¹⁴C ages and δ¹³C of sclerotium grains found in forest soils

Abstract

¹⁴C ages and δ¹³C were examined for sclerotium grains to elucidate the characteristics of these grains distributed in forest soils. The ages of the grains from surface A horizons and buried A horizons were ca 100–200 bp and ca 300–1,200 bp, respectively. In comparison with humic acid extracts, the ¹⁴C ages were in the increasing order: humic acid fraction < humic acid Pg fraction < sclerotium grains. The δ¹³C values for sclerotium grains in surface A horizons and buried A horizons were approximately −31‰ to −28‰, and these values were approximately 2–4‰ smaller than those of humic acids and soils. The C content of the grains had a tendency to decrease with increasing ¹⁴C ages, while the C content of humic acid was constant with age. The ¹⁴C ages of sclerotium grains indicate the individual age of grain formation, which are more likely to assign closer ages to the beginning of soil forming than the ¹⁴C ages of humic acid. The low δ¹³C values for sclerotium grains have presumably originated from characteristically biological organics, which may be protected from attack in soils because of their structure.

Key words:

• ¹⁴C age
• δ¹³C
• humic acid
• humic acid Pg fraction
• sclerotium grain

INTRODUCTION

Mycelium of fungal species such as Deuteromycetes, Ascomycetes and Basidiomycetes are known to form resting bodies called sclerotia. Sclerotia have small spherical black bodies (with a diameter of ∼0.5–1.5 mm) protected by thick, melanin-pigmented walls, and are considered to be resistant to attack and desiccation during the period of inactivity (Cochrane 1958; Fox 1986; Gray and Williams 1971). Sclerotium grains (Fig. 1) found in forest soils are assumed as the resting structure of an ectomycorrhizal fungus, ‘Cenococcume geophilum’, and as one of the sources of the green fraction of P type humic acid (so called ‘Pg’) by Kumada and Hurst (1967). Kumada and Hurst (1967) examined sclerotium grains in British and Swedish Podzols and showed that the distribution density of the grains tended to be greater in B horizon than in A horizon. Retallack (1990) introduced similar spherical grains found in paleosol as indicators for reducing condition. Hormes et al. (2004) examined C. geophilum spores in paleosols of moraines in Lapland, northern Sweden, and reported that the ¹⁴C ages of the spores were 5,000–6,000 bp.

Watanabe et al. (2001) reported high concentrations of Al in sclerotium grains collected from volcanic ash soils and the possible occurrence of Al–humus complexes inside the grains. Sclerotium grains had a tendency to exist in soils with high ratios (> 0.5) of organic bonding Al (Al_p: pyrophosphate-extractable Al) to amorphous Al (Al_o: acid ammonium oxalate-extractable Al) regardless of soil type and the maximum density of the sclerotium grains was approximately 2.5 g kg⁻¹ soil (Watanabe et al. 2002, 2004b). Furthermore, a highly positive relationship was observed between the mean weight of the sclerotium grains and the content of exchangeable aluminum (Al³⁺) in the soils. Watanabe et al. (2006) examined the characteristics of major and trace elements in sclerotium grains collected from volcanic ash soils at Mount Myoko. The content of the major elements C, H, N, O and Al were approximately 47.6, 3.32, 0.78,

Figure 1  Sclerotium grains collected from Myoko Asadaira soil.

30.2 and 1.4% by mass, respectively, and trace elements such as Ti, Cr, Mn, Cu, Zn, Br and Pb were detected at concentrations between 10 to 100 µg g⁻¹. Carbon in sclerotium grains was mainly composed of O-alkyl C and the contribution of aromatic C was less important than that in humic acid, which showed strongly a characteristically biological origin and completely different features to humic and fulvic acids from an allophanic Andosol.

In the present study, ¹⁴C ages and stable isotope ratio δ¹³C were examined for sclerotium grains and for humic acid extracts in equivalent layers to obtain further knowledge regarding the characteristics of sclerotium grains as an organic component in forest soils and to discuss the interpretation of ¹⁴C ages assigned for the remaining grains in soils.

MATERIALS AND METHODS

Samples of sclerotium grains were collected from Myoko Asadaira soil, central Japan (Fulvic Andosol, World Reference Base/Food and Agriculture Organization–Unesco; location, 36°54′N 138°8′E; elevation, 1,330 m; vegetation, Fagus crenata). As described in Fig. 2, sclerotium grains were collected from the surface A horizon and the buried humic horizons beneath KG-b (Koyaike ash-b) and KG-c (Koyaike ash-c), the tephra deposits derived from the Yakeyama volcano in 650 ± 100 bp and 950 ± 80 bp (Hayatsu et al. 1994), respectively. Sclerotium grains larger than 1 mm in diameter were picked up from air-dried soils using sterilized tweezers. Grains smaller than 1 mm in diameter were collected from the float of distilled water. Sclerotium grain samples were exposed to ultrasonic treatment for 5 min to remove soil from their surface and then dried at room temperature. The weight and diameter of the grains were measured using an electronic balance and a digital high-density video microscope (VH-7000, Keyence, Osaka, Japan), respectively. Samples were fractionated into three grain sizes; large (> 2 mm), medium (∼1–2 mm) and small (< 1 mm). Soil samples for ¹⁴C age determination were prepared by removing sclerotium grains larger than 0.2 mm in diameter. Soil humic acids were extracted from horizons 1A, 2A and 3A of the Myoko Asadaira soil using the repeating Acid–Alkaline–Acid (AAA) treatment developed by Nakamura and Iwahana (1990). The humic acid Pg fraction was prepared using the following procedure. First, humic acid from Myoko soil was prepared using the International Humic Substances Society (IHSS) method (Swift 1996). A 50-mg sample of humic acid was dissolved in 3 mL of 0.2 mol L⁻¹ NaOH, and then 0.3 mol L⁻¹ NaCl (3 mL) was added to the solution. The solution was filtered through a membrane filter (0.2 µm, PTFE, Billerica Corp., Milford, MA, USA), and the filtrate was applied onto a glass column of Sephadex G-50 gel (Amersham Bioscience, Piscataway, NJ, USA; column size, ϕ50 mm × 30 cm). The column was eluted with 0.1 mol L⁻¹ NaOH at a flow rate of 220 mL h⁻¹. The first brown fraction and the second greenish brown fraction were discarded, and then the green fraction, which was finally eluted, was collected. The green fraction was immediately acidified with 1 mol L⁻¹ HCl to below pH 2. The same purification procedure was repeated several times. The combined acidified solutions were placed in the dark at 4°C for 3 days, and the supernatant was removed by centrifugation (22,000 g, 30 min). The precipitate was dialyzed with distilled water for 1 week using a Spectra/Por dialysis membrane (MWCO 3500, Spectrum Laboratories, Houston TX, USA), and then it was freeze-dried. The obtained powder was then used as a Pg sample.

Accelerator mass spectrometry (AMS) measurements for ¹⁴C/¹²C ratios were carried out at Micro Analysis

Figure 2  Profile of Myoko Asadaira soil. KG-b, Koyaike ash-b; KG-c, Koyaike ash-c.

Laboratory (MALT) (5 MV Tandem accelerator), at the University of Tokyo, Japan, according to Matsuzaki et al. (2004). As 1 mg C is required for the AMS measurement, the number of grains used for AMS measurement was determined by grain size as noted in Table 1. All samples were oxidized to CO₂ gas. The CO₂ gas was purified and then graphitized using hydrogen gas and Fe powder as catalysis. Graphite samples were pressed with Fe powder into cathode cones to be set at the ion source of the AMS system. The ¹⁴C/¹²C data of samples were normalized using the ¹⁴C/¹²C of the standards. New oxalic acid provided by National Institute of Standards and Technology (NIST) (SRM 4990C) was used as a standard material. ¹⁴C ages were calculated according to the procedures of Stuiver and Polach (1977) and calibration for calendar age was carried out using the INTCAL98 and OxCal calibration program v3.9 of Ramsey (1995, 2001). The AMS measurement was repeated four times for each sample. The accurate δ¹³C values of sclerotium grains were determined using an isotopic ratio mass spectrometer (IRMS) according to the following procedure. Samples containing approximately 0.1 mg of C were weighed into tin capsules. Each sample was prepared by repeating the measurements three times. Samples were combusted in a C/N analyzer (EA-1108, Fison Milan, Italy), and the combustion product (CO₂) was directly introduced into an IRMS (Finigan Mat 252 Thermo, Waltham, MA, USA). The δ¹³C was expressed as follows:

where PDB refers to the belemnite carbonate standard of the Peedee Formation, South Carolina, USA, which is accepted as the international standard.

RESULTS AND DISCUSSION

Table 1 shows the results of the ¹⁴C age measurement for sclerotium grains and humic acids. Figure 3 represents the distribution of ¹⁴C ages of sclerotium in the profile of Asadaira, Myoko. Grains found from horizon 1A, 2A and 3A were 95 ± 40 to approximately 240 ± 35 bp, 305 ± 30 to approximately 1,000 ± 95 bp, and 1,020 ± 35 to approximately 1,200 ± 35 bp, respectively. The obtained ages were not calibrated for calendar age in discussions hereafter because of the large variation in CO₂ in air in the past 200 years. The ¹⁴C ages for the

Table 1 14C ages determined using accelerator mass spectrometry

Sample				No. applied grains	Mean weight (mg/grain)	Mean diameter (mm)	C (%)	^14C-Age (bp)	Labo Code
No.	Material	Horizon/grain size	Weight (mg)
1	SG	1A/small	2.11	39	0.05	0.6	39.8	95 ± 40	MTC-05517
2	SG	1A/medium	2.84	4	0.71	1.4	49.5	180 ± 35	MTC-04230
3	SG	1A/large	3.14	1	3.14	3.0	44.5	240 ± 35	MTC-04231
4	SG	2A/small	2.98	43	0.07	0.6	42.8	305 ± 30	MTC-04715
5	SG	2A/medium	3.53	5	0.71	1.3	40.9	750 ± 35	MTC-04716
6	SG	2A/medium	2.84	4	0.71	1.4	37.5	1000 ± 95	MTC-03640
7	SG	2A/medium	4.46	5	0.89	1.3	44.8	370 ± 40	MTC-04232
8	SG	2A/medium	3.59	4	0.90	1.2	46.2	350 ± 50	MTC-04233
9	SG	2A/large	2.58	1	2.58	2.6	42.0	530 ± 95	MTC-03641
10	SG	3A/small	2.56	42	0.06	0.6	40.2	1095 ± 35	MTC-04717
11	SG	3A/medium	4.01	4	1.00	1.5	41.0	1165 ± 35	MTC-04718
12	SG	3A/medium	3.17	4	0.79	1.4	38.8	1200 ± 35	MTC-04227
13	SG	3A/large	2.43	1	2.43	3.2	42.5	1170 ± 90	MTC-03642
14	SG	3A/large	3.79	1	3.79	3.2	42.0	1020 ± 35	MTC-04228
15	HA	1A	3.37				47.7	−674 ± 89	MTC-05518
16	HA	2A	4.24				44.3	340 ± 45	MTC-05519
17	HA	3A	4.00				44.8	920 ± 50	MTC-05520
18	HAPg	3A	2.39				44.1	959 ± 37	MTC-06005
SG, sclerotium grain; HA, humic acid; HAPg, humic acid green fraction.

grains in horizons 1A and 3A harmonized with the ages of tephras, although some grains in 2A horizons had younger ages than the deposit age of the overlaid tephra KG-b (650 ± 100 bp). As the thickness of the KG-b tephra was 3 cm or less and the depth of the tephra was 12–15 cm from surface, it was considered that relatively young grains could be formed by the extended plant roots into the 2A horizon.

Figure 3  Distribution of 14C ages of sclerotium in the profile of Myoko Asadaira soil. KG-b, Koyaike ash-b; KG-c, Koyaike ash-c.

The average ¹⁴C ages for the soil humic acids extracted from horizons A, 2A and 3A in Myoko Asadaira soil were modern (< 0 bp), 340 ± 45 bp and 920 ± 50 bp, respectively. These ages were younger than those of sclerotium grains found in equivalent horizons. The green fraction of humic acid (so called Pg) extracted from the 3A horizon was 959 ± 37 bp. Although sclerotium grains are considered to be a possible origin of humic acid Pg (Kumada and Hurst 1967), the age of the Pg extract was slightly older than humic acid, but still younger than sclerotium grains.

The relationships between ¹⁴C age and grain size obtained for the Myoko profile are shown in Fig. 4. Grains younger than 600 bp, collected from horizons 1A and 2A, showed a slight tendency to increase their ages with an increase in diameter, while there was no clear relationship between age and size for grains older than 600 bp. Relatively small grains (1–2 mm) had a spherical shape and larger grains (> 2 mm) frequently found in horizon 3A of Myoko soil were likely to have a distorted shape (as previously shown in Fig. 1). It could be inferred that several small grains conjunct to form a single large grain and this might be one of the reasons that the relationship between ¹⁴C age and grain size was not clear for larger grains in buried A horizons.

Although difficulties remain in comparing ¹⁴C ages between humus extracts and sclerotium grains, it was considered that the average age of organic materials responsible for forming sclerotium grains is older than

Figure 4  The relationship between 14C age and grain size obtained for the Myoko Asadaira profile.

that of humic substances. Most of the sclerotium grains examined in our paper are supposedly equivalent to “dead” sclerotia of C. geophilum, which were characterized as hard and brittle, and evidently persist as spores in the soil in contrast to the “live” sclerotia of C. geophilum recorded by Trappe (1969) and Massicotte and Trappe (1992). In studies of sclerotium germination and formation in vitro, initial growth of the white hyphal tip from field collected “live” sclerotia on agar elapsed within 10 days (Trappe 1969) or at the latest within 20 days (Massicotte and Trappe 1992). Hyphal growth was detected on most sclerotia that proved to be viable within 30 days, but some sclerotia sat on the agar for up to 75 days before showing germination and at maturity, after the formation of a dark rind, they resembled sclerotia formed in soil (Massicotte and Trappe 1992). Based on these studies, it is plausible that sclerotium formation in soils may also terminate within several months. Humic acids continuously exposed to metabolism are composed of heterogeneous material. In contrast, sclerotium grains, existing as a spore structure derived from the cell walls of fungals, are likely to be composed of homogeneous material. Consequently, the AMS ¹⁴C ages of sclerotium grains are the remaining ages of individual grain and assign closer ages to the beginning of soil formation compared with humic acids, which may assign the average age of soil humus.

As a high concentration of Al is detected in the cortex cell wall of ectomycorrhizas (Brunner 2001; Brunner and Frey 2000), the homogeneous distribution of Al in the transverse wall of ignited grains observed by Watanabe et al. (2004a) suggests that the grains themselves are the

Figure 5  The relationship between 14C ages of sclerotium grains and carbon content (%) measured using a CO2 pressure gauge in the process of graphite preparation.

biosynthetic product of ectomycorrhizas associated with Al retention. Furthermore, the mean weight of sclerotium grains is regulated by the amount of exchangeable Al in soils, regardless of the soil type (Watanabe et al. 2002, 2004a). Additional basic understanding was obtained using the element analysis carried out quantitatively for the Myoko sclerotium by Watanabe et al. (2006), which clarified that the concentrations of C and O, the major elements regulating the weight of grains, were 48 and 30 wt%, respectively, and that of Al was 1.4 wt%, with no significant difference in element concentrations between grain sizes. It is interesting to extend the investigation to various forest soils and compare the turnover of the grains in special consideration of the relationship between soil chemical properties and ¹⁴C ages of the grains.

According to Table 1, in which C content was measured using a CO₂ pressure gauge through the process of preparing graphite, C content for sclerotium grains in surface A horizons was approximately 40–50%, while the content in buried A soils was approximately 38–46%. The values show a slight decrease in C content in the older grains as represented in Fig. 5, and the same tendency is recognized for the C content measured by IRMS in Fig. 6, where C content of the grains is represented by the horizons. These results suggest that sclerotium grains in buried humic soils are in the process of decomposition.

The δ¹³C measurements for Myoko sclerotium grains demonstrated that grains from 1A had a δ¹³C value ranging from −28.88 ± 0.20 to approximately −31.36 ± 0.16‰, while the δ¹³C values for the grains from buried horizons 2A and 3A were −28.63 ± 0.19 to approximately

Figure 6  (a) δ13C and (b) carbon content for Myoko Asadaira sclerotium grains measured using an isotopic ratio mass spectrometer.

−30.05 ± 0.03‰. The δ¹³C values for humic acids and soil samples were approximately −28‰ and −27‰, respectively, and these values were higher than the values recorded for sclerotium grains by approximately 2–4‰. Referring to the δ¹³C values obtained for soil humus components by Hiradate et al. (2004), the δ¹³C values of sclerotium grains are definitely lower than the soil humus components. Smith and Epstein (1971) recorded δ¹³C values for Fagus leaves as −28.9‰ and Hobbie et al. (1999) reported that δ¹³C values of ectomycorrhizal fungi are higher than that of tree leaves, according to biological isotope fractionation. Although it is unclear why low δ¹³C values were recorded for sclerotium grains (−28‰ to approximately −32‰), which were lower than humic acid and fungal bodies, the following points need to be considered.

Watanabe and Takada (2006) reported an association between chemical structure and δ¹³C of humic acids in buried volcanic ash soils and clarified the increase in δ¹³C values in older humic acids, which had a relatively high content of aromatic C. The solid-state cross polarization and magic angle spinning (CPMAS) 13 C-NMR spectrum observed by Watanabe et al. (2006) for the Myoko sclerotium grains showed intense signals of O-alkyl C at 75 and 105 p.p.m., which were assigned to C2 to C6 and C1 positions of sugar chains, respectively, and a less intense signal at 130 p.p.m. (aromatic C), accompanied by minor signals of 175 p.p.m. (carboxylic C), 60 p.p.m. (methoxyl C) and 20 p.p.m. (methyl C). Considering the above knowledge together with the results of the ¹⁴C ages obtained in the present study, sclerotium grains are probably formed in advance of humic acid formation and their initial chemical structure is preserved in the grain. According to Trappe (1969) and Massicotte and Trappe (1992), the firm live sclerotia contained a large amount of ethanol-soluble oil. The low δ¹³C values obtained from sclerotium grains may originate from characteristic organic compounds that are difficult to purify from soil humus.

Studies on microbial communities of sclerotium grains by Ohta et al. (2003) found Sphingomonas spp., which could use benzoic acid and phenolic acid compounds, to be a dominant bacteria existing in the cell structures of grains collected from Myoko soil. Further investigation may explain the change in δ¹³C with age, which may occur because of activities of microorganisms in the grain. A high precision isotope micro-imaging technique using a secondary ion mass spectrometry (SIMS) with a solid-state ion imager (Kunihiro et al. 2005; Nagashima et al. 2004; Yurimoto et al. 2003) might be able to assist in observing the low δ¹³C part on the surface of cell structures in the grain.

CONCLUSION

Sclerotium grains of C. geophilum in Japanese forest soils were characterized by AMS ¹⁴C age determination and IRMS δ¹³C measurement and compared with soil humic acid. The ¹⁴C ages of sclerotium grains were older than those of humic acid extracts in equivalent soil horizons. The ¹⁴C ages of humic acids are more likely to reveal the mean age of humus, while the ¹⁴C ages of sclerotium grains indicate the individual age of grain formation. Older grains remaining in soils may provide an age close to the start of soil formation, or preserve past records of the soil environment. The decrease in C content for sclerotium grains suggested a process of decomposition or degeneration of C in the grains mainly because of internal microbial activities. The low δ¹³C values for sclerotium grains (approximately −28 to −32‰) compared with soil and humic acids presumably originate from characteristically biological organics.

ACKNOWLEDGMENTS

The authors thank the Sumitomo Science Foundation and the Japan Society for the Promotion of Science for their financial support for this study.