A method for sampling the living wood microbiome

Abstract

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.

A diverse and distinct microbiome inside living trees

“…Based on the prokaryotic abundances we observed in wood—averaging 10 6 . 35 copies per gram—and assuming four 16S copies per average cell 45 , we assert that there is approximately 1 bacterial cell for every 20 plant cells within a gram of dry wood. Given that 16S abundances vary considerably by tree species, this ratio would shift in accordance, from as low as 1:600 in Prunus serotina to as high as 1:2 in Acer saccharum .…”

“…DNA from the homogenized powder was then extracted using a MagMAX Microbiome Ultra Kit in concert with a KingFisher Apex automated extraction system. More details of this protocol, and critical validation, are presented in Arnold and Gewirtzman et al, 2024 35 . For the fine and coarse root samples taken from the black oak, roots were first washed and debrided in DI water prior to processing.…”

“…For 11 of the 16 species studied, there was no significant difference in prokaryotic abundance between heartwood and sapwood (Wilcoxon, p>0.05) tissues. As we examined a range of species with variable patterns of heartwood formation, we operationally defined "heartwood" as the innermost 5 cm of wood tissue and "sapwood" as the outermost 5 cm of wood tissue 35 . Despite an overall lack of difference between tissues, two species, Acer saccharum (red maple) and Betula lenta (black birch) displayed significantly higher prokaryotic abundances in their heartwood, whereas three species, Fraxinus americana (white ash), Pinus strobus (Eastern white pine), and Quercus rubra (red oak) displayed significantly higher prokaryotic abundances in their sapwood (p<0.05).…”

“…We estimate that there is approximately 1 bacterial cell for every 20 plant cells within the wood of the average living tree (based on observed prokaryotic abundances, which averaged 10 6 . 35 16S rRNA copies per dry gram wood). For the human microbiome, the ratio of bacterial cells to human cells is closer to parity (∼1:1) For a tree weighing approximately 5000 kg (live mass), the number of prokaryotes in the wood alone (notably, excluding the rhizosphere and external surfaces) is approximately 1 trillion cells, or one to two orders of magnitude less the number resident in the average human 36 .…”

“…In this study, we surveyed the wood-associated microbiome in more than 150 living trees across 16 species from the northeastern United States, representing 11 genera which have a global distribution across 151 countries ( SI Figure 1 ). Using newly developed, novel sample processing methods 35 , and quantitative and qualitative characterizations of microbiomes, we evaluated how wood-borne communities—both prokaryotic and fungal—vary within and among tree species, and how these consortia relate across a tree’s tissues and with neighboring environments. Through this microbial census, we illuminate the unexplored microbial biodiversity in the wood of living trees and provide insights into future forest health and ecosystem dynamics, which may lead to innovative strategies to conserve and enhance the vital functions of trees in ways that support global ecosystems and the communities that rely on them.…”