A method for sampling the living wood microbiome

Efforts to characterize microbial life across diverse environments have progressed tremendously, yet the microbiome of Earth's largest biomass reservoir-the wood of living trees-has been largely unexplored. Current understanding of the tree microbiome is largely confined to roots and leaves, with little attention given to the endophytic microbiome of wood, even though emergent studies have indicated this zone as a niche for unique taxa, of consequence for ecosystem health and global biogeochemical cycles. The lack of investigation derives partly from the physical recalcitrance of wood, which presents challenges during sampling, homogenization, and the extraction of nucleic acids. In response to these issues, we present an optimized method for processing wood for use in microbial analyses, from sampling through to downstream analyses. Using methane-cycling taxa as model endophytes, we assess losses in recovery during our method, and determine a limit-of-detection of approximately 500 cells per 100 mg of (dry) wood. For all six species evaluated-which represented several diverse taxa of hardwoods and softwoods-PCR inhibition proved minimal, and we expect this method to be applicable for a majority of tree species. The methods presented herein can facilitate future investigation into the wood microbiome and global microbial ecology of methane cycling.