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

Area of Science:

• Microbiology
• Plant Science
• Ecology

Background:

• Comprehensive microbial culture collections are vital for understanding microbiota diversity.
• Plant-associated microbiota, especially in forest trees, remain understudied.
• Effective microbial isolation methods are needed to capture this diversity.

Purpose of the Study:

• To compare agar plating and dilution-to-extinction methods for isolating oak tree-associated microbiota.
• To characterize the microbial communities using culture-dependent and culture-independent approaches.
• To determine the optimal isolation strategy for building rich microbial culture collections.

Main Methods:

• Isolation of microbiota from oak leaf, stem, and root/rhizosphere tissues using agar plating and dilution-to-extinction.
• Characterization of microbial isolates and source tissues via 16S rRNA gene and ITS community profiling.
• Comparison of culture-dependent and culture-independent microbial community data.

Main Results:

• Growth medium, incubation conditions, and sample type significantly impacted isolate yield and taxonomic richness.
• Each isolation method captured unique bacterial and fungal taxa, with only 12% overlap between methods.
• Isolation methods identified microorganisms missed by culture-independent analyses, enriching low-abundance taxa.

Conclusions:

• Agar plating and dilution-to-extinction capture distinct microbial fractions from oak trees.
• A combination of both isolation methods is necessary for creating taxonomically rich microbial culture collections.
• These methods complement culture-independent analyses by enriching for diverse microbial taxa.