Relative Resistance of Grapevine Rootstocks to Armillaria Root Disease
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408 – Baumgartner and Rizzo Relative Resistance of Grapevine Rootstocks to Armillaria Root Disease Kendra Baumgartner1* and David M. Rizzo2 Abstract: Grapevine rootstocks were screened for resistance to infection by Armillaria mellea, the pathogen that causes Armillaria root disease. The first objective was to determine which factors hasten colonization of grape- vines by A. mellea in order to develop the most rapid inoculation technique. Results showed that wounding the root collar bark and vascular cambium did not significantly increase infection rate (p = 1.0), that young vines were infected significantly faster than older vines (p = 0.03), and that fine roots (≤5 mm diam) were not susceptible to infection. Based on these findings, the inoculation technique was modified and used to inoculate dormant rootings of eight rootstocks in the greenhouse. After two years, the root collars were examined for the presence of myce- lial fans of A. mellea and infection was confirmed by culture. The rootstock Freedom had the lowest frequency of infection (7%) and was the most resistant rootstock (p = 0.0016). Rootstocks O39-16, 5C, Riparia Gloire, and 3309C had the highest frequencies of infection with 63%, 73%, 79%, and 85%, respectively. Rootstocks St. George, Ramsey, and 110R had intermediate frequencies of infection. Results suggest that Freedom and, to a lesser extent, St. George, Ramsey, and 110R will be useful components of an integrated management strategy for Armillaria root disease. Resistant rootstocks may decrease the rate of colonization of grapevines in A. mellea-infested vineyards, thereby reducing yield losses from Armillaria root disease. Key words: Armillaria mellea, disease resistance, screening technique, Vitis vinifera Armillaria root disease of grapevine (Vitis vinifera L.) is cronutrients, delayed ripening of fruit, and eventual death caused by Armillaria mellea (Vahl:Fr.) P. Kumm., a fungal of infected vines (Baumgartner and Warnock 2006). Foliar pathogen that also infects the roots of many forest trees symptoms include stunted shoots, dwarfed leaves, wilting, in California (Baumgartner and Rizzo 2001b). The pathogen and premature defoliation. survives as a saprobe on woody roots after the host dies Armillaria root disease affects vineyards in all major (Redfern and Filip 1991). Its mycelium decomposes cellu- grapegrowing regions of California, including the Central lose and other cell wall components for nutrients as it Valley, the North Coast, and the Central Coast (Baum- grows, decaying the root wood. Where forests are con- gartner and Rizzo 2001a). Because of the broad host range verted to vineyards, mycelium can survive in partially de- of A. mellea, which includes many species of fruit and cayed tree roots that remain buried in the soil and can in- nut trees (Raabe 1962), planting a different perennial crop fect grapevines for up to several years after planting in place of diseased vines is unlikely to eliminate the (Baumgartner and Rizzo 2002). Infection occurs when vine Armillaria root disease problem. The common name of A. roots come in direct contact with residual tree roots and mellea, “oak root fungus,” misleads many growers to be- are subsequently colonized by A. mellea mycelium. Armil- lieve that establishing vineyards on sites without oaks laria root disease causes significantly decreased yield and minimizes the risk of Armillaria root disease. Avoidance of growth (Baumgartner 2004), lower root absorption of ma- infested forest sites is extremely difficult because A. mel- lea infects many different tree species in addition to oaks and aboveground symptoms are rare (Baumgartner and Rizzo 2001b). Furthermore, the mycelial fans and mush- 1Research plant pathologist, USDA–Agricultural Research Service, and 2Pro- rooms of other Armillaria species, none of which cause fessor, Department of Plant Pathology, University of California, Davis, Armillaria root disease on agronomic hosts, can be mis- CA 95616. taken for A. mellea (Baumgartner and Rizzo 2001b). *Corresponding author [email: [email protected]; tel: 530 754- 7461; fax: 530 754-7195] Pesticides are not the best means of controlling Acknowledgments: Research was supported by the USDA–Viticulture Con- Armillaria root disease. Methyl bromide can kill A. mellea sortium and the USDA–Agricultural Research Service. Grapevines were in residual roots (Munnecke et al. 1981), but it does not generously donated by Sunridge Nurseries Inc., Bakersfield, CA. penetrate the soil deeply enough to reach all infected The authors thank J. Warren, B. Craughwell, and A. Euliss for greenhouse roots (Bliss 1951). Vines planted in fumigated soil are typi- and laboratory assistance and S. Krebs (Napa Valley College, CA), P. Cous- cally healthy for several years, but they eventually be- ins (USDA-ARS, Geneva, NY), and G. Schnabel (Clemson University, SC) for their comments on this manuscript. come infected when their roots contact inoculum that es- Manuscript submitted April 2006; revised May 2006 capes the effects of the fumigant (Gubler 1992). Fungicides Copyright © 2006 by the American Society for Enology and Viticulture. examined for control of Armillaria root disease also do not All rights reserved. provide long-term control (Adaskaveg et al. 1999). These 408 Am. J. Enol. Vitic. 57:4 (2006) Relative Resistance to Armillaria – 409 findings are consistent with the biology of the pathogen. made on 1.5% water agar (WA) with benomyl (4 μg a.i./ Armillaria mellea is a wood decay fungus that lives be- mL) and streptomycin sulfate (100 μg/mL) added after au- neath the bark of infected roots, where it decomposes root toclaving. Isolation plates were incubated at 24°C for 2 wood and creates breaks in the vascular system (Redfern wk. Subcultures were transferred to 1% malt extract agar and Filip 1991). Eradicating A. mellea from a living host (MEA) with an overlay of sterile cellophane. Identity was requires the difficult task of delivering a fungicide to root confirmed by PCR amplification of the nuclear rDNA wood that is decomposed and, thus, not connected to the intergenic spacer I (IGS-I) (Harrington and Wingfield vine’s vascular system. Indeed, fungicides injected into 1995). DNA was amplified directly from mycelium by gently infected almond trees were effective only to the extent scraping a pipette tip over the surface of the mycelium that they actually reached the A. mellea infections (Ad- and dipping it into the PCR vial. Digestions were per- askaveg et al. 1999). formed with AluI added directly to the PCR vial and incu- In contrast to soil fumigation or fungicide injections, a bation at 37°C for 2 hr. The isolate had a restriction pat- more sustainable approach to the control of Armillaria tern of two fragments, 322 and 155 bp, which is diagnostic root disease of grapevine may be the use of resistant of A. mellea. rootstocks. In a previous study, Vitis germplasm with re- Inoculum was prepared by inoculating segments (1.5 cm sistance to Armillaria root disease was identified through diam x 5 cm length) of dormant pear shoots collected from inoculation of potted plants in the greenhouse (Raabe our experimental orchard (Armstrong Experimental Station, 1979). However, the identity of the Armillaria isolates is Plant Pathology Department, UC Davis). Glass jars were unknown because the study was completed before the filled with 30 segments per jar and autoclaved for two 1.5- discovery that A. mellea consists of multiple biological hr periods, separated by 12 hr at 24°C. After cooling, 60 species (Anderson and Ullrich 1979), which have since agar plugs from A. mellea cultures were added per jar. been named as distinct Armillaria species (Guillaumin et Screw-top lids were tightened atop each jar and sealed al. 1991, Volk et al. 1996). Furthermore, the 30 examined with parafilm to prevent contamination. Jars were incu- selections (Raabe 1979) consisted primarily of Vitis spe- bated in the dark at 24°C for 6 mo (months). cies (such as V. girdiana and V. treleasei) that are neither Wounding and inoculation rate. The factors examined used as rootstocks nor definitively resistant to grape in the first experiment were artificial wounding (wounded phylloxera, Daktulosphaira vitifoliae (Fitch), a wide- or intact bark) and inoculation rate (one pear segment spread vineyard pest. Our goal was to identify A. mellea- colonized by A. mellea at 1x inoculum or three pear seg- resistant rootstocks that are both widely available to ments at 3x inoculum). There were four experimental treat- growers and are resistant to phylloxera. ments: intact bark–1x inoculum, intact bark–3x inoculum, Our first objective was to identify the best technique wounded bark–1x inoculum, and wounded bark–3x inocu- to hasten infection by conducting a series of experiments lum. Inoculum was prepared 6 mo before inoculating the on St. George, a rootstock identified as susceptible (Raabe plants. One-yr-old, field-grown dormant rootings of St. 1979). Raabe’s technique of inoculating vines by burying George rootstock were potted in 11.4-L pots in sterile inoculum beneath their root systems was successful at potting mix. Potting mix consisted of a 1:1:1 v/v ratio of causing infection, but it took many months to complete peat moss, perlite, and Supersoil (peat moss, ground fir the experiment. To address the problem of slow coloniza- bark, redwood compost, sand, 2-4-2 slow release fertilizer, tion for which greenhouse experiments with Armillaria oyster shell flour, and ground dolomite; Rod McClellan species are known (Singh 1980), modifications of Raabe’s Co., South San Francisco, CA) that was autoclaved for 1 inoculation technique were tested. Included in the testing hr. Plants were fertilized with Hoagland’s nutrient solution were the use of artificial wounding, inoculation of younger twice per month. plants, and placement of inoculum directly on the root Treatments were imposed on plants 2 mo after planting. collar, all factors that affect infection of other hosts by Wounds 30 mm x 5 mm were made with a sterile scalpel other Armillaria species (Garrett 1956, Sinclair et al.