Co-Inoculation of Bacillus subtilis and Priestia megaterium Promotes Growth and Shapes Rhizosphere Microbial Community of Rosa × Hybrida ‘Ruby’ Under Multiple Substrate Formulations

Efficient cultivation is essential for the rose industry. Both substrate formulation and plant growth-promoting rhizobacteria (PGPR) application both critical, yet their synergistic effects remain limited. This study investigated the synergistic effects of Bacillus subtilis (Bs) and Priestia megaterium (Pm) combined with five substrate formulations on the growth physiology, photosynthetic characteristics, and soil properties of Rosa × hybrida ‘Ruby’. Two-way ANOVA revealed significant interactions between substrate and PGPR treatments for most growth and physiological indicators. Orthogonal experiments demonstrated that specific PGPR–substrate combinations significantly enhanced plant growth and photosynthetic performance of the studied cultivar, as well as soil quality. Principal component analysis and membership function analysis identified four substrate–PGPR combinations as optimal, with the T4 substrate (humus/perlite/vermiculite/coconut coir/peat/biochar = 5:1.5:1:1:1:0.5) showing the most pronounced effects. In this T4 substrate, PGPR inoculation significantly altered the rhizobacterial community structure. LEfSe analysis revealed 67 enriched microbial biomarkers—substantially more than single-strain treatments. The relative abundance of beneficial genera such as Acidibacter and Chryseotalea increased, and the combined bacterial treatment enhanced functional pathways associated with signal transduction, cell motility, and RNA processing. Compared to single-strain treatments, the combined bacterial application demonstrated superior regulatory effects on plant growth. The optimal combined treatments increased plant height by up to 42.7%, root activity by 103.0%, soluble protein content by 302.8%, and soil ammonium nitrogen by 168.8%. These findings demonstrated that tailored combinations of PGPR and cultivation substrates highlight the potential for optimizing rose cultivation and improving the rhizosphere microecological environment.