| Abstract |
Chinese fir (Cunninghamia lanceolata) is an important timber species that has been widely cultivated in southern China. It is extensively applied in medicine, environmental monitoring, furniture, urban (e.g., street trees) and rural landscaping, windbreak forest, and soil and water conservation. In January 2022, distinct leaf spot symptoms were observed in Chinese fir in Hongya Forest Farm (29°45′N, 103°11′E), Meishan City, Sichuan Province, China. Field surveys showed that the disease was widespread, with around 70% disease incidence. The typical symptoms initially appeared as yellowish-brown necrotic lesions on the margin of the leaves. Subsequently, lesions gradually expanded and developed into larger irregular red-brown necrotic areas. The lesions later expanded throughout the leaf. Infected leaves turned dark brown and wilted, leading to seedling death. Diseased leaves with typical symptoms were collected for pathogen isolation and identification. Infected tissues from 10 samples were cut into small pieces (2 × 2 mm). Infected tissues were surface disinfected with 3% sodium hypochlorite and 75% ethanol for 30 and 60 s, respectively, and rinsed with sterile water three times. They were blotted dry with autoclaved paper towels and incubated on potato dextrose agar (PDA) with streptomycin sulfate (50 μg/ml) for 5 to 8 days at 25°C and a 12-h light/dark period. The diameter of the colonies reached 65.7 to 75.9 mm, with a gray-to-black center and white edges, while the reverse sides were gray to orange. Conidia were single celled, colorless, straight, cylindrical, and bluntly rounded at both ends. Conidia dimensions varied from 7.3 to 15.7 μm in length and 3.3 to 6.1 μm in width (n = 100). For molecular identification, the genomic DNA of the isolates SM2290708, SM229070801, and SM229070802 was extracted using a fungal genomic DNA extraction kit (Beijing Solarbio Science and Technology, Beijing, China). The internal transcribed spacer (ITS) region of ribosomal RNA (ITS1/ITS4; White et al. 1990), calmodulin (CAL; Weir et al. 2012), β-tubulin (TUB2; OʼDonnell and Cigelnik 1997), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Templeton et al. 1992) were amplified. Sequences were deposited in GenBank (ITS: ON564877, OQ535027, and OQ535028; CAL: ON583827, OQ538101, and OQ538102; TUB2: ON583830, OQ538104, and OQ538105; and GAPDH: ON583831, OQ538108, and OQ538109). BLAST results showed that our ITS, CAL, TUB2, and GAPDH sequences were >99% identical to the corresponding sequences of Colletotrichum kahawae deposited at NCBI (GenBank nos. JX010231, JX009642, JX010444, and JX010012). Identification was confirmed by Bayesian inference using MrBayes. The conidial suspension (1 × 10⁶ conidia/ml) was inoculated by spraying on leaves of ten 3-year-old Chinese fir plants for the pathogenicity test. Fifteen leaves of each plant were inoculated. An equal number of control leaves were sprayed with sterilized distilled water as a control. Finally, all potted plants were placed in a greenhouse at 28°C under a 16/8-h photoperiod with 73 to 79% relative humidity. After 15 days, the symptoms observed on the inoculated plants were similar to those of the original diseased plants, but the controls remained asymptomatic. C. kahawae was reisolated from the infected leaves and identified by both morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times, which showed similar results, confirming Koch’s postulates. To our knowledge, this is the first report of brown leaf spot on C. lanceolata caused by C. kahawae in China. The results of this study provide basic information for diagnosis of the pathogen and developing prevention strategies to manage C. lanceolata leaf spot disease.
|
