Synergistic effects from combining foliar and soil inoculation of plant growth promoting microbes: enhanced sugarcane physiology and rhizosphere modulation via organic acid exudation and nitrogen - cycling

Abstract Sugarcane ( Saccharum officinarum L.) is a strategic crop for the energy sector, but its intensive cultivation requires sustainable management practices. Plant growth-promoting bacteria (PGPB) such as Azospirillum brasilense and Nitrospirillum amazonense are known for their ability to fix atmospheric nitrogen and stimulate growth in grasses. This study evaluated the effects of inoculation with A. brasilense ( Ab ) and N. amazonense ( Na ), applied individually or in combination ( Ab + Na ), on sugarcane physiology, nitrogen use efficiency, root exudation, and rhizospheric microbial communities. The experiment was conducted under controlled greenhouse conditions with four treatments: Control (no inoculation), Ab , Na , and Ab + Na . The Ab + Na treatment promoted the highest photosynthetic rate, carboxylation efficiency, and water use efficiency. These physiological responses were associated with greater nitrogen accumulation in roots, rhizomes, and leaves, as well as increased total biomass and 15 N fertilizer recovery. Nitrogen losses were significantly reduced in Ab and Ab + Na treatments. At the molecular level, rhizosphere soil analysis showed a reduced abundance of microbial functional genes related to nitrification and denitrification ( amoA , nirK , and nosZ ) in inoculated treatments, indicating a lower potential for nitrogen losses. However, gene abundances were higher in the Ab + Na than in the individual inoculations, likely due to greater organic acid exudation stimulating soil organic matter mineralization. Only Ab and Ab + Na increased nifH abundance, indicating enhanced biological nitrogen fixation, predominantly associated with A. brasilense . The Ab + Na treatment also led to a distinct rhizospheric microbial profile and promoted more uniform microbial community structure. These findings suggest that combining microbial inoculants improve plant nitrogen efficiency and modify rhizosphere functioning through coordinated physiological and microbial mechanisms. Foliar inoculation with compatible PGPB offers a promising strategy for sustainable sugarcane intensification.