Publication Type:Journal Article
Source:Plant Disease, Volume 100, Issue 12, p.2537 - 2537 (2016)
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.