First Report of Phytophthora alni subsp. uniformis on Black Alder in Spain.

Phytophthora alni is the causal organism responsible for devastating losses occurring on riparian alders stands in Europe. This emergent hybrid pathogen has multiple variants that have been placed in three subspecies (1). P. alni subsp. uniformis and P. alni subsp. multiformis are reported to be less aggressive than P. alni subsp. alni, though all are considered pathogenic. In Spain, P. alni subsp. alni was detected for the first time in 2009 in Galicia (northwestern Spain) causing root and collar rot on riparian alder populations (3,4), but other subspecies had not been identified. In April 2011, a survey along the Deza River in Galicia was carried out to clarify the Phytophthora sp. associated with the alder decline. Thirty riparian Alnus glutinosa stands, from both sides of the river, were surveyed. Samples of bark and roots of 18 alder stands that showed symptoms of Phytophthora rot and soil from all 30 stands were collected. Roots and tissue from fresh, active, inner bark lesions from 54 trees were transferred to selective medium V8-PARPH agar and incubated for 7 days at 22°C in the dark. P. alni subsp. alni (1) was isolated from roots, bark, or soil in five alder stands. Another Phytophthora sp. was isolated from the bark of one symptomatic tree located in Silleda (Pontevedra), transferred to carrot agar (CA), and incubated in the dark. On CA, the isolate produced irregular and appressed colonies with an optimum growth temperature of 22 to 23°C. The isolate was homothallic with smooth-walled oogonia with a diameter ranging from 36 to 50 μm and two-celled, amphigynous antheridia (1). In soil extract, noncaducous, nonpapillate, ellipsoid-to-ovoid sporangia were produced. Average sporangium were 43.4 × 30.1 μm with a length/breadth ratio of 1.43. Internal proliferation occurred. Amplification of DNA was accomplished by sequence characterized amplified region (SCAR)-PCR primers (2). The amplicon sizes obtained were identical to P. alni subsp. uniformis. Internal transcribed spacer (ITS) (DC6-ITS6/ITS4) and nadh1 (NADHF1/NADHR1) mitochondrial gene regions were also amplified and deposited in GenBank (Nos. JN880411 and JN880410). Comparison of the sequences showed 100% homology with P. alni subsp. uniformis (GenBank Nos. GU259293 and DQ202489). Pathogenicity was tested on 10 3-year-old black alder plants grown in pots. A shallow wound was made with a scalpel at the root collar level of each plant. A 5-mm-diameter mycelia plug, taken from the margin of a 7-day-old culture grown on CA, was inserted in every wound and sealed with Parafilm. Five black alder control plants received only sterile CA agar plugs. Plants were kept at 24°C and 80% humidity. After 3 months, wilting of shoots, dead leaves, and dark stained necroses of the bark tissue varying in length from 0.8 to 5 cm were observed on inoculated plants. Control plants remained healthy. P. alni subsp. uniformis was recovered from inoculated plants, but not from controls. To our knowledge, this is the first time that P. alni subsp. uniformis has been reported in Spain. The presence of a new subspecies in a new region can result in hybridization between individuals of different species or subspecies. This process may allow the rapid evolution and adaptation of these species to new hosts or environmental conditions. References: (1) C. M. Brasier et al. Mycol. Res. 108:1172, 2004. (2) R. Ioos et al. Eur. J. Plant Pathol. 112:323, 2005. (3) C. Pintos et al. Plant Dis. 94:273, 2010. (4) A. Solla et al. Plant Pathol.59:78, 2010.