Leaf mold extract agar facilitates recovery of soil bacterial diversity beyond Bacillus dominance

Dominance of Bacillus species on conventional agar often hampers the recovery of diverse soil bacteria ("Bacillus dominance"). To address this, we developed an agar medium containing leaf mold extract (LME) and evaluated its effectiveness for soil microorganism isolation compared to conventional yeast malt extract agar (YME agar). The 16S rRNA gene sequencing revealed that YME agar cultures were dominated by Bacillus-related bacteria, while LME agar enabled the growth of a broader range of taxa, especially actinomycetes like Streptomyces. LME agar supported higher bacterial diversity even in samples with high Bacillus abundance. Some LME isolates had very low 16S rRNA gene similarity to known species or could not be amplified with universal primers, suggesting potentially novel taxa. One unidentified isolate proliferated only in liquid LME, indicating a unique nutrient requirement. We further screened LME-derived isolates (569 strains) for therapeutic activity using an insect (the silkworm, Bombyx mori) model infected with three pathogenic fungi (Rhizopus arrhizus, Aspergillus fumigatus, Candida albicans). Distinct extracts showed significant efficacy in each infection model, and the overall hit rate was 6.2%. Notably, the extract of the rare species Ancylobacter sonchi showed marked efficacy against R. arrhizus infection, underscoring LME agar's potential for accessing novel bioresources. These results suggest that LME agar suppresses Bacillus dominance and expands access to diverse, rare, and previously uncultured soil bacteria, providing a powerful platform for microbial library construction and novel drug discovery. KEY POINTS: • LME agar prevents Bacillus dominance, enabling recovery of diverse microbial colonies. • LME agar accesses rarer and more diverse microbiota than nutrient-rich media. • A strain from LME agar showed therapeutic activity in silkworms infected with R. arrhizus.