Lead (Pb) tolerance in the ectomycorrhizal fungi Suillus brevipes and S. tomentosus

doi:10.21203/rs.3.rs-4876672/v1

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Lead (Pb) tolerance in the ectomycorrhizal fungi Suillus brevipes and S. tomentosus

https://doi.org/10.21203/rs.3.rs-4876672/v1

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Lead (Pb) is a highly toxic metal and a contaminant of many soils across the world. Some fungi are known to be Pb tolerant, persisting in environments with high Pb levels. Here we investigate Pb tolerance in Suillus brevipes and S. tomentosus, two widespread ectomycorrhizal fungal species in the American West where soil contamination due to mining is common. We conducted in vitro growth assays, exposing previously studied isolates to a range of Pb concentrations. We found S. tomentosus to be more Pb tolerant compared to S. brevipes and that isolates from the two species showed both high and low Pb tolerance. Surprisingly, there were no correlations between Pb tolerance and Pb soil concentration, isolate growth rate, or Zn tolerance. Pb tolerance was positively correlated with Cd tolerance in S. tomentosus. Our research contributes for understanding fungal metal tolerance and paves the way for future work addressing the mechanisms of Pb tolerance and the potential for using Suillus in the recovery of contaminated sites.

Suillus

heavy metal

stress

ectomycorrhizal adaptation

Soil lead (Pb) contamination is a global issue resulting from human activity, including mining, battery manufacturing, lead-based gasoline, and lead-based paint (Tian et al. 2019). Pb is a highly toxic cumulative contaminant that persists in the environment (Ma and Rao 1999; Naik and Dubey 2013; Gadd 1993). It has no biological role in and is toxic to cells even in trace amounts (Feng et al. 2023) due to its ability to block functional groups of biologically important molecules, induce conformational modifications, and denature/inactivate enzymes (Gadd 1993). Pb negatively affects fungal cells and reduces fungal growth and spore production (Pawlowska and Charvat 2004; Feng et al. 2023).

Despite Pb being highly toxic, some fungal species display tolerance to this metal and can persist in high Pb concentrations. Examples of fungal Pb tolerance include nematode-trapping Dactylellina ellipsosporum and Arthropbothrys oligospora (Mo et al. 2008), isolates of the endophyte genera Alternaria (Li et al. 2012) and Curvularia (Feng et al. 2023), ectomycorrhizal (ECM) Paxillus involutus (Marschner et al. 1999), Rhodotorula mucilaginosa and Saccharomyces cerevisiae yeasts (Chen et al. 2022; Chang and Leu 2011), and saprotrophic Pleurotus ostreatus (Wang et al. 2019), Aspergillus niger, and Penicillium oxalicum (Tian et al. 2019). Pb tolerance in fungi is associated with intracellular chelation, accumulation, and compartmentalization, as well as with Pb biomineralization into non-soluble form and cell wall sequestration often through the segregation of organic acids that react with the metal to form insoluble mineral crystals (Tian et al. 2019; Rhee et al. 2012; Fomina et al. 2005; Cheng et al. 2024; Wang et al. 2019).

Here, we investigate Pb tolerance in Suillus brevipes and S. tomentosus, two widespread ECM fungal species in the American West where Pb is a common soil contaminant. Suillus is a common temperate genus that forms ECM partnerships with conifers and is well-known for metal tolerance, including Cu, Cd, and Zn (Branco et al. 2022; Lofgren et al. 2024; Fletcher et al. 2024). Metal tolerance in Suillus is achieved through a combination of signaling and metal immobilization, detoxification, and sequestration (Bazzicalupo et al. 2020; Lofgren et al. 2024; Smith et al. 2024). Previous work by Fletcher et al. (2024) showed S. brevipes and S. tomentosus from the Colorado Rocky Mountains display Zn and Cd tolerance inter- and inter-specific variation, with S. brevipes being more tolerant to Cd but more sensitive to Zn and isolates in the two species showing a wide range of tolerance to these metals. In addition, S. brevipes and S. tomentosus Zn-tolerant isolates were not found to be tolerant to Cd and vice versa, suggesting these species activate distinct mechanisms to tolerate Zn and Cd. Surprisingly, and contrary to other Suillus species (Jan V. Colpaert et al. 2004; 2005; J. V. Colpaert and van Assche 1987), S. brevipes and S. tomentosus Zn and Cd tolerance were not correlated with soil metal content, however, Cd tolerance was negatively correlated with growth rate in S. tomentosus (Fletcher et al. 2024). In this species, faster-growing isolates were more sensitive to Cd presumably due to higher metal intake compared to slower-growing isolates.

We investigated Pb tolerance in S. brevipes and S. tomentosus isolates previously shown to have wide variation in Cd and Zn tolerance (Fletcher et al. 2024) by exposing them to a range of Pb concentrations in vitro. Based on previous research, we anticipated finding S. brevipes and S. tomentosus isolates to display variation in Pb tolerance. We also expected Pb tolerance to be positively correlated with Pb soil content and negatively correlated with growth rate. We found inter- and intra-specific Pb tolerance, but no correlations with soil Pb content or growth rate.

Field sites, isolate collection and identification

We investigated Pb tolerance in S. brevipes and S. tomentosus isolates previously studied by Fletcher et al. (2024). In summary, we obtained 40 S. brevipes and 33 S. tomentosus isolates between July 2021 and September 2022 (Table S1) from native conifer forests dominated by Pinus contorta and P. ponderosa in Colorado (USA) with a range of soil Pb, Zn, and Cd levels (Table S2). We selected sites based on proximity to disused mines and government superfund sites that likely experienced contamination through mining activity and metal leaching. Details on soil chemistry analyses and fungal culturing can be found in Fletcher et al. (2024). In summary, we obtained pure cultures from sporocarps in Fries medium amended with streptomycin and chloramphenicol at 23°C in the dark for 2 weeks (Lofgren et al. 2024). We collected mushrooms at least 10 m apart to increase the chance of sampling different genets. Successful isolates were then transferred to plain Fries medium, grown at 23°C in the dark for 2 weeks, and stored at 4°C. We deposited the dried fruitbodies at the Denver Botanic Garden Sam Mitchel Herbarium of Fungi (Table S1).

Metal tolerance assays

To test for S. tomentosus and S. brevipes Pb tolerance, we set up in vitro growth assays on Fries medium with increasing Pb concentrations (0, 1, 2, 3, 5 mM, and when warranted 7 and 9 mM). We added Pb as a nitrate salt (Pb(NO₃)₂) to the Fries stock solution and adjusted pH to 4.5 prior to autoclaving. We plated each isolate onto each concentration in triplicate and left to grow in the dark at 23℃. After two weeks, we measured isolate radius outwards from the point of original inoculation in four quadrants, for a total of twelve measurements per isolate per concentration. We used the drc package (v3.0-1) in RStudio (R version 4.2.2) to calculate isolate EC₅₀, the amount of Pb that reduces isolate growth by 50%. To compare the mean EC₅₀ of each species we used a two-sample t-test. To determine correlations between Pb tolerance and soil metal content, Zn tolerance, Cd tolerance, and growth rate, we used the ggscatter function within the ggpubr package (v0.4.0), using the stat_cor function to generate regression lines and respective equations, R² values, and p values.

Inter- and intra-specific variation in Pb tolerance

We found S. tomentosus (mean = 2.4 mM ± 8.8) was significantly more tolerant to Pb than S. brevipes (mean = 1.7 mM ± 9.7; p = 0.0034, Fig. 1A and Table S1). We also found Pb tolerance intraspecific variation in in both species, with isolates of S. brevipes displaying EC₅₀ values ranging from 38.9 µM to 6 mM and S. tomentosus isolates ranging from 1.3 mM to 4.5 mM.

No correlation between Pb tolerance and soil Pb concentration or growth rate

There was no correlation between S. brevipes and S. tomentosus Pb tolerance and the Pb concentration of the soil of origin (Fig. 1B). The most Pb-tolerant isolate was a S. brevipes isolate with an EC₅₀ of 6 mM that was collected from a relatively low Pb concentration site (Little Vasquez Creek, 19.3 mg/kg Pb). Notably, isolates from the most contaminated site (California Gulch; 91214 mg/kg Pb) displayed a range of tolerance levels, with EC₅₀ ranging from 1.2 to 4.4 mM. There was also no correlation between Pb tolerance and growth rate in either species (Fig. 1C).

Pb tolerance is correlated with Cd tolerance in S. tomentosus

We compared isolate Pb tolerance to their previously described Cd and Zn tolerance (Fletcher et al. 2024) and found a significant positive correlation between isolate Pb tolerance and Cd tolerance in S. tomentosus but not S. brevipes (Fig. 2A). There was no correlation between Pb and Zn tolerance in either species (Fig. 2B).

We investigated Pb tolerance in S. brevipes and S. tomentosus and found S. tomentosus is more Pb tolerant than S. brevipes and that isolates from each species showed both high and low Pb tolerance. Tolerance was not correlated to Pb soil concentration, isolate growth rate, or Zn tolerance, but was positively correlated with Cd tolerance only in S. tomentosus.

Inter- and intra-specific metal tolerance is well-known in Suillus. Previous studies showed Cu, Cd, and Zn tolerance across species and isolates and as documented here, Suillus isolates belonging to the same species displayed very different abilities to endure metal stress (Colpaert and van Assche 1987; Colpaert et al. 2000; Fletcher et al. 2024). These results suggest the existence of local adaptation, with the evolution of metal tolerance tied to fitness advantage under soil metal contamination. Under this scenario, one would expect metal sensitive isolates to be restricted to non-contaminated soils and metal tolerant isolates to contaminated soils.

Interestingly, different species and isolates with distinct metal tolerances have been detected the in the same field sites (Fletcher et al. 2024; Jan V. Colpaert et al. 2004; Blaudez et al. 2000; Fomina et al. 2005). In fact, metal tolerance is often but not always positively correlated to soil contamination (Jan V. Colpaert et al. 2005; J. V. Colpaert and van Assche 1987; Jan V. Colpaert et al. 2000). For example, Zn- and Cd- tolerant S. luteus, S. brevipes and S. tomentosus isolates can be found in non-contaminated soils and sensitive isolates in contaminated soils (Smith et al. 2024; Bazzicalupo et al. 2020; Fletcher et al. 2024). These findings reflect our S. brevipes and S. tomentosus Pb results and challenge the metal tolerance as local adaptation hypothesis. Explanations for the mismatch between metal tolerance and soil metal content include the existence of soil heterogeneity, with undetected localized pockets of low contamination that allow for sensitive isolates to persist, and the absence of fitness trade-offs associated with metal tolerance allowing metal tolerant isolates to persist in non-contaminated soils (Smith et al. 2024; Fletcher et al. 2024; Jan V. Colpaert et al. 2004; Blaudez, Botton, and Chalot 2000; Blaudez et al. 2000; Adriaensen et al. 2005). In fact, a population genomics study in S. luteus reported metal tolerance is a highly polygenic trait and no evidence of population structure associated with metal contamination. Isolates from contaminated and non-contaminated sites belonged to the same population and shared considerable genetic variation despite showing differentiation in genes involved in metal homeostasis (Bazzicalupo et al. 2020). These results show that soil contamination does not necessarily lead to population differentiation and suggest metal tolerance can result from ancestral variation kept through mating across the population. Further genetic research will clarify whether S. brevipes and S. tomentosus Pb tolerance follow this same trend.

Metal tolerance can be associated with trade-offs, with metal tolerant individuals showing distinct phenotypes such as morphological features, nutrient requirements, or growth (Colpaert et al. 2005) that can have fitness consequences. For example, Cd tolerance is associated with slow growth in Saccharomyces cerevisiae (Chang and Leu 2011) and S. tomentosus (Fletcher et al. 2024). However, as we show here, such trade-off is not widespread in Suillus. There are other examples of metal tolerance being decoupled from growth in the genus (Fletcher et al. 2024; Colpaert et al. 2005), indicating that metal tolerance is a phenotype that is beneficial under metal exposure and does not always impose costs in other conditions.

Fungal metal tolerance is achieved through metal exclusion, immobilization, and detoxification (Branco et al. 2022; Bazzicalupo et al. 2020; Smith et al. 2024) and it would be reasonable to expect that the same mechanisms would be involved in tolerating different metals. However, previous work in Suillus showed no correlation between Zn and Cd tolerance (Fletcher et al. 2024; Jan V. Colpaert et al. 2000), indicating that tolerance to these metals is likely achieved through different mechanisms. Our S. brevipes findings are in line with this previous work. However, we found a positive correlation between Cd and Pb tolerance in S. tomentosus, suggesting that contrary to other Suillus this species relies on similar mechanisms to tolerate Cd and Pb. These are two toxic metals with no biological functions, and it is possible that S. tomentosus uses the same pathways and genes to regulate and mitigate Cd and Pb toxicity. These are likely to differ from the mechanisms regulating micronutrients such as Zn that are required for cell metabolism. It is interesting that there was no correlation between Cd and Pb tolerance in S. brevipes. Further research will certainly clarify the mechanisms of tolerance to different metals on these two species.

In summary, we have unveiled wide variation in Pb tolerance in Suillus and contribute for understanding the patterns of metal tolerance in this genus. Fungal metal responses are complex, and more work is needed to understand how S. brevipes and S. tomentosus tolerate Pb. Future experiments should measure Pb tissue accumulation, as well as investigate the genetic and physiological bases of Pb tolerance. In addition, it would be interesting to investigate the role of Suillus Pb tolerance on their ectomycorrhizal plant partners, as the genus is known to protect against metal toxicity and there is the potential for using Suillus as a tool for restoring Pb contaminated sites.

Funding

This research was supported by National Science Foundation IOS-PBI 2029168 awarded to S.B. W.L. and M.J. were supported by the University of Colorado Denver EURēCA! program.

Authors and Affiliations

Department of Integrative Biology, University of Colorado Denver, Denver, CO, USA

William Leary, Matthew Johnson, Jessica Fletcher, Sara Branco

Contributions

SB & WL designed the study, WL, MJ & JF performed research, JF analyzed data, WL, MJ & SB wrote the paper. All authors reviewed the manuscript.

Corresponding author

Correspondence to Sara Branco.

Conflicts of interest

The authors declare no conflict of interest.

Funding

This research was supported by National Science Foundation IOS-PBI 2029168 awarded to S.B. W.L. and M.J. were supported by the University of Colorado Denver EURēCA! program.

Author Contribution

SB & WL designed the study, WL, MJ & JF performed research, JF analyzed data, WL, MJ & SB wrote the paper. All authors reviewed the manuscript.

Acknowledgement

We thank Cody Lewis, Annie Schauster, Maham Malik, Alex Smith and the North American Mycological Association for helping with collecting, isolating, and culturing Suillus, and Andrew Wilson (Denver Botanic Gardens) for assistance with voucher submission. We also thank the Mountain Research Station/Niwot Ridge (University of Colorado Boulder, CO, USA), the United States Department of the Interior Bureau of Land Management (Gunnison and Royal Gorge Field Offices), the United States Department of Agriculture Forest Service, and the Clear Creek Open Space Commission, for granting permission to collect field samples.

Data Availability

Data is provided within the manuscript or supplementary information files.

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You are reading this latest preprint version

Lead (Pb) tolerance in the ectomycorrhizal fungi Suillus brevipes and S. tomentosus

Status:

Version 1

Abstract

Figures

Introduction

Methods

Field sites, isolate collection and identification

Metal tolerance assays

Results

Inter- and intra-specific variation in Pb tolerance

No correlation between Pb tolerance and soil Pb concentration or growth rate

Pb tolerance is correlated with Cd tolerance in S. tomentosus

Discussion

Declarations

Funding

Conflicts of interest

Funding

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1