Accumulation of aluminium and arsenic in Cenococcum geophilum sclerotia from forest soil affected by mining smoke

ABSTRACT Aluminium toxicity may increase under low soil pH, and an increase in arsenic concentration in the soil may inhibit inorganic P uptake and influence fungal growth. This study investigated metals/metalloids accumulation in Cenococcum geophilum sclerotia collected from highly polluted forest soils affected by mining activities and smoke hazards. The Al and As concentrations in sclerotia were 11,700 ± 823 and 10.0 ± 1.29 mg kg−1, respectively. TOF-SIMS ion mapping confirmed the predominant presence of Al as Al oxalate, acetate, and hydroxides, and the segregation of As showed higher intensities for organic As ions than inorganic As. Ion fragments of Al and As compounds were widely observed in sclerotial medulla and were generally elevated towards the central part coexisting with phosphoric acid ions. The mechanism of Al and As accumulation has been discussed to involve two biotransformation pathways in terms of sclerotial development using 14C dating. Because the sclerotia were significantly older than the historical smoke hazard and Al and As concentrations in sclerotia were high regardless of age, their accumulation were more likely to be promoted in the mature stage. This study provides insight into the contribution of ectomycorrhizal fungi and their sclerotia to soil ecosystems under metals/metalloids toxicity.

Aluminium influences the growth of ectomycorrhizal fungi in acidic soils.For example, Thompson and Medve [12] demonstrated that the order of fungi for tolerance to Al toxicity in an acidic environment was Suillus luteus > Pisolithius tinctorius > Cenococcum graniforme > Thelephora terrestris.In addition, the growth of Cenococcum graniforme significantly reduced at various concentrations of Al sulfate [12].Aluminium has been reported to be the predominant element in the sclerotia of C. geophilum formed in acidic soils with a content of 1.4% [13].In addition, the Al concentration in Cenococcum geophilum sclerotia has been suggested to depend on the remaining period of the grains in the soil and reduce the toxicity of exchangeable Al for host plants [14].
Previous studies have demonstrated that inorganic arsenic (iAs), dimethylarsinate (DMA), methylarsonate (MA), and arsenobetaine are present in the fruiting bodies of various fungal species, often at significant concentrations [15][16][17][18][19][20].One of the latest studies focusing on the ECM fungus Hebeloma cylindrosporum discussed the molecular mechanisms responsible for As detoxification [21].Interestingly, several studies have shown that Cenococcum geophilum has distinct abilities, such as tolerance to high heavy metal concentrations, compared to other ECM fungi [22][23][24].The prolific occurrence of Cenococcum geophilum in heavy metal-contaminated areas has been reported [11,25].The tolerance of Cenococcum geophilum to AsO 4 3− when grown in liquid culture under the lowest PO 4 3− supply, has also been reported.This behaviour is in contrast to those of basidiomycetous fungi such as Hebeloma crustuliniforme and Suillus variegatus [26].Future studies should consider the life cycle of Cenococcum geophilum along with the formation of sclerotia to understand the mechanisms underlying its survival.The objective of this study was to determine the presence and existing state of Al and As species inside sclerotia and to acquire knowledge on the mechanisms of Al and As accumulation in Cenococcum geophilum sclerotia in the soil environment.Regarding studies on arsenic speciation in solid-state samples, X-ray absorption near-edge spectroscopy [27] and combinations of HPLC/ICP MS and ion-exchange chromatography instrumental neutron activation analysis have been performed [15].Time-of-flight secondary ion mass spectrometry (TOF-SIMS) analysis was used to study Hg and As species (AsO 2 − , AsO 3 − , AsO 4 H 2 − , and KAsO 4 H − ) in oral bacteria [28].In this study, TOF-SIMS was used to examine the states of Al and As in Cenococcum geophilum sclerotium grains.This technique is expected to determine the enrichment of chemical species in the fungal sclerotia.High-resolution spectrometry may enhance our knowledge of the distribution of highly toxic metalloids/metals in the sclerotia collected from contaminated soils, thereby contributing to the development of effective remediation strategies.

Sample collection
In the late nineteenth century, smoke pollution from copper mines became a serious health and environmental issue at copper-smelting sites in Japan.Through primitive mining and smelting operations, sulfurous anhydride was discharged into the air together with metal-containing dust from refinery smoke.The Ashio copper mine, Japan's main domestic copper mine, operated for approximately 400 years until its closure in 1973.It was also the site of Japan's first major pollution disaster in the 1880s, during the Meiji era.Smoking hazards affected the environment between 1897 and 1901.Leeward areas experienced forest damage and heavy metal pollution of the soil [29][30][31].The reservoir sediments in the watershed of the Ashio mine retain high concentrations of heavy metals [32].
In this study, we collected soil samples in a Tsuga diversifolia forest with Sasa nipponnica forest floor vegetation at Hangetsu (HAN), Tochigi prefecture, Japan (36°42 ′ 30.0 ′′ N, 139°28 ′ 34.6 ′′ E, 1680 m ASL), 6-km northward of the Ashio Copper Mine area, in September, 2020 (Figure 1(a-c)).The forest comprised dead Tsuga trees (Figure 2(a)).Surface soil samples were randomly collected from 15 points within a 30 m × 30 m area using a soil sampler (φ 80 mm × 100 mm), as shown in Figure 2(b).Based on their melanic and spherical morphological features (Figure 2(c)), approximately 128 grains were collected from 500 g of soil in the study area for analysis.The grains were washed with distilled water and then air-dried at 25 °C for 5 h.
The 15 sample points were divided into four groups (HAN-A, HAN-B, HAN-C, and HAN-D) to obtain sufficient amounts of sclerotia (>20 mg per group) for elemental analysis (Figure 1(c)).

Soil analyses
Soil texture and colour, evaluated using a Munsell soil colour chart, were examined in the field.Soil pH values for air-dried samples (<2 mm) were measured in a suspension mixture of soil with a 2.5 times volume of solution (H 2 O and 1M KCl) using the glass electrode method (S20, Mettler Toledo, Columbus, OH).The total carbon and nitrogen contents (g kg −1 ) were examined using the dry combustion method (NC-22F, Sumika Chemical Analysis Service Ltd., Tokyo, Japan).
Elemental composition analysis was performed using wavelength-dispersive X-ray fluorescence (AxiosmAX, Malvern Panalytical Ltd., Almelo, Netherlands) under the same measurement conditions as those described by Nyamsanjaa et al. [14].The analytical precision and accuracy were calibrated against a certified reference material (Nepheline syenite LNS (CGL 006); the Central Geological Laboratory, Ulaanbaatar, Mongolia).

Elemental analysis
Eight samples, four, three, and one from HAN-A, HAN-B, and HAN-C, respectively, were used for water content and elemental analyses.Subsequently, the oven-dried sclerotium grains were ground to a fine powder using an agate mortar to measure the TC and TN content using an NC analyser.
Approximately 20 mg of powdered sclerotia samples were collected from HAN-A, HAN-B, HAN-C, and HAN-D.The digestion procedure used to produce a transparent solution for elemental analysis was the same as that described by Nyamsanjaa et al. [14].A blank sample was prepared in the same manner as for preparing the sclerotia.The concentrations of elements in the sclerotia were determined using ICP-OES (iCAP 6000, Thermo Fisher Scientific) and ICP-MS (XSERIES II, Thermo Fisher Scientific).The measurement conditions for the ICP-OES and ICP-MS analyses in this study were based on those reported by Nyamsanjaa et al. [14].

Line scan analysis by scanning electron microscopy and energy dispersive Xray spectroscopy
Scanning electron microscopy (SEM) observations were conducted at magnifications of up to 250 × using one grain from the HAN-A site to examine the interior of the sclerotium sample.The sample was sliced in half to provide one half for SEM with energy-dispersive X-ray spectroscopy (SEM-EDS) and the other half for TOF-SIMS analysis, as discussed in the following section.The internal structure of the sclerotia sample was observed using a digital microscope (VHX-1000; Keyence, Osaka, Japan) (Figure 2(d)).The surface of the sample section was mounted on double-sided carbon tape, and the elemental composition of the sclerotia was determined using a thermal field emission scanning electron microscope (JSM-IT800; JEOL, Tokyo, Japan) and EDS (JED-2300 Analysis Station, JEOL) at an accelerating voltage of 15 kV in the low-vacuum mode.The scan distance was 500 µm to obtain the cross-section from the outer part comprising the rind, cortex, and medulla to the interior hollow part.

Ion fragment mapping by time-of-flight secondary ion mass spectrometry
The remaining half of the sclerotia sample used for SEM-EDS analysis was subjected to TOF-SIMS experiments using a TOF-SIMS V 5-100-AD (ION-TOF Japan, Yokohama, Japan) instrument equipped with a Bi3 LMIG/Mn liquid metal primary ion source.Pulsed 60-keV primary ions were used in spectrometry mode for acquisition.The Bi 3 + + -LMIG (liquid metal ion gun) was set in the high-current bunched mode with a target current of 0.2 pA.Both the positively and negatively charged species ejected from the surface were detected using a time-of-flight mass analyser.The surface concentration of each ion species was characterised using the emission intensity (number of recorded counts) of each ion.Spectra were collected using both positive and negative secondary ion polarities from four separate 500 µm × 500 µm areas on each sample surface (256 × 256-pixel density).Four ions and 22 fragment ions were selected for analysis, comprising 12 positive and 14 negative ions and fragment ions (Table 1). 14C dating Approximately 3 mg of each sclerotium sample was powdered and used for accelerator mass spectrometry analysis at the Institute of Accelerator Analysis Ltd. (Shirakawa, Japan) to measure the 14 C, 13 C, and 12 C concentrations.The number of grains used for the measurements is listed in Table 2. Six measurements were performed.The 14 C ages were calculated according to the procedures of Stuiver and Polach [33], and the calendar age was calibrated using the Bomb13 calibration curve [34,35] and OxCal calibration program (Version 4.4) [36].

Statistical analysis
Unpaired t-tests were performed using GraphPad Prism (Version 10.0.2, GraphPad Software, San Diego, CA, USA) to the compare concentrations of elements in the soil and sclerotia with those reported by Nyamsanjaa et al. [14].

Soil properties
The soil colour and texture were 5YR 2/2 (brownish black) and SiC (silty clay), respectively.The soil pH in H 2 O and KCl were 4.39 ± 0.12 and 3.56 ± 0.09, respectively.The TC content, TN content, and ratio of C to N were 159 ± 6.72 g kg −1 , 9.5 ± 0.28 g kg −1 and 16.6 ± 0.43, respectively.

Elemental concentrations in sclerotia and soil
The A comparison of the minor element concentrations in the sclerotia between HAN and non-mining sites in central and northern Japan is shown in Figure 3(b).A previous study by Nyamsanjaa et al. [14] reported concentrations of metals in sclerotia from volcanic ash soils in non-mining areas; these were recorded from 38.4 to 106 mg kg −1 for Cu, 67.4 to 124 mg kg −1 for Zn, and 123 to 312 mg kg −1 for Pb.Moreover, the concentrations were 0.16-0.99mg kg −1 for Cd, 0.50-33.7 mg kg −1 for Cr, and 0.64-3.91mg kg −1 for Co. Arsenic concentrations were below 5 mg kg −1 in the sclerotia collected from volcanic ash soils in non-mining areas of central and northern Japan [14].
According to the unpaired t-test, the As concentration of HAN sclerotia was significantly higher (p < 0.05) than that in the studied non-mining areas.There was no significant difference in Pb between the HAN sclerotia and non-mining areas.Lower (p < 0.05) Zn concentration was recorded in HAN sclerotia than that in non-mining areas.We compared the minor element concentrations in sclerotia between HAN sites and non-mining sites in the volcanic ash soil area in Japan, as shown in Figure 3(b); the concentrations of minor elements in HAN sclerotia can be ordered as follows: As, Cu > Pb, Zn > Cr, Co, Cd.

C, O, and Al distribution in sclerotia
The distributions of C, O, and Al in the sclerotia were obtained by conducting an EDS line scan analysis on the cut surface of the sclerotia, as shown in Figure 4.The SEM-EDS line results showed that the spectral intensities of C Kα and O Kα fluctuated widely but were homogeneously distributed from the edge of the grain to the centre in sclerotia (Figure 4  (c,d)).By contrast, the intensity of Al Kα gradually increased from the outer part towards the inner part (Figure 4(b)).These results suggest a relatively higher Al accumulation in the central part of the sclerotia than that in the outer part.
EDS line-scan analysis was not feasible for As in the sclerotia because the mass percentage of As in the targeted area (0.01%) was below the detection limit.Moreover, as the peak detection positions of As Lα overlap with those of Mg Kα, semi-quantitative results for the trace content of As could be overestimated.

TOF-SIMS ion mapping of sclerotia
The secondary ion counts in TOF-SIMS measurements are not directly proportional to the actual abundance of the measured constituents [37].Therefore, the actual concentrations of the inorganic elements were quantified in advance using ICP-OES and ICP-MS analyses.The quantification results were compared with secondary ion counts obtained using TOF-SIMS.
Positive and negative fragment ion spectra are shown in Figure 5(a,b), respectively.Assignments of the fragment ions are shown in the figure.Some distinct peaks, such as the positive m/z:130 and negative m/z:145 peaks, could not be assigned in this study (Figure 5(a,b)).
Negative and positive fragment ion maps are shown in Figures 6 and 7, respectively.The distribution of C − was homogeneous throughout the measured area, including rind, cortex, and medulla of sclerotia.Al + showed an increase in intensity from the outer part towards the inner part of the sclerotia, which agreed with the line analysis results obtained by SEM-EDS.
The segregation of fragment ions of AlO   Moreover, the intensity of C 2 O 4 Al + (m/z:114.98)was stronger in the central region than that in the outer region (Figure 7).Among the As fragments proposed for TOF-SIMS analysis (Table 1), inorganic As, such as AsO Although the Mg and As peaks could not be separated by SEM-EDS, the low contribution of Mg + was confirmed by TOF-SIMS.

Discussion
The TOF-SIMS ion mapping images in this study showed a higher intensity of Al fragments in the central part of the sclerotial medulla than that in the outer part, which coincides with the results of the Al content obtained by line scan analysis using SEM-EDS.In this study, oxalic and acetic acids were also detected in the sclerotia of Cenococcum geophilum using TOF-SIMS analysis.The distribution of Al fragments suggests that Al compounds, such as acetates, oxalates, hydroxides, and phosphates, dominate the central part of the sclerotia.Hollow structures have been frequently observed in the central part of Cenococcum geophilum sclerotia associated with fungal mycelia and Al hydroxide minerals [38].Based on the optical microscopy results, whitish mycelia-like features were observed in the hollows of the HAN sclerotia (Figure 2(d)).The existing state of Al and its coexistence with phosphate in the sclerotia are likely to have common attributes observed in several studies on ECM fungi, which mention that oxalate produced by fungi may chelate Al 3+ in insoluble phosphates, such as Al phosphate, and release P [39][40][41][42].
High As accumulation in fungi may inhibit inorganic P uptake and influence fungal growth, P accumulation, and storage for utilisation owing to the similarity in the chemical analogy of phosphate and As(V) [43,44].The oxalic acid produced by fungi helps solubilise Pi under As stress [44].Thus, Cenococcum geophilum may acquire nutrients under AsO 4 3− stress [26].Although P was not detected by either SEM-EDS or ICP analyses, the localisation of the PO 3 − fragment in HAN sclerotia determined by TOF-SIMS ion mapping suggested the uptake of As and P uptake by Cenococcum geophilum sclerotia in As-polluted soils.Based on these results, we hypothesised that organic As is more likely to be present in the sclerotia than inorganic As species.How, the mechanism and function of As accumulation in the sclerotia are not well understood yet.The behaviour of As retention and formation in macrofungi has been studied with attention to their different life stages [45,46].An in vitro study on As in mushrooms suggested that the mycelial age might influence their ability to transform As species [47].In accordance with these studies, the pathways of As transfer in the sclerotia can be discussed along with the different stages of sclerotial development in the soil.Briefly, two stages were distinguished: the early stage (mycelium, with gemination function) and the mature stage (black, durable bodies, without germination function).Microorganisms methylate inorganic As compounds into organic As under oxidising conditions, such as in the presence of MA and DMA.These two methylated trivalent arsenic species have been shown to be more toxic than inorganic arsenic [48].One assumption is that the presence of MA and DMA in sclerotia may suggest metabolic processes occurring within the mature or early stages of sclerotia, whereby iAs is absorbed from the soil and then methylated.
During in vivo studies, it is not possible to control the duration of sclerotia formation from mycelia to sclerotia, and reconstructing past soil properties such as soil pH during the smoke hazard period is challenging.However, it is plausible that As transformation and/or translocation may occur during both the early and mature stages of sclerotia formation.For example, an increase in As concentration decrease ECM fungal biomass [21], and the growth of Cenococcum geophilum decreases with increasing As concentrations [26].Based on these observations, we speculated that sclerotia formation is an important life stage for Cenococcum geophilum to survive in As-contaminated soil and protect host plants from As toxicity.Combined with the 14 C dating results, the As concentration in aged sclerotia was as high as that in sclerotia formed during the smoke hazard period.This suggests that the biotransformation pathways of sclerotial As are more likely derived during the mature stages than during the early mycelial stages.
The toxicity of Al by exchangeable Al (Al 3+ release) may increase under considerably lowered soil pH conditions, caused by the discharge of sulfurous anhydride during the smoke hazard period.When the soil pH is below 5, Al 3+ released into the soil penetrates the cells of the root tips, affecting plant development [49].HAN sclerotia exhibited significantly higher (p < 0.05) Al concentrations than that in non-mining areas: 5901-9836 mg kg −1 in surface A horizons, with a modern Libby age, but lower than that of the buried A horizon (Myoko IIIA): 21,684 mg kg −1 , with an age of 1000 cal yrBP [14].It has been suggested that older sclerotia, with longer archives in the soil, may exhibit higher Al concentrations [14].Nevertheless, the Al concentration in the HAN sclerotia samples was approximately 11,000 mg kg −1 , despite having a wide sclerotial age range of 80-1120 cal yrBP.Consequently, we concluded that Al accumulation in sclerotia, initially driven by intense soil acidity in their active mycelial stage and then regulated by the duration of their existence in the soil, may accelerate Al 3+ /Al oxalate/Al acetate translocation from the soil to sclerotia in their mature stage when critically low pH stress occurs in the soil ecosystem.

Conclusion
The combination of ICP-OES, ICP-MS, SEM-EDS, and TOF-SIMS analyses provided a complementary methodology for understanding the transformation process of Al and As accumulated in Cenococcum geophilum sclerotia.
The enrichment of Al and As in the sclerotia was confirmed in forest soils affected by past smoke hazards in historical mining areas.TOF-SIMS analysis confirmed the dominant presence of Al in the Al oxalate, acetate, and hydroxides, and the presence of As species was reported for the first time in Cenococcum geophilum sclerotia.Segregation of As chemical species in the sclerotia was successfully confirmed using TOF-SIMS ion mapping analysis, and the As speciation profile of the sclerotia was dominated by organic As.Because of the recalcitrant structure of Cenococcum geophilum sclerotia, we speculate that As transfer from soil into sclerotia may occur as long as the grain structure is preserved and that sclerotia may act as an effective acceptor or sink of organic As in highly polluted soil.Our results will aid in understanding the ecological significance of Al and As accumulation in sclerotia in forest soils.

Figure 1 .
Figure 1.(a) Location of the study site.(b) The area affected by smoke hazards from Ashio mine based on 1897 survey and the location of Mt.Hangetsu.The figure was obtained from United Nation University, https://archive.unu.edu/unupress/unupbooks/uu35ie/uu35ie06.htm(Last view date: 2022/02/ 07) with revision and edit.(c) Location of the sample points and sample groups (HAN-A, B, C, D).Base map was obtained from Google Earth.

Figure 2 .
Figure 2. (a) View of HAN site conspicuous of dead Tsuga trees.(b) Core sample for analyses (c) Cg Sclerotia collected from soil.(d) The internal structure of the sclerotia applied for SEM-EDS and TOF-SIMS analyses.

Figure 3 .
Figure 3. (a)The elemental concentrations in soil and sclerotia at Hangetsu (HAN) site.Error bars are based on standard error (SE).Cd concentration in HAN soil was not determined.(b) Comparison of minor element concentrations in sclerotia between HAN site and non-mining sites: CH, IWK and MYK.Data for the non-mining sites: CH, IWK and MYK are obtained from Nyamsanjaa et al.(2021).Error bars are based on standard error (SE).Asterisk mark (*) denotes significant difference between HAN sclerotia and non-mining areas sclerotia at p < 0.05.

Figure 4 .
Figure 4.The cross-sectional SEM micrographs of HAN sclerotia with red line indicating the EDS line scan position (a), representative EDS line scans for Al-Kα (b), C-Kα (c), and O-Kα (d).

Figure 5 .
Figure 5. (a) Negative and (b) positive ion TOF-SIMS spectrum of sclerotia showing peaks of major ions and studied fragment ions.Enlarged spectrum assigned as m/z:58.98,AlO 2 − and m/z:59 C 2 H 3 O 2 − is denoted.

3 H 2 −
(iAs III ) and AsO 4 H 2 − (iAs v ), could not be confirmed in the spectra and images.Segregation of C 2 H 6 OAs − (dimethylarsinite, DMA III , m/z:120.92),C 2 H 6 O 2 As − (dimethylarsinate, DMA V , m/z:136.92), and CH 4 O 3 As − (methylarsonate, MA, m/z:138.92) was confirmed in the sclerotia (Figure 6(h,i,j)), suggesting that organic As was the dominant form of As fragment ions in the sclerotia.Interestingly, fragment ions of C 2 H 6 O 2 As − and CH 4 O 3 As − showed a similar localisation as PO 3 − .

Table 1 .
Studied negative and positive ions and fragment ions in sclerotia for TOF-SIMS analysis.