First Report of Conioselinum anthriscoides Leaf Spot Caused by Septoria anthrisci in Gansu Province, China
Conioselinum anthriscoides is a perennial herb belonging to the Apiaceae family. It grows wild on mountain slopes and forest edges, primarily at elevations between 1,300 to 1,800 meters. It is mainly cultivated in Sichuan, Shaanxi, Shandong and Xinjiang provinces. Its rhizomes are used in traditional medicine for dispelling wind and relieving pain (Committee of China Pharmacopoeia, 2020). In June 2019, severe leaf spot disease was observed on the leaves of C. anthriscoides in a 150 m2 field at a medicinal plant garden in Min County, Gansu Province, China (35°17’N, 104°2’E, altitude 2489.3 m). Initial symptoms on infected leaves appeared as small, irregular, brown necrotic spots. These lesions gradually turned dark brown to grayish-white, developing small black dots and exhibiting pronounced dark brown margins. Disease incidence was estimated at 60 to 75% across the field based on a 5-point random sampling survey of 300 leaves. The pathogen was obtained using the tissue isolation method on potato dextrose agar (PDA) at 25°C, while no fungus was isolated from asymptomatic leaves processed similarly. Isolate TCM-22, obtained with an isolation frequency of 53%, was selected as the representative isolate for further analyses.The colonies on PDA exhibit well-defined margins, dense aerial mycelium initially appears white, becomes grayish-olive with age. Pycnidia were black, spherical to subspherical, produced on PDA after 3 weeks, measuring 52.8 to 112.7 × 43.5 to 107.5 (avg. 84.8 × 75.9) μm , with ostioles 25.1 to 35.8 × 22.7 to 33.6 (avg. 32.4 × 28.9) μm in diameter. Conidia were hyaline, acicular (needle-like), straight or slightly curved, with 2 to 4 septa, measuring 18.3 to 52.8 × 1.4 to 3.8 (avg. 35.7 × 2.7) μm. These morphological characteristics were consistent with the genus Septoria. S. anthrisci TCM-22 exhibits mycelial growth at temperatures ranging from 4 to 30°C, with an optimum at 20°C. Continuous illumination enhanced growth relative to darkness. The fungus grows at pH 5.0 to 10.0, with optimal growth at pH 5.0. After culturing isolate TCM-22 on PDA medium for 20 days, 10 mL of sterile 0.1% Tween-80 solution was added to each petri dish, and the fungal culture was scraped with a spatula to prepare a spore suspension. Adjust the suspension concentration to 1 × 105 spores/mL using sterile water. Thirty healthy one-year-old plants were selected for the pathogenicity test. Fifteen plants were inoculated by spraying with the spore suspension until it runs off the edges of the leaves. The remaining sprayed with sterile 0.1% Tween-80 solution were treated as control. All plants were covered with transparent plastic bags to maintain 80% relative humidity and placed at 20℃ in the Climate Chamber for 72 hours under a 12-hour photoperiod. Then the plastic bags were removed. Twenty days post-inoculation, all leaves inoculated with spore suspension exhibited dark brown necrotic lesions with black margins, resembling the original field symptoms. No symptoms were observed in the control group. The experiment was repeated three times, and the pathogen was again isolated from the inoculated leaves, with consistent results. This satisfied Koch's postulates, confirming the pathogen's pathogenicity. The DNA of isolate TCM-22 was extracted using the FastDNA kit (SK1375) (Sangon Biotech Co. Ltd. , Shanghai, China) and 3 loci: ITS, β-tubulin and EF1α were amplified and sequenced with primers ITS1/ITS4, T1/β-Sandy-R and EF1-728F/EF-2 respectively (Verkley et al. 2013). The obtained sequences of ITS, β-tubulin, EF1α were deposited in GenBank (accession nos. PQ815052, PQ827513, and PQ827514, respectively. BLAST results of the three gene loci indicated 99% to 100% sequence identity with S. anthrisci TCM-11, CBS 109020 and CBS 109020. A phylogenetic tree based on the concatenated sequences of ITS, β-tubulin, and EF1α was constructed using the Maximum Likelihood method. The strain TCM-22 formed a single clade with S. anthrisci TCM-11. Based on morphological and molecular data, the isolate was identified as S. anthrisci. To our knowledge, this is the first report of S. anthrisci causing leaf spot on C. anthriscoides in China.