Seasonal Drought Reshapes Root Exudate Chemistry and Microbial Associations in a Mixed Mediterranean Forests

Tree root exudates and rhizosphere microbe interactions are a key pathway influencing tree health in the forest. However, the chemical mechanisms mediating these belowground interactions, particularly under climate driven drought stress, remain poorly understood. Mediterranean forests experience recurrent seasonal drought, providing a natural system to examine how water limitation alters root derived carbon inputs and associated microbial responses. We conducted a two-year field experiment in a mixed Mediterranean forest comprising mature trees of Pinus halepensis, Quercus calliprinos, and Pistacia lentiscus. Across four seasons, we simultaneously quantified root exudation rates and metabolite composition and characterized soil and root associated microbiomes using 16S rRNA gene sequencing at species level resolution. This integrative approach allowed us to directly link drought-induced changes in root chemistry with microbial community structure and interaction patterns. Root exudation rates increased on average 2.7-fold during the dry compared to the wet season, reaching up to 21.7 μg C cm⁻² day⁻¹ across all three tree species. Metabolomic analyses identified 89 drought responsive compounds, dominated by amino acids (24), phenolics (22), carbohydrates (11), and terpenoids (8). While metabolite profiles varied strongly with both tree species and season, eight metabolites consistently responded to drought across all species, suggesting conserved metabolic responses to water stress. In contrast to the pronounced chemical shifts, rhizosphere microbial community composition remained largely stable across seasons, although it differed among host tree species. Despite this taxonomic stability, correlation analyses revealed multiple bacterial taxa that were positively or negatively associated with drought responsive metabolites. Notably, 19 actinobacterial species correlated with compounds such as the terpenoid glaucocalyxin A, deoxyribose, and a C5 sugar alcohol, highlighting diverse microbial strategies for exploiting drought altered root exudates. Together, our results demonstrate that seasonal drought reshapes belowground interactions primarily through changes in root exudate chemistry rather than large scale microbial turnover. We propose that drought-induced shifts in root derived metabolites act as finely tuned metabolic signals that selectively modulate microbial interactions while preserving the overall structural stability of the rhizosphere community in Mediterranean forests.