Dynamics of 13C natural abundance in wood decomposing fungi and their ecophysiological implications

Soil Biology & Biochemistry 37 (2005) 1598–1607 www.elsevier.com/locate/soilbio

Dynamics of

13

C natural abundance in wood decomposing fungi and their ecophysiological implications

Ayato Kohzua,*, Toshihiro Miyajimab, Takahiro Tateishic, Takashi Watanabed, Munezoh Takahashid, Eitaro Wadae a

Japan Science and Technology Agency, 509-3, Hirano-Nichome, Otsu, Shiga 520-2113, Japan b Ocean Research Institute, University of Tokyo, Tokyo 164-8639, Japan c Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan d Research Institute for Sustainable Humanosphere, Kyoto University, Uji, Kyoto 611-0011, Japan e Frontier Research Center for Global Change, 3173-25 Showamachi, Kanazawa-ku, Yokohama 236-0001, Japan Received 16 December 2003; received in revised form 22 September 2004; accepted 27 January 2005

Abstract Factors that affect the d13C values of fungi need to be analyzed for the progress of isotope-based studies of food-chain or organic matter dynamics in soils. To analyze the factors that control d13C values of the fungal body, basidiomycete and ascomycete species were grown on a beechwood substrate (six species) and in glucose medium (nine species), and the d13C value of produced fungal body was compared to that of the carbon source. The 13C enrichment (Dd13C) in the fungal aggregates compared to the decomposed wood varied from 1.2 to 6.3‰ among six species. In the glucose substrate experiment, the degree of 13C enrichment in the hyphal mat was relatively small and varied from K0.1 to 2.8‰ among nine basidiomycetes species depending on their growth stage. Calculated d13C values of the respired CO2 were lower than those of the hyphal mat, organic metabolites and the glucose used. The degree of 13C enrichment was affected by fungal species, substrate and growth stage. Fungal internal metabolic processes are the plausible mechanism for the observed isotopic discrimination between fungal bodies and substrates. Especially, dark fixation of ambient CO2 and kinetic isotope fractionation during assimilation and dissimilation reactions could well explain Dd13C dynamics in our experiments. Through the analysis of field Dd13C, we could know undisturbed fungal status about starvation, aeration and type of decomposition. q 2005 Elsevier Ltd. All rights reserved. Keywords: Carbon isotopes; Detritus food chain; Fungi; Invertebrates; Isotope fractionation; Beech wood; White rot; Brown rot

1. Introduction Fungi inhabit primarily in the aerobic terrestrial soil and play two kinds of important ecological roles which cannot be replaced by any other class of organisms. One is the decomposition of plant-derived lignin-rich polymers and humus by the saprophytic fungi (Rayner and Boddy, 1988). The other is the symbiotic nutritional relationships with vascular plants by the mycorrhizal fungi and endophytes (Smith and Read, 1997). Carbon, nitrogen and phosphorus cyclings in forest cannot be maintained without these

* Corresponding author. Tel.: C81 77 549 8213; fax: C81 77 549 8201. E-mail address: [email protected] (A. Kohzu).

0038-0717/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2005.01.021

saprophytic and symbiotic fungi. In many terrestrial ecosystems, biomass of fungal hyphae exceeds that of bacteria, and fungal respiration largely contributes to the soil respiration (West et al., 1987; Alphei et al., 1995). Hyphal biomass in the soil is one of the primary food sources for diverse soil invertebrates (Beare et al., 1992). However, dynamics of fungal biomass in the field and the relative importance of hyphae for the food source of the invertebrates have been unclear. In the grazing food chains of aquatic communities, carbon isotope ratios (13C/12C; d13C) in animal tissues are known to reflect the d13C of their diet, although small 13C enrichments (%1.0‰) between dietary and body sometimes occurred (DeNiro and Epstein, 1978; Fry and Sherr, 1984; Wada et al., 1998). So, d13C of animal bodies has been used to infer their primary food sources. d13C indices were applied also to the soil invertebrates in the detritus food

A. Kohzu et al. / Soil Biology & Biochemistry 37 (2005) 1598–1607

chains to infer their energy and carbon sources (Neilson et al., 1998; Ponsard and Arditi, 2000; Santruckova´ et al., 2000; McNabb et al., 2001). Ponsard and Arditi (2000) showed that soil detritivores were enriched in 13C relative to litter by 2.0–5.0‰ on monthly average. The large 13C enrichments of soil detritivores relative to litter suggested the possibilities of the selective feeding of the 13C enriched microbial fraction on the litter. Santruckova´ et al. (2000) showed that litter-feeding soil invertebrates respire CO2 that is enriched in 13C relatively to average litter carbon, and suggested that they selectively absorb some 13C-enriched organic fraction out of the whole ingested litter. What is the 13C enriched fraction of litter utilized by soil invertebrates? The most plausible answer to this question would be fungal hyphae and/or bacterial cells colonized on and inside the litter. The information about the d13C values of the fungal sporocarps in the field and of the incubated hyphal aggregates and yeast cells has been accumulated. Basidiocarps of wood decomposing and litter decomposing fungi are known to be enriched in 13C relative to wood and litter. Hobbie et al. (1999) and Kohzu et al. (1999) showed that the degree of 13C enrichment of these saprophytic fungal sporocarps relative to their substrates (Dd13C:d13CfungiKd13Csubstrate) were larger than C3.0‰ on average. This Dd13C is large enough to explain the d13C difference between soil invertebrate and litter by the selective feeding on fungal hyphae. In fact, Tayasu (1998) reported the significant 13C enrichment in the termite relative to the litter with which they cultivates fungus comb enriched in 13C. However, the degree of the carbon isotope discrimination during fungal growth can be highly variable in natural environment, and knowledge about the patterns of variation in Dd13C values and the mechanisms responsible for the variation is lacked at present. Therefore, in order to effectively apply the d13C indices to the soil detrital food webs, we must know what controls the d13C values of the fungal body in natural environments. The variations in Dd13C values depend primarily upon the carbon isotope effects accompanied with fungal metabolic reactions. Some possible factors that affect the Dd13C variation are substrate quality (C:N ratio, lignin content, etc.) (Fernandez and Cadisch, 2003), saprophytic fungal species (e.g. brown rot vs. white rot fungi) (Henn et al., 2002; Abraham and Hesse, 2003), and the growth conditions of fungi (Henn et al., 2002), because these factors control the flux distribution among the fungal internal metabolic pathways. However, such factors have not been thoroughly considered in these previous studies of fungal d13C dynamics. In the present study, nine basidiomycetes and one ascomycetes species were used in the experiments to elucidate the interspecies d13C variation. Two carbon sources (glucose and beechwood) were used as substrates to examine the effect of the quality of carbon source on fungal body d13C. In the glucose substrate experiment, d13C values of the fungal

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body were compared between growth and autolysis period to know the effect of growth condition.

2. Materials and methods 2.1. Fungal strains We used the following 10 different wood-decomposing fungi in the wood decomposing experiments and in the glucose substrate experiments. White rot fungi: Daedaleopsis tricolor (Bull.: Fr.) Bond. et Sing. (IFO 6269); Ganoderma applanatum (Pers.) Pat. (IFO 8346); Lenzites betulina (L.: Fr.) Fr. (FFPRI L5b); Panus rudis Fr. (IFO 8994); Polyporus anceps Peck (Dichomitus squalens) (CBS432.34); Poria subvermispora Pila´t (Ceriporiopsis subvermispora) (FP90031); Trametes versicolor (L.: Fr.) Que´l. (FFPRI 1030). Brown rot fungi: Laetiporus sulphureus (Fr.) Murrill var. miniatus (Jungh.) Imaz. (IFO 6497) Tyromyces palustris (Berk.: Curt.) Murr. (FFPRI 0507). Soft rot fungi: Chaetomium globosum Kunze ex Fr. (IFO6347) Three species of white rot fungi (L. betulina, P. rudis, T. versicolor), two species of brown rot fungi (L. sulphureus, T. palustris) and one soft rot fungus (C. globosum) were used in the wood-decomposing experiment. In the glucose substrate experiment, all species except L. sulphureus were used. 2.2. Wood degradation experiment Beech (Fagus crenata Blume) wood was collected at Ashiu (35822 0 N, 135855 0 E), Japan, and cut into blocks (20!20!5 mm, 1.2–1.5 g dry weight). The wood blocks were sterilized with ethylene oxide gas (ES-15, Hirayama Manuf. Co., Kasukabe, Japan) and placed on a layer of quartz sand saturated with modified Trion’s synthetic medium (85 ml per incubation bottle): KH2PO4$613 mg, MgSO4$7H2O 246 mg, K2HPO4 114 mg, CaCl2 55.5 mg, FeSO 4 20 mg, ZnSO 4$7H 2O 3.52 mg, CuSO4 $5H 2O 0.38 mg, MnSO 4$H 2O 0.031 mg, Na 2MoO 4 $2H 2O 0.025 mg and thiamine hydrochloride 5 mg in 1000 ml distilled water (pH 6.0). Organic carbon content of this medium, consisting of thiamine, was negligible compared with that of wood (more than 500 mg C). Fungal pellets for inoculation were prepared from submerged culture using JIS9501 medium: glucose 40 g, peptone (Mikuni Co., Tokyo, Japan) 3 g and malt extract (Nacalai Co., Kyoto, Japan) 15 g in 1000 ml distilled water. The amount of fungal pellets inoculated on each wood block was less than 2.0 mg dry weight. Each wood block on the medium-saturated quartz sand was aseptically placed in a sterilized widemouthed bottle (900 ml) and incubated at 26 8C in the dark. Inside the bottle, aseptic conditions were maintained, although fresh air could pass through the loosely shut cap.

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2.3. Glucose substrate experiment In the glucose substrate experiments, glucose of known d13CZK11.9‰ (Code 168-05, Nacalai Co.) was used in the liquid culture (Chelator-free growth medium): glucose 10 g, (NH4)2SO4 3.96 g, MgSO4$7H2O 530 mg, CaCl2$ 2H2O 100 mg, KH2PO4 2 g, MnSO4$5H2O 5 mg, NaCl 10 mg, FeSO4$7H2O 1 mg, CoSO4$7H2O 1 mg, ZnSO4$ 7H2O 1 mg, AlK(SO4)2$12H2O 0.1 mg, H3BO3 0.1 mg, NaMoO4$2H2O 0.1 mg and thiamine hydrochloride 1 mg in 1000 ml distilled water adjusted to pH 6.5 with KOH (Enoki et al., 1999). Fifteen flasks per species were prepared with 50 ml of the liquid culture in each flask. The amount of organic carbon other than glucose (!0.5 mg per incubation flask), consisting of thiamine hydrochloride, was small compared with that of glucose (200 mg C) and can be ignored. Fungal pellets (!2.0 mg d.w.) were prepared as described above and inoculated in the medium, and the cultures were incubated at 25 8C in the dark under aseptic conditions. 2.4. Sample preparation During the wood-decomposing experiments, samplings were carried out at indicated intervals (4–6 times, nZ3 at each time) throughout the experiment periods (0–308 days). From the incubation bottle, we took out a beechwood block that was covered with fungal aggregates, and the fungal aggregates were taken from the wood block. After scraping the fungal aggregates off the surface, the wood block and the fungal aggregates were dried at 70 8C for 48 h. The remaining quartz sands saturated with medium were filtered under a vacuum. The filtrates were acidic at the end of the experiment in white rot and brown rot fungi; pH 3.3G0.1 (T. versicolor, nZ3), 2.5G0.2 (T. palustris, nZ3). In the glucose substrate experiment, we collected the hyphal mat and filtered medium from each flask at indicated intervals (4–6 times, nZ3 at each time except 18 points over the whole set of data with only one or two replicates). In the flask, the hyphae usually grew into a single hyphal mat and were spread over the liquid surface. After collecting these mycelial mats, the liquid medium was filtered with filter paper (No. 2). The dry weight, carbon concentration and d13C of the mycelial mat and the filtrates were analyzed after being dried at 70 8C for 72 h. Dried samples were ground in a vibrating sample mill (TI-100, C.M.T., Fukushima, Japan) for at least three minutes before analyses. 2.5. Carbon content and carbon isotope analyses Carbon content and natural 13C abundance in the samples (2–6 mg D.W.) were analyzed using an on-line C and N analyzer coupled with an isotope ratio mass spectrometer (EA1108-ConfloII-deltaS system, Finnigan, Bremen, Germany). D,L-a-Alanine (C(wt.%)Z40.45%,

d13CZK23.5‰) was used as the working standard. The 13 C abundance is expressed as follows: d13 C Z ðRsample K RPDBstandard Þ=RPDBstandard !1000ð‰Þ; where R is the ratio 13C/12C. The d13C standard deviation based on replicate analysis was less than 0.2‰. For comparing the d13C values between fungal bodies and their substrate, Dd13C parameter (Zd13Cfungal bodyK d13Csubstrate) was used. The glucose concentration of the filtrate medium was analyzed by colorimetric analysis (wave length 505 nm, Glucose CII-test, Wako Co., Osaka, Japan). 2.6. Mass balance calculation in the glucose substrate experiment Before discussing the d13C values of the fungal body, we must consider the isotopic difference between the substrates and the fungal pellets for inoculation. The d13C values of the fungal pellets were within the range of K17.5 to K15.0‰, and the carbon amount of each fungal pellets inoculated was from 0.004 to 0.437 mg among the nine species. From the simple mixing model, except the early growth stage of four species (i.e. D. tricolor 8 day, P. anceps 8 day, P. rudis 8 day, T. palustris 8 day), the isotopic effects of the carried-over fungal pellets were less than 0.1‰, which is smaller than the analytical error. Considering that the turnover times of the fungal body at the exponetial growth stage is relatively short, the isotopic effects of the fungal pellets inoculated were negligible for interpreting the d13C values of the fungal body. From the five kinds of data (the residual glucose concentrations, dry weight and carbon concentrations of the mycelial mat and filtrate), we calculated the amounts of carbon in the glucose consumed by fungi, assimilated to the fungal body and transformed into the metabolites, respectively. That is, the amount of carbon in the metabolites was calculated by the Eq. (1). CðmetabolitesÞ Z CðfiltrateÞ K Cðresidual glucoseÞ

(1)

The amount of the respired CO2 and its carbon amount were also calculated with the mass balance Eq. (2). CðCO2 Þ Z Cðabsorbed glucoseÞ K Cðhyphal matÞ K CðmetabolitesÞ

(2)

The d13C values of the metabolites and the respired CO2 were calculated by the mass balance Eqs. (3) and (4) with the calculated amount of carbon in the absorbed glucose, hyphal mat, metabolites and CO2 and with the d13C value of the glucose (K11.9‰), hyphal mat and filtrate medium. Here, we assumed that the kinetic isotope fractionation in the uptake of large molecules such as glucose was negligible. However, exchange with metabolic glucose could make the d13C value of the remained glucose somewhat different from that of the initial glucose (K11.9‰) when glucose was

A. Kohzu et al. / Soil Biology & Biochemistry 37 (2005) 1598–1607

remained as in the case of C. globosum, T. palustris, and of all species in the first steps of glucose consumption. CðfiltrateÞ !d13 CðfiltrateÞ Z Cðresidual glucoseÞ !ðK11:9Þ C CðmetabolitesÞ !d13 CðmetabolitesÞ

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Exponential, stationary and autolysis stages correspond to the increasing, constant and decreasing period of the fungal biomass, respectively. The border between stationary and autolysis stage was determined from the growth curve of fungi shown in Fig. 3. Growth period was defined as exponential and/or stationary stage, while autolysis period was the periods of autolysis stage. Yield coefficient was calculated only for the growth period.

Z Cðresidual glucoseÞ !ðK11:9Þ C ðCðfiltrateÞ K Cðresidual glucoseÞÞ !d13 CðmetabolitesÞ

2.7. Field sampling of basidiocarps (3) From the deciduous forests around Kyoto, we collected 13 basidiocarps of T. versicolor together with their associated decomposed wood in October, 1999. The basidiocarps and the corresponding wood were dried at 70 8C for 72 h. Samples were then powdered and later combusted for d13C analyses.

Cðabsorbed glucoseÞ !ðK11:9Þ Z Cðhyphal matÞ !d13 Cðhyphal matÞ C CðmetabolitesÞ !d13 CðmetabolitesÞ C CðCO2 Þ !d13 CðCO2 Þ

(4)

The amount of carbon and the 13C abundance of the total organic matter produced by fungi were also calculated by Eqs. (5) and (6). Cðtotal organic matterÞ Z Cðhyphal matÞ C CðmetabolitesÞ

The d13C of the basidiocarps collected in the deciduous forests around Kyoto ranged from K25.3 to K21.8‰ and that of the decomposed wood was from K28.3 to K25.0‰. The degree of 13C enrichment in the basidiocarps relative to the decomposed wood ranged from 2.5 to 3.9‰ (Fig. 1).

(6)

3.2. d13C dynamics in the wood degradation

Z Cðhyphal matÞ !d13 Cðhyphal matÞ

Yield coefficient shown in Table 1 as an indicator of growth efficiency in each fungus was defined by the Eq. (7). Yield coefficientð%Þ Z Cðhyphal matÞ=Cðabsorbed glucoseÞ !100

3.1. d13C of the basidiocarps of T. versicolor and that of decomposed wood

(5)

Cðtotal organic matterÞ !d13 Cðtotal organic matterÞ

C CðmetabolitesÞ !d13 CðmetabolitesÞ

3. Results

(7)

The d13C value of the fungal pellets for inoculation was high because of the less negative d13C of the C sources used for preincubation (e.g. the d13C of the glucose is K11.9‰). However, within three weeks the d13C of the mycelium decreased, and became constant thereafter (Fig. 2).

Table 1 d13C and Dd13C values (meanGSD) of the saprophytic fungi cultured in the glucose carbon source Fungal species

C. globosum D. tricolor G. applanata L. betulina P. anceps P. rudis P. subvermi spora T. palustris T. versicolor

Definition of growth and autolysis period

Yield coefficient (%, meanGSD)

d13Chyphal

Growth period (day)

Autolysis period (day)

Growth period

Growth period

Autolysis period

Growth period

13–24 8–24 24–66 8–30 8–30 8–24 62–121

n.d. 38–66 n.d. 38–95 38 30–50 207

34.4G7.2(4) 24.1G4.0(4) 42.1G9.4(12) 40.7G8.1(10) 26.2G4.6(11) 36.2G4.7(7) 34.3G6.9(9)

K11.9G0.1(4) K11.2G1.2(4) K11.5G0.2(13) K10.6G0.3(10) K11.0G0.7(12) K10.8G0.5(7) K10.9G0.3(9)

n.d. K9.4G0.3*(9) n.d. K10.1G0.3*(5) K10.3G0.1(3) K10.0G0.3*(8) K10.6G0.0(3)

0.0G0.1(4) 0.7G1.2(4) 0.4G0.2(13) 1.3G0.3(10) 0.9G0.7(12) 1.1G0.5(7) 1.0G0.3(9)

24–95 8–13

n.d. 21–90

17.1G4.4(13) 39.0G7.0(6)

K12.0G0.7(13) n.d. K10.3G0.2(6) K10.2G0.1(6)

mat

Dd13C (‰): d13Chyphal matK d13Cglucose

(‰)

K0.1G0.7(13) 1.6G0.2(6)

Autolysis period n.d. 2.5G0.3*(9) n.d. 1.8G0.3*(5) 1.6G0.1(3) 1.9G0.3*(8) 1.3G0.0(3) n.d. 1.7G0.1(6)

Growth period was defied as exponential and/or stationary stage, with autolysis period was the periods of autolysis stage. The number of samples for each period was shown in parentheses. d13C and Dd13C values of fungi in the autolysis period followed by an asterisk are significantly (by t-test P!0.01) different from those in the growth stage.

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δ13 C of the basidiocarps of T. versicolor (‰) -21

13

Y = X + 3.9

Y = X + 2.5

Y =X

-23

-25

-27

-29 -29

-27

-25

-23

-21

δ13C of the decomposed wood (‰) Fig. 1. Comparison of the d13C value between basidiocarps of T. versicolor and the decomposed wood collected from deciduous forests around Kyoto, Japan. d13C values of T. versicolor were significantly correlated with those of the wood substrate (P!0.01). Symbols distribute in the xC2.5!y!xC 3.9 area, which means that the enrichment factors were from 2.5 to 3.9‰.

The Dd13C (meanGSD throughout the degradation periods) in the mycelium relative to the decomposed wood (K25.7G0.2‰) was 1.6G0.2‰ (C. globosum), 3.5G 0.6‰ (L. betulina), 5.4G0.5‰ (L. sulphureus), 2.8G 0.2‰ (P. rudis), 3.7G0.5‰ (T. palustris) and 3.3G0.3‰ (T. versicolor) (Fig. 2). Dd13C in brown rot fungi (L. sulphureus, T. palustris) was significantly larger than that in soft rot fungi (C. globosum) (ANOVA, P!0.001). d13C values of the mycelium and decomposed wood remained constant, and their variations were less than 1.0‰ throughout the wood decomposition except for the first drop stage. 3.3. d13C dynamics in the glucose substrate experiment The biomass of the hyphal mat became maximal just before the glucose was exhausted and then decreased in the following autolysis period (Fig. 3). The total carbon of the metabolites was lower than that in the hyphal mat except T. palustris. Unlike the other species, T. palustris excreted about twice as much carbon as it assimilated. Yield coefficient in the growth period varied from 17 to 42% among species as shown in Table 1. Except C. globosum, the d13C values of the hyphal mat, metabolites and CO2 were different in each species. Among these components, the d13C of the hyphal mat was the highest, while the calculated d13C value of the CO2 was the lowest (Fig. 3). The d13C of the metabolites was between the other two components. Judging from the d13C value of the glucose (K11.9‰), Dd13C was K0.1 to C2.5‰ while

C depletion in CO2 was 0.5 to 1.3‰ on average, respectively. Intra- and inter-species variations in the hyphal mat d13C were observed (Fig. 3). In C. globosum, T. versicolor, P. subvermispora and G. applanatum, the intra-species variation in the hyphal mat d13C was relatively small and within 0.5‰. However, large intra-species variations, up to 2.5‰, were found in the incubation systems of D. tricolor, P. anceps, L. betulina, P. rudis and T. palustris. In these species, hyphal mat d13C was low at the growth period, then increased more than 1.0‰ as the glucose consumption and the autolysis of the hyphal mat proceeded (Table 1). The calculated d13C value of the CO2 showed a similar increasing pattern throughout the whole incubation period. The d13C value of the CO2 was low at the growth period and then increased and approached to a fixed value (from K12.4 to K12.2‰) slightly more depleted than that of the glucose. In all species except T. palustris, the amount of the total organic matter (hyphal matCmetabolites) decreased after the substrate glucose was exhausted. Also, the d13C values of the total organic matter increased when the carbon amount of the total organic matter decreased (see between arrows in Fig. 3). Long after the exhaustion of glucose, this significant relation was not found, and the d13C value of the hyphal mat decreased again in D. tricolor and L. betulina.

4. Discussion 4.1. Factors which affect fungal d13C 13

C enrichment in the fruit bodies collected in the field and in the mycelium of the wood decomposing experiment was observed across species (Figs. 1 and 2). In the six species among nine species, hyphal mats cultured in the glucose substrate were also enriched in 13C relative to the glucose (Fig. 3). However, Dd13C values were different corresponding to the fungal species, kinds of substrate and fungal growth stage (Table 1; Henn et al., 2002). In addition, Dd13C was negative in the hyphal mat and metabolites of C. globosum (ascomycetes) grown in the glucose substrate experiment (Fig. 3). During the early growth periods of D. tricolor and T. palustris, negative Dd13C values from K1.0 to K0.5‰ were also observed. Previous studies also showed that d13C values of cells were lower than that of the substrate glucose by 1.1–2.1‰ in the eight Ustilago species (protobasidiomycetes) (Will et al., 1986; Will et al., 1989), by 1.0G0.5‰ in Candida lipolytica (Harrison) Diddens et Lodder (Zyakun, 1996), by 1.4G0.1‰ in Zygomycetes and Hyphomycetes species (Abraham and Hesse, 2003). From these reports, the positive Dd13C value is characteristic to the eubasidiomycetes decomposing lignocellulose and not always associated with the other fungal classes or fungi decomposing non-ligneous substrates. Thus, fungal species, d13C value and the quality of substrate, and the growth stage

A. Kohzu et al. / Soil Biology & Biochemistry 37 (2005) 1598–1607

δ C (‰) 13

Fungal aggregates Beech wood

–18

L. sulphureus

L. betulina

C. globosum –14 –16

–14

–14

–16

–16

–18

–18 –20

–20

–20 –22 –24 –26 –28

–22 –24

–22

–26

–26

–24 –28

–28 Residual mass (%)

0

80

160

240

80

80

80

60

60

60

40

40

40

20

20

20

0 320

0

–18 –20

–28

–18

–20

–20 –22

240

–24 –26 –28

80

80

80

60

60

60

40

40

40

20

20

20 0 120 160 200

Incubation period (day)

160

T. palustris

–18

–26 –28

80

80

Incubation period (day)

–16

–26

40

0

–14 –16

–22 –24

0

0

0 120 160 200

–14

–22 –24

Residual mass (%)

80

T. versicolor

P. rudis 13

δ C (‰) Fungal aggregates Beech wood

–16

40

Incubation period (day)

Incubation period (day)

–14

1603

0

80

160

0 240

Incubation period (day)

0

40

80

0 120 160 200

Incubation period (day)

13

Fig. 2. Variations in d C value of the fungal aggregates and the decomposed wood (upper figure) and percentages of residual mass (lower figure) during the wood decomposition. Mean values for the fungal aggregates and the decomposed wood were plotted with solid and open circles, respectively. The mean value of the residual mass ratios is indicated with cross symbols. The standard deviations of both d13C and percentages are shown for each point with bars.

are the important factors that affect the d13C value of the fungal body. Either of the following mechanisms should be involved in the increase of d13C in the fungal body relative to their substrate: (1) selective incorporation of 13C enriched fraction in the substrate by the fungi, (2) 13C enrichment in the fungal body due to fungal internal metabolic processes. In the case of wood decomposing experiment and field surveys, first mechanism should be considered because there is relatively large d13C difference between lignin and other components in wood (Wilson and Grinsted, 1977; Gleixner et al., 1993). However, the 13C enrichment in the fungal body was also observed when grown in the isotopically homogeneous glucose (Table 1). In addition, we could not detect any significant change in wood d13C throughout the degradation, which implies that the process (1) was of minor importance. Therefore, fungal internal metabolic processes are more plausible mechanism for

the observed isotopic discrimination between fungal bodies and substrates. Possible metabolic processes that can lead to the enrichment in 13C in fungal mycelia include (2–1) dark fixation of ambient CO2 (d13CZK7.5‰), (2–2) kinetic isotope fractionation during assimilation and dissimilation reactions, and (2–3) cleavage and differential incorporation of substrates that contain intramolecular heterogeneity in carbon isotopic composition. Among the results we obtained in this study, the following observations are particularly relevant to the isotopic discrimination by internal metabolic processes. (1) Much more positive Dd13C values on the beechwood substrate than those on the glucose substrate (Figs. 2 and 3): during the decomposition experiments, the incubation bottles were always open to the atmosphere and the fungi could freely access to the atmospheric CO2. Significant fixation of CO2 by the anaplerotic pathway was reported in arbuscular mycorrhizal fungi (Bago et al., 1999) and in

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glucose (d13CZK11.9‰). This may explain why the observed Dd13C values were higher in the beechwood decomposition experiment than in the glucose substrate experiment. (2) d13C values of the fungal bodies and metabolites on the glucose substrate increased on the autolysis period (Fig. 3): in the autolysis period, the importance of the gluconeogenesis relative to the glycolysis in the fungal metabolism will increase because of the depletion of available carbohydrates in the medium (Quain and Haslam, 1979; Wilson and Bhattacharjee, 1986). Although there is little isotopic fractionation in the glycolysis

parasitic fungi (Ermakova et al., 1986). Fixation of CO2 by this pathway is catalyzed by pyruvate carboxylase, in which the equilibrium carbon isotope effect between oxalacetate (the reactant) and pyruvateCCO2 (the products) was reported as 1.0075 (Attwood et al., 1986). Thus, the fungi can assimilate atmospheric CO2 without large isotope fractionation. However, the effect of the incorporation of 13 C-rich CO2 on the fungal d13C should be relatively large for the fungi that have originally low d13C values. Therefore, the anaplerotic uptake of atmospheric CO2 should have produced larger increments in d13C of the fungi fed with beechwood (d13CZK25.7‰) than the fungi fed with C. globosum

(‰) –9.5

D. tricolor

(‰) –9.5

(mg) 100

G. applanatum

(‰) –9.5

–10.0

80

–10.0

(mg) 100 80

–10.5

60

–10.5

60

–10.5

60

–11.0

40

–11.0

40

–11.0

40

–11.5 (‰) –9.0 δ13C (‰) –10.0 –11.0 –12.0 –13.0 –14.0 –15.0 –16.0 Carbon amount

20

–11.5 (‰) –9.0 –10.0 –11.0 –12.0 –13.0 –14.0 –15.0 –16.0

20

–11.5 (‰) –9.0 –10.0 –11.0 –12.0 –13.0 –14.0 –15.0 –16.0

20

(mg)

Total organic matter 100 80

–10.0

0

0

(mg) 160

0

(mg) 160

0

(mg) 160

120

120

120

80

80

80

40

40

40

0 20 40 60 80 100

0

Growth period (day)

0 20 40 60 80 100

0

Growth period (day)

Growth period (day)

(mg) 100 80

–10.0

80

–10.0

80

–10.5

60

–10.5

60

–10.5

60

–11.0

40

–11.0

40

–11.0

40

–11.5 (‰) –9.0 –10.0 –11.0 –12.0 –13.0 –14.0 –15.0 –16.0

20

–11.5 (‰) –9.0 –10.0 –11.0 –12.0 –13.0 –14.0 –15.0 –16.0

20

–11.5 (‰) –9.0 –10.0 –11.0 –12.0 –13.0 –14.0 –15.0 –16.0

20

0

13

δ C (‰)

Carbon amount

(mg) 160

P. anceps

(mg) 100

0

(mg) 160

20

40

60

(‰) –9.5

P. rudis

(mg) 100

(mg) 160

120

120

80

80

80

40

40

40

80 100

Growth period (day)

0 0

10

20

30

40

Growth period (day)

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0

120

0 0

Glucose Hyphal mat Metabolite CO2

0 20 40 60 80 100

L. betulina (‰) –9.5 Total organic matter –10.0

(‰) –9.5

Carbon amount (mg) δ13C (‰)

Glucose Hyphal mat Metabolite CO2

0 0 10 20 30 40 50 60

Growth period (day)

Fig. 3. Time course of the glucose substrate experiments: d13C values (solid square) and carbon amounts (open square) of the total organic matter (i.e. hyphal matCmetabolites) were plotted against the experiment period in the upper figure. d13C values of the hyphal mat (solid circle), metabolite (open circle) and CO2 (triangle) were plotted against the experiment period in the middle figure. Amount of carbon in the residual glucose (cross), hyphal mat, metabolite and CO2 were plotted against the experiment period in the lower figure. The standard deviations of both d13C and carbon amount are shown for each point with bars. Two vertical arrows plotted on the x axis in the upper figure of six species correspond to the starting point and ending point of the simultaneous increase in the d13C values and decrease in the amount of the total organic carbon.

A. Kohzu et al. / Soil Biology & Biochemistry 37 (2005) 1598–1607

(‰) P. subvermispora (mg) –9.5 100 Total organic matter 80 –10.0

T. palustris

(‰) –9.5

T. versicolor

1605

(mg)

(mg) 100

(‰) –9.5

–10.0

80

–10.0

80

100

–10.5

60

–10.5

60

–10.5

60

–11.0

40

–11.0

40

–11.0

40

–11.5 (‰) –9.0 δ13C (‰) –10.0 –11.0 –12.0 –13.0 –14.0 –15.0 –16.0 Carbon amount

20

–11.5 (‰) 0 –9.0 –10.0 –11.0 –12.0 –13.0 –14.0 (mg) –15.0 160 –16.0

20

20

(mg) 160

–11.5 (‰) –9.0 –10.0 –11.0 –12.0 –13.0 –14.0 –15.0 –16.0

120

120

120

80

80

80

40

40

40

0 40 80 120 160 200

Growth period (day)

0

0

0

0 20 40 60 80 100

Growth period (day)

Carbon amount (mg) δ13C (‰)

0

(mg) 160

0

Glucose Hyphal mat Metabolite CO2

0 20 40 60 80 100

Growth period (day)

Fig. 3 (continued)

(Abelson and Hoering, 1961; DeNiro and Epstein, 1978; Monson and Hayes, 1982), the gluconeogenesis that is the reverse flow of glycolysis prefer 13C-rich monomers for the synthesis of hexoses through aldolase reactions. Thus, d13C values of fungal hyphae during this period, which are produced from the hexoses synthesized by the gluconeogenesis, are likely to become more positive than that during the growth period. Gleixner et al. (1993) reported 2.3–3.0‰ 13 C enrichment in the fungal chitin compared to the infected wood. They proposed that kinetic isotope fractionation in aldolase reactions caused the 13C enrichment in fungi also in this case. Increased gluconeogenesis activity during the autolysis period can explain the increase in the d13C values of the total organic matter after the exhaustion of the glucose (Fig. 3, Table 1). Considering that the d13C values of the fungal aggregates did not increase during the wood decomposing experiment, starvation of organic carbon would not be so severe as to change the carbon metabolic flux even at the end of the experiment (Fig. 2). (3) Larger Dd13C values in brown rot fungi than in white rot fungi (Fig. 2): since lignin comprises the relatively 13 C-depleted fraction of the whole wood material, the brown rot fungi, which cannot decompose lignin, should be more enriched in 13C than the white rot fungi, which can degrade lignin and assimilate lignin-derived carbon. However, if the utilization of the 13C enriched fraction in wood (process 1) were the sole reason for the significantly higher d13C values of the brown rot fungi (Fig. 2), mass balance calculations should indicate that the wood d13C at the end of the experiment should be more depleted by 8.1‰ (L. sulphureus) and 1.6‰ (T. palustris), respectively, than the initial d13C. However, this is not the case in our experiments (Fig. 2). There should be alternative explanations for the difference of

d13C between brown rot and white rot fungi. A possible explanation for this difference is the difference in the activity of oxalic acid production between these two groups of fungi. Oxalic acid synthesis is a normal metabolic activity of wooddecomposing fungi, and proceeds through the C–C bond cleavage reaction which is normally accompanied with kinetic isotope fractionation (Dutton and Evans, 1996; Shimada et al., 1997); that is, 13C-depleted oxalic acid is produced with relatively 13C-enriched carbon being left behind in the fungal hyphae. Net production of oxalic acid is generally higher in the brown rot fungi than the white rot fungi, because of the well-developed oxalate recycling enzymatic systems exclusively present in the latter (Dutton and Evans, 1996; Shimada et al., 1997; Munir et al., 2001). In addition, Akamatsu et al. (1994) showed that oxalate biosynthesis of the brown rot fungi (L. sulphureus and T. palustris) was enhanced both under the nitrogen-depleted conditions and in the presence of xylose as the carbon source. Considering that beechwood contains little nitrogen (! 0.1 wt.%N) and a large amount of xylose, 13C enrichment of the fungal hyphae accompanied by oxalate production presumably occurred in the wood decomposing experiment and resulted in higher d13C values of the brown rot fungi (process 2–2). 4.2. Implication for field applications of d13C indices Combining our results with existing knowledge, we postulate the following six factors that can significantly affect the Dd13C and possibly operate in the natural habitats of the fungi. (Factor 1) The predominant ecological type in the fungal community: fungal community that is dominated by

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the symbiotic mycorrhizal species should have a lower average Dd13C value than that dominated by saprotrophic fungi, because mycorrhizal fungi are known to be depleted in d13C (Hobbie et al., 1999; Kohzu et al., 1999). (Factor 2) Phylogenetic trait of the predominant saprotrophic fungi: when eubasidiomycetes become dominant in the saprotrophic fungal community, the average Dd13C values would increase. In this sense, successional stage during litter decomposition would be important for the Dd13C values because eubasidiomycetes generally predominate in late successional stages (Frankland, 1992). (Factor 3) The type of decomposition: results shown in Figs. 2 and 3 suggested large Dd13C difference among different types of decomposition (i.e. soft rot, white rot and brown rot) through different activity of lignin degradation and oxalate production. The average Dd13C in the field has the information about the predominance type of decomposition. (Factor 4) Exchange rate of fungal ambient CO2 with atmospheric CO2: the d13C value of the CO2 around fungal body depends on the exchange of the ambient air with outer atmosphere. With increasing exchange rate, the respiratory CO2 with low d13C values (normally !K20‰) occupies a smaller part of the CO2 pool around fungal body, and d13C values of the ambient CO2 increase. As a result, the air exchange rate can exert a significant effect on the Dd13C values of the fungi that have a significant dark CO2 fixing activity. (Factor 5) The substrate d13C values for fungi: d13C values of the fungal body in the field dominated by C4 or CAM plants may be high, while those of fungi in the C3 plants dominated fields may be low. In the fields where the d13C values of the fungal body are lower, dark CO2 fixation by fungi should increase the Dd13C value more. (Factor 6) Depletion in energy source for fungi: when carbohydrates are depleted, the activity of gluconeogenesis increases, and the d13C values of the fungal cell wall components (glucan and chitin) increase. Fungi which survive under carbon-limited conditions would show large Dd13C value (see between arrows in Fig. 3). In the field, the biome types, such as grassland vs. forest, coniferous vs. broad-leaved forests and deciduous vs. evergreen forests, are the major factors that control the predominant ecological type in the fungal community (Factor 1), the predominant type of decomposition (Factor 3), and the d13C values of substrate organic matter (Factor 5). On the other hand, seasonal changes, especially those associated with rainy and litter-accumulating seasons, should affect the successional stage of the fungal community (Factor 2) and the degree of limitation of available carbohydrates (Factor 6). In addition, fungal habitat structure, including the depth in the soils, can affect the air exchange rates (Factor 4). In future studies, it should be elaborated how the difference in each of these ecological conditions actually controls the d13C

signature of fungi as well as that of the litter-feeding soil invertebrates.

Acknowledgements We are indebted to I. Tayasu and K. Koba for their critical comments on the early manuscripts of this study. We also thank the students at Research Institute for Sustainable Humanosphere, Kyoto University for their logistical support. This work was supported by grants from the Research for the Future Program, Japan Society for the Promotion of Science and from CREST project, Japan Science and Technology Agency.

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