Quality Assessment and Validation of High-Throughput Sequencing for Grapevine Virus Diagnostics.

Development of High-Throughput Sequencing (HTS), also known as next generation sequencing, revolutionized diagnostic research of plant viruses. HTS outperforms bioassays and molecular diagnostic assays that are used to screen domestic and quarantine grapevine materials in data throughput, cost, scalability, and detection of novel and highly variant virus species. However, before HTS-based assays can be routinely used for plant virus diagnostics, performance specifications need to be developed and assessed. In this study, we selected 18 virus-infected grapevines as a test panel for measuring performance characteristics of an HTS-based diagnostic assay. Total nucleic acid (TNA) was extracted from petioles and dormant canes of individual samples and constructed libraries were run on Illumina NextSeq 500 instrument using a 75-bp single-end read platform. Sensitivity was 98% measured over 264 distinct virus and viroid infections with a false discovery rate (FDR) of approximately 1 in 5 positives. The results also showed that combining a spring petiole test with a fall cane test increased sensitivity to 100% for this TNA HTS assay. To evaluate extraction methodology, these results were compared to parallel dsRNA extractions. In addition, in a more detailed dilution study, the TNA HTS assay described here consistently performed well down to a dilution of 5%. In that range, sensitivity was 98% with a corresponding FDR of approximately 1 in 5. Repeatability and reproducibility were assessed at 99% and 93%, respectively. The protocol, criteria, and performance levels described here may help to standardize HTS for quality assurance and accreditation purposes in plant quarantine or certification programs.

Rootstock influences the effect of grapevine leafroll-associated viruses on berry development and metabolism via abscisic acid signalling.

Grapevine leafroll-associated virus (GLRaV) infections are accompanied by symptoms influenced by host genotype, rootstock, environment, and which individual or combination of GLRaVs is present. Using a dedicated experimental vineyard, we studied the responses to GLRaVs in ripening berries from Cabernet Franc grapevines grafted to different rootstocks and with zero, one, or pairs of leafroll infection(s). RNA sequencing data were mapped to a high-quality Cabernet Franc genome reference assembled to carry out this study and integrated with hormone and metabolite abundance data. This study characterized conserved and condition-dependent responses to GLRaV infection(s). Common responses to GLRaVs were reproduced in two consecutive years and occurred in plants grafted to different rootstocks in more than one infection condition. Though different infections were inconsistently distinguishable from one another, the effects of infections in plants grafted to different rootstocks were distinct at each developmental stage. Conserved responses included the modulation of genes related to pathogen detection, abscisic acid (ABA) signalling, phenylpropanoid biosynthesis, and cytoskeleton remodelling. ABA, ABA glucose ester, ABA and hormone signalling-related gene expression, and the expression of genes in several transcription factor families differentiated the effects of GLRaVs in berries from Cabernet Franc grapevines grafted to different rootstocks. These results support that ABA participates in the shared responses to GLRaV infection and differentiates the responses observed in grapevines grafted to different rootstocks.

Safeguarding Fruit Crops in the Age of Agricultural Globalization.

The expansion of fruit production and markets into new geographic areas provides novel opportunities and challenges for the agricultural and marketing industries. Evidence that fruit consumption helps prevent nutrient deficiencies and reduces the risk of cardiovascular disease and cancer has assisted in the expansion of all aspects of the fruit industry. In today's competitive global market environment, producers need access to the best plant material available in terms of genetics and health if they are to maintain a competitive advantage in the market. An ever-increasing amount of plant material in the form of produce, nursery plants, and breeding stock moves vast distances, and this has resulted in an increased risk of pest and disease introductions into new areas. One of the primary concerns of the global fruit industry is a group of systemic pathogens for which there are no effective remedies once plants are infected. These pathogens and diseases require expensive management and control procedures at nurseries and by producers locally and nationally. Here, we review (i) the characteristics of some of these pathogens, (ii) the history and economic consequences of some notable disease epidemics caused by these pathogens, (iii) the changes in agricultural trade that have exacerbated the risk of pathogen introduction, (iv) the path to production of healthy plants through the U.S. National Clean Plant Network and state certification programs, (v) the economic value of clean stock to nurseries and fruit growers in the United States, and (vi) current efforts to develop and harmonize effective nursery certification programs within the United States as well as with global trading partners.

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.

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.

Rose spring dwarf-associated virus has RNA structural and gene-expression features like those of Barley yellow dwarf virus.

We determined the complete nucleotide sequence of the Rose spring dwarf-associated virus (RSDaV) genomic RNA (GenBank accession no. EU024678) and compared its predicted RNA structural characteristics affecting gene expression. A cDNA library was derived from RSDaV double-stranded RNAs (dsRNAs) purified from infected tissue. Nucleotide sequence analysis of the cloned cDNAs, plus for clones generated by 5'- and 3'-RACE showed the RSDaV genomic RNA to be 5808 nucleotides. The genomic RNA contains five major open reading frames (ORFs), and three small ORFs in the 3'-terminal 800 nucleotides, typical for viruses of genus Luteovirus in the family Luteoviridae. Northern blot hybridization analysis revealed the genomic RNA and two prominent subgenomic RNAs of approximately 3 kb and 1 kb. Putative 5' ends of the sgRNAs were predicted by identification of conserved sequences and secondary structures which resembled the Barley yellow dwarf virus (BYDV) genomic RNA 5' end and subgenomic RNA promoter sequences. Secondary structures of the BYDV-like ribosomal frameshift elements and cap-independent translation elements, including long-distance base pairing spanning four kb were identified. These contain similarities but also informative differences with the BYDV structures, including a strikingly different structure predicted for the 3' cap-independent translation element. These analyses of the RSDaV genomic RNA show more complexity for the RNA structural elements for members of the Luteoviridae.

Complete nucleotide sequences and genome characterization of a novel double-stranded RNA virus infecting Rosa multiflora.

The three double-stranded (ds) RNAs were detected in Rosa multiflora plants showing rose spring dwarf (RSD) symptoms. Northern blot analysis revealed three dsRNAs in preparations of both dsRNA and total RNA from R. multiflora plants. The complete sequences of the dsRNAs (referred to as dsRNA 1, dsRNA 2 and dsRNA 3) were determined based on a combination of shotgun cloning of dsRNA cDNAs and reverse transcription-polymerase chain reaction (RT-PCR). The largest dsRNA (dsRNA 1) was 1,762 bp long with a single open reading frame (ORF) that encoded a putative polypeptide containing 479 amino acid residues with a molecular mass of 55.9 kDa. This polypeptide contains amino acid sequence motifs conserved in the RNA-dependent RNA polymerases (RdRp) of members of the family Partitiviridae. Both dsRNA 2 (1,475 bp) and dsRNA 3 (1,384 bp) contained single ORFs, encoding putative proteins of unknown function. The 5' untranslated regions (UTR) of all three segments shared regions of high sequence homology. Phylogenetic analysis using the RdRp sequences of the various partitiviruses revealed that the new sequences would constitute the genome of a virus in family Partitiviridae. This virus would cluster with Fragaria chiloensis cryptic virus and Raphanus sativus cryptic virus 2. We suggest that the three dsRNA segments constitute the genome of a novel cryptic virus infecting roses; we propose the name Rosa multiflora cryptic virus (RMCV). Detection primers were developed and used for RT-PCR detection of RMCV in rose plants.

Identification and Partial Characterization of a New Luteovirus Associated with Rose Spring Dwarf Disease.

A number of viruses in the genera Ilarvirus and Nepovirus have been shown to be associated with specific diseases in rose, but many graft-transmissible rose diseases still have unknown etiologies. One of these diseases originally was detected by grafting from nonsymptomatic roses to Rosa multiflora indicator plants. Double-stranded RNAs (dsRNAs) were recovered and used as templates for cDNA synthesis and generating a cDNA library. Analysis of deduced amino acid sequences clearly positioned this virus as a member of the family Luteoviridae. The name rose spring dwarf associated virus (RSDaV) is tentatively proposed for the novel virus because the symptoms of this virus on R. multiflora are consistent with previous descriptions of rose spring dwarf disease (RSD). Phylogenetic analysis revealed a close relationship of RSDaV with members of the genus Luteovirus. Aphid transmission studies identified the rose-grass aphid (Metapolophium dirhodum) and yellow rose aphid (Rhodobium porosum) as vectors for this new virus. Host range data showed that RSDaV has a host range including both monocots and dicots. A specific reverse-transcription polymerase chain reaction assay was developed and revealed the presence of the RSDaV in several rose cultivars. RSDaV-inoculated rose plants developed RSD symptoms, confirming its role in the etiology of the disease.

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.

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.

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.