Calibración de los parámetros del modelo de directividad de los transductores de la ecosonda Simrad EK60

The scientific echo sounder Simrad EK60 could be used for quantification of fisheries resources in marine science research campaigns. They use different frequency channels (from 18 to 200 KHz), emitted by underwater sound transducers with circular aperture geometries. Calibration is essential to obtain reliable values of Ts (Target Strength) and Sv (Volume Backscattering Coefficient) parameters, which are used to estimate fisheries stocks using echo volume integration. In this paper, manufacturer’s calibration method is presented, together a new proposal for the acquisition and post-processing of calibration data to obtain more accurate results.La ecosonda científica Simrad EK60 se utiliza para cuantificar los recursos pesqueros en campañas de investigación oceanográfica, utilizando diferentes canales de frecuencias (de 18 a 200KHz) mediante transductores acústicos de apertura con geometría circular. Su calibración resulta esencial para obtener los valores de Ts (‘target strength’) y Sv (‘Volume backscattering coefficient’), necesarios para estimar los tamaños de los stocks de las pesquerías mediante integración de los ecos. En este trabajo se presenta el método de calibración propuesto por el fabricante, así como una nueva propuesta de obtención y postproceso de datos que permite obtener calibraciones fiable

Turnip yellow mosaic virus in Chinese cabbage in Spain: Commercial seed transmission and molecular characterization

[EN] Seed transmission of Turnip yellow mosaic virus (TYMV, genus Tymovirus) was evaluated in the whole seeds and seedlings that emerged from three commercial Chinese cabbage (Brassica pekinensis) seed batches. Seedlings in the cotyledon stage and adult plants were assayed for TYMV by DAS-ELISA and confirmed by RT-PCR. The proportion of whole seeds infected with TYMV was at least 0.15 %. The seeds of the three seed batches were grown in Petri dishes, and surveyed in the cotyledon stage in trays that contained a peat:sand mixture grown in greenhouses or growth chambers, which were analysed in the cotyledon and adult stages. The seed-to-seedling transmission rate ranged from 2.5 % to 2.9 % in two different seed batches (lot-08 and lot-09, respectively). Spanish isolates derived from turnip (Sp-03) and Chinese cabbage (Sp-09 and Sp-13), collected in 2003, 2009 and 2013 in two different Spanish regions, were molecularly characterised by analysing the partial nucleotide sequences of three TYMV genome regions: partial RNA-dependent RNA polymerase (RdRp), methyltransferase (MTR) and coat protein (CP) genes. Phylogenetic analyses showed that the CP gene represented two different groups: TYMV-1 and TYMV-2. The first was subdivided into three subclades: European, Australian and Japanese. Spanish isolate Sp-03 clustered together with European TYMV group, whereas Sp-09 and Sp-13 grouped with the Japanese TYMV group, and all differed from group TYMV-2. The sequences of the three different genomic regions examined clustered into the same groups. The results suggested that Spanish isolates grouped according to the original hosts from which they were isolated. The inoculation of the Spanish TYMV isolates to four crucifer plants species (turnip, broccoli, Brunswick cabbage and radish) revealed that all the isolates infected turnip with typical symptoms, although differences were observed in other hosts.Alfaro Fernández, AO.; Serrano, A.; Tornos, T.; Cebrian Mico, MC.; Córdoba-Sellés, MDC.; Jordá, C.; Font San Ambrosio, MI. (2016). Turnip yellow mosaic virus in Chinese cabbage in Spain: Commercial seed transmission and molecular characterization. EUROPEAN JOURNAL OF PLANT PATHOLOGY. 146(2):433-442. doi:10.1007/s10658-016-0929-3S4334421462Assis Filho, M., & Sherwood, J. L. (2000). Evaluation of seed transmission of Turnip yellow mosaic virus and Tobacco mosaic virus in Arabidopsis thaliana. Phytopathology, 90, 1233–1238.Benetti, M. P., & Kaswalder, F. (1983). Trasmisione per seme del virus del mosaico giallo rapa. Annali dell Istituto Sperimentale per la Patologia Vegetale, 8, 67–70.Blok, J., Mackenzie, A., Guy, P., & Gibbs, A. (1987). Nucleotide sequence comparisons of Turnip yellow mosaic virus isolates from Australia and Europe. Archives of Virology, 97, 283–295.Brunt, A., Crabtree, K., Dallwitz, M., Gibbs, A., Watson, L., & Zurcher, E.J. (1996). Plant Viruses Online: Descriptions and Lists from the VIDE Database. Version: 20th August 1996. URL http://biology.anu.edu.au/Groups/MES/vide/ .Campbell, R. N., Wipf-Scheibel, C., & Lecoq, H. (1996). Vector-assissted seed transmission of melon necrotic spot virus in melon. Phytopathology, 86, 1294–1298.Dreher, T. W., & Bransom, K. L. (1992). Genomic RNA sequence of Turnip yellow mosaic virus isolate TYMC, a cDNA-based clone with verified infectivity. Plant Molecular Biology, 18, 403–406.Fakhro, A., Von Bargen, S., Bandte, M., Büttner, C., Franken, P., & Schwarz, D. (2011). Susceptibility of different plant species and tomato cultivars to two isolates of Pepino mosaic virus. European Journal of Plant Pathology, 129, 579–590.Gibbs, A. J., & Gower, J. C. (1960). The use of a multiple-transfer method in plant virus transmission studies: some statistical points arising in the analysis of results. Annals of Applied Biology, 48, 75–83.Hayden, C. M., Mackenzie, A. M., & Gibbs, A. J. (1998a). Virion protein sequence variation among Australian isolates of turnip yellow mosaic tymovirus. Archives of Virology, 143, 191–201.Hayden, C. M., Mackenzie, A. M., Skotnicki, M. L., & Gibbs, A. (1998b). Turnip yellow mosaic virus isolates with experimentally produced recombinant virion proteins. Journal of General Virology, 79, 395–403.Hein, A. (1984). Transmission of Turnip yellow mosaic virus through seed of Camelina sativa gold of pleasure. Journal of Plant Diseases and Protection, 91, 549–551.Herrera-Vásquez, J. A., Córdoba-Sellés, M. C., Cebrián, M. C., Alfaro-Fernández, A., & Jordá, C. (2009). Seed transmission of Melon necrotic spot virus and efficacy of seed-disinfection treatments. Plant Pathology, 58, 436–452.Hull, R. (2002). Matthews’ plant virology (4a ed.1001 pp). San Diego: Academic Press.Johansen, E., Edwards, M. C., & Hampton, R. O. (1994). Seed transmission of viruses: current perspectives. Annual Review of Phytopathology, 32, 363–386.Kirino, N., Inoue, K., Tanina, K., Yamazaki, Y., & Ohki, S. T. (2008). Turnip yellow mosaic virus isolated from Chinese cabbage in Japan. Journal of General Plant Pathology, 74, 331–334.Markham, R., & Smith, K. S. (1949). Studies on the virus of turnip yellow mosaic. Parasitology, 39, 330–342.Mathews, R. E. F. (1980). Turnip yellow mosaic virus, CMI/AAB Descriptions of plant virus No. 230 (No. 2 revised). Kew: Commonwealth Mycology Institute/Association of Applied Biologists.Mitchell, E. J., & Bond, J. M. (2005). Variation in the coat protein sequence of British isolates of Turnip yellow mosaic virus and comparison with previously published isolates. Archives of Virology, 150, 2347–2355.Pagán, I., Fraile, A., Fernández-Fueyo, E., Montes, N., Alonso-Blanco, C., & García-Arenal, F. (2010). Arabidopsis thaliana as a model for the study of plant-virus co-evolution. Philosophical Transations of the Royal Society Biological Sciences, 365, 1983–1995.Paul, H. L., Gibbs, A., & Wittman-Liebold, B. (1980). The relationships of certain Tymoviruses assessed from the amino acid composition of their coat proteins. Intervirology, 13, 99–109.Pelikanova, J. (1990). Garlic mustard a spontaneous host of TYMV. Ochrana Rostlin, 26, 17–22.Procházková, Z. (1980). Host range and symptom differences between isolates of Turnip mosaic virus obtained from Sisymbrium loeselii. Biologia Plantarum, 22, 341–347.Rimmer, S. R., Shtattuck, V. I., & Buchwaldt, L. (2007). Compendium of brassica diseases (1ª Edición ed.p. 117). USA: APS press.Rot, M. E., & Jelkman, W. (2001). Characterization and detection of several filamentous viruses of cherry: Adaptation of an alternative cloning method (DOP-PCR), and modification of an RNA extraction protocol. European Journal of Plant Pathology, 107, 411–420.Sabanadzovic, S., Abou-Ghanem, N., Castellano, M. A., Digiaero, M., & Martelli, G. P. (2000). Grapevine fleck virus-like in Vitis. Archives of Virology, 145, 553–565.Špack, J., & Kubelková, D. (2000). Serological variability among European isolates of Radish mosaic virus. Plant Pathology, 49, 295–301.Špack, J., Kubelková, D., & Hnilicka, E. (1993). Seed transmission of Turnip yellow mosaic virus in winter turnip and winter oilseed rapes. Annals of Applied Biology, 123, 33–35.Stobbs, L. W., Cerkauskas, R. F., Lowery, T., & VanDriel, L. (1998). Occurrence of Turnip yellow mosaic virus on oriental cruciferours vegetables in Southern Ontario, Canada. Plant Disease, 82, 351.Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28, 2731–2739

Host range and symptomatology of Pepino mosaic virus strains occurring in Europe

Pepino mosaic virus (PepMV) has caused great concern in the greenhouse tomato industry after it was found causing a new disease in tomato in 1999. The objective of this paper is to investigate alternative hosts and compare important biological characteristics of the three PepMV strains occurring in Europe when tested under different environmental conditions. To this end we compared the infectivity and symptom development of three, well characterized isolates belonging to three different PepMV strains, EU-tom, Ch2 and US1, by inoculating them on tomato, possible alternative host plants in the family Solanaceae and selected test plants. The inoculation experiments were done in 10 countries from south to north in Europe. The importance of alternative hosts among the solanaceous crops and the usefulness of test plants in the biological characterization of PepMV isolates are discussed. Our data for the three strains tested at 10 different European locations with both international and local cultivars showed that eggplant is an alternative host of PepMV. Sweet pepper is not an important host of PepMV, but potato can be infected when the right isolate is matched with a specific cultivar. Nicotiana occidentalis 37B is a useful indicator plant for PepMV studies, since it reacts with a different symptomatology to each one of the PepMV strains.Ravnikar, M.; Blystad, D.; Van Der Vlugt, R.; Alfaro Fernández, AO.; Del Carmen Cordoba, M.; Bese, G.; Hristova, D.... (2015). Host range and symptomatology of Pepino mosaic virus strains occurring in Europe. European Journal of Plant Pathology. 143(1):43-56. doi:10.1007/s10658-015-0664-1S43561431Alfaro-Fernández, A., Córdoba-Sellés, M. C., Herrera-Vásquez, J. A., Cebrián, M. C., & Jordá, C. (2009). Transmission of Pepino mosaic virus by the fungal vector Olpidium virulentus. Journal of Phytopathology, 158, 217–226.Charmichael, D. J., Rey, M. E. C., Naidoo, S., Cook, G., & van Heerden, S. W. (2011). First report of Pepino mosaic virus infecting tomato in South Africa. Plant Disease, 95(6), 767.2.Córdoba, M. C., Martínez-Priego, L., & Jordá, C. (2004). New natural hosts of Pepino mosaic virus in Spain. Plant Disease, 88, 906.Córdoba-Sellés, M. C., García-Rández, A., Alfaro-Fernández, A., & Jordá-Gutiérrez, C. (2007). Seed transmission of pepino mosaic virus and efficacy of tomato seed disinfection treatments. Plant Disease, 91, 1250–1254.Efthimiou, K. E., Gatsios, A. P., Aretakis, K. C., Papayannis, L. C., & Katis, N. I. (2011). First report of Pepino mosaic virus infecting greenhouse cherry tomato in Greece. Plant Disease, 95(1), 78.2.Fakhro, A., von Bargen, S., Bandte, M., Büttner, C., Franken, P., & Schwarz, D. (2011). Susceptibility of different plant species and tomato cultivars to two isolates of Pepino mosaic virus. European Journal of Plant Pathology, 129, 579–590.Gómez, P., Sempere, R. N., Elena, S. F., & Aranda, M. A. (2009). Mixed infections of Pepino mosaic virus strains modulate the evolutionary dynamics of this emergent virus. Journal of Virology, 83, 12378–12387.Hanssen, I. M., Paeleman, A., Wittemans, L., Goen, K., Lievens, B., Bragard, C., Vanachter, A. C. R. C., & Thomma, B. P. H. J. (2008). Genetic characterization of Pepino mosaic virus isolates from Belgian greenhouse tomatoes reveals genetic recombination. European Journal of Plant Pathology, 121, 131–146.Hanssen, I. M., Paeleman, A., Vandewoestijne, E., Van Bergen, L., Bragard, C., Lievens, B., Vanachter, A. C. R. C., & Thomma, B. P. H. J. (2009). Pepino mosaic virus isolates and differential symptomatology in tomato. Plant Pathology, 58, 450–460.Hanssen, I. M., Mumford, R., Blystad, D.-G., Cortez, I., Hasiów-Jaroszewska, B., Hristova, D., Pagán, I., Pereira, A.-M., Peters, J., Pospieszny, H., Ravnikar, M., Stijger, I., Tomassoli, L., Varveri, C., van der Vlugt, R., & Nielsen, S. L. (2010). Seed transmission of Pepino mosaic virus in tomato. European Journal of Plant Pathology, 126, 145–152.Hasiów-Jaroszewska, B., Borodynko, N., Jackowiak, P., Figlerowicz, M., & Pospieszny, H. (2010a). Pepino mosaic virus – a pathogen of tomato crops in Poland: biology, evolution and diagnostics. Journal of Plant Protection Research, 50, 470–476.Hasiów-Jaroszewska, B., Jackowiak, P., Borodynko, N., Figlerowicz, M., & Pospieszny, H. (2010b). Quasispecies nature of Pepino mosaic virus and its evolutionary dynamics. Virus Genes, 41, 260–267.Jeffries, C. J. (1998). FAO/IPGRI technical guidelines for the safe movement of germplasm no. 19. Potato. Food and agriculture organization of the United Nations, Rome/International Plant Genetic Resources Institute, Rome pp 177Jones, R. A. C., Koenig, R., & Lesemann, D. E. (1980). Pepino mosaic virus, a new potexvirus from pepino (Solanum muricatum). Annals of Applied Biology, 94, 61–68.Jordá, C., Lázaro Pérez, A., & Martínez Culebras, P. (2001). First report of Pepino mosaic virus on natural hosts. Plant Disease, 85, 1292.King, A. M. Q., Adams, M. J., Carstens, E. B., Lefkowitz, E. J., (eds). (2012). potexvirus, pp 912–915, in virus taxonomy, classification and nomenclature of viruses; ninth report of the international committee on taxonomy of viruses (p 1327) London, UK: Elsevier Academic PressLing, K.-S., & Zhang, W. (2011). First report of Pepino mosaic virus infecting tomato in Mexico. Plant Disease, 95(8), 1035.Martin, J., & Mousserion, C. (2002). Potato varieties which are sensitive to the tomato strains of Pepino mosaic virus (PepMV). Phytoma Défence Végétaux, 552, 26–28.Mehle, N., Gutierrez-Aguirre, I., Prezelj, N., Delić, D., Vidic, U., & Ravnikar, M. (2014). Survival and transmission of potato virus Y, pepino mosaic virus, and potato spindle tuber viroid in water. Applied and Environmental Microbiology, 80(4), 1455–1462.Moreno-Pérez, M. G., Pagán, I., Aragón-Caballero, L., Cáceres, F., Aurora Fraile, A., & García-Arenal, F. (2014). Ecological and genetic determinants of Pepino mosaic virus emergence. Journal of Virology, 88(6), 3359–3368.Noël, P., Hance, T., & Bragard, C. (2014). Transmission of the pepino mosaic virus by whitefly. European Journal of Plant Pathology, 138, 23–27.Pagan, I., Cordoba-Selles, M. D., Martinez-Priego, L., Fraile, A., Malpica, J. M., Jorda, C., & Garcia-Arenal, F. (2006). Genetic structure of the population of pepino mosaic virus infecting tomato crops in Spain. Phytopathology, 96, 274–279.Papayiannis, L. C., Kokkinos, C. D., & Alfaro-Fernández, A. (2012). Detection, characterization and host range studies of Pepino mosaic virus in Cyprus. European Journal of Plant Pathology, 132, 1–7.Pospieszny, H., Haslow, B., & Borodynko, N. (2008). Characterization of two Polish isolates of Pepino mosaic virus. European Journal of Plant Pathology, 122, 443–445.Salomone, A., & Roggero, P. (2002). Host range, seed transmission and detection by ELISA and lateral flow of an Italian isolate of Pepino mosaic virus. Journal of Plant Pathology, 84, 65–68.Samson, R. G., Allen, T. C., & Whitworth, J. L. (1993). Evaluation of direct tissue blotting to detect potato viruses. American Potato Journal, 70, 257–265.Schwarz, D., Beuch, U., Bandte, M., Fakhro, A., Büttner, C., & Obermeier, C. (2010). Spread and interaction of pepino mosaic virus (PepMV) and pythium aphanidermatum in a closed nutrient solution recirculation system: effects on tomato growth and yield. Plant Pathology, 59(3), 443–452.Shipp, J. L., Buitenhuis, R., Stobbs, L., Wang, K., Kim, W. S., & Ferguson, G. (2008). Vectoring of pepino mosaic virus by bumble-bees in tomato greenhouses. Annals of Applied Biology, 153, 149–155.Van der Vlugt, R. A. A. (2009). Pepino mosaic virus (review). Hellenic Plant Protection Journal, 2, 47–56.Van der Vlugt, R. A. A., & Stijger, C. C. M. M. (2008). Pepino mosaic virus. In B. W. J. Mahy & M. H. V. Van Regenmortel (Eds.), Encyclopedia of virology (5th ed., pp. 103–108). Wageningen: Oxford Elsevier.Van der Vlugt, R. A. A., Stijger, C. C. M. M., Verhoeven, J. T. J., & Lesemann, D.-E. (2000). First report of Pepino mosaic virus on tomato. Plant Disease, 84, 103.Van der Vlugt, R. A. A., Cuperus, C., Vink, J., Stijger, I. C. M. M., Lesemann, D.-E., Verhoeven, J. T. J., & Roenhorst, J. W. (2002). Identification and characterization of Pepino mosaic potexvirus in tomato. Bulletin EPPO/EPPO Bulletin, 32, 503–508.Verchot-Lubicz, J., Chang-Ming, Y., & Bamunusinghe, D. (2007). Molecular biology of potexviruses: recent advances. Journal of General Virology, 88(6), 1643–1655.Verhoeven, J. T. H. J., van der Vlugt, R., & Roenhorst, J. W. (2003). High similarity between tomato isolates of pepino mosaic virus suggests a common origin. European Journal of Plant Pathology, 109, 419–425.Werkman, A.W., & Sansford, C.E. (2010). Pest risk analysis for pepino mosaic virus for the EU. Deliverable Report 4.3. EU Sixth Framework project PEPEIRA. http:// www.pepeira.com .Wright, D., & Mumford, R. (1999). Pepino mosaic potexvirus (PepMV): first records in tomato in the United Kingdom. Plant disease notice (89th ed.). York, UK: Central Science Laboratory

Transmission of Pepino mosaic virus by the fungal vector Olpidium virulentus

This is the peer reviewed version of the following article: Alfaro-Fernández, A., Del Carmen Córdoba-Sellés, M., Herrera-Vásquez, J., Cebrián, M.d.C. and Jordá, C. (2010), Transmission of Pepino mosaic virus by the Fungal Vector Olpidium virulentus. Journal of Phytopathology, 158: 217-226, which has been published in final form at https://doi.org/10.1111/j.1439-0434.2009.01605.x. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.[EN] Transmission of Pepino mosaic virus (PepMV) by the fungal vector Olpidium virulentus was studied in two experiments. Two characterized cultures of the fungus were used as stock cultures for the assay: culture A was from lettuce roots collected in Castellon (Spain), and culture B was from tomato roots collected in Murcia (Spain). These fungal cultures were maintained in their original host and irrigated with sterile water. The drainage water collected from irrigating these stock cultures was used for watering PepMV-infected and non-infected tomato plants to constitute the acquisition-source plants of the assay, which were divided into six different plots: plants containing fungal culture A (non-infected and PepMV-infected); plants containing fungal culture B (non-infected and PepMV-infected); PepMV-infected plants without the fungus; and plants non-infected either with PepMV and the fungus. Thirty-six healthy plants grouped into six plots, which constituted the virus acquisition-transmission plants of the assay, were irrigated with different drainage waters obtained by watering the different plots of the acquisition-source plants. PepMV was only transmitted to plants irrigated with the drainage water collected from PepMV-infected plants whose roots contained the fungal culture B from tomato with a transmission rate of 8%. No infection was detected in plants irrigated with the drainage water collected from plots with only a fungus or virus infection. Both the virus and fungus were detected in water samples collected from the drainage water of the acquisition-source plants of the assay. These transmission assays demonstrated the possibility of PepMV transmission by O. virulentus collected from tomato crops.This work was supported by Grant AGL2005-06682-C03-01 from the Spanish Ministry of Education and Science (MEC, Spain). We also thank Instituto Agroforestal Mediterraneo (UPV, Valencia) for fellowship support to A. Alfaro-Fernandez.Alfaro Fernández, AO.; Córdoba-Sellés, MDC.; Herrera-Vasquez, JA.; Cebrián, MDC.; Jordá, C. (2010). Transmission of Pepino mosaic virus by the fungal vector Olpidium virulentus. Journal of Phytopathology. 158(4):217-226. https://doi.org/10.1111/j.1439-0434.2009.01605.x217226158

Multiplex PCR assay for the simultaneous detection and differentiation of Olpidium bornovanus, O. brassicae, and O. virulentus

[EN] A multiplex PCR method has been developed to detect, differentiate, and confirm the morphological identification of three root infecting Olpidium spp.: O. bornovanus, O. brassicae, and O. virulentus. Of the 132 root samples examined, 101 samples were infected by Olpidium spp.. Based on the morphology of resting spores, the presence of O. bornovanus was confirmed in 20.5 % of the samples, whereas species identity could not be determined for the remaining samples because they failed to reproduce sexually. With multiplex PCR, it was possible to determine the Olpidium identity of all the infected samples, even when resting spores were not formed. This method was also effective for detecting Olpidium spp. in water samples. In addition, the specificity and sensitivity of multiplex PCR were evaluated. The multiplex PCR method was validated with samples of 9 different crops from 11 countries of America, Europe, and Africa.Jose Angel Herrera Vasquez was supported by a predoctoral fellowship from the Agencia Espanola de Cooperacion Internacional Para el Desarrollo (AECID, Spain, 2004-2007 period) and the Instituto Para la Formacion y Aprovechamiento de Recursos Humanos and the Secretaria Nacional de Ciencia y Tecnologia (IFARHU-SENACYT, Panama, 2007-2010 period). We thank the Direccion Nacional de Sanidad Vegetal-Ministerio de Desarrollo Agropecuario (DNSV-MIDA, Panama) for their technical assistance. We also thank Dr. M. L Herrero (Bioforsk, Plant Health and Plant Protection Division, Norway) and Dr. J. C. Tello (Plant Production Department, University of Almeria, Spain) for providing the fungal isolates used in the specificity assays, and Dr. R. Beltran for providing the soil samples from Brazil. This work was supported by grants AGL 2005-06682-C03-01 from the Comision Interministerial de Ciencia y Tecnologia CICYT, Spain.Herrera-Vásquez, JA.; Cebrián, MDC.; Alfaro Fernández, AO.; Córdoba-Sellés, MDC.; Jordá, C. (2009). Multiplex PCR assay for the simultaneous detection and differentiation of Olpidium bornovanus, O. brassicae, and O. virulentus. Mycological Research. 113:602-610. https://doi.org/10.1016/j.mycres.2009.01.00760261011

Simultaneous detection and identification of Pepino mosaic virus (PepMV) isolates by multiplex one-step RT-PCR

[EN] A RT-PCR was developed for the simultaneous detection and identification of three groups of Pepino mosaic virus (PepMV): European/Peruvian, Chilean 1/US1 and Chilean 2/US2 groups, followed by a restriction analysis that allowed the separation of the European, Peruvian, Chilean 2 and US2 isolates (patent pending). The multiplex RT-PCR reaction was performed by a mix of six primers that amplified a part of the RNA-dependent RNA polymerase gene of PepMV plus an internal control. Amplifications resulted in a 980 bp, 703 bp or 549 bp PCR product for European/Peruvian, Chilean 1/US1 or Chilean 2/US2 groups, respectively. For the identification of the isolates present within the European/Peruvian and Chilean 2/US2 groups, the amplified PCR fragments were directly digested with SacI enzyme. The multiplex RT-PCR method presented higher sensitivity to detect CH1/US1 isolates in field samples than the RFLP-PCR method described by Hanssen et al. (European Journal of Plant Pathology 121:131-146, 2008). The detection limit observed with the multiplex RT-PCR was equal to or 3,125 times higher when compared to single RT-PCR or ELISA-DAS and molecular hybridisation methods, respectively. The use of the multiplex RT-PCR method in routine analysis of field tomato samples allowed the detection of 36.2 and 33.4% more positives when compared to the serological and molecular hybridisation methods, respectively, and the identification of plants infected with one, two or three isolates of PepMV.This work was supported by grants AGL2005-06682-C03-01 and BIO2005-07331-C02-01 from the Ministerio de Educación y Ciencia (MEC, Spain). We thank H. Pospieszny (Institute of Plant Protection, Pozna, Poland) and M. Aranda (CEBAS-CSIC, Murcia, Spain) for providing us with the PK and Sp-13 isolates of PepMV, respectively. We thank A. Espino, R. Martín (Sanidad Vegetal, Canarias) and M. Juarez (Universidad Miguel Hernández, Orihuela) for their assistance in collecting the field samples. We thank Lorena Corachán and Lorena Latorre for excellent technical assistance and Graciela Martínez for reviewing the English text.Alfaro Fernández, AO.; Sanchez Navarro, JA.; Cebrián, MDC.; Córdoba-Sellés, MDC.; Pallás Benet, V.; Jordá, C. (2009). Simultaneous detection and identification of Pepino mosaic virus (PepMV) isolates by multiplex one-step RT-PCR. European Journal of Plant Pathology. 125:143-158. https://doi.org/10.1007/s10658-009-9466-714315812

Occurrence and geographical distribution of torrado disease in Spain

This is the peer reviewed version of the following article: Alfaro-Fernández, A., Córdoba-Sellés, M.D.C., Juárez, M., Herrera-Vásquez, J., Sánchez-Navarro, J., Cebrián, M.D.C., Font, M.I. and Jordá, C. (2010), Occurrence and Geographical Distribution of the `Torrado¿ Disease in Spain. Journal of Phytopathology, 158: 457-469, which has been published in final form at https://doi.org/10.1111/j.1439-0434.2009.01639.x. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.[EN] In surveys to determine the occurrence and distribution of the torrado disease (Tomato torrado virus, ToTV) in the main Spanish tomato growing areas from 2001 to 2008, a total of 584 samples from symptomatic and asymptomatic plants were collected from 92 greenhouses. The tests showed that 451 plants from 85 greenhouses of different areas were infected with ToTV. The majority of the positive samples showed typical symptoms of the disease. However, plants showing different symptoms of necrosis and even asymptomatic plants were infected with the virus. Co-infection of ToTV with Pepino mosaic virus (PepMV) occurred in a large number of samples (60.5%), and several samples were infected with other tomatoinfecting viruses, including Cucumber mosaic virus (CMV), Potato virus Y (PVY), Tomato spotted wilt virus (TSWV), Tomato mosaic virus (ToMV), Parietaria mottle virus (PMoV), Tomato chlorosis virus (ToCV) and Tomato yellow leaf curl virus (TYLCV). Tomato apex necrosis virus (ToANV) was not detected in any of those samples with similar symptoms to those described for that virus. Additional tests revealed that (i) ToTV whitefly transmission is highly efficient and variety-dependent in tomato plants, (ii) Datura stramonium is another solanaceous species susceptible to this virus and (iii) the tissue-printing hybridization is a reliable technique which could facilitate the routine diagnosis and large-scale analysis of ToTV.This work was supported by grants AGL2005-06682-C03-01 from the Spanish Ministry of Education and Science (MEC, Spain). We thank the Instituto Agroforestal Mediterraneo (UPV, Valencia) for fellowship support to A. Alfaro-Fernandez. We also thank Dr V. Pallas (IBMCP, Valencia, Spain) and Dr M. Turina for kindly providing us the dig-RNA probe for PMoV detection and a positive control of ToANV, respectively. We also thank A. Espino and R. Martin (Sanidad Vegetal, the Canary Islands), Dr Alfredo Lacasa (IMIDA, the Murcia Region) and Dr J. Tello (Universidad de Almeria) for their advice and assistance in the field surveys.Alfaro Fernández, AO.; Córdoba-Sellés, MDC.; Juárez, M.; Herrera-Vasquez, JA.; Sanchez Navarro, JA.; Cebrián, MDC.; Font San Ambrosio, MI.... (2010). Occurrence and geographical distribution of torrado disease in Spain. Journal of Phytopathology. 158:457-469. https://doi.org/10.1111/j.1439-0434.2009.01639.x45746915