Genome Sequences and Structures of Two Biologically Distinct Strains of Grapevine leafroll - associated virus 2 and Sequence Analysis

Grapevine leafroll-associated virus 2 (GLRaV-2), a member of the genus Closterovirus within Closteroviridae, is implicated in several important diseases of grapevines including "leafroll”, "graft-incompatibility”, and "quick decline” worldwide. Several GLRaV-2 isolates have been detected from different grapevine genotypes. However, the genomes of these isolates were not sequenced or only partially sequenced. Consequently, the relationship of these viral isolates at the molecular level has not been determined. Here, we group the various GLRaV-2 isolates into four strains based on their coat protein gene sequences. We show that isolates "PN” (originated from Vitis vinifera cv. "Pinot noir”), "Sem” (from V. vinifera cv. "Semillon”) and "94/970” (from V. vinifera cv. "Muscat of Alexandria”) belong to the same strain, "93/955” (from hybrid "LN-33”) and "H4” (from V. rupestris "St. George”) each represents a distinct strain, while Grapevine rootstock stem lesion-associated viru

Research Progress in the Formation of Quality of Dry-Aged Beef and Approaches for Its Added Value

Dry aging is an effective method to improve the quality and value of meat. Meat quality characteristics such as tenderness and flavor can be significantly improved through a series of biochemical reactions. In recent years, dry-aged beef has gained huge popularity among consumers and research attention due to its unique flavor. However, there are some problems with the dry-aged beef industry, such as large quality differences, high losses during maturation, and high production costs. This article systematically summarizes quality characteristics of dry-aged beef, including flavor, tenderness, safety, color and water-holding capacity (WHC). The mechanism of formation of its characteristic flavor is briefly described. The effects of different dry-aging conditions on beef quality are summarized. New dry aging technologies and value-added ways to reuse the crust are introduced. This article will hopefully provide theoretical guidance for the standardized, efficient and high-value development of the dry-aged beef industry

Virus and Virus-like Pathogens in the Grapevine Virus Collection of Croatian Autochthonous Grapevine Cultivars

Grapevine collections play an important role, especially in the study of viruses and virus-like pathogens. In 2009, after an initial ELISA screening for eight viruses (arabis mosaic virus, grapevine fanleaf virus, grapevine fleck virus, grapevine leafroll-associated viruses 1, 2, and 3, and grapevine viruses A and B), a collection of 368 grapevine accessions representing 14 different Croatian autochthonous cultivars and containing single or mixed infection of viruses was established to further characterize the viral pathogens. Subsequently, Western blot, RT-PCR, cloning, and sequencing revealed that grapevine rupestris stem pitting-associated virus was frequently found in accessions of the collection, with isolates showing substantial genetic diversity in the helicase and coat protein regions. High-throughput sequencing of 22 grapevine accessions provides additional insight into the viruses and viroids present in the collection and confirms the fact that Croatian autochthonous grapevine cultivars have high infection rates and high virome diversity. The recent spread of “flavescence dorée” phytoplasma in Europe has not spared the collection. After the first symptoms observed in 2020 and 2021, the presence of phytoplasma was confirmed by LAMP in six grapevine accessions and some of them were lost. Single or multiple viruses and viroids, as well as own rooted grapevines in the collection, make the plants susceptible to various abiotic factors, which, together with the recent occurrence of “flavescence dorée”, makes the maintenance of the collection a challenge. Future efforts will be directed towards renewing the collection, as 56% of the original collection has been lost in the last 13 years

Molecular and Metagenomic Analyses Reveal High Prevalence and Complexity of Viral Infections in French-American Hybrids and North American Grapes

French-American hybrids and North American grape species play a significant role in Canada’s grape and wine industry. Unfortunately, the occurrence of viruses and viral diseases among these locally important non-vinifera grapes remains understudied. We report here the results from a large-scale survey to assess the prevalence of 14 viruses among 533 composite samples representing 2665 vines from seven French-American hybrid wine grape cultivars, two North American juice grape cultivars (Concord and Niagara), and the table grape cultivar Sovereign coronation. Based on reverse transcription polymerase chain reaction (RT-PCR) assays, ten viruses were detected. Grapevine rupestris stem pitting-associated virus, grapevine leafroll-associated virus 3, grapevine Pinot gris virus and grapevine red blotch virus were detected with the highest frequency. As expected, mixed infections were common; 62% of the samples contained two or more viruses. Overall, hybrid wine grapes were infected with more viruses and a higher prevalence of individual viruses than juice and table grapes. To validate these findings and to refine the virome of these non-European grapes, high-throughput sequencing (HTS) analyses of five composite samples representing each category of grapevine cultivars was performed. Results from HTS agreed with those from RT-PCR. Importantly, Vidal, a widely grown white-wine grape with international recognition due to its use in the award-winning icewine, is host to 14 viruses, four of which comprise multiple and distinct genetic variants. This comprehensive survey represents the most extensive examination of viruses among French-American hybrids and North American grapes to date

Probing into the Effects of Grapevine Leafroll-Associated Viruses on the Physiology, Fruit Quality and Gene Expression of Grapes

Grapevine leafroll is one of the most widespread and highly destructive grapevine diseases that is responsible for great economic losses to the grape and wine industries throughout the world. Six distinct viruses have been implicated in this disease complex. They belong to three genera, all in the family Closteroviridae. For the sake of convenience, these viruses are named as grapevine leafroll-associated viruses (GLRaV-1, -2, -3, -4, -7, and -13). However, their etiological role in the disease has yet to be established. Furthermore, how infections with each GLRaV induce the characteristic disease symptoms remains unresolved. Here, we first provide a brief overview on each of these GLRaVs with a focus on genome structure, expression strategies and gene functions, where available. We then provide a review on the effects of GLRaV infection on the physiology, fruit quality, fruit chemical composition, and gene expression of grapevine based on the limited information so far reported in the literature. We outline key methodologies that have been used to study how GLRaV infections alter gene expression in the grapevine host at the transcriptomic level. Finally, we present a working model as an initial attempt to explain how infections with GLRaVs lead to the characteristic symptoms of grapevine leafroll disease: leaf discoloration and downward rolling. It is our hope that this review will serve as a starting point for grapevine virology and the related research community to tackle this vastly important and yet virtually uncharted territory in virus-host interactions involving woody and perennial fruit crops

Transcriptomic Analyses of Grapevine Leafroll-Associated Virus 3 Infection in Leaves and Berries of ‘Cabernet Franc’

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the most important viruses affecting global grape and wine production. GLRaV-3 is the chief agent associated with grapevine leafroll disease (GLRD), the most prevalent and economically destructive grapevine viral disease complex. Response of grapevine to GLRaV-3 infection at the gene expression level is poorly characterized, limiting the understanding of GLRaV-3 pathogenesis and viral-associated symptom development. In this research, we used RNA-Seq to profile the changes in global gene expression of Cabernet franc, a premium red wine grape, analyzing leaf and berry tissues at three key different developmental stages. We have identified 1457 differentially expressed genes (DEGs) in leaves and 1181 DEGs in berries. The expression profiles of a subset of DEGs were validated through RT-qPCR, including those involved in photosynthesis (VvPSBP1), carbohydrate partitioning (VvSUT2, VvHT5, VvGBSS1, and VvSUS), flavonoid biosynthesis (VvUFGT, VvLAR1, and VvFLS), defense response (VvPR-10.3, and VvPR-10.7), and mitochondrial activities (ETFB, TIM13, and NDUFA1). GLRaV-3 infection altered source–sink relationship between leaves and berries. Photosynthesis and photosynthate assimilation were inhibited in mature leaves while increased in young berries. The expression of genes involved in anthocyanin biosynthesis increased in GLRaV-3-infected leaves, correlating with interveinal tissue reddening, a hallmark of GLRD symptoms. Notably, we identified changes in gene expression that suggest a compromised sugar export and increased sugar retrieval in GLRaV-3-infected leaves. Genes associated with mitochondria were down-regulated in both leaves and berries of Cabernet franc infected with GLRaV-3. Results of the present study suggest that GLRaV-3 infection may disrupt mitochondrial function in grapevine leaves, leading to repressed sugar export and accumulation of sugar in mature leaf tissues. The excessive sugar accumulation in GLRaV-3-infected leaves may trigger downstream GLRD symptom development and negatively impact berry quality. We propose a working model to account for the molecular events underlying the pathogenesis of GLRaV-3 and symptom development

Association of Grapevine fanleaf virus, Tomato ringspot virus and Grapevine rupestris stem pitting-associated virus with a grapevine vein-clearing complex on var. Chardonnay

A disease complex was observed on grapevine var. Chardonnay (Vitis vinifera) in a commercial vineyard in Missouri that destroyed the affected vineyard. Conspicuous vein-clearing symptoms on the leaves of originally diseased Chardonnay vines and bud-grafted Chardonnay, V. vinifera \u27Cabernet Franc\u27, V. vinifera \u27Baco Blanc\u27, and hybrid \u27LN-33\u27 vines are characteristics of the disease complex, which is referred to as the grapevine vein-clearing complex (GVCC). By applying reverse-transcription polymerase chain reaction (RT-PCR) using virus-specific primers, we detected combinations of Grapevine fanleaf virus (GFLV), Tomato ringspot virus (ToRSV) and Grapevine rupestris stem pitting-associated virus (GRSPaV), in symptomatic Chardonnay vines. Sequencing of RT-PCR amplified DNA fragments confirmed the identity of each virus, indicating the occurrence of ToRSV yellow vein strain, and two distinct strains of GRSPaV in the GVCC-affected Chardonnay vines. This is the first report of the co-infection of two nepoviruses and GRSPaV in var. Chardonnay. This study demonstrated that mixed infections of grapevine viruses belonging to different taxonomic groups pose a great threat to vineyards under certain climatic and soil conditions

Survey for major viruses in commercial Vitis vinifera wine grapes in Ontario

Abstract Background In recent years, the Ontario grape and wine industry has experienced outbreaks of viral diseases across the province. Little is known about the prevalence of viruses and viral diseases in Ontario. Since 2015, we have conducted large-scale surveys for major viruses in commercial wine grapes in order to obtain a comprehensive understanding of the prevalence and severity of viral diseases in Ontario. Methods A total of 657 composite leaf samples representing 3285 vines collected from 137 vine blocks of 33 vineyards from three appellations: Niagara Peninsula, Lake Erie North Shore and Prince Edward County. These samples covered six major red cultivars and five major white grape cultivars. Using a multiplex RT-PCR format, we tested these samples for 17 viruses including those involved in all major viral diseases of the grapevine, such as five grapevine leafroll-associated viruses (GLRaV-1, 2, 3, 4, 7), grapevine red blotch virus (GRBV), grapevine Pinot gris virus (GPGV), grapevine rupestris stem sitting-associated virus (GRSPaV), grapevine virus A (GVA), grapevine virus B (GVB), grapevine fleck virus (GFkV), arabis mosaic virus (ArMV), tomato ringspot virus (ToRSV), trapevine fanleaf virus (GFLV), among others. Results Fourteen of the 17 viruses were detected from these samples and the predominant viruses are GRSPaV, GLRaV-3, GFkV, GPGV and GRBaV with an incidence of 84.0, 47.9, 21.8, 21.6 and 18.3%, respectively. As expected, mixed infections with multiple viruses are common. 95.6% of the samples included in the survey were infected with at least one virus; 67% of the samples with 2–4 viruses and 4.7% of the samples with 5–6 viruses. The major grape cultivars all tested positive for these major viruses. The results also suggested that the use of infected planting material may have been one of the chief factors responsible for the recent outbreaks of viral diseases across the province. Conclusions This is the first such comprehensive survey for grapevine viruses in Ontario and one of the most extensive surveys ever conducted in Canada. The recent outbreaks of viral diseases in Ontario vineyards were likely caused by GLRaV-3, GRBV and GPGV. Findings from this survey provides a baseline for the grape and wine industry in developing strategies for managing grapevine viral diseases in Ontario vineyards

Additional file 2: Figure S1. of A highly effective and versatile technology for the isolation of RNAs from grapevines and other woody perennials for use in virus diagnostics

RT-PCR detection of GRSPaV and ubiquitin gene using RNAs isolated from old grapevine leaves with Spectrum™ Plant Total RNA kit (Sigma) with standard method (A and C) or modified method (B and D). (A) and (B): Agarose gel analysis of RT-PCR products amplified with primers RSP35 and RSP36 (Additional file 1: Table S1) on 11 total RNA extracts listed in Table 1 with Method A or B respectively. (C) and (D): Agarose gel analysis of RT-PCR products amplified with UBI primers on these 11 total RNA extracts using Method A and Method B respectively. M: molecular size marker (bp); lane 12: water. (PPT 1035 kb

The complete chloroplast genome and phylogenetic analysis of Gentiana manshurica Kitag from China

Gentiana manshurica Kitag is a perennial medicinal plant with high economic value. In this study, we first reported the complete chloroplast (cp) genome of G. manshurica. The total length of the cp genome was 149,185 bp in length, consisting of a large single-copy (LSC) region of 81,347 bp, a small single-copy (SSC) region of 17,268 bp, and a pair of inverted repeats (IRs) regions of 25,285 bp each. The cp genome contained 130 genes, including 85 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. The overall GC content was 37.61%. The phylogenetic analysis indicated that the G. manshurica belongs to the tribe Gentianeae, and showed a close relationship with other Gentiana species