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

Abstract

Ailanthus altissima (Mill.) Swingle, commonly known as tree-of-heaven, is an invasive tree species that has spread throughout the United States since its introduction in 1784 (2). During a survey in July 2009, approximately 1,100 A. altissima trees were observed at two locations in western Virginia (a roadside in Montgomery Co. and a wooded area adjacent to a railroad in Bedford Co.) exhibiting foliar wilt symptoms, defoliation, yellowish vascular discoloration, or death at an incidence of ~77%. Similar symptoms on A. altissima were reported in Roanoke, VA in the early 1930s and after 2005 in Pennsylvania, attributed to a Verticillium sp. (1,2). To identify the causal agent, discolored xylem tissue samples were excised from 10 symptomatic A. altissima trees at both locations, soaked in 1% NaOCl for 2 min, rinsed with sterilized distilled water for 5 min, and placed onto plum extract agar. Cultures were incubated in the dark at 22°C for 7 to 14 days. The resultant colonies (three to four per location) were subcultured and identified putatively as a Verticillium sp. closely related to Verticillium albo-atrum Reinke and Berthold (3), based on melanized, thick-walled, resting mycelia and phialides arranged in verticillate whorls that amassed round, oval-shaped conidia (5.1 ± 1.2 μm × 2.8 ± 0.4 μm, n = 100). Molecular identification of two fungal isolates (one per location) was determined by amplification of the protein coding genes elongation factor 1-alpha (EF), glyceraldehyde-3-phosphate dehydrogenase (GPD), and tryptophan synthase (TS), using PCR primers developed recently for Verticillium (3). A BLAST search on the edited contigs revealed 100% sequence similarity for all three protein coding genes among the two isolates and reference sequences of isolates PD592 (GenBank Accessions JN188227, JN188163, and JN188035 for EF, GPD, and TS, respectively) and VnAaPA140 (KC307764, KC307766, and KC307768 for EF, GPD, and TS, respectively) of the newly-proposed species, V. nonalfalfae (formerly V. albo-atrum). Aligned sequences from one representative isolate, VnAaVA2 (Bedford Co.), were deposited into GenBank as KC307758 (EF), KC307759 (GPD), and KC307760 (TS). To confirm pathogenicity to A. altissima, the two molecularly characterized isolates (one per location) were inoculated into 18 10-week old A. altissima stems that were grown in an environmental chamber at 24°C, 60% RH, and a 12-h photoperiod from seeds collected in Blacksburg, VA. A conidial suspension of each isolate was injected into each stem (0.1 ml of 1 × 108 CFU/ml/stem). All 36 seedlings inoculated with the proposed V. nonalfalfae isolates developed wilting of leaflets within 2 weeks post-inoculation (WPI), defoliation of leaflets by 6 WPI, and were dead by 9 WPI. Eighteen control seedlings were inoculated similarly with distilled water, and remained asymptomatic. Fungi resembling the proposed species V. nonalfalfae were reisolated from all inoculated stems except the control plants, and the species confirmed morphologically as described above. V. nonalfalfae is a recently proposed species that can infect a variety of plant species (3). To our knowledge, this is the first report of this proposed species on A. altissima in Virginia. New state reports of this pathogen on A. altissima are important for regulatory issues associated with using this pathogen as a potential biological control agent.