Penetration of 99 Richter Grapevine Roots by Phytophthora cinnamomi
South African Journal of Enology and Viticulture
The mechanism of penetration of 99 Richter grapevine roots by Phytophthora cinnamomi was studied by light, scanning and transmission electron microscopy. Zoospores encysted on roots within 30 min after inoculation. More spores encysted and germinated near roots than further away, indicating a response to some stimulus exuded by the roots. Germ tubes were usually not swollen at the point of entry into the roots. Penetration occurred mostly down the anticlinal walls separating epidermal cells and
... hyphae developed intercellularly. Swollen germ tubes were sometimes observed where intracellular hyphal growth was preceded by direct penetration of epidermal cells. Some evidence of hydrolysis of epidermal cell walls was found, whereas hydrolysis of cortical cell walls occurred more frequently. Shortly after penetration a plug of amorphous material formed in the germ tube sealing off the penetration peg within the roots. Within 48 hours after inoculation hyphae were observed in the endodermis. Invaded epidermal, cortical and endodermal cells were disrupted and underwent plasmolysis. Phytophthora cinnamomi Rands is the causal agent of root rot associated with the decline and ultimate death of grapevines grafted on 99 Richter (Van der Merwe, Joubert & Matthee, 1972; Marais, 1978a) . Different grapevine rootstock cultivars have been shown, both in the field and pot trials, to vary in susceptibility to P. cinnamomi (Marais, 1979) . The pathogen apparently attacks susceptible as well as tolerant rootstock (Marais, 1978b ), but the mechanism of penetration and infection in grapevine roots is largely unknown. Additional information is required to broaden existing knowledge of resistance to P. cinnamomi. Infection and penetration of 99 Richter roots by P. cinnamomi zoospores and vegetative hyphae were, therefore, studied. MATERIAL AND METHODS Zoospore production: To obtain sporangia of P. cinnamomi an isolate from grapevine roots was initially cultured on potato dextrose agar. Inoculum disks were cut from the advancing margin of the culture after 7 d, placed in petri dishes containing V-8 juice broth (Chen & Zentmyer, 1970) and incubated for 2 d at 25°C. The resulting mycelial growth was washed with sterile distilled water and incubated at 20°C in petri dishes containing liquid soil extract (James, 1958) . To achieve synchronized release of zoospores, mycelia bearing sporangia were chilled at 10°C for 15 min and returned to room temperature. Zoospore concentration was determined with a haemacytometer. . Preparation and inoculation of plant material: Lengths (40 mm) of actively growing shoots were cut from 99 Richter grapevines, surface-sterilized in 0,5% sodium hypochlorite, washed in sterile distilled water, transferred aseptically onto filter paper bridges in 2,5 cm diameter test tubes containing 0,5% Hoagland's nutrient solution, and incubated at 22°C to induce root formation. Root tips (20-30 mm) were excised from rooted shoots, washed in distilled water and placed in a suspension containing 10 2 zoospores/ml in an observation cell designed by Khew & Zentmyer (1973) . The number of encysted and *Part of a Ph.D. (Agric) thesis to be submitted by the senior author to the University of Stellenbosch, Stellenbosch 7600 germinating zoospore cysts present in 0,5 mm zones extending 2 mm from the root surface was determined microscopically ( 100 X). Each test tube containing rooted shoots was inoculated with 30 ml of the zoospore suspension ( 10 2 zoos pores/ ml). Control tubes received 30 ml distilled water. For observation of vegetative hyphal penetration, roots were placed on water agar plates containing 3d old P. cinnamomi colonies and incubated in the dark at 25°C. Light and electron microscopy: Root specimens for light and electron microscopy were removed at intervals, 2-12 h after inoculation. For scanning electron microscopy, root sections (1 mm long) were dissected from the inoculated roots and fixed in 4% glutaraldehyde, (pH 4), for 24 h at 4°C. The roots were afterwards washed twice for 15 min periods in a 0,2 M sodium cacodylate buffer (pH 7 ,2), dehydrated in a graded acetone-water series, subjected to critical point drying with C0 2 and coated with a gold-palladium alloy. For light and transmission electron microscopy, root sections ( 1 mm long) were fixed for 4 h in 6% glutaraldehyde, buffered to pH 7 ,2 in 0,2 M sodium cacodylate. The sections were postfixed for 12 h in 1% sodium tetroxide and subsequently dehydrated through the acetone-water sen es. Tissue was infiltrated with 30% and 60% spurr/acetone mixtures for 6 h periods, followed by Spurr's mixture for 24 h. The material was embedded in Spurr's epoxy resin (Spurr. 1969 ) and polymerized at 70°C for 8 h. Sections for electron microscopy were cut on a Reichert ultramicrotome and stained with 4% uranyl acetate and lead citrate (Reynolds, 1963) . Sections (2,0 µm) for light microscopy were stained with 0,2% toluidine blue, pH 9,0. RESULTS AND DISCUSSION Zoos pores were clustered around the zone of root elongation within 30 min of exposure to the zoospore suspension. After 30 min, more zoospores could be found in the zones closest to the roots (Table 1 ). More zoospores encysted and germinated on and near the root than further away and germ tubes were directed towards the root (Fig. 1 ) .