We completed a comprehensive study comparing virus detection between high throughput sequencing (HTS) and standard protocols in 30 berry selections (12 Fragaria, 10 Vaccinium, and eight Rubus) with known virus profiles. The study examined temporal detection of viruses at four sampling times encompassing two growing seasons. Within the standard protocols, reverse transcription (RT) PCR proved better than biological indexing. Detection of known viruses by HTS and RT-PCR nearly mirrored each other. HTS provided superior detection compared with RT-PCR on a wide spectrum of variants and discovery of novel viruses. More importantly, in most cases in which the two protocols showed parallel virus detection, 11 viruses in 16 selections were not consistently detected by both methods at all sampling points. Based on these data, we propose a testing requirement of four sampling times over two growing seasons for berry and potentially other crops, to ensure that no virus remains undetected independent of titer, distribution, or other virus-virus or virus-host interactions.

Blueberry mosaic disease was first reported in 1957 and is now known to occur in most blueberry producing areas around the world. A new virus, closely associated with the disease has been recently identified. The virus, tentatively named Blueberry mosaic associated virus (BlMaV), is a putative member of the genus Ophiovirus with a genome comprised of three negative-sense RNAs (RNAs 1-3). Genome organization and phylogenetic analysis indicates that BlMaV resembles Citrus psorosis virus, the type member of the family Ophioviridae. RNA 1 encodes two proteins, a 23 kDa protein of unknown function and a 272 kDa replicase. RNAs 2 and 3 encode for a putative 58 kDa movement protein and the 50 kDa nucleocapsid protein, respectively. Phylogenetic analyses conducted on 1200 nucleotides of coding regions in RNAs 2 and 3 of 59 North American and European isolates revealed significant amino acid conservation among isolates.

Cuttings of nine elderberry (Sambucus spp.) cultivars were sent from Missouri (USA) to the USDA-ARS laboratory in Corvallis, Oregon (USA) to be tested for the presence of viruses. Double-stranded RNA (dsRNA) was extracted from the nine cultivars and all showed a similar electrophoretic banding pattern, with bands of about 8000 base pairs (bp) in addition to several smaller bands. The dsRNA from 'Bob Gordon' was used as template for next generation sequencing (NGS) and further analysis. DsRNA was converted to cDNA using reverse transcription with degenerate oligonucleotide primers. The cDNA was further amplified by PCR, prepared for NGS using the Illumina format. Sequencing yielded approximately 75 million 80 bp, paired end reads. Reads were assembled into contigs using SCRAPE.pl and two large contigs of 8521 and 8425 nucleotides were obtained. Each contig displayed significant levels of identity to several carlaviruses and about 50% nucleotide sequence identity between each other. From nucleotide sequence and translated amino acid sequence data, it is clear that there are two distinct carlaviruses infecting elderberry. Elderberry carlavirus 145 (EBCV145) showed less homology to known carlaviruses both at the nucleotide and amino acid level. Elderberry carlavirus 153 (EBCV153) had numerous coding regions that were homologous to American hop latent virus. Specific primers were developed for each of the two carlaviruses. All nine cultivars tested were positive for both viruses with the exception of 'Marge', which only had EBCV145. Meristem tips were collected from 'Wyldewood' and used to regenerate plants in tissue culture. Plants were obtained that tested negative for one or both carlaviruses, demonstrating the ability to produce elderberries free of these viruses.

Pea (Pisum sativum L.) has a genome of about 4 Gb that appears to share conserved synteny with model legumes having genomes of 0.2-0.4 Gb despite extensive intergenic expansion. Pea plant inventory (PI) accession 269818 has been used to introgress genetic diversity into the cultivated germplasm pool. The aim here was to develop pea bacterial artificial chromosome (BAC) libraries that would enable the isolation of genes involved in plant disease resistance or control of economically important traits. The BAC libraries encompassed about 3.2 haploid genome equivalents consisting of partially HindIII-digested DNA fragments with a mean size of 105 kb that were inserted in 1 of 2 vectors. The low-copy oriT-based T-DNA vector (pCLD04541) library contained 55 680 clones. The single-copy oriS-based vector (pIndigoBAC-5) library contained 65 280 clones. Colony hybridization of a universal chloroplast probe indicated that about 1% of clones in the libraries were of chloroplast origin. The presence of about 0.1% empty vectors was inferred by white/blue colony plate counts. The usefulness of the libraries was tested by 2 replicated methods. First, high-density filters were probed with low copy number sequences. Second, BAC plate-pool DNA was used successfully to PCR amplify 7 of 9 published pea resistance gene analogs (RGAs) and several other low copy number pea sequences. Individual BAC clones encoding specific sequences were identified. Therefore, the HindIII BAC libraries of pea, based on germplasm accession PI 269818, will be useful for the isolation of genes underlying disease resistance and other economically important traits.

Molecular characterization of eight distinct, difficult-to-clone RNA plant viruses was accomplished after the development of a reverse transcriptase-based first- and second-strand cDNA synthesis method. Double-stranded (ds) RNA templates isolated from strawberry and blackberry and several herbaceous hosts (mint, pea and tobacco) were cloned using this method. Templates, combined with random primers, were denatured with methyl mercuric hydroxide. Reverse transcriptase was added followed by the addition of RNase H. The resulting dsDNA was then digested with restriction endonucleases to produce shorter fragments that could be cloned efficiently into a T-tailed vector after adding an A-overhang using Taq polymerase. This procedure resulted in a high number of cloned fragments and allowed insert sizes up to three kilobase-pairs. Unlike traditional cDNA construction methods, there is no need for additional enzymes/steps for second-strand synthesis, PCR amplification or prior sequence information. Synthesis and cloning of cDNA derived from dsRNA templates is much more efficient than with previously described methods. This procedure also worked well for cloning gel-purified dsRNA and with single-stranded RNA templates.

HomePlant DiseaseVol. 82, No. 2Beet Western Yellows Luteovirus in Western Oregon: Pathosystem Relationships in a Vegetable-Sugar Beet Seed Production Region PreviousNext OPENOpen Access licenseBeet Western Yellows Luteovirus in Western Oregon: Pathosystem Relationships in a Vegetable-Sugar Beet Seed Production RegionR. O. Hampton, K. E. Keller, and J. R. BaggettR. O. HamptonSearch for more papers by this author, K. E. KellerSearch for more papers by this author, and J. R. BaggettSearch for more papers by this authorAffiliationsAuthors and Affiliations R. O. Hampton K. E. Keller , U.S. Department of Agriculture, Agricultural Research Service J. R. Baggett , Oregon State University, Corvallis Published Online:22 Feb 2007https://doi.org/10.1094/PDIS.1998.82.2.140AboutSectionsPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat DetailsFiguresLiterature CitedRelated Vol. 82, No. 2 February 1998SubscribeISSN:0191-2917e-ISSN:1943-7692 Metrics Article History Issue Date: 25 Jan 2008Published: 22 Feb 2007 Pages: 140-148 InformationThe American Phytopathological Society, 1998PDF downloadCited byBeet western yellows virus (turnip (mild) yellows)CABI Compendium, Vol. CABI CompendiumSpinacia oleracea (Spinach)6 June 2020Occurence of beet western yellows virus on squash and broad bean in Turkey9 April 2018 | Journal of Plant Pathology, Vol. 100, No. 2Wild Lactuca species, their genetic diversity, resistance to diseases and pests, and exploitation in lettuce breeding2 August 2013 | European Journal of Plant Pathology, Vol. 138, No. 3Occurrence of Beet western yellows virus and its aphid vectors in over-summering broad-leafed weeds and volunteer crop plants in the grainbelt region of south-western AustraliaAustralian Journal of Agricultural Research, Vol. 57, No. 9Management of Diseases in Seed Crops27 February 2004Biological, Serological, and Molecular Variability Suggest Three Distinct Polerovirus Species Infecting Beet or RapeSébastien Hauser, Mark Stevens, Christophe Mougel, Helen G. Smith, Christiane Fritsch, Etienne Herrbach, and Olivier Lemaire22 February 2007 | Phytopathology®, Vol. 90, No. 5

The mechanism of Pisum sativum pathotype-specific resistance to pea seed-borne mosaic potyvirus (PSbMV) was investigated and the coding region determinant of PSbMV virulence was defined. Homozygous recessive sbm-1 peas are unable to support replication of PSbMV pathotype 1 (P-1), whereas biochemically and serologically related pathotype 4 (P-4) is fully infectious in the sbm-1/sbm-1 genotype. We were unable to detect viral coat protein or RNA with double antibody sandwich-enzyme-linked im-munosorbent assay and reverse transcription-polymerase chain reaction in sbm-1/sbm-1 P-1-inoculated protoplasts and plants. Lack of viral coat protein or RNA in P-1 trans-fected sbm-1/sbm-1 protoplasts suggests that sbm-1 resistance is occurring at the cellular level and that inhibition of cell-to-cell virus movement is not the operating form of resistance. In addition, because virus products were not detected at any time post-inoculation, resistance must either be constitutive or expressed very early in the virus infection process. P-1-resistant peas challenged with full-length, infectious P-1/P-4 recombinant clones demonstrated that a specific P-4 coding region, the 21-kDa, genome-linked protein (VPg), was capable of overcoming sbm-1 resistance, whereas clones containing the P-1 VPg coding region were noninfectious to sbm-1/sbm-1 peas. VPg is believed to be involved in potyvirus replication and its identification as the PSbMV determinant of infectivity in sbm-1/sbm-1 peas is consistent with disruption of an early P-1 replication event.

A tobacco etch virus (TEV)-based expression vector has been used for insertion of several ORFs derived from the unrelated beet yellows virus (BYV). Hybrid TEV variants expressing the BYV capsid protein, 20-kDa protein, or HSP70 homolog systemically infected Nicotiana tabacum and stably retained BYV sequences. In contrast, insertion of the ORF encoding BYV leader proteinase (L-Pro) resulted in severely impaired systemic transport and accumulation of recombinant TEV. Progeny of this virus underwent various deletions affecting the L-Pro sequence and mitigating the defects in virus spread. Model experiments involving several spontaneous and engineered mutants indicated that the central domain of BYV L-Pro was responsible for the defect in hybrid virus accumulation, whereas full-size L-Pro was required for maximal debilitation of systemic transport. Strikingly, BYV L-Pro expression did not debilitate systemic infection of hybrid TEV in Nicotiana benthamiana plants. No major defects in replication or encapsidation of recombinant RNA were revealed in N. tabacum protoplasts. These results indicated that BYV L-Pro specifically interfered with TEV systemic transport and accumulation in a host-dependent manner and suggested a potential utility of closterovirus L-Pro as an inhibitor of potyvirus infection. In addition, it was demonstrated that the 107-amino-acid-residues-long N-terminal part of the TEV helper component proteinase is not essential for systemic infection.