Autumn drought drives deterministic bacterial filtering and network destabilization in a phenotype-related manner in Pinus halepensis seedlings

Mediterranean forest restoration depends critically on autumn seedling outplanting and establishment, a period increasingly threatened by delayed and irregular precipitation. Although critical, drought at moderate temperatures, decoupled from summer heat stress, remains poorly characterized in terms of plant physiology and root microbiome responses. In the present study we simulated a short but severe drought under moderate air temperatures (5-25{degrees}C) in Pinus halepensis Mill. seedlings to examine the independent effects of water deficit on physiology and root-associated microbial communities. Drought reduced stomatal conductance to one-third of control values and induced a decoupling between stomatal conductance and net photosynthesis upon rewatering. This decoupling is rather due to the residual hydraulic and biochemical limitations rather than transpirational cooling demands. Drought-treated seedlings diverged into distinct phenotypic classes differing in recovery capacity, with a subset failing to recover despite being phenotypically identical to the controls. Root microbiome restructuring was phenotype-dependent and differed between active and resident fractions: bacterial richness and evenness increased while bacterial assembly shifted progressively toward determinism with increasing phenotype severity (29% to 37%), whereas fungal communities shifted toward stochastic drift (up to 89%). Functionally, drought disrupted symbiotic associations and drove a shift toward a fungal saprotrophic lifestyle. Network analyses revealed displacement of Rhizobium from its central hub position, reducing connectivity and compromising functional resilience. These results demonstrate that short, severe drought at moderate temperatures fundamentally affect plant-microbiome interactions through phenotype-dependent assembly processes, with direct consequences for seedling establishment and Mediterranean reforestation under climate change.