First report of Phytophthora tentaculata on Verbena sp. in Spain

The oomycete Phytophthora tentaculata causes root and stalk rot of Chrysanthemum spp., Delphinium ajacis and Verbena spp. in nurseries in the Netherlands and Germany (Kröber & Marwitz, 1993). In June 2001, P. tentaculata was isolated from a young potted Verbena hybrid, showing a collar and stalk rot, in a nursery in Majorca (Balearic Islands, Spain). It was initially recovered by plating ∼10 mm pieces of necrotic tissue from the leading lesion on to a phytophthora selective medium, P₅ARP (Erwin & Ribeiro, 1996). A pure culture (isolate CBS 115458) was obtained by transferring aseptically a hyphal tip onto corn meal agar (CMA) and was first identified from morphological characters.

The colony surface texture was uniform and formed sparse, loosely branched mycelium on carrot piece agar (CPA: 50 g carrot pieces and 20 g agar per 1000 mL distilled water) and CMA. The radial growth rate was 2–3 mm day⁻¹ at 20°C on CMA. Sporangia did not appear on either agar media but formed readily in soil extract (50 g soil from a holm oak forest suspended in 1 L ionized water for 24 h at 20°C and then filtered and autoclaved). The sporangia were ovoid to globose, 27–52 (36·9) × 17–31 (24·6) µm, length:breadth ratio 1·4, papillate with a narrow exit pore, and some were caducous with a short pedicel (< 5 µm). Hyphal swellings were present in water. Chlamydospores were only seen on CMA after 2 weeks. Oogonia, readily produced on CPA in pure culture, were globose, mostly terminal or a few lateral, and ranged from 17 to 41 (34·0) µm in diameter. Single paragynous, monoclinous or diclinous, usually long-stalked antheridia were club-shaped or spherical, 9–16 (12·7) µm in diameter and many had appendages. Occasionally two paragynous antheridia per oogonium, as well as some amphigynous antheridia, were observed. Oospores were aplerotic 16–33 (28·4) µm in diameter and thin-walled.

To further confirm its identity, isozyme analysis based on malate dehydrogenase (EC 1.1.1.37) and malic enzymes (EC 1.1.1.40) was performed. Isozyme profiles fitted exactly those of three P. tentaculata strains: two strains ex-Chrysanthemum (including CBS 552.96 paratype) and one strain ex-Verbena; and differing from those of all other papillate species (Oudemans & Coffey, 1991a,b). Pathogenicity was assessed by flooding three potted Verbena plants with a 10⁴ mL⁻¹ zoospore suspension for 48 h at 20–22°C. As controls, two potted Verbena plants were flooded with ionized water. Controls remained healthy 15 days after inoculation. All three inoculated Verbena plants exhibited collar rot after 15 days, from which the pathogen was reisolated using PARP medium, thus confirming Koch's postulates. This is the first report of P. tentaculata in Spain.