Abstract
The effect of selective virus elimination on vigor, yield, and fruit quality of Vitis vinifera cv. Chardonnay was evaluated over six years in a replicate field trial. Test material was propagated from three clones infected by multiple virus combinations (GLRaV-1, GVB, and GRSPaV; GFkV, GLRaV-1, GVB, and GRSPaV; GLRaV-2, GLRaV-3, and GRSPaV) and virus-like diseases (vein mosaic and vein necrosis), and selected progeny were obtained after virus elimination by heat therapy and in vitro apex grafting. Some but not all viruses were eradicated in the progeny subjected to virus elimination, as shown by graft indexing and DAS-ELISA using appropriate γ-globulins, allowing for comparative viticultural performance analysis of clonal material with distinct viral infectious status. Elimination of GLRaV-2 had the highest beneficial impact with a marked increase in cumulative weight growth (21%), fresh fruit yield (22%), and sugar concentration of fruit juice (9%). Selective elimination of GLRaV-1, GLRaV-3, and GVB had also a beneficial impact, with an increase in vigor (0, 6, and 35%, respectively) and production (12, 3, and 16%, respectively), while fruit maturity indices were variably affected (0, 3, and 0% increase in fruit juice sugar and 13, 0, and 0% reduction in titratable acidity, respectively). These results offer new insights into the impact of GLRaV-2 on vigor, yield, and fruit quality of V. vinifera and provide strong evidence for the need to include detection assays for this detrimental virus in certification programs.
Grapevine is one of the most important fruit crops worldwide, with over 65 billion tons produced essentially as table, wine, and raisin grapes on 7.6 million ha in 2004 (FAO 2005). Numerous pathogens, including viruses and virus-like disease agents, can have a detrimental impact on grape production by reducing yield, fruit quality, vigor, and longevity (Andret-Link et al. 2004, Bovey et al. 1980, Goheen 1990), and grapevine fanleaf virus alone is responsible for losses of over $1.5 billion in France (M. Fuchs, unpublished). Over 53 virus species that belong to 20 different genera have been described in grapevines (Martelli 2006) and mixed viral infections are common (Al Tamini et al. 1998, Borgo et al. 2005, Credi and Babini 1997, Credi et al. 2003).
In the early 1980s, an apparent new graft-transmissible disease, vein yellowing leafroll, was described on Vitis vinifera cv. Chardonnay in the Champagne region in France (Caudwell et al. 1983, 1985). Untypical leafroll-type symptoms were observed on this variety as leaf blades showed downward rolling but primary veins turned yellow instead of remaining green. At first, a phytoplasm similar to Bois noir was suspected as causal agent of the disease based on epidemiological observations (Caudwell et al. 1983). Subsequent work showed the presence of several viruses and virus-like diseases, including leafroll, fleck, corky bark, vein mosaic, vein necrosis, and stem pitting in Chardonnay vines affected by vein yellowing leafroll disease (Legin et al. 1989). Despite significant progress in understanding the biology and etiology of this syndrome, limited information is available on virus species implicated and their specific impact on vigor and yield of V. vinifera cv. Chardonnay.
We determined the sanitary status of three clones of V. vinifera cv. Chardonnay affected by vein yellowing leafroll disease. Various combinations of Grapevine leafroll-associated virus 1 (GLRaV-1), Grapevine leafroll-associated virus 2 (GLRaV-2), Grapevine leafroll-associated virus 3 (GLRaV-3), grapevine fleck virus (GFkV), grapevine virus B (GVB), and Grapevine rupestris stem pitting-associated virus (GRSPaV) were identified in diseased material. Following heat therapy and apex grafting for virus eradication, the effect of selective virus elimination on the viticultural performance of clonal material was examined. The largest beneficial impact on vigor, yield, and sugar concentration of fruit juice was observed after elimination of GLRaV-2. Our findings offer new insights into the effect of GLRaV-2 on the viticultural performance of V. vinifera and provide strong evidence for the need to include detection assays for this virus in grapevine certification schemes.
Materials and Methods
Plant material.
Three clones (75, 76, and 95) of V. vinifera cv. Chardonnay affected by vein yellowing leafroll disease (Legin et al. 1989) were used in this study. These were initially from commercial vineyards in the Champagne region, France (Caudwell et al. 1983). Cuttings from each clone were propagated at the Institut National de la Recherche Agronomique (INRA), Colmar, France, and subsequently grafted onto the certified rootstock SO4 (V. berlandieri x V. riparia) clone 762. Grafted plants were established in an experimental field site and used as controls in comparative performance analysis.
Virus elimination.
Rooted cuttings of V. vinifera cv. Chardonnay clones 75, 76, and 95 were subjected to heat treatment at 32°C. Apices from heat-treated plants were periodically collected during a 30- to 60-day treatment period and grafted in vitro onto fragments of hypocotyls from seedlings of the grapevine rootstock V. labrusca x V. riparia cv. Vialla (Bass and Vuittenez 1977). A few apices were directly propagated in tissue culture without heat treatment (Barlass et al. 1982). Plantlets were derived from actively growing apices and propagated in tissue culture for subsequent establishment in the greenhouse. Graft indexing and double antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA) were used to test for the presence of viruses and virus-like diseases before and after virus elimination.
Graft indexing.
Virus-like diseases were assessed by graft indexing in the greenhouse using herbaceous material (Walter et al. 1990) and the following indicators: V. vinifera cv. Pinot noir for leafroll, V. rupestris du Lot for V. rupestris stem pitting, Kober 5BB (V. berlandieri x V. riparia) for Kober stem grooving, LN33 (1613 C x Sultanine) for corky bark, V. vinifera cv. Gloire de Montpellier for fleck and vein mosaic, and 110R (V. rupestris x V. berlandieri) for vein necrosis.
DAS-ELISA.
DAS-ELISA was carried out with plant crude extracts from leaves homogenized in 200 mM Tris-HCl pH 8.2, 140 mM NaCl, 2% polyvinylpyrrolidone 40, and 0.05% Tween 20 at a 1:5 ratio (w:v) (Walter and Etienne 1987). Samples were incubated overnight at 4°C in microtiter plates precoated with diluted specific immunoglobulins either produced in our laboratory or provided to us by Dr. Dennis Gonsalves. Diluted biotinylated γ-globulins were then added for 4 hr at 37°C. A 1:20,000 dilution of streptavidin-labeled alkaline phosphatase was incubated for 30 min at 37°C and p-nitrophenyl phosphate was added at 1 mg/mL. Substrate hydrolysis was recorded at 405 nm with a Titertek Multiscan MCC/340 reader (Labsystems, Les Ulis, France). Three washings with phosphate buffer saline (150 mM NaCl, 1.5 mM KH2PO4, 10 mM Na2HPO4, 2 mM KCl, pH 7.4) containing 0.05% Tween 20 preceded each step of the DAS-ELISA procedure. Biotinylated γ-globulins were sometimes saturated with 5% crude extracts from healthy grapevine leaves to reduce background OD405nm values. Samples were considered positive if their OD405nm readings were at least twice those of healthy controls ± 20%. Serological tests were performed with different accessions from the European virus reference collection at INRA-Colmar (Greif and Walter 1997) as positive controls and the heat-treated accession TG64 (V. vinifera cv. Klevener de Heiligenstein) (Bass and Vuittenez 1977) as healthy control.
Field trial and experimental approach.
An area of homogeneous topographic and soil condition was selected as field site at the Huben farm, INRA-Colmar. The site was a loam soil. Chardonnay test material consisted of the three initial infected clones (75, 76, and 95) and their selected progeny that had undergone virus elimination: 75A and 75B, 76A and 76B, and 95A and 95B, respectively. All Chardonnay test material was grafted onto certified root-stock SO4 clone 762. This rootstock was purposely chosen, over Kober 5BB (V. berlandieri x V. riparia) for instance, to avoid graft-incompatibility problems related to GLRaV-2 (Golino 2003, Greif et al. 1995). Forty vines were planted for each of the three initially infected clones (75, 76, and 95) and their progeny (75A, 75B, 76A, 76B, 95A, and 95B), making a total of 360 (9 x 40) plants. Treatments (three untreated clones and six clones treated for virus elimination) were assigned to two blocks in a complete randomized block design with 20 experimental units per block. Experimental units were planted in groups of five plants each. For each block, 180 vines were planted in three rows of 60 plants each, spaced 1 m apart within rows and 2 m between rows. Two guard vines were planted at the ends and on each side of the six trial rows. A vertical single-plane trellis system was used, with a fruiting wire ~1.0 m aboveground and an upper foliage wire at ~1.70 m (Schneider et al. 1989). Chardonnay was trained to a bilateral cordon and cane-pruned. The vine culture was conventional for a vineyard in Alsace.
Data collection.
At harvest, the number of clusters was counted and clusters were weighted for each clone/treatment group. In addition, berry samples were randomly collected for each clone/treatment group and crushed with a hand press to measure sugar content with a hand temperature-compensating refractometer and titratable acidity by titration with 0.1 N NaOH to pH 7.0. Pruning wood was weighted in winter for each clone/treatment group as a measure of growth during the preceding season. Data on pruning wood, fresh fruit yield, cluster number, and fruit maturity indices were expressed as increase or decrease relative to the initial clonal material.
Statistical analysis.
Data collected on cane pruning weight, yield, number of clusters, fruit sugar content, and fruit juice titratable acidity were processed for statistical analysis. Analysis of variance was used to detect treatment differences and relationship between variables with SAS (Statistical Analysis System, SAS Institute Inc., Cary, NC). Contrasts among treatment means were used to compare differential effects on field performance due to selective virus elimination at 5% least significant difference (LSD). The first contrast allowed us to compare the performance of the initial material (for example, clone 75) with that of the first progeny obtained after virus elimination (for example, clone 75A) for each parameter measured. The second contrast was used to compare performance of the two progeny obtained after virus elimination (for example, clones 75A and 75B) for each parameter measured.
Beneficial impact assessment of virus elimination.
In order to assess comparatively the relative beneficial impact of virus elimination, target viruses were ranked according to their effect on the performance of V. vinifera cv. Chardonnay by scoring each clone/treatment group using an impact index. The impact index was calculated by integrating significant positive effects on vigor, yield, and fruit maturity indices of each clone/treatment group relative to the corresponding untreated clone on a scale of 0 (no significant impact) to 6 (highest beneficial impact). Impact indices of each variable were added to calculate a total impact index for each clone/treatment group.
Results
Sanitary status of test material.
Test vines were assessed for virus status at the beginning of the experiment by graft indexing and at the end of the experiment by DAS-ELISA. Plants of V. vinifera cv. Chardonnay clones 75, 76, and 95 were initially infected by leafroll, rupestris stem pitting, vein mosaic, and vein necrosis, as shown by biological indexing (Table 1⇓). Additionally, plants of clones 75 and 95 were initially infected by corky bark, and fleck was also present in clone 75. Furthermore, clone 76 incited an incompatibility disorder on Kober 5BB. In viruses for which serological reagents are available, DAS-ELISA indicated the presence of GLRaV-1, -2, and -3 in leafroll-infected material, GFkV in fleck-infected material, GVB in corky bark-infected material, and GRSPaV in rupestris stem pitting-infected material. DAS-ELISA OD405nm readings were prominent in infected plants (0.34–0.45 for GLRaV-1; 0.74–1.09 for GLRaV-2; 1.72–2.14 for GLRaV-3; 0.32–0.58 for GFkV; 1.29–1.53 for GVB; and 0.61–1.08 for GRSPaV) relative to healthy controls (0.05–0.10 for most γ-globulins, except 0.51–0.58 for GVB γ-globulins and 0.21–0.27 for GRSPaV γ-globulins) after 1 to 2 hr substrate hydrolysis. DAS-ELISA further revealed a differential infectious status of the test material with the presence of GLRaV-1 and GVB in clones 75 and 95, GLRaV-2 and GLRaV-3 in clone 76, and GRSPaV in the three clones (Table 1⇓). GFkV was also present in clone 75.
Selective virus elimination.
Elimination of leafroll, fleck, and corky bark was achieved in some of the Chardonnay progeny by heat therapy and apex grafting or apex culture, as shown by biological indexing (Table 1⇑). However, rupestris stem pitting, vein mosaic, and vein necrosis were not eliminated. DAS-ELISA confirmed the presence of GRSPaV in all the Chardonnay progeny and the selective elimination of GLRaV-1, GLRaV-2, GLRaV-3, GFkV, and GVB in some progeny.
Field site.
Chardonnay material that was untreated or treated for virus elimination (Table 1⇑) was grafted onto the certified rootstock SO4 and test plants were established in the field on 21 Aug 1990. Mealybug and soft-scale insect vectors of GVB, GLRaV-1, GLRaV-3, and other virus species from the genera Vitivirus and Ampelovirus (Gugerli 2003) were not known to occur in the field site and surrounding vineyards and were not detected throughout the trial. Therefore, opportunities for plant-to-plant transmission of these viruses during the trial period were limited.
Effect of selective virus elimination on vigor.
The cumulative pruning wood weight data over five years (1996–2000) indicated a 19 to 35% increase in vigor after selective virus elimination (Table 2⇓). The highest increase in vigor after eradicating a single virus was observed when GLRaV-2 was eliminated in clone 76A (21% increase), while the elimination of GLRaV-3 in clone 76B only slightly increased growth (6% = 27 – 21). Elimination of GLRaV-1 in association to GFkV caused a 19% increase in growth in clone 75A, while elimination of GVB caused a 10% (29 -19) increase in clone 75B. Unexpectedly, the elimination of GLRaV-1 had no positive impact on the cumulative weight of growth in clone 95A, but elimination of GLRaV-1 in association to GVB caused a 35% increase in clone 95B. This increase was the highest combined effect observed after elimination of two viruses. Differences between clone/treatment groups were significant (5% LSD), but no difference was detected due to block (0.0248 < F < 10.641).
Effect of selective virus elimination on yield.
Cumulative fresh fruit yield over six years (1996–2001) increased by 12 to 34% after selective virus elimination (Table 3⇓). The highest increase in yield was obtained when GLRaV-1 in association to GFkV (34%) was eliminated in clone 75A. For elimination of a single virus, the highest increase in yield was observed when GLRaV-2 was eliminated in clone 76A (22%), while elimination of GLRaV-3 induced a 3% (25 - 22) yield increase in clone 76A. A 12% yield increase was obtained after elimination of GLRaV-1 in clone 95A and an additional 16% (28 - 12) increase was obtained in clone 95B when GVB was eliminated. Differences between clone/treatment groups were significant at 5% LSD but no significant difference was observed for block (0.0007 < F < 34.895).
Remarkably, the mean cluster number increased by 9 to 29% over six years (1996–2001), with 13% increase after elimination of GLRaV-2 in clone 76A or GLRaV-1 and GVB in clone 95B and 29% increase after elimination of GLRaV-1 in association to GFkV in clone 75A (Table 4⇓). As a consequence, the cumulative mean cluster weight increased by 12 to 17% over six years (1996–2001) after selective sanitation (Table 5⇓). Significant differences were obtained after elimination of GLRaV-2 in clone 76A (12%), GLRaV-1 in clone 95A (14%), and GLRaV-1 in association to GFkV in clone 75A (15%). Although elimination of GLRaV-1 in clone 95A had no effect on mean cluster number (Table 4⇓), cluster weight significantly increased by 14% (Table 5⇓). For mean cluster weight, significant differences at 5% LSD were found for clone/treatment groups but not for blocks (0.0036 < F < 46.7706).
Effect of selective virus elimination on fruit maturity.
When averaged over three years (1999–2001), the juice sugar content increased significantly by 3 (12 - 9) to 9% when GLRaV-3 and GLRV-2 were eliminated from clones 76B and 76A, respectively (Table 6⇓). Elimination of other target viruses did not significantly impact soluble solids in fruit juice. Also, when averaged over three years (1999–2001), a significant difference in titratable juice acidity was measured after eradication of GLRaV-1 (−13%) in clone 95A, as well as GLRaV-1 in association to GFkV (−6%) in clone 75A (Table 7⇓). Elimination of the other target viruses had no significant impact on titratable juice acidity. Again, significant differences were due to clone/treatment groups, not to block (0.0028 < F < 17.2957) at 5% LSD.
Beneficial impact assessment of virus elimination.
An impact index was calculated for each clone/treatment group and each measured variable (vigor, yield, fruit maturity) to compare the relative beneficial impact of virus elimination (Table 8⇓). For vigor, a 6 value was attributed to clone 95B for which elimination of GLRaV-1 and GVB had the highest beneficial impact (+35%) relative to the other clone/treatment groups; a 5 value to clone 76B with a 21% increase in relation to elimination of GLRaV-2; a 4 value to clone 75A with a 19% increase in relation to elimination of GLRaV-1 in association to GFkV; a 3 value to clone 75B with a 10% increase in relation to elimination of GVB; a 2 value to clone 76B with a 6% increase in relation to elimination of GLRaV-3; and a 0 value to clone 95A with no significant beneficial impact in relation to elimination of GLRaV-1. The same scoring sequence was applied to each clone/treatment group for yield and fruit maturity indices. Impact indices of each variable were then added to calculate a total impact index for each clone/treatment group (Table 8⇓).
The highest total impact index was obtained for clone 76A when GLRaV-2 was eliminated (16), followed by clone 75A with elimination of GLRaV-1 in association to GFkV (15), clone 95A with elimination of GLRaV-1 (10), clone 76B with elimination of GLRaV-2 and GLRaV-3, and clone 95B with elimination of GLRaV-1 and GVB (9), and, lastly, clone 75B with elimination of GLRaV-1, GFkV, and GVB (3–8) (Table 8⇑). Overall, these results indicated that eradication of GLRaV-2 had the most prominent beneficial impact across variables and target viruses.
Discussion
Analysis of the sanitary status of V. vinifera cv. Chardonnay affected by vein yellowing leafroll disease by indexing and ELISA confirmed the presence of several combinations of viruses and virus-like diseases, including the three major leafroll-associated viruses: GLRaV-1, GLRaV-2, and GLRaV-3. The selective elimination of individual viruses from combination mixtures allowed for a comparative impact analysis of the viticultural performance of V. vinifera cv. Chardonnay in a replicated field trial over six years. The selective elimination of GLRaV-1, GLRaV-2, GLRaV-3, GVB, and GLRaV-1 in association to GFkV increased vigor and yield and modified fruit maturity indices. In particular, the elimination of GLRaV-2 had the greatest beneficial influence. When averaged over five years, eradicating GLRaV-2 elevated significantly cumulative weight growth (21%) and fresh fruit yield (22%). Sugar concentration of fruit juice increased by 9% while titratable acidity was not significantly affected over three years. To the best of our knowledge, this is the first direct evidence of the significant detrimental impact of GLRaV-2 on the viticultural performance of V. vinifera L.
The association of GLRaV-2 with vine decline and graft incompatibility disorder on certain rootstocks is well documented (Golino 2003, Greif et al. 1995, Rowhani et al. 2005). However, the effect on plant vigor and fruit yield and quality has not been reported. We were able to assess the impact of GLRaV-2 by comparing mixed infected material that contained or did not contain this virus after sanitation of Chardonnay clone 76. The International Council for the Study of Viruses and Virus-like Diseases of the Grapevine (ICVG) recommends GLRaV-2 to be considered a class I pathogen and assayed as part of phytosanitary requirements in official certification schemes for nursery stock to be eligible to move between regulated areas (ICVG 2003). This recommendation is based on circumstantial evidence on the viticultural performance of material free of and infected by GLRaV-2 across varieties and countries. Our study provides the first evidence on the marked negative impact of GLRaV-2 and supports ICVG’s earlier predictions.
GRSPaV, vein mosaic, and vein necrosis were present in all Chardonnay progeny obtained after sanitation, in particular in clones 75B, 76B, and 95B with no other virus(es). Interestingly, these latter three progeny performed similarly, with total impact indices of 3–9 (Table 8⇑). No marked negative effect on vigor, yield, and fruit maturity indices was documented previously for vein mosaic, vein necrosis, and rupestris stem pitting in V. vinifera cv. Albana and Trebbiano Romagnolo (Credi and Babini 1997). Limited impact on growth and yield was also reported for rupestris stem pitting in five Vitis cultivars (Reynolds et al. 1997) and for vein mosaic in two clones of V. vinifera cv. Chardonnay and Klevener de Heiligenstein (Legin et al. 1993). Thus, assuming vein mosaic, vein necrosis, and GRSPaV react similarly in different grapevines, as suggested by previous studies (Credi and Babini 1997, Legin et al. 1993, Reynolds et al. 1997), these viral agents had likely very limited impact, if any, on the performance of V. vinifera cv. Chardonnay clones in our study.
No significant difference was noticed between Chardonnay clones 76A and 76B for most of the variables measured. Therefore, no apparent antagonistic or synergistic effect likely occurred between GRSPaV, vein necrosis, and vein mosaic on one hand and GLRaV-3 on the other. However, based on our field data, a synergism could have occurred between GLRaV-1 and GFkV since the elimination of these two viruses in clone 75 had a greater beneficial impact than the elimination of GLRaV-1 in clone 95 (total index impact of 15 versus 10). Interestingly, a greater negative impact on fruit quality of GFkV in association to GLRaV-3 relative to GLRaV-3 by itself was documented in French-American hybrids, although a synergy between these two viruses was not reported (Kovacs et al. 2001). Similarly, a synergistic interaction between GLRaV-2 and GLRaV-3 cannot be ruled out in Chardonnay clone 76.
Elimination of GVB had a significant beneficial impact on vigor and yield (in clone 95 but not 75) but not on fruit maturity indices. It will be interesting to assess if these results translate into similar performance of Vitis sp. other than V. vinifera cv. Chardonnay. Noteworthy, GVB had limited, if any, impact in clone 75 with a total impact index of 3, whereas it had a more pronounced influence in clone 95 with a total impact index of 9. This differential reaction is not too unexpected since numerous factors, including the clonal material or the effect of co-infecting viruses, such as GLRaV-1 in our study, can influence the impact of viruses and virus-like diseases.
Our findings on the effect of GLRaV-1 and GLRaV-3 on vigor, yield, and fruit maturity of V. vinifera cv. Chardonnay agree with published data in other Vitis spp. (Mannini 2003), although with an overall lower impact, particularly for GLRaV-3. Interestingly, elimination of GLRaV-1 but not GLRaV-3 depressed titratable juice acidity, and elimination of GLRaV-3 but not GLRaV-1 increased sugar concentration of fruit juice, despite a similar total impact index (10 for GLRaV-1 and 9 for GLRaV-3).
Assessing the effect of viruses and virus-like diseases on the viticultural performance of grapevines relies usually on the comparative performance of virus-free and virus-infected material. In such studies, virus-infected material is often produced by graft inoculation of healthy vines with bud material from different virus-infected source vines. This approach is powerful; however, it does not take into account the potential presence of uncharacterized viruses or virus-like agents in the bud material infected with viruses and virus-like diseases of interest. Such uncharacterized viral entities can be readily graft transmitted, and thus, potentially interfere with the effect of the targeted virus(es) and/or virus-like diseases. The test material used in this study was obtained from clonal material by heat therapy in combination with in vitro apex grafting or apex culture. Therefore, uncharacterized viruses, if present, should have limited, if any, impact on the performance of the test material. Also, in most comparative studies for impact assessment, experiments are not always carried out with a given clonal material. Instead, various grapevines cultivars or clones are often used. Thus, a certain degree of variability cannot be excluded because of a lack of genetic uniformity of the healthy and infected test material. Our study was conducted with clonal grapevine material because progeny obtained after virus elimination represented the same clonal genotype. In addition, there is little evidence, if any, that heat treatment and apex grafting cause genetic changes. Therefore, limited, if any, variability in performance likely occurred in our study due to plant material because our test vines were genetically homogenous.
Conclusion
Our study documented the beneficial effect of selective virus elimination (GLRaV-1, GLRaV-2, GLRaV-3, GVB, and GLRaV-1 in association to GFkV), in particular of GLRaV-2, on Chardonnay growth, yield, and fruit maturity indices. These findings strongly support the need for GLRaV-2 detection assays to be included in grapevine certification programs, as recommended by ICVG.
Footnotes
Acknowledgments: We are grateful to Dennis Gonsalves for providing antiserum to GRSPaV, J. Barnard for advice with statistical analyses, L.M. Yepes for critically reading the manuscript, and Paul Bass and René Legin for graft indexing and producing plant material. We thank Peggy Andret-Link, Marlène Henry, Marie-Louise Bechler, and Daniela Dancea for assistance at harvest.
- Received September 2006.
- Revision received October 2006.
- Copyright © 2007 by the American Society for Enology and Viticulture