First Report of Neoascochyta cylindrispora Causing Leaf Spot on Taraxacum mongolicum in China

Taraxacum mongolicum, which is valued for its both feeding and medicinal values (Lin et al. 2022; Wang et al. 2022), is one of the main associated species in the alpine meadow of China. In August 2023, a new leaf spot disease of T. mongolicum with an incidence rate of about 90% was observed on natural grassland in Hongyuan County, Aba (Ngawa) Tibetan and Qiang Autonomous Prefecture, China (32°48' N, 102°33' E, alt. 3500 m). The symptoms appeared as grayish white spots in the center with dark brown or purple black margins. For isolation, 18 tissue pieces (5mm × 5mm) from 2 symptomatic plants were surface sterilized with 70% ethanol for 30 s and rinsed three times with sterilized distilled water. Then, these tissues were placed on potato dextrose agar (PDA) at 25°C and incubated in the dark for 2 to 7 days. Finally, six pure strains with consistent colony characteristics were obtained from hyphal tips. Colonies on PDA were dark brown or green black with abundant aerial mycelia on the upper side, and black on the reverse side. Hyphae were hyaline to brown, septate, 3 to 6 μm wide. Conidia were hyaline, cylindrical, 10.7 to 15.4 × 3.0 to 4.9 μm (average 12.8 × 3.8 μm; n = 50), guttulate. For further identification, the internal transcribed spacer region (ITS), 28S nrRNA gene (LSU), partial beta-tubulin (tub2) and RNA polymerase II subunit 2 (rpb2) genes of the six strains were amplified with primers described by Valenzuela-Lopez et al. (2018). Sequences were deposited in GenBank (PP345804, PV840064-PV840068 for ITS; PQ002493, PV840076-PV840080 for LSU; PQ015093, PV854533-PV854537 for rpb2; and PQ015094, PV854538-PV854542 for tub2). A maximum likelihood (ML) phylogenetic tree based on the combined ITS, LSU, rpb2, and tub2 alignments showed the six strains, and UTHSC: DI16-352 and ex-type CBS 142456 of N. cylindrispora (Valenzuela-Lopez et al. 2018) formed a subclade with 97% bootstrap support. Based on the above morphological and molecular features, the six strains were finally confirmed as Neoascochyta cylindrispora. For pathogenicity tests, 7-week-old healthy plants were obtained by growing T. mongolicum seeds in pots (two plants per pot). Above six pure cultures were subsequently used to inoculate healthy plants as follows: for each strain, four pots were spray inoculated with a conidial suspension (about 1 × 10 6 conidia/ml); in addition, four pots considered as non-inoculated controls were sprayed with sterilized distilled water. All pots were individually covered with transparent polyethylene bags for 5 days to maintain high relative humidity and placed in a greenhouse with a temperature of 23 to 29°C, humidity of 60 to 70%, 12 hours of light and 12 hours of darkness. After incubation for 10 days, plants developed symptoms similar to those observed in the field. No symptoms were observed on the non-inoculated control plants. Pathogenicity tests were conducted thrice using the same method. The same fungus was re-isolated from the lesions and confirmed by the morphological and molecular methods described in this note, thus fulfilling Koch’s postulates. N. desmazieri, closely phylogenetically related to N. cylindrispora, has been recorded on Lolium perenne (Poaceae) in Germany (Chen et al. 2015; Valenzuela-Lopez et al. 2018). To our knowledge, this is the first report of N. cylindrispora as pathogen on T. mongolicum worldwide. This information would be benefit for the diagnosis, detection and pathogen identification of leaf spot disease on T. mongolicum in natural grassland.