Introduction

Manto 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; Cretazzo et al., 2010a). Despite these findings, there are very few studies on the elimination of GFkV and even less information about its effects on photosynthesis or other physiological parameters. In general, the consequences of biotic stress on Vitis vinifera L. are still far from being completely understood; several studies on the effects of virus and other pathogens on grapevine physiology showed highly variable patterns (Balachandran et al., 1997; González et al., 1997; Christov et al., 2001; Sampol et al., 2003; Petit et al., 2006; Komar et al., 2007; Christov et al., 2007; Basso et al., 2010). In particular, a number of viruses are known to affect grape yield and quality (González et al., 1997; Guidoni et al., 1997; Cabaleiro et al., 1999; Cretazzo et al., 2010a; Lee and Martin, 2009; Lee et al., 2009; Basso et al., 2010). However, there is only scarce information about the effects of GFkV on plant physiology.

The aim of this study was to develop an effective procedure to eliminate GFkV in order to obtain clones suitable for certification. Additionally, the effects of virus elimination on different photosynthetic parameters were evaluated.

Materials and methods

1. Plant material and treatments

A candidate clone (MPL15.01) of Manto Negro cv. was tested for GFLV, GLRaV-1, GLRaV-2, GLRaV-3 and GFkV. It displayed a single infection with GFkV (Cretazzo et al., 2010b). Two sanitation methodologies were compared: (1) field thermotherapy and shoot tip culture and (2) chamber thermotherapy and shoot tip culture. Both thermotherapy treatments occurred in vivo. In (1), the naturally high and increasing temperature in the vineyard during the experiment acted as a natural thermotherapy; in (2), the thermotherapy treatment was applied in a growth chamber. Two different media were tested for the establishment of explants: Murashige and Skoog’s basal medium (Murashige and Skoog, 1962), 3% sucrose and 0.7% agar without any hormonal supplement (MS-0) and supplemented with 2 mg/L of 6-benzyladenine (MS-BA). The same basal media with half strength of macro and microelements were used as rooting media.

1.1. Field thermotherapy and shoot tip culture

Shoots from clone MPL15.01 grown in field trials were taken at its maximum growth period (July 2009). Plants were well irrigated and some shoots removed, ensuring maximum new shoot growth. High temperatures were predicted through the web site www.wetterzentrale.de/topkarten/fsavneur.html. During the week prior to sampling, the average maximum temperature did not fall below 30ºC, reaching over 39ºC three days before sampling. The average night-time temperature for the week before the experiment was 19.7ºC and did not fall below 16ºC. Shoots were stripped of leaves and washed with tap water. Growing shoot tips were rinsed in 70% ethanol for 30 s and soaked for 15 min in an aqueous solution of sodium hypochlorite (1% active chlorine) plus a few drops of Tween-20. After surface sterilization, the plant material was washed three times in sterile distilled water to remove sterilizing agents. Shoot tips (1-3 mm) were isolated and cultivated on MS-0 and MS-BA. Glass test tubes (150 mm x 24 mm) with 25-mm diameter plastic stoppers (Kap-Uts K25, Bellco) and containing 20 ml of medium were used. Cultures were incubated in a Fitoclima S600PLH chamber (Aralab) at 23ºC under a 16-h photoperiod and light intensity of 56 μmol e^- m^-2 s^-1 provided by white fluorescent tubes. Relative humidity (RH) was around 60%. After 5 weeks of culture, explants were transferred to rooting media.

1.2. Chamber thermotherapy

Plants were obtained by direct rooting of 0.2-m dormant canes from selected field-grown MPL15.01 vines. Plants were maintained in the greenhouse until sprouting. After 5 weeks, plants with actively growing shoots were subjected to chamber thermotherapy. Plants were transferred into a growth chamber at 26ºC/22ºC (day/night) under a 16-h photoperiod and light intensity of 56 μmol e^- m^-2 s^-1 provided by white fluorescent tubes. Temperature was gradually increased by 4ºC per week. Final day/night temperatures (37.5ºC/34.0ºC) were maintained for 40 days. After this period, apical and axillary buds were excised, surface-sterilized and cultivated as described above.

2. Transfer to ex vitro conditions

Rooted plantlets were transferred to sterile soil and maintained in a growth chamber at 25ºC, low light intensity and 90% RH for 15 days. Plantlets were transferred into a greenhouse under higher light intensity but still with high RH. RH was gradually decreased to obtain full ex vitro acclimation and active growth. Two months later, plants were placed outdoors under a shading mesh to protect them from direct sunlight. Plants were grown outdoors in 5-L pots filled with organic substrate and perlite mixture (3:1). Approximately one year after acclimation, when plants had sufficient lignified material, grapevines were sampled to check their sanitary status. Plants were irrigated daily from May to October as follows: 5 times a week with water and twice a week with 50% Hoagland solution (Hoagland and Arnon, 1950). Gas exchange measurements were performed during the last week of July as described below.3.

3. Virus detection

3.1 ELISA

Plants were tested for GFkV before and after treatments. Petiole or leaf samples were extracted and analyzed by indirect DAS-ELISA (Clark and Adams, 1977) to detect the presence of GFkV using a commercial kit (Bioreba) according to manufacturer’s instructions.

3.2 RT-PCR

Total RNA was extracted from grapevines (50 mg of phloem scraped from mature canes or leaves) using Spectrum Plant Total RNA Kit (Sigma-Aldrich) according to manufacturer’s instructions. First-strand cDNA synthesis was performed using 500 ng of total RNA, 200 units of recombinant Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (Invitrogen Life Technologies), 40 units of RNase inhibitor (RNase Out, Invitrogen Life Technologies), 0.4 mM of dNTPs, and 2 mM of random nonamers (Takara Bio). The mixture for reverse transcriptase (20 μl) was incubated for 50 min at 37ºC and the reaction was heat-inactivated at 70ºC for 15 min.

GFkV-specific primer pairs GFk V1/F and GFk C1/R (Osman et al., 2008) were used for PCR amplification of a 344-bp fragment. The PCR reaction mix (25 μl) contained 2 μl of cDNA, 1 mM of dNTPs, 0.5 mM of each primer and 5 units of Taq polymerase (Takara Taq^TM, Takara Bio). Thermo-cycling was performed as follows: 30 min at 52ºC followed by 35 cycles of 94ºC for 30 s, 58ºC for 45 s and 72ºC for 60 s, final extension at 72ºC for 7 min, and storage at -20ºC. Finally, 10 μl of amplification product was electrophoresed on a 2% agarose gel in TBE buffer [90 mM Tris–borate, 2 mM EDTA, pH (8.0)], stained with ethidium bromide, visualized on an UV transilluminator and photographed. Positive and negative GFkV controls were included in the experiment.

4. Gas exchange and chlorophyll fluorescence measurements

GFkV-free plants (sanitized) and GFkV-infected (not sanitized) acclimated plants coming from the GFkV elimination experiment were used to determine the possible effects of GFkV on photosynthetic parameters. Four plants per treatment were selected to perform measurements without distinction between the two techniques used for virus elimination. Leaf gas exchange and chlorophyll a (Chl a) fluorescence were simultaneously measured with an open infrared gas exchange analyser system equipped with a leaf chamber fluorometer (Li-6400-40, Li-Cor, Lincoln NE, USA). All measurements were taken between noon and 1 P.M. local time at 1500 μmol e^- m^-2 s^-1, with a CO₂ concentration in the leaf cuvette of 400 μmol CO₂ mol^-1 air. Five to eight young, fully expanded leaves from four different plants per treatment were analyzed.

5. Pigment analysis and protein content

Pigment analysis and protein content were performed in eight samples per treatment (two replicates per plant). Leaf discs (5.3 cm²) were freeze-clamped in liquid nitrogen and stored at -70°C until assay. Samples were ground to fine powder in a liquid nitrogen-chilled mortar and homogenised in 1 ml of extraction medium. The extraction medium was optimised for this cultivar by testing different components. The extraction medium used contained 0.5 M Tris (pH 7.5), 10 mM EDTA, 1% Triton X-100 and 2% b-Mercaptoethanol. For pigment determinations, 50 μl of crude extract were diluted in 950 μl of ethanol and after 10 min in the dark, extracts were clarified by centrifugation (12000 rpm at 4ºC for 2 min) and Chl a, Chl b and carotenoids were quantitatively determined by spectrophotometry according to Lichtenthaler and Wellburn (1983). For protein content determination, extracts were clarified by centrifugation (12000 rpm at 4ºC for 2 min) and supernatants assayed for total soluble protein according to the method of Bradford (1976).

6. Determination of leaf number and leaf area

Total number of leaves per plant and total leaf area were determined during the same week as gas exchange and chlorophyll fluorescence measurements. Leaf area was measured using public domain image analysis software (ImageJ version 1.32; http://rsb.info.nih.gov/ij/). Twenty leaves of different sizes were used to develop a regression equation for leaf area estimation based on linear measurements (leaf length).

7. Statistical analysis

The influence of media and treatments on survival and virus eradication rates was analyzed by a Chi-Square Test using the SPSS statistical package v12 (SPSS Inc., Chicago IL, USA). One-way ANOVA was also performed for physiological parameters to compare GFkV-free and GFkV-infected plants.

Results

1. Virus detection

The candidate clone MPL15.01 displayed an infection by GFkV (Cretazzo et al., 2010b). Virus detection in mother plants grown in a commercial vineyard was carried out by DAS-ELISA. Neither leaf nor other symptoms were observed in these plants, as GFkV is symptomless in Vitis vinifera L. Virus infection of mother plants was confirmed by RT-PCR. The expected 344-bp amplification product was observed in GFkV-infected plants (mother and non-sanitized plants), while no amplification was obtained in GFkV-free ones (certified GFkV-free clone used as negative control and sanitized plants) (Figures 1 and 2).

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Figure 1. Agarose gel analysis of RT-PCR assays with specific primers for GFkV. Lane 1. Positive sample originated from field thermotherapy and shoot tip culture (non-sanitized plant). Lane 2. Negative sample originated from field thermotherapy and shoot tip culture (sanitized plant). Lane 3. Field-grown original plant material, positive control. Lane 4. Certified clone, negative control. Lane 5. Blank. M. 100-bp DNA ladder.

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Figure 2. Agarose gel analysis of RT-PCR assays with specific primers for GFkV. Lane 1. Negative sample originated from chamber thermotherapy and shoot tip culture (sanitized plant). Lane 2. Positive sample originated from chamber thermotherapy and shoot tip culture (non-sanitized plant). Lane 3. Field-grown original plant material, positive control. Lane 4. Certified clone, negative control. Lane 5. Blank. M. 100-bp DNA ladder.

2. Explant survival and virus elimination

Regeneration rate from shoot tip culture was significantly higher in the MS-0 medium than in the MS-BA medium in both treatments (Table 1). In spite of the efforts for virus elimination using shoot tips instead of meristems, the survival rates were low. Values for shoot tip survival were 33% in MS-0 and 16% in MS-BA for the field thermotherapy treatment. Similar values were obtained in plants undergoing heat treatment in a growth chamber (38% in MS-0 and 10% in MS-BA). The addition of BA to the medium resulted in considerable shoot proliferation and hyperhydricity, also known as vitrification (Figure 3). These results indicate that BA is not necessary for proliferation and rooting and may even be detrimental.

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Figure 3. 6-Benzyladenine (BA) effect on shoot proliferation. (A) Explant established on MS-BA medium. (B) Explant established on MS-0 medium.

Based on ELISA tests, GFkV was eliminated from 25% of the regenerated plantlets in the field thermotherapy and shoot tip culture treatment (Table 1). In order to confirm these results, RT-PCR amplification was carried out for ELISA-negative samples. No PCR products were obtained from these samples, therefore confirming the immunoenzymatic assay results (Figure 1).

The percentage of sanitized plants was not significantly increased when the heat treatment was performed in a growth chamber prior to shoot tip culture, with 20% of the plants testing negative (Table 1). Figure 2 shows the RT-PCR products obtained from ELISA-negative samples and controls (positive and negative) using specific primers for GFkV. The RT-PCR results confirm the percentage of sanitized plants, as no RNA amplification was obtained in lanes corresponding to ELISA-negative samples coming from the chamber thermotherapy and shoot tip culture treatment.

3. Effects of virus elimination on leaf area and photosynthetic parameters

GFkV-free plants presented fewer but larger leaves than infected ones; therefore total leaf area was not significantly different (Table 2).

3.1. Gas exchange

GFkV-infected plants showed lower photosynthetic rate (A_N), although the difference was not statistically significant (Table 2). However, significant reductions were observed in stomatal conductance (g_s) and transpiration rate (E). Intercellular CO₂ concentration (Ci) was lower in GFkV-infected plants; this decrease indicates that non-stomatal limitations are not present, which is consistent with the absence of variation in protein content (Table 2).

3.2. Chlorophyll fluorescence and pigment content

No differences were observed between GFkV-free and GFkV-infected plants in the chlorophyll fluorescence parameters Fm’, ΦPSII and ETR (Table 2). This suggests that PSII was not impaired. There were no visible leaf symptoms in infected plants. Chlorophyll and carotenoid contents did not change with viral infection, which confirms that pigment degradation did not occur by GFkV infection (Table 2).

Discussion

The high incidence of virus infection in Manto Negro cv. requires the elimination of harmful viruses from selected clones using the most appropriate technique. Sanitation is the only way to generate a virus-free clone to be included in clonal selection process.

Several techniques have been used for grapevine virus elimination. Both meristem culture (Sampol et al., 2003; Maliogka et al., 2009; Youssef et al., 2009) and somatic embryogenesis (Gambino et al., 2006; Borroto-Fernandez et al., 2009; Gambino et al., 2009) are reported to eliminate different virus infections from Vitis vinifera L. The lack of vascular connections between parent tissue and meristems or embryos can be the reason for the sanitation occurring during cultures, especially for phloem-limited virus like GFkV. To maximize virus elimination, these methods may be combined with heat treatment before or after the establishment of tissue culture (Krizan et al., 2009; Gribaudo et al., 2006). Valero et al. (2003) claimed that a form of “natural thermotherapy” can occur during summer when the vineyard temperatures reach more than 38ºC. In vitro culture of shoot tips under these conditions was used with success for GLRaV-3 and GFLV (Valero et al., 2003), but none of these methodologies were tested for GFkV elimination. In the present study, the combination of either high temperature during summer in the field or growth chamber thermotherapy treatment with shoot tip culture was effective for the elimination of GFkV (25% and 20%, respectively). This contrasts with Panattoni and Triolo (2010), who recently showed that GFkV was insensitive to heat stress. Komar et al. (2007) managed to eliminate GFkV from a clone of Chardonnay using only shoot apex culture, however, the elimination rate was not presented. In general, it seems that the success of a method not only depends on grapevine genotype but also on virus species and the specific virus-grapevine interactions (Maliogka et al., 2009). It is likely that the application of other elimination techniques such as somatic embryogenesis or meristem culture to GFkV-infected vines will provide higher sanitation rates, as shown for other virus infections (Gambino et al., 2006; Gribaudo et al., 2006; Borroto-Fernandez et al., 2009; Gambino et al., 2009). However, these virus elimination techniques are much more difficult, laborious, time consuming and present the risk of genetic variation (Popescu et al., 2002).

The possibility of somaclonal variation should also be taken into account. Although genetic changes in the grapevine genome after in vitro or thermotherapy treatments are minimal or null (Baránek et al., 2009; 2010), the use of larger explants (>1 mm) in the present experiment gives additional security to minimize genetic or phenotypic changes induced by tissue culture. In this sense, a similar method with a reduced subculture number and the use of hormone-free culture medium during growth phase was used in the sanitation of Malvasia de Banyalbufar cv. (Sampol et al., 2003) and showed to be efficient in the non-appearance of juvenile characters (Carámbula et al., 2009). Therefore, it should be possible to achieve GFkV elimination from grapevine by using relatively large explants (1-3 mm) and avoiding the difficulties of meristem tip culture or somatic embryogenesis. A culture medium without any hormone supplement was effective for proliferation and rooting. The lower survival of plantlets in MS-BA medium could be attributed to vitrification. Vitrified plantlets have high water content in leaf and stem tissues and poor epicuticular wax production (Pâques and Boxus, 1987; Majada et al., 2001), which lowers survival under ex vitro conditions. The use of a culture medium without hormone supplements really simplifies the production of GFkV-free plants.

GFkV-free and still GFkV-infected plants were compared in order to assess the effects of virus elimination on grapevine physiology. Results clearly showed that GFkV infection induced a significant g_s reduction and lower internal CO₂ concentration. Consequently, lower photosynthesis was observed in GFkV-infected plants, although the reduction was not statistically significant, as observed in other grapevine varieties and virus infections (Guidoni et al., 1997; Cabaleiro et al., 1999; Sampol et al., 2003) or other grapevine pathogens as Esca (Petit et al., 2006) and Pierce’s disease (Goodwin et al., 1988). It seems that the effects are mostly at the stomatal level, since neither fluorescence parameters nor the pigment content or ETR were affected. In contrast to previous suggestions (Sampol et al., 2003), the smaller reduction in photosynthesis with respect to g_s suggests that, despite the reduction of CO₂ availability at the substomatal level, Rubisco activity seems unaffected. This is consistent with the non-alteration of total protein content. A possible explanation for the maintenance of photosynthetic activity despite the significant reduction in g_s could be an increase in mesophyll conductance (g_mes), which somehow compensates a lower CO₂ input. The lack of co regulation between g_s and g_mes has been demonstrated under stress conditions (Perez-Martin et al., 2009). This hypothesis disagrees with findings in Malvasia de Banyalbufar vines infected by GFLV and GLRaV (Sampol et al., 2003) since g_mes was lower in virus-infected than in non-infected plants and the reduction of CO₂ diffusion through the mesophyll was considered an important factor limiting photosynthesis in virus-infected plants. Genotype, virus type and degree of virus infection may play an essential role in the severity of biotic stress. Further studies with different grape cultivars and/or virus infections will provide a better understanding of the mechanisms of virus-vine interactions.

Conclusion

Our study described a simple and rapid method that requires only one medium for virus elimination (GFkV). The method has the potential to produce GFkV-free rooted plantlets in a shorter time than other methods and is potentially safer with respect to maintaining the genetic stability of the regenerated clone. Furthermore, the data presented here suggest that GFkV can affect physiological processes, especially g_s, but further studies on these disturbances are needed to better understand the influence of the virus on photosynthesis.

Acknowledgements: This work was supported through research projects RTA2008-00085-C02-01 and RTA2010-00118-00-00 funded by INIA, Spain. We gratefully acknowledge D. Francis and M. Ribas-Carbó for grammar correction. We are also grateful to C. Padilla for helping in data collection and H. El Aououad for translating the abstract into French.

Abbreviations: BA, 6-benzyladenine; DAS-ELISA, double antibody sandwich-enzyme-linked immunosorbent assay; ETR, electron transport rate; GFkV, Grapevine fleck virus; GFLV, Grapevine fanleaf virus; GLRaV-1 and -3, Grapevine leafroll-associated virus 1 and 3; MS, Murashige and Skoog’s basal medium; RH, relative humidity; RT-PCR, reverse transcription-polymerase chain reaction; g_s, stomatal conductance

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