Abstract
The mycorrhizal field inoculation of 161-49 Couderc (Vitis riparia Michx. x Vitis berlandieri Planch.) and 140 Ruggeri (Vitis rupestris L. x V. berlandieri) grapevine rootstock plants grafted with Cabernet Sauvignon was evaluated in two replant soils of a Mediterranean production area in northeastern Spain. The first soil (vineyard 1) had been under tillage for 10 years. The second soil (vineyard 2) had been cultivated for one year before setting up the new plantation and was infested by the white root rot fungus Armillaria mellea (Vahl:Fr.) P. Kumm. The number of infective mycorrhizal propagules estimated in the soil before planting was 1 per 100 mL soil in vineyard 1 and none in vineyard 2. Half of the vines for each rootstock were inoculated with the arbuscular mycorrhizal fungus (AMF) Glomus intraradices Schenck and Smith (BEG 72). In vineyard 2, lacking mycorrhizal propagules and infested with A. mellea, soil was also inoculated before planting with Thymus vulgaris L. and Lavandula officinalis Mill. plants colonized by G. intraradices in two rows. At the end of the second growing season, 19 months after planting, the direct field inoculation showed a beneficial effect on the growth of 140 Ruggeri vines in vineyard 1. The total biomass of inoculated 161-49 Couderc vines increased in vineyard 2 but not in vineyard 1. Precropping with mycorrhizal carrier plants increased the mycorrhizal potential of the replant soil. Grapevine response to AMF inoculation was influenced by the intrinsic characteristics of the vineyard soil and by the rootstock used and the time after planting.
Much information is available on the agronomic performance of grapevine rootstocks and their compatibility with varieties grown in Spain (Hidalgo 2002); however, experimental data on grapevine replant disease are more difficult to find. Grapevine replant disease is a consequence of continuous land exploitation and repeated cropping in which stress problems, mainly of pathological origin, play an important role (Westphal et al. 2002). Related symptoms are failures following transplant and increased vine mortality, lack of vigor, mild chlorosis, and severe stunting, particularly during the summer months (De Luca et al. 2003).
Causal agents contributing to the severity of the disease in woody crops can be of biotic or abiotic origin and include soilborne plant pathogens and stress factors such as extreme pH, poor drainage, and toxic metabolites (Calvet et al. 2000). Among soilborne plant pathogens, the root rot fungus Armillaria mellea (Vahl:Fr.) P. Kumm is considered the principal cause of replant disease in Spanish vineyards, although several species of Armillaria have been associated with the disease (Aguín et al. 2006). In the Mediterranean forest, A. mellea lives on the roots of trees and is particularly common in orchards and vineyards previously inhabited by Quercus species. Armillaria mellea lives as a parasite on living host roots but can survive as a saprophyte in dead wood tissue (Rishbeth 1985). Roots and rhizomorphs remain in the replant soil after Quercus trees are removed and are capable of infecting newly planted grapevines. The disease occurs in localized “centers” where infected roots from diseased trees are concentrated below ground. Vines develop varying degrees of symptoms as their roots grow to contact infected tree roots at different times and are colonized. Few control measures are available, as there are no commercial grapevine rootstocks conferring resistance against A. mellea. Soil fumigation is banned in vineyard soils and probably inefficient because remnant roots are buried deeply. The strongly recommended practice of leaving the land fallow long term is not performed by growers in intensive production areas. Alternative biological and cultural control methods are under study (Baumgartner and Rizzo 2006).
Some specific groups of microorganisms protect the plant against pathogens through several mechanisms. Among these organisms, arbuscular mycorrhizal fungi (AMF) are promising because of their ubiquity in natural and agricultural terrestrial ecosystems, especially in crops managed with sustainable practices (Jeffries and Barea 2001). Arbuscular mycorrhizal fungi form symbiotic associations with root systems of most agricultural, horticultural, and woody crop species. Many authors have reported that AMF symbiosis can reduce damage caused by several soilborne pathogens such as nematodes (Calvet et al. 2001) and fungal pathogens causing root rots (Rosendahl and Rosendahl 1990) and vascular damage (Garmendia et al. 2004).
Previous studies demonstrated that inoculation of grapevine plants with AMF can reduce disease incidence, as reported for damage caused by Cylindrocarpon macrodidyum on Vitis rupestris (Petit and Gubler 2006). Mycorrhizal inoculation increased tolerance to A. mellea in several grapevine rootstocks under greenhouse conditions (Aguín et al. 2006, Nogales et al. 2009).
Mycorrhizal status in replant situations is usually not evaluated as a contributing factor to the severity of replant disease. Vineyard management practices often have a negative impact on the biological activity of soil, including mycorrhizal symbiosis, and can decrease the populations of indigenous AMF (Thompson 1994). The importance of AMF symbiosis in plant survival and health is generally accepted and multiple benefits of the artificial inoculation of grapevines with AMF are reported under controlled conditions (Schubert et al. 1988, Linderman and Davis 2001, Aguín et al. 2006) and in the field (Menge et al. 1983, Calvet et al. 2007, Camprubí et al. 2008). In each case, inoculation with AMF improved the short-term growth of vines, indicating that the introduction of selected AM fungi can be significant in replanted vineyards, by optimizing nutrition and enhancing plant survival. The growth response can vary with the rootstock/cultivar combination (Aguín et al. 2004) and the fungal isolate used (Camprubí et al. 2008). Soil biological activity and chemical characteristics also influence mycorrhizal performance (Schreiner 2003).
In this paper, field application of a selected mycorrhizal fungal inoculum was evaluated in two rootstocks suitable for Mediterranean soils and climate, grafted with the same cultivar, established in two replant vineyards in the same geographical area, and with similar physical and chemical characteristics. One vineyard had an identified disease causal agent, A. mellea, and the other had no specific cause of replant syndrome. Both rootstocks, 161-49 Couderc (161-49C) and 140 Ruggeri, are commercially available and resistant to high lime content, drought stress, and phylloxera (Hidalgo 2002). Moreover, their mycorrhizal status has been previously assessed (Karagiannidis et al. 1997, Aguín et al. 2006). They are both commonly used in the wine production area in northeastern Spain.
Inoculation methods that favor the dispersal of mycorrhizal propagules should be considered before planting vines, including the precropping of mycorrhizal carrier aromatic plants, as recommended for citrus nurseries (Camprubí and Calvet 1996a). This method was evaluated in the vineyard soil in which no native propagules had been detected.
Materials and Methods
Soils analysis.
Five composite soil samples were taken from each vineyard before planting to determine their physical and chemical properties (Applus Agroambiental, Lleida, Spain) and to estimate the number of infective mycorrhizal propagules by the most probable number technique. The estimation was done in a 10-fold soil dilution series using autoclaved (120°C, 1 hr) coarse sand as diluent and leek (Allium porrum L.) plantlets as hosts (Powell 1980, Porter 1979).
Identification of pathogenic agents.
Grapevine root debris were collected from vineyards 1 and 2 and plated on malt agar with streptomycine and benomyl (Mansilla et al. 2000) to determine the presence of root rot fungi. White mycelium was observed on the fresh root samples from vineyard 2. Plates were incubated at 25°C for two weeks and the isolated fungal cultures were incubated in the same medium for DNA extraction. To determine the identity of the isolates, DNA was extracted from 40–60 mg of fungal mycelia from fungal cultures or fresh vegetal material. Extraction was performed using EZNA fungal DNA miniprep kit (Omega Bio-Tek, Norcross, GA) following the manufacturer’s instructions with modifications as previously described (Martin and Torres 2001). A PCR technique was subsequently performed with the specific primers LR12R (5′-CTGAACGCCTCTAAGTCAGAA-3′) and O-1 (5′-AGTCCTATGGCCGTGGAT-3′) (Mansilla et al. 2000) using a Ready-to-Go PCR bead kit (Amersham-Pharmacia, Piscataway, NJ), 1 μL fungal DNA extract diluted 1/500 as template, and 0.5 μL of a 10 μM solution of each primer to achieve a final volume of 25 μL. PCR conditions were: initial denaturation at 94°C for 95 sec followed by 35 cycles of annealing at 60°C for 60 sec, extension at 72°C for 120 sec, and denaturation at 60°C for 95 sec. The last cycle was followed by a final extension at 72°C for 10 min. The amplicons obtained were used to conduct a restriction fragment length polymorphism (RFLP) test with Alu1 (37°C, 1 hr) for A. mellea pattern characterization (Harrington and Wingfield 1995).
Direct field inoculation.
Cabernet Sauvignon plants grafted on the vine rootstocks 161-49C (Vitis riparia Michx. x V. berlandieri Planch.) and 140 Ruggeri (Vitis rupestris x V. berlandieri) were obtained from a commercial nursery and planted in early May 2005 in two different replant vineyards in northeastern Spain. The soil from vineyard 1 had been under tillage for 10 years and the soil from vineyard 2 had been cultivated until the extraction of old vines one year before the establishment of the new plantation.
After plowing, 100 and 120 vines from each rootstock were established in the first and second replant soils, with 1.2 m and 2.8 m between vines and rows, respectively. In both locations, half of the plants were inoculated with the AMF Glomus intraradices Schenck and Smith. The experiment used a randomized block design with three and four blocks per treatment (inoculation with G. intraradices and no inoculation) and rootstock (161-49C and 140 Ruggeri) in vineyards 1 and 2, respectively. There were 12 plants per block but only six plants per block, chosen at random, were measured at each sampling when growth data were recorded.
The isolate of G. intraradices, registered in the International Bank for the Glomeromycota as BEG 72, is a native arbuscular fungus from the same Mediterranean area. It was isolated from a citrus nursery (Camprubí and Calvet 1996a) and has proved effective in many agricultural and landscape restoration situations (Calvet et al. 2001, Estaún et al. 2007). The mycorrhizal inoculum was obtained from greenhouse Allium porrum L. cultures on Terragreen (Oil-Dri Company, Wisbech, UK), and 200 mL homogenized rhizosphere substrate including spores and root fragments was introduced under each inoculated vine at planting.
In December 2005, at the end of the first growing season (seven months after planting), vines were pruned and pruning weight was recorded from six plants per block. In December 2006 (19 months after planting), the vines were pruned again and plant biomass produced during the second growing season was determined.
Data were analyzed (SPSS version 9.0; SPSS Inc., Chicago, IL) in a four-way ANOVA considering the main factors inoculation, location, rootstock, and year and their respective interactions. Due to significant interactions among factors, eight independent Student t tests were performed comparing inoculated or noninoculated plants for each combination of location, rootstock, and year. The probability level was established at α = 0.10.
Indirect field inoculation.
In February 2005 seeds of Lavandula officinalis Mill. (Eurogarden) and Thymus vulgaris L. (Semillas Fitó) were surface sterilized as described (Camprubí and Calvet 1996b) and planted in seedbeds filled with Terragreen. A layer of G. intraradices (BEG 72) inoculum (1.5 L per seedbed) was applied on top and covered by the same substrate before sowing seeds. Allium porrum mycorrhizal roots and G. intraradices propagules in Terragreen (27 propagules in 100 mL Terragreen) were used as inoculum.
One month later, mixed root samples from each plant species were stained with Trypan Blue (Koske and Gemma 1989) and the internal root colonization of G. intraradices was estimated following the gridline method (Giovannetti and Mosse 1980). The percentage of root colonization was 69% for L. officinalis and 62% for T. vulgaris. The mycorrhizal plants were transferred to 20 × 40 × 15 cm containers filled with a pasteurized mixture of sandy soil, quartz sand, and sphagnum peat (3:2:1 v/v) and kept under greenhouse conditions for 4.5 months.
Two field rows were plowed and prepared for planting in vineyard 2, where no indigenous AMF had been identified. In June 2005 the mycorrhizal lavender and thyme plants were planted alternately, 30 cm apart in three lines within the row. Three and 11 months after planting (September 2005 and May 2006, respectively), eight soil samples were taken at random from each row to estimate the number of infective propagules present in the soil. After the second soil sampling, plants were harvested and the root systems plowed into the soil. Twenty Cabernet Sauvignon vines grafted on 161-49C were then planted in the central line of each row, 1.2 m apart. In February 2008, soil samples were taken again to estimate the number of infective propagules present.
Results
Soils analysis.
The chemical properties of the soils in the two vineyards were similar: the pH was alkaline, because of high carbonate concentration, and the organic matter content was low (Table 1⇓). Only the phosphorus (P) and potassium (K) levels differed slightly. Vineyard 1 had normal P and low K; vineyard 2 had low P and normal K. The physical properties analyzed indicated that vineyard 1 had a silty clay loam soil and that vineyard 2 had a clay loam. In vineyard 1, the number of infective mycorrhizal propagules estimated by the most probable number technique was 1 propagule per 100 mL soil; in vineyard 2, there were no mycorrhizal propagules.
Chemical and physical soil analysis of replant vineyard soils.
Identification of pathogenic agents.
No pathogenic agents were recovered from the root debris in vineyard 1. Isolates of Armillaria spp. obtained from old vine roots buried in the soil of vineyard 2 were identified as Armillaria mellea pattern mel 1, as three restriction fragments of 320, 180, and 135 bp were obtained with the RFLP test performed with Alu 1 (Pérez et al. 1999).
Direct field inoculation.
Vine growth was statistically different depending on the inoculation treatment (inoculated and noninoculated with G. intraradices), the rootstock (161-49C or 140 Ruggeri), the location (vineyard 1 or 2), and the year (first or second growing year). Significant interactions were detected between location and year (p = 0.019), rootstock and year (p = 0.057), and location, root-stock, and year (p = 0.096). Therefore, factors were analyzed separately.
In vineyard 1, where A. mellea was not recovered from the soil and mycorrhizal propagules were present, 161-49C plants inoculated with G. intraradices had greater biomass than noninoculated plants in the first year (Table 2⇓). However, in the second year, there were no significant differences among treatments. In vineyard 2, where A. mellea was recovered from the soil and no mycorrhizal propagules were detected, the growth response of 161-49C plants was different: in 2005 no differences were reported between inoculated and noninoculated plants, but in 2006 a higher shoot biomass was recorded in G. intraradices inoculated plants (Table 2⇓). In vineyard 1, G. intraradices inoculation enhanced growth of vines grafted on 140 Ruggeri in both 2005 and 2006. In vineyard 2, although G. intraradices enhanced plant growth in 2005, in 2006 no differences in pruning weight were found among treatments (Table 2⇓).
Growth of Cabernet Sauvignon plants grafted on 161-49C and 140 Ruggeri rootstocks inoculated or not with Glomus intraradices at the end of the first (2005) and second (2006) growing seasons in two replant vineyards.
Indirect field inoculation.
In the rows planted with mycorrhizal aromatic carrier plants in vineyard 2, the number of AMF propagules in the soil slightly increased at the end of 2005, from 0 to 9 propagules per 100 mL (Figure 1⇓). In May 2006, 11 months after planting, the number of propagules drastically increased, from 9 to 46 propagules per 100 mL. In 2008, when vines had been growing for two years, the number of mycorrhizal propagules had again increased slightly in the rows, from 46 to 58 propagules per 100 mL.
Number of arbuscular mycorrhizal fungal propagules per 100 mL soil in vineyard 2 before planting aromatic carrier plants (June 2005), three months after planting (September 2005), at harvest and vine planting (May 2006), and after two years growth of vines in the field (May 2008). Bars are means of 16 soil samples ± standard error.
Discussion
In vineyard 1, 161-49C and 140 Ruggeri plants inoculated with G. intraradices showed a significant growth enhancement the first year. Thus, initially, the mycorrhizal inoculum applied at planting was more effective than the propagules of the native fungus or fungi. However in the second year, while there was still a significant growth increase in inoculated 140 Ruggeri plants, no differences due to inoculation were detected in 161-49C plants, perhaps due to a different response of the rootstocks to soil and/or climate conditions. 140 Ruggeri is less tolerant to extreme moisture or drought (Hidalgo 2002) and can thus benefit more from mycorrhizal colonization to withstand stress conditions. Rootstock 161-49C confers slower growth and thus the mycorrhizal potential of the soil might not be a growth limiting factor for the vines. This observation could explain why there were no significant differences in the growth of inoculated and noninoculated 161-49C vines after two years in vineyard 1. The different affinity for mycorrhizal infection of different cultivars within the same plant species has been reported previously (Calvet et al. 2004). Here, vines grafted on 161-49C may have a high affinity for the indigenous AMF, as in 2006 no differences were detected in plant growth among treatments in vineyard 1, by which time noninoculated plants could already be colonized by native AMF. Plants can probably achieve optimal growth when colonized by the native fungal propagules and do not benefit from the additional artificial inoculum. However, despite the presence of natural AMF propagules, artificial inoculation with the selected fungus was useful when vines were grafted on the highly vigorous 140 Ruggeri rootstock, as G. intraradices BEG 72 inoculated plants outgrew plants from the noninoculated treatment in both 2005 and 2006.
The ability of mycorrhizal fungi to enhance grapevine growth varies with rootstock and fungus (Schubert et al. 1988, Linderman and Davis 2001, Aguín et al. 2004). A comparison of the responses of several grapevine root-stocks and cultivars to AMF found that all isolates were effective at promoting plant growth, but that in most vines, the largest increase was found after inoculation with G. intraradices (Linderman and Davis 2001). Another study under field conditions showed that only G. intraradices BEG 72 significantly increased plant growth when compared with two native AMF isolated from replant vineyards (Camprubí et al. 2008). The G. intraradices BEG 72 isolate used in our vineyards is thus an aggressive AMF that can probably infect grapevines more rapidly than the native isolates present in the vineyard soil. Localized inoculation with G. intraradices BEG 72 significantly increased growth in vineyard 1 in the first year. Glomus intraradices has a high capacity to colonize and form an extensive and effective network of external hyphae around the root for nutrient acquisition (Abbott and Robson 1985). Moreover, the high concentration of AMF propagules placed in direct contact with the vine roots probably helped to induce a more rapid colonization.
In vineyard 2, soil conditions were different: no mycorrhizal fungal propagules were found and the soil was infested with the root rot fungus A. mellea. Accordingly, significant differences due to location were observed in plant growth, as vines on both rootstocks had lower aerial biomass after two years in vineyard 2 as compared with vineyard 1, regardless of AMF inoculation. The differences in soil chemical and physical characteristics were not important enough to justify the differences in plant growth.
The response of both rootstocks also varied depending on mycorrhizal inoculation. In the first year, no differences in plant biomass were found in inoculated and non-inoculated 161-49C vines, but in the second year growth enhancement because of G. intraradices was significant, suggesting better adaptation of inoculated plants in replant situations. The mycorrhizal condition of some crop plants could be crucial in providing the rootstocks an increased capacity to withstand stress conditions, such as nutrient deficiencies and drought (Linderman and Davis 2001), and to tolerate soilborne pathogenic fungi and nematodes (Azcón-Aguilar et al. 2002, Calvet et al. 2004). Previous work demonstrated that prior inoculation of 161-49C rootstock vines in containers with Glomus aggregatum increased growth in a soil artificially inoculated with A. mellea and possibly a higher tolerance to the disease (Aguín et al. 2006).
On the other hand, although growth increased in inoculated 140 Ruggeri plants in 2005, the next year there were no significant differences among treatments in vineyard 2. The relative resistance of various grapevine rootstocks to root rot disease caused by Armillaria has been studied (Baumgartner and Rizzo 2006), but to our knowledge, no information is available concerning the susceptibility of 140 Ruggeri to A. mellea. Our results suggest its response is similar to the response of 161-49C, as the growth decrease of both rootstocks in vineyard 2 versus vineyard 1 was ~65–75%. However, inoculation with G. intraradices BEG 72 did not enhance growth of vines grafted on 140 Ruggeri after two years in the field. The amount of mycorrhizal inoculum may have been insufficient for 140 Ruggeri vines. The latter rootstock confers more vigor than 161-49C and its root growth rate is likely much higher. The localized inoculum may not be effective for 140 Ruggeri plants in a soil with no native fungal propagules and a pathogenic agent. In vineyard 1, despite the higher soil P, the amount of inoculum was high enough to enhance plant growth, thus the limiting factor on growth in vineyard 2 may be the presence of A. mellea. Nevertheless, other limiting factors such as different microbial communities or a higher flooding possibility in the soil of vineyard 2 cannot be discarded.
Nursery production of mycorrhizal vines in containers previous to their establishment in a replant soil could be more effective at increasing tolerance to replant conditions. Early mycorrhizal inoculation of Merlot vines grafted on SO4 rootstock in containers significantly increased plant growth and survival eight months after planting in a replant vineyard (Calvet et al. 2007). However, this alternative is not compatible with traditional agronomic practices in Spain, where growers transplant grafted dormant cuttings directly into the soil. In vineyard 2, inoculation through precropping the mycorrhizal carriers lavender and thyme increased the number of AMF propagules in the soil drastically after 11 months. One study reported that cover crops had no effect on the mycorrhizal status of grapevine roots (Baumgartner et al. 2005), but in this case there was a lack of contact between grapevine and cover crop roots in the vine rows. Our proposal increased the mycorrhizal potential of a soil lacking mycorrhizal propagules by maintaining artificially inoculated vegetation before establishing the vineyard in the same rows.
Conclusion
Much remains to be learned about the field application of AMF inoculum. Nevertheless, it seems clear that the physical, chemical, and biological composition of replant soils, generally disturbed, contribute to poor development of plants in the field and that introduction of selected AMF inocula can produce long-term benefits. Such benefits were found here for grapevines grafted on 140 Ruggeri regardless of indigenous AMF and for grapevines grafted on 161-49C in a vineyard without indigenous AMF. A preliminary study of agronomic conditions is essential before considering the suitability of either AMF application or a particular inoculation procedure.
Footnotes
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Acknowledgments: Research was financed by the INIA Instituto de Investigaciones Agroalimentarias, contract grant RTA 04-027-C2. Amaia Nogales received funding from AGAUR, Generalitat de Catalunya, Spain.
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The authors acknowledge the helpful collaboration of Marc Vidal in the agronomic management of the vineyards.
- Received December 2008.
- Revision received April 2009.
- Accepted July 2009.
- Copyright © 2009 by the American Society for Enology and Viticulture
Literature Cited
Vol 60 Issue 4
