Deborah A. Golino

Deep sequencing analysis of RNAs from a grapevine showing Syrah decline symptoms reveals a multiple virus infection that includes a novel virus.

In a search for viruses associated with decline symptoms of Syrah grapevines, we have undertaken an analysis of total plant RNA sequences using Life Sciences 454 high-throughput sequencing. 67.5 megabases of sequence data were derived from reverse-transcribed cDNA fragments, and screened for sequences of viral or viroid origin. The data revealed that a vine showing decline symptoms supported a mixed infection that included seven different RNA genomes. Fragments identified as derived from viruses or viroids spanned a approximately ten thousand fold range in relative prevalence, from 48,278 fragments derived from Rupestris stem pitting-associated virus to 4 fragments from Australian grapevine viroid. 1527 fragments were identified as derived from an unknown marafivirus. Its complete genome was sequenced and characterized, and an RT-PCR test was developed to analyze its field distribution and to demonstrate its presence in leafhoppers (vector for marafiviruses) collected from diseased vines. Initial surveys detected a limited presence of the virus in grape-growing regions of California.

Mealybug transmission of Grapevine leafroll viruses: an analysis of virus-vector specificity.

To understand ecological factors mediating the spread of insect-borne plant pathogens, vector species for these pathogens need to be identified. Grapevine leafroll disease is caused by a complex of phylogenetically related closteroviruses, some of which are transmitted by insect vectors; however, the specificities of these complex virus-vector interactions are poorly understood thus far. Through biological assays and phylogenetic analyses, we studied the role of vector-pathogen specificity in the transmission of several grapevine leafroll-associated viruses (GLRaVs) by their mealybug vectors. Using plants with multiple virus infections, several virus species were screened for vector transmission by the mealybug species Planococcus ficus and Pseudococcus longispinus. We report that two GLRaVs (-4 and -9), for which no vector transmission evidence was available, are mealybug-borne. The analyses performed indicated no evidence of mealybug-GLRaV specificity; for example, different vector species transmitted GLRaV-3 and one vector species, Planococcus ficus, transmitted five GLRaVs. Based on available data, there is no compelling evidence of vector-virus specificity in the mealybug transmission of GLRaVs. However, more studies aimed at increasing the number of mealybug species tested as vectors of different GLRaVs are necessary. This is especially important given the increasing importance of grapevine leafroll disease spread by mealybugs in vineyards worldwide.

Grapevine leafroll-associated virus 3

Grapevine leafroll disease (GLD) is one of the most important grapevine viral diseases affecting grapevines worldwide. The impact on vine health, crop yield, and quality is difficult to assess due to a high number of variables, but significant economic losses are consistently reported over the lifespan of a vineyard if intervention strategies are not implemented. Several viruses from the family Closteroviridae are associated with GLD. However, Grapevine leafroll-associated virus 3 (GLRaV-3), the type species for the genus Ampelovirus, is regarded as the most important causative agent. Here we provide a general overview on various aspects of GLRaV-3, with an emphasis on the latest advances in the characterization of the genome. The full genome of several isolates have recently been sequenced and annotated, revealing the existence of several genetic variants. The classification of these variants, based on their genome sequence, will be discussed and a guideline is presented to facilitate future comparative studies. The characterization of sgRNAs produced during the infection cycle of GLRaV-3 has given some insight into the replication strategy and the putative functionality of the ORFs. The latest nucleotide sequence based molecular diagnostic techniques were shown to be more sensitive than conventional serological assays and although ELISA is not as sensitive it remains valuable for high-throughput screening and complementary to molecular diagnostics. The application of next-generation sequencing is proving to be a valuable tool to study the complexity of viral infection as well as plant pathogen interaction. Next-generation sequencing data can provide information regarding disease complexes, variants of viral species, and abundance of particular viruses. This information can be used to develop more accurate diagnostic assays. Reliable virus screening in support of robust grapevine certification programs remains the cornerstone of GLD management.

Comparison of low-density arrays, RT-PCR and real-time TaqMan® RT-PCR in detection of grapevine viruses

Low-density arrays (LDA) have been designed based on the real-time RT-PCR (TaqMan) assays for the specific detection of 13 viruses that infect Grapevines in addition to the housekeeping gene 18S rRNA. The viruses included in the study are Grapevine leafroll associated viruses 1, 2, 3, 4, 5, and 9, Grapevine leafroll associated virus-2 Redglobe (GLRaV-2RG) strain, Ruspestris stem pitting associated virus, Grapevine vitivirus A, Grapevine vitivirus B, Grapevine fanleaf virus, Tomato ringspot virus (ToRSV), and Grapevine fleck virus (GFkV). This study includes three new TaqMan RT-PCR assays that have been developed for GLRaV-2RG, GFkV and ToRSV and have been included in the TaqMan RT-PCR and LDA detection. The LDAs were evaluated against a wide range of isolates distributed geographically. Geographical locations included Africa, Europe, Australia, Asia, Latin America and the United States. High-throughput detection of these viruses using LDAs was compared to RT-PCR and real-time TaqMan RT-PCR. The efficiency of different RNA extraction methodologies and buffers were compared for use in low-density array detection. In addition improving the RNA extraction technique and testing the quality of the RNA using the 18S ribosomal RNA TaqMan assay as an RNA specific internal control proved to generate better diagnostic assays. This is the first report on the use of LDA for the detection of plant viruses.

Grapevine Leafroll: A Complex Viral Disease Affecting a High-Value Fruit Crop

Grapevine (Vitis spp.) is one of the most widely grown fruit crops in the world. It is a deciduous woody perennial vine for which the cultivation of domesticated species began approximately 6,000 to 8,000 years ago in the Near East. Grapevines are broadly classified into red- and white-berried cultivars based on their fruit skin color, although yellow, pink, crimson, dark blue, and black-berried cultivars also exist. Grapevines can be subject to attacks by many different pests and pathogens, including graft-transmissible agents such as viruses, viroids, and phytoplasmas. Among the virus and virus-like diseases, grapevine leafroll disease (GLD) is by far the most widespread and economically damaging viral disease of grapevines in many regions around the world. The global expansion of the grape and wine industry has seen a parallel increase in the incidence and economic impact of GLD. Despite the fact that GLD was recognized as a potential threat to grape production for several decades, our knowledge of the nature of the disease is still quite limited due to a variety of challenges related to the complexity of this virus disease, the association of several distinct GLD-associated viruses, and contrasting symptoms in red- and white-berried cultivars. In view of the growing significance of GLD to wine grape production worldwide, this feature article provides an overview of the state of knowledge on the biology and epidemiology of the disease and describes management strategies currently deployed in vineyards.

Comparison of Next-Generation Sequencing Versus Biological Indexing for the Optimal Detection of Viral Pathogens in Grapevine

A bioassay is routinely used to determine the viral phytosanitary status of commercial grapevine propagation material in many countries around the world. That test is based on the symptoms developed in the field by specific indicator host plants that are graft-inoculated from the vines being tested. We compared the bioassay against next-generation sequencing (NGS) analysis of grapevine material. NGS is a laboratory procedure that catalogs the genomic sequences of the viruses and other pathogens extracted as DNA and RNA from infected vines. NGS analysis was found to be superior to the standard bioassay in detection of viruses of agronomic significance, including virus infections at low titers. NGS was also found to be superior to the bioassay in its comprehensiveness, the speed of its analysis, and for the discovery of novel, uncharacterized viruses.

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.

Vitis californica and Vitis californica × Vitis vinifera Hybrids are Hosts for Grapevine leafroll-associated virus-2 and -3 and Grapevine virus A and B

Vitis and non-Vitis spp. surrounding nine Napa Valley vineyards were surveyed for Grapevine leafroll-associated virus (GLRaV)-1 to -5 and -9, Grapevine virus A (GVA), Grapevine virus B (GVB), and Grapevine virus D (GVD). Vitis spp. from three riparian areas not adjacent to vineyards were also included. DNA fingerprinting and probability analyses indicated that the Vitis samples consisted primarily of Vitis californica followed by V. californica × V. vinifera hybrids. Single and mixed infections of GLRaV-2, -3, GVA, or GVB were detected by conventional or quantitative reverse-transcription polymerase chain reaction in 6 of the 66 V. californica and 11 of the 19 V. californica × V. vinifera hybrids. GLRaV-1, -4, -5, -9, and GVD were not detected. Phylogenetic analysis of GLRaV-2 and -3 partial coat protein gene nucleotide sequences indicated that the isolates from V. californica and V. californica × V. vinifera hybrids were closely related to isolates from V. vinifera.

Comparative procedures for sample processing and quantitative PCR detection of grapevine viruses.

In this study different instruments and methods used for tissue homogenization, RNA extraction and quantitative PCR (qPCR) based detection of grapevine RNA viruses were evaluated. Semi-automated and automated homogenization techniques were compared to process samples from grapevine petioles and cambial tissue. Four different high throughput automated nucleic acid extraction platforms were compared with the RNeasy plant extraction kit for their capacity and efficiency of extracting viral RNA from grapevine infected tissues. The RNA prepared from each extraction platform was then used as template for a comparative analysis of qPCR by One Step RT-qPCR, Two Step RT-qPCR and low density array (LDA) detection. This study showed that a thorough homogenization of grapevine tissues using the Tissue Lyser as well as DNase digestion of the purified RNA prior to cDNA synthesis improved the virus detection and yielded the lowest quantitation cycle (Cq) values in RT-qPCR. Comparison of different RNA extraction methods showed that methods implementing the magnetic bead-based technology were superior to other methods used. Comparing different qPCR detection methods, One Step RT-qPCR showed the lowest Cq values for the same sample tested compared to Two Step RT-qPCR and LDA.

Development of a sensitive colorimetric-PCR assay for detection of viruses in woody plants

Diagnostic methods employing the polymerase chain reaction (PCR) provide the most sensitive means currently available for detecting viruses in woody plants. A new technique has been tested that does not rely on gel electrophoresis or molecular hybridization to detect virus-specific PCR products. This colorimetric method for detection of PCR products from woody plants was demonstrated to be at least as sensitive as gel analysis. When combined with immunocapture of virions from plant sap, colorimetric detection provides a means to apply PCR technology to a large number of samples. Here, we report on the use of this technique for detection and quantitation of a walnut isolate of cherry leafroll virus (CLRV-W), citrus tristeza virus (CTV), prune dwarf virus (PDV), prunus necrotic ringspot virus (PNRSV), and tomato ringspot virus (ToRSV) in woody and herbaceous plants. For purified virus preparations, detection limits ranged from 100 pg/ml to 100 ag/ml. We also describe the colorimetric PCR detection of CTV in pooled samples.