Comparative metabolomic analysis of resistant and susceptible Actinidia rufa and Actinidia chinensis var. chinensis hybrid germplasms in response to Pseudomonas syringae pv. actinidiae infection

【Objective】Actinidia Lindl., a deciduous perennial liana genus endemic to China, is recognized globally as a functional fruit crop owing to its unique organoleptic characteristics, exceptional nutritional profile (notably distinguished as the ascorbate hyperaccumulator within Rosidae clade), and pharmacological properties. The Chinese kiwifruit industry has undergone remarkable expansion, with cultivation areas reaching 300 000 hectares and annual production achieving 4.346 million metric tons by 2024. This growth has significantly contributed to regional economic development, poverty alleviation initiatives, and agroecosystem rehabilitation. Pseudomonas syringae pv. actinidiae (Psa), a phytopathogen with rapid dissemination capacity and high virulence, poses severe threats to global kiwifruit production, causing epidemic canker outbreaks. Identifying excellent disease-resistant germplasms, elucidating the underlying mechanisms of resistance formation, and developing resistant cultivars are crucial approaches to addressing the occurrence of canker. Plant metabolite alterations provide insights into metabolic states, gene expression, and protein function. By conducting metabolomics research on the resistant genotype E2545 and susceptible genotype E674 at different infection time points, we can identify differential metabolites, determine associated metabolic pathways, and reveal their roles in biological processes. This study offers a metabolic perspective on the molecular mechanisms underlying the response of kiwifruit materials with differential resistance to Psa infection.【Methods】In vitro stem inoculation assays were conducted on Psa-resistant (E2545) and susceptible (E674) kiwifruit genotypes under controlled phytosanitary conditions. Longitudinal sections (1 cm) of xylem-differentiated tissues were sampled proximal and distal from the inoculation site at defined intervals (0, 3, 7, and 14 days post-inoculation, dpi). Untargeted metabolomic profiling was performed using the ultra-high-performance liquid chromatography coupled with quadrupole time- of- flight tandem mass spectrometry (UHPLC-QTOF-MS/MS). Raw metabolite data was acquired using MassLynx V4.2. Data processing including peak extraction and alignment was performed using Progenesis QI. Metabolite identification was achieved against a self-built database (BMK) using MS/MS data, with quantification via multiple reaction monitoring on a triple quadrupole mass spectrometer. Differential metabolites were screened using thresholds of variable importance in projection (VIP)≥1, P- value＜0.05, and log2|FC|≥1.【Results】Comparative metabolomic profiling revealed distinct clustering of specialized metabolites between P. syringae pv. actinidiae (Psa)-resistant and susceptible Actinidia genotypes. Differential metabolites were predominantly categorized as benzene and substituted derivatives, coumarins and derivatives, prenol lipids, carboxylic acids and derivatives. Quantitative analysis demonstrated significant accumulation of 87 defense- related metabolites in resistant germplasm, including key phytoalexins and signaling molecules linked to systemic acquired resistance. Four metabolites exhibiting dual antimicrobial and immune- priming activities were prioritized: aspirin, liquoric acid, pestalactam B, and valtratum. KEGG pathway enrichment analysis revealed significant activation of phenylpropanoid biosynthesis and phenylpropanoid metabolism, indicating their critical roles in kiwifruit defense against Psa infection.【Conclusion】This study conducted comparative metabolomic profiling of resistant and susceptible germplasms in Actinidia rufa and Actinidia chinensis var. chinensis subjected to varying P. syringae pv. actinidiae (Psa) infection periods. Building on these findings, further in-depth research on the key differential metabolites between resistant and susceptible kiwifruit germplasms could elucidate the disease resistance mechanisms of germplasms, providing a theoretical basis for screening novel kiwifruit germplasms resistant to bacterial canker. Additionally, antimicrobial activity assays and field trials should be conducted to validate the antibacterial properties of these metabolites, facilitating the development of innovative biocontrol agents. This integrated approach would advance both the theoretical framework and practical applications in kiwifruit disease resistance breeding and sustainable disease management.