First report of Alternaria alternata causing leaf spot on Aesculus chinensis in China

Chinese horse chestnut (Aesculus chinensis Bunge) is an important medicinal and ornamental tree species that is widely planted throughout China (Sun et al. 2023). In June 2024, A. chinensis plants in and around Dushan Forest Park (33°04′13″N, 112°35′11″E) in Nanyang exhibited numerous foliar lesions. Across an 8.1-ha area, 100 A. chinensis trees were inspected, and leaf spot symptoms were observed on 59 trees. Early symptoms appeared as small black spots on the leaf margin, which gradually expanded toward the center. Dark halos subsequently developed around the lesions. Five diseased leaves were collected from each of ten individual plants (n = 50). Pieces were excised from the lesion margin and placed on potato dextrose agar. In total, 33 purified fungal strains were obtained from symptomatic leaves. Three isolates (Q-1, Q-2, and Q-5) from different sampling sites were selected for additional analysis. On PDA, colonies produced abundant aerial mycelia. The colony surface was olivaceous to dark green, and the reverse was greyish brown to dark brown (Figure S1c, d). Conidia were produced in unbranched chains of 5 to 11, obclavate or spheroidal, dark brown, with 2 to 5 transverse septa and 0 to 3 longitudinal septa, measuring 11.5 to 37.2 × 7.3 to 11.8 μm (n = 100) (Figure S1e, f). These morphological characteristics are consistent with Alternaria spp. (Simmons 2007). Genomic DNA was extracted from fresh mycelia using the CTAB method. The internal transcribed spacer (ITS) region, translation elongation factor 1-alpha gene (tef1), glyceraldehyde 3-phosphate dehydrogenase gene (gapdh), and the second largest subunit of RNA polymerase II (rpb2) were amplified by PCR using primer pairs ITS1/ITS4, EF1-728F/EF1-986R (Carbone and Kohn 1999), gpd1/gpd2 (Berbee et al. 1999), and fRPB2-5F/fRPB2-7cR (Liu et al. 1999), respectively, following the PCR protocols described in the corresponding references. Sequences were deposited in GenBank: ITS (PV342512, PV342513, PV412997), tef1 (PV417283–PV417285), gapdh (PV528059, PV528060, PV543653), and rpb2 (PV528056–PV528058). A maximum-likelihood phylogeny based on the concatenated ITS-tef1-gapdh-rpb2 dataset was inferred in MEGA12. The Kimura 2-parameter model with gamma-distributed rate variation and a proportion of invariant sites (K2+G+I) was selected as the best-fit nucleotide substitution model. It placed all three isolates within the Alternaria alternata clade (Figure S2), clearly separated from other congeners (Woudenberg et al. 2015). Together, morphological characteristics and multilocus evidence identified the causal agents as A. alternata. Pathogenicity tests were conducted using three representative isolates. For each isolate, five healthy 6-month-old potted A. chinensis seedlings (~30 cm tall) were inoculated (15 seeding in total), and five seedlings sprayed with sterile distilled water served as controls. Inoculation was performed by spraying 50 μL of a conidial suspension onto the leaf surfaces, and the inoculated plants were maintained in a greenhouse at 25 °C and 90% relative humidity. Typical symptoms developed on incubated leaves within 7 days and were consistent with field observations, whereas negative controls remained symptomless (Figure S1g,h). The inoculated fungus was reisolated from symptomatic tissues and confirmed as A. alternata based on morphology, thereby fulfilling Koch’s postulates. Previously reported diseases of A. chinensis in China include leaf blotch caused by Colletotrichum sp. (Sun et al. 2020) and leaf blight caused by Botryosphaeria dothidea (Wang et al. 2025). Our current results constitute the first report of A. alternata as the causal agent of leaf spot on A. chinensis in China. Given the high ornamental value, leaf spot symptoms can reduce aesthetic quality and may lead to premature defoliation under favorable conditions, highlighting the need for timely diagnosis and management. At present, there is no direct evidence demonstrating a host jump from another Aesculus plant. Resolving this question will require broader sampling across hosts and locations, coupled with cross-inoculation assays.