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.
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.
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 specific profiles of individual organisms through downstream diversity and functional analyses due to the availability of pure fungal cultures.
Despite rarely exceeding thousands of cells per gram of soil, yeasts, broadly defined as unicellular fungi, are essential decomposers and food sources for other soil dwellers4,5. In fact, yeasts may be the predominant soil fungi in cold biospheres such as continental Antarctica6,7. Soil is also a primary reservoir of medically relevant yeasts that cause serious opportunistic infections in humans and other mammals8. Despite morphological similarities, yeast species are phylogenetically diverse and occur among filamentous fungi in two major phyla, Ascomycota and Basidiomycota, within the fungal kingdom9. Yeasts lack a defining DNA signature at the fungal barcoding gene, the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster10, making them indistinguishable from other fungi in metagenomics investigations and thus necessitating the use of culture-dependent methods to isolate yeast species.
The protocol below was implemented to characterize soil yeast communities of nine countries and identify global trends and patterns in soil yeast diversity9,11,12. Metagenomics approaches are of limited use when studying targeted groups of organisms such as yeasts2,3. Due to their phylogenetic diversity, yeasts cannot be distinguished from other fungi based on DNA sequence alone. Thus, studying yeast populations requires the continued use of culture-dependent isolation. However, culturing is often significantly more time-consuming and requires more personnel to perform the experiments. Therefore, the protocol has been optimized and streamlined for faster processing with limited personnel. The main advantage of culturing is that the yeast species identified are living yeasts and not dead ones, and thus are more likely to be true soil dwellers rather than transient cells present in the soils. It has been estimated that approximately 40% of fungal DNA in soil are either contaminants from other environments, extracellular, or come from cells that are no longer intact, causing high-throughput sequencing approaches to overestimate fungal richness by as much as 55%13. Culture-dependent isolation can readily confirm yeast species identity with the added benefit of securing pure culture to be used in downstream analyses. Indeed, pure cultures of 44 putative new yeast species were identified using this soil isolation protocol that allowed the use of a range of methods to study their taxonomic and functional properties in detail14.
The protocol below can also be used to isolate molds present within soil, such as A. fumigatus. Aspergillus fumigatus is a thermophilic and saprophytic mold with a wide, global distribution in soil15. It has been isolated from numerous clinical and non-clinical environments. Non-clinical sampling commonly includes air, organic debris (compost, saw dust, tulip bulb waste), and soil (agricultural, garden, and natural soils)16,17,18,19. Aspergillus fumigatus is a human opportunistic pathogen causing a range of infections collectively termed aspergillosis, affecting over 8 million people worldwide16,20. Approximately 300,000 people around the globe suffer from invasive aspergillosis, which is the most severe form of aspergillosis16. Depending on factors such as the patient population, site of infection, and efficacy of antifungal therapy, mortality rate can be as high as 90%. Over the past several decades, resistance to antifungal therapies has increased, requiring global surveillance efforts in both clinical and environmental populations to track these resistance genotypes21,22,23. Given its ability to grow at temperatures upward of 50 °C, this temperature can be exploited to select for A. fumigatus isolates from soil using culture-dependent methods. Aspergillus fumigatus isolates are commonly genotyped at nine highly polymorphic short tandem repeat (STR) loci, shown to have high discriminatory power between strains24. These STR genotypes can be compared to other previously surveyed populations to track the spread of A. fumigatus genotypes, including drug-resistance genes, around the world.
Below we describe a protocol for the speedy isolation of yeasts and A. fumigatus from soil samples in a culture-dependent manner. Depending on the amount of soil obtained per sample, the soil samples can be shared between the two protocols. In comparison to similar methods that isolate yeast and A. fumigatus from soil, this protocol uses 10x less soil per isolate obtained. Studies attempting to isolate A. fumigatus from soil require between 1 and 2 g of soil per isolate, whereas this protocol requires only 0.1-0.2 g of soil18,19,25. This protocol utilizes smaller plastics and containers that facilitate its high-throughput design. Therefore, a larger number of samples can be processed using less space for equipment such as incubators and roller drums. Soil samples can be fully processed to obtain isolates in as little as 7 days. This protocol has been optimized to allow processing of up to 150-200 samples per day per person.
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
2. Isolation of Aspergillus fumigatus from soil
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 the slower yeast growth at 14 °C, soil samples from Iceland were incubated on a roller drum for an additional 48 h (96-120 h total). Following 2-3 days of incubation on solid medium, microbial growth was visible on plates for all samples (Figure 1A). A randomly selected colony for each yeast morphology present on a plate was streaked for single colonies on fresh plates (Figure 1B).
The rate of successful yeast isolation differed between countries (Table 1). For example, 97 yeast strains were cultured from 562 Saudi Arabian soil samples (17.3%), whereas 261 yeasts were cultured from 300 Canadian soil samples (87%). A rarefaction analysis should be performed to determine if sufficient soil sampling has been conducted to obtain accurate estimates of the true yeast diversity in sampled locations. An example rarefaction analysis for each country was preformed where the Shannon diversity index was used as a measure of soil yeast diversity (Figure 2). The resulting rarefaction curves approached the saturation asymptote indicating that additional sampling was not likely to have yielded more yeast diversity.
Sequencing of the ITS region revealed that the 1,473 yeast isolates can be categorized into 134 distinct species. This included 90 known species and 44 potentially novel species. We applied a mixed-effects model to identify predictors of global culturable soil yeast diversity, as quantified by the Shannon diversity index31. In this model, mean annual precipitation, mean annual temperature, elevation, and distance to equator of the sampling locations were fitted as fixed effects, while sampling country was set as a random effect9. This model identified mean annual precipitation to be significantly correlated with the Shannon diversity index (p = 0.012), while no significant correlations were found between other predictor variables and the Shannon diversity index9.
To compare this culture-based protocol to culture-independent methods, we compared these findings to a previous study by Tedersoo and colleagues that investigated global diversity of soil fungi using metagenomics2. Tedersoo and colleagues performed high-throughput sequencing of the ITS region on DNA directly extracted from soil samples of 39 countries, four of which, namely Cameroon, Canada, China, and New Zealand, were also sampled in this study. We performed BLAST searches to identify ITS sequences present in both datasets32, and found 26% of the ITS sequences had a significant match (>98.41% nucleotide identity) in the metagenomics dataset. However, <3% matched a fungal sequence from the same country, while the remaining 23% matched a fungal sequence found in a different country9. We were more successful than the metagenomics study in annotating the ITS sequences with yeast species identity. Tedersoo and colleagues reported a total of 50,589 fungal operational taxonomic units (OTUs), with the number of yeast OTUs not known. Of these fungal OTUs, 33% were merely annotated as environmental_sequence (724, 1.4%), uncultured_soil_fungus (2,405, 4.8%), uncultured_ectomycorrhizal_fungus (1,407, 2.8%), or uncultured_fungus (11,898, 23.5%).
Aspergillus fumigatus isolation from soil
After 3 days of soil incubation at 50 °C in 1 mL of SDB in a microcentrifuge tube, mycelia may be found growing within the SDB, on the SDB to air boundary, and/or on the inside walls. Aspergillus fumigatus mycelia are typically found growing on the SDB to air boundary but can also be harvested from just below the SDB surface. After this step, the mycelia are transferred to MEA and grown for 3 days at 37 °C. Mycelial growth on plates may only form the green suede morphology typical of A. fumigatus (Figure 3A). More commonly, other thermotolerant molds may be present within the same soil and grow with A. fumigatus on the same plate or by themselves (Figure3B–D). Aspergillus fumigatus isolation rates may vary between geographic locations, similar to what has been found for soil yeasts. For example, within Vancouver, Canada, 251 isolates were obtained through this method from 540 soil samples (46.5%) (unpublished results). By contrast, within Cameroon, 51 A. fumigatus isolates were harvested from 495 soil samples (10.3%)33.
A. fumigatus conidiophore structure can be viewed and identified using light microscopy. Conidiophores have a ball on stick morphology (Figure 4). Additionally, A. fumigatus conidiophores are uniseriate, where the phialides, attached to the conidia chains, are attached directly to the spherical vesicle. In other Aspergillus species, conidiophores are biseriate, where the phialides are connected to metulae attached to the vesicle.
Several software programs are available to perform fragment analysis of the raw data from capillary electrophoresis, converting the capillary electrophoresis spectrum to fragment sizes. Figure 5 is a chromatogram generated by the program Osiris. The three channels visualizing the fragment lengths of A. fumigatus dinucleotide (2A, 2B, and 2C), trinucleotide (3A, 3B, and 3C), and tetranucleotide (4A, 4B, and 4C) STR loci are shown. In addition, PCR artifacts that occur if the sample DNA concentration during PCR is too high are also shown. The highest peaks of each color represent the fragment sizes of the three STR loci in this plot.
The genetic variation present between A. fumigatus STR genotypes can be visualized using the R package poppr. The R script bruvo.msn creates a pairwise Bruvo's genetic distances matrix between each pair of genotypes. This matrix is then used to generate a minimum spanning network (MSN). A high-quality MSN can then be generated using the R script plot_poppr_msn or imsn (Figure 6). A discriminatory analysis of principal components (DAPC) is another method to visualize the genetic relationships among strains (Figure 7). The R script dapc in the R package adegenet is used to perform DAPC and can be used with known or unknown group priors. With unknown group priors, it uses K-means clustering to identify the likely number of groups of individuals.
Figure 1: Yeast isolation from soil samples. (A) Microbial growth is visible on solid agar following 2-3 days of incubation. Yeast colonies can be seen interspersed among other fungal/bacterial colonies. One representative yeast colony for each morphological type on a plate is selected to streak for single colonies. (B) Three streaks are performed to obtain single colonies of yeast isolates. One plate was used to obtain each soil isolate. Please click here to view a larger version of this figure.
Figure 2: Rarefaction analysis of soil sampling. In each of the nine sampled countries, soil yeast diversity, as quantified by the Shannon diversity index, approaches saturation as the number of soil sample increases. This figure was adapted from 9. Please click here to view a larger version of this figure.
Figure 3: Aspergillus fumigatus morphology on Sabouraud dextroseagar. (A) Green suede-like A. fumigatus growth morphology. (B–D) A. fumigatus growth with other thermophilic molds present within the soil sample. A. fumigatus conidiation is visibly reduced in B and D. Please click here to view a larger version of this figure.
Figure 4: Photo of an Aspergillus fumigatus conidiophore under a light microscope. Scale bar = 10 µm. Please click here to view a larger version of this figure.
Figure 5: Osiris output of nine microsatellite loci from an Aspergillus fumigatus strain. The outputs correspond to the (A) Dinucleotide (2A, 2B, and 2C), (B) trinucleotide (3A, 3B, and 3C), and (C) tetranucleotide (4A, 4B, and 4C) STR loci. Forward primer 5` fluorescent labels are: A = 6-FAM; B = HEX; C = ATTO550. Multiplex reaction products were diluted 30x prior to capillary electrophoresis. The LIZ600 dye standard was used during capillary electrophoresis. Raw data was analyzed using the peak analysis software Osiris identifying potential peaks between 60 and 400 bp. Several PCR artifacts are off-scale peaks that cause fluorescence bleeding, stutter peaks, and N-1 peaks (C). Abbreviations: 6-FAM = 6-carboxyfluorescein; HEX = hexachlorofluorescein; STR = short tandem repeats; RFU = relative fluorescence units; BPS = base pairs. Please click here to view a larger version of this figure.
Figure 6: Minimum-spanning network showing the genetic relationship between MLGs of A. fumigatus from Iceland and Northwest Territories in Canada. Each node represents one MLG, where node size corresponds to the number of strains for each MLG. Nodes that are more genetically similar have darker and thicker edges, whereas nodes genetically distant have lighter and thinner edges. This figure was adapted from 34. Abbreviations: ISL = Iceland; NWT = Northwest Territories in Canada; MLG = multilocus genotype. Please click here to view a larger version of this figure.
Figure 7: Genetic clustering using Discriminant Analysis of Principal Components (DAPC) of Iceland, NWT, Eurasian, North American, and Oceanian A. fumigatus populations. Isolates were genotyped at nine microsatellite loci and clone-corrected, totaling 1,703 unique multilocus genotypes. Genotypes were colored according to geographic origins. This figure was adapted from 34. Abbreviations: DAPC = discriminant analysis of principal components; NWT = Northwest Territories; ISL = Iceland; CMR = Cameroon; CAN = Hamilton, Ontario, Canada; BEL = Belgium; FRA = France; DEU = Germany; IND = India; NLD = Netherlands; NOR = Norway; NZL = New Zealand; ESP = Spain; CHE = Switzerland; USA = United States. Please click here to view a larger version of this figure.
Country | Incubation temperature (°C) | Soil samples | Yeast isolates | Known species/ Novel species |
Cameroon | 30 | 493 | 110 | 10/9 |
Canada | 23 | 300 | 261 | 34/12 |
China | 23 | 340 | 230 | 23/5 |
Costa Rica | 30 | 388 | 95 | 20/2 |
France | 23 | 327 | 175 | 12/2 |
Iceland | 14 | 316 | 211 | 11/0 |
New Zealand | 23 | 610 | 155 | 14/4 |
Peru | 23 | 490 | 139 | 30/9 |
Saudi Arabia | 30 | 562 | 97 | 8/1 |
Total | 3826 | 1473 | 90/44 |
Table 1: Soil yeast isolation from nine countries in six continents. Incubation temperature for soil samples from each country was determined based on its mean annual temperature. Results presented here are adapted from 9, 12.
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 laboratory settings using standard microbiology and cell culturing equipment. However, when isolating yeast or molds from international soil, extra precautionary measures are required. All non-disposable plastic equipment, such as plastic trays and cell spreaders, should be sterilized by submersion in 10% bleach, followed by autoclaving. The used bench coat should be sealed in a plastic bag and sterilized in an autoclave prior to discarding.
To note, the number and identity of yeasts isolated using the protocol above can be limited by the incubating conditions and media used. For example, 24 h incubation in the roller drum can favor the isolation of fast-growing aerobic yeasts over slow-growing species. In addition, the use of nutrient-rich YEPD medium for yeast isolation may have excluded yeast species with different nutrient requirements and preferences. Furthermore, most locations sampled here experience seasonal variations in temperature and other climatic conditions throughout the year. If time and resources permit, the same soil samples can be processed under a range of different temperatures to allow for isolation of a broader range of yeasts that inhabit these soils.
While culture-independent methods are crucial for elucidating large-scale patterns in environmental fungal diversity, they fail to be sufficiently informative for targeted groups of fungi such as yeasts. The use of culture-dependent isolation allowed for a comprehensive investigation of global soil yeast diversity and its predictors9. While the metagenomics study conducted by Tedersoo and colleagues identified global predictors and patterns in soil fungal diversity, the extent to which these findings apply specifically to yeast diversity could not be elucidated2. Tedersoo and colleagues identified mean annual precipitation as a major climatic driver of soil fungal diversity worldwide2. This study found a similar correlation between mean annual precipitation and culturable yeast diversity in global soils, highlighting that culture-dependent methods can complement and expand on findings of high-throughput sequencing approaches9.
The addition of chloramphenicol is an important step in the preparation of both solid and liquid media to prevent fungal growth interference by bacteria. The addition of chloramphenicol to liquid media is crucial as the lack of antibiotics will allow the bacteria present in the soil to ferment. This will lead to the production of gases that will forcibly open microcentrifuge tubes or 13 mL culture tubes used during soil incubation in the protocol. If bacterial contamination in chloramphenicol containing agar/broth is an issue, chloramphenicol may be substituted or supplemented with stronger antibiotics. When isolating A. fumigatus from soil, SD broth may be substituted with a Tween 20 solution (0.2 M NaCl with 1% Tween 20) to help suspend the hydrophobic A. fumigatus conidia35,36. Following suspension, 100 µL can be plated onto SD agar and incubated at 50 °C.
Aspergillus fumigatus grows quickly and sporulates abundantly when incubated alone on both SD agar and MEA media between 22 °C and 50 °C. However, depending on what other thermotolerant species are present in the soil sample, A. fumigatus growth and sporulation may be hindered (Figure 3A,D). Under such a situation, one to several subculturing steps may be required to obtain a pure colony for subsequent harvest and characterization.
The fragment analysis of A. fumigatus strains in Figure 5 was conducted using the program Osiris. Several PCR artifacts have been highlighted in Figure 5C and are discussed in detail by De Valk et al.30. B-1 stutter peaks that have shorter repeat values are caused by strand slippage during the synthesis of the antisense strand by the DNA polymerase. N-1 peaks occur if the DNA concentration is too high during PCR. The recommended DNA concentration is 0.1 ng as stated in the protocol above. Another artifact is dye bleeding that can be remediated using fluorescent labels with non-overlapping absorbance wavelengths. For example, the fluorescent labels 6-FAM, HEX, and ATTO550, as well as the LIZ600 dye standard, generate distinct fragment peaks (Figure 5). Lastly, to prevent off-scale peaks, dilute each PCR reaction (in this case, it was ~50x dilution) prior to capillary electrophoresis. To further reduce fluorescence of the unused forward primers in the reaction mixture, halve the forward primer concentrations relative to the reverse primers when creating the master mix.
The high throughput and conservative nature of this protocol allows for a relatively quick and easy acquisition of many yeasts and molds from soils. However, there are two main limitations present with the sampling methodology. First, the morphological characteristics of yeast colonies grown from soil were used to select for unique species. These were then subcultured and used for species identification via ITS sequencing. This was done to maximize the representation of yeast species present. However, yeast species that shared similar or identical morphologies to the selected colonies may fail to be subcultured. Second, during A. fumigatus isolation, as only one individual colony is selected per soil sample, the presence of multiple individuals within the soil sample will be missed. This may lead to an underrepresentation of the true genetic diversity present within the sample population, as unique genotypes present within the same soil sample will not be collected. To mitigate this issue, during the streak for single colony step following the first culturing on solid media, several single colonies can be collected to obtain additional genotypes. The small volume of soil used per sample helps minimize this sampling limitation when compared to methods that used larger quantities of soil per sample.
The use of this high-throughput and labor-efficient protocol for the isolation of yeast and mold will increase the number of individuals within soil population while using less effort per sample. The increase in statistical power will provide a better picture of culturable yeast communities within the soil and help characterize soil populations of A. fumigatus.
The authors have nothing to disclose.
This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada (Grant No. ALLRP 570780-2021) and McMaster University.
1.5 mL microcentrifuge tube | Sarstedt Inc | 72.690.001 | |
Benomyl powder | Toronto Research Chemicals | B161380 | |
Chloramphenicol powder | Sigma-Aldrich | SKU: C0378-5G | |
Dextrose | Sigma-Aldrich | SKU: D9434-500G | |
Fragment Analysis Software | NCBI's Osiris | https://www.ncbi.nlm.nih.gov/osiris/ | |
ITS sequence database | NCBI GenBank | https://www.ncbi.nlm.nih.gov/genbank/ | |
ITS sequence database | UNITE | https://unite.ut.ee/ | |
Peptone | Sigma-Aldrich | SKU: P5905-500G | |
Reusable cell spreaders | Fisher Scientific | 08-100-12 | |
Sterile 10 cm diameter Petri dishes | Sarstedt Inc | 83.3902 | |
Sterile 13 mL culture tubes | Sarstedt Inc | 62.515.006 | |
Wooden plain-tipped applicator sticks | Fisher Scientific | 23-400-112 | |
Yeast extract | Sigma-Aldrich | SKU: Y1625-250G |