Vitis 35 (2), 95-98 (1996) Aseptic dual culture of grape (Vitis spp.) and grape phylloxera (Daktulosphaira vitifoliae FITCH). by AsTRID FORNECK 1 ), M. A. WALKER 2 ) and N. MERKT 1 ) 1 ) Universitiit Hohenheim, Institut filr Obst-, Gemiise- und Weinbau, Stuttgart, Deutschland 2 ) University of California, Department of Viticulture and Enology, Davis, California S u m m a r y : An aseptic dual culture of grape phylloxera (Daktulosphaira vitifoliae FJTCH) and grape vine (Vitis spp.) was developed. This method permits continuous observation ofphylloxera feeding and the whole plant response on a dynamic basis. The plant/parasite interaction of three testplants (V. vinifera L., var. Riesling, SO 4 (V. berlandieri PLANCH. x V. riparia L.) and V. riparia, var. Gloire de Montpellier) are demonstrated by observing post-infectious reactions of the host- and population dynamics of the parasite. Different stages of phylloxera could be observed including nymphs, winged phylloxera (alatae) and sexual male phylloxera. Several potential applications for this aseptic dual culture are demonstrated. K e y w o r d s : Daktulosphaira vitifoliae, phylloxera, Vitis, aseptic dual culture. Introduction Grape phylloxera (Daktulosphaira vitifoliae FITCH) has been recognized as the major pest insect problem for grapes since its accidental introduction to Europe in the 1860s. The planting of vines grafted on resistant rootstocks de- rived from resistant Vitis species is the only long-term con- trol of phylloxera. The breeding and use of resistant root- stocks began near the end of the 19th century and the best of those rootstocks remain in use today. The fact that phylloxera continues to impact viticulture in California (WALKER 1992), Germany (ROHL 1995) and Switzerland (REMUND and BoLLER 1994) and the need for rootstocks with resistance to other soil-borne problems in addition to phylloxera, necessitates further studies of the phylloxera I grape interaction. Past studies have investigated this interaction in field trials, with potted and containerized greenhouse plants, in excised root studies in laboratory bioassays and aseptic tissue culture of roots, callus or foliage. These methods allow the study of the host and parasite interaction. How- ever, none of these methods allows continuous observa- tion of the phylloxera feeding and whole plant response. This paper presents a method to observe the interaction of plant roots and shoots with phylloxera on a dynamic basis. It is a rapid technique which is seasonably independent, provides controlled conditions, and requires less space as well as lower inoculum volumes than conventional green- house methods. Materials and methods G rap e m i c r o p r o p a g at i o n : Green cuttings of Vitis vinifera L., var. Riesling, and the rootstock SO 4 (V. berlandieri PLANCH. x V. riparia L.) and V. riparia, var. Gloire de Montpellier, were cut into one-node-segments of 3-4 cm length and 0.5 cm diameter. After harvesting, these cuttings were immediately surface-sterilized by immersion in a 1.5 % NaOCl-solution containing a drop of liquid deter- gent. Treated shoot pieces were rinsed in sterile water six times and inserted into culture tubes (25 mm x 100 mm) con- taining 20 ml of media and closed with a cap (Kap-UTS (K25), Bellco, USA). The medium consisted of 50 % MS (MURASHIGE and SKooa 1962), mineral salts (Gibco Lab, Grand Island, NY), supplemented with 50 % MS vitamins (Gibco Lab), 10 g sucrose, 1 mg/1 indole-3-acetic acid (Sigma) and 7 g/1 plant tissue agar (A-1296, Sigma). The pH was adjusted with KOH or HCl to 5.7. Tubes containing plants were kept in a growth chamber at 27 °C with a 16-h light period. Ph y 11 o x er a e g g s our c e : Cultured plants were inoculated with phylloxera eggs gathered from a mixture of laboratory based colonies. These phylloxera strains were collected from a variety of rootstocks and locations through- out California and maintained in the laboratory on root pieces of V. vinifera, var. Cabernet Sauvignon (DE BENEDICTIS and GRANETT 1992). The eggs ranged from one- to 4-day-old when used for inoculation. D u a l c u l t u r e : The rooted nodal cultures were placed in magenta culture vessels (Magenta GA-7 Vessel, Sigma) prior to inoculation with phylloxera. The vessels were filled with 25 ml modified MS media supplemented with additional 2 g/1 agar (Sigma). As the agar cooled and solidified after autoclaving the vessels were stored at an 45° angle resulting in a media corpus with a sloped surface covering 50% of the bottom (Fig. 1). A two-node segment of the micropropagated plants was inserted in the media filled vessels and incubated in a growth chamber as de- scribed above. After two weeks the containers were tipped by 90° in order to promote root growth towards the media- free side of the vessel. Optimal host plants for inoculation Correspondence to: AsTRID FoRNECK, Universitiit Hohenheim, Institut filr Obst-, Gemiise- und Weinbau (370), D-70599 Stuttgart, Germany. Fax: (0711) 4593496.