First report of alder Phytophthora closely related to P. uniformis on Alnus glutinosa seedling in Finland.
A. Poimala, S. Werres, T. Pennanen, and J. Hantula' Plant Disease 0 0:0, PDIS-03-17-0322-PDN
The allotriploid Phytophthora × alni (Brasier & S.A. Kirk) Husson, Ioos & Marcais and its progenitors, the diploid P. uniformis (Brasier & S.A. Kirk) Husson, Ioos & Aguayo and the allotetraploid P. × multiformis (Brasier & S.A. Kirk) Husson, Ioos & Frey are the causal agents of alder decline in Europe. In June 2015, a dark, ∼40 mm stem lesion was found on one out of 100 inspected Alnus glutinosa (L.) Gaertn seedlings in Mäntyharju, Finland. Surface wood pieces from the lesion edges were plated onto malt agar. Cultures on carrot piece agar (CPA) showed an appressed colony with woolly aerial mycelium. Oogonia (Ø mean 44.8 µm, n = 50) and oospores (Ø mean 40.0 µm, n = 50) developed after 7 days at 20°C in the dark. They were commonly smooth walled, and 49 out of 50 of the amphigynous antheridia were single celled. Sporangia (21.7 × 18.1 µm, n = 50) were produced on pea broth, with 2-day flooding with soil extract, and they were commonly unpapillate and obpyriform-ellipsoidal. DNA was extracted, and the internal transcribed spacer (ITS) rDNA was amplified (ITS6 and ITS4; Cooke et al. 2000). The PCR products were then cloned because of double peaks in the sequence. Among 33 clones, four alleles with five polymorphic bases were obtained (A1, 55%; A2, 6%; A3, 15%; and A4, 24%; these were deposited in GenBank as accession nos. MF356294, MF356295, MF356296, and MF356297, respectively). The closest match to the ITS allele sequences in GenBank was the very closely related species P. cambivora AF087479 (differences, 7 to 9 bases). All other sequenced regions showed single alleles. The mitochondrial cox spacer (MF356298; amplified by FMPhy8 and FMPhy10, Martin et al. 2004) matched 100% with P. cambivora P1431 (GU221955) and P. alni P16202 (GU221933) in GenBank. A partial beta tubulin gene (MF356299; amplified by 901F and 1401R, Bilodeau et al. 2007) matched 100% with P. uniformis ALN58 (KU899249). ASF-like, GPA1, RAS-Ypt, and TRP1 genes were amplified with primers by Ioos et al. (2006). GPA1 (MF356301) matched 100% with P. uniformis PAU84 (DQ092849), and RAS-Ypt (MF356302) had exact matches with four P. uniformis isolates in GenBank (e.g., PANM53 and EU371549). The TRP1 gene (MF356303) differed by 1 base from P. uniformis alleles PAU60 (DQ202480) and PAU89 (DQ202481). The ASF-like gene (MF356300) matched 100% with PAU84 (DQ092815). Nine 1-year-old seedlings of A. glutinosa and of Betula pendula Roth were inoculated with plugs from 10-day-old mycelial culture on CPA after making a bark incision. Nine control seedlings of each species received a sterile CPA plug. Inoculations were wrapped in damp cotton wool, and the plants were kept outdoors at 10 to 22°C. After 15 days, eight out of nine (89%) A. glutinosa seedlings had developed lesions (mean length 22 mm), as well as six out of nine (67%) B. pendula seedlings (mean length 4 mm). No lesions were observed in control seedlings. The pathogen was reisolated from two symptomatic seedlings of both hosts. This is the first report of an alder Phytophthora in Finland. The sequence data suggested the isolate to be closely related but dissimilar to P. uniformis. The morphology corresponded to that previously reported for P. uniformis (Brasier et al. 2004), except for the antheridia, which were almost all single celled. Multiple ITS alleles could also refer to the initially reported P. uniformis karyotype 2n+2 (Brasier et al. 2004). These findings add to our knowledge on the variation among the alder Phytophthora group. Furthermore, they demonstrate the risk that the pathogen could be transported in new hosts.