Multi-omics analysis revealed the comprehensive regulatory pattern of chestnut development on pathogenic defense

• First systematic study on pathogen colonization and defense mechanisms in developing Chinese chestnuts (from pollination to maturity). • Alternaria is identified as the dominant pathogen, with Filobasidium and Methylobacterium as key microbial taxa during development. • A shift from non-enzymatic to enzymatic antioxidants reveals a dynamic, balanced defense strategy in the kernel. • WGCNA pinpoints critical defense genes ( CML, CPK, RPM1 ) and pathway genes ( COMT, POD ) involved in pathogen resistance. Chestnut ( Castanea ) a globally cultivated crop, which have been threating the chestnut quality by the postharvest pathogen infections. It keeps the health status during their developmental stages, but the inner regulatory mechanism remains largely unexplored. In this study, microbiome, transcriptome, metabolomics, and defense substances were investigated in the burs, seeds, shells, and kernels collecting from developing Chinese chestnuts ( C. mollissima ) at 10-day intervals, spanning from the pollination to the maturation stage. Pathogen infection patterns show that during the development of chestnuts, various pathogenic fungi can colonize the developing chestnuts, with Alternaria emerging as the dominant pathogen. Notably, Filobasidium and Methylobacterium were identified as key microbial taxa throughout development. Defense substance profiling revealed a progressive decline in non-enzymatic antioxidants (polyphenol, flavonoid and tannin), contrasted with an increase in enzymatic antioxidants (SOD, POD, CAT), indicating a complementary defense strategy. The heatmap of differentially expressed genes indicates that chestnut fruit development is mainly focused on plant-pathogen interactions and genes related to chestnut growth and defense; Weighted gene co-expression network analysis highlighted critical defense genes, including CML, CPK, RPM1 , and RPS3 , involved in plant-pathogen interactions, as well as COMT and POD in the phenylpropanoid biosynthesis pathway. This study offers new insights into an inner defense mechanism of pathogen during chestnut development, providing a theoretical basis for enhanced chestnut cultivation and disease management.