Bridging the gap

Seeds represent unique microenvironments for endophytic microorganisms. These microbes have likely co-evolved with their host and expanding our knowledge about them has crucial implications for microbiome-targeted plant breeding, seed viability, and early plant establishment. While metagenomic studies have substantially contributed to uncovering the diverse roles of uncultured microbes, advancing culture-based approaches remains essential to harness the full potential of microbial diversity. Here, we provide a comprehensive perspective on the cultured and uncultured bacterial fractions of the Cannabis seed microbiota, emphasizing phylogenetic diversity, cultivability patterns, and microbial interactions. Our cultivation efforts recovered bacteria predominantly from well-represented classes in the Cannabis seed microbiome, including Gammaproteobacteria, Bacilli, Actinobacteria, Alphaproteobacteria, and Bacteroidia. The use of different nutrient media, including such that was supplemented with Cannabis extracts, expanded the diversity of cultivated taxa compared to previous studies, resulting in the isolation of 36 bacterial genera. However, overall cultivability was still limited to 6.32% of the microbiota, with nearly half of the uncultured diversity comprising phylogenetically distant bacteria. Rare taxa such as Acidobacteriae and Verrucomicrobiae, known for plant growth-promoting traits, remained uncultured, highlighting the challenges associated with recovering functionally significant but low-abundance microbes. Comparative network analyses revealed structural differences between cultured and uncultured fractions. Cultured taxa were more peripheral, with lower connectivity when compared to uncultured taxa. This suggests that uncultured taxa, particularly those serving as network hubs, may play pivotal roles in microbial community organization and functionality, potentially due to syntrophic interdependencies, requiring co-cultivation approaches to become cultured. This work provides a framework for integrating cultured and uncultured microbiota fractions to enhance our understanding of seed-associated microbial ecosystems and unlock the uncultured microbial potential.