Cucumber Green Mottle Mosaic Virus Isolates Research Articles

First report of cucumber green mottle mosaic virus infecting Cynanchum rostellatum in China.

Cucumber green mottle mosaic virus (CGMMV) was first discovered on cucumber in the United Kingdom in 1935 (Ainsworth, 1935), and has spread worldwide except to Antarctica (Jones, 2021). Given its extensive damage, it is considered an important pathogen on global cucurbit plants and fruit crops. In China, CGMMV was first reported on pumpkin in Guangxi Province in 2003 (Qin et al., 2005), and occurred on 34 plants species across 23 provinces (Liu et al., 2016). Cynanchum rostellatum is a member of the family Apocynaceae. In July 2021, leaves of C. rostellatum exhibiting virus-like symptoms (yellowing, severe crinkling, deformation) were observed and collected in Liaoning Province, China. Aphids were also observed on the leaves and stems (Fig. S1) of the plants and were collected. Total RNA was extracted from diseased leaves following the CTAB method, followed by the depletion of ribosomal RNAs (rRNA) with TIANSeq rRNA Depletion Kit (Tiangen, China). The RNAs were, then processed into a DNBSEQ LncRNA-Seq library, and sequenced on the MGISEQ-2000 platform at BGI Genomics (Wuhan, China). A total of 106.98 M clean reads were obtained after data filtering using SOAPnuke software (BGI, China). The clean reads were assembled into contigs using CLC Genomics Workbench 11 (Qiagen, USA) and Trinity v2.0.6 (Haas et al., 2013). A contig (4,760 reads, average coverage:73.76) of 6,391 nucleotides was found to share the highest sequence identity (99.83%) with CGMMV isolate GDLZ (MK933286), irrespective of other virus-like contigs related to Polerovirus and Totivirus. Based on the genome of GDLZ isolate, seven specific primers (Table S1) were designed to amplify the full viral genomic sequences using a PrimeScriptTM One-Step RT-PCR Kit. Seven expected amplicons were obtained, cloned, and sequenced. The complete genome was determined to be 6,423 nucleotides (GenBank accession number OR854819) in length and designated as LNMJ isolate. LNMJ shared 96.8%-99.7% nucleotide sequence identities with CGMMV isolates from China. Phylogenetic analysis based on the complete genome sequences showed that LNMJ clustered together with CGMMV isolates hn (GenBank accession number KC851866), GDLZ (GenBank accession number MK933286), and JD8 (GenBank accession number KM873784) from China. The specific primers LM-TJ-3F/3R were designed to determine the virus-symptom association for LNMJ, and all twelve symptomatic C. rostellatum plants collected from fields tested positive for LNMJ. Two out of six randomly selected aphids from the diseased plants also tested positive. To further prove its infectivity, LNMJ was inoculated mechanically onto ten healthy Nicotiana benthamiana plants, and the results indicated a high infection rate of 80% (8/10), at 30 days post-inoculation despite no distinct symptoms observed. To our knowledge, this is the first report of the natural infection of C. rostellatum plants with CGMMV. C. rostellatum is a widespread herb in China (Wei et al., 2019) and more surveys are needed to determine the distribution of CGMMV. The habitats of C. rostellatum span diverse agroecological zones, and thus our study underscores the potential spillover of CGMMV to neighboring crops as a significant risk.

Genome and Phylogenetic Analysis of Cucumber Green Mottle Mosaic Virus Global Isolates and Validation of a Highly Sensitive RT-qPCR Assay.

The last two decades have seen exponential growth in the international movement of seeds for annual food crops, from a gross U.S. import value of $349 million in 1999 to $1.05 billion in 2019. This has led to the proportionate growth of seedborne pathogens dispersed with seed stocks. One such viral pathogen is cucumber green mottle mosaic virus (CGMMV), a tobamovirus that infects cucurbit crops such as melon, watermelon, cucumber, pumpkin, and squash. The first CGMMV introduction to California occurred in 2013, with subsequent annual outbreaks or detections since then. Here, we describe the use of next-generation sequencing to characterize the full genomes of 25 CGMMV isolates collected between 2013 and 2020 in California, either from CGMMV field detections or seed lots identified as CGMMV positive. We sequenced an additional 31 CGMMV isolates collected in Europe, Israel, and southeast Asia that were provided by industry collaborators. We also performed an in silico nucleotide database search in GenBank for full genome CGMMV sequences to include in all in silico analyses. Based on conserved regions within the coat protein gene, we then developed a quantitative reverse-transcription PCR assay for the sensitive and specific detection of CGMMV in seed and plant samples. Finally, based on our sequence and phylogenetic analysis, our data support that CGMMV has been introduced multiple times into California.

Genetic diversity of cucumber green mottle mosaic virus (CGMMV) infecting cucurbits

Cucumber green mottle mosaic virus (CGMMV), a well-known Tobamovirus, infects cucurbits across the globe. To determine its current status, molecular characterization, genetic recombination, gene flow and selection pressure, 10 districts from Punjab province of Pakistan were surveyed and a total of 2561 cucurbits samples were collected during 2019–2020. These samples were subjected to virus-specific double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) for the detection of CGMMV. The results revealed that viral disease was prevalent in all surveyed districts of Punjab with an overall 25.69% disease incidence. ELISA positive samples were further confirmed through RT-PCR and sequencing of coat protein (CP) cistron. Sequence analysis showed that the present studied CGMMV isolates have 96–99.5% nucleotide and 94.40–99.50% amino acid identities with those already available in GenBank. Phylogenetic analysis also revealed that understudied isolates were closely related with South Korean (AB369274) and Japanese (V01551) isolates and clustered in a separate clad. Sequence polymorphisms were observed in 663 bp of sequence within 31 CGMMV isolates covering complete CP gene. Total number of sites were 662, of which 610 and 52 sites were monomorphic and polymorphic (segregating), respectively. Of these polymorphic, 24 were singleton variable and 28 were parsimony informative. Overall nucleotide diversity (π) in all the understudied 31 isolates was 0.00010 while a total of 1 InDel event was observed and InDel Diversity (k) was 0.065. Haplotype diversity analysis revealed that there was a total 29 haplotypes with haplotype diversity (Hd) of 0.993458 in all the 31 isolates which provide evidence of less diversity among Pakistani isolates. The statistical analysis revealed the values 2.568, 5.31304 and 4.86698 of Tajima's D, Fu, & Li’s F* and D*, respectively, which witnessed the population of CGMMV was under balanced selection pressure.

First Report of Natural Infection of Siraitia grosvenorii by Cucumber Green Mottle Mosaic Virus in China

Fruit of Siraitia grosvenorii (family Cucurbitaceae) are used as a sweetener in beverage and traditional herbal medicine production in China. Some viruses have been known to infect S. grosvenorii, such as papaya ringspot virus (PRSV), watermelon mosaic virus (WMV), and zucchini yellow mosaic virus (ZYMV) (Cai et al. 2001; Liao et al. 2005; Qin et al. 2005). In August 2017, six S. grosvenorii samples (nos. LHG-1 to LHG-6) that showed mosaic, chlorosis, and mottling of leaves were collected from Yongfu, Guangxi province, China. All symptomatic samples were tested by enzyme-linked immunosorbent assay (ELISA) in double antibody sandwich format for 10 known cucurbit-infecting viruses, including cucumber green mottle mosaic virus (CGMMV), cucumber mosaic virus, kyuri green mottle mosaic virus, PRSV, squash mosaic virus, tobacco mosaic virus (TMV), watermelon silver mottle virus, WMV, zucchini green mottle mosaic virus, and ZYMV. The sample LHG-1 with chlorosis and mottle symptoms reacted positively against the antisera of CGMMV and TMV (Adgen, U.K.) but negatively against antisera of the other viruses. The other samples were negative in ELISA for all viruses tested. Total RNA was extracted from the LHG-1 sample using TRIzol reagent (Takara Dalian, China) following the manufacturer’s instructions. Reverse transcription polymerase chain reaction (RT-PCR) assays using CGMMV-specific primer pair CGM1/CGM2 (Huang et al. 2007) and TMV-specific primers (Li et al. 2000) showed a positive RT-PCR product of the expected size (650 bp) for CGMMV but not for TMV. The amplicon was further cloned into pMD-18-T vector (Takara Dalian) and sequenced (GenBank accession no. MK858182). Sequence analysis revealed that the RT-PCR product showed 99% nucleotide identity to the sequences of CGMMV isolates available in GenBank (accession nos. HM008919, KY753928, KX883833, KP868654, KM873786, and MH271443). The virus in LHG-1 was therefore identified as an isolate of CGMMV and named as CGMMV-LHG. Additionally, to confirm the infectivity of CGMMV-LHG, leaf sap extract in phosphate buffer (0.1 M, pH 7.0) of LHG-1 was mechanically inoculated on leaves of S. grosvenorii and Lagenaria siceraria seedlings. Ten days after inoculation, S. grosvenorii produced symptoms similar to the diseased plants in the field, and systemic mosaic and vein-banding were observed on L. siceraria. The control seedlings inoculated with phosphate buffer remained symptomless. Presence of CGMMV in inoculated plants was confirmed by RT-PCR. To the best of our knowledge, this is the first report of CGMMV infection on S. grosvenorii under natural conditions. The virus can be transmitted by seeds, infects various cucurbit species, and causes yield losses all over the world (Liu et al. 2014; Wu et al. 2011). Use of virus-free seeds and seedlings will be helpful to control CGMMV in S. grosvenorii production areas.

Genetic diversity of the Cucumber green mottle mosaic virus and the development of RT-LAMP assay for its detection

To analyse the genetic diversity of the Cucumber green mottle mosaic virus (CGMMV) population in Poland and to establish the phylogenetic relationships between the Polish and other isolates described to date, 91 isolates were collected from cucumber plants. The analysis, based on coat protein (CP) gene, revealed the presence of two phylogenetic groups: one consisting of the Polish isolates collected in 2017 and those originated mainly from Asia region and the second including the Polish isolates collected in 2016 and the others from European countries. The sensitive, specific, and rapid one-step loop-mediated isothermal amplification assay was developed for the early detection of genetically diverse CGMMV isolates in seeds and plant material.

Biological and Molecular Diversity of Cucumber green mottle mosaic virus in Spain.

The complete RNA genome from Cucumber green mottle mosaic virus (CGMMV) (Alm08), collected during 2009 in cucumber crops located in Spain, was found to be 6,422 nucleotides long. The nucleotide sequence shared the highest identity with isolates from Russia (GQ495274, GQ495275, FJ848666) as do nucleotide sequences of partial CP and MP genes described in Spain since 2005. All the partial genome sequences including RdRp, CP, and MP from 26 isolates collected from 2013 to 2015 in the southeast of Spain, and from seven isolates of other parts of the world, suggest that they grouped in two major clusters: one cluster (I) included 14 isolates collected between 2013 and 2014, and also reference isolates from France, the Netherlands, and Uzbekistan. A second cluster (II) grouped 12 isolates, which were mostly collected in 2015 together with those from Japan, South Korea, and Canada. For the first time, CGMMV isolates of different geographical origin were found coinfecting the same crop and territory. A host range study revealed that representative isolates of cluster II, but not from cluster I, produced local lesions in Chenopodium amaranticolor. RT-PCR using a common primer pair for CGMMV followed by restriction enzyme analysis with KpnI allowed distinguishing cluster I from II CGMMV isolates.

Genetic variation and population structure of Cucumber green mottle mosaic virus

Cucumber green mottle mosaic virus (CGMMV) is a single-stranded, positive sense RNA virus infecting cucurbitaceous plants. In recent years, CGMMV has become an important pathogen of cucurbitaceous crops including watermelon, pumpkin, cucumber and bottle gourd in China, causing serious losses to their production. In this study, we surveyed CGMMV infection in various cucurbitaceous crops grown in Zhejiang Province and in several seed lots purchased from local stores with the dot enzyme-linked immunosorbent assay (dot-ELISA), using a CGMMV specific monoclonal antibody. Seven CGMMV isolates obtained from watermelon, grafted watermelon or oriental melon samples were cloned and sequenced. Identity analysis showed that the nucleotide identities of the seven complete genome sequences ranged from 99.2 to 100%. Phylogenetic analysis of seven CGMMV isolates as well as 24 other CGMMV isolates from the GenBank database showed that all CGMMV isolates could be grouped into two distinct monophyletic clades according to geographic distribution, i.e. Asian isolates for subtype I and European isolates for subtype II, indicating that population diversification of CGMMV isolates may be affected by geographical distribution. Site variation rate analysis of CGMMV found that the overall variation rate was below 8% and mainly ranged from 2 to 5%, indicating that the CGMMV genomic sequence was conservative. Base substitution type analysis of CGMMV showed a mutational bias, with more transitions (A↔G and C↔T) than transversions (A↔C, A↔T, G↔C and G↔T). Most of the variation occurring in the CGMMV genome resulted in non-synonymous substitutions, and the variation rate of some sites was higher than 30% because of this mutational bias. Selection constraint analysis of CGMMV ORFs showed strong negative selection acting on the replication-associated protein, similar to what occurs for other plant RNA viruses. Finally, potential recombination analysis identified isolate Ec as a recombinant with a low degree of confidence.

Complete nucleotide sequences and construction of full-length infectious cDNA clones of cucumber green mottle mosaic virus (CGMMV) in a versatile newly developed binary vector including both 35S and T7 promoters.

Seed-transmitted viruses have caused significant damage to watermelon crops in Korea in recent years, with cucumber green mottle mosaic virus (CGMMV) infection widespread as a result of infected seed lots. To determine the likely origin of CGMMV infection, we collected CGMMV isolates from watermelon and melon fields and generated full-length infectious cDNA clones. The full-length cDNAs were cloned into newly constructed binary vector pJY, which includes both the 35S and T7 promoters for versatile usage (agroinfiltration and in vitro RNA transcription) and a modified hepatitis delta virus ribozyme sequence to precisely cleave RNA transcripts at the 3' end of the tobamovirus genome. Three CGMMV isolates (OMpj, Wpj, and Mpj) were separately evaluated for infectivity in Nicotiana benthamiana, demonstrated by either Agroinfiltration or inoculation with in vitro RNA transcripts. CGMMV nucleotide identities to other tobamoviruses were calculated from pairwise alignments using DNAMAN. CGMMV identities were 49.89% to tobacco mosaic virus; 49.85% to pepper mild mottle virus; 50.47% to tomato mosaic virus; 60.9% to zucchini green mottle mosaic virus; and 60.96% to kyuri green mottle mosaic virus, confirming that CGMMV is a distinct species most similar to other cucurbit-infecting tobamoviruses. We further performed phylogenetic analysis to determine relationships of our new Korean CGMMV isolates to previously characterized isolates from Canada, China, India, Israel, Japan, Korea, Russia, Spain, and Taiwan available from NCBI. Analysis of CGMMV amino acid sequences showed three major clades, broadly typified as 'Russian,' 'Israeli,' and 'Asian' groups. All of our new Korean isolates fell within the 'Asian' clade. Neither the 128 nor 186kDa RdRps of the three new isolates showed any detectable gene silencing suppressor function.

Establishment of an Agrobacterium-mediated Inoculation System for Cucumber Green Mottle Mosaic Virus.

The infectious full-length cDNA clones of Cucumber green mottle mosaic virus (CGMMV) isolates KW and KOM, which were isolated from watermelon and oriental melon, respectively, were constructed under the control of the cauliflower mosaic virus 35S promoter. We successfully inoculated Nicotiana benthamiana with the cloned CGMMV isolates KW and KOM by Agrobacterium-mediated infiltration. Virulence and symptomatic characteristics of the cloned CGMMV isolates KW and KOM were tested on several indicator plants. No obvious differences between two cloned isolates in disease development were observed on the tested indicator plants. We also determined full genome sequences of the cloned CGMMV isolates KW and KOM. Sequence comparison revealed that only four amino acids (at positions 228, 699, 1212, and 1238 of the replicase protein region) differ between the cloned isolates KW and KOM. A previous study reported that the isolate KOM could not infect Chenopodium amaranticolor, but the cloned KOM induced chlorotic spots on the inoculated leaves. When compared with the previously reported sequence of the original KOM isolate, the cloned KOM contained one amino acid mutation (Ala to Thr) at position 228 of the replicase protein, suggesting that this mutation might be responsible for induction of chlorotic spots on the inoculated leaves of C. amaranticolor.

First Report of Cucumber green mottle mosaic virus on Melon in the United States

In July 2013, a melon (Cucumis melo var. Saski) field in Yolo County, California, was inspected as part of a phytosanitary inspection for seed production. The leaves of the plants showed mosaic, green mottle, and blotches. When plant sap was examined using a transmission electron microscope, rigid rod-shaped particles were observed. Melon plant samples were analyzed by both CDFA and USDA APHIS PPQ laboratories and tested positive using DAS-ELISA against Cucumber green mottle mosaic virus (CGMMV) (Agdia, Elkhart, IN). To confirm the presence of CGMMV, total RNA was analyzed by RT-PCR using primers CGMMV-F5370 5'-CTAATTATTCTGTCGTGGCTGCGGATGC-3' and CGMMV-R6390 5'-CTTGCAGAATTACTGCCCATA-3' designed by PPQ based on 21 genomic sequences of CGMMV found worldwide. The 976-bp amplicon was sequenced (GenBank Accession No. KJ453559) and BLAST analysis showed the sequence was 95% identical to MP and CP region of CGMMV isolates reported from Russia (GQ495274, FJ848666), Spain (GQ411361), and Israel (KF155231), and 92% to the isolates from China (KC852074), Korea (AF417243), India (DQ767631), and Japan (D12505). These analyses confirm the virus was CGMMV. To our knowledge, this is the first report of CGMMV in the United States. Based on our sequence data, a second set of primers (CGMMV-F5796 5'-TTGCGTTTAGTGCTTCTTATGT-3' and CGMMV-R6237 5'-GAGGTGGTAGCCTCTGACCAGA-3'), which amplified a 440-bp amplicon from CGMMV CP region, was designed and used for testing all the subsequent field and seed samples. Thirty-seven out of 40 randomly collected Saski melon samples tested positive for CGMMV, suggesting the virus was widespread in the field. All the melon samples also tested positive for Squash mosaic virus (SqMV) using DAS-ELISA (Agdia). Therefore, the symptoms observed likely resulted from a mixed infection. The melon field affected by CGMMV was immediately adjacent to fields of cucumber (Cucumis sativus var. Marketmore 76) and watermelon (Citrullus lanatus var. Sugar Baby) crops, both for seed production with no barrier between the crops. CGMMV was also detected from symptomatic plants from both fields. Seed lots used for planting all three crops were tested and only the melon seed was positive for CGMMV, suggesting the seed as the source of infection. The sequenced 440-bp RT-PCR amplicons from CGMMV-infected cucumber and watermelon plants and melon seeds were 99% identical to the CGMMV from the field melon. A cucumber plant infected with CGMMV but not SqMV was used for mechanical inoculation at the Contained Research Facility at University of California, Davis. Inoculated cucumber, melon, and watermelon plants showed green mottle and mosaic similar to that observed in the field. CGMMV is a highly contagious virus and damage by this virus on cucurbit crops has been reported in regions where CGMMV is present (2). CGMMV was detected on cucumber grown in greenhouses in Canada with 10 to 15% yield losses reported due to this virus (1). The three cucurbit crops in Yolo County were planted in an isolated area with no other cucurbits nearby. Measures, including destroying all the cucurbit plant material, have been taken to eradicate the virus. Use of CGMMV free cucurbit seed is necessary for prevention of this disease.

First Report of Cucumber green mottle mosaic virus Infecting Greenhouse Cucumber in Canada.

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.

First Report and the Genetic Variability of Cucumber green mottle mosaic virus Occurring on Bottle Gourd in Myanmar

AbstractBottle gourd [Lagenaria siceraria (Mol.) Standl.] plants showing leaf mosaic and mottle were observed in Myanmar in 2007 and shown by RT‐PCR and ELISA to be infected with Cucumber green mottle mosaic virus (CGMMV). This is the first report of the virus occurring in Myanmar. Despite considerable differences in geographical origins, natural host species and year of sampling of 22 CGMMV isolates, we found low genetic variation of the CP gene except for isolates GR3 and GR5, which showed similarity higher than 97%; based on the MP gene, and 16 CGMMV isolates showed similarity higher than 94% in nucleotide identities by pairwise comparison. Using MluI restriction endonuclease for CP genes, the CGMMV isolates fell into three types: Type I and Type II were included in the SH group and Type III in the W group. The two CGMMV isolates from Myanmar were found to belong to Type I and Type III, respectively.

Occurrence of Cucumber green mottle mosaic virus on Cucurbitaceous Plants in China.

Cucumber green mottle mosaic virus (CGMMV) was first discovered in 2003 in China (2) and developed an epidemic during 2005 (1). To know the occurrence scale and damage level of CGMMV, a survey was carried out in the main areas of cucurbitaceous plant production and seed trading in 2006 and 2007 in Mainland China. Samples of 739 plants of 16 types (seed samples from Cucurbita moschata, Cucumis melo, Lagenaria siceraria, Cucumis sativus, Momordica charantia, L. siceraria var. clavata, and C. pepo, leaf samples from C. moschata, Cucumis melo, L. siceraria, Cucumis sativus, and M. charantia, fruit samples from C. moschata, Citrullus lanatus, and L. siceraria, and seedling samples from Citrullus lanatus) were collected from 13 regions and analyzed by a double antibody sandwich (DAS)-ELISA. CGMMV was detected in Citrullus lanatus, L. siceraria, Cucumis melo, and C. moschata from six regions in 2006. Citrullus vulgaris, Cucumis melo, and C. moschata were infected most frequently; in 76, 60, and 30 of the leaf samples, respectively. CGMMV was tested positive in the samples of 8 seedlings, 23 seeds, and 1 fruit. In contrast, all samples tested negative in 2007. To confirm CGMMV identification, ELISA-positive samples were detected by reverse transcription-PCR assay using virus-specific primers that amplified a 524-bp fragment in the coat protein coding region. The nucleotide sequence of the PCR product (GenBank Accession No. DQ997778) isolated form L. siceraria in Liaoning Province (LHP) showed 100% identity with the Japanese watermelon strain (Japan W) and 91% identity with the Greece GR5 strain. On the basis of the symptoms of indicator plants (chlorotic spot and systemic mosaic were observed in L. siceraria, Chenopodium amaranticolor, and Cucumis sativus; no infection was observed in Datura stramonium, Nicotiana tabacum, and Chenopodium quinoa) and coat protein nucleotide sequence comparisons, the two CGMMV isolates of Wcn (1) and LHP from China should be grouped into the watermelon strain. The results showed that the government should establish effective quarantine strategy and the growers take proper planting measures to avoid further spreading of this virus.

Molecular and Biological Diversities ofCucumber green mottle mosaic virusfrom Cucurbitaceous Crops in Korea

AbstractCucumber green mottle mosaic virus(CGMMV) is the first‐known cucurbit‐infecting tobamovirus. To analyse its biological and molecular diversities, 36 isolates of CGMMV were collected from watermelon (Citrullus vulgaris) and cucumber (Cucumis sativus) plants in Korea. The cDNAs of coat protein (CP) and partial movement protein genes of the isolates were synthesized by reverse transcription and polymerase chain reaction (RT‐PCR) and subjected to single‐strand conformation polymorphism (SSCP) analysis with 530 bp‐synthesized RT‐PCR products. CGMMV isolates were categorized into three subgroups by SSCP analysis, designated types I, II and III. All watermelon isolates of CGMMV belonged to types I and II, whereas cucumber isolates were subgrouped into types II and III. These three type isolates of CGMMV were divided by RT‐PCR‐restriction fragment length polymorphism (RFLP) using full‐length cDNAs of three types of CGMMV. Biological properties of each subgroup‐type of CGMMV isolates were determined by mechanical inoculation of common host plants of CGMMV. Among the virus isolates, isolate NW‐1, belonging to type I, did not infect cucumber (cv. Baegbong), whereas all CGMMV isolates (types II and III) infected the same host species. In addition, all isolates of CGMMV types could infectNicotiana debneyii, except that isolate KC1‐2, subgrouped into type III, failed to infect. Nucleotide and predicted amino acid sequences ofCPgene of CGMMV isolates were compared to those of reported CGMMVs and other cucurbit‐infecting tobamoviruses. In phylogenetic tree analyses between CGMMV isolates and other cucurbit‐infecting tobamoviruses, CGMMV was subgrouped as a cluster and no remarkable molecular diversity was found among the CGMMV isolates.

Etiology of Leaf Chlorosis and Deterioration of the Fruit Interior of Watermelon Plants

AbstractA severe disease with combined symptomatology affecting only grafted watermelon plants appeared in several regions of northern and central Greece during the summers of 1999 and 2000. Disease symptoms included chlorotic mottling followed by pedicel necrosis at a later more mature fruit stage, and finally decomposition of fruit interior. Electron microscopy, double antibody sandwich enzyme‐linked immunosorbent assay tests and reverse transcriptase‐polymerase chain reaction assays identified Cucumber green mottle mosaic virus (CGMMV) as the causal agent. Our studies indicated that the CGMMV isolates from watermelon are closely related to the Israeli variant of CGMMV (CGMMV‐Is). However, bioassays revealed that the Greek isolates elicit symptoms of higher severity and have a wider host range, compared with other described CGMMV isolates. Nucleotide sequence comparison of the movement protein gene of the Greek isolates showed a 97–99% identity with other CGMMV strains. This paper reports the presence of the virus in arable weeds such as Amaranthus blitoides, Amaranthus retroflexus, Heliotropium europaeum, Portulaca oleracea and Solanum nigrum.