First Report of Verticillium Wilt on Ailanthus altissima in Europe Caused by Verticillium nonalfalfae.

In the course of taxonomic changes, Verticillium albo-atrum sensu lato, a causal agent of Verticillium wilt, was split into the three different species, V. albo-atrum Reinke & Berthold, V. alfalfae Inderb. et al., and V. nonalfalfae Inderb. et al. (Inderbitzin et al. 2011), which questions all prior morphology-based identifications of this ascomycete as a pathogen on multiple hosts worldwide. Thus, genetically confirmed new reports of the newly described species will provide essential information on the geographical distribution and host range of these pathogens. In order to investigate the natural distribution of Verticillium spp. on Tree of Heaven, Ailanthus altissima (Mill.) Swingle, and to obtain isolates that might have potential as biological control agents for this nonnative, invasive tree species (Schall and Davis 2009, Kasson et al. 2014), a survey of Verticillium wilt and its causal agents on A. altissima was conducted from May to August 2011 in the distribution range of the tree in eastern and southern Austria. During this survey, symptomatic A. altissima trees exhibiting foliar wilt symptoms, yellowish vascular discoloration, epicormic shoots on the stem, dieback, and premature death were observed at 12 out of the 22 investigated sites. Similar symptoms were reported on A. altissima in southern Styria in autumn 1997, attributed to Verticillium spp. and other fungi causing bark canker (Cech 1998). To identify the causal organism, 50-cm-long branch samples were taken from symptomatic A. altissima and cut into 5-cm pieces, surface sterilized with 96% ethanol for 1 min, and then rinsed with sterile water. After removing the bark, tissue samples were excised from discolored sapwood and placed onto 2% malt extract agar plates supplemented with 100 mg/liter streptomycin sulfate. Plates were incubated at 22°C in the dark for 10 days. At two out of the 22 sites (Bad Radkersburg, province Styria, and Gänserndorf, province Lower Austria) with symptomatic A. altissima, cultures morphologically resembling Verticillium albo-atrum sensu lato were isolated, whereas V. dahliae or no Verticillium spp. were obtained from the other sites. Morphological characteristics of the isolates corresponded to the description of V. nonalfalfae (Inderbitzin et al. 2011), which is, however, morphologically indistinguishable from V. alfalfae. Thus, identification was confirmed by DNA sequencing, using primer pair ITS1-F and ITS4, and a BLAST search against reference ITS sequences at GenBank, which revealed a 100% homology between the two Austrian isolates and the type specimen of V. nonalfalfae (GenBank Accession No. JN187973). Aligned sequences from both isolates (G1/5 and I3/2), were deposited into GenBank (Accession Nos. KT223526 and KT223527). To confirm pathogenicity, 20 canopy trees were stem-inoculated with a conidial suspension (1 × 107 spores/ml, 3 ml per tree) of V. nonalfalfae isolate G1/5 in August 2011; six canopy trees treated with sterile water served as controls. Four weeks after inoculation, all fungus-inoculated trees developed wilting symptoms and were dead by 2013, whereas all six control trees remained asymptomatic. V. nonalfalfae was consistently reisolated from symptomatic trees. To our knowledge, this is the first report of V. nonalfalfae causing Verticillium wilt on A. altissima in Europe. © 2016 The American Phytopathological Society.