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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol is an effective, speedy method of culturing yeasts and the mold Aspergillus fumigatus from large sets of soil samples in as little as 7 days. The methods can be easily modified to accommodate a range of incubation media and temperatures as needed for experiments.

Abstract

Soil is host to an incredible amount of microbial life, with each gram containing up to billions of bacterial, archaeal, and fungal cells. Multicellular fungi such as molds and unicellular fungi, broadly defined as yeasts, fulfill essential roles in soil ecosystems as decomposers of organic material and as food sources for other soil dwellers. Fungal species diversity in soil is dependent on a multitude of climatic factors such as rainfall and temperature, as well as soil properties including organic matter, pH, and moisture. Lack of adequate environmental sampling, especially in regions of Asia, Africa, South America, and Central America, hinders the characterization of soil fungal communities and the discovery of novel species.

We characterized soil fungal communities in nine countries across six continents using ~4,000 soil samples and a protocol developed in the laboratory for the isolation of yeasts and molds. This protocol begins with separate selective enrichment for yeasts and the medically relevant mold Aspergillus fumigatus, in liquid media while inhibiting bacterial growth. Resulting colonies are then transferred to solid media and further processed to obtain pure cultures, followed by downstream genetic characterization. Yeast species identity is established via sequencing of their internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster, while global population structure of A. fumigatus is explored via microsatellite marker analysis.

The protocol was successfully applied to isolate and characterize soil yeast and A. fumigatus populations in Cameroon, Canada, China, Costa Rica, Iceland, Peru, New Zealand, and Saudi Arabia. These findings revealed much-needed insights on global patterns in soil yeast diversity, as well as global population structure and antifungal resistance profiles of A. fumigatus. This paper presents the method of isolating both yeasts and A. fumigatus from international soil samples.

Introduction

Fungi in soil ecosystems play essential roles in organic matter decomposition, nutrient cycling, and soil fertilization1. Both culture-independent (i.e., high-throughput sequencing) and culture-dependent approaches are widely used in the study of soil fungi2,3. While the large amount of data generated by high-throughput metabarcode sequencing is useful for elucidating broad-scale patterns in community structure and diversity, the culture-dependent approach can provide highly complementary information on the taxonomic and functional structures of fungal communities, as well as more speci....

Protocol

NOTE: Any steps utilizing international soil samples and/or A. fumigatus spores and mycelia require working within a biosafety cabinet for level 2 organisms (BSCII).

1. Isolation of yeast from soil

  1. Preparation of antibacterial and antifungal solutions
    1. Suspend chloramphenicol powder in 70% ethanol to prepare a 50 g/L stock solution. Sterilize by syringe filtration and store at 4 °C.
      ​NOTE: This antibiotic will prevent the grow.......

Representative Results

Yeast isolation from soil
The above yeast isolation protocol was implemented to culture yeasts from soil samples originating from 53 locations in nine countries9,12. In total, 1,473 yeast strains were isolated from 3,826 soil samples. Given the different climatic conditions of the nine originating countries, the best incubation temperature for each country was determined based on its mean annual temperature (Table 1). Given.......

Discussion

The protocol developed for isolating yeasts and A. fumigatus from soil is a fast and efficient method for high-throughput soil processing and fungal isolation. The protocol only requires a small amount of soil (0.1-0.2 g) per sample, allowing for more sites to be sampled with similar effort. The quick turnaround time ensures that results can be obtained within a short timeframe and allows time for troubleshooting and repeating experiments if necessary. This protocol can be easily replicated across many laborator.......

Acknowledgements

This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada (Grant No. ALLRP 570780-2021) and McMaster University.

....

Materials

NameCompanyCatalog NumberComments
1.5 mL microcentrifuge tubeSarstedt Inc72.690.001
Benomyl powder Toronto Research ChemicalsB161380
Chloramphenicol powder Sigma-AldrichSKU: C0378-5G
DextroseSigma-AldrichSKU: D9434-500G
Fragment Analysis SoftwareNCBI's Osirishttps://www.ncbi.nlm.nih.gov/osiris/
ITS sequence databaseNCBI GenBank https://www.ncbi.nlm.nih.gov/genbank/
ITS sequence databaseUNITE https://unite.ut.ee/
PeptoneSigma-AldrichSKU: P5905-500G
Reusable cell spreaders Fisher Scientific08-100-12
Sterile 10 cm diameter Petri dishes Sarstedt Inc83.3902
Sterile 13 mL culture tubes Sarstedt Inc62.515.006
Wooden plain-tipped applicator sticks Fisher Scientific23-400-112
Yeast extractSigma-AldrichSKU: Y1625-250G

References

  1. Frac, M., Hannula, S. E., Belka, M., JÈ©dryczka, M. Fungal biodiversity and their role in soil health. Frontiers in Microbiology. 9, 707 (2018).
  2. Tedersoo, L., et al. Global diversity and geography of soil fungi. Science....

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