Metabolomics Reveals Resistance-Related Secondary Metabolism in Sweet Cherry Infected by Alternaria alternata

Sweet cherry (<i>Prunus avium</i> L.) is a tree species cultivated worldwide with high economic value. During its growth, it is frequently threatened by pathogenic fungi, leading to reduced yield and deteriorated quality. However, in sweet cherry, the disease-resistant cultivars against brown spot disease (BSD) caused by <i>Alternaria alternata</i>, as well as the associated disease-resistant metabolic pathways and metabolites, remain limited. In this study, we investigated the disease-resistant germplasm and associated metabolic pathways of sweet cherries using field disease resistance screening, physiological analyses, and metabolomics. The results showed that sweet cherry cultivar Q8 exhibited relatively strong resistance to BSD, while cultivar Q9 demonstrated the weakest resistance. Physiological experiments revealed that the changes in relative electrical conductivity (REC), malondialdehyde (MDA) content, and relative water content (RWC) of the disease-resistant cultivar Q8 were smaller than those of Q9 within 9 days under <i>A. alternata</i> infection. At 9 days post-infection (dpi), the relative electrical conductivity (REC) of Q9 (57.78%) was significantly (<i>p</i> < 0.05) higher than that of Q8 (49.01%), whereas the relative water content (RWC) of Q8 (78.29%) was significantly (<i>p</i> < 0.05) greater than that of Q9 (67.88%). For malondialdehyde (MDA) levels, Q8 reached its peak value (27.81 nmol/g) at 3 dpi, while Q9 attained its maximum (27.80 nmol/g) at 9 dpi. At 3 dpi and 9 dpi, Q8 consistently maintained a significantly (<i>p</i> < 0.05) higher RWC than Q9. Linolenic acid metabolism and unsaturated fatty acids were found to be involved in the disease resistance process, and Pinellic acid might be a bioactive metabolite conferring disease resistance. The disease-resistant metabolic pathways and bioactive metabolites identified in this study may be conserved in plants beyond sweet cherry, providing a theoretical basis for disease-resistant breeding of sweet cherry.