Grapevine fleck virus (GFkV) elimination in a selected clone of Vitis vinifera L. cv. Manto Negro and its effects on photosynthesis

摘要

Aims: The use of healthy propagating material is required to control grapevine viruses. The aim of this work was to eliminate Grapevine fleck virus (GFkV) from a Manto Negro clone, a local grapevine variety, in order to include this material in certification programs. Additionally, the effects of virus elimination on photosynthesis and related parameters were evaluated.Methods and results: Two method combinations for virus elimination were evaluated: (1) field thermotherapy and shoot tip culture and (2) chamber thermotherapy and shoot tip culture. GFkV elimination was tested by double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). The results suggest that a natural field thermotherapy prior to shoot tip culture is effective, making unnecessary the chamber thermotherapy treatment. Additionally, the effects of virus elimination on gas exchanges, chlorophyll fluorescence, electron transport rate (ETR), protein and pigment content were evaluated. The results indicate that GFkV infection affects physiological processes, especially stomatal conductance (gs), whereas photosynthesis, protein, pigment content, ETR, and fluorescence parameters were not significantly changed.?Conclusion: This study described a simple and rapid method that requires only one medium for virus elimination (GFkV). Beyond its sanitation potential, the use of larger explants (1-3 mm) ensures the integrity of the clone. The presence of the virus affects physiological processes, especially gs, demonstrating the beneficial effect of eliminating GFkV. Significance and impact of the study: The described method has the potential to produce GFkV-free rooted plantlets faster than other methods while being potentially safer in maintaining the genetic and phenotypic stability of the regenerated clone. The beneficial effects of GFkV elimination provide evidence for the importance to detect this virus prior to the inclusion of clones in certification programs. IntroductionManto Negro is the most widely cultivated variety in Majorca (Spain) with approx. 20% of the total grape cultivation area. The importance of this variety in the Balearic Islands has stimulated a clonal selection process for its characterization, improvement and conservation. Clonal selection is the most common method to improve grapevine varieties (Walter and Martelli, 1997; Mannini, 2000), and usually the genetic and sanitary selections are performed simultaneously (Mannini, 2000). Previous studies have demonstrated that the phytosanitary status of vineyards in the Balearic Islands is strongly degraded due to the high degree of virus infection. Cretazzo et al. (2010b) showed that only 6.4% of over 200 Manto Negro vines, sited in 33 different vineyards, were virus-free. Among the candidate clones chosen for evaluation in homologation fields, only one Manto Negro vine displayed the ?virus-free certified? status. No other ?virus-free certified? vines could be selected as candidate clone because they did not meet the minimum agronomic requirements. Infection by multiple virus strains was detected in 58.4% of Manto Negro vines. Given this scenario, it was very difficult to identify virus-free clones with suitable agronomic characteristics to be considered as a reference for the grape market.The final aim of any clonal selection process is the achievement of clones free from the most harmful grapevine viruses (certified clones) while possessing varietal identity. According to the Commission Directive 2005/43/EC amending the Annexes to Council Directive 68/193/EEC on the marketing of grapevine propagation material, each member state should ensure the absence of Grapevine fanleaf virus (GFLV), Grapevine leafroll-associated virus 1 and 3 (GLRaV-1 and GLRaV-3), Arabis mosaic virus (ArMV) and Grapevine fleck virus (GFkV) (for rootstocks only) in grapevine nursery plants. A candidate clone, MPL15.01, met the minimum agronomic requirements but was infected with GFkV, a phloem-limited virus responsible for grapevine fleck, a widespread graft-transmissible disease of grapevine (Martelli, 1993). The use of healthy propagating material is strongly advised and represents an important measure to control grapevine viruses, since there is no treatment or cure for viral diseases established in the field. In endemic areas, sanitation becomes the main focus of the clonal selection process. High infection degrees among selected autochthonous clones entail the need for a quick and easy technique in order to "clean up" large amounts of material. Although certification regulations require the absence of GFkV in rootstocks only, it is inappropriate to include a clone infected with GFkV in a certification program. In fact, GFkV can easily move between scion and rootstock through phloem and may negatively affect yield and grape quality, usually in combination with other viruses (Teray, 1990; Kovacs et al., 2001; Komar et al., 2007; Cret