First Report of Alternaria tenuissima Causing Leaf Spot on Magnolia figo in China

Magnolia figo (banana shrub) is an evergreen shrub in the family Magnoliaceae that is widely cultivated in China for its fragrant flowers and ornamental value in landscaping and nursery production. In July 2025, typical leaf spot symptoms were observed on Magnolia figo plants in an outdoor ornamental field at Nanjing Xiaozhuang University, Jiangsu Province, China (118°54′6.56″E, 31°53′34.26″N). The plants were cultivated at a density of approximately 2–3 individuals per 10 m ² . At the time of symptom observation, the weather was hot and humid, with an average daily temperature of 20–30 °C and an average relative humidity of 75–85%. A total of 60 M. figo plants were randomly surveyed across the field; among these, 12 plants exhibited typical leaf spot symptoms, resulting in an overall disease incidence of 20%. The disease induces early defoliation and reduces ornamental value, and causes small, irregular yellow-brown to dark-brown leaf spots that expand into large necrotic lesions, ultimately leading to severe leaf drop. Symptomatic leaf lesions (5 × 5 mm; five plants, three lesions per plant) were surface-sterilized in 75% ethanol (30 s) and 2% NaOCl (5 min), rinsed with sterile distilled water, and plated onto potato dextrose agar (PDA). After 5 days of incubation at 25 °C in the dark, fungal colonies were transferred to fresh PDA and purified by hyphal tip transfer, yielding 10 morphologically similar isolates from 15 leaf pieces (66.7% isolation frequency). Among these isolates, isolate NXU-HX4 was selected as the representative isolate for further morphological characterization, molecular identification, and pathogenicity assays because it showed typical colony characteristics. Colonies on PDA were circular, initially white and cottony with loose, radial mycelia; after 13 d of incubation at 25°C, they became grayish-black to black with dense, velvety mycelia and a dark brown reverse. Hyphae were septate and smooth. Conidia were solitary, obclavate or ellipsoid, pale brown, with smooth walls, 2-3 transverse septa, and constricted at the septa, measuring 10.2–18.5 × 7.1–10.3 μm (n=50), a truncated hilum and no distinct beak. The rDNA internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1-α), and RNA polymerase II second largest subunit (RPB2) regions were amplified with primers ITS1/ITS4, gpd1/gpd2, EF1-728F/EF1-986R, and RPB2-5F2/RPB2-7cR (White et al. 1990; Berbee et al. 1999; Carbone and Kohn 1999; Sung et al. 2007). All sequences were deposited in GenBank (ITS: PX643372; GAPDH: PX843854; TEF1-α: PX843855; RPB2: PX843856). BLAST analysis revealed high sequence homology (99.75%–100%) with Alternaria tenuissima reference sequences (GenBank accession nos. AF347032 for ITS, AY278809 for GAPDH, KC584693 for TEF1-α, and KC584435 for RPB2). A maximum-likelihood phylogenetic tree was constructed in MEGA 5 using concatenated sequences of four loci (ITS, GAPDH, TEF1-α, and RPB2) from isolate NXU-HX-4 and related Alternaria reference isolates, with 1000 bootstrap replicates. This analysis revealed that NXU-HX-4 clustered with previously reported A. tenuissima reference isolates, forming a well-supported clade. To fulfill Koch’s postulates, three healthy 1-year-old potted M. figo plants were used. Five mature leaves per plant were surface-sterilized, wounded with a sterile needle, and inoculated with 20 μL of a spore suspension of isolate NXU-HX4 from 7-day-old PDA cultures. Leaves treated with sterile water served as controls. All inoculation sites were covered with moist plastic film to maintain humidity. At 7 days post-inoculation (dpi), brown spots similar to field symptoms appeared on treated leaves; by 10 dpi, spots expanded into typical necrotic lesions, with a disease incidence of 75% on inoculated leaves. Control plants remained asymptomatic, and the assay was repeated twice with consistent results. The fungus was re-isolated from lesion margins of symptomatic leaves and identified as Alternaria tenuissima based on morphological characteristics and sequence analyses, thereby fulfilling Koch’s postulates. To our knowledge, this is the first report of A. tenuissima causing leaf spot on M. figo in China.