Simple SummaryMicroorganisms are tightly associated with crops and can be pathogens or beneficials. Rose is the main ornamental crop worldwide. During production, rose varieties (over 100 in a single farm) are permanently grafted in a unique genotype as rootstock to improve plant performance. This work aimed to study the effect of grafting on root-associated microorganisms using next-generation DNA sequencing analysis. To this end, we have demonstrated that root-associated microorganisms of the rootstock (bacteria and fungi) will depend on the grafted genotype. In other words, a change in the variety will drive unique changes in the assembly of microorganisms at the root. This manuscript discusses differences in the bacterial and fungal communities when grafted and non-grafted and their potential impact on plant performance and agriculture.AbstractThe root microbiome is vital in plant development and health and is highly influenced by crop cultural practices. Rose (Rosasp.) is the most popular cut flower worldwide. Grafting in rose production is a standard practice to increase yield, improve flower quality, or reduce root-associated pests and diseases. ‘Natal Brier’ is a standard rootstock used in most commercial operations in Ecuador and Colombia, leading countries in producing and exporting ornamentals. It is known that the rose scion genotype affects root biomass and the root exudate profile of grafted plants. However, little is known about the influence of the rose scion genotype on the rhizosphere microbiome. We examined the influence of grafting and scion genotype on the rhizosphere microbiome of the rootstock ‘Natal Brier’. The microbiomes of the non-grafted rootstock and the rootstock grafted with two red rose cultivars were assessed using 16S rRNA and ITS sequencing. Grafting changed microbial community structure and function. Further, analysis of grafted plant samples revealed that the scion genotype highly influences the rootstock microbiome. Under the presented experimental conditions, the rootstock ‘Natal Brier’ core microbiome consisted of 16 bacterial and 40 fungal taxa. Our results highlight that the scion genotype influences root microbe’s recruitment, which might also influence the functionality of assembled microbiomes.