04285nas a2200157 4500008004100000022001400041245011100055210006900166260001600235300002000251520373900271100001704010700002004027700001404047856006604061 2016 eng d a0191-291700aFirst Report of Phytophthora uniformis and P. plurivora Causing Stem Cankers on Alnus glutinosa in Denmark0 aFirst Report of Phytophthora uniformis and P plurivora Causing S cAug-12-2017 aPDIS-09-16-12873 a

Phytophthora uniformis (Brasier & S.A. Kirk) Husson, Ioos & Aguayo, comb. nov. is an aggressive pathogen associated with root and collar rot of alder trees (Alnus spp.) in Europe (Husson et al. 2015). In summer 2016, symptomatic trees were observed in the banks of a backwater lake area of the river Gurre Å (56°1′46.24″N, 12°27′15.30″E, 29 m above sea level). Several trees displayed chlorosis, reduced leaf size, defoliation, and bleeding cankers on the base of the stem. Inspections upstream near Gurre forest failed in finding other trees with similar crown symptoms, whereas a tree with a bleeding canker was observed downstream (56°3′10.97″N, 12°26′4.40″E, 23 m above sea level). Bark from six trees in the first site and from the tree in the second site was removed from the canker area. As described in Redondo et al. (2015), pieces of 1 cm × 0.5 cm × 1 mm of necrotic cambial tissue from the infection front were plated directly into CMA-PARPBH selective medium. Growing hyphae were transferred onto V8 juice agar medium and incubated at 20°C. Isolates from the first site grew 5 mm/day and formed cottony uniform colonies. After 1 week, spherical smooth-walled oogonia often showing two-celled amphigynous antheridia developed profusely (Ø 39.6 μm; SE = 0.84). Sporangia were ellipsoid and nonpapillate, often with nested proliferation. Colonies from the second site grew 5.5 mm/day, and formed a stellate colony pattern with limited aerial mycelium. Abundant spherical (Ø 29.8 μm; SE = 0.53) oogonia with paragynous antheridia formed after 1 week. Sporangia were commonly ovoid and semipapillate. DNA was extracted from all isolates and the ITS region was amplified using the ITS4 and ITS6f primers. BLAST searches showed 99% similarity with P. uniformis sequence no. AF139367.1 for the six isolates from the first site (GenBank accession nos. KX822022 to KX822027), and 99% similarity with P. plurivora sequence no. FJ665227.1 for the isolate from the second site (KX822028). As morphological characteristics of the first six isolates corresponded to those of the P. alni species complex, primers TRP-PAU-F/-R and RAS-PAM1-F/-R (Ioos et al. 2006) were used to classify them as P. uniformis. ITS patterns from Jung and Burgess (2009) were used to classify the P. plurivora-like isolate as P. plurivora. Pathogenicity of two P. uniformis isolates and the P. plurivora isolate was confirmed by immersing 30 alder seedlings, grown in vermiculite for 3 weeks, in a zoospore suspension of each isolate adjusted to 3 × 104 zoospores ml–1 for 3 h. Seedlings were later kept in dark Eppendorf tubes in sterile water, and rewatered daily. After 10 days, mortality was 76.6 and 86.6% among those inoculated with P. uniformis, and 60% for those inoculated with P. plurivora. Surviving seedlings after 10 days displayed wilting symptoms, while seedlings immersed in water were symptomless. The roots of the dead seedlings were plated onto CMA-PARPBH selective medium. Growing hyphae were observed in all plated roots, and were transferred to V8 juice agar medium. The reisolated Phytophthora colonies were morphologically similar to P. uniformis and P. plurivora, fulfilling Koch’s postulates. Our finding raises concern about the future health of alder trees in Denmark, a country from which P. alni has not yet been reported. However, symptoms of Phytophthora disease have been observed on alder since 1995 (Thinggaard 1996). Further monitoring should investigate the extent of the outbreak.

1 aRedondo, Á.1 aThomsen, I., M.1 aOliva, J. uhttp://apsjournals.apsnet.org/doi/10.1094/PDIS-09-16-1287-PDN03785nas a2200157 4500008004100000022001400041245009600055210006900151260001600220300001600236490000800252520327000260100001703530700001403547856006603561 2016 eng d a0191-291700aFirst Report of Phytophthora pseudosyringae Causing Stem Canker on Fagus sylvatica in Spain0 aFirst Report of Phytophthora pseudosyringae Causing Stem Canker  cJan-07-2016 a1508 - 15080 v1003 a

Phytophthora pseudosyringae T. Jung & Delatour was described in 2003 as a pathogen associated with root and collar rot of several tree species (Fagus sylvatica, Alnus glutinosa, Quercus spp.) (Jung et al. 2003), and as the causal agent of stem lesions on beech in Europe (Scanu and Webber 2016). In August 2015, a beech (Fagus sylvatica L.) tree displaying 25% crown dieback and a bleeding canker at the base of the stem was observed in a forested area used for recreational purpose in Central Pyrenees (42°36.87′ N, 0°46.09′ E, 1,546 m above sea level). Coarse sections of wood surrounding the bleeding patches were sampled and kept moist and cold for 5 days until processed. Once in the laboratory, small phloem pieces from the margin of the lesion were plated onto corn meal agar-PARPBH medium (Jeffers and Martin 1986) and incubated at 20°C. Growing hyphae were transferred to V8 agar and incubated at 20°C in darkness. Colony growth on V8 medium averaged 3.4 cm/week and had a distinct stellate pattern. Sporangia were sympodial, semipapillate, and ovoid, with a length/breadth average ratio of 1.41 μm (SE = 0.02). Oogonia were 21.9 μm (SE = 0.49) in diameter, smoothly walled, spherical, and with paraginous antheridia, although some amphiginous antheridia were observed. Morphological features and growth appearance on V8 corresponded to those reported for P. pseudosyringae (Jung et al. 2003). DNA was extracted from mycelium, and the ITS region was amplified using the ITS4 and ITS6 primers (Cooke et al. 2000). A BLAST search of the sequenced isolate (submitted to GenBank, Accession No. KU321521) showed 100% identity with P. pseudosyringae ITS sequence (AY230190) from Jung et al. (2003). An under-bark inoculation test with mycelia plugs were performed on 16 excised shoots of 1 cm of diameter obtained from four different F. sylvatica trees. After 4 weeks, inoculated shoots displayed longer necrotic lesions than control inoculations with agar (3.55 cm SE: 2.2 versus 0.59 cm SE: 2.8, P < 0.0001). The P. pseudosyringae inoculated shoots exhibited lesions that were dark red underneath the bark, whereas the control lesions did not. P. pseudosyringae, which was identified morphologically, was reisolated from 40% of the infected shoots. No reisolation of the pathogen occurred from any of the six control shoots. Although previously detected in nursery stock on sweet chestnut (Castanea sativa) (Pintos Varela et al. 2007), our report represents the first report of P. pseudosyringae naturally affecting trees in Spain, and widens the geographical distribution of this pathogen in southwestern Europe. In Spain, F. sylvatica is the third species in terms of standing volume, where it covers 400,000 ha, mainly in the Pyrenees. Our finding proves the capacity of this pathogen to establish in nature, and it raises concern over the potential impact on beech forests in Spain.

1 aRedondo, Á.1 aOliva, J. uhttp://apsjournals.apsnet.org/doi/10.1094/PDIS-12-15-1497-PDN