Genetic diversity of bacterial endophytes associated with Crotalaria (Rattlepods)

Crotalaria is a diverse legume genus valued for its ecological and agronomic roles. However, its associated endophytic microbiome remains minimally characterized. This study examined the genetic diversity and community structure of bacterial endophytes inhabiting domesticated and wild Crotalaria species. Twenty-nine healthy Crotalaria plant samples were analyzed using the culture-independent 16S rRNA gene amplicon sequencing approach. Genomic DNA was extracted using the Phenol-Chloroform method from both domesticated and wild samples. Sequencing of 16S ribosomal RNA gene was performed using Illumina MiSeq to profile endophytic bacterial communities. The sequence data was imported into R and processed using Divisive Amplicon Denoising Algorithm 2 (DADA2) pipeline. In total, 1,546,153 high-quality sequence reads were obtained and grouped into 611 amplicon sequence variants (ASVs) at 97% sequence similarity threshold. Domesticated samples exhibited higher observed species richness (median of 39 ASVs) than wild samples (median of 36 ASVs), while wild samples showed greater microbial community evenness (median ~1.08) compared to domesticated samples (median ~0.93). Beta diversity analyses showed clear separation between domesticated and wild endophytic bacterial communities, with domestication status explaining most of the observed variations (PERMANOVA: Bray-Curtis R 2 = 0.826; Jaccard R 2 = 0.846), but PERMANOVA detected no statistically significant difference (Bray-Curtis and Jaccard; p = 0.067). Overall, five bacteria phyla were detected in the Crotalaria samples. Pseudomonadota was the dominant phylum (64.27%) followed by Cyanobacteriota (35.5%). Enterobacteriaceae was the most abundant family (81.06%) with Enterobacter emerging as the most abundant genus in both domesticated (38.3%) and wild (89.4%) accessions. Notably, Pseudomonas was relatively enriched in domesticated plants while several bacterial genera were restricted to either wild or cultivated hosts, indicating habitat-associated endophytic specialization. This study provides the first comprehensive assessment of endophytic bacterial diversity in Kenyan Crotalaria species and highlights wild germplasm as an important reservoir of microbial diversity with potential biotechnological value.