Meta-transcriptomic identification of groundnut RNA viruses in western Kenya and the novel detection of groundnut as a host for Cauliflower mosaic virus.

BACKGROUND: Groundnut (Arachis hypogaea L.) is the 13th most important global crop grown throughout the tropical and subtropical regions of the world. One of the major constraints to groundnut production is viruses, which are also the most economically important and most abundant pathogens among cultivated legumes. Only a few studies have reported the characterization of RNA viruses in cultivated groundnuts in western Kenya, most of which deployed classical methods of detecting known viruses. METHODS: We sampled twenty-one symptomatic and three asymptomatic groundnut leaf samples from farmers' fields in western Kenya. Total RNA was extracted from the samples followed by First-strand cDNA synthesis and sequencing on the Illumina HiSeq 2500 platform. After removing host and rRNA sequences, high-quality viral RNA sequences were de novo assembled and viral genomes annotated using the publicly available NCBI virus database. Multiple sequence alignment and phylogenetic analysis were done using MEGA X. RESULTS: Bioinformatics analyses using as low as ∼3.5 million reads yielded complete and partial genomes for Cauliflower mosaic virus (CaMV), Cowpea polerovirus 2 (CPPV2), Groundnut rosette assistor virus (GRAV), Groundnut rosette virus (GRV), Groundnut rosette virus satellite RNA (satRNA) and Peanut mottle virus (PeMoV) falling within the species demarcation criteria. This is the first report of CaMV and the second report of CPPV2 on groundnut hosts in the world. Confirmation of the detected viruses was further verified through phylogenetic analyses alongside reported publicly available highly similar viruses. PeMoV was the only seed-borne virus reported. CONCLUSION: Our findings demonstrate the power of Next Generation Sequencing in the discovery and identification of novel viruses in groundnuts. The detection of the new viruses indicates the complexity of virus diseases in groundnuts and would require more focus in future studies to establish the effect of the viruses as sole or mixed infections on the crop. The detection of PeMoV with potential origin from Malawi indicates the importance of seed certification and cross-boundary seed health testing.

Metatranscriptome analysis of symptomatic bitter apple plants revealed mixed viral infections with a putative novel polerovirus.

BACKGROUND: Next-generation Sequencing (NGS) combined with bioinformatic analyses constitutes a powerful approach for identifying and characterizing previously unknown viral genomes. In this study, leaf samples from bitter apple plants (Citrullus colocynthis (L.) Schrad) exhibiting symptoms such as dwarfing, leaf crinkling, and chlorosis were collected from the southern part of Kerman province, Iran. RESULTS: Putative infecting viruses were identified through de novo assembly of sequencing reads using various tools, followed by BLAST analysis. Complete genomes for Squash vein yellowing virus (SqVYV), Citrus-associated rhabdovirus (CiaRV), and a novel polerovirus-related strain termed Bitter apple aphid-borne yellows virus (BaABYV) were assembled and characterized. Additionally, a partial genome for Watermelon mosaic virus (WMV) was assembled. The genomic organization of the BaABYV was determined to be 5'-ORF0-ORF1-ORF1,2-ORF3a-ORF3-ORF3,5-ORF4-3'. Amino acid sequence identities for inferred proteins (P0 and P1, P1,2) with known poleroviruses were found to be the 90% species delineation limit, implying that BaABYV should be considered a new member of the genus Polerovirus. Recombination events were observed in the BaABYV and WMV strains; such events were not found in the CiaRV strain. CONCLUSIONS: Molecular evidence from this study suggests that C. colocynthis is a reservoir host of several plant viruses. Among them, BaABYV is proposed as a new member of the genus Polerovirus. Furthermore, the CiaRV strain has been reported for the first time from Iran.

Genomic variation in pepper vein yellows viruses in Australia, including a new putative variant, PeVYV-10.

Since the first identification and full sequence of the polerovirus pepper vein yellows virus in Australia in 2016, virus surveys of crops and weeds have sporadically identified PeVYV in different hosts and locations. Genomic comparisons of 14 PeVYV-like isolates using RT-PCR products spanning the 3' end of the RdRp region (ORF 2), the intergenic region, ORF 3a, ORF 4, and ORF 3 (1388 nt) showed that four of the PeVYV isolates might be a new variant or PeVYV-like virus. From six PeVYV-positive plants, eight PeVYV-like sequences were obtained by high-throughput sequencing, as two hosts, 5352 and 5634, contained two slightly different PeVYV-like isolates. Three of the PeVYV-like isolates were most closely related to PeVYV-6 and PeVYV-5, and two isolates were closely related to PeVYV-9 and PeVYV-2. The other three isolates shared only 69-74% nucleotide sequence identity across the whole genome with any of the other PeVYVs, despite sharing 73-98%, 87-91%, and 84-87% amino acid sequence identity in ORF 3a, ORF 3, and the RdRp (ORF 2), respectively, suggesting that this virus is a new PeVYV-like virus, which we have tentatively called PeVYV-10. This is also the first report of a PeVYV-like virus infecting garlic.

Complete nucleotide sequence of chrysanthemum virus D, a polero-like virus.

A putative new polerovirus, named "chrysanthemum virus D" (ChVD), was detected in a Chrysanthemum morifolium plant in South Korea. The virus was identified by high-throughput sequencing and confirmed by reverse transcription polymerase chain reaction. The entire ChVD genome is composed of 5,963 nucleotides and contains seven open reading frames (ORF0-5 and ORF3a), which are arranged similarly to those of other poleroviruses. These ORFs encode the putative proteins P0-5 and P3a, respectively. Pairwise amino acid sequence comparisons showed that the ChVD P0-5 and P3a proteins have 30.45-75% sequence identity to the corresponding proteins of other members of the genus Polerovirus. Since one of the species demarcation criteria for the genus Polerovirus is > 10% difference in the amino acid sequence of any gene product, the sequence comparisons indicate that ChVD represents a new species in this genus. Phylogenetic analysis of the P1-P2 and P3 amino acid sequences further indicate that ChVD is a novel polerovirus.

Molecular Characterization of a Novel Enamovirus Infecting Raspberry.

Raspberry plants, valued for their fruits, are vulnerable to a range of viruses that adversely affect their yield and quality. Utilizing high-throughput sequencing (HTS), we identified a novel virus, tentatively named raspberry enamovirus 1 (RaEV1), in three distinct raspberry plants. This study provides a comprehensive characterization of RaEV1, focusing on its genomic structure, phylogeny, and possible transmission routes. Analysis of nearly complete genomes from 14 RaEV1 isolates highlighted regions of variance, particularly marked by indel events. The evidence from phylogenetic and sequence analyses supports the classification of RaEV1 as a distinct species within the Enamovirus genus. Among the 289 plant and 168 invertebrate samples analyzed, RaEV1 was detected in 10.4% and 0.4%, respectively. Most detections occurred in plants that were also infected with other common raspberry viruses. The virus was present in both commercial and wild raspberries, indicating the potential of wild plants to act as viral reservoirs. Experiments involving aphids as potential vectors demonstrated their ability to acquire RaEV1 but not to successfully transmit it to plants.

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.

Differential gene expression between viruliferous and non-viruliferous Schizaphis graminum (Rondani).

An experiment was performed to measure the effect of Cereal Yellow-Dwarf Virus (CYDV), strain CYDV-RPV, on gene expression in its insect vector, greenbug aphid (Schizaphis graminum (Rondani)). RNA was sampled in three replicates from four treatments (biotypes B and H with or without carried CYDV), at 0, 1, 2, 3, 5, 10, 15 and 20 days from the introduction of carrier and virus-free greenbugs to uninfected wheat cv. 'Newton'. Illumina paired-end sequencing produced 1,840,820,000,000 raw reads that yielded 1,089,950,000 clean reads, which were aligned to two greenbug, Trinity transcriptome assemblies with bowtie2. Read counts to contigs were analyzed with principal components and with DESeq2 after removing contaminating contigs of wheat or microbial origin. Likelihood ratio tests with one transcriptome showed that CYDV influenced gene expression about seven-fold less than time or biotype, which were approximately equal. With the other transcriptome, virus, time, and biotype were about equally important. Pairwise comparisons of virus to no virus for each timepoint yielded estimates of fold-change that comprised expression profiles for each contig when ordered by timepoint. Hierarchical clustering separated expression profiles into 20 groups of contigs that were significantly differentially expressed for at least one timepoint. Contigs were also sorted by timepoint of maximally differential expression between virus and no virus. All contigs that were significantly differentially expressed at FDR = 0.05 were annotated by blast searches against NCBI nr and nt databases. Interesting examples of up-regulation with virus included a lysosomal-trafficking regulator, peptidylprolylisomerase, RNA helicase, and two secreted effector proteins. However, carried virus did not consistently change aphid gene expression overall. Instead there was complex interaction of time, biotype, host response, and virus.

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.

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.

Screening and Identification of Host Factors Interacting with the Virulence Factor P0 Encoded by Sugarcane Yellow Leaf Virus by Yeast Two-Hybrid Assay.

Sugarcane yellow leaf virus (SCYLV), a member of the genus Polerovirus in the family Luteoviridae, causes severe damage and represents a great threat to sugarcane cultivation and sugar industry development. In this study, inoculation of Nicotiana benthamiana plants with a potato virus X (PVX)-based vector carrying the SCYLV P0 gene induced typical mosaic, leaf rolling symptoms and was associated with a hypersensitive-like response (HLR) necrosis symptom, which is accompanied with a systemic burst of H2O2 and also leads to higher PVX viral genome accumulation levels. Our results demonstrate that SCYLV P0 is a pathogenicity determinant and plays important roles in disease development. To further explore its function in pathogenic processes, a yeast two-hybrid assay was performed to screen the putative P0-interacting host factors. The recombinant plasmid pGBKT7-P0 was constructed as a bait and transformed into the yeast strain Y2HGold. The ROC22 cultivar (an important parental resource of the main cultivar in China) cDNA prey library was constructed and screened by co-transformation with the P0 bait. We identified 28 potential interacting partners including those involved in the optical signal path, plant growth and development, transcriptional regulation, host defense response, and viral replication. To our knowledge, this is the first time we have reported the host proteins interacting with the P0 virulence factor encoded by sugarcane yellow leaf virus. This study not only provides valuable insights into elucidating the molecular mechanism of the pathogenicity of SCYLV, but also sheds light on revealing the probable new pathogenesis of Polerovirus in the future.

Genomic characterization of two new viruses infecting Ageratum conyzoides in China.

Two new RNA viruses were identified in Ageratum conyzoides in China using high-throughput sequencing, and their genome sequences were determined using PCR and rapid amplification of cDNA ends. The new viruses, which have positive-sense, single-stranded RNA genomes, were provisionally named "ageratum virus 1" (AgV1) and "ageratum virus 2" (AgV2). AgV1 has a genome of 3,526 nucleotides with three open reading frames (ORFs) and shares 49.9% nucleotide sequence identity with the complete genome of Ethiopian tobacco bushy top virus (genus Umbravirus, family Tombusviridae). The genome of AgV2 consists of 5,523 nucleotides and contains five ORFs that are commonly observed in members of the genus Enamovirus of the family Solemoviridae. Proteins encoded by AgV2 exhibited the highest amino acid sequence similarity (31.7-75.0% identity) to the corresponding proteins of pepper enamovirus R1 (an unclassified enamovirus) and citrus vein enation virus (genus Enamovirus). Based on their genome organization, sequence, and phylogenetic relationships, AgV1 is proposed to be a new umbra-like virus of the family Tombusviridae, and AgV2 is proposed to be a new member of the genus Enamovirus of the family Solemoviridae.

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, cucumber mosaic virus, Moroccan watermelon mosaic virus, and papaya ring spot virus, 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 from CABYV and WMV isolates by using the Pacific Biosciences single-molecule real-time (PacBio) high-throughput technology to assess the genetic variation and structure of their populations. Our results showed that the CABYV population displayed seven codons under positive selection, and although most isolates clustered in the Mediterranean clade, a subsequent analysis of molecular variance revealed a significant, 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 and under purifying selection. These results underlie the epidemiological relevance of mixed infections for CABYV and provide a link between genetic diversity and CABYV dynamics at the whole-genome level.

Complete genome sequence of triticum yellow stripe virus, a new polerovirus infecting wheat (triticum aestivum) in China.

Wheat plants with yellow stripes on their leaves were collected in the city of Tai'an (Shandong province, China). High-throughput sequencing analysis of the collected plants showed that they were coinfected with wheat leaf yellowing-associated virus (WLYaV) and an unidentified polerovirus. The genome of the unidentified virus, tentatively named "triticum yellow stripe virus" (TriYSV), comprises 5,595 nucleotides and contains seven open reading frames (ORFs), with a typical polerovirus genome structure. Analysis by sequence alignment showed that TriYSV had the highest sequence similarity to wheat yellow dwarf virus (WYDV, a tentative member of the genus Polerovirus), with 87.3% nucleotide sequence identity over the whole genome. Except for P3a and the coat protein (CP), all of the proteins encoded by TriYSV showed < 90% amino acid identity to those of other poleroviruses. Phylogenetic analysis based on RNA-dependent RNA polymerase and CP amino acid sequences and complete genome nucleotide sequences showed that the poleroviruses WYDV, cereal yellow dwarf virus RPS (CYDV-RPS), CYDV-RPV, and barley yellow dwarf virus GPV are the most closely related to TriYSV. Thus, TriYSV is proposed to be a new member of the genus Polerovirus.

Naturally occurring substitution of an amino acid in a plant virus gene-silencing suppressor enhances viral adaptation to increasing thermal stress.

Cereal yellow dwarf virus (CYDV-RPV) encodes a P0 protein that functions as a viral suppressor of RNA silencing (VSR). The strength of silencing suppression is highly variable among CYDV-RPV isolates. In this study, comparison of the P0 sequences of CYDV-RPV isolates and mutational analysis identified a single C-terminal amino acid that influenced P0 RNA-silencing suppressor activity. A serine at position 247 was associated with strong suppressor activity, whereas a proline at position 247 was associated with weak suppressor activity. Amino acid changes at position 247 did not affect the interaction of P0 with SKP1 proteins from Hordeum vulgare (barley) or Nicotiana benthamiana. Subsequent studies found P0 proteins containing a P247 residue were less stable than the P0 proteins containing an S247 residue. Higher temperatures contributed to the lower stability and in planta and the P247 P0 proteins were subject to degradation via the autophagy-mediated pathway. A P247S amino acid residue substitution in P0 increased CYDV-RPV replication after expression in agroinfiltrated plant leaves and increased viral pathogenicity of P0 generated from the heterologous Potato virus X expression vector system. Moreover, an S247 CYDV-RPV could outcompete the P247 CYDV-RPV in a mixed infection in natural host at higher temperature. These traits contributed to increased transmission by aphid vectors and could play a significant role in virus competition in warming climates. Our findings underscore the capacity of a plant RNA virus to adapt to climate warming through minor genetic changes in gene-silencing suppressor, resulting in the potential for disease persistence and prevalence.

Cotton leafroll dwarf disease: An enigmatic viral disease in cotton.

TAXONOMY: Cotton leafroll dwarf virus (CLRDV) is a member of the genus Polerovirus, family Solemoviridae. Geographical Distribution: CLRDV is present in most cotton-producing regions worldwide, prominently in North and South America. PHYSICAL PROPERTIES: The virion is a nonenveloped icosahedron with T = 3 icosahedral lattice symmetry that has a diameter of 26-34 nm and comprises 180 molecules of the capsid protein. The CsCl buoyant density of the virion is 1.39-1.42 g/cm3 and S20w is 115-127S. Genome: CLRDV shares genomic features with other poleroviruses; its genome consists of monopartite, single-stranded, positive-sense RNA, is approximately 5.7-5.8 kb in length, and is composed of seven open reading frames (ORFs) with an intergenic region between ORF2 and ORF3a. TRANSMISSION: CLRDV is transmitted efficiently by the cotton aphid (Aphis gossypii Glover) in a circulative and nonpropagative manner. Host: CLRDV has a limited host range. Cotton is the primary host, and it has also been detected in different weeds in and around commercial cotton fields in Georgia, USA. SYMPTOMS: Cotton plants infected early in the growth stage exhibit reddening or bronzing of foliage, maroon stems and petioles, and drooping. Plants infected in later growth stages exhibit intense green foliage with leaf rugosity, moderate to severe stunting, shortened internodes, and increased boll shedding/abortion, resulting in poor boll retention. These symptoms are variable and are probably influenced by the time of infection, plant growth stage, varieties, soil health, and geographical location. CLRDV is also often detected in symptomless plants. CONTROL: Vector management with the application of chemical insecticides is ineffective. Some host plant varieties grown in South America are resistant, but all varieties grown in the United States are susceptible. Integrated disease management strategies, including weed management and removal of volunteer stalks, could reduce the abundance of virus inoculum in the field.

High-throughput RNA sequencing analysis of Mallotus japonicus revealed novel polerovirus and amalgavirus.

High-throughput RNA sequencing (RNA-seq) analysis of samples from Mallotus japonicus, a traditional medicinal plant, yielded two novel RNA viruses tentatively named Mallotus japonicus virus A (MjVA) and Mallotus japonicus virus B (MjVB). The MjVA and MjVB genomes encode proteins showing amino acid sequence similarities to those of poleroviruses (the genus Polerovirus, the family Solemoviridae) and amalgaviruses (the genus Amalgavirus, the family Amalgaviridae), respectively. The MjVA genome contains seven highly overlapping open reading frames, which are translated to seven proteins through various translational mechanisms, including -1 programmed ribosomal frameshifting (PRF) at the slippery motif GGGAAAC, non-AUG translational initiation, and stop codon readthrough. The MjVB genome encodes two proteins; one of which is translated by +1 PRF mechanism at the slippery motif UUUCGN. The abundance analysis of virus-derived RNA fragments revealed that MjVA is highly concentrated in plant parts with well-developed phloem tissues as previously demonstrated in other poleroviruses, which are transmitted by phloem feeders, such as aphids. MjVB, an amalgavirus generally transmitted by seeds, is distributed in all samples at low concentrations. Thus, this study demonstrates the effectiveness and usefulness of RNA-seq analysis of plant samples for the identification of novel RNA viruses and analysis of their tissue distribution. Keywords: Polerovirus; Amalgavirus; Mallotus japonicus; RNA virus; viral genome; programmed ribosomal frameshifting.

Complete genome sequence of a novel member of the genus Polerovirus from Cnidium officinale in South Korea.

The complete genome sequence of a novel virus found infecting Cnidium officinale, which we have named "cnidium polerovirus 1" (CnPV1), is 6,090 nucleotides in length, similar to those of other poleroviruses. Seven open reading frames (ORF0-5 and ORF3a) were predicted in this genome. CnPV1 shares 32.4%-38.9% full-length nucleotide sequence identity with other known polerovirus genome sequences. The putative P0, P1-2, P3-5, P3, and P4 proteins share 11.3%-19.5%, 37.1%-49.8%, 26.7%-39.5%, 40.8%-49.7%, and 40.8%-49.7% amino acid sequence identity, respectively, with homologous inferred protein sequences from known poleroviruses. Phylogenetic analysis of P1-2 and P3 sequences places CnPV1 with other members of the genus Polerovirus, indicating that it should be classified in a new distinct species.

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".

Analysis of Cotton Leafroll Dwarf Virus P0 Gene Sequences from South Carolina Reveals Low Variability Among Isolates.

Cotton leafroll dwarf virus (CLRDV) is emerging across the major cotton-producing states of the southern United States. Because it was detected in nearly all cotton-producing states within a few years of its initial detection in the United States, the spread of the virus has apparently occurred rapidly. In this study spanning three growing seasons in South Carolina, we collected CLRDV isolates from symptomatic and asymptomatic cotton plants in 10 counties. The genomic region encoding P0, the viral suppressor of RNA silencing, was sequenced and compared among CLRDV isolates. Low variability among CLRDV P0 sequences from South Carolina isolates with similarities to other United States isolates was revealed by amino acid sequence alignment and phylogenetic analysis. Low variability among South Carolina isolates was also confirmed by sequencing a subset of eight near-complete genomes of CLRDV isolates. Although sequence variability was low among South Carolina isolates, this data should be taken in the context of all United States isolates, for which diversity may be higher than initially expected. Sequences gathered in this study add to the body of knowledge on CLRDV diversity in the United States.

A new simple and effective method for PLRV infection to screen for virus resistance in potato.

Effective screening of plant germplasm collections for resistance to plant viruses requires that there is a rapid and efficient system in place to challenge individual plants with the virus. Potato leafroll virus (PLRV), a commercially important pathogen of potato, is able naturally to infect only the phloem-associated tissue of plants and is delivered to this tissue by feeding aphids. Mechanical (non-vector-mediated) infection by PLRV does not occur thus screening for PLRV resistance is currently laborious and time consuming. We constructed an infectious cDNA clone of a new (Hutton) isolate of PLRV in the binary vector pDIVA and transformed it into Agrobacterium tumefaciens strain LBA4404. Infiltration of this culture into leaves of Nicotiana benthamiana, a highly susceptible model plant, produced a systemic infection with PLRV, although this approach was not successful for potato. However, a very efficient and reproducible systemic infection of potato was achieved when we submerged cut stems of the plant into the agrobacterium cell suspension and then transplanted the stems into compost to grow roots and new apical leaves. Using a standardised protocol developed for this new PLRV inoculation method we have confirmed the previously described resistance to the virus in the JHI breeding line G8107(1) and identified 62 plant accessions from the Commonwealth Potato Collection in which no PLRV infection was detected.