Enright, D. J. | Search bioRxiv

bioRxiv2024

Evaluating Best Practices for Isolating Pyrophilous Bacteria and Fungi from Burned Soil

A live microbial culture is invaluable to assess traits and functions, yet culturing immediately from fresh soil is logistically challenging and media selection is not trivial. Deeper ecological understanding of pyrophilous microbes and their traits is hampered by a lack of culture-based work causing researchers to rely heavily on community sequence analysis. To improve our understanding of isolating pyrophilous bacteria and fungi after wildfires from burned soil, we tested which: 1) soil storage method and 2) media retained highest genera richness and highest culturable viability as measured by CFUs retained. We tested four soil storage methods (dried, refrigerated at 4{degrees}C, stored at -80{degrees}C with or without glycerol) using a well-homogenized soil sample, plated microbes on rich media, and compared to fresh soil obtained 6 months after a severe California shrubland wildfire. From the fresh soils, we tested 3 media types: rich (Lysogeny Broth (LB) for bacteria; Malt Yeast Agar (MYA) for fungi), oligotrophic (Reasoners 2 Agar (R2A)) and media made from pyrogenic organic matter (PyOM). For bacteria, storing soil frozen at -80{degrees}C without glycerol yielded the highest genera richness and with glycerol preserved the most species. For fungi, storing soil at -80{degrees}C without glycerol preserved the most species but preserved equivalent richness of genera as fresh and 4{degrees}C soil. R2A yielded the highest bacterial and fungal genus richness but some species of interest were only captured with PyOM. Using a combination of these storage and media methods along with a few additional techniques (mushroom cultivation, smoke cultivation, etc.) from 2018-2022, we cultured >500 isolates (286 bacteria; 258 fungi) after 7 California wildfires for testing pyrophilous microbial traits. ImportanceFires are increasing in frequency and severity across the globe, making the understanding of the traits of the pyrophilous microbes that survive and thrive post-fire and influence post-fire ecosystem regeneration critical and pressing. Yet, our ability to characterize the traits of pyrophilous microbes through genomics, transcriptomics, and phenotypic assays is hampered by lack of organisms in culture. Here, we debut a new medium created from pyrogenic organic matter that helps to cultivate fastidious pyrophilous microbes, and also describe a large-scale culture collection of pyrophilous bacteria and fungi (>500 isolates) that can be used for future collaborative research. We comprehensively test how soil storage and media type affect the cultivation of both bacteria and fungi, and for the first time, we test the best method to store soil to retain fungal diversity. Our work can be applied to improve culture collections of fastidious microbes from a variety of environments.

microbiology↗

bioRxiv2021

Mega-fire in Redwood Tanoak Forest Reduces Bacterial and Fungal Richness and Selects for Pyrophilous Taxa and Traits that are Phylogenetically Conserved

Mega-fires of unprecedented size, intensity, and socio-economic impacts have surged globally due to climate change, fire suppression, and development. Soil microbiomes are critical for post-fire plant regeneration and nutrient cycling, yet how mega-fires impact the soil microbiome remains unclear. We had a serendipitous opportunity to obtain pre- and post-fire soils from the same sampling locations because the 2016 Soberanes Fire, a mega-fire burning >500 Km2, burned with high severity throughout several of our established redwood-tanoak plots. This makes our study the first to examine microbial fire response in redwood-tanoak forests. We re-sampled soils immediately post-fire from two burned plots and one unburned plot to elucidate the effect of mega-fire on soil microbiomes. We used Illumina MiSeq sequencing of 16S and ITS1 to determine that both bacterial and fungal richness were reduced by 38-70% in burned plots, with richness unchanged in the unburned plot. Fire altered composition by 27% for bacteria and 24% for fungi, whereas the unburned plots experienced no change in fungal and negligible change in bacterial composition. We observed several pyrophilous taxa previously observed in Pinaceae forests, indicating that these microbes are likely general fire-responders across forest types. Further, the pyrophilous taxa that positively responded to fire were phylogenetically conserved, suggesting shared evolutionary traits. For bacteria, fire selected for increased Firmicutes and Actinobacteria. For fungi, fire selected for the Ascomycota classes Pezizomycetes and Eurotiomycetes and for a Basidiomycota class of heat-resistant Geminibasidiomycete yeasts. We hypothesize that microbes share analogous fire response to plants and propose a trait-based conceptual model of microbial response to fire that builds from Grimes Competitor-Stress tolerator-Ruderal framework (C-S-R) and its recent applications to microbes. Using this framework and established literature on several microbial species, we hypothesize some generalizable principals to predict which microbial taxa will respond to fire.

ecology↗