First Report of Leaf Spot Disease Caused by Alternaria brassicae on Cercidiphyllum japonicum in China

Cercidiphyllum japonicum is a deciduous tree belonging to the genus Cercidiphyllum of the family Cercidiphyllaceae (Li et al., 2008). Fossil records indicated that this tree was once distributed throughout the Northern hemisphere during the tertiary period, whereas it is now only found in Japan and China as a consequence of quaternary glaciation. In 1989, C. japonicum was listed as a Rare and Endangered plant in China (Song et al., 1989). It is also highly valued for use in ornamental, medicinal, and research contexts, leading to its widespread planting and cultivation throughout China. In September 2021, a severe leaf spot infection (FigS1.A) was first detected on C. japonicum trees in Meigu County, Sichuan Province, China (N 28°33', E 103°14'). In a survey of twenty 100-year-old C. japonicum trees in this region, the incidence of such leaf spot was found to be approximately 95%. During the early stages of disease, infected leaves exhibited small punctate spots along the leaf center or margins. These spots were brown in the center with black edges. As the disease progressed, these spots expanded until they coalesced to yield large circular or irregularly shaped regions of necrotic tissue, and finally produced mildew. Samples of leaf tissue between symptomatic and healthy regions (5 mm×5 mm) were excised from five symptomatic leaves, surface disinfected for 30 s with 75% ethanol, soaked for 2 min in 3% NaClO, rinsed then plated on potato dextrose agar (PDA) medium supplemented with ampicillin and carbenicillin (50 μg/ml each). After cultured for 3 days in the dark at 25°C, emergent hyphae were purified by subculturing them on fresh PDA medium. In total, single spore culturing was performed by collecting and purifying seven fungal isolates. These isolates exhibited largely comparable morphological characteristics. Aerial hyphae had a cotton-like appearance and were white to pale gray in color (FigS1.B), turning pale reddish-brown with profuse sporulation (FigS1.C). Conidia were present in long chains, with conidiophores being present in clusters or in isolation (FigS1.D), with 1-5 transverse septa, 0-3 oblique and longitudinal septa and an ellipsoidal to obpyriform structure, measuring 9.0-38.6 μm in length and 5.1-12.6 μm in width (n = 40) (FigS1.E). These seven isolates thus exhibited morphological characteristics consistent with those of members of the Alternaria genus (Simmons, 2008). Molecular identification of a representative isolate (LGB9) was performed by amplifying the internal transcribed spacer (ITS) rDNA, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1), and partial RNA polymerase II largest subunit (RPB2) gene sequences with the ITS1/ITS4 (White et al.,1990), GDF/GDR (Templeton et al., 1992), TEF-728F/TEF-986R (Carbone & Kohn 1999) and RPB2-5F2/RPB2-7cR (Sung et al., 1990; Liu et al., 1999), and Bt-2a/Bt-2b (Glass and Donaldson 1995) primer pairs, respectively. The resultant sequences were deposited in GenBank (ITS, OL659190; GAPDH, OL685343; TEF, ON340848; RPB2, OL685344). Further phylogenetic analyses of isolate LGB9 revealed it to cluster in the A. brassicae clade with 97% bootstrap support. To confirm the pathogenicity of isolate LGB9, 15 healthy leaves from five one-year-old C. japonicum plants were spray-inoculated with a suspension containing 3×105 LGB9 conidia/mL, with control leaves instead being sprayed with distilled water. After 8 days, inoculated leaves exhibited symptoms similar to those observed on naturally infected leaves (FigS1.F-I), whereas the mock leaves were free of any symptoms. This is the first report to our knowledge of a case of leaf spot disease caused by A. brassicae affecting C. japonicum in China or anywhere else in the world. To ensure the protection of this living fossil species, appropriate interventional measures should be adopted to manage the development and spread of this disease.