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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.

Transmission of Pepino mosaic virus by the Fungal Vector Olpidium virulentus A Ana na A Alfaro lfaro-F -Fernandez erná ndez, , M Marıa aría del del C Carmen armen C Cordoba ó rdoba-S -Selles ellés, , J Jose osé A Á ngel ngel H Herrera errera-V -Vasquez á squez, , M Marıa del aría del C Carmen armen C Cebrian ebriá n and and C Concepcion oncepció n J Abstract 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 Castello´n (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 PepMVinfected); 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 acquisitionsource plants of the assay. These transmission assays demonstrated the possibility of PepMV transmission by O. virulentus collected from tomato crops.

In surveys to determine the occurrence and distribution of the ÔtorradoÕ 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.

European Journal of Plant Pathology Turnip yellow mosaic virus in Chinese cabbage in Spain: commercial seed transmission and molecular characterisation.--Manuscript Draft-- Abstract: 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 seedto-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) Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporationrevealed that all the isolates infected turnip with typical symptoms, although differences were observed in other hosts.Response to Reviewers: Reviewer # 1: Firstly, the reviewer pointed that there were no significant details about the seed origin. As he/she correctly stated the seeds of Chinese cabagge var. Sumiko are a F1 hybrid variety developed by Bejo Zaden, however no further information was available. They were probably produced in France, Italy, The Netherlands, Australia or the USA, and imported to Spain, but this information is not supplied. Secondly, we have included more details in the seedling tests and corrected the Table 3, because the footnote (b) was incomplete. In the revised version, we have explained how was performed the Petri dishes sown and in the Table 3 clarified the groups of samples: 10 seedlings were considered a single sample in the Petri dishes assay and 4 in the trays. Thirdly, we think that testing the plants individually probably gives a realistic data of TYMV se...