Spatiotemporal dynamics of the barley rhizosphere prokaryotic community following repeated inoculation with Ensifer meliloti Rm2011 in a three-year field study

Abstract Background and aims Microbial inoculants are increasingly recognized for enhancing crop productivity and sustainable agriculture. We assessed the rhizosphere competence of Ensifer meliloti Rm2011 in different barley genotypes following repeated inoculation in three growing seasons, tracking its establishment and influence on the indigenous rhizosphere prokaryotic communities. We aimed to understand how consistent field application affects prokaryotic dynamics and whether environmental conditions or plant genotype modulate its establishment within the rhizosphere community. Methods Three-year field trials evaluated the establishment of E. meliloti Rm2011 in barley rhizosphere and its impact on the prokaryotic community. Rhizosphere samples were collected at several time points throughout the vegetation period and analyzed using colony-forming units and 16S rRNA gene amplicon sequencing to further evaluate the effects of barley genotype and environmental conditions. Results E. meliloti Rm2011 demonstrated robust persistence throughout the growing season, successfully integrating into the rhizosphere community. Despite repeated inoculations, its impact on the native prokaryota was negligible across all years. Neither environmental conditions nor barley genotype had a significant effect on its establishment. Notably, a set of core bacterial taxa was consistently detected, indicating microbiome stability. Further, one ASV assigned to Ensifer was significantly enriched after inoculation in all three years. Conclusion E. meliloti Rm2011 survives and establishes in the barley rhizosphere without modulating the native prokaryotic community composition. Its ecological compatibility and persistence underscore its potential as a microbial inoculant in sustainable crop management. This study highlights the importance of long-term field studies in assessing inoculants' performance and impact on prokaryotic communities.