Our protocol investigates yeast and mold diversity in soil using soil samples obtained from different climatic regions. This provides insights into environmental yeast diversity and helps track pathogenic species. The protocol offers a cost-effective and speedy method of harvesting yeasts and mold from the soil.
Soil samples can be fully processed to obtain isolates in as little as seven days. Well organized experimental setup, such as labeling culture tubes and preparing media one day in advance is the key to avoid feeling overwhelmed during the experiment. Start with preparing a set of sterile 13 milliliter culture tubes.
By labeling them with soil sample ID, then use a serological pipette to add five milliliters of Yeast Extract-Peptone-Dextrose broth supplemented with chloramphenicol and benomyl in each tube. Inside a biosafety cabinet for level two organisms, transfer approximately 0.1 gram of soil into the appropriate culture tube using a sterile wooden plane tip applicator. Cap the tube securely to the first stop, preventing spillage while allowing the air exchange during incubation.
Next incubate the culture tubes in a roller drum for 24 hours at a temperature deemed optimum for maximizing yeast growth. Then label the chloramphenicol and benomyl supplemented yeast extract peptone-dextrose agar plates with the soil sample ID before transferring the supernatant to the solid medium. Remove the culture tubes from the roller drum.
Then inside a biosafety cabinet for level two organisms, briefly vortex the tube to draw soil particles and cells that may have settled at the bottom back into suspension. Next, transfer 100 microliters of the supernatant onto a plate using a micropipette. Use a sterile reusable cell spreader to spread the liquid thoroughly and evenly over the agar surface.
After allowing for sufficient incubation time of two to three days, inspect the plates for any yeast growth inside a biosafety cabinet for level two organisms. Look for creamy, round, matte-like yeasts that can be easily distinguished from bacterial and mold colonies. Select one yeast-like colony from each plate using sterile wooden plain tipped applicator sticks.
Transfer each selected colony onto a fresh yeast extract peptone-dextro agar plates supplemented with chloramphenicol and benomyl and streak for single colonies. Perform three separate streaks and use a new applicator stick for every streak. Next, use fresh cells developed on a plate to perform a colony polymerase chain reaction.
Use internal transcribed spacers ITS1 and ITS4 primers to amplify the fungal bar coding gene. Compare the obtained internal transcribed spacer sequence of the yeast strains, two sequences deposited in public databases, such as National Center for Biotechnology Information GenBank and UNITE to establish species identity. Add soil to 1.5 milliliter microcentrifuge tubes containing one milliliter of Sabouraud dextrose broth and incubate at 50 degrees Celsius for two to four days.
To transfer the mycelia from the soil inoculated broth onto malt extract agar plates, first, identify the soil inoculums that have visible mycelial growth add the Sabouraud dextrose broth to air boundary. Then use sterilized wooden plain tip applicator sticks to transfer the mycelia to the center of a malt extract agar plate and incubate these malt extract agar plate set 37 degrees Celsius for three days. For the selection of mycelia, with Aspergillus fumigatus morphological properties, identify the mold colonies that have characteristic green swayed-like growth.
Inside a biosafety cabinet for level two organisms, harvest conidia or mycelia by scraping the surface once using sterilized wooden plane tip applicator sticks or an inoculation loop and transfer it to the center of a malt extract agar plate by streaking onto the agar for single colonies. Incubate the plates at 37 degrees Celsius for two days. Using a sterile application stick or inoculation loop, subculture a single colony generated onto malt extract agar by streaking the colony once.
Spread the harvested spores into the center of the plate. Prepare a sterile 30%glycerol solution to harvest the Aspergillus fumigatus spores or mycelia for culture storage. After the two days of incubation of the agar plates at 37 degrees Celsius, aspirate one milliliter of the 30%glycerol solution using a pipette and disperse it onto the Aspergillus fumigatus colony.
For phenotypic identification of Aspergillus fumigatus strains, aspirate 10 microliters of the mycelial and spore stocks to add in 990 microliters of water. Vortex this diluted spore suspension and dispense 10 microliters of the suspension onto a standard microscope slide. Then using a compound microscope at 400X magnification view the suspension and locate conidiophores.
Compare the observed conidiophore morphology with Aspergillus fumigatus conidiophore morphology. To determine the correct fragment size for each of the nine micro-satellite loci, use software capable of fragment analysis. Retrieve the raw data obtained from capillary electrophoresis and score the fragment sizes based on the largest peak using the fragmented analysis software.
Next, convert the fragment sizes to repeat numbers for each of the nine low use the fragment sizes of the repeat numbers of the reference strain Af293. From 3, 826 soil samples collected from 53 locations in nine countries, 1, 473 yeast strains were isolated. The best incubation temperature for each country was determined based on its mean annual temperature.
In rarefaction analysis for each country, the Shannon diversity index was used as a measure of soil yeast diversity. The resulting rarefaction curves approached the saturation asymptote indicating that additional sampling was not likely to have yielded more yeast diversity. Mycelial growth on plates formed the green swayed morphology typical of Aspergillus fumigatus.
Other thermotolerant fungal species could be present within the same soil sample and grow the Aspergillus fumigatus on the same plate or by themselves. Aspergillus fumigatus conidiophore structure viewed and identified using light microscopy revealed that conidiophores have a ball on the stick morphology and conidiophores are uniseriate, where the phialides attached to the conidia chains are attached directly to the spherical vesicle. A chromatogram generated biseriate revealed the three channels visualizing the fragment lengths of Aspergillus fumigatus dinucleotide, trinucleotide, and tetranucleotide short tandem repeats loci.
A high quality minimum spanning network can be generated using the R script plot_poppr_msn or imsn. A discriminatory analysis of principle components is another method to visualize the genetic relationships among strains. Adding benomyl and chloramphenicol to growth media prevents mold and bacterial growth, respectively, and favors yeast selection.
Chloramphenicol also reduces fermentation in media, lowering gas buildup in incubating tubes. Performing metagenomics on soil samples will provide additional information on cultural diversity. Susceptible testing of A.fumigatus strains will identify the presence of resistance genotypes.
This protocol can be applied to other yeast and mold systems with little modification. It facilitated the investigation of cultural soil yeast diversity and its predictors on a global scale.
