Abstract
The dagger nematode Xiphinema index specifically transmits Grapevine fanleaf virus (GFLV), which causes progressive grape degeneration in vineyards worldwide. Nematode-resistant rootstocks are a promising alternative to the ban of nematicides. We report the evaluation, under controlled conditions in two independent experiments, of the host suitability (reproduction factor, RF) of 40 Vitis and Vitis-Muscadinia accessions to the isofemale line Fréjus of X. index. In the first experiment, 17 accessions, grown from either in vitro culture or cuttings, were evaluated after 1 and 3 years and, for four accessions, also after 4 years. The nematode multiplied early and late on reference rootstocks V. rupestris du Lot and V. riparia Gloire de Montpellier, respectively. Among Vitis-Muscadinia material, after 1 and 3 years, accession VRH 8624 had high RFs, while VRH 8771 and NC 35–50 had RFs close to 1. The nematode-resistant candidate rootstock accession RPG1 (=VRH 8771 x Vitis rootstock 140Ru) and the intergeneric hybrid VRH 97-99-79 had RFs significantly higher at 4 years than at 1 year, suggesting a progressive adaptation of the nematode to the plant resistance factors. Most Vitis botanic accessions had high RFs at both 1-year and 3-year durations. Low RFs, observed for the remaining accessions, were probably due to the poor root development and the subsequent difficult access of nematodes to feeding on this material. In the second experiment, 23 Vitis accessions, evaluated only at 3 years, had high RFs, except for a few accessions that expressed low RFs and poor development. Thus some muscadine-derived intergeneric hybrids are promising resistance sources that must be confirmed using nematode lines from other geographic origins.
The dagger nematode Xiphinema index is the vector of Grapevine fanleaf nepovirus, which is the most economically important virus of grapevine worldwide (Martelli 1978, Andret-Link et al. 2004). This nematode belongs to the family Longidoridae and is present in all major grapegrowing regions. Its frequency is generally high in vineyards with a long history of grape cultivation. Together with Grapevine fanleaf virus (GFLV) grape prophylaxy, control of the nematode has been the most common strategy against the virus and has been mainly based on preplant fumigants or nonvolatiles combined with fallow periods between successive grape plantings (Raski et al. 1983, Esmenjaud et al. 1988). Because the active ingredients of the chemicals are the most toxic in agriculture, they are being progressively removed from the market, and because of the economically unacceptable interval (>7 years) required for nematode eradication between vine plantings (Esmenjaud 2008), alternative nematode management strategies must be developed. In vineyards, those alternatives face two major constraints. The first is nematode location in deep soil layers, as previously described in the Champagne region of France (Esmenjaud et al. 1992) and confirmed in the Bordeaux vineyard (Villate et al. 2008). The second constraint is the long retention time of the virus by the nematode in the soil between two successive vines: GFLV was still detected by RT-PCR in groups of individuals kept starving in soil for 4 years (Demangeat et al. 2005). In the context of these spatial and temporal constraints, using nematode-resistant grapevine rootstocks (showing a significantly delayed viral transmission) appears the most promising control method, even though it may require complementary cultivation of nonhost or nematicidal crops between two vine plantings. But as for any control experiment, the pest variability has to be taken into account in sustainable management strategies. Xiphinema index reproduces by meiotic parthenogenesis (Dalmasso and Younes 1969), but its genetic diversity has not been investigated and studies are in progress. Single female progenies have been created using populations introduced from the worldwide geographic range of the nematode, and such so-called isofemale lines constitute a genetically homogeneous material for further studies (Wang et al. 2003).
Field and container evaluations have shown that host suitability of Vitis species and accessions commonly used as rootstocks range from poor to excellent hosts to the nematode and major differences have been reported (Boubals and Pistre 1978, Meredith et al. 1982, Harris 1983, Malan and Meyer 1993, Walker et al. 1994b, Sopp et al. 1998, McKenry et al. 2001). Although results are highly variable, some clones from V. riparia (Bouquet and Pistre 1978), V. rufotomentosa, and V. arizonica (Meredith et al. 1982) have been reported as having greater resistance than the rest of Vitis material. Recently, forms of V. arizonica from Mexico were shown to carry resistance to X. index and Pierce’s disease (Riaz et al. 2007, Xu et al. 2008). Because of the limited success of the screening for nematode-resistant Vitis material, resistance has been sought in closely related genera. Some Muscadinia material has been reported as carrying greater resistance than Vitis spp. (Boubals and Pistre 1978). Muscadine grapes (M. rotundifolia) are currently the best potential sources for X. index resistance and are also immune to GFLV transmission (Bouquet 1981, Bouquet and Danglot 1983). Intergeneric material V. vinifera x M. rotundifolia has been created (Bouquet 1980, Walker et al. 1991, 1994a). In particular, such hybrids obtained in France have shown a high level of resistance but major cultural defects, which render another generation of crosses necessary despite their low fertility (Bouquet et al. 2000). An individual obtained by crossing a F1 hybrid with a Vitis rootstock is a candidate rootstock in highly nematode-infected plots, as it has shown good resistance under field conditions (Bouquet et al. 2003). In parallel, a breeding project for hybrid rootstocks is being developed at INRA (Ollat et al. 2005) that aims to associate the high nematode resistance from M. rotundifolia with partial resistance from some Vitis species.
We report experiments on the host suitability of diverse material comprising intergeneric hybrids involving M. rotundifolia sources together with new Vitis clonal and reference rootstock accessions. This study also assessed some new experimental techniques, as the evaluation has been performed with an isofemale line of X. index over a duration from 1 to 4 years. For some accessions, evaluations were conducted at 1, 3, and 4 years to give a good estimate of resistance over time.
Materials and Methods
Plant material.
Plant material was comprised of seven accessions grown from in vitro culture and 33 Vitis accessions grown from hardwood cuttings (Table 1⇓). Plant material grown from in vitro culture comprised the reference Vitis material, V. rupestris cv. du Lot (RL), V. riparia cv. Gloire de Montpellier (RGM), and some Vitis x M. rotundifolia F1 hybrids obtained in France (VRH 11-6-76 = VRH 8771, VRH 97-99-79), California (T6-38 = VRH 8624), and North Carolina (NC 35–50). It also included the R1 individual VMH 3146-1-87 (= RPG1) obtained by crossing the intergeneric F1 hybrid VRH 8771 by the Vitis rootstock 140Ru (Vitis berlandieri x V. rupestris).
The Vitis material grown from cuttings grouped diverse botanic accessions belonging to the species V. aestivalis, V. amurensis, V. bayleyana, V. berlandieri, V. bicolor, V. candicans, V. cinerea, V. doaniana, V. flexuosa, V. lincecumii, V. piazeski, V. riparia, V. rubra, V. rupestris, and V. simpsonii. The material was obtained from the INRA germplasm conservatory in Bordeaux (France).
Nematode isofemale line.
The experiment was conducted with an isofemale line obtained from the population X. index Fréjus. This line is one of the seven single female descendencies of X. index created for genetic and pathogenic studies (Wang et al. 2003). The original population was obtained from a vineyard severely infected by GFLV in Fréjus (Var) in southeastern France. The Fréjus population was grown on grape in the greenhouse and the line was created from a single female inoculated on a fig plant grown in vitro. The original female progeny was then multiplied on fig plants grown in containers filled with steam-sterilized soil. Although original nematodes grown on grape were viruliferous, the single-female progeny and subsequent nematode generations were virus-free as confirmed by the RT-PCR assay for GFLV (Esmenjaud et al. 1994, Demangeat et al. 2004).
Evaluation of plant material.
Plant material (Table 1⇑) was evaluated in two experiments. The first experiment, conducted in 2-L containers, included the seven accessions grown from in vitro culture and 10 of the 33 botanical Vitis accessions grown from hardwood cuttings. This material was divided into four subsets. The first subset was evaluated after 3 months (two replicates), the second subset after 1 year (six replicates), the third subset after 3 years (four replicates), and the fourth subset, limited to four accessions, after 4 years (six replicates). Plants of this fourth subset (RL, RGM, VRH 97-99-79, and RPG1) were repotted after 2 years in 10-L containers. In the second experiment, the remaining 23 botanical Vitis accessions (grown from cuttings) grouped plants with a more heterogeneous development that were evaluated in 5-L containers (three replicates) and analyzed after 3 years.
All material was planted into containers in the greenhouse on May 2002. In vitro material showed equivalent initial root development. However, that was not true for Vitis accessions grown from cuttings, which were consequently rated for root development on a 1 to 3 scale before planting (Table 1⇑). On 20 July 2002, each container was inoculated with 180 X. index individuals obtained by soil extraction from fig multiplication containers. Containers without plant (bare soil) were included as a control for nematode survival. Pots were arranged in a completely randomized block design on greenhouse benches.
At harvest, total soil of each 2-L container was recovered in a 10-L bucket over which plant roots were washed individually with caution under tap water. For 10-L containers, the total soil was divided into five 2-L volumes that were analyzed individually. For 5-L containers, plants were delicately pulled out, total soil was weighed and mixed, and a representative 1 kg soil from the total amount was sampled and suspended into a 10-L bucket as above. Then X. index nematodes were extracted from suspended soil using an adapted Oostenbrink method (Hooper 1986). Nematode developmental stages were counted under a binocular to obtain total numbers (directly for 2-L containers or by summing partial 2-L extractions for 10-L containers) or estimated total numbers (5-L containers) of nematodes. The ratio between total X. index number and initial inoculum was calculated for each container and the mean of all replicates taken to evaluate the reproduction factor (RF) of the nematode isofemale line for each accession.
Data from the four accessions evaluated at 1-year and 4-year durations (six replicates) were analyzed using a one-way analysis of variance (ANOVA). Nematode numbers were log10(x+1) transformed before ANOVA (Noe 1985). Means of transformed nematode numbers were then compared by Fisher’s (LSD) multiple range test at p ≤ 0.05.
Results
First experiment.
Estimation of nematode survival and multiplication after 3 months showed that the reproduction factor was already >1 in grown material and V. candicans 10189, V. rubra 10168, and V. amurensis 10151 for material grown from cuttings (data not shown). Multiplication rates were highly variable after 1 year (Figure 1⇓). Natural survival of the nematode in plantless pots was only 7% at 1 year. High RFs (>10) were reached by VRH 8624, RL, V. candicans 10189, V. rubra 10168, and V. amurensis 10151, which confirmed their early good host status. The accessions V. candicans 10196 and V. cinerea 10139 also had high RF values. Accessions V. riparia Gloire de Montpellier (RGM), VRH 97-99-79, V. aestivalis 11051, and V. rubra 10919 had a RF between 1 and 5. VRH 8771, RPG1, and NC 35–50 had a RF slightly less than 1. Vitis aestivalis 11053 and V. bicolor 11219, with a RF <1, had very poor root development.
Multiplication rates evaluated after 3 years were also quite variable (Figure 1⇑). No nematodes survived in bare soil. Generally, accessions that were highly infested after 1 year tended to have fewer nematodes after 3 years, particularly RL, with a RF that decreased from 90 to 3.7. The reverse situation was observed for RGM, with a RF that increased from 3.5 to 13.2. Among the Vitis x Muscadinia accessions with a RF close to 1 after 1 year, there was little variation after 3 years except for RPG1, on which the RF increased from 0.8 to 2.2. Within Vitis accessions, the RF remained high except in the three poorly developed accessions V. rubra 10919, V. aestivalis 11053, and V. bicolor 11219. The latter two died between 1 and 3 years.
Multiplication after 4 years in the four accessions RL, RGM, VRH97-99-79, and RPG1 confirmed previous trends at 1 and 3 years. For RGM, the RF reached 28.8 (Figure 2⇓). For the other reference rootstock RL, the RF decreased to 0 as a consequence of the death of the plants between 3 and 4 years, presumably consecutive to the severe nematode attacks. VRH97-99-79 and RPG1 had RFs of 5.5 and 7.6, respectively, values significantly higher than those previously observed.
Second experiment.
Plant material was divided and evaluated in two separate series harvested after 3 years (Table 2⇓). Nematode recovery in plantless containers was 5%. RF values of the 23 Vitis sp. accessions ranged from 0.85 to 138. Nineteen of those tested clones had RF >5. The four accessions with RF <5 had low vigor throughout the experiment.
Discussion
Our study provided data on both the method for evaluation of plant susceptibility to X. index and the host suitability of diverse accessions for this dagger nematode. The first aspect concerns methodological issues such as the amount of inoculum and the duration of the tests. The amount of inoculum applied allowed differentiation of plant material with high and low RF values after only 3 months, that is, approximately the duration of the nematode life cycle (Cohn and Mordechai 1969) and numbers comparable to the initial inoculum were already observed at that date. In bare soils, nematode numbers decreased rapidly and individuals were only recovered in 5-L containers, possibly because the soil conditions were more stable than in 2-L containers. Differences were assessed until 4 years among certain accessions. The rootstock RL had a very high RF after 1 year, dropped to a very low RF after 3 years, and to a null RF after 4 years as, presumably because of the severity of the attacks, all plants of this rootstock died between 3 and 4 years. Conversely, the rootstock RGM increased regularly from a low RF after 1 year, to an intermediate RF after 3 years, and to a high RF after 4 years. Early evaluation may be useful in revealing very susceptible plant material such as RL that is subsequently severely affected, which leads to a marked decrease in nematode numbers. In contrast, our data for RGM illustrate that this accession, although having few nematodes after one year, can support increasing numbers in the long term, suggesting a tolerant behavior of RGM with potentially more damaging consequences than expected. The wide use of these two rootstocks in France together with their opposite response to X. index makes them suitable as references for further Vitis material evaluations. Given that environmental conditions, particularly in greenhouses, are often difficult to reproduce perfectly between successive experiments, including such material in the tests permits us to track the evolution of nematode multiplication at different times, thus allowing consistent standards in each experiment.
We evaluated the RF at two times (1 and 3 years) for 17 accessions that were either Vitis x Muscadinia intergeneric hybrids or Vitis species from very diverse origins, and at three time points (1, 3, and 4 years) for four accessions, including the candidate rootstock RPG1. Among Vitis x Muscadinia hybrids, different Muscadinia parents were involved. The susceptible VRH 8624 (=T6-38) is a cross between the V. vinifera accession F2-35 (Carignan x Cabernet Sauvignon) and the muscadine cultivar Trayshed from UC Davis (Jelenkovic and Olmo 1968). Under field conditions, this hybrid is very susceptible to GFLV transmission by X. index (Bouquet et al. 2000). The hybrid VRH 8771 is a cross between the V. vinifera accession 43–35 (Cabernet Sauvignon x Alicante Bouschet) and the muscadine NC 184-4 (Bouquet 1980). In contrast to VRH 8624, this hybrid is highly resistant to GFLV transmission by X. index (Bouquet et al. 2000). For 1- and 3-year tests, another intergeneric hybrid, NC 35–50—obtained by crossing an open-pollinated seedling of T6-38 with the muscadine cultivar Dixie—had a RF <1. The resistance levels of VRH 8771 and NC 35–50 were the highest at both time periods and confirm these Vitis x M. rotundifolia accessions as resistance genitors (RF close to 1). Nevertheless, RPG1, a cross of VRH 8771 by the Vitis rootstock 140Ru, had a RF that increased from 2.2 to 7.6 between 3 and 4 years. To a lesser extent, the accession VRH 97-99-79 also had increasing nematode numbers over the same time. This apparent erosion of resistance may indicate a progressive adaptation of the nematode to resistance pressure from the plant. Nevertheless, such intergeneric hybrids also appear to be reliable standards for evaluating new Vitis material.
Pure Vitis material evaluated after 1 and 3 years showed diverse results. Many accessions supported high multiplication rates associated with vigorous growth. They confirm the nematode susceptibility commonly observed among Vitis material. Low RFs have only been observed among poorly developed material with limited rooting, which may provide fewer resources for X. index. This cannot be considered a resistance phenomenon, as a high proportion of those plants did not survive until the 3-year final rating. A single putative resistant accession, V. rubra 10919, has been detected. Nevertheless, this accession, although surviving throughout the test duration, grew slowly at first and had more vigorous root growth later, which might suggest that most nematodes did not reach the roots as easily as in material with earlier and more marked rooting.
In the second experiment, 23 other Vitis accessions were evaluated at a single time point, 3 years, to eliminate nematode-susceptible material and prescreen for putative resistance sources. Of them, 19 accessions had a RF >5, and only four had a RF close to 1. Unfortunately, these latter four accessions had very weak root development, which might explain the lower RF. Consequently, none of these Vitis accessions has been retained for further consideration for nematode-resistance breeding.
Conclusion
We confirm that accessions VRH 8771, VRH 97-99-79, and NC 35–50 are promising resistance sources to the French isofemale line Fréjus. Such resistance sources will also have to be evaluated against other lines of X. index. Six other isofemale lines, obtained from populations originating from diverse vineyards worldwide (Israel, Cyprus, Italy, Mesnil in the French Champagne region, Spain, and Napa in California) are available and their pathogenicity will be assessed to determine whether these resistance sources are isolate-dependent.
Finally, we show the importance of characterizing the plant host suitability to the dagger nematode X. index in both short- and long-duration tests. Short durations allow discrimination of highly susceptible plant material before pathogen numbers are reduced because of plant damage, and longer durations allow separation of early temporary resistance responses from those that are more durable. Such long evaluations also discriminate the false resistance resulting from poor root development from truly resistant material that expresses a real antagonistic effect to nematode multiplication. This latter plant resistance response has to be monitored to establish the ability of nematode individuals to adapt to resistance factors over time. Recently, microsatellite markers have been developed for X. index that might provide tools to investigate nematode adaptation to the resistant material used in this study.
Footnotes
Acknowledgment: This article is dedicated to the memory of Alain Bouquet, who died on 12 May 2009. His coauthors wish to honor him for his excellent work in grape breeding.
- Received January 2009.
- Revision received June 2009.
- Revision received September 2009.
- Accepted October 2009.
- Published online March 2010
- Copyright © 2010 by the American Society for Enology and Viticulture