First Report of Verticillium Wilt of Ailanthus altissima in Hungary Caused by Verticillium dahliae

Ailanthus altissima (Mill) Swingle, commonly known as tree of heaven, was brought to Hungary as an ornamental tree between 1841 and 1843 and became widespread in the country in the middle of the 20th century. Because of its ability to transform its environment and reduce biodiversity, it became one of the most dangerous invasive plant species in Hungary (Udvardy and Zagyvai 2012). Verticillium wilt of A. altissima has been reported in the U.S.A. (Rebbeck et al. 2013; Snyder et al. 2013) and Europe (Maschek and Halmschlager 2017) in the last few years. According to these studies, two Verticillium species can cause wilt symptoms on A. altissima: Verticillium nonalfalfae (formerly V. albo-atrum) and V. dahliae Kleb. During the summer of 2016, dying A. altissima trees were observed in Nyíregyháza (northeastern Hungary) and in several parts of Budapest that exhibited wilt symptoms, defoliation, and yellowish vascular discoloration. To identify the causal agent, samples were collected from symptomatic A. altissima trees and cut into 10-cm-long pieces, rinsed with tap water, then surface sterilized with 70% ethanol. Bark was removed from samples and small pieces of discolored xylem tissue were excised and placed onto potato dextrose agar (PDA) amended with chloramphenicol (25 mg/liter). Cultures were incubated at 22°C in the dark for 10 days. Five isolates were collected from the two investigated sites, all of which were putatively identified as V. dahliae based on the presence of microsclerotia and hyaline, nonseptate, cylindrical or ellipsoid conidia developed on verticillate conidiophores (2.8 to 8.0 × 1.8 to 4.6 μm, n = 100) (Inderbitzin et al. 2011). DNA was extracted from the mycelium of two isolates (one per location) and molecular identification was performed using primers coding for ribosomal internal transcribed spacer (ITS), elongation factor 1-alpha (EF), and actin (ACT) gene regions (Inderbitzin et al. 2011). The obtained sequences of Hungarian isolates Vd 16-2 and Vd 16-9 were deposited into GenBank as accession nos. MG585718 and MG585719 (ITS), MG587000 and MG587002 (EF), and MG586999 and MG587001 (ACT), and showed 100% similarity to several V. dahliae isolates (HE972023 and AF104926 for ITS; KY039313 and KY039314 for EF; HQ206923, HQ206921, and HQ206926 for ACT) including the epitype strain PD322. To confirm pathogenicity, seedlings of tree of heaven were collected in Budapest and planted into individual pots. After planting, seedlings were grown in an environmental chamber at 24°C and a 12-h photoperiod. Ten 12-week-old seedlings (five per isolate) were stem-inoculated with a conidial suspension (1 × 10⁸ spores/ml) of the two isolates. Five control plants were injected with sterile distilled water. All inoculated plants exhibited vascular discoloration, wilt symptoms,w and defoliation within 4 weeks while control plants remained asymptomatic. The pathogen was consistently reisolated from symptomatic seedlings, and morphological characteristics of the resultant colonies were identical to the two Hungarian V. dahliae isolates. To our knowledge, this is the first report of V. dahliae causing Verticillium wilt on A. altissima in Hungary.