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Rhododendron hybridum Ker Gawl, a widely cultivated horticultural species in China, is highly valued for its ornamental and medicinal properties. However, with the expansion of its cultivation, leaf spot disease has become more prevalent, significantly affecting the ornamental value of R. hybridum Ker Gawl. In this study, R. hybridum Ker Gawl from the Kunming area was selected as the experimental material. The tissue isolation method was employed in this study to isolate pathogenic strains. The biological characteristics of the pathogens were determined using the mycelial growth rate method. The pathogens’ influence on the host plant’s ultrastructure was investigated using transmission electron microscopy (TEM). Colletotrichum nymphaeae was identified as the pathogen implicated in the development of leaf spot disease in R. hybridum Ker Gawl across three regions in Kunming City through the integration of morphological traits and phylogenetic analyses of multiple genes (ITS, ACT, GAPDH, HIS3, CHS1, and TUB2). Its mycelial growth is most effective at a temperature of 25 °C. pH and light have relatively minor effects on the growth of mycelium. The preferred carbon and nitrogen sources were identified as mannitol and yeast extract, respectively. Additionally, TEM observations revealed significant damage to the cell structure of R. hybridum Ker Gawl leaves infected by the pathogen. The cell walls were dissolved, the number of chloroplasts decreased markedly, starch granules within chloroplasts were largely absent, and the number of osmiophilic granules increased. This is the first report of leaf spot disease in R. hybridum Ker Gawl caused by C. nymphaeae. The results of this study provide valuable insights for future research on the prevention and control of this disease. Full article