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.

Knowledge about the host range and genetic structure of emerging plant viruses provides insights into fundamental ecological and evolutionary processes, and from an applied perspective, facilitates the design and implementation of sustainable disease control measures. Tomato leaf curl New Delhi virus (ToLCNDV) is an emerging whitefly transmitted begomovirus that is rapidly spreading and inciting economically important diseases in cucurbit crops of the Mediterranean basin. Genetic characterization of the ToLCNDV Mediterranean populations has shown that they are monophyletic in cucurbit plants. However, the extent to which other alternative (cultivated and wild) hosts may affect ToLCNDV genetic population structure and virus prevalence remains unknown. In this study a total of 683 samples from 13 cultivated species, and 203 samples from 24 wild species from three major cucurbit-producing areas of Spain (Murcia, Alicante and Castilla-La Mancha) from five cropping seasons (2012–2016) were analyzed for ToLCNDV infection. Except for watermelon, ToLCNDV was detected in all cultivated-cucurbit species as well as in tomato. Among weeds, Ecballium elaterium, Datura stramonium, Sonchus oleraceus , and Solanum nigrum were identified as alternative ToLCNDV plant hosts, which could act as new potential sources of virus inoculum. Furthermore, we performed full-genome deep-sequencing of 80 ToLCNDV isolates from different hosts, location and cropping year. Our phylogenetic analysis supports a Mediterranean virus population that is genetically very homogeneous, with no clustering pattern, and clearly different from Asian virus populations. Additionally, D. stramonium displayed higher levels of within-host genetic diversity than cultivated plants, and this variability appeared to increase with time. These results suggest that the potential ToLCNDV adaptive evolution occurring in wild plant hosts could serve as a source of virus genetic variability, thereby affecting the genetic structure and spatial-temporal dynamics of the viral population.

Despite the importance of field-grown cucurbits in Spain, only limited information is available about the impact of disease on their production. During the 2003 and 2004 growing seasons, systematic surveys were carried out in open field melon (Cucumis melo) and squash (Cucurbita pepo) crops of Murcia Province (Spain). The fields were chosen with no previous information regarding their sanitation status, and samples were taken from plants showing viruslike symptoms. Samples were analyzed using molecular hybridization to detect Beet pseudo-yellows virus (BPYV), Cucurbit aphid-borne yellows virus (CABYV), Cucumber mosaic virus (CMV), Cucumber vein yellowing virus (CVYV), Cucurbit yellow stunting disorder virus (CYSDV), Melon necrotic spot virus (MNSV), Papaya ringspot virus (PRSV), Watermelon mosaic virus (WMV), and Zucchini yellow mosaic virus (ZYMV). We collected 924 samples from 48 field plots. Out of these, almost 90% were infected by at least one of the viruses considered, usually CABYV, which was present in 83 and 66% of the melon and squash samples, respectively. In the case of melon, CYSDV, BPYV, and WMV followed CABYV in relative importance, with frequencies of around 20 to 30%, while in squash, CVYV and BPYY showed frequencies between 28 and 21%. The number of multiple infections was very high, 66 and 56% of the infected samples of melon and squash, respectively, being afflicted. CABYV was present in all multiple infections. The high incidence of CABYV in single and multiple infections suggests that this virus may well become an important threat for cucurbit crops in the region. Restriction fragment length polymorphism (RFLP) analysis revealed that CABYV isolates can be grouped into two genetic types, both of which seemed to be present during the 2003 epidemic episode, but only one of the types was found in 2004.

Viral diseases that could cause important economic losses often affect cucurbits, but only limited information on the incidence and spatial distribution of specific viruses is currently available. During the 2005 and 2006 growing seasons, systematic surveys were carried out in open field melon (Cucumis melo), squash and pumpkin (Cucurbita pepo), watermelon (Citrullus lanatus) and cucumber (Cucumis sativus) crops of the Spanish Community of Valencia (eastern Spain), where several counties have a long standing tradition of cucurbit cultivation and production. Surveyed fields were chosen with no previous information as to their sanitation status, and samples were taken from plants that showed virus-like symptoms. Samples were analysed using molecular hybridisation to detect Beet pseudo-yellows virus (BPYV), Cucurbit aphid-borne yellows virus (CABYV), Cucumber mosaic virus (CMV), Cucumber vein yellowing virus (CVYV), Cucurbit yellow stunting disorder virus (CYSDV), Melon necrotic spot virus (MNSV), Papaya ring spot virus (PRSV), Watermelon mosaic virus (WMV) and Zucchini yellow mosaic virus (ZYMV). We collected 1767 samples from 122 independent field plots; out of these, approximately 94% of the samples were infected by at least one of these viruses. Percentages for the more frequently detected viruses were 35.8%, 27.0%, 16.5% and 7.2% for CABYV, WMV, PRSV and ZYMV, respectively, and significant deviations were found on the frequency distributions based on either the area or the host sampled. The number of multiple infections was high (average 36%), particularly for squash (more than 57%), with the most frequent combination being WMV + PRSV (12%) followed by WMV + CABYV (10%). Sequencing of WMV complementary DNA suggested that 'emerging' isolates have replaced the 'classic' ones, as described in southern regions of France, leading us to believe that cucurbit cultivation could be severely affected by these new, emerging isolates. Analysis of WMV nucleotide sequencesNucleic and amino acid sequences were aligned using ClustalX (Thompson et al., 1997) and BioEdit (Biological sequence alignment editor, Tom Hall, Ibis biosciences, Ann Appl Biol 162 (2013) 362-370

The genetic variability of a Cucurbit aphid-borne yellows virus (CABYV) (genus Polerovirus, family Luteoviridae) population was evaluated by determining the nucleotide sequences of two genomic regions of CABYV isolates collected in open-field melon and squash crops during three consecutive years in Murcia (southeastern Spain). A phylogenetic analysis showed the existence of two major clades. The sequences did not cluster according to host, year, or locality of collection, and nucleotide similarities among isolates were 97 to 100 and 94 to 97% within and between clades, respectively. The ratio of nonsynonymous to synonymous nucleotide substitutions reflected that all open reading frames have been under purifying selection. Estimates of the population's genetic diversity were of the same magnitude as those previously reported for other plant virus populations sampled at larger spatial and temporal scales, suggesting either the presence of CABYV in the surveyed area long before it was first described, multiple introductions, or a particularly rapid diversification. We also determined the full-length sequences of three isolates, identifying the occurrence and location of recombination events along the CABYV genome. Furthermore, our field surveys indicated that Aphis gossypii was the major vector species of CABYV and the most abundant aphid species colonizing melon fields in the Murcia (Spain) region. Our surveys also suggested the importance of the weed species Ecballium elaterium as an alternative host and potential virus reservoir.