First Report of Eutypella Canker Caused by Eutypella parasitica on Acer campestre in Italy

Abstract

In autumn 2016, typical Eutypella canker symptoms were first observed on ∼30-year-old field maple (Acer campestre L.) trees growing in a mixed ash-maple seminatural forest in northeastern Italy (Paciuch/Pacuh, Udine/Videm, 46°10′43″N, 13°38′13″E). In subsequent surveys carried out in 2017, the disease was observed in another 20 trees along a 36-km-long transect parallel to the Italian-Slovenian border (Drenchia/Dreka to Gorizia/Gorica, 46°10′20″N, 13°38′30″E to 45°57′18″N, 13°36′40″E). Symptomatic trees showed approximately 80-cm-long elongated cankers with the bark remaining firmly attached to the stem and with the presence of subcortical white mycelial fans and black perithecial necks. The morphological features of the teleomorph—perithecia 502 (365 to 638) µm, asci 87.9 (65.3 to 106.5) × 9.0 (7.3 to 11.5) µm, and ascospores 9.3 (7.8 to 11.2) × 3.2 (2.5 to 3.7) µm (n = 40)—matched the description of Eutypella parasitica R.W. Davidson & R.C. Lorenz reported by Davidson and Lorenz (1938). To isolate the pathogen, small fragments of mycelial fans and symptomatic inner bark and woody tissues (4 to 5 mm²) taken from the margin of 20 cankers were placed into Petri dishes (90 mm O) containing potato dextrose agar (PDA, 39 g/liter, Oxoid, Basingstoke, U.K.). All isolates developed white colonies with a dense mycelium appressed to the agar surface that became light gray after 20 to 25 days at 25°C in the dark. The hyaline, filiform, irregularly curved, and aseptate conidia were exuded in a yellow matrix and measured 37.8 (26.9 to 48.5) × 2.0 (1.4 to 2.6) μm (n = 50). The identity of the 20 fungal isolates obtained (one from each canker) was confirmed by sequence analysis of the internal transcribed spacer region (ITS1-5.8S-ITS2) of rDNA. Fungal DNA extraction, PCR amplification reactions, and DNA sequencing were carried out as reported by Linaldeddu et al. (2016). BLAST searches against GenBank showed 100% identity with reference sequences of E. parasitica, including that of the ex-type culture CBS 210.39 (GenBank accession no. NR145214). The ITS sequence of a representative strain of E. parasitica, PC3, was deposited in GenBank (accession no. MN227194). The strain PC3 was stored on PDA slants under oil in the culture collection of the Dipartimento Territorio e Sistemi Agro-Forestali, University di Padua (Italy). Testing the pathogenicity of the strain (PC3) was undertaken by inoculating ten 3-year-old field maple seedlings. Ten additional seedlings were inoculated with a Slovenian strain of E. parasitica (T1-1/3NO) obtained from Acer pseudoplatanus L. (GenBank accession no. MN607439). The part of the stem to be inoculated was surface disinfected with 90% ethanol, and a small piece (4 × 4 mm) of bark was removed with a flamed scalpel. An agar mycelium plug (3 × 3 mm) taken from the margin of an actively growing colony on PDA was placed on the wound, covered with cotton wool soaked in sterile water, and finally wrapped in a piece of aluminum foil. A sterile PDA plug was used instead of the mycelial inoculum in 10 control seedlings. Plants were kept under field conditions at 13 to 37°C and watered regularly for 2 months. At the end of the experiment, all plants inoculated with E. parasitica were symptomatic and showed open cankers and dark brown lesions in the xylem spreading up and down from the inoculation site. The average lesion size was 2.40 ± 0.71 cm for the Italian strain (PC3) and 2.70 ± 0.55 cm for the Slovenian strain (T1-1/3NO). Both strains were successfully recovered from all the inoculated plants, fulfilling Koch’s postulates. No disease symptoms were detected on control seedlings, and the wounds had begun to heal. E. parasitica, a pathogen probably native to North America, was detected for the first time in Europe (Slovenia) in 2005 (Jurc et al. 2006) and thereafter reported in Austria, the Czech Republic, Croatia, Germany, Hungary, and Poland (Cech et al. 2016; Cerný et al. 2017). This is the first record of E. parasitica in Italy, where maple tree species (mainly A. campestre, A. pseudoplatanus, and A. platanoides L.) are naturally present in the mountain forests in the Alps and intensively cultivated for timber production. The wide spread and virulence of this pathogen together with its recent detection in the same sites on A. pseudoplatanus represents a serious threat to maple ecosystems in the Alpine region.