Microbial Interactions and Culturability of Seed Bacteria

Seed-associated microorganisms contribute to plant health by influencing nutrient acquisition, disease resistance, and stress adaptation. Culturing microbes is an essential step in microbiome research, allowing for direct functional analysis and potential agricultural applications. However, not all microbes can be readily cultured in laboratory settings, limiting our understanding of their ecological roles. In our latest study, we compared the cultured and uncultured bacterial fractions of Cannabis seeds, shedding light on the factors that determine which microbes can be grown in the lab and which remain elusive.

To investigate which bacteria could be recovered from Cannabis seeds, we worked with 54 Cannabis accessions, employing both standard and plant-based media, including those supplemented with Cannabis extracts, which aimed at optimizing the growth of slow-growing and highly adapted bacterial taxa. Through this approach, we successfully cultivated bacteria from 36 genera within the five major existing classes in Cannabis seeds: Gammaproteobacteria, Bacilli, Actinobacteria, Alphaproteobacteria, and Bacteroidia. Despite representing only 6.3% of the total microbial diversity detected in the seeds, these cultured bacteria accounted for 89.2% of the overall microbial abundance. This means that while we can recover many of the dominant bacterial groups, the true extent of microbial diversity within Cannabis seeds remains largely unattainable.

Culture-based methods revealed that a subset of the microbial community, consisting of nearly 60% of the bacterial taxa detected by sequencing, had no close cultured relatives. This included many rare and functionally significant bacteria, such as members of Acidobacteriae and Verrucomicrobiae. These groups are known to play critical roles in plant growth and nutrient cycling, yet their presence in seeds has been largely unexplored.

Do Microbial Interactions Have a Role in Culturability?

To further investigate why some microbes remain uncultured despite of their abundance, we conducted a network analyses based on bacterial co-occurrence patterns. The results revealed that uncultured seed bacteria tend to be more central within the microbial network, exhibiting strong connections mainly to other uncultured taxa. This suggests that interspecies interactions — such as nutrient exchange or signaling mechanisms — may play a crucial role in determining whether a microbe can grow independently.

This study highlights the limitations of current culturing methods but also provides a framework for improving microbial isolation strategies. Our findings suggest that microbial culturability is strongly influenced by ecological interactions, not just individual metabolic traits. The proposed implications are expected to be extended beyond Cannabis research. Seeds from diverse plant species likely harbor similarly complex microbial communities, with critical yet uncultured members influencing plant health. By refining cultivation techniques and leveraging microbial interactions, we may be able to recover missing bacterial taxa with potential applications in plant health and disease resistance.

Keywords: Cannabis • Culturing • Endophytes • Microbial Dark-matter • Microbial Interactions • Seed microbiome

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