Brown leaf spot, a severe fungal disease in Rhododendron, substantially impairs its ornamental and economic value. However, the molecular mechanisms governing the interaction between Rhododendron leaves and the pathogen are still poorly understood. In this study, the less susceptible Rhododendron hybridum ‘Yangmeihong’ (YMH) and the highly susceptible R. hybridum ‘Xiaotaohong’ (XTH) leaves were selected for transcriptomic and widely targeted metabolomic analyses. We compared constitutive metabolite profiles prior to inoculation as well as differentially expressed gene (DEGs) and differentially accumulated metabolites (DAMs) at two days post-inoculation (dpi) with Neopestalotiopsis clavispora. Results showed that YMH leaves exhibited 273 upregulated constitutive metabolites, whereas XTH had only 46. After infection, DEGs in YMH leaves were significantly enriched in pathways including flavonoid biosynthesis and glycerolipid metabolism, while XTH showed enrichment in vitamin B6 metabolism and glutathione metabolism. Metabolomic analysis identified 788 metabolites at 2 dpi, with YMH accumulating 49 DAMs and XTH accumulating 115 DAMs. Integrated transcriptomic and metabolomic analyses further revealed distinctions in reactive oxygen species (ROS) scavenging mechanisms between the two cultivars. These findings offer valuable insights for developing disease-resistant breeding and control strategies in Rhododendron.

Petal blight caused by Neopestalotiopsis clavispora infection in Rhododendron delavayi petals severely reduces their ornamental value; however, the metabolic and transcriptional responses of petals to this pathogen remain unclear. In this study, we integrated widely targeted metabolomic and transcriptomic analyses to systematically characterize the response mechanisms of R. delavayi petals at early (1 day post-infection), middle (2 days post-infection), and late (4 days post-infection) stages of N. clavispora infection. A total of 1251 metabolites were identified, and K-means clustering analysis revealed that the differentially accumulated metabolites (DAMs) in Subclass 1 were significantly enriched in flavonoid, phenylpropanoid, and glutathione metabolic pathways. Transcriptomic analysis identified 3744, 6986, and 5407 differentially expressed genes (DEGs) at early, middle and late stages, respectively. Weighted gene co-expression network analysis (WGCNA) indicated that the key hub genes at early, middle, and late stages were mainly involved in maintaining reactive oxygen species (ROS) homeostasis, plant–pathogen interaction signal transduction, and cell wall remodeling, respectively. Joint metabolomic and transcriptomic analysis showed that phenylpropanoid, flavonoid, and glutathione pathways were commonly and significantly enriched. In vitro antifungal assays demonstrated that 2000 mg/L phloretin significantly inhibited mycelial growth of N. clavispora and effectively alleviated petal infection. Collectively, R. delavayi petals exhibit stage-specific defense strategies against N. clavispora: in the early stage, by inducing H₂O₂ accumulation and ROS homeostasis; in the middle stage, by activating immune signaling pathways; and in the late stage, by enhancing cell wall remodeling and antioxidant capacity. This study provides a theoretical basis and a candidate compound for green control of petal blight.