Specific detection of Malus- and Pyrus-infecting viroids by real-time reverse-transcription quantitative PCR assays.

Viroids present a number of issues for their detection and diagnosis because of the absence of symptom expression in many hosts and their low titers in infected plants. However, quarantine programs rely on symptom observations and routine diagnostic testing to reduce the risk of spreading viroid-infected materials to situations where they might affect crop health and production. Sensitive, accurate, and specific assays for viroid detection from both asymptomatic and symptomatic hosts are necessary for managing viroids in post-entry quarantine and certification schemes. The aim of this study was to develop and optimize superior assays based on the reverse-transcription quantitative polymerase chain reaction (RT-qPCR) for the specific detection of apple hammerhead viroid (AHVd), apple scar skin viroid (ASSVd) and pear blister canker viroid (PBCVd). The real-time RT-qPCR assays thus developed detected a greater range of viroid isolates and with greater sensitivity than the current endpoint RT-PCR assays, down to 101 copies per reaction without any amplification of the non-target viroid or virus sequences tested.

Development of RT-qPCR assays for the detection and quantification of three carlaviruses infecting hop.

American hop latent virus (AHLV), hop latent virus (HLV) and hop mosaic virus (HMV) infect members of the Humulus genus worldwide, but very little is known of the biology and etiology of these viruses. A better understanding of these viruses from the molecular level to their economic impact relies on efficient diagnostic assays. Therefore, in this study we developed reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays for the detection of AHLV, HLV, and HMV through an alignment of representative sequences from the National Center for Biotechnology Information (NCBI) database. These assays demonstrated unambiguously their high sensitivity by detecting the respective targets from as low as 102 copies of transcripts per reaction without any amplification from non-targets.

Development of RT-qPCR assays for the detection of three latent viruses of pome.

Latent fruit tree viruses present economic threat to the industry and nurseries as diseases they cause not only reduce fruit quality and production yield, but can also be spread inadvertently through propagation due to the lack of viral symptoms on an infected mother plant. As a result, these viruses require appropriate detection tools for effective management. In this study we developed RT-qPCR assays for the detection of three latent viruses of pome, apple chlorotic leaf spot virus (ACLSV), apple stem pitting virus (ASPV), and apple mosaic virus (ApMV), using the alignment of representative sequences from the NCBI database. The optimized assays were shown to be specific by successfully amplifying the target from positive controls without showing any detectable amplification in negative and non-target controls, and revealed high sensitivity by reliably detecting as low as 101 copies per reaction. The results also demonstrated that both the choice of extraction method and the reagents used for RT-qPCRcould play a critical role in virus detection outcome. These assays were both reliable and robust compared to the extant RT-PCR methods, and they could be a viable tool for making informed management decisions.

Detection and quantification of four viruses in Prunus pollen: Implications for biosecurity.

Pollen transmitted viruses require accurate detection and identification to minimize the risk of spread through the global import and export of pollen. Therefore in this study we developed RT-qPCR assays for the detection of Cherry leaf roll virus (CLRV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV), and Cherry virus A (CVA), four viruses that infect pollen of Prunus species. Assays were designed against alignments of extant sequences, optimized, and specificity was tested against known positive, negative, and non-target controls. An examination of assay sensitivity showed that detection of virus at concentrations as low as 101 copies was possible, although 102 copies was more consistent. Furthermore, comparison against extant assays showed that in both pollen and plant samples, the newly developed RT-qPCR assays were more sensitive and could detect a greater range of isolates than extant endpoint RT-PCR and ELISA assays. Use of updated assays will improve biosecurity protocols as well as the study of viruses infecting pollen.

Development of real-time RT-PCR assays for two viruses infecting pome fruit.

Apple stem grooving virus (ASGV) and Apple green crinkle-associated virus (AGCaV) negatively impact production, maintenance, and distribution of apples and other Malus species world-wide. Due to the increasing diversity of isolates found by high-throughput sequencing, we have developed real-time RT-qPCR assays for these two viruses. Primers and probes were designed against alignments of representative extant sequences from around the world, and reaction conditions optimized for sensitivity and specificity. Assays were validated against a panel of virus isolates, and compared to extant endpoint RT-PCR and ELISA assays. The new real-time RT-qPCR assays showed greater detection sensitivity than extant assays and were able to detect their target viruses from different host tissues.

Diversity of three bunya-like viruses infecting apple.

High-throughput sequencing of two trees with apple decline revealed the presence of three bunya-like viruses: apple rubbery wood-associated viruses 1 and 2 (ARWaV-1, ARWaV-2) and citrus concave gum-associated virus (CCGaV), which previously had only been observed in citrus trees. The apple and citrus CCGaV isolates shared over 97% sequence identity. A global collection of apple trees was screened by RT-PCR for these viruses. Twenty-seven of 30 trees were infected with one or more bunya-like virus. Sequence data revealed some diversity among isolates but no geographic grouping. Additional work will be needed to determine if any of these viruses contribute to apple decline.

First Report of Grapevine fleck virus in Idaho Grapevines.

Idaho has a growing viticulture industry, with nearly 1,600 acres of wine grapes (Vitis vinifera L.). Production is largely concentrated in two locations, the Snake River valley, which includes Canyon County in the southwest, and the Clearwater River valley, primarily Nez Perce County in the northwest. Grapevine fleck virus (GFkV) belongs to the genus Maculavirus, family Tymoviridae, comprising positive-sense, single-stranded RNA viruses with ca. 7.6-kb genome (3). It is one of five non-mechanically transmitted viruses associated with the fleck disease complex and has been previously documented to occur in the neighboring state of Washington (2). Main sources of wine grape nursery material imported to Idaho reside in Washington or in California, and it is important to monitor virus status of the planting material brought to the state. However, no information was available on the occurrence and prevalence of GFkV in wine grapes in Idaho. During three growing seasons in 2009 through 2011, random grapevine samples were collected in 14 vineyards in Canyon, Elmore, Ada, and Nez Perce counties. A total of 434 samples were tested by one step RT-PCR using GFkV-specific primers, GFkVf: 5'-TGACCAGCCTGCTGTCTCTA-3' and GFkVr: 5'-TGGACAGGGAGGTGTAGGAG-3' designed to amplify a fragment of the GFkV capsid protein gene (1). Twenty-four samples tested positive for GFkV by RT-PCR and produced the expected 179-bp DNA fragment. These samples came from five vineyards sampled across all surveyed counties, and represented seven wine grape cultivars, including Pinot Noir, Cabernet Sauvignon, Syrah, Lemberger, Riesling, Chardonnay, Pinot Gris, and one unknown table grape cultivar. Twelve PCR products were cloned into the pGEM-T Easy plasmid vector (Promega), sequenced (numbered ID1 to 12, available upon request), and confirmed to represent fragments of the GFkV CP gene between positions 6,453 and 6,631 in the genome of GFkV isolate MT48 (GenBank Accession No. AJ309022.1). Eight of the Idaho GFkV sequences (ID2, ID3, ID7 to 11, and ID12) matched closely with other GFkV sequences from Washington State, Italy, India, and South America, showing 97 to 99% identity at the nucleotide level in pair-wise comparisons. Four GFkV sequences from Idaho (ID1 and ID4 to 6) showed only modest (90 to 92%) identity in pair-wise comparisons with GFkV sequences available in GenBank. Consequently, in phylogenetic reconstructions eight Idaho GFkV sequences clustered in the same lineage with the six GFkV sequences deposited in GenBank, and four other GFkV sequences were placed outside of this main clade. It is possible that this phylogeny of the Idaho GFkV reflects different sources of the virus-infected planting material brought to the state. In the absence of symptoms expressed in wine grape cultivars infected with GFkV, laboratory methods remain the only tool to detect the virus. To our knowledge, this is the first report of GFkV found in wine grapes in Idaho demonstrating its substantial presence in production areas. References: (1) G. Gambino and I. Gribaudo. Phytopathology 96:1223, 2006. (2) R. A. Naidu et al. Plant Dis. 94:784, 2010. (3) S. Sabanadzovic et al. J. Gen. Virol. 82:2009, 2001.