First Report of Leaf Spot Disease Caused by Alternaria brassicae on Camptotheca acuminata in Sichuan, China.

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Camptotheca acuminata (C. acuminata), is belongs to a monotypic genus endemic to southwestern China and listed as the first class national protected plant in China in 1999 (Wen, et al. 2020). Camptothecin, isolated from the wood and bark of C. acuminata Decne, which exhibits clinical effects in various cancer treatments (Pommier, et al. 2006; Kang, et al. 2021). In October 2021, we investigated leaf spot disease occurrence on C. acuminata (FigS1.A) with 80% incidence in Beichuan County, Sichuan Province of China. Leaf symptoms were randomly distributed on the adaxial surfaces and consisted of punctate spots of alternating light gray and dark brown in the early stage of onset (FigS1. B, C). As the disease progressed, these spots expanded irregularly shaped regions of necrotic tissue, and gray-white mildew layers can be seen on the front and back of the lesions in a humid environment. Infected tissues from symptomatic leaves disinfected in 75% ethanol for 45 s, and with 0.1% HgCl2 for 1 min, rinsed then plated on potato dextrose agar (PDA) medium supplemented with ampicillin and carbenicillin (50 μg/ml each). Plates were incubated for 3 days at 25°C. Then prepared by transferring hyphal tips from the edges of these colonies onto fresh PDA medium for subculture. Aerial hyphae had a cotton-like appearance with white to pale gray color (FigS1.D). Conidia were present in long chains, with conidiophores being present in clusters or in isolation (FigS1E), with 1-6 transverse septa, 0-3 oblique and longitudinal septa and an ellipsoidal to obpyriform structure, measuring 10.0-50.9 μm in length and 5.6-11.8 μm in width (n = 20) (FigS1E, G). On the basis of conidial and cultural characteristics, the fungus was consistent with those of members of the Alternaria genus (Simmons, 2008). To confirm this tentative identification, DNA was extracted from isolate XS9, the internal transcribed spacer rDNA regions (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), translation elongation factor 1-alpha (TEF1), partial RNA polymerase II largest subunit (RPB2) genes were amplified with primers pairs ITS1/ITS4 (White et al.1990), GDF/GDR (Templeton et al.), TEF-728F/TEF-986R (Carbone & Kohn 1999) and RPB2-5F2/RPB2-7cR (Sung et al. 1990; Liu et al. 1999), Bt-2a/Bt-2b (Glass and Donaldson 1995) respectively. The resulting sequences were deposited in GenBank (ITS, OP113690; GAPDH, OP120953; TEF, OP120952; RPB2, OP120954). Further phylogenetic analyses of isolate XS9 revealed it to cluster in the A. brassicae clade with 97% bootstrap support. Pathogenicity identification of isolate XS9 was carried out on the detached leaves. The pure agar plugs (as control) or spraying water on the leaf surface were inoculated on detached leaves, the controls remained healthy after 8 days (FigS1.H-J). but the leaves inoculated with other the mycelium plugs (Fig S1K, L) or the conidia suspension (2×105 conidia/mL) of isolate XS9 was sprayed on the detached leaves (Fig S1M, N), both showed brown necrotic lesions that are similar to the symptoms observed in the field. The pathogen was reisolated and confirmed to be A. brassicae. To our knowledge, this is the first report of leaf spot disease caused by A. brassicae on C. acuminata in China. Leaf spot disease causes the branches and leaves of camptotheca acuminata to wither and even the whole plant to die. To ensure the protection of the irreplaceable species, effective measures should be taken to prevent the spread of the leaf spot disease.