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

Occurrence and distribution of pome fruit viruses in Tunisia

The phytosanitary status of pome fruit trees was examined in Tunisia, in surveys conducted in spring 2009 and 2010, in the main Tunisian mother blocks. A total of 248 samples were collected (111 from apple, 106 from pear and 31 from quince), and tested for the presence of Apple chlorotic leaf spot virus (ACLSV) and Apple mosaic virus (ApMV) using ELISA and RT-PCR, and for Apple stem pitting virus (ASPV) using RT-PCR. 37% of the samples were infected by at least one virus. ACLSV was the most widespread virus (34% of samples), followed by ApMV (4%). Furthermore, molecular analysis showed that 69% of the sampled trees were infected and apple was the most infected species (80%), followed by pear (75%) and quince (10%). ASPV was the most prevalent virus (46%), followed by ACLSV (39%) and ApMV (10). Mixed infections occurred in several trees, and the most common combination was ASPV+ACLSV (23%). This is the first report on the presence of viruses infecting pome fruits in Tunisia

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

Efficacy of Tissue Culture in Virus Elimination from Caprifig and Female Fig Varieties (Ficus carica L.)

Fig mosaic disease (FMD) is a viral disease that spreads in all Tunisian fig (Ficus carica L.) orchards. RT-PCR technique was applied to leaf samples of 29 fig accessions of 15 fig varieties from the fig germplasm collection of High Agronomic Institute (I.S.A) of Chatt-Mariem, to detect viruses associated to FMD. Analysis results show that 65.5% of the accessions (19/29) and 80.0% (12/15) of the fig varieties are infected by FMD-associated viruses. From all fig accessions, 41.4% of them are with single infection (one virus) and 24.1% are with multi-infections (2 virus and more). Viruses infecting fig leaf samples are Fig mosaic virus (FMV) (20.7%), Fig milde-mottle-associated virus (FMMaV) (17.25%), Fig fleck associated virus (FFkaV) (3.45%), and Fig cryptic virus (FCV) (55.17%). A reliable protocol for FCV and FMMaV elimination from 4 local fig varieties Zidi (ZDI), Soltani (SNI), Bither Abiadh (BA), and Assafri (ASF) via in vitro culture of 3 meristem sizes was established and optimized. With this protocol, global sanitation rates of 79.46%, 65.55%, 68.75%, and 70.83% respectively for ZDI, SNI, BA, and ASF are achieved. For all sanitized varieties, the effectiveness of meristem culture for the elimination of FCV and FMMaV viruses was related to meristem size. Meristem size 0.5 mm provides the highest sanitation rates ranging from 70% to 90%

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

Diversification of Vascular Occlusions and Crystal Deposits in the Xylem Sap Flow of Five Tunisian Grapevines

Xylem vessels are essential pivotal organs in bulk hydraulic flow through the whole woody plant. However, environmental constraints generate disagreements in xylem structures, which are characterized by air emboli and occlusions formations, compromising water conductivity in grapevines. The aim of this work was to explore xylem morphology dynamics through the xylem sap flow of five Tunisian grapevine cultivars during the natural bleeding sap periods of 2019, 2021, and 2022. In fact, Sakasly, Khamri, Hencha, Razegui1, and Razegui2 rain-fed grapevine cultivars revealed differential responses towards xylem sap movement. The results demonstrated that the xylem sap flow was significantly more abundant in 2019 than 2021 and 2022 bleeding sap campaigns. A variation was revealed between the cultivars regarding the xylem sap flow. In fact, Sakasly gave the best xylem flow during the three campaigns. Razegui1 and Razegui2 registered approximately similar xylem sap flow, while Hencha and Khamri present the lowest sap fluxes during the three campaigns. Moreover, several vascular occlusions forms were identified from stem cross sections using environmental scanning electron microscopy (ESEM), including tyloses, gels, starch, and gum deposits. The highest occlusion number was observed in Sakasly, Razegui1, and Razegui2 cultivars. Among different biogenic calcium shapes, several were observed for the first time in grapevine, including multi-faceted druse, cubic, crystalline sand, styloids, spherical, or drop-like structures. Considering their lower flow and totally blocked vessels, both Hencha and Khamri confirmed their susceptibility to environmental constraints. However, Sakasly, Razegui1, and Razegui2 cultivars presented higher tolerance according to their sap flow and xylem morphology

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