A New Era in Federal Quarantine and State Certification Diagnostics at Clean Plant Centers in the USA.

Quarantine and certification programs exist to prevent the entry or spread of harmful pests and pathogens into agricultural systems. Their common objective is to identify pathogen-free source material through the application of validated testing methods for subsequent release for propagation. Tests must be accurate, efficient and cost-effective.

Characterization of Genetic Diversity in the Capsid Protein Gene of Grapevine Fleck Virus and Development of a New Real-Time RT-PCR Assay.

The grapevine fleck virus (GFkV) is a ubiquitous grapevine-infecting virus found worldwide, is associated with the grapevine fleck complex, and is often found in mixed infections with viruses of the grapevine leafroll complex and/or vitiviruses. Although GFkV has been studied for a long time, limited sequence information is available in the public databases. In this study, the GFkV sequence data available in GenBank and data generated at the Foundation Plant Services, University of California, Davis, were used to perform nucleotide sequence comparisons, construct a phylogenetic tree, and develop a new RT-qPCR assay.

Development of a one-step RT-qPCR assay for the detection of Grapevine leafroll-associated virus 7.

Grapevine leafroll disease (GLD) is one of the most economically important viral diseases of grapevines. GLD is caused by a complex of several ssRNA (+) viruses referred to as Grapevine leafroll-associated viruses (GLRaVs). To date, five different GLRaV species have been identified.

Sequencing a Strawberry Germplasm Collection Reveals New Viral Genetic Diversity and the Basis for New RT-qPCR Assays.

Viruses are considered of major importance in strawberry ( × Duchesne) production given their negative impact on plant vigor and growth. Strawberry accessions from the National Clonal Germplasm Repository were screened for viruses using high throughput sequencing (HTS). Analyses of sequence information from 45 plants identified multiple variants of 14 known viruses, comprising strawberry mottle virus (SMoV), beet pseudo yellows virus (BPYV), strawberry pallidosis-associated virus (SPaV), tomato ringspot virus (ToRSV), strawberry mild yellow edge virus (SMYEV), strawberry vein banding virus (SVBV), strawberry crinkle virus (SCV), strawberry polerovirus 1 (SPV-1), apple mosaic virus (ApMV), strawberry chlorotic fleck virus (SCFaV), strawberry crinivirus 4 (SCrV-4), strawberry crinivirus 3 (SCrV-3), Fragaria chiloensis latent virus (FClLV) and Fragaria chiloensis cryptic virus (FCCV).

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.

Comprehensive Real-Time RT-PCR Assays for the Detection of Fifteen Viruses Infecting spp.

Viruses can cause economic losses in fruit trees, including spp., by reducing yield and marketable fruit. Given the genetic diversity of viruses, reliable diagnostic methods relying on PCR are critical in determining viral infection in fruit trees.

Characterization of grapevine leafroll-associated virus 3 genetic variants and application towards RT-qPCR assay design.

Grapevine leafroll-associated virus 3 (GLRaV-3) is the most widely prevalent and economically important of the complex of RNA viruses associated with grapevine leafroll disease (GLD). Phylogenetic studies have grouped GLRaV-3 isolates into nine different monophyletic groups and four supergroups, making GLRaV-3 a genetically highly diverse virus species. In addition, new divergent variants have been discovered recently around the world.

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.

In vitro transcripts from cloned cDNAs of the lettuce infectious yellows closterovirus bipartite genomic RNAs are competent for replication in Nicotiana benthamiana protoplasts.

Full-length cloned cDNAs of lettuce infectious yellows closterovirus (LIYV) RNAs 1 and 2 were constructed and fused to the bacteriophage T3 RNA polymerase promoter. To assess RNA replication, Nicotiana benthamiana protoplasts were inoculated with LIYV virion RNAs and LIYV cDNA-derived in vitro transcripts. Analysis of protoplasts inoculated with LIYV virion RNAs or capped (m7GpppG) in vitro transcripts from LIYV RNA 1 and 2 cDNAs showed accumulation of LIYV genomic and putative subgenomic RNAs (sgRNAs), synthesis of LIYV coat protein, and formation of LIYV virions.

Genome structure and phylogenetic analysis of lettuce infectious yellows virus, a whitefly-transmitted, bipartite closterovirus.

We report the complete nucleotide sequences of lettuce infectious yellows virus (LIYV) RNAs 1 and 2. LIYV RNA 1 is 8118 nucleotides and includes three open reading frames (ORFs). Computer-assisted analysis of LIYV RNA 1 ORFs identified domains for a papain-like protease, methyltransferase (MTR), RNA helicase (HEL), and RNA-dependent RNA polymerase (RdRp).

Partial characterization of the lettuce infectious yellows virus genomic RNAs, identification of the coat protein gene and comparison of its amino acid sequence with those of other filamentous RNA plant viruses.

Purified virions of lettuce infectious yellows virus (LIYV), a tentative member of the closterovirus group, contained two RNAs of approximately 8500 and 7300 nucleotides (RNAs 1 and 2 respectively) and a single coat protein species with M(r) of approximately 28,000. LIYV-infected plants contained multiple dsRNAs. The two largest were the correct size for the replicative forms of LIYV virion RNAs 1 and 2.

Nucleotide sequence and RNA hybridization analyses reveal an ambisense coding strategy for maize stripe virus RNA3.

The 2357-nt sequence of maize stripe virus (MStV) RNA3 was determined. Two nonoverlapping open reading frames (ORFs) of opposite polarities are contained in RNA3. A 591-nt ORF is located near the 5' end of MStV RNA3, while a second ORF of 948 nt is located near the 3' end in the viral complementary RNA (vcRNA).

Identification and sequence analysis of the maize stripe virus major noncapsid protein gene.

The maize stripe virus (MStV) major noncapsid protein (NCP) gene was characterized, and the location of the NCP gene was identified among the 5-RNA, 18-kb genome. A 12-amino-acid sequence of the NCP was compared with nucleotide sequence data for MStV RNAs 3 and 4 and was found to align perfectly within a 528-nucleotide open reading frame (ORF) of RNA 4. The amino acid composition of purified NCP was almost identical to that deduced from the putative coding region.

Complementary DNA cloning and hybridization analysis of maize stripe virus RNAs.

Cloned cDNAs were used to assess the relationships of the RNAs isolated from maize stripe virus (MStV) virion-like nucleoproteins. Northern blot hybridization analysis showed no detectable sequence homology between the five different sized RNAs; however, cDNAs which hybridized with a specific ssRNA also hybridized with a specific dsRNA. Hybridization analysis of denatured MStV RNAs using ssRNA riboprobes of opposite polarities showed that for each of the five MStV RNAs, opposite, complementary polarity RNAs were present.