Evaluating agar-plating and dilution-to-extinction isolation methods for generating oak-associated microbial culture collections.

Microbial isolation methods are crucial for producing comprehensive microbial culture collections that reflect the richness and diversity of natural microbiotas. Few studies have focused on isolation of plant-associated microbiota, with even less focus on forest trees. Here, we tested two isolation methods, (i) agar plating and (ii) dilution-to-extinction, for isolation of microbiota from leaf, stem, and root/rhizosphere tissues of oak trees. Microbial isolates obtained (culture-dependent) and the endogenous oak microbiota of the source tissue samples (culture-independent) were characterized by 16S rRNA gene and ITS community profiling. We found that the type of growth medium, incubation conditions, and sample type inoculated onto agar influenced the number of isolates and taxonomic richness of the isolates obtained. Most bacterial and fungal ASVs obtained from isolation-based approaches were only obtained using one of the two isolation methods, with only 12% of the ASVs detected in both. Moreover, the isolation methods captured microorganisms not detected by culture-independent analysis of the microbiota, suggesting these approaches can complement culture-independent analysis by enriching low-abundant taxa. Our results suggest that dilution-to-extinction and agar-plating approaches captured distinct fractions of the oak microbiota, and that a combination of both isolation methods was required to produce taxonomically richer microbial culture collections.