Influence of Gliricidia sepium living support on rhizosphere bacterial recruitment and fruit metabolomics in black pepper (Piper nigrum)

The ability of the rhizosphere to recruit beneficial microorganisms is central to the sustainable management of crops. Here, we investigated, through shotgun metagenomic sequencing and mass spectrometry techniques, the microbial composition, antimicrobial resistance (AMRs), and metabolomic profiles of Piper nigrum cultivated under two support systems: living Gliricidia sepium support (SL/RL) and dead wooden support (SD/RD). Bulk and rhizosphere soils were sequenced to assess taxonomic structure and ARGs, with emphasis on Actinobacteria due to their relevance in antibiotic production. Actinobacteria were highly represented across the samples (~ 35%). Dead-support rhizospheres exhibited a broader ARG repertoire and greater abundance of taxa known to include antibiotic-producing members, whereas living-support rhizospheres showed reduced ARG diversity. Metabolomic analysis further revealed support-dependent physiological responses in the host plant. PLS-DA distinguished the two cultivation systems, and three defense-related flavonoids, such as sakuranetin, isovitexin, and orientin, which were enriched in fruits of plants grown with dead support, indicating activation of stress - and defense - associated metabolic pathways. Their low abundance in living-support plants suggests reduced physiological stress, likely linked to improved nutrient cycling and rhizosphere stability provided by G. sepium. However, the volatile and total phenolic profile did not display significant chemical alterations in the samples. Together, these results suggest that support type influences both microbial functions and plant secondary metabolism. Dead support creates a more stressful ecological context that enriches antimicrobial resistance genes ARGs and defense metabolites, whereas living support promotes a more balanced soil-plant-microbe environment, supporting the use of G. sepium as a sustainable and biologically advantageous cultivation strategy for black pepper.