03912nas a2200169   4500008004100000022001400041245007100055210006900126260001200195300000800207490000700215520340000222100002203622700002403644700002303668856005103691       2013                        eng d  a0191-291700aFirst Report of Phytophthora Taxon Walnut in Lombardy, North Italy0 aFirst Report of Phytophthora Taxon Walnut in Lombardy North Ital  c03/2014  a4240 v983 a<p>The park Boscoincittà, Milan, North Italy (136 m a.s.l., 45º 29' 06" N, 9º 5' 32" E) has an area of 110 hectares and includes tree stands, wood clearings, trails and watercourses. Recently, common walnut (Jugland regia) trees in the park have begun to suffer from a progressive dieback that has caused roughly 90% mortality. Aerial symptoms were: stunted growth, loss of vigor, crown thinning and bark cankers with tarry exudates on the lower stem. The xylem tissue of trees showed large necrosis and flame-shaped discolouration below the bark. Since the dieback seemed caused by Phytophthora, samples were taken from 3 symptomatic trees and, by baiting, from the nearby soil and watercourses. Isolations from apple baits were carried out after a week. Isolations taken from tissue at the edge of active lesions of the trees were transferred on the selective medium V8A-PARPNH (1) and incubated at 24ºC. Cottony colonies appeared after 3 days and single hyphal tip derivatives were transferred to V8A for a further 4-7 days. Fragments (1 cm2) of mycelium of the subcultured colonies were then placed in filtered (Ø 0.22 μm nitrocellulose filters, Millipore) pond water. Three isolates were retrieved within 24 h, 2 from tree tissue and one from water. These produced ovoid, non-papillate sporangia (Fig A), thirty of which per isolate were measured. Sporangia averaged 52.5 ± 9.6 x 32.9 ± 4.7 µm (range 30.8 – 67 x 22.5 – 42.8) with a l:b ratio of 1.59 ± 0.19 (range 1.27 – 2.05), and exit pores of 11.1 ± 1.7 µm (range 7.31 – 14.21). External proliferation from previously emptied sporangia and hyphal swellings were observed on V8A. On V8A, colonies had optimal growth at 32°C (5.7 ± 0.8 mm d-1) with a maximum at 37°C. Colonies had a chrysanthemum-shaped, scanty fluffy aspect on PDA (Fig B). Isolates were identified as Phytophthora taxon walnut on the basis of macro- and micro-morphology and sequence information from the ITS-rDNA region, that was amplified with primers ITS6 and ITS4 (2) after DNA extraction with a commercial kit (Sigma Aldrich). A region of the cox1 gene of isolate B164 was also amplified with primers OomCoxILevup and Fm85mod (3) and sequenced (GenBank acc. no. KC291584) but this was irrelevant for identification purposes because that gene region has not been sequenced for other isolates of this taxon. A BLAST search in GenBank and the Phytophthora database revealed a 99% identity of the ITS-rDNA from our isolate B164 (GenBank acc. no. KC291550) with the P. taxon walnut isolate P532 (AF541910) (4). Inoculation trials were conducted on 10 detached leaves. A little lesion was produced with a sterile scalpel on the lower leaf surfaces and a 0.5 cm Ø agar plug was placed over the wounds. Necrotic lesions averaged 3.7±1.6 x 2.0±0.5 cm after 1 week of incubation at 20°C in the dark (Fig C). Control leaves showed no symptoms. Reisolations on V8-PARPNH agar confirmed P. taxon walnut as the causal agent. Members of the Phytophthora genus grouping with the P. taxon walnut in clade 6 are mainly reported as saprophytes or pathogens from riparian ecosystems and forests (4). This is the first report of P. taxon walnut from Italy. Since the oomycete proved in our growth trial to be distinctly thermophilic, we hypothesize that its spread is being favored by the rising temperatures observed during the last decades in the area.</p>1 aGinetti, Beatrice1 aRagazzi, Alessandro1 aMoricca, Salvatore  uhttp://dx.doi.org/10.1094/PDIS-07-13-0766-PDN 