Deciphering genotype and geography dependent microbiome composition and its role in disease suppression in Ashwagandha

Ashwagandha, Withania somnifera (L.) Dunal is a perennial evergreen shrub widely used to treat mental health disorders and physical debility, and to enhance overall physiological function. Variations in genotype and geographic origin significantly influence rhizospheric microbial communities by altering soil physicochemical properties. This study applied shotgun metagenomic sequencing to investigate microbial community shifts in the rhizosphere of Nagori Ashwagandha (RN) from Rajasthan, Vallabh Ashwagandha-1 (GV) from Gujarat, and Nagori Ashwagandha from Rajasthan cultivated in Gujarat (GN). Fusarium wilt incidence was 67%, affecting the roots, which represent the most economically important part of ashwagandha. Taxonomic analysis identified Actinomycetota (46–60%) and Pseudomonadota (35–42%) as the predominant phyla, with Nocardioides (3.1–8.8%), Streptomyces (4.5–6.5%), and Bradyrhizobium (1–1.6%) as dominant genera across all groups in metagenomic analysis. Alpha-diversity analysis revealed higher species richness and Simpson's index in the GV group compared to the GN and RN groups. Beta-diversity assessment using Bray–Curtis distances showed partial clustering of GN and RN relative to GV in principal coordinate analysis and hierarchical dendrograms. Functional profiling based on KEGG annotation indicated that core metabolic and cellular pathways predominated across all genotypes, with no significant differences in Tier 1 and Tier 2 functional categories. To our knowledge, this represents the first shotgun metagenomic analysis of ashwagandha. Culturomics analysis yielded seventeen isolates from two rhizospheric locations; among these, Bacillus subtilis DMA1 exhibited the highest mycelial inhibition against Fusarium solani (64%), with a germination rate of 98%, root length of 2.1 cm, shoot length of 1.3 cm, seed vigor index of 333.2, and maximum fresh biomass of 1.12 g. Co-inoculation with F. solani and Bacillus subtilis DMA1 in pot trials significantly increased root length (20.1 cm), shoot length (39.5 cm), root girth (14.9 mm), and total biomass (51.1 g) compared to control and Fusarium -only treatments. These findings indicate that Bacillus subtilis DMA1 reduced wilt incidence by 70% and enhanced plant growth under pathogen-stress conditions.