamiento & purificación , Enfermedades de las Plantas/virología , Reacción en Cadena de la Polimerasa/métodos , Solanum lycopersicum/virología , Animales , Begomovirus/genética , Capsicum/virología , Hemípteros/virología , Insectos Vectores/virología , Nylons , Extractos Vegetales/análisis , Sensibilidad y Especificidad , Alineación de Secuencia , Moldes Genéticos

RESUMEN

A real-time PCR assay based on TaqMan technology was developed and evaluated for the rapid detection of the B and Q biotypes of Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). A survey was conducted during 2005-2007 in order to identify the distribution and prevalence of B. tabaci biotypes in Cyprus using the real-time PCR assay. More than 700 adult whiteflies collected from 35 cultivated and weed plant species were individually haplotyped using TaqMan PCR, and the results of the assay were validated by restriction fragment length polymorphism analysis and DNA sequencing of the mitochondrial cytochrome oxidase I (mtCOI) gene. Two biotypes, B and Q, were identified in the collected plant species on the island. The real-time PCR and RFLP assay consistently yielded the same results, although the real-time assay was more sensitive and less time consuming. Phylogenetic analysis of the mtCOI DNA sequences corroborated the identity of the B and Q biotypes 100% of the time and by phylogenetic analysis the haplotypes grouped, as expected, in the major North African-Mediterranean-Middle Eastern clade of the B. tabaci complex.

Asunto(s)

Hemípteros/clasificación , Animales , Chipre , Complejo IV de Transporte de Electrones/química , Complejo IV de Transporte de Electrones/genética , Genes Mitocondriales , Haplotipos , Hemípteros/genética , Filogenia , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de Secuencia de ADN

RESUMEN

Tomato yellow leaf curl is one of the most devastating virus diseases of tomato (Lycopersicon esculentum Mill) crops worldwide. Several whitefly-transmitted viruses are associated with the disease and all are assigned to the genus Begomovirus, family Geminiviridae. In Greece, Tomato yellow leaf curl virus (TYLCV) was first reported to infect greenhouse and open-field tomatoes in 2000 (2). During 2006, a survey was conducted in the southwestern part of Peloponnese (mainland) within the areas of Kyparissia and Filiatra (Perfecture of Messinia) to identify the prevalence and natural hosts of the disease. During this survey, yellow mosaic, severe leaf curling, and leaf crumple symptoms were observed in greenhouse bean plants (Phaseolus vulgaris) that were cultivated together with tomatoes showing typical TYLCV symptoms. In all affected greenhouses, the incidence of the disease ranged from 1 to 5% in beans and 90 to 100% in tomato plants. Both bean and tomato plants were highly infested with Bemisia tabaci (Gennadius) populations and produced unmarketable fruits. Twenty-four symptomatic bean plants were collected from four greenhouses that tested positive by triple-antibody sandwich-ELISA using TYLCV-specific antibodies purchased from NEOGEN, EUROPE, Ltd. DNA was extracted from all infected bean plants, and a 580-bp fragment of the coat protein gene was amplified by PCR using the TY(+)/TY(-) primer pair (1). Amplified fragments were then analyzed by restriction fragment length polymorphism with Ava II cutter enzyme. Two DNA fragments of 277 and 302 bp in agarose gels were produced from all isolates and the restriction pattern corresponded to TYLCV. The amplified DNA from four infected bean plants was cloned and sequenced. All four sequences were 100% identical (EMBL Accession No. AM418398) and showed 99% nucleotide identity to a TYLCV isolate from Italy (EMBL Accession No. DQ144621). To our knowledge, this is the first report of TYLCV infection of P. vulgaris, which is an important commercial crop in Messinia, Greece. Within the last decade, TYLCV has emerged as an important pathogen for several cultivated plants in many regions and different TYLCV variants have been reported to infect P. vulgaris (3). Bean is often used as an intercrop between tomato crops, and thus, infected plants may serve as a potential reservoir for virus survival and spread (4). References: (1) G. P. Accotto et al. Eur. J. Plant Pathol. 106:179, 2000. (2) A. D. Avgelis et al. Plant Dis. 85:678, 2001. (3) J. Morris et al. EPPO Bull. 32:41, 2002. (4) J. Navas-Castillo et al. Plant Dis. 83:29, 1999.