Geographic, Temporal and Genetic Factors Shaping the Structure and Function of Walnut Rhizosphere Microbiome

Walnut (Juglans regia L.) performance and sustainability are closely linked to soil–plant–microbe interactions; nowadays, the combined influence of edaphic context, plantation development and rootstock genotype on walnut-associated microbiomes remains insufficiently resolved. Here, we integrated soil physicochemical characterization, community-level physiological profiling and 16S rRNA gene amplicon sequencing across walnut plantations in four Spanish regions. The design included 14-year clonal stands (Galicia, Gerona, Toledo), an age gradient in Galicia (4, 9 and 14 years), and four rootstocks (MJ209, Vlach, own-rooted ‘Chandler’ and J. regia seedling) in the Córdoba plantation. At the community-level, rhizospheres exhibited higher overall metabolic activity, displaying substrate-specific functional fingerprints across regions. Regarding stand ages, a functional peak was observed at middle age, with a decline in richness and diversity with age. Moreover, rootstock genotype further modulated rhizosphere metabolic function. Sequencing supported compositional differences among regions, ages and rootstocks, identifying a bacterial core of Juglans spp. rhizosphere and detecting 36 putative Plant Growth-Promoting Rhizobacteria (PGPR) genera, suggesting a potential reservoir and possible uses in plant biotechnology. Overall, walnut-associated microbiomes are jointly structured by soil gradients, plantation development and rootstock genotype, supporting site and genotype-tailored microbiome management.