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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.

Selmi, I., Elbeaino, T., Arezki, L., El Air, M., Digiaro, M., and Mahfoudhi, N. 2021. Prevalence and genetic diversity of grapevine virus D in Tunisia. Tunisian Journal of Plant Protection 16 (2): 19-27.

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.

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.

A survey was conducted of central and western Algerian grape cultivars and a germplasm collection to detect the presence of Grapevine leafroll-associated virus 1 (GLRaV-1), using DAS-ELISA. The virus was found in 26 of 484 sample (5.4%). No infection was found in the germplasm collection. Analysis of the sequence of a coat protein gene region revealed that of the 17 recognised phylogenetic groups, the Algerian isolates belong to Groups I, II and XVI. This is the first study of genetic diversity of GLRaV-1 in Algeria.

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.

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.

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%).