Computational protocol: Cd and Zn interactions and toxicity in ectomycorrhizal basidiomycetes in axenic culture

Similar protocols

Protocol publication

[…] Toxicity trials were performed in vitro using five ECM species originated from non-polluted environments: Hebeloma subsaponaceum (from a Boreal Forest, Norway); H. cylindrosporum (from under pine trees, France); H. crustuliniforme (from Sitka spruce, Brown Earth); Scleroderma sp. (woodlands, Western Australia) and Austroboletus occidentalis (Western Australia), a species recently found to be a non-colonizing fungal partner (). These species were selected from our in-house collection due to their growth rates observed previously in agar medium. Methods were based on a previous study by . Four circular plugs (1 mm) were cut out from the edges of actively growing colonies (five weeks old) and transferred to Petri dishes with 25 ml of Melin-Norkrans liquid medium (MMN). The medium composition was: 6.51 mM NH4NO3, 0.57 mM MgSO4 ⋅ 7H2O, 0.23 mM CaCl2, 0.015 mM ZnSO4, 0.3 mM Thiamine, 5.55 mM d-glucose, 2 mM KH2PO4, 0.035 mM Ferric EDTA; pH was adjusted to 5.5. No Zn (ZnSO4) was added to the initial MMN medium used for the Zn treatments, as this metal was added later to make up the desired range of concentrations.Cd and Zn concentrations were added via CdCl2 and ZnSO4 solutions to the final medium, and the final concentrations were (in mg L−1): 0; 1; 3; 9; 27; 81; 243 for the Cd treatments, and 0; 1; 30; 90; 270; 810; 2,430 for the Zn treatments. Such concentrations were selected based on similar toxicity experiments with mycorrhizal fungi found in the literature (; ; ; ; ; ).The fungal cultures were incubated in the dark at 20 °C for 30 days, each treatment had four replicates. The mycelial mats were then removed from the medium, placed on small aluminum envelopes (weighed previously) and oven-dried overnight at 60 °C. The dry weight (DW) was assessed gravimetrically. The Tolerance Index (TI%) was used to express the tolerance results (), calculated by the equation: (1)TI%=DWtreatedDWcontrol×100. In which DW is the dry weight obtained from the fungal biomass.Statistical analysis was performed on the dry weight data using STATISTICA 12®. To attain normal distribution (Shapiro–Wilk), box-cox transformation was applied. However, the data did not meet the assumption of homogeneity of variances (Levene’s test). Thus, analysis of variance was carried out using Welch’s test (), followed by Dunnett’s test to determine the LOAEC values (Lowest Observed Adverse Effects Concentration), which also does not require equal variances (). The Dunnett’s for Zn toxicity considered the treatment of 1 mg L−1 Zn as the control. [...] To verify the effect of Zn in preventing Cd toxicity in ECM fungi, a second experiment was carried out using the same methods described above, except no basal Zn was added to the basic MMN medium in all treatments, and was added later to make up the desired range of concentrations; growth period of 21 days. However, because H. cylindrosporum had lower or similar performance as H. subsaponaceum, the former was excluded from this experiment. In this case, ECM species were exposed to Cd and Zn together, with concentrations added in different combinations: 0, 1 and 9 mg L−1 for Cd, and 0, 1, 9 and 30 mg L−1 for Zn. Therefore, this assay was comprised of 12 treatments (Cd × Zn: 0 × 0, 0 × 1, 0 × 9, 0 × 30, 1 × 0, 1 × 1, 1 × 9, 1 × 30, 9 × 0, 9 × 1, 9 × 9, 9 × 30 mg L−1).Relative dry weight was calculated with , and ANOVA followed by Tukey’s test were performed to verify significant differences among the Zn treatments (0; 1; 9 and 30 mg L−1). For attaining normality and homoscedasticity in two variables (1 mg L−1 Cd in H. crustuliniforme and 0 mg L−1 Cd in Scleroderma sp.), data were transformed by the equation: 1∕x.Due to the high Cd toxicity observed, this experiment was repeated subsequently with only Scleroderma sp. and Hebeloma subsaponaceum, but using another range of concentrations (0; 1; 9 mg L−1 Cd and 0; 30; 60; 120 mg L−1 Zn) and two types of MMN media, a solid medium containing 2% agar, and a liquid medium as described previously, with four replicates. Plates were incubated in the dark, at 20 ± 2 °C for 30 days. By the end of the growth period, treatments with solid media were measured for radial growth (a mean between vertical and horizontal diameters, in centimeters). After which the agar was cut and removed from the plates and melted in a microwave in short 15 s burst for no more than one minute in total (); the mycelium was removed and blotted dry with absorbent paper until it was free of all agar medium, the mycelium was then washed with deionized water, oven-dried overnight (60 °C) and weighed. Liquid media treatments were handled as described previously. Statistical analyses were performed following the same steps as the previous experiments. Contour plots were achieved by linear interpolation (using SigmaPlot®; Systat Software Inc., San Jose, CA, USA) of the fungal Tolerance Indexes (TI%, but in this case considering 100% as the treatment with the highest biomass production: i.e., Cd × Zn (0 × 30 mg L−1 in liquid cultures and Cd × Zn (1 × 30 mg L−1) in solid cultures); using 12 Zn × Cd co-ordinates, based on publications by and . […]

Pipeline specifications

Software tools Statistica, SigmaPlot
Application Miscellaneous
Chemicals Cadmium, Zinc