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Three temperate forest soil types with mean annual precipitation ranging from 1,500 to >5,000 mm were chosen. The first soil sampled was a loamy Inceptisol (Soil Survey Staff, 2014) which was derived from intrusive granodiorite rocks in Nahuelbuta National Park (37°47′S, 72°12′W) (Bernhard et al., 2018). This soil was formed by ancient Araucaria araucana and Nothofagus pumilio forests. The second soil sampled in Alerce Costero National Park in the Coastal range under Nothofagus spp. and Fitzroya cupressoides (40°22′S 73°38′W) was a loamy clay Ultisol derived from metamorphic mica-schist materials with illite-kaolinite as dominant clays (Luzio et al., 2003). The final soil was collected in the Andes Cordillera from a primary temperate rainforest of Nothofagus betuloides in Puyehue National Park (40°47′S, 72°12′W) as Andisol derived from recent volcanic ash and basaltic scoria deposits with a high content of allophane, imogolite, and ferrihydrite (Neculman et al., 2013).

Four composite soil samples were extracted from the top Ah mineral horizon (0–15 cm) at each site after the litter layer was removed. The samples were cleaned in the laboratory to remove coarse organic debris and separated into two parts: one was stored at 4°C for a microcosm experiment and microbial analyses, and the other was air-dried for further physico-chemical analyses.

The pH and electrical conductivity were measured in an aliquot of soil in a 1:2.5 suspension of soil:water. TOC-VCSH (Shimadzu, Kyoto, Japan) was used to determine soil organic C, and total N was determined using Kjeldahl distillation (VELP, Usmate, Italy). The aluminum, iron and manganese were extracted by oxalate; Al_o, Fe_o and Mn_o respectively, using 0.2 M ammonium oxalate at pH 3 (Sadzawka et al., 2006). The aluminium, iron and Mn complexed with SOM, extracted by pyrophosphate, Al_p, Fe_p and Mn_p, respectively were obtained using a solution of 0.1 M sodium pyrophosphate (Van Reeuwijk, 2002). To identify exchangeable, crystalline, and complexed-SOM metals in soil samples, dithionite-citrate-bicarbonate (Fe_d) was used. Atomic absorption spectroscopy (Perkin Elmer 3110, Waltham, Massachusetts, United States) was used to determine Fe and Mn concentrations at 248.3 nm for Fe and 279.5 nm for Mn using a nitrous oxide acetylene flame. Cation exchange capacity (CEC) and nutrient characterization were carried out as Sadzawka et al. (2006) suggested. The total Fe concentration in 100 mg of dry soil was determined by adding 0.9 mL 0.28 M hydroxylamine hydrochloride and 1 mL 0.28 M HCl (Stookey, 1970). Approximately 100 μL of the extract was added to 4 mL of ferrozine color reagent (1 g ferrozine in 6.5 M ammonium acetate solution). Iron (II) concentration was determined in 100 mg of soil by adding 1 mL of 0.5 M HCl and vigorously shaking. Ferrozine reagent was added as previously described, and absorbance at 562 nm (ferrozine-Fe(II) complex standard) was measured in a UV spectrophotometer (Lovley and Phillips, 1987). The difference between total Fe and extractable Fe(II)-HCl forms was used to calculate the Fe(III) (oxyhydr) oxide content.

In the same areas where soil samples were taken, white rot fungi were isolated from small fragments of decayed wood or fruiting body pieces. The fragments were placed in sterile tubes and stored at 4°C until they were analyzed. Small fragments of fungi fruiting bodies or decayed wood colonized by fungi were incubated on acidified glucose malt extract agar plates (15 g/L agar, 3.5 g/L malt extract, 10 g/L glucose, pH 5.5). Under aseptic conditions, pure mycelial cultures were obtained, and strains were identified using ITS sequencing. The DNA from each strain was extracted using the E.Z.N.A.® SP Fungal DNA Mini Kit D5524-01 (Omega, Bio-Tek, Norcross, GA, United States). The ITS1-5.8S—ITS2 rDNA was amplified using ITS1 and ITS4 primers (White et al., 1990). PCR was carried out in a total volume of 25 μL with 0.1 mM dNTPs, 0.1 mM of each primer, 5 U of Taq DNA polymerase, and the supplied reaction buffer (Promega Inc., Seoul, Korea). An ABI PRISM 3730 l DNA Analyzer System was used to sequence the PCR products (Macrogen, Seoul, Korea). The nucleotide sequences were compared in the GenBank database (Horisawa et al., 2013). Schizophyllum commune was identified in Nahuebuta soil, Stereum hirsutum in Alerce Costero soil, and Galerina patagonica in Puyehue soil (see Supplementary Table S1, for more information). In each microcosm, the isolates were inoculated at a final concentration of 3 × 10⁸ CFU gr soil⁻¹ in 100 μL of sterilized water.

Under anaerobic conditions, the contribution of and ROS production and consumption from soils at different pH levels was determined in a sterilized portion of soil at field capacity (80%) from each soil (oxygen-free by N₂ purge). In a destructive sampling design, soil samples were incubated in 120 mL serum bottles for 36 h at 12°C to monitor CO₂ evolution, ROS production, and enzymatic activity. To remove the microbial resistant structures, the soil samples were sterilized for 20 min at 121°C (in an autoclave) on four consecutive days. The soils were then fumigated with chloroform vapor for 24 h in a vacuum chamber (Trevors, 1996). Autoclaving was chosen because it has little effect on the structure of SOM, primarily causing changes in the carbohydrate and N-alkyl domains (Berns et al., 2008). Because it causes Fe reduction and oxidation, gamma radiation was avoided (Bank et al., 2008; Abedini et al., 2014; Sutherland et al., 2017). Gamma radiation also increased the bioavailability of Fe(III) (oxyhydr)oxide minerals, which helped in Fe(III) reduction (Brown et al., 2012). There were three treatments (WRF, Fenton reaction, and WRF + Fenton) and four replicates with four induced pH in each soil; 2.5, 3, 4, and the natural pH of each soil (3.6, 4.5, and 5.1 for Nahuelbuta, Alerce Costero, and Puyehue, respectively). A H₂O₂:Fe(II) ratio was also added to induce the Fenton reaction, as previously reported by Merino et al. (2020). This was achieved by adding 120–143 mL of H₂O₂ (0.1 M) and 1.29 g Fe(II) kg⁻¹ soil as FeCl₂ to the soil (Sigma Aldrich, United States). A total of 144 serum bottles with septums for gas sampling were used. CO₂ was collected at 0.5, 4, 8, 12, 24, and 36 days of incubation. Approximately 10 mL of CO₂ gas sample was extracted and injected into a gas chromatograph with Flame Ionization Detector (GC/FID) (Thermo Fisher ScientificTM, Austin, TX) with a 30 mm DB1-MS column and selected ion mode at each sampling time. At the end of the incubation period, 72 microcosm bottles were harvested, and soil was homogenized and quickly subsampled for ROS analysis and enzymatic activity at each sampling.

Superoxide anion (O₂•-) production was measured in 0.5 g of soil, which was extracted with 12 mL of potassium phosphate buffer (PPB, 65 mM, pH = 7.8) and centrifuged at 10,000×g for 15 min at 4°C. The supernatant (5 mL) was combined with 0.9 mL of PPB, 1 mL of sulfanilamide (17 mM), 1 mL of hydroxylamine hydrochloride (10 mM), and 1 mL of naphthylamine (7 mM) and incubated in the dark for 20 min at 25°C. At 530 nm, the absorbance of the samples was measured (Greenwald, 2018). Hydrogen peroxide (H₂O₂) was determined using the iodometric titration method (Velikova et al., 2000). The concentration of H₂O₂ in mg L⁻¹ was determined using a calibration curve as the standard. Although iodometric titration is less precise than permanganate titration, it produces fewer interferences with SOM (Liang and He, 2018; Merino et al., 2020). Page et al. (2013) previously used terephthalic acid (TPA) in soil to measure •OH production. Approximately 200 l of soil water (1:2.5 soil:water suspension) was injected into 3 mL aliquots of deoxygenated water for the blank (in triplicate) and 3 mL of TPA for the test (in triplicate) (1.5 mM, in triplicate). TPA-treated soil suspension was incubated for 24 h in the dark to oxidize into hydroxyterephthalic acid (hTPA). Finally, the sample was filtered at 0.22 m and the fluorescence was measured in a microplate reader at λex = 310 nm, λem = 425 nm (Synergy HT, Biotek). The fluorescence intensity was compared to a standard curve with 2-hydroxyterephthalic acid (0, 2, 20, 40, and 80 nM) (hTPA, Sigma Aldrich).

Exoenzymatic activities were spectrophotometrically measured under pH variations after 36 h of incubation. The formation of Mn(III)-tartrate complex during the oxidation of 0.1 mM MnSO₄ in 100 mM tartrate buffer at pH 5 was used to determine manganese peroxidase (MnP) activity. At 238 nm, manganese peroxidase was measured spectrophotometrically (Xu et al., 2018). The oxidation of 2,2′-azino-bis (3-ethylthiazoline-6-sulfonate) (ABTS) (2.5 mM) to its cation radical in 100 mM tartrate buffer at pH 5 was followed by dye-decolorizing peroxidase (DyP) activity. At 418 nm, dye-decolorizing peroxidase was measured spectrophotometrically (Salvachúa et al., 2013). Veratryl alcohol (2 mM) was used as a substrate for LiP during the oxidation of 0.21 mL of 50 mM sodium tartrate buffer at pH 2.5. At 310 nm, lignin peroxidase was measured. The enzymatic activity tests were all performed in the presence of 0.1 mM H₂O₂. All oxidative enzymatic activities were measured in units (U) per milliliter (i.e., one millimole of substrate oxidized per minute). The measured H₂O₂ was normalized in relation to the treatment values in terms of representation.

The normal data distribution and variance homogeneity were tested for each treatment and soil type. One-way ANOVA was used for cumulative gas sampling (CO₂). Using a repeated-measures ANOVA test, the enzyme activity, ROS production, and Fenton contribution were tested over a 40-day incubation period. The last three variables measured were plotted as a six-sample time average. Duncan’s multiple range test was used for multiple comparison means because all ANOVA tests were significant at p 0.05. The RStudio software was used for all analyses (1.1.442).

The soils studied are formed from a variety of parent materials, including Granitic (Nahuelbuta), Metamorphic (Alerce Costero), and Volcanic-allophanic (Puyehue); their textures range from sandy to clay. The pH ranged from 3.6 to 5.1, with organic C levels ranging from 9.2 ± 0.1 to 11.4% ± 0.3. Natural H₂O₂ levels in soil range between 25.6 ± 0.7 and 33.7 ± 0.5 μM g⁻¹ soil (Nahuelbuta<Alerce Costero< Puyehue soils). Puyehue soil had the highest content of Al complexed with SOM (Al_p) (11 g kg⁻¹ soil ±1.5), while Nahuelbuta soil had the lowest (0.7 g kg⁻¹ soil ±0.1). The Al_o + 1/2 Fe_o > 2%, which indicates Andic properties <2, was found at higher levels of 3.8% in Puyehue soil. The Al_o symbol represents Al-oxides associated with amorphous structures, such as Alp with SOM. Puyehue soil had the highest value of 3.1 ± 0.2 g kg⁻¹ soil. The Puyehue soil had the lowest CEC (5.3 cmol(+) kg⁻¹), while Alerce Costero soil had the highest (19.7 cmol(+) kg⁻¹). The total Fe content was determined by combining the soluble ferrous iron Fe(II) and ferric iron Fe(III) forms, which ranged between 8.4 ± 0.5 and 11.8 ± 0.5 g kg⁻¹. Alerce Costero soil had a higher total Fe content than other soils (Table 1).

Over the course of 36 days, increasing patterns of CO₂ efflux were observed in all evaluated soils (Figures 1A–C). The highest values were found in Alerce Costero soil during anaerobic incubation of sterilized samples inoculated with WRF, at pH of 4.5 (217.45 mg C kg⁻¹) (Figure 1B). The lowest levels of C mineralization (82 mg C kg⁻¹) with the lowest pH 4 were found in Nahuelbuta soil (Figure 1A). Alerce Costero and Puyehue soils followed a similar pattern, with the highest CO₂ release occurring at the highest pH levels, 4.5 and 5.1, and the lowest at induced pH 2.5 (Figures 1B,C). Figure 1A depicts the inverse trends for Nahuelbuta soil. Furthermore, all the soils treated with WRF without Fenton, and soils treated without inoculum, showed a lower average rate values than the combined treatments (WRF + Fenton). Similarly, the lowest average rate of mineralization was observed in sterile soils treated with Fenton, followed by WRF without Fenton induction (Figures 1A,C).

After 36 days of incubation, the activity of LiP, MnP, and DyP was measured. When compared to non-induced Fenton groups, all WRF plus induced-Fenton reactions increased proportionally to enzyme activity. The responses of enzyme activities to pH changes in the soils studied varied (Figure 2). Except for Puyehue soil, only the Nahuelbuta microcosm showed a decrease in activity as the pH increased, with the highest activity observed at pH 2.5 (Figures 2A,D,G). LiP, MnP, and DyP were one, two, and eight times higher, respectively, than at natural pH. The CO₂ released was distributed uniformly across the soil pH gradient. With the exception of Alerce Costero with LiP and Puyehue, the highest pH showed the lowest enzymatic activity and CO2 release after 36 days of incubation. LiP activity in Alerce Costero soil displayed a distinct pattern of activity when compared to MnP and DyP. The activity of the LiP increased as the pH increased from acidic to moderate acidic, with higher activity observed at pH (4.5) (Figure 2B). MnP and DyP, on the other hand, significantly reduced their activity from pH 2.5 to 4.5, while CO₂ increased at day 36 (Figures 2E,H). The pH variation in Puyehue soil was observed to have a non-linear distribution. Lip, MnP, and DyP activity were found to be lower in acidic conditions (pH 2.5), with the highest activity at pH 3 (Figures 2C,F,I), followed by a decrease in activity at natural pH (4.5).

pH had an effect on O₂•- production, and the variation was significant between treatments and soils, ranging from 0.5 to 1.3 μmol g⁻¹ soil (Figures 3A,D,G). The highest values in Nahuelbuta soil were obtained at higher pH, in the Fenton + WRF group, and in the independent treatments (Figures 3A–C). This pattern was also observed in Alerce Costero soil, where pH 2.5 and 3 had the highest levels of O₂•-, decreasing to 4 and 4.5 (Figure 3A). In comparison to the other soils, the values in Puyehue soil were inverse to the trend (Figure 3G). The lowest levels of O₂•- were found in the most acidic values (2.5), but at pH 3, the maximum production was reached, and as the pH increased, the O₂•- content decreased (Figure 3G). As the pH changed, the production of H₂O₂ varied without a clear trend (Figures 3B,C,H). The highest value of 3.4 μmol g⁻¹ of soil was discovered in Alerce Costero at a natural pH of 4.5 (Figure 3E). Puyehue had the lowest H₂O₂ production of 1.3 μmol g⁻¹ soil and the lowest pH of 2.5 (Figure 3H). These values take into account the subtraction of the added H₂O₂ to initiate the Fenton H₂O₂/Fe(II) reaction (10,1 ratio). When compared to the other reactive oxygen species, the production of hydroxyl radicals (•OH) was the highest (ROS) (Figures 3C,F,I). Similar to H₂O₂ production, there was no discernible trend in •OH production, with the highest value of 4.3 μmol g⁻¹ soil being recorded at the lowest pH 2.5 in Nahuelbuta (Figure 3C). The almost undetectable •OH 0.7 μmol g⁻¹ soil was recorded in Puyehue with the lowest pH of 2.5 (Figure 3I). The WRF + Fenton treatment produced the highest total ROS, followed by Fenton and the WRF treatments with lower contributions (Figure 3). Furthermore, the cumulative production of ROS (the sum of the values of each ROS in all soils) was found to be lowest for O₂•-, followed by •OH, and H₂O₂ had the highest abundance.

Figure 4 depicts the consumption of H₂O₂ by exoenzyme and Fenton reaction after 36 days of incubation due to their high oxidative content, allowing them to be used as a cofactor in catalysis and iron oxidation (Fenton). When WRF was added to Nahuelbuta soil, this increased H₂O₂ consumption between pH 2.5 and 3.6, but decreased at pH 4. The Fenton reaction, on the other hand, consumes more H₂O₂ at higher pH levels (pH 4) (Figure 4A). This was not the case with Alerce Costero soil. At moderate acidity pH 4.5 and acid condition (2.5), the exoenzyme consumes more H₂O₂, whereas consumption is similar but significantly reduced at pH 3 and 4. The Fenton reaction, on the other hand, exhibits higher consumption values between pH 3 and 4, but lower consumption values between pH 4.5 and 2.5 (Figure 4B). At pH 3.6, similar consumption values were observed in both exoenzyme and Fenton reactions, but not at the other pH tested. When the pH is raised, exoenzymes consume the least. At pH 2.5, the Fenton reaction produced the lowest values (Figure 4C).

The consumption of CO₂ and H₂O₂ was found to have a positive and significant relationship (Figures 5A–C). CO₂ production in WRF-treated soils was strongly related to peroxide consumption (p < 0.01, R2 > 0.85). Puyehue soil had a lower CO₂ evolution trend associated with H₂O₂ consumption, but the tendency was lower in comparison to Alerce Costero soil, which had a higher CO₂ production and H₂O₂ consumption at pH 3, with a steeper slope (Figures 5B,C).