Characterization of alfalfa virus F, a new member of the genus Marafivirus.

Viral infections of alfalfa are widespread in major cultivation areas and their impact on alfalfa production may be underestimated. A new viral species, provisionally named alfalfa virus F (AVF), was identified using a virion-associated nucleic acid (VANA) metagenomics-based approach in alfalfa (Medicago sativa L.) samples collected in Southern France. The nucleotide sequence of the viral genome was determined by de-novo assembly of VANA reads and by 5'/3' RACE with viral RNA extracted from enriched viral particles or with total RNA, respectively. The virus shares the greatest degree of overall sequence identity (~78%) with Medicago sativa marafivirus 1 (MsMV1) recently deduced from alfalfa transcriptomic data. The tentative nucleotide sequence of the AVF coat protein shares ~83% identity with the corresponding region of MsMV1. A sequence search of the predicted single large ORF encoding a polyprotein of 235kDa in the Pfam database resulted in identification of five domains, characteristic of the genus Marafivirus, family Tymoviridae. The AVF genome also contains a conserved "marafibox", a 16-nt consensus sequence present in all known marafiviruses. Phylogenetic analysis of the complete nucleotide sequences of AVF and other viruses of the family Tymoviridae grouped AVF in the same cluster with MsMV1. In addition to 5' and 3' terminal extensions, the identity of the virus was confirmed by RT-PCRs with primers derived from VANA-contigs, transmission electron microscopy with virus-infected tissues and transient expression of the viral coat protein gene using a heterologous virus-based vector. Based on the criteria demarcating species in the genus Marafivirus that include overall sequence identity less than 80% and coat protein identity less than 90%, we propose that AVF represents a distinct viral species in the genus Marafivirus, family Tymoviridae.

Molecular and biological characterization of ϕRs551, a filamentous bacteriophage isolated from a race 3 biovar 2 strain of Ralstonia solanacearum.

A filamentous bacteriophage, designated ϕRs551, was isolated and purified from the quarantine and select agent phytopathogen Ralstonia solanacearum race 3 biovar 2 strain UW551 (phylotype IIB sequevar 1) grown under normal culture conditions. Electron microscopy suggested that ϕRs551 is a member of the family Inoviridae, and is about 1200 nm long and 7 nm wide. ϕRs551 has a genome of 7929 nucleotides containing 14 open reading frames, and is the first isolated virion that contains a resolvase (ORF13) and putative type-2 phage repressor (ORF14). Unlike other R. solanacearum phages isolated from soil, the genome sequence of ϕRs551 is not only 100% identical to its prophage sequence in the deposited genome of R. solanacearum strain UW551 from which the phage was isolated, but is also surprisingly found with 100% identity in the deposited genomes of 10 other phylotype II sequevar 1 strains of R. solanacearum. Furthermore, it is homologous to genome RS-09-161, resulting in the identification of a new prophage, designated RSM10, in a R. solanacearum strain from India. When ORF13 and a core attP site of ϕRs551 were either deleted individually or in combination, phage integration was not observed, suggesting that similar to other filamentous R. solanacearum ϕRSM phages, ϕRs551 relies on its resolvase and the core att sequence for site-directed integration into its susceptible R. solanacearum strain. The integration occurred four hours after phage infection. Infection of a susceptible R. solanacearum strain RUN302 by ϕRs551 resulted in less fluidal colonies and EPS production, and reduced motilities of the bacterium. Interestingly, infection of RUN302 by ϕRs551 also resulted in reduced virulence, rather than enhanced or loss of virulence caused by other ϕRSM phages. Study of bacteriophages of R. solanacearum would contribute to a better understanding of the phage-bacterium-environment interactions in order to develop integrated management strategies to combat R. solanacearum.

First Report and Complete Genome Sequence of Alfalfa Enamovirus from Sudan.

We report the complete genome sequence of a viral pathogen detected in alfalfa samples from Sudan and provisionally named alfalfa enamovirus 2 (AEV-2). Based on high nucleotide and amino acid identities, AEV-2 represents a strain of a newly discovered alfalfa enamovirus 1 that has only been described in Argentina.

Alfalfa virus S, a new species in the family Alphaflexiviridae.

A new species of the family Alphaflexiviridae provisionally named alfalfa virus S (AVS) was discovered in alfalfa samples originating from Sudan. A complete nucleotide sequence of the viral genome consisting of 8,349 nucleotides excluding the 3' poly(A) tail was determined by high throughput sequencing (HTS) on an Illumina platform. NCBI BLAST searches revealed that the virus shares the greatest degree of sequence identity with members of the family Alphaflexiviridae, genus Allexivirus. The AVS genome contains six computationally-predicted open reading frames (ORF) encoding viral replication protein, triple gene block protein 1 (TGB1), TGB2, TGB3-like protein, unknown 38.4 kDa protein resembling serine-rich 40 kDa protein characteristic for allexiviruses, and coat protein (CP). AVS lacks a clear 3' proximal ORF that encodes a nucleic acid-binding protein typical for allexiviruses. The identity of the virus was confirmed by RT-PCR with primers derived from the HTS-generated sequence, dot blot hybridization with DIG-labeled virus-specific RNA probes, and Western blot analysis with antibodies produced against a peptide derived from the CP sequence. Transmission electron microscopic observations of the infected tissues showed the presence of filamentous particles similar to allexiviruses in their length and appearance. To the best of our knowledge, this is the first report on the identification of a putative allexivirus in alfalfa (Medicago sativa). The genome sequence of AVS has been deposited in NCBI GenBank on 03/02/2016 as accession № KY696659.

Higher copy numbers of the potato RB transgene correspond to enhanced transcript and late blight resistance levels.

Late blight of potato ranks among the costliest of crop diseases worldwide. Host resistance offers the best means for controlling late blight, but previously deployed single resistance genes have been short-lived in their effectiveness. The foliar blight resistance gene RB, previously cloned from the wild potato Solanum bulbocastanum, has proven effective in greenhouse tests of transgenic cultivated potato. In this study, we examined the effects of the RB transgene on foliar late blight resistance in transgenic cultivated potato under field production conditions. In a two-year replicated trial, the RB transgene, under the control of its endogenous promoter, provided effective disease resistance in various genetic backgrounds, including commercially prominent potato cultivars, without fungicides. RB copy numbers and transcript levels were estimated with transgene-specific assays. Disease resistance was enhanced as copy numbers and transcript levels increased. The RB gene, like many other disease resistance genes, is constitutively transcribed at low levels. Transgenic potato lines with an estimated 15 copies of the RB transgene maintain high RB transcript levels and were ranked among the most resistant of 57 lines tested. We conclude that even in these ultra-high copy number lines, innate RNA silencing mechanisms have not been fully activated. Our findings suggest resistance-gene transcript levels may have to surpass a threshold before triggering RNA silencing. Strategies for the deployment of RB are discussed in light of the current research.

Changes in disease resistance phenotypes associated with plant physiological age are not caused by variation in R gene transcript abundance.

Foliar late blight is one of the most important diseases of potato. Foliar blight resistance has been shown to change as a plant ages. In other pathosystems, resistance (R) gene transcript levels appear to be correlated to disease resistance. The cloning of the broad-spectrum, foliar blight resistance gene RB provided the opportunity to explore how foliar blight resistance and R-gene transcript levels vary with plant age. Plants of Solanum bulbocastanum PT29, from which RB, including the native promoter and other flanking regions, was cloned, and S. tuberosum cv. Dark Red Norland (nontransformed and RB-transformed) representing three different developmental stages were screened for resistance to late blight and RB transcript levels. Preflowering plants of all genotypes exhibited the highest levels of resistance, followed by postflowering and near-senescing plants. The RB transgene significantly affected resistance, enhancing resistance levels of all RB-containing lines, especially in younger plants. RB transgene transcripts were detected at all plant ages, despite weak correlation with disease resistance. Consistent transcript levels in plants of different physiological ages with variable levels of disease resistance demonstrate that changes in disease-resistance phenotypes associated with plant age cannot be attributed to changes in R-gene transcript abundance.

Identification and Characterization of a Carlavirus Causing Veinal Necrosis of Coleus.

A filamentous virus identified in coleus (Coleus × hybrida) in Minnesota and New York was found to cause veinal necrosis in coleus, although this symptom was observed only under certain conditions. The virus was transmitted readily by mechanical inoculation to coleus and Nicotiana spp. and was not transmitted by Myzus persicae. The particles of the coleus virus had a modal length of 640 nm and a single capsid protein with an estimated molecular mass of 34 kDa. The amino acid sequence of the coat protein region of the coleus virus genome had significant similarities only to the corresponding domain of carlaviruses. Based on virion morphology, capsid protein size, genome size and organization, amino acid sequence, and phylogenetic analyses, the coleus virus, which was named provisionally Coleus vein necrosis virus (CVNV), was concluded to be a new definitive member of the genus Carlavirus. A 2-kb fragment of the 3' terminus of the CVNV genome sequence is accessible under accession number DQ915963 in GenBank.