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Melon necrotic spot virus (MNSV) was detected in field-grown Cucumis melo (rockmelon) and Citrullus lanatus (watermelon) plants in the Sunraysia district of New South Wales and Victoria, Australia, in 2012, 2013, and 2016, and in two watermelon seed lots tested at the Australian border in 2016. High-throughput sequencing was used to generate near full-length genomes of six isolates detected during the incursions and seed testing. Phylogenetic analysis of the genomes suggests that there have been at least two incursions of MNSV into Australia and none of the field isolates were the same as the isolates detected in seeds. The analysis indicated that one watermelon field sample (L10), the Victorian rockmelon field sample, and two seed interception samples may have European origins. The results showed that two isolates (L8 and L9) from watermelon were divergent from the type MNSV strain (MNSV-GA, D12536.2) and had 99% nucleotide identity to two MNSV isolates from human stool collected in the United States (KY124135.1, KY124136.1). These isolates also had high nucleotide pairwise identity (96%) to a partial sequence from a Spanish MNSV isolate (KT962848.1). The analysis supported the identification of three previously described MNSV genotype groups: EU-LA, Japan melon, and Japan watermelon. To account for the greater diversity of hosts and geographic regions of the MNSV isolates used in this study, it is suggested that the genotype groups EU-LA, Japan melon, and Japan watermelon be renamed to groups I, II, and III, respectively. The divergent isolates L8 and L9 from this study and the stool isolates from the United States formed a fourth genotype group, group IV. Soil collected from the site of the Victorian rockmelon MNSV outbreak was found to contain viable MNSV and the virus vector, a chytrid fungus, Olpidium bornovanus (Sahtiyanci) Karling, 18 months after the initial MNSV detection. This is a first report of O. bornovanus from soil sampled from an MNSV-contaminated site in Australia.

[EN] Plant defense against melon necrotic spot virus (MNSV) is triggered by the viral auxiliary replicase p29 that is targeted to mitochondrial membranes causing morphological alterations, oxidative burst and necrosis. Here we show that MNSV coat protein (CP) was also targeted to mitochondria and mitochondrial-derived replication complexes [viral replication factories or complex (VRC)], in close association with p29, in addition to chloroplasts. CP import resulted in the cleavage of the R/arm domain previously implicated in genome binding during encapsidation and RNA silencing suppression (RSS). We also show that CP organelle import inhibition enhanced RSS activity, CP accumulation and VRC biogenesis but resulted in inhibition of systemic spreading, indicating that MNSV whole-plant infection requires CP organelle import. We hypothesize that to alleviate the p29 impact on host physiology, MNSV could moderate its replication and p29 accumulation by regulating CP RSS activity through organelle targeting and, consequently, eluding early-triggered antiviral response. Cellular and molecular events also suggested that S/P domains, which correspond to processed CP in chloroplast stroma or mitochondrion matrix, could mitigate host response inhibiting p29-induced necrosis. S/P deletion mainly resulted in a precarious balance between defense and counter-defense responses, generating either cytopathic alterations and MNSV cell-to-cell movement restriction or some degree of local movement. In addition, local necrosis and defense responses were dampened when RSS activity but not S/P organelle targeting was affected. Based on a robust biochemical and cellular analysis, we established that the mitochondrial and chloroplast dual targeting of MNSV CP profoundly impacts the viral infection cycle., The authors thank L. Corachan-Valencia for technical assistance. This work was funded by grant BIO2017¿88321-R from the Spanish Agencia Estatal de Investigacion (AEI) and Fondo Europeo de Desarrollo Regional (FEDER). J.A.N. and M.S.-B. are the recipients of a postdoctoral contract and a PhD fellowship from the Ministerio de Ciencia, Innovacion y Universidades of Spain, respectively. Ministerio de Ciencia e Innovacion (PID2020-115571RB-I00), European Regional Development Fund

Seeds of three important cucurbit species (cucumber, melon and zucchini) were obtained through on-line mail-order providers and tested for selected viruses of biosecurity and agronomic concern. One or more of the targeted viruses were detected in 23 of the 31 seed lots tested. The quarantine pest melon necrotic spot virus was detected by ELISA in melon seeds. Multiple instances of contamination with the quarantine pest cucumber green mottle mosaic virus were also detected, and its presence in seeds of all three plant species was confirmed by inoculation and re-isolation from indicator plants. Cucumber mosaic virus, squash mosaic virus and potyviruses were repeatedly detected, and instances of insect pest and plant trash contamination were also observed. Seeds imported into Australia from some on-line sources have potential to introduce organisms of biosecurity concern.

In December 2018, virus-like symptoms (yellowing, vein clearing) were observed on 2% of muskmelon (Cucumis melo L.) plants in plastic houses on a farm in Gyeongsang province, Korea Total RNA from two symptomatic and two asymptomatic plants was extracted using RNeasy Plant Mini Kit (Qiagen, Germany) for high throughput sequencing (HTS). After pre-processing and Ribo-Zero rRNA removal, a cDNA library was prepared (Illumina TruSeq Stranded Total RNA kit) and sequenced (Illumina NovaSeq 6000 system: Macrogen Inc. Korea). De novo assembly of 88,222,684 HTS reads with Trinity software (r20140717) yielded 146,269 contigs of 201-28,442 bp, which were screened against the NCBI viral genome database by BLASTn. Contigs from cucumber mosaic virus (CMV), melon necrotic spot virus (MNSV), tobacco mosaic virus (TMV) and watermelon mosaic virus (WMV) were identified, all previously reported in Korea. Two contigs (8,539 and 8,040 bp) with 99.9% sequence identity to distinct cucurbit chlorotic yellows virus (CCYV) isolates (JN641883, RNA1, Taiwan; MH819191, RNA2, China) were also identified. The ten sequences most closely related to each RNA of the Korean isolate (≥99% coverage, ≥99.6% nt identity) were from Japan, China, Taiwan, or Israel. CCYV presence was confirmed by reverse transcription-PCR (RT-PCR) using newly designed specific primers, RdRp-F/RdRp-R (5'-ACCGAACACTTGGCTATCCAA-3'/5'-CTTAATGCCGCGTATGAACTCA-3') span style="font-family:'Times New Roman'; letter-spacing:-0.5pt">and HSP-F/HSP-R (5'-TGAACGACACTGAGTTCATTCCTA-3'/5'-CGCCAAGATCGTACATGAGGAA-3'), against RNA dependent RNA polymerase (RdRp; RNA1) and the heat shock protein 70 homolog (HSP70h; RNA2). Symptomatic samples yielded products of expected sizes (RdRp,450 bp; HSP70h, 510 bp) while asymptomatic samples did not. The amplicons were cloned, and two clones of each were sequenced (BIONEER, Korea; GenBank acc. nos. LC592226 and LC592227) showing 100% and 99.2% nt identity with RdRp and HSP70h genes of Chinese CCYV isolate SD (MH819190 and MH819191, respectively) and other Asian isolates. Primers specific for CMV, WMV, beet pseudo-yellows virus (BPYV) (Okuda et al., 2007), TMV (Kim et al., 2018), MNSV (F/R, 5'-ATCTCGCATTTGGCATTACTC-3'/5'-ATTTGTAGAGATGCCAACGTA-3'), cucurbit yellow stunting disorder virus (CYSDV; Zeng et al., 2011) and cucurbit aphid-borne yellows virus (CABYV; F/R, 5'-CGGTCTATTGTCTGCAGTACCA-3'/5'- GTAGAGGATCTTGAATTGGTCCTCA-3') were also used. None of these viruses were detected in the symptomatic samples, but both asymptomatic plants were positive for CMV and WMV, and one also for MNSV. In June and September 2020, muskmelon and oriental melon (Cucumis melo L. var. makuwa) plants with yellowing disease (incidence 80-90%) and whiteflies were observed in all investigated plastic houses of one muskmelon and one oriental melon farm in Gyeonggi and Jeolla provinces. Symptomatic samples (14 muskmelon; 6 oriental melon) were collected and RT-PCR tested as above; 19/20 samples were positive for CCYV, but none for the other viruses. The oriental melon sequence (LC592895, LC592230) showed 99.7% and 100% nt identity with the RdRp and HSP70h genes of Chinese isolate SD, respectively. CCYV was first reported in Japan (Okuda et al., 2010), Taiwan, and China (Huang et al., 2010; Gu et al., 2011); to our knowledge, this is the first report of CCYV infecting muskmelon and oriental melon in Korea. Whitefly-transmitted CCYV could present a serious threat of yield losses to cucurbit crops in Korea, requiring control of vector populations to prevent spread of CCYV.

[Background] Viruses are among the most destructive and difficult to control plant pathogens. Melon (Cucumis melo L.) has become the model species for the agriculturally important Cucurbitaceae family. Approaches that take advantage of recently developed genomic tools in melon have been extremely useful for understanding viral pathogenesis and can contribute to the identification of target genes for breeding new resistant cultivars. In this work, we have used a recently described melon microarray for transcriptome profiling of two melon cultivars infected with two strains of Melon necrotic spot virus (MNSV) that only differ on their 3′-untranslated regions., [Results] Melon plant tissues from the cultivars Tendral or Planters Jumbo were locally infected with either MNSV-Mα5 or MNSV-Mα5/3’264 and analysed in a time-course experiment. Principal component and hierarchical clustering analyses identified treatment (healthy vs. infected) and sampling date (3 vs. 5 dpi) as the primary and secondary variables, respectively. Out of 7566 and 7074 genes deregulated by MNSV-Mα5 and MNSV-Mα5/3’264, 1851 and 1356, respectively, were strain-specific. Likewise, MNSV-Mα5/3’264 specifically deregulated 2925 and 1618 genes in Tendral and Planters Jumbo, respectively. The GO categories that were significantly affected were clearly different for the different virus/host combinations. Grouping genes according to their patterns of expression allowed for the identification of two groups that were specifically deregulated by MNSV-Mα5/3’264 with respect to MNSV-Mα5 in Tendral, and one group that was antagonistically regulated in Planters Jumbo vs. Tendral after MNSV-Mα5/3’264 infection. Genes in these three groups belonged to diverse functional classes, and no obvious regulatory commonalities were identified. When data on MNSV-Mα5/Tendral infections were compared to equivalent data on cucumber mosaic virus or watermelon mosaic virus infections, cytokinin-O-glucosyltransferase2 was identified as the only gene that was deregulated by all three viruses, with infection dynamics correlating with the amplitude of transcriptome remodeling., [Conclusions] Strain-specific changes, as well as cultivar-specific changes, were identified by profiling the transcriptomes of plants from two melon cultivars infected with two MNSV strains. No obvious regulatory features shared among deregulated genes have been identified, pointing toward regulation through differential functional pathways., This work was supported by grants AGL2012-37390 and PCIN-2013-043 (Ministerio de Economía y Competitividad, Spain)

Melon necrotic spot virus (MNSV) infects various species of cucurbitaceous plants (Cucurbitaceae), including commercially grown crops like greenhouse cucumber in Poland. The virus is found in various climate zones and is widespread worldwide. In most cases, only reports of the first detection of the virus in a country are available, and there is no information on its potential further spread. MNSV is efficiently transmitted by the pathogenic fungus Olpidium radicale (Olpidium bornovanus) and with melon seeds. The virus is also transmitted mechanically with the sap of infected plants, which can result in unknowing spread of the virus within and between crops, especially when there are no visible disease symptoms. To date, no MNSV has been found in the PRA area and it is not subject to special regulations. Phytosanitary procedures include usually visual inspection of plants that may be infected with the virus., PL; pl; PDF; kwarantanna@iorpib.poznan.pl

México es uno de los principales productores de sandía a nivel mundial. Pero la presencia de patógenos del suelo como nematodos, Fusarium oxysporum f. sp. niveum y el virus de la mancha necrótica del melón (MNSV) representan un serio problema para los productores. El uso de plantas injertadas es una buena alternativa para hacer frente a estos problemas, ayudando a incrementar el rendimiento y calidad en sandía. Los portainjertos de sandía silvestre (Citrullus lanatus var. citroides) han demostrado ser eficaces para el control de la mayoría de las enfermedades que se transmiten a través del suelo, además de tener alta resistencia a nematodos; no obstante, no se tiene registro sobre su resistencia al MNSV. Se realizaron dos experimentos para evaluar la resistencia al MNSV de portainjertos para sandía. En el experimento 1 se evaluaron los portainjertos de híbridos interespecíficos “RS-841” “Ercole” (Cucurbita maxima×Cucurbita moschata) y de sandía silvestre los portainjertos “Robusta”, “RS0272” y “RS1833”. En el experimento 2 se evaluaron los portainjertos “RS-841”, “Ercole” y “Robusta”. En ambos casos en suelos infestados con MNSV. Los portainjertos de sandía silvestre fueron altamente susceptibles al MNSV, en cambio los híbridos interespecíficos mostraron resistencia a esta enfermedad. El portainjertos “RS-841” fue el que produjo los frutos de mejor calidad. El rendimiento fue inferior con diferencia significativa cuando se utilizaron los portainjertos de sandía silvestre.

Melon necrotic spot virus (MNSV) is endemic in cucurbit crops worldwide, causing epidemic outbreaks from time to time. MNSV is transmitted in nature by a soil-inhabiting fungus and also through seeds, making its detection in seed certification programs a necessity. Polyclonal antisera and RT-PCR-based detection assays have been developed for MNSV, but up to now no monoclonal antibodies (mAbs) have been described for this virus. In this study, we have produced mAbs in BALB/c mice against the MNSV over-expressed coat protein (CP). Titers of the antibodies produced against the recombinant MNSV CP ranged around 10-3 - 10-4 and the IgG yields for each mAb from ascitic fluids ranged from 1.51 to 6 mg/mL. Supernatants from ten hybridoma cell lines were evaluated in Western blot analysis and seven of them efficiently recognized the MNSV CP in crude extracts of MNSV-infected leaf material; the 2D4H4 hybridoma cell line was selected for further purification and characterization. The isotype of the 2D4H4 immunoglobulin class was identified as IgG2a and kappa light-chain. Western-blot analyses showed that mAb 2D4H4 provided sensitive and specific detection of MNSV. A TAS-ELISA protocol was developed for mAb 2D4H4. Using this protocol, limits of detection of 1:20,480 and 1:10,240 (g/mL, w/v) were attained for the homologous isolate and a heterologous MNSV isolate, respectively. Moreover, mAb 2D4H4 was used successfully to localize the MNSV CP in infected cells by immunocytochemistry/transmission electron microscopy, illustrating the usefulness of this mAb for advanced cellular studies.

At the beginning of 1999, 30 melon (Cucumis melo L.) plots on several farms (1,500 ha) in the Zacapa Valley of Guatemala were visited, and melon plants with two different symptomologies were observed. One group of plants exhibited stem necrosis at the crown level, and less frequently, small necrotic spots on leaves. Some plants exhibited necrosis of veins and yellow areas that evolved into interveinal necrosis and often expanded into large necrotic interveinal lesions. Roots were poor and lacked secondary rootlets. In some cases, wilt and plant death were detected. Affected plants appeared as localized patches in various areas of the plots on farms that were visited. Double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) serological analyses were carried out with 34 symptomatic plants. In these plants, a mixture of the crown and root were analyzed with two repetitions and two lots of different Melon necrotic spot virus (MNSV) polyclonal antisera (Loewe No. 07097 and Sanofi No. 70217). All 34 plants were positive for this virus. These results were confirmed using reverse transcription-polymerase chain reaction (RT-PCR) with specific MNSV primers (1). Spores of Olpidium bornovanus, the vector of MNSV, were seen on all ELISA-positive plants after staining rootlets with potassium hydroxide and neutralization with hydrochloric acid. In the same fields, another group of melon plants showed yellowing, curling, and mottling of leaves. Leaves collected from five symptomatic plants gave positive results in triple-antibody sandwich-ELISA using a Tomato yellow leaf curl begomovirus antiserum (DSMZ AS-0421 and DSMZ AS-0546/2). In 2001, these results were confirmed using PCR with degenerate primers that amplify the core region of most begomovirus coat protein genes (P. Martínez-Culebras, M. I. Font, and C. Jordá, unpublished). A 560-bp DNA fragment was amplified in these symptomatic melon samples. Three of the PCR products were sequenced and each showed 99% identity with the Melon chlorotic leaf curl virus isolate from Guatemala (GenBank Accession No. AF325497). Only one mixed infection of MNSV and MCLCV was found. During the four years subsequent to 1999, the number of melon plants showing both types of symptoms has increased. This study provides information on the current status of virus diseases in melon crops in Guatemala, and to our knowledge, this is the first report of MNSV in Guatemala. Reference: (1) B. Gosalvez et al. J. Virol. Methods 113:87.

Melon (Cucumis melo L.) represents an important crop in Panama where 1,449 ha were cultivated in 2005 with 920.4 ha of this crop planted in Los Santos Province (southeast region of Panama). During April 2005 and January 2006, several melon plants in commercial fields in that area showed stem necrosis at the crown level, and less frequently, small necrotic spots on leaves. In some cases, wilting and plant death were observed. Symptoms were similar to those caused by the carmovirus Melon necrotic spot virus (MNSV). Cysts of Olpidium bornovanus also were observed in the roots of all affected melon plants. Roots from eight symptomatic plants collected in seven fields were positive using doubleantibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with an antiserum specific for MNSV (BIO-RAD, Life Sciences, Barcelona, Spain). To confirm these results, total RNA was extracted from symptomatic plants and used in one-step reverse transcription-polymerase chain reaction (RT-PCR) with Platinum Taq (Invitrogen Life Technologies, Barcelona, Spain). MNSV specific primers designed to amplify a region of the coat protein gene were used in the assays. Amplicons of the expected size (651 bp) were generated from symptomatic plant tissue, but were not produced from healthy plants or the water used as negative controls. To establish the authenticity of this virus, RT-PCR products were purified with the High Pure PCR Product Purification Kit (Roche Diagnostics, Mannheim, Germany) and directly sequenced. Nucleotide sequences were analyzed by using the basic local alignment search tool (BLAST) (1). The primers produced two amplicons with different but similar sequences. One sequence (GenBank Accession No. DQ443546) showed 92% identity to the coat protein gene of the MNSV Spanish isolate (GenBank Accession No. AY330700) and the MNSV Dutch isolate (GenBank Accession No. M29671) and 88% identity to the Japanese isolate (GenBank Accession No. AB189944). The second sequence (GenBank Accession No. DQ443547) was 93% identical with the Spanish and Dutch MNSV isolates, 88% identical with the Japanese isolate, and 100% identical with sequences from commercial melon seed previously isolated in our laboratory (GenBank Accession No. DQ443545). Infected seed may be a concern with regard to long distance spread of the virus independent of the vector (3) and should be considered in disease management strategies. MNSV has been previously reported in Japan, the Netherlands, the United Kingdom, the United States (2), Guatemala (4), Mexico, Honduras, and Uruguay (C. Jordá, unpublished). To our knowledge, this is the first report of MNSV in Panama. References: (1) S. F. Altschul et al. Nucleic Acids Res. 25:3389, 1997. (2) A. A. Brunt et al. Plant Viruses Online: Descriptions and Lists from the VIDE Database. Online Publication, 1996. (3) R. N. Campbell et al. Phytopathology 86:1294, 1996. (4) C. Jordá et al. Plant Dis. 89:338, 2005.

Ali, A., Mohammad, O., and Khattab, A. 2012. Distribution of viruses infecting cucurbit crops and isolation of potential new virus-like sequences from weeds in Oklahoma. Plant Dis. 96:243-248. Field surveys were conducted from 2008 to 2010 to detect and determine the incidence of viruses in the major cucurbit-growing areas of Oklahoma. In total, 1,049 symptomatic leaf samples (890 from cucurbits, 109 from weed species, and 50 from crop plants [agricultural crops]) were collected from 90 fields in four counties (Atoka, Blaine, Jefferson, and Tulsa) of Oklahoma. Samples were tested against seven viruses, including Cucumber mosaic virus (CMV), Cucumber green mottle mosaic virus (CGMMV), Melon necrotic spot virus (MNSV), Papaya ringspot virus-watermelon strain (PRSV-W, formerly known as Watermelon mosaic virus-1), Squash mosaic virus (SqMV), Watermelon mosaic virus-2 (WMV-2), and Zucchini yellow mosaic virus (ZYMV), using dot-immunobinding assay (DIBA). Results showed the highest incidence for PRSV (51%), followed by WMV-2 (14%) and ZYMV (10%) among the collected samples. SqMV, MNSV, and CMV were detected in 3.8, 3.3, and 1.1% of the samples, respectively. None of the samples collected during surveys reacted positively against the antiserum of CGMMV. Mixed virus infections were common involving two (5.18%) or three (4.61%) viruses in various combinations. New weed host species were found to be infected with PRSV when confirmed by both DIBA and reverse-transcription polymerase chain reaction (RT-PCR). Some weed species contained possible new viruses when analyzed by random RT-PCR, followed by cloning, sequencing, and BLAST analysis with sequences in GenBank. Cucurbits (cucumber, cantaloupe, pumpkin, watermelon, and zucchini squash) are economically important cash crops worldwide, including the United States. Virus diseases are a major constraint in commercial cucurbit production (21,27), causing sporadic epidemics. More than 39 different viruses have been reported to cause cucurbit diseases and many are responsible for economic losses in the quality and quantity of cucurbit crops (15,18,27). In the United States, cucurbit crops are grown in several states and are commonly infected by a number of viruses. Several of these viruses, particularly the Potyvirus spp. Papaya ringspot virus-watermelon strain (PRSV-W, formerly known as Watermelon mosaic virus-1) (29), Watermelon mosaic virus-2 (WMV-2) (30), Zucchini yellow mosaic virus (ZYMV) (19,20), and Cucumber mosaic virus (CMV) (9), are the most frequently encountered and economically important (24,27). These viruses have been shown to infect cucurbit crops in major cucurbit-growing areas of California and Connecticut (25,28), Florida (1,31), Hawaii (35), Illinois (14)

In early 2013, greenhouse cucumber growers in Alberta, Canada, observed virus-like disease symptoms on mini-cucumber (Cucumis sativus) crops (e.g., ‘Picowell’). Two types of symptoms were commonly observed, green mottle mosaic and necrotic spots. In the early infection, young leaves of infected cucumber plants displayed light green mottle and blisters. The infected plants were stunted in growth, with darker green blisters and green mottle mosaic symptoms on mature leaves. Disease incidence varied from one greenhouse to another. In some severe cases, diseased plants were widely distributed inside the greenhouse, resulting in 10 to 15% yield losses based on grower's estimation. Nine symptomatic samples were collected and subjected to total RNA isolation using the TRIzol reagent (Invitrogen, Carlsbad, CA). Laboratory analyses were conducted using real-time RT-PCR systems for Cucumber green mottle mosaic virus (CGMMV) (1), Melon necrotic spot virus (MNSV, Ling, unpublished), and Squash mosaic virus (SqMV) (3). All nine samples were positive for CGMMV and seven of them were in mixed infections with MNSV. Two samples were selected for validation for the presence of CGMMV using conventional RT-PCR (2) with a new primer set (CGMMVMP F1: 5′-ATGTCTCTAAGTAAGGTGTC-3′ and CGMMV3′UTR R1: 5′-TGGGCCCCTACCCGGGG-3′) and two previous online published primer sets, one for CGMMV MP (5′ TAAGTTTGCTAGGTGTGATC-3′, GenBank Accession No. AJ250104 and 5′ ACATAGATGTCTCTAAGTAAG-3′, AJ250105), and another for CGMMV CP (5′ ACCCTCGAAACTAAGCTTTC-3′, AJ243351 and 5′ GAAGAGTCCAGTTCTGTTTC-3′, AJ243352). The expected sizes of RT-PCR products were obtained and sequenced directly. Sequences from these three products overlapped and generated a 1,282-bp contig (KF683202). BLASTn analysis to the NCBI database showed 99% sequence identity to CGMMV isolates identified in Asia, including China (GQ277655, KC852074), India (DQ767631), Korea (AF417243), Myanmar (AB510355), and Taiwan (HQ692886), but only 92% sequence identity to other CGMMV isolates identified in Europe, including Spain (GQ411361) and Russia (GQ495274), and 95% to CGMMV isolate from Israel (KF155231). The strong sequence identity to the CGMMV Asian isolates suggests that the Canadian CGMMV isolate identified in Alberta was likely of Asian origin. In two bioassay experiments using one sample prepared in 0.01 M phosphate buffer, the similar green mottle mosaic symptoms were observed on systemic leaves in the mechanically inoculated plants and the presence of CGMMV, but not MNSV, was confirmed through real-time RT-PCR on four different cucurbits, including three Cucumis sativus cultivars (six plants in ‘Marketer,’ five plants in ‘Poinsett 76,’ six plants in ‘Straight 8’), seven plants of C. melo ‘Athena,’ six plants of C. metulifer (PI201681), and two plants of Citrullus lanatus ‘Charleston Gray.’ To our knowledge, CGMMV has only been reported in Asia, Europe, and the Middle East, and this is the first report of CGMMV in the American continents. CGMMV is highly contagious and is seed borne on cucurbits. With the increasing trend in growing grafted watermelon and other cucurbits in the United States and elsewhere, it is even more important now that a vigilant seed health test program for CGMMV should be implemented. References: (1) H. Chen et al. J. Virol. Methods 149:326, 2008. (2) K.-S. Ling et al. Plant Dis. 92:1683, 2008. (3) K.-S. Ling et al. J. Phytopathol. 159:649, 2011.

Three RNA viruses&mdash, Cucumis melo cryptic virus (CmCV), Cucumis melo amalgavirus 1 (CmAV1), and melon necrotic spot virus (MNSV)&mdash, were identified from a melon (Cucumis melo) transcriptome dataset. CmCV has two dsRNA genome segments, dsRNA-1 is 1592 bp in size, containing a conserved RNA-dependent RNA polymerase (RdRp), and dsRNA-2 is 1715 bp in size, and encodes a coat protein (CP). The sequence alignment and phylogenetic analyses of the CmCV RdRp and CP indicated CmCV clusters with approved or putative deltapartitiviruses in well-supported monophyletic clade. The RdRp of CmCV shared an amino acid sequence identity of 60.7% with the closest RdRp of beet cryptic virus 3, and is &lt, 57% identical to other partitiviruses. CmAV1 is a nonsegmented dsRNA virus with a genome of 3424 bp, including two partially overlapping open reading frames (ORFs) encoding a putative CP and RdRp. The sequence alignment and phylogenetic analyses of CmAV1 RdRp revealed that it belongs to the genus Amalgavirus in the family Amalgaviridae. The RdRp of CmAV1 shares 57.7% of its amino acid sequence identity with the most closely related RdRp of Phalaenopsis equestris amalgavirus 1, and is &lt, 47% identical to the other reported amalgaviruses. These analyses suggest that CmCV and CmAV1 are novel species in the genera Amalgavirus and Deltapartitivirus, respectively. These findings enrich our understanding of new plant dsRNA virus species.

The coat protein (CP) of Melon necrotic spot virus (MNSV) is structurally composed of three major domains. The middle S-domain builds a robust protein shell around the viral genome, whereas the C-terminal protruding domain, or P-domain, is involved in the attachment of virions to the transmission vector. Here, we have shown that the N-terminal domain, or R-domain, and the arm region, which connects the R-domain and S-domain, are involved in different key steps of the viral cycle, such as cell-to-cell movement and the suppression of RNA silencing and pathogenesis through their RNA-binding capabilities. Deletion mutants revealed that the CP RNA-binding ability was abolished only after complete, but not partial, deletion of the R-domain and the arm region. However, a comparison of the apparent dissociation constants for the CP RNA-binding reaction of several partial deletion mutants showed that the arm region played a more relevant role than the R-domain in in vitro RNA binding. Similar results were obtained in in vivo assays, although, in this case, full-length CPs were required to encapsidate full-length genomes. We also found that the R-domain carboxyl portion and the arm region were essential for efficient cell-to-cell movement, for enhancement of Potato virus X pathogenicity, for suppression of systemic RNA silencing and for binding of small RNAs. Therefore, unlike other carmovirus CPs, the R-domain and the arm region of MNSV CP have acquired, in addition to other essential functions such as genome binding and encapsidation functions, the ability to suppress RNA silencing by preventing systemic small RNA transport.

We have shown previously that the translation of Melon necrotic spot virus (MNSV, family Tombusviridae, genus Carmovirus) RNAs is controlled by a 3'-cap-independent translation enhancer (CITE), which is genetically and functionally dependent on the eukaryotic translation initiation factor (eIF) 4E. Here, we describe structural and functional analyses of the MNSV-Mα5 3'-CITE and its translation initiation factor partner. We first mapped the minimal 3'-CITE (Ma5TE) to a 45-nucleotide sequence, which consists of a stem-loop structure with two internal loops, similar to other I-shaped 3'-CITEs. UV crosslinking, followed by gel retardation assays, indicated that Ma5TE interacts in vitro with the complex formed by eIF4E + eIF4G980-1159 (eIF4Fp20 ), but not with each subunit alone or with eIF4E + eIF4G1003-1092 , suggesting binding either through interaction with eIF4E following a conformational change induced by its binding to eIF4G980-1159 , or through a double interaction with eIF4E and eIF4G980-1159 . Critical residues for this interaction reside in an internal bulge of Ma5TE, so that their mutation abolished binding to eIF4E + eIF4G1003-1092 and cap-independent translation. We also developed an in vivo system to test the effect of mutations in eIF4E in Ma5TE-driven cap-independent translation, showing that conserved amino acids in a positively charged RNA-binding motif around amino acid position 228, implicated in eIF4E-eIF4G binding or belonging to the cap-recognition pocket, are essential for cap-independent translation controlled by Ma5TE, and thus for the multiplication of MNSV.

Melon necrotic spot virus (MNSV) can cause significant economic losses due to decreased quality in cucurbit crops. The current study is the first to use reverse transcription loop-mediated isothermal amplification (RT-LAMP) for detection of MNSV. A set of four LAMP primers was designed based on the coat protein gene sequence of MNSV, and a RT-LAMP reaction was successfully performed for 1 h at 62°C. The results of RT-LAMP showed high specificity for MNSV and no cross-reaction with other viruses. Compared to traditional reverse transcription-PCR (RT-PCR), the RT-LAMP assay was 103-fold more sensitive in detecting MNSV. Due to its sensitivity, speed and visual assessment, RT-LAMP is appropriate for detecting MNSV in the laboratory.

Frontiers and its users benefit from a Creative Commons CC-BY licence over all content., In eukaryotes, the formation of a 5'-cap and 3'-poly(A) dependent protein-protein bridge is required for translation of its mRNAs. In contrast, several plant virus RNA genomes lack both of these mRNA features, but instead have a 3'-CITE (for cap-independent translation enhancer), a RNA element present in their 3'-untranslated region that recruits translation initiation factors and is able to control its cap-independent translation. For several 3'-CITEs, direct RNA-RNA long-distance interactions based on sequence complementarity between the 5'- and 3'-ends are required for efficient translation, as they bring the translation initiation factors bound to the 3'-CITE to the 5'-end. For the carmovirus melon necrotic spot virus (MNSV), a 3'-CITE has been identified, and the presence of its 5'-end in cis has been shown to be required for its activity. Here, we analyze the secondary structure of the 5'-end of the MNSV RNA genome and identify two highly conserved nucleotide sequence stretches that are complementary to the apical loop of its 3'-CITE. In in vivo cap-independent translation assays with mutant constructs, by disrupting and restoring sequence complementarity, we show that the interaction between the 3'-CITE and at least one complementary sequence in the 5'-end is essential for virus RNA translation, although efficient virus translation and multiplication requires both connections. The complementary sequence stretches are invariant in all MNSV isolates, suggesting that the dual 5'-3' RNA: RNA interactions are required for optimal MNSV cap-independent translation and multiplication., This work was supported by grants AGL2009-07552/AGR, AGL2015-72804-EXP, and AGL2015-65838 (MINECO, Spain) and 19252/PI/2014 (Fundacion Seneca, Spain). AR-H was supported by grants from the National Council of Science and Technology (CONACyT, Mexico) and the Spanish Agency for International Development Cooperation (AECID). MM was recipient of a fellowship from the Spanish Ministerio de Ciencia e Innovacion (BES-2010-032827).

Surveys of yellowing viruses in plastic tunnels and in open field crops of melon (Cucumis melo cultivar catalupo), oriental melon (C. melo cultivar oriental melon), and cucumber (C. sativus) were carried out in two melon-growing areas in 2014, Korea. Severe yellowing symptoms on older leaves of melon and chlorotic spots on younger leaves of melon were observed in the plastic tunnels. The symptoms were widespread and included initial chlorotic lesions followed by yellowing of whole leaves and thickening of older leaves. RT-PCR analysis using total RNA extracted from diseased leaves did not show any synthesized products for four cucurbit-infecting viruses; Beet pseudo-yellows virus, Cucumber mosaic virus, Cucurbit yellows stunting disorder virus, and Melon necrotic spot virus. Virus identification using RT-PCR showed Cucurbit aphid-borne yellows Virus (CABYV) was largely distributed in melon, oriental melon and cucumber. This result was verified by aphid (Aphis gossypii) transmission of CABYV. The complete coat protein (CP) gene amplified from melon was cloned and sequenced. The CP gene nucleotide and the deduced amino acid sequence comparisons as well as phylogenetic tree analysis of CABYV CPs showed that the CABYV isolates were undivided into subgroups. Although the low incidence of CABYV in infections to cucurbit crops in this survey, CABYV may become an important treat for cucurbit crops in many different regions in Korea, suggesting that CABYV should be taken into account in disease control of cucurbit crops in Korea.

The full-length nucleotide sequence and genomic organization of a melon necrotic spot virus isolate from Haimen, China (MNSV-HM), were determined. The MNSV-HM genome consists of a positive sense single-stranded RNA, 4267 nt in length, with at least five open reading frames (ORFs) encoding p29, p89, p42, and two small 7-kDa proteins (p7A and p7B). p89 shares a common start codon with p29 and continues through the amber stop codon of p29 to produce an 89-kDa protein. The p7A ORF terminates in an amber codon whose read-through could generate a 14-kDa protein. Phylogenetic analyses based on the p42 amino acid sequence and complete genomic sequence placed MNSV-HM and Spanish isolates of the virus in a major cluster, indicating a close genetic relationship. In conclusion, we report the full-length sequence of MNSV-HM and its translation strategy. The obtained genomic organization and phylogenetic trees indicate that MNSV-HM belongs to the MNSV genus Carmovirus. To the best of our knowledge, this is the first demonstration of the complete nucleotide sequence of an MNSV isolate from China.

[EN] Despite harboring the smallest genomes among plant RNA viruses, carmoviruses have emerged as an ideal model system for studying essential steps of the viral cycle including intracellular and intercellular trafficking. Two small movement proteins, formerly known as double gene block proteins (DGBp1 and DGBp2), have been involved in the movement throughout the plant of some members of carmovirus genera. DGBp1 RNA-binding capability was indispensable for cell-to-cell movement indicating that viral genomes must interact with DGBp1 to be transported. Further investigation on Melon necrotic spot virus (MNSV) DGBp1 subcellular localization and dynamics also supported this idea as this protein showed an actin-dependent movement along microfilaments and accumulated at the cellular periphery. Regarding DGBp2, subcellular localization studies showed that MNSV and Pelargonium flower break virus DGBp2s were inserted into the endoplasmic reticulum (ER) membrane but only MNSV DGBp2 trafficked to plasmodesmata (PD) via the Golgi apparatus through a COPII-dependent pathway. DGBp2 function is still unknown but its localization at PD was a requisite for an efficient cell-to-cell movement. It is also known that MNSV infection can induce a dramatic reorganization of mitochondria resulting in anomalous organelles containing viral RNAs. These putative viral factories were frequently found associated with the ER near the PD leading to the possibility that MNSV movement and replication could be spatially linked. Here, we update the current knowledge of the plant endomembrane system involvement in carmovirus intra-and intercellular movement and the tentative model proposed for MNSV transport within plant cells., This work was funded by grant BIO2014-54862-R from the Spanish Direccion General de Investigacion Cientifica y Tecnica (DGICYT) and the Prometeo Program GV2014/010 from the Generalitat Valenciana.

Unlike fungal and bacterial diseases, no direct method is available to control viral diseases. The use of resistance-inducing compounds can be an alternative strategy for plant viruses. Here we studied the basal response of melon to Melon necrotic spot virus (MNSV) and demonstrated the efficacy of hexanoic acid (Hx) priming, which prevents the virus from systemically spreading. We analysed callose deposition and the hormonal profile and gene expression at the whole plant level. This allowed us to determine hormonal homeostasis in the melon roots, cotyledons, hypocotyls, stems and leaves involved in basal and hexanoic acid-induced resistance (Hx-IR) to MNSV. Our data indicate important roles of salicylic acid (SA), 12-oxo-phytodienoic acid (OPDA), jasmonic-isoleucine, and ferulic acid in both responses to MNSV. The hormonal and metabolites balance, depending on the time and location associated with basal and Hx-IR, demonstrated the reprogramming of plant metabolism in MNSV-inoculated plants. The treatment with both SA and OPDA prior to virus infection significantly reduced MNSV systemic movement by inducing callose deposition. This demonstrates their relevance in Hx-IR against MNSV and a high correlation with callose deposition. Our data also provide valuable evidence to unravel priming mechanisms by natural compounds., This work has been supported by grants from the Spanish Ministry of Science and Innovation (AGL2010-22300-C03-01-02, AGL2013-49023-C03-01-02-R and BIO2014-54862-R), co-funded by the European Regional Development Fund. The authors are grateful to the Serveis Centrals d’Instrumentació Científica (SCIC) of the Universitat Jaume I (UJI, Castellón, Spain).

The genus Carmovirus of the family Tombusviridae consists of the members having isometric virion particles with positive sense ssRNA genome of 4.0–4.8 kb encoding five proteins. In India, till date four carmoviruses were reported viz., blackgram mottle virus (BMoV), carnation mottle virus (CarMV), melon necrotic spot virus (MNSV) and soybean yellow mottle mosaic virus (SYMMV). BMoV and SYMMV are the legume infecting carmoviruses, which differs by serology and symptomatology. The Indian isolate of CarMV is a wide spread and distinct from other isolates. The Indian isoate of SYMMV is distinct from the SYMMV isolates occurring in the other countries being highly sap transmissible to guarbean, French bean, mungbean, soybean and urdbean with the distinct symptoms. Polyclonal antiserum developed against the recombinant coat protein of the Indian isolate of SYMMV can be utilized for successful detection of SYMMV in various plant samples. However, there is a great need to exploit these carmoviruses for further understanding the process of replication, gene expression, and exploiting them as gene expression vector for the expression of heterologous proteins in plant and as virus induced gene silencing vector for studying gene functions in legume crops. This chapter summarises the research work conducted on carmoviruses occurring in India.

Cucurbits are economic crops in Nigeria which serve as additional nutritional supplements and also good sources of income for farmers. Viral diseases are a worldwide problem of cucurbits and a major limiting factor for cucurbit production. A survey of farmer’s fields where cucurbit crops were grown was carried out to assess the incidence and severity of virus symptoms and viruses associated with the crops and weeds in selected locations in Ogun and Osun, in southwest Nigeria, in June, 2012. In all, 38 leaf samples were collected in Ogun state and 52 in Osun state from cucurbit crops and weeds. Leaf samples were tested against Cucumber mosaic virus (CMV), Melon necrotic spot virus (MNSV), Papaya ringspot virus (PRSV), Watermelon mosaic virus (WMV),Zucchini yellow mosaic virus (ZYMV) and Cucumber green mottle mosaic virus (CGMMV) using Double Antibody Sandwich (DAS) enzyme-linked immunosorbent assay (ELISA). All the fields surveyed had virus symptom incidences of 100% except for melon fields in Osun state with incidences of between 10 and 30%. In Ogun state, the occurrence of CMV was 5/31 (16.1%) while MNSV was detected in Lagenaria siceraria and T. occidentalis and it occurred in 6.5% of the leaf samples. In Osun state, CMV was detected in watermelon, melon and weeds found in all locations surveyed. The occurrence of CMV was 9/38 (23.7%) in the cucurbit crops and in 78.6% (11/14) of the weeds. PRSV and WMV also occurred in mixed infection with CMV in 7.1% respectively. CMV was the most widespread and prevalent virus infecting cucurbit crops and weeds.Cucurbits are economic crops in Nigeria which serve as additional nutritional supplements and also good sources of income for farmers. Viral diseases are a worldwide problem of cucurbits and a major limiting factor for cucurbit production. A survey of farmer’s fields where cucurbit crops were grown was carried out to assess the incidence and severity of virus symptoms and viruses associated with the crops and weeds in selected locations in Ogun and Osun, in southwest Nigeria, in June, 2012. In all, 38 leaf samples were collected in Ogun state and 52 in Osun state from cucurbit crops and weeds. Leaf samples were tested against Cucumber mosaic virus (CMV), Melon necrotic spot virus (MNSV), Papaya ringspot virus (PRSV), Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV) and Cucumber green mottle mosaic virus (CGMMV) using Double Antibody Sandwich (DAS) enzyme-linked immunosorbent assay (ELISA). All the fields surveyed had virus symptom incidences of 100% except for melon fields in Osun state with incidences of between 10 and 30%. In Ogun state, the occurrence of CMV was 5/31 (16.1%) while MNSV was detected in Lagenaria siceraria and T. occidentalis and it occurred in 6.5% of the leaf samples. In Osun state, CMV was detected in watermelon, melon and weeds found in all locations surveyed. The occurrence of CMV was 9/38 (23.7%) in the cucurbit crops and in 78.6% (11/14) of the weeds. PRSV and WMV also occurred in mixed infection with CMV in 7.1% respectively. CMV was the most widespread and prevalent virus infecting cucurbit crops and weeds.

Extremes of environmental conditions, such as biotic stresses, strongly affect plant growth and development and may adversely affect photosynthetic process. Virus infection is especially problematic in crops, because unlike other diseases, its impact cannot be reduced by phytosanitary treatments. The vegetable crops ( Solanum lycopеrsicum L, Cucurbita melo L., Cucumis sativus L., Piper longum L., Solanum melongena L., Vicia faba L.) showing virus-like symptoms were collected from fields located in the main crop production provinces of Azerbaijan. Infection of the plants were confirmed by Enzyme-linked immunosorbent assay using commercial kits for the following viruses: Tomato yellow leaf curl virus , Tomato mosaic virus , Tomato chlorosis virus , Melon necrotic spot virus and Cucumber mosaic virus , Bean common mosaic virus and Bean yellow mosaic virus . Generation sites of superoxide and hydrogen peroxide radicals and activities of enzymes involved in the detoxification of reactive oxygen species (catalase, glutathione reductase, ascorbate peroxidase, guaiacol peroxidase and superoxide dismutase) were examined in uninfected leaves and in leaves infected with viruses. High accumulation of superoxide and hydrogen peroxide radicals was visualized in infected leaves as a purple discoloration of nitro blue tetrazolium and 3,3′-diaminobenzidine tetrahydrochloride. It was found that the activities of APX and CAT significantly increased in all infected samples compared with non-infected ones. Dynamics of GR and Cu/Zn-SOD activities differed from those of CAT and APX, and slightly increased in stressed samples. Electrophoretic mobility profiling of APX, GPX and CAT isoenzymes was also studied.

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.

International audience; This article lists the changes to virus taxonomy approved and ratified by the International Committee on Taxonomy of Viruses (ICTV) in April 2016.Changes to virus taxonomy (the Universal Scheme of Virus Classification of the International Committee on Taxonomy of Viruses [ICTV]) now take place annually and are the result of a multi-stage process. In accordance with the ICTV Statutes ( http://www.ictvonline.org/statutes.asp ), proposals submitted to the ICTV Executive Committee (EC) undergo a review process that involves input from the ICTV Study Groups (SGs) and Subcommittees (SCs), other interested virologists, and the EC. After final approval by the EC, proposals are then presented for ratification to the full ICTV membership by publication on an ICTV web site ( http://www.ictvonline.org/ ) followed by an electronic vote. The latest set of proposals approved by the EC was made available on the ICTV website by January 2016 ( https://talk.ictvonline.org/files/proposals/ ). A list of these proposals was then emailed on 28 March 2016 to the 148 members of ICTV, namely the EC Members, Life Members, ICTV Subcommittee Members (including the SG chairs) and ICTV National Representatives. Members were then requested to vote on whether to ratify the taxonomic proposals (voting closed on 29 April 2016).

En este trabajo se ha demostrado que el NH4+ actúa como inductor de resistencia frente a estrés salino en cítricos, observándose acumulación de ABA, poliaminas (PAs), H2O2 y prolina. También se ha demostrado que plantas de tomate crecidas con NH4+ muestran una reducción de los síntomas de la enfermedad producida por Pst. El estudio del modo de acción de la NH4+-IR reveló que los mecanismos inducidos en las plantas en respuesta a NH4+, tales como la acumulación de ABA, PAs y H2O2 son clave para la inducción de aclimatación sistémica adquirida (SAA) que confiere a las plantas de tomate resistencia frente a Pst. Por último, se ha demostrado que el virus MNSV induce en plantas de melón una compleja red hormonal de respuesta, así como la acumulación de calosa y ROS. El Hx resultó ser efectivo como inductor de resistencia frente a virus evitando el paso del virus al floema., In this work, we reveal that NH4+ nutrition in citrange Carrizo plants acts as an inducer of resistance against salinity conditions. We investigated its mode of action and provide evidence that NH4+ confers resistance by priming abscisic acid and polyamines, just as enhancing H2O2 and proline basal content. Moreover, we demonstrated the NH4+ nutrition induces-resistance (NH4+-IR) against Pseudomonas syringae pv tomato DC3000 (Pst) in tomato plants. N-NH4+ plants displayed basal H2O2, abscisic acid (ABA) and putrescine (Put) accumulation. H2O2 accumulation acted as a signal to induce ABA-dependent signalling pathways required to prevent NH4+ toxicity. This acclimatory event provoked an increase of resistance against later pathogen infection. We studied the basal response of melon (Cucumis melo) to Melon necrotic spot virus (MNSV) and demonstrated the efficacy of the hexanoic acid priming that blocks the virus systemic spread. We analyzed callose deposition and ROS production, as well as the hormonal profile and gene expression at the whole-plant level.

National Institute of Horticultural and Herbal Science, Horticultural and Herbal Environmental Division,Suwon 441-707, Korea(Received on September 11, 2012; Revised on October 11, 2012; Accepted on October 12, 2012)The total number of requests and associated specimens for the diagnosis of virus infection were 573 and 2,992,respectively, on crops from agricultural places of farmers, Agricultural extension services and so forth for 5years from 2007. The total number of virus tests was 13,325. The number of species of viruses infected on thesubmitted crops was 21 in 2007, 15 in 2008, 23 in 2009, 21 in 2010 and 17 in 2011. The newly recorded viruseswere Tobacco leaf curl virus (TbLCV) in 2007, Tomato yellow leaf curl virus (TYLCV) in 2008, Impatiencenecrotic spot virus (INSV) and Radish mosaic virus (RaMV) in 2009, and Beet western yellows virus (BWYV) in2010. Forty virus species including Alfalfa mosaic virus were detected over 5 years. The ten most frequentlydetected virus species were Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV), Tomato leaf curlvirus (TYLCV), Cucumber green mottle mosaic virus (CGMMV), Broad bean wilt virus 2 (BBWV2), Zucchiniyellow mosaic virus (ZYMV), Melon necrotic spot virus (MNSV), Pepper mild mottle virus (PMMoV),Watermelon mosaic virus (WMV) and Pepper mottle virus (PepMoV). The types of crops submitted fromagricultural places were 51 in total and the ten most frequently submitted crops were red pepper, tomato,paprika, watermelon, melon, rice, cucumber, corn, radish and gourd. The total request rate for the top 10crops and top 20 crops was 81.6% and 94.2%, respectively. Eight pepper infecting virus species includedCMV, and the average infection rate was 24.6% for CMV, 18.9% for PMMoV and 14.7% for TSWV. Sevenkinds of double infection were detected in pepper including BBWV2+CMV at 14.7% on average, and fourtypes of triple infection including BBWV2+CMV+PepMoV at 0.9% on average. Six virus species detected ontomato including TYLCV, and the average infection rate was 50.6% for TYLCV, 14.5% for TSWV and10.9% for Tobacco leaf curl virus (TbLCV). The mixed infection of CMV+TSWV on tomato was 3.9% onaverage and of Tomato mosaic virus (ToMV)+TYLCV was 0.4% on average. Five viruses detected onwatermelon included MNSV and the average infection rate was 37.0% for MNSV, 20.4% for CGMMV,18.1% for ZYMV and 17.8% for WMV. The mixed infection rate on watermelon was CMV+MNSV andWMV+ZYMV having an average infection rate of 0.7% and 5.0%, respectively. The average infection rateson melon were 77.6% for MNSV, 5.6% for CMV and 3.3% for WMV. Mixed infections of CMV+MNSVoccurred on melon with an average infection rate of 13.5%. Keywords : BBWV2, BWVY, CGMMV, CMV, MNSV, Plant virus, TYLCV, ZYMV

Efficient and sustainable control of plant viruses may be achieved using genetically resistant crop varieties, although resistance genes are not always available for each pathogen; in this regard, the identification of new genes that are able to confer broad-spectrum and durable resistance is highly desirable. Recently, the cloning and characterization of recessive resistance genes from different plant species has pointed towards eukaryotic translation initiation factors (eIF) of the 4E family as factors required for the multiplication of many different viruses. Thus, we hypothesized that eIF4E may control the susceptibility of melon (Cucumis melo L.) to a broad range of viruses. To test this hypothesis, Cm-eIF4E knockdown melon plants were generated by the transformation of explants with a construct that was designed to induce the silencing of this gene, and the plants from T2 generations were genetically and phenotypically characterized. In transformed plants, Cm-eIF4E was specifically silenced, as identified by the decreased accumulation of Cm-eIF4E mRNA and the appearance of small interfering RNAs derived from the transgene, whereas the Cm-eIF(iso)4E mRNA levels remained unaffected. We challenged these transgenic melon plants with eight agronomically important melon-infecting viruses, and identified that they were resistant to Cucumber vein yellowing virus (CVYV), Melon necrotic spot virus (MNSV), Moroccan watermelon mosaic virus (MWMV) and Zucchini yellow mosaic virus (ZYMV), indicating that Cm-eIF4E controls melon susceptibility to these four viruses. Therefore, Cm-eIF4E is an efficient target for the identification of new resistance alleles able to confer broad-spectrum virus resistance in melon.

The chytrid fungus Olpidium bornovanus is an obligate plant parasite that acts as a vector to transmit Melon necrotic spot virus in cultivated soil. Here, we conducted a molecular phylogenetic analysis of 16 isolates of O. bornovanus taken from soil in which Cucurbitaceae plants had been grown in various locations throughout Japan. The ribosomal DNA and internal transcribed spacer region sequences of the 16 O. bornovanus isolates were divided into four molecular phylogenetic groups, designated O.bor-A to O.bor-D. Biological features of O. bornovanus isolates such as host specificity varied consistently with their molecular phylogenetic groups.

Present address: Centro Nacional de Biotecnologia (CNB), Consejo Superior de Investigaciones Cientificas (CSIC), 28049 Cantoblanco, Madrid, Spain. SUMMARY Nicotiana benthamiana has been described as non-host for Melon necrotic spot virus (MNSV). We investigated the basis of this resistance using the unique opportunity provided by strain MNSV-264, a recombinant virus that is able to overcome the resistance. Analysis of chimeric MNSV mutants showed that virulence in N. benthamiana is conferred by a 49 nucleotide section of the MNSV-264 3¢-UTR, which acts in this host as a cap-independent translational enhancer (3¢-CITE). Although the 3¢-CITE of non-adapted MNSV-Ma5 is active in susceptible melon, it does not promote efficient translation in N. benthamiana, thus preventing expression of proteins required for virus replication. However, MNSV-Ma5 gains the ability to multiply in N. benthamiana cells if eIF4E from a susceptible melon variety (Cm-eIF4E-S) is supplied in trans. These data show that N. benthamiana resistance to MNSV-Ma5 results from incompatibility between the MNSV-Ma 53 ¢-CITE and N. benthamiana eIF4E in initiating efficient translation of the viral genome. Therefore, non-host resistance conferred by the inability of a host susceptibility factor to support viral multiplication may be a possible mechanism for this type of resistance to viruses.

One of the major challenges of melon ( Cucumis melo L.) yield is the decrease of fruit yield and quality caused by soil diseases. Soil disinfection with methyl bromide (MB) has been used to prevent soil-borne diseases damage; however, use of MB is being restricted because this substance damages the ozone layer. Searching for new MB alternatives, field experiments were carried out in open fields in soils infested with Fusarium oxysporum f. sp. melonis , Olpidium bornovanus , and Melon Necrotic Spot Virus (MNSV) in Colima, Mexico, where melons had only been grown previously using soil fumigation with MB. Yield and quality of melon cultivar Cruiser F1 (Harris Moran seed company) grafted on two rootstocks of Cucurbita maxima Duchesne × Cucurbita moschata Duchesne (“RS841”; Seminis seed company and “Shintosa Camelforce”; Nunhens seed company) were evaluated during two consecutive years. Each experiment had five treatments, two of which with non-grafted plants at a density of 18,519 plants/ha, with and without MB fumigation. The other three treatments consisted of grafted plants in non-fumigated soil with plant densities of 14,815, 11,111, and 9260 plants/ha. The use of melon grafted on “RS841” and “Shintosa Camelforce” rootstocks significantly increased the average fruit weight and the total yield of melons in soil without the need for MB. Grafting may be considered an alternative to MB fumigation. With the use of grafted melon plants, planting density may be reduced by 60%, obtaining higher yields than those obtained from non-grafted plants grown on fumigated land. Fruits harvested from grafted plants had equal firmness than those harvested from non-grafted plants, without affecting the content of soluble solids.

Survival of Pepper mild mottle virus (PMMV) and Melon necrotic spot virus (MNSV) in diseased plant residues of pepper and melon, respectively, was studied in several experiments investigating drying and composting conducted in experimental heaps. The viability of the soil-borne fungus Olpidium bornovanus, the vector of MNSV, in infected melon root residues was also evaluated. Maximum temperatures in the heap reached 61.9-73.8 °C during composting processes. Drying of plant residues reduced the infectivity of MNSV whereas the infectivity of PMMV and O. bornovanus was unaffected by drying. Neither the virus nor the fungus was viable after composting. PMMV antigens where detected by serology after 30 and 60 days composting in the four experiments, but virus infectivity was detected only in one process after 30 days. Our results show that the use of composts using plant residues from PMMV and MNSV-infected crops as organic amendments in SE Spain do not appear to pose a risk of re-introducing pathogens to soil. Environmental conditions in that area are suitable for the adequate elimination of these pathogens from infected vegetable residues through composting even during winter.

The geographic incidence, genetic diversity and phylogenetic relationships of Melon necrotic spot virus (MNSV) and Olpidium isolates were studied in three cucurbit species from several Latin American and European countries on different collecting dates. Of the 112 cucurbit samples analysed, 69 from Guatemala, Honduras, Mexico, Panama and Spain were DAS-ELISA-positive for MNSV. Olpidium bornovanus and O. virulentus infections, and MNSV infections mixed with these Olpidium species, were observed for all these countries. Twenty-nine MNSV isolates from all the origins where the virus was detected were selected and amplified by RT-PCR. The resulting RT-PCR of the p29, p89, p7A, p7B and p42 proteins was used to estimate the genetic diversity and the phylogenetic relationships of the MNSV population. The sequences obtained in this study were compared with the MNSV sequences of the NCBI database, and three groups were recovered by nucleotide composition according to geographical origins: the EU-LA genotype group (with two subgroups: EU and LA, European and Latin American isolates, respectively), the JP melon genotype group (Japanese melon reference isolates) and the JP watermelon genotype group (Japanese watermelon reference isolates). The genetic diversity in the entire p7A and p7B proteins of MNSV suggests that these coding regions are under strong selective pressure. Additionally, the rDNA-ITS region was analysed in 40 O. bornovanus and O. virulentus isolates associated with each geographical location and host examined. Phylogenetic analysis showed two groups for each Olpidium species, and these groupings were related to the host from which they were originally isolated.

Plant viruses hijack endogenous host transport machinery to aid their intracellular spread. Here, we study the localization of the p7B, the membrane-associated viral movement protein (MP) of the Melon necrotic spot virus (MNSV), and also the potential involvement of the secretory pathway on the p7B targeting and intra- and intercellular virus movements. p7B fused to fluorescent proteins was located throughout the endoplasmic reticulum (ER) at motile Golgi apparatus (GA) stacks that actively tracked the actin microfilaments, and at the plasmodesmata (PD). Hence, the secretory pathway inhibitor, Brefeldin A (BFA), and the overexpression of the GTPase-defective mutant of Sar1p, Sar1[H74L], fully retained the p7B within the ER, revealing that the protein is delivered to PD in a BFA-sensitive and COPII-dependent manner. Disruption of the actin cytoskeleton with latrunculin B led to the accumulation of p7B in the ER, which strongly suggests that p7B is also targeted to the cell periphery in an actin-dependent manner. Remarkably, the local spread of the viral infection was significantly restricted either with the presence of BFA or under the overexpression of Sar1[H74L], thus revealing the involvement of an active secretory pathway in the intracellular movement of MNSV. Overall, these findings support a novel route for the intracellular transport of a plant virus led by the GA.