First Report of Phytophthora inundata Associated with Decline and Death of Walnut (Juglans regia L.) in Italy

Persian walnut (Juglans regia L.) is an important nut crop in Italy. In recent years, incidence of walnut decline and death has increased in many Italian commercial orchards. In early summer 2020, we observed a serious decline in approximately 5% of trees in a waterlogged area of a Veneto-region walnut orchard (J. regia, cv Lara). Symptoms included extensive foliar wilt and canopy decline associated with collar and root rot. Symptomatic tissues excised from larger roots of affected trees 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 (Jeffers and Martin 1986). A Phytophthora-like organism was consistently isolated. Pure cultures were obtained by single-hyphal transfers onto potato dextrose agar (PDA). Isolates were identified as Phytophthora inundata based on morphological characteristics (Brasier et al. 2003), sequences of internal transcribed spacer (ITS) amplicons from universal primers ITS6 (Cooke et al. 2000) and ITS4 (White et al. 1990) and sequences of cytochrome c oxidase, subunit II (Cox II) from Fm75 and Fm78 primer pair (Martin and Tooley 2003). On carrot agar (CA), colonies had a characteristic stellate to broad lobed patterns. On this medium, optimal growth was at 28-30 °C (7,3 mm/day) and the upper temperature limit for mycelial growth was 37°C. Mycelial disks of isolate CREADC-Om306, grown on CA, were floated in Petri plates with soil extract solution and incubated under continuous fluorescent light at room temperature (25+/-2 °C). Within 48 to 72 h, sporangia were produced that were persistent, non-papillate, ovoid or ovoid-obpyriform, measuring 55.0 to 80.7 (length) x 41.3 to 65.2 (width) µm (averages 64.3+/-10.2 x 47.9+/-9.7 µm). Oospores and chlamydospores were absent. BLAST analysis of the amplicons from CREADC-Om306 revealed ITS sequences (854-bp; GenBank accession no. OK342200) and Cox II sequences (568-bp; GenBank accession no. OK349677) that shared 100% identity with published P. inundata sequences available in GenBank (acc. n. AF266791 for ITS; MT458994 for Cox II). Pathogenicity tests were conducted in the greenhouse on six 2-year-old walnut (J. regia, cv Lara) plants. Four of the plants were inoculated with CREADC-Om306 on two opposite sides of each plant's stem at 1-2 cm above soil line. A cork borer was used to remove a 5-mm disk of bark that was replaced by a 5-mm diameter mycelial plug from 10-day-old cultures of the pathogen on PDA. Two control plants were treated in the same way except the bark wounds were inoculated with sterile PDA plugs. Plants were kept in greenhouse at 24 ± 2°C. After 3 months, lesions had developed from all points of inoculation with. P. inundata (mean lesion length 55,25+/-6,22 mm) and the pathogen was reisolated from the lesion margins of all inoculated plants. The control plants remained symptomless and did not yield the pathogen. P. inundata is widely distributed across the world as a plant pathogen on several native as well as horticultural crops, especially in riparian or other areas subject to flooding or waterlogging. This report is the first to document P. inundata as a pathogen on Persian walnut and adds it to the diverse list of known susceptible perennial native, ornamental, and agricultural hosts of this organism. In addition to P. inundata, which belongs to the major Phytophthora ITS Clade 6, other members of the clade including P. megasperma (Belisario et al. 2012) and P. gonapodyides (Belisario et al. 2016) have been described as walnut pathogens. References: Belisario, A., et al. 2012. Plant Dis. 96 (11):1695. https://doi.org/10.1094/PDIS-05-12-0470-PDN. Belisario, A., et al. 2016. Plant Dis. 100 (12):2537. https://doi.org/10.1094/PDIS-03-16-0394-PDN. Brasier, C.M., et al. 2003. Mycol. Res. 107 (4):477. DOI: 10.1017/S0953756203007548. Cooke, D. E. L., et al. 2000. Fungal Genet. Biol. 30:17. https://doi.org/10.1006/fgbi.2000.1202. Jeffers SN, Martin SB. (1986) Plant Dis70:1038. Martin, F. N., and Tooley, P. W. 2003. Mycologia 95:269. https://pubmed.ncbi.nlm.nih.gov/21156613/. Schena, L., et al. 2008. Plant Pathol. 57:64. https://doi.org/10.1111/j.1365-3059.2007.01689.x. White, T.J. et al. 1990. In PCR Protocols: A Guide to Methods and Applications; Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J., Eds.; Academic Press, (USA,) 18: 315-322.