Wilt of Shantung Maple Caused by Verticillium dahliae in China

Shantung maple (Acer truncatum Bunge) is an important economic plant, widely grown in northern China. The seed oils contain nervonic acid, which can reduce the risk of many mental illnesses (Hu et al. 2017). In June 2016, some trees showed symptoms of wilting, leaf fall, branch and twig dieback, and discoloration of vascular tissues in Jining, Shandong. The leaves of the diseased trees only showed dehydration without yellowing. The xylem also became black. The incidence of diseased plants was more than 30% on two farms (total of 4,882 trees) where eggplants and soybeans were previously planted. As we know, eggplants tend to be infected by Verticillium dahliae in China (Zhu and Ye 2006). To identify the pathogen, five stems (each approximately 70 mm in diameter and 10 cm long) sampled from five symptomatic plants were flushed with tap water for 30 min, surface-sterilized with 75% ethanol for 5 min, rinsed in sterilized water three times, and dried. The discolored vascular tissues were cut into 0.5-cm-long segments, and then placed on V-8 juice agar (V-8) medium. A fungus was isolated from diseased vascular tissues of each sample after the tissues were cultured on V-8 medium in darkness at 25°C after 5 days. Due to the production of microsclerotia after 4 to 5 days, the colonies changed from white to black. Microscopic observations showed many irregularly shaped, dark microsclerotia measuring 31.22 to 90.74 × 28.12 to 49.47 µm (average 57.24 × 34.48 µm) distributed on the medium. Hyaline, elliptical, single-celled conidia of dimensions 2.26 to 5.00 × 1.46 to 2.96 µm (average 3.18 × 1.99 µm) developed on hyaline and verticillate conidiophores (150 data are measured in each structure). Each node of conidiophores had two to four phialides. The fungus isolated was morphologically identified as V. dahliae (Pegg and Brady 2002). Koch’s postulates were checked using 20 A. truncatum seedlings (3 years old), 10 for inoculation and the rest for control. The pathogenicity test was an artificial inoculation in which the treated plants were drenched with 50 ml of conidial suspension (1.0 × 10⁷ conidia/ml) and the controls with sterilized water. Disease symptoms began to show on three seedlings 35 days after inoculation and two of these seedlings died on the 42nd day. Another four seedlings displayed the same symptoms 60 days after inoculation and died on the 67th day. All of the visible symptoms and death happened 70 and 78 days after inoculation, respectively. However, control seedlings did not show symptoms. The pathogen was recovered from the discolored vascular tissues of symptomatic seedlings but not control seedlings. For molecular identification, the universal primers ITS1/ITS4 were used to amplify the internal transcribed spacer (ITS) region (∼500 bp) of the nuclear ribosomal DNA (White et al. 1990). The ITS region was amplified, sequenced, and submitted to GenBank (accession no. MF149108). Based on the morphological characteristics and a BLAST search with 100% identity to an isolate of V. dahliae (GU461623), the fungus was identified as V. dahliae, which is known for causing diseases in many maple species, such as A. rubrum, A. negundo, and others (Chandelier et al. 2003). To our knowledge, this is the first report of wilt on A. truncatum caused by V. dahliae in China.