Sampling took place in the Stockholm-Uppsala area of central Sweden during the interval of May 2023 to April 2024 (Table 1). The only exception was Marasmius oreades MW66 which originates from a mushroom which was collected, isolated as monokaryotic single basidiospore isolates in 2021, and subsequently stored at 4 °C. The sampling effort was designed to isolate strains from as many different fungal species as possible. Conservation was considered when mushrooms were sampled. We limited the impact on the organism and the environment by never sampling more than three mushrooms per site, and no threatened species was sampled (Naturvårdsverket 2016; IUCN 2023).

For the sampled wild strains, we applied either of two methods to obtain a pure mycelium culture (Suppl. material 1: fig. S1; Table 1). Culturing attempts were in all of these cases done within 48 hours of sampling. In the “tissue isolation method” (Suppl. material 1: fig. S1), the mushroom stipe was first surface sterilized by submergence for 10 seconds in 70% ethanol. Afterwards, inner stipe tissue was placed onto fresh potato dextrose agar (PDA, NutriSelect® Basic, Merck KGaA, Darmstadt, Germany; 4 g L^-1 potato extract, 20 g L^-1 dextrose, 15 g L^-1 agar) plates supplemented with 10 ml L^-1 Penicillin-Streptomycin (BioReagent, Sigma-Aldrich, Burlington, USA; 10’000 U ml^-1 penicillin, 10 mg ml^-1 streptomycin) and incubated at 20 °C without light in a MLR-351H incubator (Sanyo, Moriguchi, Japan). When hyphae emerged from the tissue, they were transferred onto a new PDA plate to evade contaminants. After several rounds of subculturing, we obtained a pure monohyphal culture by cutting off and transferring a single hyphal tip from the subculture mycelium growth front to a fresh plate. For the second method, the “spore print isolation method” (Suppl. material 1: fig. S1), mushroom caps were placed with the spore-bearing structure facing downwards onto a sterile petri dish (92 mm, polystyrene, Sarstedt, Nümbrecht, Germany). After 12–48 hours, the collected basidiospores were suspended in sterilized water, and 100 µl of the suspension were streaked onto a PDA plate with 10 ml L^-1 Penicillin-Streptomycin. The germinated spores were identified by eye under a stereo microscope and isolated by picking with a sterile needle and transferring to a fresh PDA plate. To create a pure dikaryotic culture, mycelium from two single-basidiospore monokaryons was transferred onto the same PDA plate. Their compatibility and hyphal fusion to a dikaryotic mycelium was confirmed by the observation of clamp connections, which is a common method to distinguish a dikaryon from a monokaryon in basidiomycetes (Miles and Chang 2004).

For M. oreades, we continued with the single-spore isolates as for the other strains with the spore print isolation method. The only ascomycete strains in this project, Morchella importuna Zebra-I and Zebra-II, were isolated from dry morel fruiting body tissue collected in May 2023. For both M. importuna strains, we revitalized the dry tissue by dipping it for 10 seconds in sterilized water, then surface sterilized it with 70% ethanol, and washed it with sterilized water three times for 10 seconds. The sterilized morel tissue was then further treated as other wild mushroom tissue in the above-mentioned tissue isolation method.

To ensure that the cultures were pure, and that the mycelium was from the mushroom origin, we amplified and sequenced the internal transcribed spacer region (ITS); the official barcoding marker for fungi (Schoch et al. 2012; Raja et al. 2017). For this purpose, we first grew the pure culture mycelium on PDA covered with cellophane, and harvested the mycelial tissue by scraping it off the plate with a sterile cell scraper. DNA was extracted from the tissue by using the Quick-DNA Fungal/Bacterial Miniprep kit (Zymo Research Corporation, Irvine, USA) and the extracted DNA was used to amplify the ITS sequence with ITS1 and ITS4 primers (White et al. 1990). The PCR reaction contained 1 μl DNA, 4 μl 5× Phusion HF Buffer, 0.4 μl 10 mM dNTP, 0.5 μl of each primer (20 μM), 0.6 μl DMSO, 0.2 μl Phusion Polymerase, and milliQ water to a final volume of 20 μl per reaction. The PCR program consisted of initial denaturation for 1 min at 98 °C; 35 cycles of 15 s at 98 °C, 30 s at 58 °C, and 45 s at 72 °C; and a 7 min extension phase at 72 °C.

PCR products were cleaned using the QIAquick PCR Purification kit (QIAGEN, Venlo, The Netherlands). Cleaned PCR products were sequenced by Eurofins Genomics Germany GmbH, using Sanger technology and the same primers as were used for amplification. The resulting chromatograms were visually inspected, and regions of low quality were removed. A consensus ITS-sequence was generated for each strain using BioEdit (Hall 1999). Sequences similar to the obtained ITS sequences were searched for in the NCBI GenBank NT database using BLASTN (Sayers et al. 2021). The result from the BLAST search was compared with the mushroom morphology-based species preliminary determination in the field, to check whether a mycelium from other than the target species was isolated. Finally, the newly generated ITS sequences were submitted to NCBI together with information on the strain (Table 1).

To test whether our experimental setup for growth and fruiting body formation was suitable, and for comparative purposes, we included six control strains from five species of basidiomycetes in our study (Coprinopsis cinerea, Flammulina velutipes, Pleurotus ostreatus, Pleurotus pulmonarius, and Schizophyllum commune; Suppl. material 2: table S1). These are all established species in mushroom production or research, and thus have documented cultivation protocols (Stamets 2000; Miles and Chang 2004; Kamada et al. 2010; Sánchez 2010; Kües et al. 2016; Grimm and Wösten 2018; Sakamoto 2018; Singh et al. 2021). The control strains were provided by research institutions or companies (Suppl. material 2: table S1).

We investigated the optimal growth temperature (OGT) by comparing mycelial growth on PDA plates incubated at different temperatures, and identified OGT as a temperature, or a range of temperatures, that resulted in significantly higher growth rates compared to both higher and lower temperatures. First, we tested mycelial growth of each strain on PDA at 20, 25, and 30 °C. Based on the result of this initial screen, we also, when needed, investigated growth at lower or higher temperatures (10, 15, or 35 °C) to identify the growth optimum.

For the inoculation, approximately 5 × 5 mm cubes of the pure cultures’ growth front were cut with a lancet-shaped dissection needle and placed in the center of fresh PDA plates. We inoculated three replicate plates per strain for each temperature, i.e. we used three biological replicates for each measurement. We marked the center of the inoculation point and three measurement directions (Fig. 1a), whereafter the plates were sealed with Parafilm®, stacked and placed in poly propylene boxes (40 × 30 × 18 cm, Orthex Sweden AB, Tingsryd, Sweden) together with a glass beaker filled with 300 ml of water to avoid dry conditions, and incubated at the designated temperature.

Mycelial growth was assessed with the radial mycelial growth rate. To minimize the effect of inoculum size and to enter the phase of linear growth, we made a second marking to indicate the starting of the assay when the mycelial growth front on the petri dish had expanded at least three millimeters from the inoculum (Fig. 1b; Brancato and Golding 1953; Taniwaki et al. 2006). A third marking was made after the growth front moved at least another five millimeters outwards (Fig. 1c). The distances between the second and the third marking were measured and averaged over the three directions. We divided this average distance by the number of days to get the radial growth rate for each plate, i.e. each biological replicate.

Figure 1. 

Marking of the mycelial growth front on the plate bottom a plate after inoculation, with marking of inoculation center with three previously defined measuring directions (black) b second marking (green) showing mycelium front after initial growth c third marking (red) showing mycelium front at end of experiment.

The statistical analysis was performed in R 4.1.1 (R Core Team 2021). The growth rate data separated by strain were tested for non-normality with the Shapiro-Wilk test (Shapiro and Wilk 1965). An insignificant Shapiro-Wilk test with the confidence level of 95% indicated that the growth rate was normally distributed, and for these strains, we performed a linear model and analysis of variance (ANOVA) to infer differences in growth between each temperature. The model was then tested for significant between-temperature differences with the Tukey multiple comparison of means test (Tukey 1959). We plotted the results with significance letter grouping using the multcompView package in R (Graves et al. 2023). For strains for which the Shapiro-Wilk test indicated non-normal data distribution, we chose a cluster analysis to infer the OGT. The cluster analysis was done with the kmeans and silhouette function of the cluster package (Maechler et al. 2023). The best fitting cluster number was determined by comparing average silhouette coefficients for each possible cluster number. To find the OGT, we then plotted and analyzed the clusters visually.

To investigate the conditions suitable for fruiting of the strains, we investigated both the formation of primordia, i.e. the very early developmental stage of mushrooms, and the formation of mature mushrooms. In this study we focused on fruiting body formation of basidiomycetes that form fruiting bodies directly from the mycelium, and thus the ascomycete Morchella importuna wild strains were not included in the fruiting experiments. For all basidiomycete strains, we first investigated primordial formation when grown on PDA and the basidiomycete strains sampled before 2024 (Table 1) were also investigated when grown on straw. In addition to this, we investigated primordia and fruiting body formation using customized protocols for individual strains.

The PDA approach was performed with the triplicates of each temperature from the mycelial growth phase. For the approach with straw, all basidiomycete strains were grown in duplicates at their OGT. We hydrated straw pellets (Svamphuset, Stockholm, Sweden) with 200 ml deionized water per 100 g pellets before sterilization. 9 g of the straw substrate were filled into petri dishes. The inoculation and cultivation conditions were the same as for the mycelial growth on PDA.

We chose the customized protocols with specific substrates (Table 2) based on the species’ ecology. Autoclaved substrates were filled into sterilized pipette tip boxes (13 × 9.5 × 6 cm, Sarstedt, Nümbrecht, Germany) and then sealed with micropore tape (Micropore, 3M, Saint Paul, USA). Per tip box, we used either 60.9 g sowing soil (Hasselfors Garden AB, Örebro, Sweden), 22.5 g alder chips (Millamore Premium, Harjumaa, Estonia), 43.5 g birch pellets (Svamphuset, Stockholm, Sweden) soaked with 2 ml deionized water per gram pellets, or 69 g rye flakes (Ekologiska Rågflingor, Axfood AB, Stockholm, Sweden). For this experiment, taking an exploratory approach, only one replicate was used. Inoculation and cultivation conditions were the same as in the other approaches.

All three approaches had the same change to fruiting conditions regarding temperature and light. The temperature was shifted down 5 °C after mycelial growth to induce fruiting initiation (Stamets 2000; Ford et al. 2015; Sakamoto 2018). After growing the mycelia in the dark, they were exposed to light at either 500 or 1’500 lux for fruiting (Perkins 1969; Ford et al. 2015; Sakamoto 2018). We chose a 12 h day and 12 h night regime and white light (Kamada et al. 2010; Wang et al. 2020). The cultures were switched to fruiting conditions when the mycelium was 0.5–1 mm away from the edge (Kües et al. 2016). The last condition switched for the fruiting phase, which only applied to the PDA and straw plates, was to remove the Parafilm® sealing to increase gas exchange (Kinugawa et al. 1994; Sakamoto 2018).

After switching plates or boxes to fruiting conditions, we observed on a daily basis whether primordia formation was present or not. As soon as primordia were visible, we documented the number of days since switching from mycelial growth to fruiting conditions. In case the primordia developed into mushrooms, we harvested them, dried them for 24 h at 70 °C, and weighed the total dry mass. This resulted in the three measures: 1) primordia presence, 2) fruiting time, and 3) mushroom dry mass. As not enough replicates were tested for each strain and condition, we could not perform a relevant statistical analysis.

To maintain the strains, we used the established glycerol cryopreservation method (Linde et al. 2018). We added sterile glycerol (MP Biomedicals, Santa Ana, USA) to sterile ddH2O up to a 15% (v/v) solution. Mycelial cultures on PDA were cut to 3 × 3 mm cubes and five cubes were added to each tube. The cryotubes were then stored at -80 °C. Our newly isolated wild strains were also submitted to the Westerdijk Fungal Biodiversity Institute (CBS) culture collection (Table 1).

Of 33 mushrooms initially used for isolation, 17 resulted in isolated monohyphal cultures. Contamination by bacteria and different mold-forming fungi caused the exclusion of some isolation attempts. Molds were detected by the dust-like appearance of the often colored asexual spores (conidia) that are formed, and by a much faster growth on PDA compared to the target mushroom-forming species. From the ITS sequence analysis, all 17 wild strains were identified with high confidence (i.e., over 99% identity of the ITS sequence with a single species reported in GenBank) (Table 1), and the preliminary morphological species determination was confirmed in all cases. The distinct mycelium and mushroom characteristics of the 17 newly isolated wild strains are shown in Fig. 2.

Figure 2. 

Mycelia on PDA (left) and mushrooms (right) of the 15 basidiomycete wild strains a Agaricus arvensis MW41 b Armillaria borealis MW40 c Armillaria borealis MW82 d Coprinus comatus MW21 e Hericium coralloides MW80 f Hypholoma lateritium MW49 g Lycoperdon perlatum MW73 h Macrolepiota procera MW90 i Marasmius oreades MW66 j Paralepista flaccida MW6 k Pleurotus ostreatus MW97 l Pleurotus pulmonarius MW44 m Schizophyllum commune KK98 n Schizophyllum commune MW99 o Stropharia aeruginosa MW56. Photos taken by Mario Walthert.

To find their optimal growth temperatures (OGT), the radial mycelial growth rate was measured at 20, 25, and 30 °C, and for most strains also at higher or lower temperatures (Fig. 3). Significant letter grouping from the Tukey test revealed the OGT for 15 strains. The remaining seven strains showed non-normal distributed growth rate data (Suppl. material 2: table S2). Therefore, their OGTs were determined with the cluster analysis (Fig. 4). Overall, we found a considerable variation between the growth rate of the newly isolated wild strains when grown at different temperatures (Fig. 3 and Suppl. material 2: table S3). The growth rates ranged from 0 mm d^-1 (SD = 0) for S. aeruginosa MW56 at 30 °C up to 9.22 mm d^-1 (SD = 0.22) for S. commune MW99 at 30 °C. In tendency, the control strains showed higher growth rates than the wild strains, particularly at 30 °C. However, the four wild strains from species also included as control strains and the only ascomycete, M. importuna, also showed fast growth and preference for warmer temperatures. Nevertheless, most wild strains grew better at 25 °C or even 20 °C (Fig. 3; Suppl. material 2: table S3).

Many strains grew faster at increasing temperatures up to certain optimal temperature, before the growth rate dropped sharply (Fig. 3; Suppl. material 2: table S3). For example, this was observed very clearly in H. coralloides MW80 and P. flaccida MW6. Focusing on the OGT, 9 out of 16 tested wild strains had their OGT at 25 °C (Figs 3, 4). Four wild strains grew better at 20 °C, and the three wild strains sampled in spring 2024 had their OGT at 30 °C. Several strains did show optimal growth over a range of multiple temperatures (Figs 3, 4). Most broad from the wild strains were H. coralloides MW80 and S. aeruginosa MW56 with an OGT range from 15 °C to 25 °C.

Figure 3. 

Average radial mycelial growth rates of all strains at tested temperatures. The strains were tested at a subset of 10, 15, 20, 25, 30, and 35 °C to find the optimal growth temperature (OGT). n/d stands for no data, i.e. temperatures that were not measured. Bars represent the average growth rate, while error bars represent the standard deviation from three replicate plates. Grouping letters based on Tukey multiple comparison of means (CL = 95%) are shown for the normal distributed strains.

Figure 4. 

Silhouette cluster analysis for growth rates of all strains with non-normal distributed data. For each strain, indicated with species name and strain number, there are two graphs. The optimal cluster number analysis on the left shows the average silhouette width as a measure of fit for each tested number of clusters (k). The cluster graph on the right shows all data points and to which cluster they belong. Different clusters are shown by colors.

As mentioned before, the fruiting phase of this study was not statistically analyzed because not all conditions and strains were tested with a substantial replicate number. Nevertheless, the three measures of primordia presence, fruiting initiation time, and mushroom dry mass indicated trends for different strains. The control strains indicated that the fruiting conditions in this project enabled primordia and mushroom development (Table 3; Suppl. material 1: fig. S2). In tendency, faster-growing control and wild strains also developed primordia more often. From the wild strains, the fast-growers P. pulmonarius MW44 (Fig. 5a), S. commune KK98 (Fig. 5b), and S. commune MW99 (Fig. 5c) developed fruiting bodies on PDA. Additionally, A. arvensis MW41, L. perlatum MW73, and M. oreades MW66 did show the growth of some hyphal aggregates. However, it was not clear whether these structures were primordia, so these plates were scored as unsuccessful fruiting. None of the other wild strains showed any development at any of the tested conditions (Suppl. material 2: table S4).

Looking at the strains forming fruiting bodies on PDA, we did not observe a clear tendency for temperature and light conditions for primordia presence, fruiting initiation time, or mushroom dry mass. The formation rather depended on a strain-level, for example, P. pulmonarius MW44 indicated that it rather fruits at 1’500 lux than 500 lux (Table 3). The strains producing primordia needed between 8 and 41 days to initiate them. The two S. commune wild strains KK98 and MW99 were the fastest, particularly at the lowest tested temperature of 15 °C. Furthermore, P. pulmonarius MW44 was also rather fast in comparison with the control strains. P. pulmonarius MW44 was the only wild strain where the primordia developed into mushrooms, here defined as primordia that grew larger than 10 mm (Miles and Chang 2004). Its mushroom dry mass was in tendency higher at lower temperatures (Table 3).

Figure 5. 

Primordia and mushroom development of wild strains. PDA plates: a Pleurotus pulmonarius MW44 b Schizophyllum commune KK98 c Schizophyllum commune MW99. Plates with straw substrate: d Pleurotus pulmonarius MW44. Boxes with birch substrate: e Hericium coralloides MW80 f Pleurotus pulmonarius MW44.

Similar to the assay from growth on PDA, the control strains showed that the chosen fruiting conditions were also enabling fruiting body formation on straw. Especially Coprinopsis cinerea AmutBmut underwent its full life cycle from inoculation to mature mushrooms within a week (Suppl. material 1: fig. S2). From the wild strains, P. pulmonarius MW44 produced coral-like mushrooms (Fig. 5d). All the other tested strains, that is all basidiomycetes, did not form fruiting bodies on straw. Some strains such as L. perlatum MW73 did not fully colonize the straw substrate and were thus not even switched to fruiting conditions.

The customized approach to fruiting consisted of testing strains of interest on sowing soil, rye flakes, alder chips, and birch pellets (Table 2). All strains tested on sowing soil colonized the substrate fully, but none developed fruiting bodies. No success resulted from rye flakes and alder wood chips. The mycelial cubes dried out within 48 h on both of these substrates and no growth was observed. The last substrate, birch pellets, was the most fruitful. All tested strains grew into the substrate, however to a varying extent. Full colonization of the birch substrate was reached by P. pulmonarius MW44 within three weeks, and by H. lateritium MW49 and H. coralloides MW80 within five weeks. From those strains, only H. lateritium MW49 did not develop fruiting bodies. P. pulmonarius MW44 produced primordia after two weeks, which developed into mushrooms (Fig. 5f). P. pulmonarius MW44 mushrooms developed only a small cap. Furthermore, fruiting body development in the H. coralloides strain MW80 was observed for the first time in this study. Its primordia grew after one week close to the tip box edge. They formed spiky structures after three more weeks and developed into the iconic and spectacular mushroom form (Fig. 5e) after another four weeks.

The authors have declared that no competing interests exist.

No ethical statement was reported.

This study was supported by the Department of Biology Education and by The Bergianus foundation/The Royal Swedish Academy of Sciences.

Conceptualization: MW, MHT, and HJ; Lab work and data analysis: MW; Writing – original draft: MW; Writing – review and editing: MW, MHT, and HJ; All authors read and approved the final manuscript.

Hanna Johannesson https://orcid.org/0000-0001-6359-9856

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.