First Report of Phytophthora × multiformis on Alnus glutinosa in Spain

Publication Type:

Journal Article


Plant Disease, Volume 101, Issue 1, p.261 - 261 (2017)



Alder species are threatened by a lethal disease caused by the oomycete Phytophthora alni, one of the most important emergent pathogens of natural ecosystems in Europe during the last 20 years (Aguayo et al. 2014). Phytophthora alder decline has caused substantial economic losses and ecological damage from riparian alder populations. Initially, three different subspecies had been described, P. alni subsp. alni, P. alni subsp. uniformis, and P. alni subsp. multiformis. Recently, they have been raised to species status and renamed P. × alni, P. uniformis, and P. × multiformis, respectively (Husson et al. 2015). P. × alni was reported to be the most aggressive and pathogenic to alders. The other two species appear to be less aggressive, but are also considered pathogenic (Brasier and Kirk 2001). In Spain, P. × alni and P. uniformis has also been detected (Pintos Varela et al. 2012). In April 2014, crown dieback and mortality of Alnus glutinosa were noted across the riparian area along the Muiños River in Galicia (northwest Spain). Affected trees, showing abnormally small, yellow, and sparse leaves and necrotic lesions in the inner bark, were surveyed. Samples of bark including the cambium from active lesions, roots, and soil were collected. Phytophthora spp. were baited from saturated rhizosphere soil using carnation petals. Roots and tissue from fresh active inner bark lesions were plated onto selective medium V8-PARPH agar and incubated for 7 days at 22°C in the dark. A Phytophthora sp. isolated from root and bark was transferred to carrot agar (CA) and incubated in the dark. Colonies on CA were irregular with upper temperature limits for growth at 30°C. The isolates were homothallic, with smooth to extremely ornamented oogonia. Oogonial diameters ranging from 42 to 59 µm and one or two celled amphigynous antheridia were observed. In soil extract, noncaducous, nonpapillate, ellipsoid to ovoid sporangia were produced. Amplification of DNA was accomplished by using SCAR-PCR primers (Ioos et al. 2005). DNA samples of Phyophthora isolates have amplified using primers pairs PAM-F/R and PA-F/R. No amplicon was obtained using PAU-F/R primers. ITS (DC6-ITS6/ITS4) and nadh1 (NADHF1/NADHR1) mitochondrial gene regions were also amplified and deposited in GenBank (accession nos. KX090045 and KX090044 isolate CECT 20954). Comparison of the sequences showed 100% homology with P. × multiformis (KJ755099 and FJ696567). Pathogenicity of P. × multiformis isolate CECT 20954 was performed by inoculating 10 3-year-old A. glutinosa plants growing in pots. One shallow cut was made at the root collar level. A colonized 5-mm mycelial agar plug from a 7-day-old culture was inserted in every wound and sealed with Parafilm. Five control plants were inoculated with a sterile agar plug. Plants were maintained in a controlled chamber at 24°C and 80% humidity for 2 months. After a 5-week incubation period, inoculated plants showed dieback symptoms and necrosis of the inner bark tissue. Lesion lengths ranged from 2 to 10 cm. Control plants remained symptomless. P. × multiformis was recovered from all inoculated plants, but not from controls. To our knowledge, this is the first report of P. × multiformis in Spain. With this report, the detection of the P. alni species complex in Spain has been completed.