Sweet cherry ( Prunus avium) is a commercially vital fruit crop in China. Hop stunt viroid (HSVd) infection in sweet cherry causes dappled fruit. This study investigated the mechanism of dappled fruit formation in HSVd-infected sweet cherry using integrated metabolomics and transcriptomics. Dappled and non-dappled peel tissues were sampled at the color change and ripening stages. Ultra-performance liquid chromatography-tandem mass spectrometry identified 181 flavonoid metabolites, with peonidin-3-O-rutinoside, cyanidin-3-O-glucoside, peonidin-3-O-glucoside, cyanidin-3-O-arabinoside, cyanidin 3-xyloside, and cinchonain Ic being significantly enriched in dappled areas. RNA-seq revealed 3,287 differentially expressed genes, with PaCHS, PaCHI, PaDFR, and PaANS upregulated in dappled areas at the early stage, correlating with anthocyanin accumulation. KEGG enrichment highlighted anthocyanin and flavonoid biosynthesis pathways as central to pigmentation. This study suggests that HSVd disrupts anthocyanin biosynthesis to induce dappled pigmentation, offering novel insights into viroid–host interactions affecting fruit color in sweet cherry.

Phytoplasmas are minute phytopathogenic bacteria that induce excessive vegetative growth, known as witches’ broom (WB), in many infected plant species during the later stages of infection. The WB structure is characterized by densely clustered little (small) leaves, which are frequently accompanied by chlorosis (yellowing). The mechanisms behind the formation of little leaves within WB structures (LL-WB) are poorly understood. To address this gap, the LL-WB formation was extensively studied using sweet cherry virescence (SCV) phytoplasma-infected sweet cherry plants. Based on morphological examinations, signs of premature leaf senescence were observed in LL-WB samples, including reduced leaf size, chlorosis, and alterations in shape. Subsequent physiological analyses indicated decreased sucrose and glucose levels and changes in hormone concentrations in LL-WB samples. Additionally, the transcriptomic analysis revealed impaired ribosome biogenesis and DNA replication. As an essential process in protein production, the compromised ribosome biogenesis and the inhibited DNA replication led to cell cycle arrest, thus affecting leaf morphogenesis and further plant development. Moreover, the expression of marker genes involved in premature leaf senescence was significantly altered. These results indicate a complicated interplay between the development of leaves, premature leaf senescence, and pathogen-induced stress responses in SCV phytoplasma-infected sweet cherry trees. The results of this study provide insight into understanding the underlying molecular mechanisms driving the formation of little leaves and interactions between plants and pathogens. The findings might help control phytoplasma diseases in sweet cherry cultivation.