Enzymatic and proteomic changes in resistant and susceptible cacao cultivars reveal distinct response mechanisms to Phytophthora citrophthora infection

Black pod rot, caused by <i>Phytophthora</i> species, is one of the most severe diseases affecting cocoa production. Among these species, <i>P. citrophthora</i> is considered one of the most aggressive, yet little is known about the molecular responses of cocoa to this pathogen. This study aimed to investigate the defense mechanisms of cacao against <i>P. citrophthora</i> through enzymatic analyses and gel-free comparative proteomics. Seedlings obtained by rooting cuttings from one resistant and one susceptible cultivar were inoculated with the pathogen, while controls received sterile distilled water. The activities of ascorbate peroxidase (APX), guaiacol peroxidase (GPX), catalase (CAT), and superoxide dismutase (SOD) were measured at 6, 12, 18, and 24 hours after inoculation (HAI). Protein abundance was evaluated at 24 HAI using mass spectrometry. The pathogen induced GPX activity from 6 HAI in the resistant and from 12 HAI in the susceptible cultivar, while APX activity increased in both cultivars after 18 HAI. A total of 1,583 proteins were identified across treatments. In the resistant cultivar, infection was associated with reduced photosynthesis, redirection of carbohydrate metabolism, and changes in the ascorbate/dehydroascorbate ratio, suggesting an efficient activation of defense responses. Constitutively abundant proteins related to antioxidant activity may also have contributed to resistance. In contrast, the susceptible cultivar showed limited protein abundance changes, with indications of increased metabolism of small molecules and accumulation of methylglyoxal, a cytotoxic compound linked to disease susceptibility. Overall, the results demonstrate that the resistant cultivar mobilizes early antioxidant defenses and metabolic reprogramming to cope with infection, whereas the susceptible exhibits inefficient responses leading to cellular damage. These findings provide new insights into cacao-<i>P. citrophthora</i> interactions, offer a foundation for future transcription-level studies, and may support the development of new pre-breeding stages for cacao cultivars.