First nucleotide sequence of a Carlavirus infecting caper

A carlavirus from asymptomatic caper plants (Capparis spinosa L.), that was provisionally considered a distinct isolate of the previously described Caper latent virus, was partially sequenced. The translated amino acid sequences of the RNA dependent RNA polymerase (RdRp) gene and coat protein (CP) gene were compared with the equivalent sequences of members of the genus Carlavirus. The isolate, named CapLV-L7, showed less than 78% and 72% amino acid identity in RdRp and CP regions, respectively, with the other carlaviruses tested. The closest sequence similarity was with Nerine latent virus and Potato virus M. The phylogenic trees showed a close relationship of CapLV-L7 with Nerine latent virus in both genes

Rapid identification of Trialeurodes vaporariorum, Bemisia tabaci (MEAM1 and MED) and tomato-infecting criniviruses in whiteflies and in tomato leaves by real-time reverse transcription-PCR assay

Abstract The whiteflies Bemisia tabaci and Trialeurodes vaporariorum (Hemiptera Aleyrodidae) are harmful pests of vegetable and ornamental crops in many countries. Also, they are vectors of emergent viruses on tomato including the criniviruses (Closteroviridae genus Crinivirus) Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV). Since different vectors are involved in the transmission of both viruses (ToCV is transmitted by B. tabaci, Trialeurodes abutiloneus and T. vaporariorum while TICV is transmitted only by T. vaporariorum), and they induce similar symptoms on tomato plants, a sensitive and specific diagnosis method is desirable. In addition, a rapid discriminating method of the vectors is essential for monitoring and control activities and epidemiological studies. For these reasons, a combined protocol based on one-step multiplex real-time reverse transcription (RT)-PCR has been developed for the identification of T. vaporariorum, two invasive species of the complex B. tabaci (MEAM1 and MED) and for the specific detection of ToCV and TICV in whiteflies and plants

In vitro thermotherapy and shoot-tip culture to eliminate Caper latent virus in Capparis spinosa

Capparis spinosa plants in the Sicilian Islands, Italy, are frequently infected with Caper latent virus (CapLV). CapLV affects the vigour and longevity of caper plants and exacerbates the effect of other adverse biotic and abiotic factors. To have CapLV-free cuttings of caper for multiplication and new planting is therefore advantageous for a revival of this crop wherever it is cultivated. An improved procedure for the in vitro thermotherapy combined with the culture of the shoot tips of C. spinosa has been applied to caper material collected from Salina (Aeolian Archipelago) and Pantelleria. More than of 60% (Salina) and 90% (Pantelleria) of the shoot tips survived, and 89–93% of the regenerated plantlets were CapLV-free when tested by RT-PCR. This is the first report on eliminating CapLV from caper plants

Guidelines for improving statistical analyses of validation datasets for plant pest diagnostic tests

peer reviewedAppropriate statistical analysis of the validation data for diagnostic tests facilitates the evaluation of the performance criteria and increases the confidence in the conclusions drawn from these data. A comprehensive approach to analysing and reporting data from validation studies and inter-laboratory comparisons such as test performance studies is described. The proposed methods, including statistical analyses, presentation and interpretation of the data, are illustrated using a real dataset generated during a test performance study conducted in the framework of the European project, VALITEST. This analytical approach uses, wherever possible and whenever applicable, statistical analyses recommended by international standards illustrating their application to plant health diagnostic tests. The present work is addressed to plant health diagnosticians and researchers interested and/or involved in the validation of plant diagnostic tests, and also aims to convey the necessary information to those without a statistical background. Detailed statistical explanations are provided in the Appendices

Inheritance analysis and identification of SNP markers associated with ZYMV resistance in Cucurbita pepo

[EN] Cucurbit crops are economically important worldwide. One of the most serious threats to cucurbit production is Zucchini yellow mosaic virus (ZYMV). Several resistant accessions were identified in Cucurbita moschata and their resistance was introgressed into Cucurbita pepo. However, the mode of inheritance of ZYMV resistance in C. pepo presents a great challenge to attempts at introgressing resistance into elite germplasm. The main goal of this work was to analyze the inheritance of ZYMV resistance and to identify markers associated with genes conferring resistance. An Illumina GoldenGate assay allowed us to assess polymorphism among nine squash genotypes and to discover six polymorphic single-nucleotide polymorphisms (SNPs) between two near-isogenic lines, "True French" (susceptible to ZYMV) and Accession 381e (resistant to ZYMV). Two F-2 and three BC1 populations obtained from crossing the ZYMV-resistant Accession 381e with two susceptible ones, the zucchini True French and the cocozelle "San Pasquale," were assayed for ZYMV resistance. Molecular analysis revealed an approximately 90% association between SNP1 and resistance, which was confirmed using High Resolution Melt (HRM) and a CAPS marker. Co-segregation up to 72% in populations segregating for resistance was observed for two other SNP markers that could be potentially linked to genes involved in resistance expression. A functional prediction of proteins involved in the resistance response was performed on genome scaffolds containing the three SNPs of interest. Indeed, 16 full-length pathogen recognition genes (PRGs) were identified around the three SNP markers. In particular, we discovered that two nucleotide-binding site leucine-rich repeat (NBS-LRR) protein-encoding genes were located near the SNP1 marker. The investigation of ZYMV resistance in squash populations and the genomic analysis performed in this work could be useful for better directing the introgression of disease resistance into elite C. pepo germplasm.This work was supported by the Ministry of University and Research (GenHORT project).Capuozzo, C.; Formisano, G.; Iovieno, P.; Andolfo, G.; Tomassoli, L.; Barbella, M.; Picó Sirvent, MB.... (2017). Inheritance analysis and identification of SNP markers associated with ZYMV resistance in Cucurbita pepo. Molecular Breeding. 37(8). https://doi.org/10.1007/s11032-017-0698-5S378Addinsoft (2007) XLSTAT, Analyse de données et statistique avec MS Excel. Addinsoft, NYAndolfo G, Ercolano MR (2015) Plant innate immunity multicomponent model. Front Plant Sci 6:987Andolfo G, Sanseverino W, Rombauts S et al (2013) Overview of tomato (Solanum lycopersicum) candidate pathogen recognition genes reveals important Solanum R locus dynamics. New Phytol 197:223–237Andolfo G, Ferriello F, Tardella L et al (2014) Tomato genome-wide transcriptional responses to fusarium wilt, and tomato mosaic virus. PLoS One 9:e94963Blanca J, Cañizares J, Roig C, Ziarsolo P, Nuez F, Picó B (2011) Transcriptome characterization and high throughput SSRs and SNPs discovery in Cucurbita pepo (Cucurbitaceae). BMC Genomics 12:104Brown RN, Bolanos-Herrera A, Myers JR, Jahn MM (2003) Inheritance of resistance to four cucurbit viruses in Cucurbita moschata. Euphytica 129:253–258Burge CB, Karlin S (1998) Finding the genes in genomic DNA. Curr Opin Struct Biol 8:346–354Cipollini D (2008) Constitutive expression of methyl jasmonate-inducible responses delays reproduction and constrains fitness responses to nutrients in Arabidopsis thaliana. Evol Ecol 24:59–68Cohen R, Hanan A, Paris HS (2003) Single-gene resistance to powdery mildew in zucchini squash (Cucurbita pepo). Euphytica 130:433–441Collum TD, Padmanabhan MS, Hsieh YC, Culver JN (2016) Tobacco mosaic virus-directed reprogramming of auxin/indole acetic acid protein transcriptional responses enhances virus phloem loading. Proc Natl Acad Sci U S A 113:E2740–E2749Desbiez C, Lecoq H (1997) Zucchini yellow mosaic virus. Plant Pathol 46:809–829Ercolano MR, Sanseverino W, Carli P, Ferriello F, Frusciante L (2012) Genetic and genomic approaches for R-gene mediated disease resistance in tomato: retrospects and prospects. Plant Cell Rep 31:973–985Esteras C, Gómez P, Monforte AJ, Blanca J, Vicente-Dólera N, Roig C, Nuez F, Picó B (2012) High-throughput SNP genotyping in Cucurbita pepo for map construction and quantitative trait loci mapping. BMC Genomics 13:80Formisano G, Paris HS, Frusciante L, Ercolano MR (2010) Commercial Cucurbita pepo squash hybrids carrying disease resistance introgressed from Cucurbita moschata have high genetic similarity. Plant Genet Resour 8:198–203Fulton TM, Chunwongse J, Tanksley SD (1995) Microprep protocol for extraction of DNA from tomato and other herbaceous plants. Plant Mol Biol Report 13:207–209Gal-On A (2007) Zucchini yellow mosaic virus: insect transmission and pathogenicity—the tails of two proteins. Mol Plant Pathol 8:139–150Gilbert-Albertini F, Lecoq H, Pitrat M, Nicolet JL (1993) Resistance of Cucurbita moschata to watermelon mosaic virus type 2 and its genetic relation to resistance to zucchini yellow mosaic virus. Euphytica 69:231–237Gómez P, Rodríguez-Hernández AM, Moury B, Aranda MA (2009) Genetic resistance for the sustainable control of plant virus diseases: breeding, mechanisms and durability. Eur J Plant Pathol 125:1–22Gong L, Stift G, Kofler R, Pachner M, Lelley T (2008a) Microsatellites for the genus Cucurbita and an SSR-based genetic linkage map of Cucurbita pepo L. Theor Appl Genet 117:37–48Gong L, Pachner M, Kalai K, Lelley T (2008b) SSR-based genetic linkage map of Cucurbita moschata and its synteny with Cucurbita pepo. Genome 51:878–887Iovieno P, Andolfo G, Schiavulli A, Catalano D, Ricciardi L, Frusciante L et al. (2015) Structure, evolution and functional inference on the MildewLocusO (MLO) gene family in three cultivated Cucurbitaceae. BMC Genomics 16:1112. doi: 10.1186/s12864-015-2325-3Ishibashi K, Kezuka Y, Kobayashi C, Kato M, Inoue T, Nonaka T et al (2014) Structural basis for the recognition–evasion arms race between Tomato mosaic virus and the resistance gene Tm-1. PNAS 111:E3486–E3495Lecoq H, Pitrat M, Clément M (1981) Identification et caractérisation d’un potyvirus provoquant la maladie du rabougrissement jaune du melon. Agronomie 1:827–834Lefebvre V, Palloix A (1996) Both epistatic and additive effects of QTLs are involved in polygenic induced resistance to disease: a case study, the interaction pepper—Phytophthora capsici Leonian. Theor Appl Genet 93:503–511Levi A, Thomas CE, Newman M, Zhan X, Xu Y, Wehner TC (2003) Massive preferential segregation and nonrandom assortment of linkage-groups produce quasi-linkage in an F2 mapping population of watermelon. Hortscience 38:782Lisa V, Lecoq H (1984) Zucchini yellow mosaic virus. Descriptions of Plant Viruses, Commonwealth Mycological Institute and Association of Applied Biologists 282Lisa V, Boccardo G, D'Agostino G, Dellavalle G, d’Aquilio M (1981) Characterization of a potyvirus that causes zucchini yellow mosaic. Phytopathology 71:667–672MacQueen A, Bergelson J (2016) Modulation of R-gene expression across environments. J Exp Bot 67:2093–2105Mantel N (1967) The detection of disease clustering and a generalized regression approach. Cancer Res 27:209–220Munger HM, Provvidenti R (1987) Inheritance of resistance to zucchini yellow mosaic virus in Cucurbita moschata. Cucurbit Genet Coop Rep 10:8–81Nameth ST, Dodds JA, Paulus AO, Laemmlen FF (1986) Cucurbit viruses of California: an ever-changing problem. Plant Dis 70:8–12Ott J, Wang J, Leal SM (2015) Genetic linkage analysis in the age of whole-genome sequencing. Nat Rev Genet 16(5):275–284Pachner M, Lelley T (2004) Different genes for resistance to zucchini yellow mosaic virus (ZYMV) in Cucurbita moschata. In: Lebeda A, Paris HS (eds) Progress in cucurbit genetics and breeding research: Proceedings of Cucurbitaceae 2004. Palacky University, Olomouc (Czech Republic), pp 237–243Pachner M, Paris HS, Lelley T (2011) Genes for resistance to zucchini yellow mosaic in tropical pumpkin. J Hered 102:330–335Pachner M, Paris HS, Winkler J, Lelley T (2015) Phenotypic and marker-assisted pyramiding of genes for resistance to zucchini yellow mosaic virus in oilseed pumpkin (Cucurbita pepo). Plant Breed 134:121–128Paris HS (1986) A proposed subspecific classification for Cucurbita pepo. Phytologia 61:133–138Paris HS (2001) Characterization of the Cucurbita pepo collection at the Newe Ya‘ar Research Center, Israel. Plant Genet Resour Newsl 126:41–45Paris HS (2008) Summer squash. In: Prohens J, Nuez F (eds) Handbook of plant breeding, Vegetables I: 351–379Paris HS, Cohen S (2000) Oligogenic inheritance for resistance to zucchini yellow mosaic virus in Cucurbita pepo. Ann Appl Biol 136:209–214Paris HS, Cohen S, Burger Y, Joseph R (1988) Single-gene resistance to zucchini yellow mosaic virus in Cucurbita moschata. Euphytica 37:27–29Peakall PE, Smouse R (2012) GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research—an update. Bioinformatics 28:2537–2539Sakamoto T, Deguchi M, Brustolini OJ, Santos AA, Silva FF, Fontes EP (2012) The tomato RLK superfamily: phylogeny and functional predictions about the role of the LRRII-RLK subfamily in antiviral defense. BMC Plant Biol 12:229Sanseverino W, Ercolano MR (2012) In silico approach to predict candidate R proteins and to define their domain architecture. BMC Res Notes 5:678Tamura 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. Mol Biol Evol 28:2731–2739Teare MD, Santibanez Koref MF (2014) Linkage analysis and the study of Mendelian disease in the era of whole exome and genome sequencing. Brief Funct Genomics 13(5):378–383Valkonen JPT, Wiegmann K, Hämäläinen JH, Marczewski W, Watanabe KN (2008) Evidence for utility of the same PCR-based markers for selection of extreme resistance to Potato virus Y controlled by Rysto of Solanum stoloniferum derived from different sources. Ann Appl Biol 152:121–130Wessel-Beaver L (2005) Cultivar and germplasm release. Release of ‘Soler’ tropical pumpkin. J Agric Univ P R 89:263–266Whitaker TW, Davis GN (1962) Cucurbits: botany, cultivation and utilization. Interscience, New York, pp 105–116Whitaker TW, Robinson RW (1986) Squash breeding. In: Bassett MJ (ed) Breeding vegetable crops. Avi, Westport, pp 209–242Xu Y, Crouch JH (2008) Marker-assisted selection in plant breeding: from publications to practice. Crop Sci 48:391–407Xu R, Zhang S, Huang J, Zheng C (2013) Genome-wide comparative in silico analysis of the RNA helicase gene family in Zea mays and Glycine max: a comparison with Arabidopsis and Oryza sativa. PLoS One 8:e78982Ye G, Smith KF (2008) Marker-assisted gene pyramiding for inbred line development: basic principles and practical guidelines. Int J Plant Breed 2:1–10Zdobnov EM, Apweiler R (2001) InterProScan—an integration platform for the signature-recognition methods in InterPro. Bioinformatics 17:847–848Zraidi A, Stift G, Pachner M, Shojaeiyan A, Gong L, Lelley T (2007) A consensus map for Cucurbita pepo. Mol Breed 20:375–38

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

Genetic variability of watermelon mosaic virus isolates infecting cucurbit crops in Italy

10 Pág.Watermelon mosaic virus (WMV; genus Potyvirus, family Potyviridae) is responsible for serious cucurbit yield losses worldwide. Different WMV genetic groups have been characterized so far. Among these, the "classical" (CL) group has been present in the Mediterranean basin for 40 years, whereas the "emergent" (EM) group includes isolates that are associated with more-severe symptoms observed since 2000. Information on the spatial and temporal evolution of WMV isolates in Italy is currently sparse. In this study, 39 WMV isolates samples collected in different regions over the last two decades were analysed at two different genomic regions that are known to be highly variable and contain recombination breakpoints. Most of the isolates collected between 2002 and 2009 were found to belong to the CL group, whereas the isolates from 2012 onwards were classified as EM, indicating that EM isolates have progressively displaced the CL population in Italy. Although genetic variability was observed within both CL and EM groups and recombinant isolates were detected, no positive selection or haplotype geographic structure were inferred. This suggest that the shift from CL to EM populations was likely due to multiple introductions of EM isolates in different regions of Italy rather than from genetic differentiation of local populations. The progressive increase in prevalence of the highly virulent EM populations is a serious concern because of their symptom severity, and the presence of multiple EM variants that include recombinants necessitates new efforts to develop durable control strategies.This work was supported by the project “EMERAMB, Emergent Viruses and Virus Vectors in Mediterranean Basin Crops”, which is funded through the ARIMNet2 2015 Call. ARIMNet2 (2014—2017) is an ERA-NET coordinated by INRA (France); it has received funding from the European Union’s Seventh Framework Programme for Research, Technological Development and Demonstration under Grant Agreement No. 618127.Peer reviewe

Survey of the distribution of Bemisia tabaci (Hemiptera: Aleyrodidae) in Lazio region (Central Italy): a threat for the northward expansion of Tomato leaf curl New Delhi virus (Begomovirus: Geminiviridae) infection

Bemisia tabaci (Hemiptera: Aleyrodidae) is responsible for severe damage to horticultural and ornamental crops worldwide, mainly for its role as virus vector. In Italy, the B. tabaci Mediterranean (MED) and Middle East–Asia Minor 1 (MEAM1) cryptic species are widespread in the Southern regions as well as in Sicily and Sardinia. During the last two decades, MED populations progressively increased, in those areas where intensive farming is applied. The recent introduction of the begomovirus Tomato leaf curl New Delhi virus (ToLCNDV) prompted extensive surveys of both vector and symptomatic plants. In 2016 and 2017, monitoring activities were carried out in Lazio region (Central Italy) where begomovirus epidemics had never occurred before and the presence of B. tabaci was thought to be only occasional. ToLCNDV-infected zucchini plants were found in Southern Lazio together with whitefly populations belonging only to the MED cryptic species. The MED-Q2 haplotype was the most abundant, likely favored by high temperatures and intensive agricultural practices. Single and mixed populations of MED and MEAM1 were found in Central and Northern Lazio, suggesting that agro-ecological factors still limit MED outbreaks in these areas. This preliminary survey indicates that B. tabaci is well established in Lazio, making ToLCNDV potentially able to spread to the rest of the region as well as to the nearby regions of Central Italy that have similar climatic and cultural conditions. The northward spread of B. tabaci is a critical issue for viral disease epidemiology and the management of whitefly-transmitted viruses in Central Italy, and must be kept under strict surveillance

Inter-Laboratory Comparison of RT-PCR-Based Methods for the Detection of Tomato Brown Rugose Fruit Virus on Tomato

In 2020, a test performance study (TPS) for the specific detection of tomato brown rugose fruit virus (ToBRFV) was organized in the frame of the H2020 Valitest project. Since no validated tests were available, all the protocols reported in the literature were at first screened, performing preliminary studies in accordance with the EPPO standard PM 7/98 (4). Five molecular tests, two conventional RT-PCR and three real-time RT-PCR were found to be suitable and were included in the TPS. Thirty-four laboratories from 18 countries worldwide took part in TPS, receiving a panel of 22 blind samples. The panel consisted of sap belonging to symptomatic or asymptomatic leaves of Solanum lycopersicum and Capsicum annuum. The results returned by each laboratory were analyzed and diagnostic parameters were assessed for each test: reproducibility, repeatability, analytical sensitivity, diagnostic sensitivity and diagnostic specificity. All the evaluated tests resulted in being reliable in detecting ToBRFV and were included in an EPPO Standard PM 7/146—Diagnostics

Development, Validation, and Application of Reverse Transcription Real-Time and Droplet Digital PCR Assays for the Detection of the Potyviruses Watermelon Mosaic Virus and Zucchini Yellow Mosaic Virus in Cucurbits

Among the cucurbit-infecting viruses, watermelon mosaic virus (WMV) and zucchini yellow mosaic virus (ZYMV) (Potyvirus: Potyviridae) are responsible for severe symptoms on cucumber, melon, watermelon, and zucchini cultivations worldwide. In this study, reverse transcription real-time PCR (real-time RT-PCR) and droplet-digital PCR (RT-ddPCR) assays targeting the coat protein (CP) genes of WMV and ZYMV were developed and validated according to the international standards of plant pest diagnosis (EPPO PM 7/98 (5)). First, the diagnostic performance of WMV-CP and ZYMV-CP real-time RT-PCRs was evaluated, and the assays displayed an analytical sensitivity of 10−5 and 10−3, respectively. The tests also showed an optimal repeatability, reproducibility and analytical specificity, and were reliable for the virus detection in naturally infected samples and across a wide range of cucurbit hosts. Based on these results, the real-time RT-PCR reactions were adapted to set up RT-ddPCR assays. These were the first RT-ddPCR assays aiming at the detection and quantification of WMV and ZYMV and showed a high sensitivity, being able to detect until 9 and 8 copies/µL of WMV or ZYMV, respectively. The RT-ddPCRs allowed the direct estimation of the virus concentrations and opened to a broad range of applications in disease management, such as the evaluation of partial resistance in breeding processes, identification of antagonistic/synergistic events, and studies on the implementation of natural compounds in the integrated management strategies