Inoculation with Bacillus subtilis and Paenibacillus sp. shapes the rhizosphere bacteriome of prickly pear cactus under water deficit

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Abstract Background and aims Prickly pear cactus ( Nopalea cochenillifera L.) plays a central role in sustaining forage production in semiarid environments, yet prolonged drought associated with climate change increasingly constrains its performance. Plant growth-promoting bacteria (PGPB), including Bacillus subtilis and Paenibacillus spp., are recognized for enhancing plant tolerance to water limitation, but their influence on the rhizosphere microbiome of cactus under drought conditions remains unclear. We tested whether inoculation with these bacteria shapes rhizosphere bacterial community composition and interaction patterns under water deficit. Methods A greenhouse experiment was conducted using a factorial design combining two water regimes (well-watered and water-deficit) with three inoculation treatments ( B. subtilis , Paenibacillus sp., and a non-inoculated control). After 150 days of water stress exposure, rhizosphere bacterial communities were assessed using 16S rRNA gene amplicon sequencing. Results Water availability was the dominant driver of rhizosphere bacterial community structure, with drought favoring Actinobacteriota and well-watered conditions enriching Proteobacteria. Inoculation with B. subtilis modified community composition and increased the prevalence of drought-associated taxa, while also promoting a higher proportion of positive microbial interactions under water deficit. In contrast, Paenibacillus sp. exerted weaker and more variable effects. Despite microbial shifts, inoculation did not significantly affect plant biomass or root traits. Conclusions Rhizosphere bacterial communities of prickly pear cactus are primarily structured by water availability, whereas PGPB inoculation induces secondary, taxa-specific effects. Under drought, B. subtilis enhances microbial network connectivity, suggesting that inoculants may influence rhizosphere functioning even when plant growth responses are limited.

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