04374nas a2200193 4500008004100000022001400041245010900055210006900164260001600233300001600249490000800265520376600273100001704039700001504056700001504071700001404086700001404100856006604114 2016 eng d a0191-291700aPhytophthora gonapodyides Causes Decline and Death of English (Persian) Walnut ( Juglans regia) in Italy0 aPhytophthora gonapodyides Causes Decline and Death of English Pe cJan-12-2016 a2537 - 25370 v1003 a

In late spring 2015, several commercially grown Persian walnut (Juglans regia L.) cv. Lara trees in northeastern Italy (Udine) exhibited extensive foliar wilt and canopy decline associated with collar and root rot. Sudden collapse was recorded in about 80% of trees facing an irrigation canal. Symptomatic tissues excised from roots and collars of affected plants were surface disinfested for 1 min in a 1% NaOCl solution, rinsed for 5 min in sterile distilled water, and placed onto P5ARPH selective medium. A Phytophthora-like organism was consistently isolated. Pure cultures, with a typical rosette pattern, were obtained by single-hyphal transfers onto potato dextrose agar (PDA). Mycelial disks of three isolates, AB260, AB261, and AB262, grown on carrot piece agar, were floated in petri plates with soil extract solution and incubated under continuous fluorescent light at room temperature. Within 48 to 72 h, sporangia were produced that were persistent, nonpapillate, tapered at the apex, and 40.0 to 102.8 × 22.8 to 45.7 µm (average 56 × 33 µm of 30 sporangia). Both internal and external proliferations were observed. Oospores and chlamydospores were absent. These morphological characteristics all corresponded to those reported for Phytophthora gonapodyides (Brasier et al. 1993; Erwin and Ribeiro 1996). Optimal growth for all three isolates was at 30°C (3.4 mm/day), with limited growth at 4°C (1.25 mm/day), and no growth at 35°C. The identity was confirmed by sequencing the internal transcribed spacer (ITS) using universal primers ITS4 and ITS6 (Cooke et al. 2000) and cytochrome c oxidase, subunit II (Cox II) (Martin and Tooley 2003). BLAST analysis of ITS 821-bp segment (GenBank accession nos. LN877760, LN901312, LN901313) showed 100% identity with published P. gonapodyides sequences available in GenBank (i.e., HQ261570, AF541889, or AF541888), as well as with a Cox II 568-bp segment (LN877762, LN894191, LN894192) against AY129197. Greenhouse pathogenicity tests were conducted in controlled conditions. A total of six 1-year-old shoots cut from J. regia plants, about 2 cm in diameter, were used and three inoculation points each were made. Mycelial plugs (6 mm in diameter) cut from margins of actively growing 10-day-old cultures on PDA were inserted through the epidermis into phloem tissue. Controls were treated as described above except that sterile PDA plugs replaced the inoculum. Shoots were incubated in test tubes with sterile water in the dark at 24 ± 2°C. After 2 weeks, lesions were evident at all inoculation points, with an average length of 26 mm. Symptoms were similar to those caused by natural infection. P. gonapodyides was consistently reisolated from lesion margins. No colonies were isolated from control plants that remained symptomless. P. gonapodyides is ubiquitous in streams and ephemeral water pools, and is capable of saprophytic existence (Erwin and Ribeiro 1996). Although it is mainly known as a minor pathogen, there are reports indicating that some isolates can be highly virulent (Orlikowski et al. 2011) as in the present study where well developed 7-year-old walnut trees were killed by the pathogen. In the current study, P. gonapodyides aggressiveness was most likely sustained by the prolonged presence of flooding water at the root level, and a cool soil environment. To our knowledge, this is the first report of P. gonapodyides on Persian walnut in Italy or elsewhere.

1 aBelisario, A1 aLuongo, L.1 aVitale, S.1 aGalli, M.1 aHaegi, A. uhttp://apsjournals.apsnet.org/doi/10.1094/PDIS-03-16-0394-PDN03368nas a2200145 4500008004100000245011600041210006900157300001400226490000700240520286100247100001703108700001703125700001803142856006203160 2006 eng d00aFirst report of Phytophthora cambivora causing bleeding cankers and dieback on beech (Fagus sylvatica) in Italy0 aFirst report of Phytophthora cambivora causing bleeding cankers  a1362-13620 v903 a

European beech (Fagus sylvatica) is an important forest tree species common in northern and central Europe. In Italy, this species is typical in mountain areas over 1,000 m above sea level. In the last decade, decline and death was reported on European beech caused by several Phytophthora species (2), and P. pseudosyringae was recently reported in Italy (3). During 2004 and 2005, seven declining and dying F. sylvatica trees, older than 20 years, were observed in the Veneto Region of Italy with symptoms of bleeding cankers at the base of trunks and on branches. Cankers on the collar showed tongue-shaped necroses of the inner bark and cambium tissues. Four trees were in a public park of Mestre and three were in a forest stand in the province of Belluno. Samples were taken from declining trees, one in the park and two in the forest. Inner bark and cambium tissue pieces were cut from the canker margins, cultured on CARPBHy-agar (corn meal agar amended with 250 μg ml-1 ampicillin, 10 μg ml-1 rifampicin, 10 μg ml-1 pimaricin, 15 μg ml-1 benomyl, 50 μg ml-1 hymexazol), and incubated at 20°C. Ten morphologically similar isolates were subcultured as single hyphal tips and characterized. These isolates produced nonpapillate sporangia that were ovoid, obpyriform or ellipsoid, and exhibited predominately internal proliferation in soil extract. Hyphal swellings with outgrowths were present under those conditions. The morphological characteristics were consistent with those of P. cambivora (1). Base sequences of the ITS region of rDNA were determined for six of the isolates, and an 832-bp fragment was amplified for each isolate and that sequence was 100% homologous with sequences DQ396418 and AY880985 of P. cambivora in the NCBI database (http://www.ncbi.nlm.nih.gov/BLAST/). The sequence of one isolate, ISPaVe 1950, was deposited in GenBank (Accession No. AM269752). Pathogenicity tests were conducted with 2-year-old potted beech seedlings. Inoculum of representative isolates was grown for 4 weeks on sterilized millet seeds moistened with V8 broth and added to soil at 3% (wt/vol). Control plants received sterilized inoculum only. The soil was flooded for 48 h. Inoculations were performed during May 2005 at 15 to 35°C with six replicates for the inoculated and control plants. The plants were maintained outdoors and assessed after 3 months. Wilt, root rot, and dark brown lesions at the collar developed on inoculated plants, but not on the controls. Symptoms were similar to those on naturally infected trees. The pathogen reisolated from the inoculated plants was morphologically identical to the original isolates, which confirmed P. cambivora as the causal agent. To our knowledge, this is the first report of P. cambivora on beech in Italy.

1 aBelisario, A1 aMaccaroni, M1 aVettorazzo, M uhttp://apsjournals.apsnet.org/doi/abs/10.1094/PD-90-1362C