Variovorax paradoxus alters the root microbiome and alleviates bicarbonate-induced Fe limitation in cotton (Gossypium hirsutum L.) with enhanced benefits from bilateral root inoculation

Alkaline and calcareous soils can induce iron (Fe) limitation in plants, yet the responses of root-associated microbial communities to beneficial rhizobacteria under these conditions remain poorly understood in cotton. Here, we investigated the effects of Variovorax paradoxus on plant performance, Fe nutrition, and root microbiome dynamics in cotton exposed to bicarbonate-induced Fe limitation. In this study, V. paradoxus inoculation under bicarbonate-induced Fe limitation significantly improved photosynthetic parameters, growth parameters, and tissue Fe status. Interestingly, V. paradoxus partially suppressed the Fe-deficiency-induced increase in root ferric-chelate reductase activity without further increasing rhizosphere siderophore activity. This response suggests that improved Fe availability reduced the demand for maximal activation of the intrinsic Strategy I response. Despite improved plant health, V. paradoxus reduced root C levels, suggesting altered belowground carbon utilization associated with bacterial inoculation and stress conditions. Split-root experiments further showed that inoculating both root compartments showed substantially greater recovery than unilateral inoculation, indicating that broader root exposure to V. paradoxus enhanced the beneficial response. Although bacterial alpha diversity remained unchanged, V. paradoxus significantly altered bacterial community composition and enriched Cellvibrio together with the fungal taxa Funneliformis and Dominikia under Fe limitation. Exploratory analysis identified the plant-beneficial fungal hubs Funneliformis and Serendipita in the V. paradoxus-treated community under indirect Fe deficiency, along with the core genera Pseudomonas, Hydrogenophaga, and Funneliformis and the indicator taxa Shinella and Aquabispora. Spearman correlation analysis further associated Streptomyces with root Fe accumulation and biomass, while Epicoccum and Sordariales were positively associated with siderophore production in cotton exposed to bicarbonate-induced Fe limitation and inoculated with V. paradoxus. These findings demonstrate the potential of V. paradoxus and identify candidate microbial partners for microbiome-informed biofertilizers to improve Fe nutrition in cotton grown in calcareous soils.