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Paeonia, one of China’s traditional ornamental plants, is also an important medicinal species (Fan et al., 2024; Ma et al., 2024). Its cultivation originated in China, with the Chinese peony group being the only source of wild species in the genus Paeonia, including Paeonia emodi Wall. ex Royle, Paeonia mairei H. Lév., and Paeonia obovata Maxim. (Ma et al., 2024). Paeonia is also widely used as a high-quality cut flower around the world. Dianjiang, Chongqing, has a long history of peony cultivation, with abundant resources. However, in recent years, a noticeable occurrence of peony leaf spot has been reported in the planting areas, with an incidence rate of approximately 30%. The primary symptoms include the appearance of round or near-round lesions on the leaves, with clear margins. The center of the lesions is lighter in color, while the edges are darker, forming a distinct ring pattern. As the disease progresses, the central tissue of the lesions dries and necroses, forming holes. Multiple lesions merge into large yellow-brown spots with concentric rings. These lesions gradually expand and may coalesce into irregular large patches. To isolate the pathogen, ten tissue samples (approximately 5×5 mm) were taken from the boundary between healthy and diseased leaf tissues. The samples were sterilized with 75% ethanol for 1 minute, followed by 30 seconds of treatment with sodium hypochlorite and three rinses with sterile water. They were then inoculated onto Potato Dextrose Agar (PDA) plates and incubated at 25°C for 7 days. Ten isolates with similar morphological characteristics were obtained. Among them, a representative isolate, designated Mb1, was selected for further morphological, molecular, and pathogenicity analyses. The colonies were initially white and fluffy, turning gray-black with numerous microsclerotia, resulting in a granular black appearance on the surface. The aerial mycelium was transparent or pale in color, and as the culture progressed, the colonies darkened to brown or black. Approximately 30 microsclerotia per colony were randomly measured, and were found to be spherical, 50–200 μm in diameter, while the pycnidia were nearly spherical and black, measuring approximately 100–200 μm in diameter. The conidia were aseptate and unicellular, produced within the black pycnidia. Conidia production was carried out on water agar supplemented with pine needles. TUB (PX910748) and ITS (PX911514) sequence fragments of the isolates were PCR-amplified and compared to sequences in the NCBI database. Phylogenetic analysis showed that isolate Mb1 clustered with Macrophomina species, specifically M. phaseolina, with a bootstrap support of 100% both markers, respectively, confirming their homology. To assess pathogenicity, a spore suspension (1.0 × 10⁶ CFU·mL⁻¹) in 55% sterilized glycerol was evenly applied to intact peony leaves without any artificial wounding using a sterile soft brush, with 30 μL of the suspension applied per leaf. A sterile water control was also included. 7 days after inoculation, the leaves treated with the spore suspension developed circular to near-circular lesions with dark margins and lighter centers, identical to those observed in the field, whereas the control leaves remained healthy. The pathogen was successfully re-isolated from the inoculated leaves, confirming its identity and fulfilling Koch’s postulates. Based on morphological and molecular phylogenetic analyses, the pathogen was identified as M. phaseolina. Biological characteristic studies showed that the optimal growth conditions for Mb1 were PDA medium, soluble starch and yeast extract as carbon and nitrogen sources, pH 5.0, and a temperature of 25°C. The fungus exhibited optimal growth under both continuous dark and light conditions. M. phaseolina is a non-specialized, soil-borne pathogen that is widely distributed in tropical and subtropical regions, capable of infecting up to 500 plant species (Marquez et al., 2021). This study represents the first report of M. phaseolina causing leaf spot in Paeonia in Dianjiang, Chongqing, providing important insights for the control of peony leaf diseases and the improvement of peony quality.