Prevalence and genetic diversity of Grapevine virus A in Tunisia

Prevalence and genetic diversity of the complete CP gene of Grapevine virus A (GVA) were assessed in isolates from rootstocks, wine and table grape varieties and autochthonous grapevines. Wine grapes were the most infected (63%), followed by table grapes (49%) and rootstocks (44%). Autochthonous grapevines were the less infected (35%). Analyses of the complete coat protein sequences of 20 GVA isolates from the main grapevine growing areas of Tunisia identified three phylogroups, accounting, respectively, for 70% (group I), 25% (group IV) and 5% (group III) of the isolates. No sequences clustered into group II. Phylogenetic analyses indicated that Tunisian GVA isolates are not grouped by the host cultivar or geographic origin

Managing the deluge of newly discovered plant viruses and viroids: an optimized scientific and regulatory framework for their characterization and risk analysis

The advances in high-throughput sequencing (HTS) technologies and bioinformatic tools have provided new opportunities for virus and viroid discovery and diagnostics. Hence, new sequences of viral origin are being discovered and published at a previously unseen rate. Therefore, a collective effort was undertaken to write and propose a framework for prioritizing the biological characterization steps needed after discovering a new plant virus to evaluate its impact at different levels. Even though the proposed approach was widely used, a revision of these guidelines was prepared to consider virus discovery and characterization trends and integrate novel approaches and tools recently published or under development. This updated framework is more adapted to the current rate of virus discovery and provides an improved prioritization for filling knowledge and data gaps. It consists of four distinct steps adapted to include a multi-stakeholder feedback loop. Key improvements include better prioritization and organization of the various steps, earlier data sharing among researchers and involved stakeholders, public database screening, and exploitation of genomic information to predict biological properties

Prevalence of Viruses Associated with Grapevine Rugose Wood Disease in Tunisia

To assess the prevalence and the distribution of viruses associated with rugose wood (RW) disease in Tunisian grapevines, surveys were conducted in the main Tunisian grapevine growing areas. A total of 403 samples were collected including autochthonous and international table and wine grapes, and rootstocks. All samples were analyzed by RT-PCR for the presence of Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine virus D (GVD), Grapevine virus E (GVE), Grapevine virus F (GVF) and Grapevine rupestris stem pitting-associated virus (GRSPaV), using specific primers. Molecular analysis showed that 80.9% (326 / 403) of the tested samples were infected with at least one virus. GRSPaV was the most widespread virus (51.3%), followed by GVA (47.9%), GVD (31.5%), GVF (22.3%), GVB (17.8%), and finally GVE (7.2%). According to grapevines typology, wine grapes were the most infected (93.9%) vines, followed by table grapes (87.8%) and rootstocks (75.0%). Autochthonous grapevine varieties were the less infected (65.9%). This is the first study on RWassociated viruses in autochthonous grapevines and rootstocks and the first report on the presence of GVE and GVF in Tunisian vines

Prevalence of Viruses Infecting Autochthonous Grapevines in Tunisia

The incidence of virus infections was conducted in the grapevine germplasm collection at the Institut National de la Recherche Agronomique de Tunisie. In this grapevine collection, 162 different autochthonous cultivars were maintained, including numerous spontaneous ecotypes coming from different Tunisian grapevine growing regions. All accessions were sampled and analyzed by DASELISA for the presence of Grapevine leafroll associated viruses 1, 2, 3 (GLRaV-1, -2, -3), Grapevine fanleaf virus (GFLV), Grapevine fleck virus (GFkV) and Arabis mosaic virus (ArMV), using commercial polyclonal antisera. Almost all the major grapevine-infecting viruses assayed, except for ArMV, were detected in the tested cultivars. Conversely, all the wild grapevine accessions were found to be free from the same viruses. Out of 141 cultivars submitted to DAS-ELISA, 40.4% were infected with at least one virus. GLRaV-3 was the prevailing virus (23.4%), followed by GLRaV-1 (19.6%), GFkV (9.2%), GLRaV-2 (4.2%), and GFLV (1.4%). Cultivars collected from northern regions (61.4%) were more infected than their homologues from southern regions (19.7%)

Sanitary Selection of Virus-Tested Fig (Ficus carica) Cultivars in Tunisia

Field surveys were carried out during autumn 2011 and spring 2012, in different fig orchards located at Rafraf, Takelsa, Mornag, Djebba, Sousse, and Sfax to select virus-free plants. A total of 202 trees representing 26 fig cultivars were prospected and sampled. Total nucleic acids were extracted from leaf veins and tested by RT-PCR for the presence of FMV,FLMaV-1, FLMaV-2, FMMaV, FCV, and FFkaV using specific primers. PCR results indicate that all these viruses were present in the Tunisian fig orchards. The average of infection level determined by RT-PCR was 63.86%. FMV was proved to be the most widespread virus (37.12%), followed by FLMaV-1 (11.9%), FFkaV (11.4%), FCV (8.9%), FMMaV (8.4%), and finally FLMaV-2 (5.9%). Among the 26 tested cultivars, only 7 (Marghrebi, Boukhobza, Zagfar, Assafri, Kahli, Chetoui, Delgane) were free from the tested viruses. By the contrary, the sanitary status of the main Tunisian cultivars Bayoudhi, Bouhouli, Soltani, Zidi and Bither, seemed heavily degraded (75, 70.37, 69.23, 66.03, and 46.15% of infection, respectively). Seven cultivars (Wahchi, Khartoumi, Thguegli, Besbessi, Bidh-Bghal, Njeli and Khedhri) were totally infected. This study allowed the identification of at least one “virus-tested” candidate clone from 19 different fig cultivars which can represent the potential mother plants for propagating materials in order to establish new fig nurseries and orchards

Elimination of Grapevine leafroll associated virus-3, Grapevine rupestris stem pitting associated virus and Grapevine virus A from a Tunisian Cultivar by Somatic Embryogenesis and Characterization of the Somaclones Using Ampelographic Descriptors

Prospecting of local grapevine (Vitis vinifera L.) germplasm revealed that Tunisia possesses a rich patrimony which presents diversified organoleptic characteristics. However, viral diseases seriously affect all local grapevine cultivars which risk a complete extinction. Sanitation programs need to be established to preserve and exploit, as a gene pool, the Tunisian vineyards areas. The presence of the Grapevine leafroll associated virus-3 (GLRaV-3), Grapevine stem pitting associated virus (GRSPaV) and Grapevine virus A (GVA), were confirmed in a Tunisian grapevine cultivar using serological and molecular analyses. The association between GRSPaV and GVA viruses induces more rugose wood symptoms and damages. For this reason the cleansing of the infected cultivar is highly advisable. Direct and recurrent somatic embryos of cv. ‘Hencha’ were successfully induced from filament, when cultured on Chée and Pool (1987). based-medium, enriched with 2 mg 1−1 of 2,4-dichlorophenoxyacetic acid and 2.5 mg 1−1 of Thidiazuron, after 36 weeks of culture. After six months of acclimatization, RT-PCR carried on 50 somaplants confirmed the absence of GVA, GRSPa-V as well as GLRaV-3 viruses in all somaplants. Ampelographic analysis, based on eight OIV descriptors, was carried out on two years acclimated somaplants, compared to the mother plant. Results demonstrated that the shape and contours of 46 somaclones leaves are identical to mother plant leaves and four phenotypically off-type plants were observed. The healthy state of 100% ‘Hencha’ somaclones and the high percentage of phenotypically true-to-type plants demonstrate that somatic embryogenesis is a promising technique to adopt for grapevine viruses elimination

Going beyond consensus genome sequences: an innovative SNP-based methodology reconstructs different Uganda cassava brown streak virus haplotypes at a nationwide scale in Rwanda.

International audienceCassava Brown Streak Disease (CBSD), which is caused by cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), represents one of the most devastating threats to cassava production in Africa, including in Rwanda where a dramatic epidemic in 2014 dropped cassava yield from 3.3 million to 900,000 tonnes (1). Studying viral genetic diversity at the genome level is essential in disease management, as it can provide valuable information on the origin and dynamics of epidemic events. To fill the current lack of genome-based diversity studies of UCBSV, we performed a nationwide survey of cassava ipomovirus genomic sequences in Rwanda by high-throughput sequencing (HTS) of pools of plants sampled from 130 cassava fields in 13 cassava-producing districts, spanning seven agro-ecological zones with contrasting climatic conditions and different cassava cultivars. HTS allowed the assembly of a nearly complete consensus genome of UCBSV in 12 districts. The phylogenetic analysis revealed high homology between UCBSV genome sequences, with a maximum of 0.8 % divergence between genomes at the nucleotide level. An in-depth investigation based on Single Nucleotide Polymorphisms (SNP) was conducted to explore the genome diversity beyond the consensus sequences. First, to ensure the validity of the result, a panel of SNPs was confirmed by independent RT-PCR and Sanger sequencing. Furthermore, the combination of fixation index (FST) calculation and Principal Component Analysis (PCA) based on SNPs patterns identified three different UCBSV haplotypes geographically clustered. The haplotype 2 (H2) was restricted to the central regions, where the NAROCAS 1 cultivar is predominantly farmed. RT-PCR and Sanger sequencing of individual NAROCAS1 plants confirmed their association with H2. Haplotype 1 was widely spread, with a 100% occurrence in the Eastern region, while Haplotype 3 was only found in the Western region. These haplotypes’ associations with specific cultivars or regions would need further confirmation. Our results prove that a much more complex picture of genetic diversity can be deciphered beyond the consensus sequences, with practical implications on virus epidemiology, evolution, and disease management. Our methodology proposes a high-resolution analysis of genome diversity beyond the consensus between and within samples. It can be used at various scales, from individual plants to pooled samples of virus-infected plants. Our findings also showed how subtle genetic differences could be informative on the potential impact of agricultural practices, as the presence and frequency of a virus haplotype could be correlated with the dissemination and adoption of improved cultivars