Cucurbit Aphid-borne Yellows Virus Research Articles

Non-Native Plant Viruses Prevalent in Remnant Natural Plant Communities Harm Native Perennial Hosts

Plant viruses are ubiquitous throughout plant communities, but research on viral impacts largely focuses on crops. Little is known about how viruses influence wild plants in their native habitats. To address this gap, we examined virus interactions with wild drought-tolerant perennials in California desert natural areas encroached upon by agriculture. We used metagenomics, targeted diagnostics, and phylogenetics to assess virus diversity and clade relationships, and experiments to investigate viral influence on hosts. We focused on three herbaceous perennials ( Cucurbita foetidissima, C. palmata, and Datura wrightii) and tested the hypothesis that these wild species accumulate virus infections typically found in crops and transmitted by polyphagous insects. We predicted that such infections might be retained across seasons and potentially impair plant performance. Virome profiling revealed a rich community of previously characterized virus species (12 total), with virus community structure varying by site and host species. The dominant viruses in the wild hosts were non-native crop pathogens, including cucurbit aphid-borne yellows virus (CABYV) and cucurbit yellow stunting disorder virus (CYSDV). Targeted testing revealed that CABYV infected as many as 88% of sampled wild Cucurbita individuals, with dual CABYV–CYSDV infections common in natural areas adjacent to desert agriculture. CABYV infections reduced shoot and root production in greenhouse experiments with the two wild Cucurbita species. Phylogenetic analyses suggest that CABYV was introduced to California multiple times from other continents. Our findings provide concerning evidence of ways in which human activities can alter virus pressure on wild plants and potentially contribute to plant decline.

First report of cucurbit yellow stunting disorder virus infecting cucurbit crops in Jamaica.

The increasing prevalence of whitefly-transmitted viruses affecting cucurbit crops has emerged as a significant concern for global cucurbit production. Two of the most widely prevalent threats in the Americas are cucurbit yellow stunting disorder virus (CYSDV) and cucurbit chlorotic yellows virus (CCYV) (Crinivirus, Closteroviridae). These viruses induce similar foliar symptoms on cucurbit crops (Mondal et al., 2023) leading to loss of photosynthetic capability and decreased yields. Cantaloupe (Cucumis melo), watermelon (Citrullus lanatus), and cucumber (Cucumis sativus) are major cucurbit crops in St. Elizabeth, Jamaica, which is the principal fruit and vegetable producing region of the country. In August 2018, foliar symptoms were observed on cantaloupe, watermelon, and cucumber plants in several commercial farms in St. Elizabeth. These symptoms, mainly on the older leaves, consisted of severe yellowing or interveinal mottle and they appeared more pronounced on cantaloupe and cucumber plants compared to watermelon. Growers noticed the production of smaller than normal fruit. Disease incidence ranged from 10 to 100% and whiteflies (Bemisia tabaci Gennadius) were observed in the fields. To identify virus(es) associated with the disease, six plants (cantaloupe [n = 3], cucumber [n = 1] and watermelon [n = 2) exhibiting symptoms were sampled from four fields for preliminary screening. Total RNA was extracted from leaf tissues as described in Tamang et al. (2021) and samples tested by a multiplex reverse transcription RT-PCR method that targeted the RNA-dependent RNA polymerase (RdRp) of the whitefly transmitted viruses, CYSDV, CCYV, squash vein yellowing virus (SqVYV), and the aphid- transmitted cucurbit aphid-borne yellows virus (CABYV) (Mondal et al. 2023). RT-PCR amplified the expected 494-bp fragment of the CYSDV RdRp gene (Mondal et al., 2023) from two symptomatic plants; one cantaloupe, one cucumber, as well as from CYSDV-infected control plants but not from healthy controls. Further testing was conducted during the June-August 2020 growing season after similar symptoms were observed on additional farms in St. Elizabeth and two regions, Manchester and Clarendon, located to the east of St. Elizabeth. Twenty-one cucurbit leaf samples (11 cantaloupe, seven watermelon and two cucumber from St. Elizabeth and one cantaloupe from Clarendon) exhibiting foliar yellowing progressing from the crown outward, and mottling were collected. Whiteflies (5) from these fields in St. Elizabeth and 20 asymptomatic weed samples were also collected and sent to the USDA-ARS laboratory at Salinas, CA. Total RNA from leaf samples was extracted as described above and tested for CYSDV, CCYV, and CABYV. Total leaf DNA was also extracted (Mondal et al. 2016) and assayed with PCR (Gilbertson 2001) to detect the presence of the whitefly-transmitted cucurbit leaf crumple virus (CuLCrV), a begomovirus, commonly found in the southeastern United States (Gadhave et al., 2018; Keinath et al., 2018). Nineteen of the 21 cucurbit samples tested positive for the presence of CYSDV by RT-PCR (Mondal et al. 2023). Of the 19 CYSDV-positive samples, 13 cantaloupe, one cucumber, and five watermelon samples were singly infected with CYSDV, and one cantaloupe sample was infected with both CYSDV and CABYV. Amplicons of the Jamaica isolate from cantaloupe were sequenced (OR399555) and a 494 nt section of the RdRp gene was found to share 100% sequence identity to the Arizona 1 isolate (EF547827.1). The presence of CYSDV, was further confirmed using a second set of primers that amplified a 394-nt portion of the CYSDV coat protein gene (Polston et al., 2008). Among the weed samples, CABYV was detected in one sample from a Leonotis nepetifolia plant (Lamiaceae) and two Cleome sp. (Capparaceae) collected from St. Elizabeth. None of the crop and weed samples tested positive for CCYV or CuLCrV. DNA from whiteflies was extracted and assayed with PCR using species specific primers (Chen et al. 2016). All whiteflies were identified as B. tabaci cryptic species MEAM1, which is widely known an efficient vector of CYSDV (Berdiales, et al. 1999). This is the first report of CYSDV in Jamaica and its first known occurrence in these hosts within the country. Further monitoring of cucurbit crops and the whitefly vector is warranted to better understand the epidemiology.

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.

Advances in Understanding the Mechanism of Cap-Independent Cucurbit Aphid-Borne Yellows Virus Protein Synthesis.

Non-canonical translation mechanisms have been described for many viral RNAs. In the case of several plant viruses, their protein synthesis is controlled by RNA elements in their genomic 3'-ends that are able to enhance cap-independent translation (3'-CITE). The proposed general mechanism of 3'-CITEs includes their binding to eukaryotic translation initiation factors (eIFs) that reach the 5'-end and AUG start codon through 5'-3'-UTR-interactions. It was previously shown that cucurbit aphid-borne yellows virus (CABYV) has a 3'-CITE, which varies in sequence and structure depending on the phylogenetic group to which the isolate belongs, possibly as a result of adaptation to the different geographical regions. In this work, the cap-independent translation mechanisms of two CABYV 3'-CITEs belonging to the Mediterranean (CMTE) and Asian (CXTE) groups, respectively, were studied. In vivo cap-independent translation assays show that these 3'-CITEs require the presence of the CABYV short genomic 5'-UTR with at least 40% adenines in cis and an accessible 5'-end for its activity. Additionally, they suggest that the eIF4E-independent CABYV 3'-CITE activities may not require either eIF4A or the eIF4F complex, but may depend on eIF4G and PABP. By pulling down host proteins using RNA baits containing both 5'- and 3'-CABYV-UTRs, 80 RNA binding proteins were identified. These interacted preferentially with either CMTE, CXTE, or both. One of these proteins, specifically interacting with the RNA containing CMTE, was HSP70.2. Preliminary results suggested that HSP70.2 may be involved in CMTE- but not CXTE-mediated cap-independent translation activity.

Global phylodynamics of two relevant aphid-transmitted viruses in cucurbit crops: cucurbit aphid-borne yellows virus and watermelon mosaic virus

AbstractCucurbit aphid-borne yellows virus (CABYV) and watermelon mosaic virus (WMV) are major plant pathogens that cause severe epidemics in cucurbit crops. While there has been an increasing interest in molecular epidemiological studies on both viruses at regional scales, their phylodynamic analysis by using the temporal data at global scale remains unexplored. In this study, we implemented the Nextstrain phylodynamic approach to comprehensively examine the coat protein gene and full-length genome sequences of the CABYV and WMV worldwide populations. Our analyses reconstructed a robust phylogeny of CABYV and confirmed the occurrence of isolates grouped into three clusters (Asian, Mediterranean, and Recombinant). Nextstrain analysis suggested that CABYV epidemics likely originated in Southeast Asia in fourteenth century, while the Mediterranean population emerged in Spain in seventeenth century. We also found a high divergence between Asian and Mediterranean isolates, with low genetic diversity and scarce evidence of selection, as reflected by the lack of gene flow. Moreover, the hierarchical analysis of molecular variance revealed a significant differentiation between CABYV populations grouped by geographical location and plant host. Additionally, the global phylogenetic reconstruction of the WMV population confirmed a clear differentiation among isolates, which grouped into two clusters (classical and emergent), and Nextstrain analysis suggested that WMV epidemics were most probably originated in USA during the sixteenth century. The initial WMV population diverged in the eighteenth century, with the origin of the emergent population in the nineteenth century. Our analysis confirmed that WMV population has a polyphyletic origin, defining an extensive genetic diversity. Overall, this work provides insights into the CABYV and WMV origin and evolutionary dynamics, gaining an understanding of the global spread of both viral diseases. Additionally, the integration of all spatio-temporal CABYV and WMV data, along with their genome sequence data by open access (https://github.com/PiR92) and the Nextstrain approach, provides a valuable tool for compiling and sharing current knowledge of these viral diseases in cucurbit crops.

Emergence of yellowing disease in cucurbitaceous vegetables caused by Crinivirus and Polerovirus in India

Totally 102 symptomatic samples of cucurbitaceous vegetables showing yellowing were collected from fields of Uttar Pradesh and screened by RT-PCR assay for the presence of Crinivirus and Polerovirus. Among them, Crinivirus (16%) and Polerovirus (23%) were tested positive with the universal primer pairs. Based on the sequence analysis of amplified product, two Crinivirus (Cucurbit chlorotic yellows virus - CCYV and Cucurbit yellow stunting disorder virus - CYSDV) and two Polerovirus (Cucurbit aphid-borne yellows virus - CABYV and Luffa aphid-borne yellows virus - LABYV) species were characterized. Phylogenetic analysis revealed less genetic distance among the Indian isolates of CCYV, CYSDV and LABYV whereas CABYV closely related to Chinese isolates. To the best of our knowledge, this study documents infection of CCYV on cucumber, round melon and muskmelon; CYSDV on satputia and sponge gourd; CABYV on ivy gourd; and LABYV on ridge gourd, satputia and muskmelon for the first time in India.

Differential seasonal prevalence of yellowing viruses infecting melon crops in southern California and Arizona determined by multiplex RT-PCR and RT-qPCR.

Viruses transmitted by the whitefly (Bemisia tabaci) are an increasing threat to cucurbit production in the southwestern United States as well as many other cucurbit production regions throughout the world. The crinivirus, cucurbit yellow stunting disorder virus (CYSDV), has severely impacted melon production in California and Arizona since its introduction to the region in 2006. Within the past few years, another crinivirus, cucurbit chlorotic yellows virus (CCYV), and the whitefly-transmitted ipomovirus, squash vein yellowing virus (SqVYV) were found infecting melon plants in California's Imperial Valley as well. Both CYSDV and CCYV, as well as an aphid transmitted polerovirus, cucurbit aphid-borne yellows virus (CABYV), occur together in the region and produce identical yellowing symptoms on cucurbit plants. Mixed infections of these four viruses both in the Sonoran Desert and other regions pose challenges for disease management and efforts to develop resistant varieties. A multiplex single-step RT-PCR method was developed that differentiates these viruses from one another, and this was used to determine the prevalence and distribution these viruses in melon samples collected from fields in the Sonoran Desert melon production region of California and Arizona, USA during spring and fall melon seasons from 2019 through 2021. TaqMan probes were also developed, optimized, and applied in a single-step multiplex RT-qPCR to quantify titers of these four viruses in plant samples, which frequently carry mixed infections. Results of the multiplex RT-PCR analysis demonstrated that CYSDV is the predominant virus during the fall season, whereas CCYV was by far the most prevalent virus during spring seasons each year. Multiplex RT-qPCR was used to evaluate differential accumulation and spatio-temporal distribution of viruses within plants and suggested differences in competitive accumulation of CCYV and CYSDV within melon. This study also provides the first official report of SqVYV in Arizona, USA, and offers an efficient method for virus detection and quantification for breeding and disease management in areas impacted by cucurbit yellowing viruses.

Genetic Variation between Asian and Mediterranean Populations of Cucurbit Aphid-Borne Yellows Virus.

Viral symptoms, such as yellowing, leaf deformation, mottling, vein clearing, and reduced yield, were observed in cucurbits in Iran. This study aimed to detect the main suspected causal agent, cucurbit aphid-borne yellows virus (CABYV), in Iran and analyze the genetic diversity among isolates. Two hundred samples were collected from different growing areas between 2019 and 2022. PCR amplification was performed on the P3 and P4 genes. The sequences of 18 Iranian isolates were obtained and deposited in GenBank. Recombination, phylogenetic, and population genetics studies were then carried out for the complete genome and all ORFs sequences, together with other isolates in GenBank. The nucleotide identities of the overlapped ORF3/4 sequences of Iranian isolates were 94.8 to 99.5% among themselves, and with other tested isolates ranging from 94.3 to 99.3%. Phylogenetic trees based on the complete genome and the overlapped ORF3/4 showed two major clades, namely Asian and Mediterranean, and the new isolates from Iran were positioned in both clades. The obtained results also suggest that all the genes and two clades of CABYV populations were under negative selection pressure. Furthermore, rare gene flow between these two clades (FST > 0.33) confirmed the high genetic separation among them.

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.

Musk mallow (Abelmoschus moschatus): a new host of Cucurbit aphid‐borne yellows virus

Musk mallow (Abelmoschus moschatus, family Malvaceae) is a medicinal plant cultivated across Asia, Africa and South America. It is found as a weed all over the Deccan region of India (Pawar & Vyawahare, 2017). The seeds of the plant are used for medicinal purposes and as a source of fragrant compounds (Gul et al., 2011). Musk mallow plants exhibiting yellowing of older leaves, midvein clearing, leaf reduction, mottling and slight downward curling (Fig. 1) were observed in October 2022 at the experimental research farm of the Indian Agricultural Research Institute in Pune, Maharashtra, India. The plants occurred as weeds and the disease incidence was 4% (c. 16 plants) in an eight-acre area. Ten diseased and four asymptomatic plants were sampled to ascertain the viruses associated with the symptoms. ELISA was done using antisera against Cucurbit aphid-borne yellows virus (CABYV) (DSMZ, Germany), Cucurbit yellow stunting disorder virus (CYSDV) (Arsh Biotech, India), Cucumber mosaic virus (CMV), Papaya ringspot virus (PRSV) and Zucchini yellow mosaic virus (ZYMV) (Agdia, USA). CABYV was detected in all diseased samples but CMV, CYSDV, PRSV and ZYMV were not detected. None of the five viruses were detected by ELISA in the asymptomatic samples. Total RNA was extracted from both diseased and asymptomatic samples using an RNeasy Plant Mini Kit (Qiagen, USA). Two-step RT-PCR assay was done using the extracted RNA and CABYV-specific primers which amplify c. 484 bp of the partial coat protein region (Boubourakas et al., 2006). RT-PCR was also performed using primers specific for CMV, CYSDV, PRSV, ZYMV, Beet pseudo-yellows virus and Cucumber vein yellowing virus. Amplicons of the expected size were obtained in all diseased plants with CABYV primers but not with primers for any of the other viruses. No amplification was obtained in the asymptomatic samples for any of the viruses. The amplicons of five samples were Sanger sequenced bidirectionally and found to be identical (GenBank Accession No. OP455156). A BLASTn search of the sequence revealed that it shared 100% identity with an Indian CABYV sequence from Praecitrullus fistulosus (OP455157). A phylogenetic tree was constructed using the neighbour-joining method (MEGA X program) with CABYV sequences from different hosts and geographies, and the sequence clustered with other Indian CABYV sequences (Fig. 2). CABYV primarily infects cucurbits but also infects other crops and weeds including three Malvaceae species, Abutilon theophrasti, Gossypium hirsutum and Malva parviflora (Lecoq et al., 2022). To our knowledge, this is the first report of CABYV infecting Abelmoschus moschatus in the world. The present finding is of epidemiological importance as it indicates the further broadening of the host range of CABYV and the possibility of infecting other economically important Malvaceae members, such as G. hirsutum which is grown for cotton in India. Furthermore, musk mallow plants could serve as an alternative / reservoir host for CABYV aiding its transmission to other commercial crops. The authors thank the Director, ICAR-IARI, New Delhi for providing lab facilities.

Incidence of Virus Diseases in Major Cultivated Areas of Watermelon and Melon in Chungbuk Province

To investigate the incidence status of viruses in major cultivated areas of watermelon and melon in Chungbuk Province, samples were collected from 2020 to 2021 in vinyl greenhouse of Jincheon and Eumseong and examined for virus infection using reverse transcription polymerase chain reaction. Of the six viruses on watermelon that was analyzed in this study, watermelons were infected with cucumber mosaic virus (CMV), watermelon mosaic virus (WMV), cucumber green mottle mosaic virus (CGMMV), and cucurbit aphid-borne yellows virus (CABYV). The incidence rate of CMV was 20.9–35.0%, WMV 0.4–15.8%, CGMMV 1.6–38.5%, and CABYV was 3.5–3.7% from 2020 to 2021. But strangely, there were no incidence of zucchini yellow mosaic virus and cucurbit chlorotic yellows virus (CCYV) during investigation. From this result, we knew the major virus was CGMMV on watermelon in Chungbuk Province. Molecular diagnosis assays of the two melon viruses, showed that melons were infected with CABYV and CCYV from 2020 to 2021. The incidence rate of CABYV was 53.9–92.2% and CCYV was 2.7–20.8%. The incidence of CABYV was high in melon cultivation of Jincheon and Eumseong, Chungbuk. Afterwards, it is necessary to establish a control management strategy for reduce the incidence of CABYV. Furthermore, we must pay attention that of CCYV even if the incidence was low.

Phylogenetic Analysis of Cucurbit Chlorotic Yellows Virus from Melon in 2020 in Chungbuk, Korea

Cucurbit chlorotic yellows virus (CCYV) is a plant virus that causes damage to cucurbit crops such as watermelon and cucumber, and is transmitted by an insect vector known as the whitefly. Since CCYV was first detected on cucumber in Chungbuk in 2018, it has been reported in other areas including Gyeongsang in Korea. In 2020, we performed field surveys of yellowing diseases in the greenhouses growing melon and watermelon in Chungbuk (Jincheon and Eumseong). Reverse transcription-polymerase chain reaction analysis of 79 collected samples including melon, watermelon, and weeds resulted in detection of CCYV in 4 samples: Three samples were singly infected with CCYV and one samples was mixed infected with CCYV, Cucurbit aphid borne yellows virus, and Watermelon mosaic virus. The complete genome sequences of the four collected CCYV melon isolates (ES 1–ES 4) were determined and genetically compared with those of previously reported CCYV isolates retrieved from GenBank. Phylogenetic analyses of RNA 1 and 2 sequences revealed that four ES isolates were clustered in one group and closely related to the CCYV isolates from China. The analysis also revealed very low genetic diversity among the CCYV ES isolates. In general, CCYV isolates showed little genetic diversity, regardless of host or geographic origins. CCYV has the potential to pose a serious threat to melon, watermelon, and cucumber production in Korea. Further studies are needed to examine the pathogenicity and transmissibility of CCYV in weeds and other cucurbits including watermelon.

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".

First report of cucurbit aphid-borne yellows virus infecting muskmelon (Cucumis melo) in India

First report of cucurbit aphid-borne yellows virus infecting muskmelon (Cucumis melo) in India

First Report of Cucurbit Aphid-Borne Yellows Virus Infecting Groundnut in Kenya

Cucurbit aphid-borne yellows virus (CABYV, genus Polerovirus, family Luteoviridae) causing cucurbit aphid-borne yellows disease (CABYD) in groundnut ( Arachis hypogaea) is characterized by interveinal yellowing symptoms in leaves. CABYV is transmitted by Aphis gossypii,  Myzus persicae, and  Macrosiphum euphorbiae. CABYV causes up to 80% yield loss, but there is no information available on its occurrence and serological and molecular characteristics in groundnuts. Two disease diagnostic surveys were conducted during the short and long rains seasons of 2020 and 2021 in Bungoma, Busia, Kakamega, and Siaya counties growing groundnut. Disease incidence and severity were scored, and collected data were analyzed using R software. Serological bioassays were performed using TAS-ELISA, and positive samples were pooled and processed with the transposon-based chemistry library preparation kit (Nextera XT, Illumina). Phylogenetic analyses and comparisons were performed using the MEGA X software. In the short rains season higher incidence (73.61%) was recorded than in the long rains season (42.65%). The CABYV isolates from Kenya clustered with other poleroviruses variedly. CABYV6-2 showed 94.5% nucleotide identity with CpPV2 isolate KX599164.1 from Burkina Faso and 83.4% identity with CABYV isolate MG257902.1 from Korea. This research provides evidence for the first report of CABYV infecting A. hypogaea relevant for development of better plant health management technologies for increased groundnut yield.

First report of Pepo aphid‐borne yellows virus on courgette in Italy

During monitoring for cucurbit viruses in greenhouses in the municipalities of Torre del Greco (Napoli province, Campania) and Aversana (Salerno province, Campania) in September 2019 and October 2022, respectively, samples of commercial hybrids of courgette (Cucurbita pepo) showing symptoms of leaf yellowing and mosaics (Figure 1) were collected and stored as dried material. Three samples from 2019 and two samples from 2022, were inoculated mechanically onto courgette. Within two weeks, typical symptoms of Tomato leaf curl New Delhi virus (ToLCNDV), consisting of leaf crumpling, yellowing and downward curling, were observed. ToLCNDV infection was confirmed by DAS-ELISA (Bioreba, Switzerland). This is not a new record for Campania since ToLCNDV was detected in courgette in 2016 and causes a serious disease problem for cucurbit crops in this region. The samples were also checked for polerovirus infection by RT-PCR as follows. Total RNA was extracted from infected tissues using TRI-reagent (Sigma-Aldrich, USA) and RT-PCR was performed with polerovirus primers Pol-G-F and Pol-G-R (Knierim et al., 2014). A 1.1 Kb fragment was obtained from two samples, one sampled in 2019 and the other in 2022. These amplicons were sent to Genoscreen (Lille, France) for direct sequencing. The sequences obtained (GenBank Accession Nos. OP156930 and OP973153, respectively), encompassing part of the polerovirus RNA-dependent RNA polymerase, intergenic region, coat protein and movement protein, shared a close relationship with Pepo aphid-borne yellows virus (PABYV) isolates from different origins. PABYV was first described in 2008 in Mali (Knierim et al., 2014), then in South Africa (Ibaba et al., 2015), Côte d'Ivoire (Kone et al., 2015), in Tanzania and Mayotte (Desbiez et al., 2016), and a polerovirus closely related to PABYV, named “pumpkin polerovirus” was described more recently in Kenya (Kidanemariam et al., 2019). In the Mediterranean area, PABYV has been detected in Syria in 2009 and in Greece in 2016 (Lotos et al., 2016). The two sequences of the Italian PABYV isolates (IT-236-19 and IT-22, respectively) were 99.2% identical and shared 98.4% identity with the isolate AE142 (LN865082),reported in Greece in 2016 (Lotos et al., 2016), and 98.3% with isolate W-8-1-Sy-09 (KC960436) reported in Syria in 2009. These four isolates formed a distinct group in a phylogenetic reconstruction (Figure 2), suggesting a common origin for the PABYV isolates spreading in the Mediterranean area. PABYV belongs to the polerovirus genus which are phloem-restricted RNA plant viruses transmitted by aphids. Nevertheless, Bemisia tabaci has been reported recently to transmit a new species of polerovirus, Pepper whitefly borne vein yellows virus, in pepper in Israel, as well as a recombinant strain of Cucurbit aphid-borne yellows virus in cucurbits in Brazil. In our case, no aphids were observed during sampling (although previous infestation could not be excluded) but abundant colonies of B. tabaci were recorded. The possibility of PABYV transmission by B. tabaci requires investigation. This is the first report of PABYV in Italy. The finding of PABYV in a new geographic area represents further evidence of the active and current spread of the virus, suggesting the need for more extensive sampling of cucurbit crops and the identification of vectors and alternate hosts to better understand the epidemiology of cucurbit-infecting poleroviruses in the Mediterranean and in Africa. This work was carried out as part of the Short Term Mobility (STM) 2020 programme financed by the National Research Council (CNR) of Italy.

First report of cucurbit aphid-borne yellows virus causing yellow spots in Momordica charantia in Korea

First report of cucurbit aphid-borne yellows virus causing yellow spots in Momordica charantia in Korea

Construction of an infectious full-length cDNA clone of a recombinant isolate of cucurbit aphid-borne yellows virus from Brazil.

In Brazil, the main viral disease of melon plant is severe yellowing disease called "Amarelão do Meloeiro," and a polerovirus, cucurbit aphid-borne yellows virus (CABYV) was considered one of the etiological agents. This virus is a recombinant strain originated from CABYV and unknown polerovirus. Due to unsuccessful mechanical inoculations of CABYV to host plants, the study of its biological characterization is hampered. Therefore, an infectious clone of the recombinant strain of CABYV was constructed using the Gibson Assembly technology. The full-length cDNA clones produced in this study showed to be infectious in three cucurbit species; melon (Cucumis melo), squash (a hybrid of Cucurbita maxima × C. moschata), and West Indian gherkin (Cucumis anguria) plants, but not in watermelon, cucumber, and zucchini plants. This insusceptibility of watermelon plants to the infectious clone corroborates the observation that this virus was never found in watermelon plants often located next to the infected melon plants. This infectious clone provides important tools for future study in developing resistant melon variety to CABYV infection.

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.

Genetic Differentiation and Migration Fluxes of Viruses from Melon Crops and Crop Edge Weeds.

ABSTRACTWeeds surrounding crops may act as alternative hosts, playing important epidemiological roles as virus reservoirs and impacting virus evolution. We used high-throughput sequencing to identify viruses in Spanish melon crops and plants belonging to three pluriannual weed species, Ecballium elaterium, Malva sylvestris, and Solanum nigrum, sampled at the edges of the crops. Melon and E. elaterium, both belonging to the family Cucurbitaceae, shared three virus species, whereas there was no virus species overlap between melon and the other two weeds. The diversity of cucurbit aphid-borne yellows virus (CABYV) and tomato leaf curl New Delhi virus (ToLCNDV), both in melon and E. elaterium, was further studied by amplicon sequencing. Phylogenetic and population genetics analyses showed that the CABYV population was structured by the host, identifying three sites in the CABYV RNA-dependent RNA polymerase under positive selection, perhaps reflecting host adaptation. The ToLCNDV population was much less diverse than the CABYV one, likely as a consequence of the relatively recent introduction of ToLCNDV in Spain. In spite of its low diversity, we identified geographical but no host differentiation for ToLCNDV. Potential virus migration fluxes between E. elaterium and melon plants were also analyzed. For CABYV, no evidence of migration between the populations of the two hosts was found, whereas important fluxes were identified between geographically distant subpopulations for each host. For ToLCNDV, in contrast, evidence of migration from melon to E. elaterium was found, but not the other way around.IMPORTANCE It has been reported that about half of the emerging diseases affecting plants are caused by viruses. Alternative hosts often play critical roles in virus emergence as virus reservoirs, bridging host species that are otherwise unconnected and/or favoring virus diversification. In spite of this, the viromes of potential alternative hosts remain largely unexplored. In the case of crops, pluriannual weeds at the crop edges may play these roles. Here, we took advantage of the power of high-throughput sequencing to characterize the viromes of three weed species frequently found at the edges of melon crops. We identified three viruses shared by melon and the cucurbit weed, with two of them being epidemiologically relevant for melon crops. Further genetic analyses showed that these two viruses had contrasting patterns of diversification and migration, providing an interesting example on the role that weeds may play in the ecology and evolution of viruses affecting crops.