Detection of a New Luteovirus in Imported Nectarine Trees: A Case Study to Propose Adoption of Metagenomics in Post-Entry Quarantine.

In spring 2013, 5-year-old nectarine (Prunus persica) trees, grafted on peach rootstock Nemaguard, were found stunted in a propagation block in California. These trees had been propagated from budwood of three nectarine cultivars imported from France and cleared through the post-entry quarantine procedure. Examination of the canopy failed to reveal any obvious symptoms. However, examination of the trunks, after stripping the bark, revealed extensive pitting on the woody cylinder. To investigate the etiological agent, double-stranded RNA was extracted from bark scrapings from the scion and rootstock portions, and a cDNA library was prepared and sequenced using the Illumina platform. BLAST analysis of the contigs generated by the de novo assembly of sequence reads indicated the presence of a novel luteovirus. Complete sequence of the viral genome was determined by sequencing of three overlapping cDNA clones generated by reverse transcription-polymerase chain reaction (RT-PCR) and by rapid amplification of the 5'- and 3'-termini. The virus genome was comprised of 4,991 nucleotides with a gene organization similar to members of the genus Luteovirus (family Luteoviridae). The presence of the virus, tentatively named Nectarine stem pitting-associated virus, was confirmed in symptomatic trees by RT-PCR. Discovery of a new virus in nectarine trees after post-entry quarantine indicates the importance of including (i) metagenomic analysis by next-generation sequencing approach as an essential tool to assess the plant health status, and (ii) examination of the woody cylinders as part of the indexing process.

Complete genome sequence of a novel vitivirus isolated from grapevine.

A novel virus-like sequence from grapevine was identified by Illumina sequencing. The complete genome is 7,551 nucleotides in length, with polyadenylation at the 3' end. Translation of the sequence revealed five open reading frames (ORFs). The genomic organization was most similar to those of vitiviruses. The polymerase (ORF1) and coat protein (ORF4) genes shared 31 to 49% nucleotide and 40 to 70% amino acid sequence identities, respectively, with other grapevine vitiviruses. The virus was tentatively named grapevine virus F (GVF).

Association of a DNA virus with grapevines affected by red blotch disease in California.

In the Napa Valley of California, vineyards of 'Cabernet Franc' (CF) clone 214, 'Cabernet Sauvignon' clone 337, and 'Zinfandel' clone 1A (Z1A) with grapevines exhibiting foliar symptoms of red blotches, marginal reddening, and red veins that were accompanied by reduced sugar accumulation in fruit at harvest were initially suspected to be infected with leafroll-associated viruses. However, reverse-transcription polymerase chain reaction (PCR) tests were negative for all known leafroll-associated viruses, with the exception of Grapevine leafroll-associated virus 2 in Z1A. Metagenomic analysis of cDNA libraries obtained from double-stranded RNA enriched nucleic acid (NA) preparations from bark scrapings of dormant canes on an Illumina platform revealed sequences having a distant relationship with members of the family Geminiviridae. Sequencing of products obtained by PCR assays using overlapping primers and rolling circle amplification (RCA) confirmed the presence of a single circular genome of 3,206 nucleotides which was nearly identical to the genome of a recently reported Grapevine cabernet franc-associated virus found in declining grapevines in New York. We propose to call this virus "Grapevine red blotch-associated virus" (GRBaV) to describe its association with grapevine red blotch disease. Primers specific to GRBaV amplified a product of expected size (557 bp) from NA preparations obtained from petioles of several diseased source vines. Chip bud inoculations successfully transmitted GRBaV to test plants of CF, as confirmed by PCR analysis. This is the first report of a DNA virus associated with red blotch disease of grapevines in California.

Biological, molecular, and serological studies of a novel strain of grapevine leafroll-associated virus 2.

In California, a novel closterovirus was detected in "Redglobe" grapevine, associated with graft incompatibility and given a trivial name "Grapevine rootstock stem lesion associated virus (GRSLaV)." The biological properties of the putative virus were ascertained when asymptomatic yet infected Redglobe scion buds were graft-inoculated onto test plants of Cabernet Sauvignon propagated on 18 different rootstocks. It proved lethal on test plants growing on rootstocks 1616C, 5BB, 5C, 3309C, and 1103 P, whereas latent infections occurred on the remaining scion-rootstock combinations. In contrast, GLRaV-2 type (type strain) produced only typical leafroll symptoms. In a different experiment, GLRaV-2 type was successfully sap-transmitted to N. benthamiana, whereas sap transmission of GRSLaV was unsuccessful. Double-stranded RNA was extracted from infected Redglobe grapevines, cloned, sequenced, and determined a genome length of 16,527 nucleotides. Computer-assisted analysis of open-reading frames (ORFs) revealed a genome organization typical of monopartite viruses in the genus Closterovirus with nine ORFs (range 71-79% identity) with GLRaV-2 type, the closest similar virus species within the family Closteroviridae. Also the 3'-UTR of GRSLaV consisted of 223 nucleotides with an extended oligo(A) tract similar to that of GLRaV-2 type, Beet yellow stunt virus, and Beet yellows virus. Recombinant GRSLaV coat protein was expressed in E. coli, purified, and immunized a rabbit to produce polyclonal antiserum. Serological data matched the molecular data, whereby exposed plant tissue extracts of grapevines infected by both viruses (GRSLaV and GLRaV-2) reacted positively with homologous and heterologous viral antisera but not with healthy grapevine extracts in ELISA and Western blot tests. Based on the comparative sequence data and shared antigens, GRSLaV is now considered a strain of GLRaV-2 and redesignated as Grapevine leafroll associated virus-2 Redglobe (GLRaV-2RG). Primers specific for GLRaV-2RG were developed, which did not amplify GLRaV-2 type strain. When both sets of specific primers were used in assays of different grapevine collections, the incidence of the respective viruses varied considerably, e.g., 1.7 and 13.5%, respectively, for GLRaV-2RG and GLRaV-2 type.

Pathogen testing and certification of Vitis and Prunus species.

Strategies to screen horticultural crops for graft-transmissible agents, particularly viruses and phytoplasmas, have advanced substantially over the past decade. Tests used for Vitis and Prunus are reviewed in detail, including both biological indexing procedures and laboratory-based assays. Despite advances in laboratory molecular-based detection techniques, a strong case is presented for the continued use of slower biological tests in programs requiring high levels of confidence in detection of pathogens that must be excluded from valuable germplasm.

A Comparison Between Serological and Biological Assays in Detecting Grapevine Leafroll Associated Viruses.

The efficacy of the serological procedure enzyme-linked immunosorbent assay (ELISA) for detecting grapevine leafroll associated viruses (GLRaV types -1, -2, -3, and -4) was compared with indexing on Vitis vinifera L. cv. Cabernet Franc. Results of the biological assays confirmed the infectious nature of all grapevine sources testing positive by ELISA for GLRaV-1 (9 sources), GLRaV-2 (14 sources), and GLRaV-4 (14 sources), and the noninfectious nature of ELISA-negative grapevines (75 sources). However, among 57 sources testing positive by ELISA for GLRaV-3, or 24 sources with multiple infections, 8 and 1 sources, respectively, were negative by Cabernet Franc assays. Serological assays were repeated on all graft-inoculated indicators and only symptomatic ones reacted positively. Also, the 8 original GLRaV-3 sources that had tested positive by ELISA and negative by bioassay were found positive using immuno-capture/reverse transcription-polymerase chain reaction (IC/RT-PCR). The single multiple-infected source was not available for retesting. The distribution of GLRaV in infected grapevines was tested by assaying 20 to 40 samples per source of 36 plants infected with GLRaV-1, -2, -3, or -4. The incidence of GLRaV-positive canes as determined by ELISA ranged from 0 to 100%, suggesting that GLRaV can be unevenly distributed in chronically infected grapevines.