Advancing microbial isolation: The impact of leaf mold extract agar on soil samples

Abstract In this study, we developed a new agar medium using leaf mold extract and evaluated its microbial cultivation performance with soil samples. As a control for performance evaluation, the general-purpose nutrient medium YME agar was used. For YME agar, isolated microbes were confirmed to belong mainly to the Bacillus genus through 16S rRNA gene sequencing. In contrast, for the leaf mold agar, bacteria belonging to either Streptomyces or Rhizobium were frequently isolated. Also, of the 51 sequenced isolates on the leaf mold agar medium, an unidentified species (i.e., not listed in databases) with 16S rRNA sequence identity below 98.7% was found. The unidentified species did not grow in standard nutrient media (YME, BHI, LB10, or TSB) but grew in 10% leaf mold extract. The findings of this study suggest that the use of leaf mold agar medium can effectively isolate soil microbes that are difficult to culture in general-purpose nutrient media such as YME. Moreover, this approach indicates a viable method for discovering unknown, previously-uncultured microbial species from soil samples.