Cucurbit Aphid-borne Yellows Virus Isolates Research Articles

First Report of Cucurbit aphid-borne yellows virus infecting five cucurbits in India.

Cucurbits are among the most popular vegetables cultivated globally. They have high economic importance, especially in India, where they are cooked and eaten as vegetables (Dhillon et al. 2016). In February 2023, yellowing symptoms were observed on cucurbitaceous species, viz. Trichosanthes cucumerina (Snake gourd - SG), Luffa acutangula (Ridge gourd - RG), Lagenaria siceraria (Bottle gourd - BG), Luffa aegyptiaca (Sponge gourd - SPG) and yellow chlorotic spots were recorded on Benincasa hispida (Ash gourd - AG) growing in the experimental farm at the Indian Agricultural Research Institute, Regional Station, Pune (Supplementary Figure 1). The average disease incidence ranged from 5% to 30%. A total of 175 leaf samples, including thirty symptomatic and five asymptomatic plants of each cucurbit, were collected and tested by DAS-ELISA using antisera against cucurbit aphid-borne yellows virus (CABYV) (DSMZ, Germany), cucurbit yellow stunting disorder virus (CYSDV) (Arsh Biotech, India), cucumber mosaic virus (CMV), zucchini yellow mosaic virus (ZYMV), and papaya ringspot virus (PRSV) (Agdia, USA). All 150 symptomatic cucurbit samples tested positive for CABYV, while five samples from SG, 14 from RG, two from AG, and 11 from SPG hosts were also positive for PRSV. Asymptomatic samples were negative for all viruses tested. In order to further confirm the presence of the virus, total RNA was extracted from ten samples of each cucurbit host that were positive only for CABYV and the asymptomatic samples using the RNeasy Plant Mini Kit (Qiagen, Germany) as per the manufacturer's protocol. Two-step RT-PCR was carried out using the extracted RNA and CABYV-specific primers, amplifying c. 484 bp of the coat protein gene region (Boubourakas et al. 2006). Amplicons of expected size were obtained in all symptomatic samples, whereas the asymptomatic samples tested negative. Three amplicons obtained from positive samples from each of the cucurbit species were directly sequenced and found to be identical to each other. A representative virus sequence obtained from each cucurbit was deposited in GenBank (Snake gourd - OQ921128, Ridge gourd - OQ921127, Bottle gourd - OQ921126, Ash gourd - OQ921125, Sponge gourd - OQ921129). In BLASTn analysis, the isolates shared from 94.23 to 100% nucleotide identities with the Indian CABYV isolates of various cucurbits and clustered closely with other Pune isolates in the phylogenetic analysis (Supplementary Figure 2). CABYV (genus Polerovirus) is a single-stranded positive-sense RNA virus, and is known to infect and cause severe economic losses in cucurbits worldwide. Previously, occurrences of CABYV have been reported in cucurbits such as watermelon, bitter gourd, cucumber, squash, teasel gourd, and muskmelon in India (Nagendran et al. 2022; Tripathi et al. 2023). It has also been reported to infect a weed species - Abelmoschus moschatus from the same geographical region (Verma et al. 2023). To our knowledge, this is the first report of the natural occurrence of CABYV in snake gourd and ridge gourd worldwide and bottle gourd, ash gourd and sponge gourd in India. The present findings have significant epidemiological importance, as they demonstrate that CABYV is spreading to other cucurbits and occurring widely in India.

Long-term monitoring of aphid-transmitted viruses in melon and zucchini crops: genetic diversity and population structure of cucurbit aphid-borne yellows virus and watermelon mosaic virus.

Understanding the emergence and prevalence of viral diseases in crops requires the systematic epidemiological monitoring of viruses, as well as the analysis of how ecological and evolutionary processes combine to shape viral population dynamics. Here, we extensively monitored the occurrence of six aphid-transmitted viruses in melon and zucchini crops in Spain for 10 consecutive cropping seasons between 2011 and 2020. The most prevalent viruses were cucurbit aphid-borne yellows virus (CABYV) and watermelon mosaic virus (WMV), found in 31 and 26% of samples with yellowing and mosaic symptoms. Other viruses, such as zucchini yellow mosaic virus (ZYMV), cucumber mosaic virus (CMV), Moroccan watermelon mosaic virus (MWMV), and papaya ring spot virus (PRSV) were detected less frequently (< 3%) and mostly in mixed infections. Notably, our statistical analysis showed a significant association between CABYV and WMV in melon and zucchini hosts, suggesting that mixed infections might be influencing the evolutionary epidemiology of these viral diseases. We then carried out a comprehensive genetic characterization of the full-length genome sequences of CABYV and WMV isolates by using the PacBio single-molecule real-time high-throughput technology, in order to assess the genetic variation and structure of their populations. Our results showed that most isolates clustered in the Mediterranean clade, with a fine-scale temporal structure, which was in part explained by the level of the variance between isolates from single and mixed infections. In contrast, the WMV population genetic analysis showed that most of the isolates grouped into the Emergent clade, with no genetic differentiation.

Characterization of Cucurbit Aphid-Borne Yellows Virus (CABYV) from Passion Fruit in Brazil: Evidence of a Complex of Species within CABYV Isolates.

High-throughput sequencing (HTS) has been an important tool for the discovery of plant viruses and their surveillance. In 2015, several virus-like symptoms were observed in passion fruit (PF) plants in Bahia state, Brazil. Using HTS technology, bioinformatics tools, RT-PCR, and Sanger sequencing, we identified the cucurbit aphid-borne yellows virus (CABYV, Polerovirus, Solemoviridae) in co-infection with cowpea aphid-borne mosaic virus (CABMV, Potyvirus, Potyviridae) in PF, in green manure, and spontaneous plants in several localities in Bahia. Complete genomes of CABYV-PF isolates were determined and analyzed with other CABYV isolates available in GenBank that have been identified in various countries. Phylogenetic analysis and pairwise identity comparison with CABYV isolates showed that CABYV-PFs are more closely related to French and Spanish isolates. Overall, analyses of all the CABYV genomes revealed that these could represent ten distinct species, and we thus proposed reclassifying these CABYV as isolates into ten species, tentatively named "Polerovirus curcubitaeprimum" to "Polerovirus curcubitaenonum", and "Polerovirus melo". CABYV-PF is a member of "Polerovirus curcubitaeprimum".

Different RNA Elements Control Viral Protein Synthesis in Polerovirus Isolates Evolved in Separate Geographical Regions.

Most plant viruses lack the 5′-cap and 3′-poly(A) structures, which are common in their host mRNAs, and are crucial for translation initiation. Thus, alternative translation initiation mechanisms were identified for viral mRNAs, one of these being controlled by an RNA element in their 3′-ends that is able to enhance mRNA cap-independent translation (3′-CITE). The 3′-CITEs are modular and transferable RNA elements. In the case of poleroviruses, the mechanism of translation initiation of their RNAs in the host cell is still unclear; thus, it was studied for one of its members, cucurbit aphid-borne yellows virus (CABYV). We determined that efficient CABYV RNA translation requires the presence of a 3′-CITE in its 3′-UTR. We showed that this 3′-CITE requires the presence of the 5′-UTR in cis for its eIF4E-independent activity. Efficient virus multiplication depended on 3′-CITE activity. In CABYV isolates belonging to the three phylogenetic groups identified so far, the 3′-CITEs differ, and recombination prediction analyses suggest that these 3′-CITEs have been acquired through recombination with an unknown donor. Since these isolates have evolved in different geographical regions, this may suggest that their respective 3′-CITEs are possibly better adapted to each region. We propose that translation of other polerovirus genomes may also be 3′-CITE-dependent.

High Prevalence of Three Potyviruses Infecting Cucurbits in Oklahoma and Phylogenetic Analysis of Cucurbit Aphid-Borne Yellows Virus Isolated from Pumpkins.

Field information about viruses infecting crops is fundamental for understanding the severity of the effects they cause in plants. To determine the status of cucurbit viruses, surveys were conducted for three consecutive years (2016–2018) in different agricultural districts of Oklahoma. A total of 1331 leaf samples from >90 fields were randomly collected from both symptomatic and asymptomatic cucurbit plants across 11 counties. All samples were tested with the dot-immunobinding assay (DIBA) against the antisera of 10 known viruses. Samples infected with papaya ringspot virus (PRSV-W), watermelon mosaic virus (WMV), zucchini yellow mosaic virus (ZYMV), and cucurbit aphid-borne-yellows virus (CABYV) were also tested by RT-PCR. Of the 10 viruses, PRSV-W was the most widespread, with an overall prevalence of 59.1%, present in all 11 counties, followed by ZYMV (27.6%), in 10 counties, and WMV (20.7%), in seven counties, while the remaining viruses were present sporadically with low incidence. Approximately 42% of the infected samples were positive, with more than one virus indicating a high proportion of mixed infections. CABYV was detected for the first time in Oklahoma, and the phylogenetic analysis of the first complete genome sequence of a CABYV isolate (BL-4) from the US showed a close relationship with Asian isolates.

The recombinant isolate of cucurbit aphid‐borne yellows virus from Brazil is a polerovirus transmitted by whiteflies

AbstractThe severe yellowing disease (amarelão) on melon plants is a serious problem in Brazil, although the causative agent remained unknown for a long time. Recently, recombinant isolates of cucurbit aphid‐borne yellows virus (CABYV) were reported as the possible causative agents of this disease on melon plants. Although aphids are known to be the vectors of the common type of CABYV isolates, almost no aphid colony was observed in the major melon fields in Brazil with high incidence of the severe yellowing disease. In contrast, whiteflies are often abundant. Based on this observation, the hypothesis of the transmission of recombinant CABYV by whiteflies was evaluated. After thorough transmission experiments, we found that this recombinant CABYV isolate was transmitted by the whitefly Bemisia tabaci MEAM1, but not by Aphis gossipii. Furthermore, the host response by whitefly‐based inoculation in cucurbits and other indicator plants showed differences in host range when compared to the common type of CABYV. Due to its transmissibility by the whitefly and the distant relationship of the P3/P5 protein to CABYV, the name “cucurbit whitefly‐borne yellows virus” is proposed for this recombinant CABYV. This is the second report of polerovirus transmission by the whitefly B. tabaci, following the report of pepper whitefly‐borne vein yellows virus.

First report of cucurbit aphid-borne yellows virus on cucumber in Java, Indonesia

Chlorosis and yellowing with green-vein banding on older leaves were observed in surveys of cultivated cucumber fields in 2016 in Java, Indonesia. Initial dot immuno-binding assays detected seven different viruses varying in frequency among over 500 samples from 27 locations. Cucurbit aphid-borne yellows virus (CABYV) was detected at a frequency approaching 100% in four provinces in Java. The coat protein nucleotide and amino acid sequences of four isolates had highest identity with that of CABYV isolate Aileu in East Timor. This is the first report of CABYV infection of cucumber in Indonesia.

First Report of Cucurbit Aphid-Borne Yellows Virus in Passion Fruit Plants Exhibiting Mosaic and Mottling in China

First Report of Cucurbit Aphid-Borne Yellows Virus in Passion Fruit Plants Exhibiting Mosaic and Mottling in China

First Complete Genome Sequence of Cucurbit Aphid-Borne Yellows Virus from Pumpkin in the United States.

Cucurbit aphid-borne yellows virus (CABYV) was first described in France in 1992 and since then has been reported in various parts of the world, including the United States. Here, we present the first complete genome sequence of a CABYV isolate (BL4) that was collected from pumpkin during the 2017 growing season in Oklahoma.

Complete Genome Sequences and Evolutionary Analysis of Cucurbit aphid-borne yellows virus Isolates from Melon in Korea.

Complete genome sequences of 22 isolates of Cucurbit aphid-borne yellows virus (CABYV), collected from melon plants showing yellowing symptom in Korea during the years 2013–2014, were determined and compared with previously reported CABYV genome sequences. The complete genomes were found to be 5,680–5,684 nucleotides in length and to encode six open reading frames (ORFs) that are separated into two regions by a non-coding internal region (IR) of 199 nucleotides. Their genomic organization is typical of the genus Polerovirus. Based on phylogenetic analyses of complete nucleotide (nt) sequences, CABYV isolates were divided into four groups: Asian, Mediterranean, Taiwanese, and R groups. The Korean CABYV isolates clustered with the Asian group with > 94% nt sequence identity. In contrast, the Korean CABYV isolates shared 87–89% sequence identities with the Mediterranean group, 88% with the Taiwanese group, 81–84% with the CABYV-R group, and 72% with another polerovirus, M.. Recombination analyses identified 24 recombination events (12 different recombination types) in the analyzed CABYV population. In the Korean CABYV isolates, four recombination types were detected from eight isolates. Two recombination types were detected in the IR and P3–P5 regions, respectively, which have been reported as hotspots for recombination of CABYV. This result suggests that recombination is an important evolutionary force in the genetic diversification of CABYV populations.

First report of Cucurbit aphid‐borne yellows virus in Vicia faba

There are 17 formally recognised virus species in the genus Polerovirus (family Luteoviridae) according to the ICTV classification released in 2014. Three polerovirus species have been described that affect production in economically important cucurbit crops: Cucurbit aphid-borne yellows virus (CABYV), Melon aphid-borne yellows virus (MABYV) and Suakwa aphid-borne yellows virus (SABYV). Two other poleroviruses, Beet western yellows virus (BWYV) and Pepper vein yellows virus (PVYV) have been detected recently in field-grown pepper plants in Turkey (Buzkan et al., 1). During surveys in March 2016, symptoms including chlorosis of young leaves and yellowing of older leaves, suggestive of polerovirus infection, were observed in broad bean (Vicia faba) plants (Fig. 1) in Tarsus, a province in the Mediterranean region of Turkey. A total of 36 diseased plants were tested for the presence of poleroviruses using DAS-ELISA (Dombrovsky et al., 2) and 26 tested positive. A 1.1 kb portion of the polerovirus genome was amplified from all 26 ELISA-positive samples using the general polerovirus primer pair Pol-G-F and Pol-G-R in RT-PCR (Knierim et al., 3). PCR amplicons were subsequently sequenced (GenBank Accession No. KY112798) and subject to a BLASTn search to identify the polerovirus species. According to an alignment of 473 nucleotides of the RdRp, the broad bean isolate showed 98% nucleotide identity with a CABYV isolate from cucumber from Iran (KF425567). To the best of our knowledge this is the first report of CABYV in V. faba in Turkey and globally. CABYV has been reported previously in a range of cucurbit crops in Turkey using DAS-ELISA (Yardımcı & Özgönen, 6). However, the virus species was not confirmed since the CABYV polyclonal antibody used cross-reacts with other poleroviruses (Dombrovsky et al., 2). Since CABYV was first identified in 1992, the virus has been detected worldwide in temperate and subtropical areas (Lecoq et al., 4). Apart from cucurbit crops, other species of agronomic importance that are CABYV hosts include Beta vulgaris and Lactuca sativa, as well as common weed species such as Capsella bursa-pastoris, Papaver rhoeas and Senecio vulgaris which are thought to be virus reservoirs (Knierim et al., 3; Mnari-Hattab et al., 2009). CABYV is a phloem-limited virus that is transmitted in a persistent, non-propagative mode by aphids, including Aphis gossypii and Myzus persicae (Lecoq et al., 4). Mixed polerovirus infections increase the risk of the emergence of recombinant viruses. Poleroviruses must be considered as important emerging pathogens in open-field and greenhouse vegetable cultivation. This work was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) (Project No: 113 O 423).

Biological and Molecular Characterization of a Korean Isolate of Cucurbit aphid-borne yellows virus Infecting Cucumis Species in Korea.

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.

Genetic Diversity and Potential Vectors and Reservoirs of Cucurbit aphid-borne yellows virus in Southeastern Spain

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

Full‐length genome sequences of four polerovirus isolates infecting cucurbits in Taiwan determined from total RNA extracted from field samples

Poleroviruses are not mechanically transmittable by sap inoculation, which makes their isolation and multiplication in laboratory hosts for virion extraction and genome sequence analysis difficult. A reverse‐transcription polymerase chain reaction (RT‐PCR) procedure was modified to amplify the entire genomes of cucurbit‐infecting poleroviruses in three sections directly from RNA extracts from infected plant field samples. The procedure was used to determine the first full‐length genome nucleotide sequences of four polerovirus isolates from field samples collected in Taiwan. TW1 was identified as Melon aphid‐borne yellows virus (MABYV), because it had &lt;10% variation in the deduced amino acid (aa) sequence in any of the encoded proteins when compared to MABYV‐China. C‐TW20 was most similar to Cucurbit aphid‐borne yellows virus (CABYV) from China with aa sequence identities of &gt;90% for P3 and P3‐5 and aa identities of 78–88% for all the other proteins. TW19 represents the first full‐length sequence for the recently described Suakwa aphid‐borne yellows virus (SABYV). The deduced aa sequences for all the proteins of TW19 showed &gt;10% variation compared to all other poleroviruses, though recombination analysis suggested MABYV as the major parent and an unidentified polerovirus as minor parent. R‐TW82 was identified as a strain of CABYV, as the deduced CP aa sequence showed &gt;90% identity to that of other CABYV isolates, and recombination analysis suggested that MABYV was the major parent with CABYV the minor parent. Comparative details of the new full‐length sequences and their implications on criteria for distinguishing polerovirus species are discussed.

Cucurbit aphid-borne yellows virus in Egypt

During 2010, yellowing symptoms were frequently observed in cultivated squash fields in Egypt. A total of 717 symptomatic squash leaf samples were collected from four regions where squash cultivation is of economic importance for the country: Kafrelsheikh, El-Behira, El-Sharkia and El-Ismailia. Serological analysis showed that 95.6% of the symptomatic squash samples were infected by Cucurbit aphid-borne yellows virus (CABYV), and visual estimation of the incidence of yellowing symptoms suggested a very high incidence of CABYV in the fields. Twelve CABYV isolates were characterized by sequencing two regions of the viral genome, open reading frame (ORF) 3 and ORFs 4/5. Overall, Egyptian isolates were very similar among them, and had higher similarity values with a French than with a Chinese isolate. The average nucleotide diversity for ORF 3 was significantly higher than for the other two regions, indicating that variability is not evenly distributed along the viral genome. The ratios between nucleotide diversity values in non-synonymous (dN) and synonymous (dS) positions (dN/dS) for each ORF showed that the three ORFs are evolving under different pressures, although predominantly under purifying selection. Phylogenetic analyses revealed that these Egyptian isolates, with only one exception, shared the same clade with a French isolate. Moreover, these analyses suggested that Egyptian isolates belong to the Mediterranean group described previously.

First Report of the Occurrence of Cucurbit aphid-borne yellows virus on Oilseed Pumpkin in Serbia

In July 2008, field-grown oilseed pumpkins (Cucurbita pepo L. 'Olinka') showing severe yellowing and thickening of older leaves were observed in the Kisač locality of Vojvodina Province, Serbia. Symptomatic plants were found only near the borders of the field. Leaf samples collected from 15 symptomatic plants were tested for the presence of four viruses causing the cucurbit yellowing disorder. Total RNAs were extracted from deep frozen plant materials with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and reverse transcription (RT)-PCR was conducted with the OneStep RT-PCR Kit (Qiagen) following the manufacturer's instructions. RNA extracted from healthy C. pepo and molecular-grade water were included as negative controls in each PCR reaction. Species-specific primers (1,2) failed to detect the presence of three viruses causing the cucurbit yellowing disorder, Cucumber vein yellowing virus, Cucumber yellow stunting disorder virus, and Beet pseudo-yellows virus, in symptomatic samples. When two different sets of CABYV-specific primer pairs, CABYVup/CABYVdown (2) and CE9/CE10 (3), for a 484-bp and a 600-bp fragment of the CP gene of Cucurbit aphid-borne yellows virus (CABYV), respectively, were used for amplification, the former amplified fragments of the expected size from all symptomatic samples, whereas the latter successfully amplified a 600-bp fragment from only 7 of 15 samples. The 600-bp amplified product derived from isolate 145-08 was purified (QIAquick PCR Purification Kit, Qiagen), sequenced in both directions, deposited in GenBank (Accession No. HQ202745), and subjected to sequence analysis by MEGA4 software. Sequence comparisons revealed a high nucleotide identity of 99.8% (100 and 99.5% amino acid identities for the CP and the overlapping MP genes, respectively) with Czech CABYV isolates from C. pepo 'Ovifera' (HM771271-73). A neighbor-joining tree obtained on a 545-bp CP fragment of CABYV isolates available in GenBank database revealed that Serbian CABYV isolate 145-08 was clustered with isolates from Spain, Italy, France, and Tunisia in the Mediterranean subgroup denoted previously (4). In a persistent type transmission test, which was carried out using Aphis gossypii Glover, the aphids were allowed to feed on leaves of the collected sample (145-08) for an acquisition access period of 2 days and then 10 aphids were transferred to each of 20 C. pepo 'Olinka' plants for a 5 day inoculation access period. Transmission was successful in 6 of 20 plants as assessed by the development of a mild yellowing symptom 2 weeks after transmission and confirmed by RT-PCR with the CABYVup/CABYVdown primers. To our knowledge, this is the first record of the occurrence of CABYV in Serbia. The discovery of CABYV on oilseed pumpkin should prompt more detailed surveys and subsequent testing of other cucurbits cultivated in Serbia to establish the distribution and incidence of CABYV in Serbia.

First Report of Cucurbit aphid-borne yellows virus in Squash in the Czech Republic

A yellowing of buttercup squash (Cucurbita pepo L. var. oleifera Pietsch) leaves was observed on plants in southern Moravia, the main squash-growing area of the Czech Republic. Forty leaf samples were collected in September 2009 and examined for the presence of possible cucurbit viruses by double-antibody sandwich-ELISA. Thirty-three samples were infected with Zucchini yellow mosaic virus and five with Cucurbit aphid-borne yellows virus (CABYV). The positive samples of CABYV originated near the villages of Josefov and Prušánky (one per sample) and Rakvice (three samples), and the virus isolates were named Jos-5, Pr-15, Rak-1, Rak-4, and Rak-5, respectively. CABYV was immediately transmitted from leaves collected in the field to summer squash (Cucurbita pepo L. convar. giromontiina Grebenšcikov) plants by aphids in a persistent manner. Green peach aphids, Myzus persicae (Sulzer), were used to inoculate squash plants with acquisition and inoculation feeding times of 2 and 5 days, respectively. Twenty-one plants were inoculated with 20 aphids per plant. Transmission was successful in 25% of the plants as assessed by ELISA. Infected plants showed very mild yellowing 2 weeks after transmission and were shorter compared with noninoculated controls. Leaf samples of newly infected plants were examined by electron microscopy and isometric particles of approximately 25 nm in diameter, corresponding in size and shape to described particles of CABYV (3), were observed. The presence of CABYV was verified by reverse transcription (RT)-PCR using a primer pair specific to the CABYV coat protein gene (2). The amplicons were sequenced (GenBank Accession Nos. HM771269-HM771273) and 100% sequence identity was found between isolates Jos-5 and Pr-15 and among the isolates Rak-1, Rak-2, and Rak-3. Sequence identity between these two groups was 99.3%. Blast analysis (4) showed that the Czech CABYV isolates are closely related to the Slovak isolates SK-1 (Accession No. FJ428797) and IR-3 (Accession No. FJ428800) with nucleotide sequence identities of 99.6 and 99.1%, respectively. These results indicate a similar origin between the Czech and Slovak isolates. To our knowledge, this is the first report of the natural occurrence of CABYV in the Czech Republic. CABYV is a widespread virus that reduces the yield of cucurbit vegetables (1). Protection against epidemics should be based on the control of aphid vectors, protecting plants with very fine mesh netting, keeping the cultivation area free of weeds, or planting cultivars resistant to CABYV.

Biological and Molecular Characterization of the Cucurbit aphid-borne yellows virus Affecting Cucurbits in Tunisia.

Surveys of yellowing viruses under nonheated and geothermal heated plastic tunnels and in open field crops of melon (Cucumis melo), cucumber (C. sativus), zucchini (Cucurbita pepo), squash (C. maxima), watermelon (Citrullus lanatus), and ware cucurbit (Ecballium elaterium) were carried out year-round during 2000-2001, 2003, and 2004 in the major cucurbit-growing areas in Tunisia. Severe yellowing symptoms on older leaves of cucurbits were observed in open fields and under plastic-tunnel production systems. These yellowing symptoms and large populations of aphids (Aphis gossypii) on a diversity of cucurbit crops in Tunisia support the hypothesis of a viral cause of the disease. Virus identification using double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), followed by reverse transcription-polymerase chain reaction (RT-PCR) and immunocapture (IC)-RT-PCR showed that Cucurbit aphid-borne yellows virus (CABYV) was largely distributed in melon, cucumber, zucchini, squash, and watermelon crops. Ware cucurbit (E. elaterium) and lettuce (Lactuca sativa) crops were identified as potential CABYV reservoirs. The RT-PCR-amplified partial coat protein (CP) and P4 genes were cloned and sequenced from nine Tunisian CABYV isolates. CP and P4 gene nucleotide and amino acid sequence comparisons as well as phylogenetic reconstructions showed that the Tunisian isolates clustered into two major subgroups. Comparisons with CABYV sequences retrieved from GenBank showed high nucleotide and CP amino acid identities, and close relationships of the Tunisian isolates with Italian and French isolates.

Distribution and molecular diversity of three cucurbit-infecting poleroviruses in China

Cucurbit aphid-borne yellows virus (CABYV) and Melon aphid-borne yellows virus (MABYV) have been found to be associated with cucurbit yellowing disease in China. Our report identifies for the first time a third distinct polerovirus, tentatively named Suakwa aphid-borne yellows virus (SABYV), infecting Suakwa vegetable sponge. To better understand the distribution and molecular diversity of these three poleroviruses infecting cucurbits, a total of 214 cucurbitaceous crop samples were collected from 25 provinces in China, and were investigated by RT-PCR and sequencing. Of these, 108 samples tested positive for CABYV, while 40 samples from five provinces were positive for MABYV, and SABYV was detected in only 4 samples which were collected in the southern part of China. Forty-one PCR-amplified fragments containing a portion of the RdRp gene, intergenic NCR and CP gene were cloned and sequenced. Sequence comparisons showed that CABYV isolates shared 78.0–79.2% nucleotide sequence identity with MABYV isolates, and 69.7–70.8% with SABYV. Sequence identity between MABYV and SABYV was 73.3–76.5%. In contrast, the nucleotide identities within each species were 93.2–98.7% (CABYV), 98.1–99.9% (MABYV), and 96.1–98.6% (SABYV). Phylogenetic analyses revealed that the polerovirus isolates fit into three distinct groups, corresponding to the three species. The CABYV group could be further divided into two subgroups: the Asia subgroup and the Mediterranean subgroup, based on CP gene and partial RdRp gene sequences. Recombination analysis suggested that MABYV may be a recombinant virus.

Molecular characterization of geographically different Cucurbit aphid-borne yellows virus isolates

Cucurbit aphid-borne yellows virus (CABYV) causes yellowing symptoms in cucurbit crops worldwide. In this work, the sequences for the coat protein (CP) gene for CABYV isolates from Iran and Slovakia are reported for the first time. Three Iranian isolates shared 96.2-99.3% and the Slovak isolate 98.9% of nucleotide identity in comparison with the reference French CABYV-N isolate. Phylogenetic analysis showed that the sequence of CP gene of examined CABYV isolates differed only slightly. Relatively close relationship and sequence similarity of geographically distant CABYV isolates could reflect the epidemic outbreak and rapid expansion of the virus throughout the world due to the host and vector abundance.