First Report of Verticillium Wilt on Ailanthus altissima (Tree-of-Heaven) in North Macedonia caused by Verticillium dahliae

Abstract

Tree-of-Heaven, a highly invasive species on all continents except Antarctica, is frequently found on the Balkan Peninsula. In 2019, Ailanthus altissima has been put on the "List of invasive alien species of Union concern"; thus, measures for eradication and control are mandatory in all EU member countries. Verticillium wilt of A. altissima, which has been reported in the USA (Schall and Davis 2009; Kasson et al. 2014; Rebbeck et al. 2013) and in Europe (Maschek and Halmschlager 2016; Moragrega et al. 2021) in the last decade, has already been successfully used for biological control of Ailanthus. In the course of an excursion of the international REUFIS meeting, young symptomatic A. altissima trees, exhibiting severe foliar wilt symptoms, brownish vascular discoloration, dieback, and premature death, were observed at two different sites in the southern part of Skopje, North Macedonia (Kluchka Sonchev: 41.973155N, 21.428846E; Vidikovec (Panorama): 41.977671N, 21.421135E) in June 2024. To identify the causal organism, 50-cm-long branch samples were taken from symptomatic A. altissima and cut into 5-cm pieces, surface sterilized by dipping into 96% ethanol for 1 min, and then breamed. After removing the bark, 23 (Kluchka Sonchev) and 8 (Vidikovec) tissue samples of about 5 mm in length were excised from discolored sapwood and placed onto 2% malt extract agar plates supplemented with 100 mg/l streptomycin sulphate. Plates were incubated at 22°C in the dark for 10 days. Fungal colonies emerged from all plated tissue samples of both sites, all of which were putatively identified as Verticillium dahliae Kleb. based on the abundant formation of microsclerotia and the hyaline, non-septate, cylindrical or ellipsoid conidia developing on verticillate conidiophores. Morphological identification was confirmed by DNA sequencing of two selected isolates (one from each site), using primers coding for ribosomal internal transcribed spacer (ITS1) and elongation factor 1-alpha (TEF) (Inderbitzin et al. 2011) and a BLAST search against the reference sequences of the type strain of V. dahliae PD322 (GenBank Accession No. NR_126124 (ITS) and HQ414624.1 (TEF)) at NCBI NLM, which revealed 100% homology (ITS1: 492 matching base pairs; TEF: 579 matching base pairs) between the type strain and the two North Macedonian isolates. Aligned sequences of both isolates (Vd-NM01, Vd-NM02) were deposited in GenBank (Accession Nos. PQ615325, PQ615358, PQ858706, PQ858707). To confirm pathogenicity, 10 two-year-old potted Ailanthus seedlings were stem-inoculated with a conidial suspension (1 x 107 spores/ml, 1 ml/tree) of V. dahliae isolate Vd-NM01 and 10 with isolate Vd-NM02 on September 12, 2024; 5 seedlings treated with sterile water and 3 untreated seedlings served as controls. Following maintenance in the greenhouse, all V. dahliae-inoculated trees developed wilting symptoms two to three weeks after inoculation and were completely defoliated or showed heavy wilting symptoms on October 31, 2024, whereas all 8 control trees remained asymptomatic or displayed minor wilting symptoms related to autumn senescence. V. dahliae was re-isolated from all treated trees, but no control tree, thus confirming Koch's postulates. Despite of the high disease severity on infected trees, natural V. dahliae infections will not allow efficient control of A. altissima without human intervention on these affected sites in North Macedonia, due to the low disease incidence (<5%) on both sites.