Transcriptomic profiling reveals the complex interaction between a bipartite begomovirus and a cucurbitaceous host plant.

BACKGROUND: Begomoviruses are major constraint in the production of many crops. Upon infection, begomoviruses may substantially modulate plant biological processes. While how monopartite begomoviruses interact with their plant hosts has been investigated extensively, bipartite begomoviruses-plant interactions are understudied. Moreover, as one of the major groups of hosts, cucurbitaceous plants have been seldom examined in the interaction with begomoviruses. RESULTS: We profiled the zucchini transcriptomic changes induced by a bipartite begomovirus squash leaf curl China virus (SLCCNV). We identified 2275 differentially-expressed genes (DEGs), of which 1310 were upregulated and 965 were downregulated. KEGG enrichment analysis of the DEGs revealed that many pathways related to primary and secondary metabolisms were enriched. qRT-PCR verified the transcriptional changes of twelve selected DEGs induced by SLCCNV infection. Close examination revealed that the expression levels of all the DEGs of the pathway Photosynthesis were downregulated upon SLCCNV infection. Most DEGs in the pathway Plant-pathogen interaction were upregulated, including some positive regulators of plant defenses. Moreover, the majority of DEGs in the MAPK signaling pathway-plant were upregulated. CONCLUSION: Our findings indicates that SLCCNV actively interact with its cucurbitaceous plant host by suppressing the conversion of light energy to chemical energy and inducing immune responses. Our study not only provides new insights into the interactions between begomoviruses and host plants, but also adds to our knowledge on virus-plant interactions in general.

Plant Virus Impacts on Yield and Plant-Pollinator Interactions Are Phylogenetically Modulated Independently of Domestication in Cucurbita spp.

Plant defenses are conserved among closely related species, but domestication can alter host genotypes through artificial selection with potential losses in host defenses. Therefore, both domestication and host phylogenetic structure may influence plant virus infection outcomes. Here, we examined the association of phylogeny and domestication with the fitness of infected plants. We inoculated three pairs of domesticated and wild/noncultivated squash (Cucurbita spp.) with a combination of two viruses commonly found to coinfect cucurbits, zucchini yellow mosaic virus and squash mosaic virus, and recorded fitness traits related to flowers, pollination, fruit, and seed viability in the field over 2 separate years. In an additional field experiment, we recorded the relative abundance of both viruses via RT-qPCR. We found a gradient of susceptibility across the six tested lineages, and phylogenetic structure, but not domestication, contributed to differences in infection outcomes and impacts on several fitness traits, including fruit number, fruit weight, and germination. Plant virus infection also impacted the quantity and quality of floral rewards and visitation rates of specialist bee pollinators. There were no detectable differences in viral load between the six host taxa for either virus individually or the ratio of zucchini yellow mosaic virus to squash mosaic virus. Our results highlight the importance of phylogenetic structure in predicting host susceptibility to disease across wild and domesticated plants and the ability of several hosts to maintain fitness in the field despite infection. Broader consequences of plant pathogens for beneficial insects, such as pollinators, should also be considered in future research.

Genome characterization of zucchini yellow mosaic virus infecting cucurbits reveals the presence of a new genotype in Trinidad and Tobago in the Caribbean region.

Zucchini yellow mosaic virus (ZYMV) is a member of the genus Potyvirus that is becoming a serious pathogen of pumpkin and other cucurbits in Trinidad and Tobago and the entire Caribbean region. In this study, four ZYMV isolates infecting pumpkin in Trinidad and Tobago were characterized by complete genome sequencing. Phylogenetic analysis showed 5.9-6.0% nt and 7.7-7.9% aa sequence divergence in comparison to the most closely related isolates NAT and AG from Israel and SE04T from Slovakia. Based on the variations in the complete genome sequence as well as individual gene sequences, a new genotype, designated ZYMV-Trini, is proposed for these isolates. Among the gene sequences of ZYMV-Trini isolates, the greatest variation was observed in the HC-Pro gene, with 20.8% aa sequence divergence from their closest relatives, whereas the least variation was observed in the NIb, P3, and CP genes, with 1.8-2.2% aa sequence divergence. This study also showed that transmission of ZYMV can occur through seeds, but this was less common than transmission via the aphid Aphis gossypii. The progression of ZYMV in pumpkin seedlings was quantified by RT-qPCR, which showed a rapid surge in viral load after 37 days. From recombination analysis, it could be concluded that the isolates SE04T from Slovakia, NAT from Israel, and AG from Israel have made major contributions to the genome architecture of ZYMV-Trini isolates.

Impact of Cultivated Hosts on the Recombination of Cucumber Mosaic Virus.

Cucumber mosaic virus (CMV) is one of the most successful viruses known, infecting over 1,200 species of plants. Like other single-stranded RNA viruses, CMV is known to have a high potential for population diversity due to error-prone replication and short generation times. Recombination is also a mechanism that allows viruses to adapt to new hosts. Host genes have been identified that impact the recombination of RNA viruses by using single-cell yeast systems. To determine the impact that the natural plant host has on virus recombination, we used a high-recombination-frequency strain of CMV, LS-CMV, which belongs to subgroup II, in three different cultivated hosts: Capsicum annuum cv. Marengo (pepper), Nicotiana tabacum cv. Xanthi nc (tobacco), and Cucurbita pepo cv. Black Beauty (zucchini). The recombination frequency was calculated by using an RNA 3 reporter carrying restriction enzyme sites created by introducing silent mutations. Our results show that the recombination frequency of LS-CMV is correlated with the infected host. The recombination events in pepper were 1.8-fold higher than those in tobacco and 5-fold higher than those in zucchini. Furthermore, we observed the generation of defective RNAs in inoculated pepper plants, but not in tobacco or zucchini. These results indicate that the host is involved in both intra- and intermolecular recombination events and that hosts like pepper could foster more rapid evolution of the virus. In addition, we report for the first time the production of defective RNAs in a CMV subgroup II isolate.IMPORTANCE Recombination is an important mechanism used by viruses for their diversification and to adapt to diverse hosts. Understanding the host role in the mechanisms of evolution is important for virus disease management and controlling the emergence of new strains. This study shows the impact that cultivated hosts are playing in the evolution of CMV. Furthermore, our results and previous studies show how some specific hosts could be an ideal environment for the emergence of new viral strains.

A rapid fluorescence-based real-time isothermal assay for the detection of Cucurbit yellow stunting disorder virus in squash and watermelon plants.

Cucurbit yellow stunting disorder virus (CYSDV) is a single-stranded positive-sense RNA virus that produces devastating disease in watermelon and squash. Foliar symptoms of CYSDV consist of interveinal yellowing, brittleness, and thickening of older leaves leading to reduced plant vigor. A rapid diagnostic method for CYSDV would facilitate early detection and implementation of best viral-based management practices. We developed a rapid isothermal reverse transcription-recombination polymerase amplification (exo RT-RPA) assay for the detection of CYSDV. The primers and a 6-fluorescein amidite (6-FAM) probe were developed to target the nucleocapsid gene. The real-time assay detected CYSDV at 2.5 pg purified total RNA extracted from CYSDV-infected leaf tissue and corresponded to 10 copies of the target molecule. The assay was specific and did not cross-react with other common cucurbit viruses found in Florida and Georgia. The performance of the exo RT-RPA was evaluated using crude extract from 21 cucurbit field samples and demonstrated that the exo RT-RPA is a rapid procedure, thus providing a promising novel alternative approach for the detection of CYSDV.

Genetic variability of watermelon mosaic virus isolates infecting cucurbit crops in Italy.

Watermelon mosaic virus (WMV; genus Potyvirus, family Potyviridae) is responsible for serious cucurbit yield losses worldwide. Different WMV genetic groups have been characterized so far. Among these, the "classical" (CL) group has been present in the Mediterranean basin for 40 years, whereas the "emergent" (EM) group includes isolates that are associated with more-severe symptoms observed since 2000. Information on the spatial and temporal evolution of WMV isolates in Italy is currently sparse. In this study, 39 WMV isolates samples collected in different regions over the last two decades were analysed at two different genomic regions that are known to be highly variable and contain recombination breakpoints. Most of the isolates collected between 2002 and 2009 were found to belong to the CL group, whereas the isolates from 2012 onwards were classified as EM, indicating that EM isolates have progressively displaced the CL population in Italy. Although genetic variability was observed within both CL and EM groups and recombinant isolates were detected, no positive selection or haplotype geographic structure were inferred. This suggest that the shift from CL to EM populations was likely due to multiple introductions of EM isolates in different regions of Italy rather than from genetic differentiation of local populations. The progressive increase in prevalence of the highly virulent EM populations is a serious concern because of their symptom severity, and the presence of multiple EM variants that include recombinants necessitates new efforts to develop durable control strategies.

Molecular characterisation of a putative new polerovirus infecting pumpkin (Cucurbita pepo) in Kenya.

Next-generation sequencing of RNA extracted from a pumpkin plant with mosaic symptoms in Kenya identified the presence of a polerovirus sequence closely related to pepo aphid-borne yellows virus (PABYV). The near-complete polerovirus sequence comprised 5,810 nucleotides and contained seven putative open reading frames (ORFs) with a genome organisation typical of poleroviruses. BLASTp analysis of the translated sequences of ORFs 0, 1 and 2 revealed that their amino acid sequences differed by more than 10% from the corresponding protein sequences of other poleroviruses. These results suggest that this virus is a putative novel member of the genus Polerovirus, which has been provisionally named "pumpkin polerovirus" (PuPV).

Detection and genome characterization of a novel member of the genus Polerovirus from zucchini (Cucurbita pepo) in China.

Here, we report a novel member of the genus Polerovirus, zucchini aphid-borne yellows virus (ZABYV), which was identified in zucchini grown for seed production in the Xinjiang Uygur Autonomous Region, China. The complete nucleotide sequence of the ZABYV genome was determined and found to be 5,792 nucleotides in length, and like those of other poleroviruses, to contain seven open reading frames (ORFs). Multiple sequence alignments and phylogenetic analysis indicated that ZABYV is a new member of the genus Polerovirus, although several regions of its genome are closely related to chickpea chlorotic stunt virus (CpCSV). Further comparative analysis suggested that ZABYV originated from a recombination event between CpCSV and another unknown virus in the genus Polerovirus.

Resistance to the Emerging Moroccan Watermelon Mosaic Virus in Squash.

Moroccan watermelon mosaic virus (MWMV) represents an emerging threat to cucurbit production in the Mediterranean Basin. We sequenced the near complete genome of MWMV-SQ10_1.1, a cloned Spanish isolate. MWMV-SQ10_1.1 has the typical potyvirus genomic structure, and phylogenetic analysis showed that it shared a common ancestor with other Mediterranean MWMV isolates. We used MWMV SQ10_1.1 to inoculate plants in a collection of commercial squash cultivars, including some described as potyvirus resistant. All inoculated plants from all cultivars showed severe infection symptoms. Twenty-four Cucurbita spp. accessions were then tested for their susceptibility to MWMV-SQ10_1.1. Plants of the C. ecuadorensis PI 432441 accession showed no symptoms and their enzyme-linked immunosorbent assay readings were similar to uninfected controls. Progeny analysis of F1 and F2 populations suggested that two recessive genes control PI 432441 resistance to MWMV. We hypothesized that this resistance could be associated with alleles of genes encoding the eukaryotic translation initiation factor 4E (eIF4E), particularly after determination of its recessive nature. A multiple sequence alignment including the two eIF4E ortholog sequences from PI 432441 (CeeIF4E1 and CeeIF4E2) identified three amino acid substitutions in CeeIF4E1 and two amino acid substitutions in CeeIF4E2 potentially involved in potyvirus resistance. Polymerase chain reaction markers for CeeIF4E1 and CeeIF4E2 were developed and used to genotype 156 F2 individuals already phenotyped; this analysis did not support an association of either CeeIF4E2 or CeeIF4E1 with MWMV resistance.

Characterization and in silico proteomic analysis of C2 and C3 proteins of squash leaf curl China virus associated with pumpkin leaf curl disease in Assam, India.

The pumpkin leaf curl disease is an emerging disease of pumpkin in Assam, India. Symptomatic pumpkin leaf samples from different locations were immunologically tested using Begomovirus specific antibody. PCR with the ELISA-positive samples, using Geminivirus universal primers amplified 1.4 kb virus-specific fragments. Sequence of these amplicons showed around 95% identity with squash leaf curl China virus-[Pumpkin: Varanasi] (SLCCV-Pumpkin: Varanasi EU573715). To investigate the possible functions of the viral proteins present in the fragment, the full-length C2 and C3 genes were conceptually translated and were subjected to in silico proteomic analyses. The phylogenetic analysis of both the proteins divulged the relationship of our isolate with related viruses and isolates. Multiple sequence alignment (MSA) of the proteins revealed the presence of the known viral conserved motifs, viz., zinc-finger (ZNF) motif [36CXCX(7)CX(6)H53], the arginine-rich nuclear localization signal (NLS) motif (28RRRR31) as well as the minimal activation domain in C2 protein. In the C3 protein, the 91LKYLD95 and the replication enhancer motif (30YFK32) were found to be conserved. Finally, 3-D models of the two proteins were predicted via ab initio approach and subsequently, the models were validated. To our knowledge, this study is a pioneering attempt to construct the ab initio 3-D models of two begomoviral proteins taking a SLCCV isolate as a model. Keywords: begomovirus; ELISA; ZNF motif; NLS motif; ab initio modelling.

The bottle gourd genome provides insights into Cucurbitaceae evolution and facilitates mapping of a Papaya ring-spot virus resistance locus.

Bottle gourd (Lagenaria siceraria) is an important vegetable crop as well as a rootstock for other cucurbit crops. In this study, we report a high-quality 313.4-Mb genome sequence of a bottle gourd inbred line, USVL1VR-Ls, with a scaffold N50 of 8.7 Mb and the longest of 19.0 Mb. About 98.3% of the assembled scaffolds are anchored to the 11 pseudomolecules. Our comparative genomic analysis identifies chromosome-level syntenic relationships between bottle gourd and other cucurbits, as well as lineage-specific gene family expansions in bottle gourd. We reconstructed the genome of the most recent common ancestor of Cucurbitaceae, which revealed that the ancestral Cucurbitaceae karyotypes consisted of 12 protochromosomes with 18 534 protogenes. The 12 protochromosomes are largely retained in the modern melon genome, while have undergone different degrees of shuffling events in other investigated cucurbit genomes. The 11 bottle gourd chromosomes derive from the ancestral Cucurbitaceae karyotypes followed by 19 chromosomal fissions and 20 fusions. The bottle gourd genome sequence has facilitated the mapping of a dominant monogenic locus, Prs, conferring Papaya ring-spot virus (PRSV) resistance in bottle gourd, to a 317.8-kb region on chromosome 1. We have developed a cleaved amplified polymorphic sequence (CAPS) marker tightly linked to the Prs locus and demonstrated its potential application in marker-assisted selection of PRSV resistance in bottle gourd. This study provides insights into the paleohistory of Cucurbitaceae genome evolution, and the high-quality genome sequence of bottle gourd provides a useful resource for plant comparative genomics studies and cucurbit improvement.

Molecular characterization reveals that squash chlorosis mottling virus and zucchini tigré mosaic virus are the same newly emerging potyvirus.

In 2013, we published the first report of a novel potyvirus isolate, which was tentatively named squash chlorosis mottling virus (SqCMV), from an infected squash plant (Cucurbita pepo) collected in the Homestead area of Florida. The purpose of the current work was to further characterize the virus isolate based on molecular properties, and to determine its relationship to other potyviruses. The complete genome sequence of the virus was 10,292 nucleotides (nt), consisting of a 5'-UTR (182 nt), a complete ORF (10,098 nt) encoding a polyprotein of 3,365 amino acids, a motif for the PIPO protein and a 3'-UTR (112 nt), but excluding the poly-(A) tail. Phylogenetic analysis based on the complete genome and amino acid sequences revealed that the virus from Florida clustered with the type isolate of the newly described potyvirus zucchini tigré mosaic virus (ZTMV) sharing 82-90% nucleotide and 83-86% amino acid identities, respectively. Recombination analysis confirmed one major recombination event in the putative P1 coding region of the virus with the putative parental sequences predicted to resemble to ZTMV-Venezuela and France isolates. In addition, genetic diversity analysis indicated that the CP gene was under the highest selection pressure compared to other genes. Together, these results suggest that SqCMV and ZTMV are both representative isolates of the same species, Zucchini tiger mosaic virus. We present the first complete genome sequence of the ZTMV-HFL isolate from the USA.

Identification and characterization of a new potyvirus infecting cucurbits.

A new potyvirus, tentatively named cucurbit vein banding virus (CVBV), was identified in crops of cucurbits in San Pedro (Buenos Aires, Argentina). The complete genome sequences of two isolates of CVBV were obtained by next-generation sequencing (Illumina). The genomic RNA consisted of 9968 and 9813 nucleotides, respectively, and displayed typical potyvirus organization. The percentage identity for these two genome sequences, using BLASTn, was 77% to sweet potato virus c and 73% to tomato necrotic stunt virus. BLASTx analysis of the complete polyprotein showed that the most closely related virus is plum pox virus, with 48% amino acid sequence identity for both isolates. Sequence comparisons and phylogenetic analyses indicate that CVBV belongs to a previously undescribed species in genus Potyvirus.

Absolute Quantification of Tomato leaf curl New Delhi virus Spain strain, ToLCNDV-ES: Virus Accumulation in a Host-Specific Manner.

Tomato leaf curl New Delhi virus (ToLCNDV) (family Geminiviridae, genus Begomovirus) has recently been introduced in western Mediterranean countries. Isolates in Spain constitute a new strain, denominated ToLCNDV-ES, that is causing losses in commercial zucchini and melon crops; however, it is also, although less often, detected in commercial tomato crops. We developed a tissue-print hybridization test to detect the two genomic components of the virus and a TaqMan quantitative polymerase chain reaction (qPCR) test to estimate the number of genome copies in plants. qPCR was approximately 104 to 106 times more sensitive than tissue-print hybridization to detect viral genomic DNA-A and DNA-B, respectively. It also detected the virus in more experimentally and naturally ToLCNDV-ES-infected zucchini squash and tomato plants. ToLCNDV-ES DNA-A titers were significantly lower in tomato than in zucchini plants, often falling below the detection limits in the hybridization test. In addition, the DNA-B accumulation was impaired in tomato when compared with zucchini. According to the data obtained in this study, the differences in viral titers of both plant species contribute to explain the dissimilarities in symptom expression, capability of detection, and transmission of the virus.

Use of Fluorescent Microsphere-Based Assay for Detection of Three Cucurbit-Infecting Viruses.

In this study, we describe multiplex polymerase chain reaction (PCR) coupled with the LiquiChip assay for the identification of Zucchini yellow mosaic virus, Cucumber green mottle mosaic virus, and Cucumber mosaic virus by coamplification with plant mRNA as an internal control. Multiplex reverse-transcription (RT)-PCR products were subjected to allele-specific primer extension, then hybridized to carboxylated microspheres with unique fluorescent identifiers followed by detection using the LiquiChip 200 workstation. This assay is highly specific for distinguishing individual viruses from a mixed viral population and is 10 times more sensitive than multiplex RT-PCR. In addition, the establishment of this method enabled the detection of cucurbit viruses in field samples.

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.

Genetic diversity, distant phylogenetic relationships and the occurrence of recombination events among cucumber mosaic virus isolates from zucchini in Poland.

In recent years, the occurrence of cucumber mosaic virus (CMV) has been noted in zucchini crops in Poland. Beside characteristic isolates, which displayed mosaics and chlorosis on infected plants, new necrotic isolates have also been identified. Here, we analysed the molecular variability of 27 isolates of CMV collected from zucchini in various regions of the country. Sequence and phylogenetic analysis based on the genes encoding the coat (CP) and movement (MP) proteins revealed that the Polish isolates belong to two subgroups: IA and II, with the prevalence of subgroup II. New recombinant variants with an IA-MP/II-CP pattern for RNA3 were also detected.

Incidence and molecular diversity of poleroviruses infecting cucurbit crops and weed plants in Thailand.

Overall, 244 samples of cucurbit crops with yellowing symptoms and selected weed species, from 15 provinces in Thailand, were screened by RT-PCR using primers Polero-CP-F and Polero-CP-R. A total of 160 samples (~66%) were infected by poleroviruses. Analysis of a 1.4 kb region covering the 3' RNA-dependent RNA polymerase (RdRp) gene, the intergenic non-coding region (iNCR), and the coat protein (CP), showed that four poleroviruses, namely, cucurbit aphid-borne yellows virus (CABYV), luffa aphid-borne yellows virus (LABYV), melon aphid-borne yellows virus (MABYV) and suakwa aphid-borne yellows virus (SABYV) were associated with the yellowing symptoms in cucurbit crops. Further analyses indicated presence of putative recombinant viruses referred to as CABYV-R and SABYV-R. CABYV-R was derived from the recombination between MABYV and the common strain of CABYV (CABYV-C). SABYV-R was derived from the recombination of MABYV and SABYV.

Pepo aphid-borne yellows virus: a new species in the genus Polerovirus.

Pepo aphid-borne yellows virus (PABYV) has been proposed as a putative representative of a new species in the genus Polerovirus in the family Luteoviridae. The genomes of two South African (SA) isolates of cucurbit-infecting PABYV were described in this record. Total RNA, extracted from a pattypan (Cucurbita pepo L.) and a baby marrow (C. pepo L.) leaf samples, was subjected to next-generation sequencing (NGS) on the HiSeq Illumina platform. Sanger sequencing was subsequently used to authenticate the integrity of PABYV's genome generated from de novo assembly of the NGS data. PABYV genome of SA isolates consists of 5813 nucleotides and displays an organisation typical of poleroviruses. Genome sequence comparisons of the SA PABYV isolates to other poleroviruses support the classification of PABYV as a new species in the genus Polerovirus. Recombination analyses showed that PABYV and Cucurbit aphid-borne yellows virus (CABYV) shared the same ancestor for the genome part situated between breaking points. Phylogenetic analyses of the RNA-dependent RNA polymerase and the coat protein genes showed that SA PABYV isolates shared distant relationship with CABYV and Suakwa aphid-borne yellows virus. Based on our results, we propose that PABYV is a distinct species in the genus Polerovirus.

The complete genome of the tospovirus Zucchini lethal chlorosis virus.

BACKGROUND: Zucchini lethal chlorosis virus (ZLCV) causes significant losses in the production of cucurbits in Brazil. This virus belongs to the genus Tospovirus (family Bunyaviridae) and seems to be exclusively transmitted by Frankliniella zucchini (Thysanoptera). Tospoviruses have a tripartite and single-stranded RNA genome classified as S (Small), M (Medium) and L (Large) RNAS. Although ZLCV was identified as a member of the genus Tospovirus in 1999, its complete genome had not been sequenced until now. FINDINGS: We sequenced the full-length genome of two ZLCV isolates named ZLCV-SP and ZLCV-DF. The phylogenetic analysis showed that ZLCV-SP and ZLCV-DF clustered with the previously reported isolate ZLCV-BR09. Their proteins were closely related, except the non-structural protein (NSm), which was highly divergent (approximately 90 % identity). All viral proteins clustered similarly in our phylogenetic analysis, excluding that these ZLCV isolates have originated from reassortment events of different tospovirus species. CONCLUSION: Here we report for the first time the complete genome of two ZLCV isolates that were found in the field infecting zucchini and cucumber.