N concentration controls decomposition rates of different strains of ectomycorrhizal fungi

fungal ecology 2 (2009) 197–202

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N concentration controls decomposition rates of different strains of ectomycorrhizal fungi Roger T. KOIDEa,b,*, Glenna M. MALCOLMb,1 a

Department of Horticulture, The Pennsylvania State University, University Park, PA 16802, USA Intercollege Graduate Degree Program in Ecology, The Pennsylvania State University, University Park, PA 16802, USA

b

article info

abstract

Article history:

The decomposition of soil organic matter in forest ecosystems is important in two ways.

Received 30 January 2009

First, soil organic matter is the largest pool of C in terrestrial ecosystems, so understanding

Revision received 8 June 2009

global carbon cycling requires an appreciation of the factors that control the size of that

Accepted 17 June 2009

pool and the fluxes through it. Among these factors are those that control the rate of

Published online 24 July 2009

organic matter decomposition. Second, organic matter decomposition is the major process

Corresponding editor: Bjo¨rn Lindahl

controlling the supply of nutrients to plants. In some ecosystems ectomycorrhizal fungi comprise a surprisingly large fraction of soil organic matter. However, little is known of the

Keywords:

rates of decomposition of ectomycorrhizal fungi, or of the factors that control those rates.

Community structure

Therefore, we set out to examine the relationship between N concentrations and decom-

Decomposition

position rates of ectomycorrhizal fungi using a wide variety of strains isolated from a Pinus

Ectomycorrhizal fungi

resinosa plantation. We found that substantial variation among strains existed in decom-

N concentration

position rate, and that decomposition rate was highly correlated with tissue N concen-

N cycling

tration. We conclude, therefore, that the structures of ectomycorrhizal fungal communities

Soil C sequestration

may be ecologically important in terms of ecosystem C and N dynamics. ª 2009 Elsevier Ltd and The British Mycological Society. All rights reserved.

Introduction The decomposition of soil organic matter in forest ecosystems is important for two major reasons. First, soil organic matter is the largest pool of C in terrestrial ecosystems, accounting for two to three times as much C as terrestrial vegetation (Dixon et al. 1994; Schimel 1995; Batjes 1996; Garten et al. 1999; Jobba´gy & Jackson 2000; Houghton 2007). The potential to store C as organic matter in soils makes it one of the most important considerations in any attempt to mitigate rising atmospheric CO2 concentrations (Batjes 1998). Therefore, we need to understand the factors that control the size of the soil carbon pool, including the factors controlling organic matter decomposition (Aber et al. 1990; King et al. 1997). Second, in

natural ecosystems organic matter decomposition is the major process controlling the supply of plant nutrients (Swift et al. 1979; Attiwill & Adams 1993). The contribution to soil organic matter by microbial biomass and necromass may be significant in forest soils (Bauhus & Barthel 1995; Ho¨gberg & Ho¨gberg 2002), but not all microbes have a net positive effect on C sequestration. The C in saprotrophic microorganisms is only a small fraction of the C originally found in the organic matter they consume, the vast majority of which is lost to the atmosphere as respired CO2. In contrast, ectomycorrhizal fungi can retard organic matter decomposition by inhibiting the activities of saprotrophic microorganisms (Koide & Wu 2003 and references therein). Moreover, the biotrophic or largely biotrophic ectomycorrhizal

* Corresponding author. Department of Horticulture, The Pennsylvania State University, 102 Tyson Building, University Park, PA 16802, USA. Tel.: þ1 814 863 0710; fax: þ1 814 863 6139. E-mail address: [email protected] (R.T. Koide). 1 Present address: Department of Plant Pathology, The Pennsylvania State University, University Park, PA 16802, USA. 1754-5048/$ – see front matter ª 2009 Elsevier Ltd and The British Mycological Society. All rights reserved. doi:10.1016/j.funeco.2009.06.001

198

fungi obtain C from live host plants (Wallander et al. 2004; Koide et al. 2008). Their biomass and necromass is, therefore, analogous to plant biomass and litter in terms of C cycling. In certain forest ecosystems, ectomycorrhizal fungi account for a third of total soil microbial biomass (Ho¨gberg & Ho¨gberg 2002) and 47–84 % of total soil fungal biomass (Ba˚a˚th et al. 2004). Wallander et al. (2004) indicated that ectomycorrhizal fungi have a standing crop of between 4 800 and 5 800 kg ha1 to a depth of 70 cm in Swedish spruce (Picea abies) and mixed spruce stands, respectively, of the same order of magnitude as the contribution made by roots. To put this in relation to aboveground allocation, the leaf:fine root ratio was shown to vary between 2.1 and 6.4 for Norway spruce (P. abies) stands in Finland (Helmisaari et al. 2007). Thus, the size of the standing crop of ectomycorrhizal fungi can be significant, requiring approximately 20 % of net photosynthesis of host trees for production and maintenance (Hobbie 2006). Ectomycorrhizal fungi, like other higher fungi, possess cell walls containing chitin and protein in relatively high concentrations (Cooke & Whipps 1993). Thus, their tissues contain considerable concentrations of nitrogen (Clinton et al. 1999; Langley & Hungate 2003; Rudawska & Leski 2005). This, coupled with a relatively large abundance in forest ecosystems, results in ectomycorrhizal fungi being a significant source of N upon their decomposition (Colpaert et al. 1996; Wallander et al. 2004). Little is known of the rates of natural decomposition of ectomycorrhizal fungi, or of the factors that control those rates. Several factors control decomposition of plant tissues, among which one of the most important in the early stages of decomposition is tissue N concentration or C:N ratio (Taylor et al. 1989; Cotrufo et al. 1994; Fenn 1991; Aerts 1997; Berg 2000). While fungal tissues can contain high concentrations of N, some evidence suggests that this N is not easily accessible and thus does not control decomposition (Langley & Hungate 2003) as it can for plant tissues. Because the extent to which N concentration of fungal tissue controls decomposition is not clear, we set out to examine this relationship using a wide variety of ectomycorrhizal fungal strains grown in nutrient solutions varying in C:N ratio.

Materials and methods Two experiments were performed in which fungi produced in liquid cultures were rinsed in distilled water, dried at 70  C, placed into nylon mesh envelopes (38 mm openings) and heat sealed prior to burial in a Pinus resinosa plantation (for a description of the plantation, see Koide & Wu 2003). In both cases an attempt was made to alter the N:C ratio of the fungal tissues. In Experiment 1, fungi were grown with higher and lower concentrations of glucose, keeping the N availability constant. In Experiment 2, fungi were grown with higher and lower concentrations of available nitrogen, keeping the glucose availability constant.

Experiment 1 Both the standard and low carbon nutrient solutions were modifications of Melin–Norkrans solutions without agar. Both

R.T. Koide, G.M. Malcolm

solutions contained (in g l1) KH2PO4, 0.5; MgSO4 $ 7H2O, 0.25; NaCl, 0.025; CaCl2, 0.05; NH4Cl, 0.25; Yeast extract, 0.4; and (in mg l1) NaFeEDTA, 8.0; KI, 0.75; MnCl2 $ 4H2O, 6.0; ZnSO4 $ 7H2O, 2.6; H3BO3, 1.5; CuSO4 $ 5H2O, 0.13; Na2MoO4 $ 2H2O, 0.0024. The standard and low glucose solutions contained, respectively, 8.0 and 2.0 g l1 glucose. Four strains were grown for various periods: Amanita brunnescens (SC040, grown for 60 d), Cenococcum geophilum (SC052, 40 d), Suillus intermedius (SC065, 44 d), and Tricholoma cf. flavovirens (SC104, 45 d), each of which was isolated from the P. resinosa plantation where the samples were buried. One subsample of tissue from each species  treatment combination was analyzed for total N and total C content by combustion (Pella 1990; Horneck & Miller 1998) using an Elementar Vario Max N/C Analyzer (Hanau, Germany). We prepared for burial three samples for each species  nutrient solution combination, for a total of 24 samples. Each sample was dried in an oven at 70 C, weighed, sealed in a 6 cm  6 cm nylon mesh envelope and, on 7 May 2008, buried 5–8 cm below the surface of the forest floor within the F-layer in three, randomly located complete blocks, each block containing both treatments of all strains. The mean starting dry weight of the samples was 63 mg. On 11 Jun. 2008, the samples were collected, dried and weighed.

Experiment 2 The standard nutrient solution in this experiment was the same as for Experiment 1. The high N nutrient solution contained three times the N concentration (0.75 g l1 NH4Cl) as in the standard solution. Six strains were grown for various periods: Amanita muscaria var. formosa (BX008, grown for 48 d), C. geophilum (SC052, 48 d), Lactarius chrysorheus (SC098, 54 d), Rozites caperata (SC099, 39 d), Scleroderma citrinum (SC038, 54 d), and S. intermedius (BX007, 44 d). Again, each of these strains had been isolated from the P. resinosa plantation where the samples were buried. One subsample of tissue from each species  treatment combination was analyzed for total N and total C content by combustion (Pella 1990; Horneck & Miller 1998) using an Elementar Vario Max N/C Analyzer (Hanau, Germany). We prepared for burial four samples for each species  nutrient solution combination, for a total of 48 samples. Each sample was prepared and buried on the same date as in Experiment 1, but this time there were 4 randomly located complete blocks. The mean starting dry weight of the samples was 208 mg. On 11 Jun. 2008, the samples were collected, dried and weighed. Decomposition (% loss) comparisons were made using analysis of variance (in the Statgraphics programs (STSC 1991). A correlation analysis was also performed for decomposition and tissue N concentration using the Statgraphics programs (STSC 1991).

Results In Experiment 1, treatment (low glucose vs. standard glucose) did not significantly affect tissue N concentration (p ¼ 0.351), tissue C concentration ( p ¼ 0.338) or C:N ratio ( p ¼ 0.705). Similarly, in Experiment 2, treatment (high N vs. standard N) did not significantly affect tissue N concentration ( p ¼ 0.509),

Decomposition of ectomycorrhizal fungal tissues

199

In Experiment 1, decomposition (% dry weight loss) was significantly affected by strain ( p < 0.0001; Fig 2A) but not by treatment (low glucose vs. standard glucose, p ¼ 0.8886). Moreover, the interaction between strain and treatment was not significant (p ¼ 0.4271). For Experiment 2, decomposition (% dry weight loss) was significantly affected by strain ( p < 0.0001), but not by treatment (high N vs. standard N, p ¼ 0.3825, Fig 2B). In this case the interaction between strain and treatment was significant ( p < 0.0001), but according to the sums of squares of the analysis of variance, the vast majority of variation (83 %) in decomposition was due to strain, and only

43.6 (0.58)b 55.7 (0.58)a 42.2 (0.58)b 42.5 (0.58)b

7.95 20.06 10.68 12.64

(1.62)b (1.62)a (1.62)b (1.62)b

B. Experiment 2 SC052 2.91 SC099 4.36 SC038 5.37 BX007 4.42 SC098 2.26 BX008 4.76

(0.38)b (0.38)a (0.38)a (0.38)a (0.38)b (0.38)a

54.9 (1.55)a 48.4 (1.55)b 47.2 (1.55)b 46.5 (1.55)b 44.6 (1.55)b 46.3 (1.55)b

18.84 11.24 8.83 10.52 22.13 9.73

(3.09)ab (3.09)bc (3.09)c (3.09)c (3.09)a (3.09)c

tissue C concentration ( p ¼ 0.675) or C:N ratio ( p ¼ 0.428). In contrast, considerable variation existed among strains in tissue N concentration in both Experiment 1 ( p ¼ 0.040, Table 1A) and Experiment 2 ( p ¼ 0.013, Table 1B). Strain SC052 (C. geophilum) had significantly greater C concentrations than all other strains in both Experiment 1 ( p ¼ 0.001, Table 1A) and Experiment 2 ( p ¼ 0.043, Table 1B). The mean C concentration of SC052 was 55.3 %, while the mean of all other strains was 45.1 %. However, much less variation occurred among strains in tissue C concentration than in tissue N concentration. Therefore, the significant variation among strains in C:N ratio (Experiment 1: p ¼ 0.044; Experiment 2: p ¼ 0.046) was principally due to variation in N concentration (Fig 1), indicating that for most species N concentration was a good proxy for C:N ratio.

Decomposition Experiments 1 & 2 70

50 40 30

100

A

a

80

60

b

b c

40

20

0

Decomposition Experiment 2, June 2008

B 80

b b

60

40

20

Growth in standard medium Growth in high N medium

a

b

b c

d d

d

d d e

0 S C C0 .g 5 eo 2 ph ilu SC m R 0 .c 9 ap 9 er a BX ta A. 0 m 08 us BX car ia S. 0 in 07 te rm ed iu SC s S. 0 ci 38 tr SC inu m L. 0 ch 98 ry so rh eu s

C concentration (%)

60

Decomposition Experiment 1, June 2008 100

iu s S A. C0 br 40 un ne sc en s S T. C1 cf 04 .f la vo vi re ns

(0.35)a (0.35)b (0.35)ab (0.35)b

m

A. Experiment 1 SC040 5.49 SC052 2.80 SC065 4.04 SC104 3.40

S S. C0 in 65 te rm ed

C:N ratio

S C C0 .g 5 eo 2 ph ilu

Nitrogen conc. (%) Carbon conc. (%)

Decomposition of ECM fungi (% loss)

Strain

Decomposition of ECM fungi (% loss)

Table 1 – Means (pooled standard errors) for N and C concentrations, and C:N ratios of tissues used in the decomposition study. Different letters indicate significantly different means according to the least significant difference method ( p < 0.05). n [ 2

20 10 0

0

1

2

3

4

5

6

N concentration (%) Fig 1 – The relationship between N and C concentrations of the tissues used in Experiments 1 and 2 of the decomposition study (mean of each treatment of each strain). The data points for strain SC052 (Cenococcum geophilum) are black to highlight its variation from all other species in C concentration. The figure indicates that the great majority of variation among strains in C:N ratio is due to variation in N concentration alone.

Fig 2 – Decomposition (% dry weight loss) of tissues of ectomycorrhizal fungi placed within the F-layer of a Pinus resinosa (Ait) plantation from 7 May to 11 Jun. 2008. Error bars are ±1 pooled standard errors of the mean. Experiments 1 and 2 were run concurrently. (A) Results from Experiment 1. n [ 6 replicates per strain. Treatments were not significantly different (p > 0.05), and hence were combined within strains. (B) Results from Experiment 2. n [ 4 per treatment per strain. Both treatments are shown for each strain due to the significant (p £ 0.05) interaction between treatment and strain. The same letter above a bar indicates no significant difference (p > 0.05).

200

R.T. Koide, G.M. Malcolm

8 % was attributable to the interaction term. Significant variation in decomposition occurred among strains in these Experiments, ranging from an average of approximately 20 % loss by SC098 (L. chrysorheus, Experiment 2, Fig 2B) to approximately 80 % loss by SC040 (A. brunnescens, Experiment 1, Fig 2A). There was a significant correlation between decomposition and tissue N concentration among the 10 strains (Fig 3). Because of the interaction between treatment and strain in Experiment 2, the means for both treatments of each strain are shown in the relationship between decomposition and tissue N concentration (Fig 3). Compared to the other strains, SC038 (S. citrinum) had a slightly higher tissue N concentration for a given decomposition rate, and the correlation coefficient was much larger without SC038 (r2 ¼ 0.82, p < 0.0001) than with SC038 in the model (r2 ¼ 0.46, p ¼ 0.001).

Discussion Fungal tissue N and C concentrations are probably determined largely by cell wall chemistry (Wallander et al. 2003) and, because cell wall function is determined by its composition, it was not entirely surprising to find that manipulation of nutrient content of the growth medium did not significantly influence mycelial N or C concentrations or C:N ratios. In fact, sporocarp C:N ratio or N concentration appears to be remarkably constant irrespective of substrate, location or fungal trophic status (Clinton et al. 1999; Hart et al. 2006). There was, however, significant variability among strains of ectomycorrhizal fungi in tissue N concentration, and significant variability among strains in mycelial decomposition rate. Because a significant proportion of the variability in decomposition was accounted for by tissue N concentration, decomposition of these fungal tissues appears to be controlled

Decomposition (% loss)

100 SC099 SC040

80 BX008 SC040 SC099

60

BX007 BX008

SC104 SC104 SC065

SC052 SC052 SC052 SC098

40

SC065 BX007

SC038 SC038

SC052

20 SC098

0

0

1

2

3

4

5

6

Tissue N concentration (%) Fig 3 – The relationship between tissue N concentration and decomposition (% dry weight loss). Data are for mean of each treatment of each strain from Experiments 1 and 2, the samples of which were placed within the F-layer alongside each other in the same blocks and at the same time. The equation of the best fit line excluding SC038: Decomposition [ 16.1(%N) L 7.9. r2 [ 0.82, p < 0.0001. The equation of the best fit line including SC038: Decomposition [ 11.3 (%N) D 6.9. r2 [ 0.46, p [ 0.001.

in a similar fashion to many plant tissues. For plant tissues, N concentration is frequently among the best predictors of decomposition rate in the early stages (Taylor et al. 1989; Fenn 1991; Enrı´quez et al. 1993; Berg 2000). Our discovery of the relationship between N concentration and decomposition for ectomycorrhizal fungal tissues suggests that many of the factors that control decomposition of plant litter probably also control decomposition of fungal litter, and this may simplify the modeling of decomposition of this important forest soil pool. Because strains vary significantly in N concentration, because N concentration controls decomposition rate, and because decomposition rate can influence the size of the C pool of each strain, the structure of the ectomycorrhizal fungal community may influence ecosystem C sequestration. At this point, however, this is difficult to determine because of our inability to measure the biomass and necromass of individual strains in the field. In any case, the fluxes of C through the C pools represented by each strain may be quite variable. The significant interaction between treatment and strain with regard to decomposition occurred because for most strains there were no significant differences in decomposition between tissues grown in high and standard N media, but for two strains (SC099-R. caperata and BX007-S. intermedius) the rate of decomposition was higher for tissues grown in high N medium and for one strain (SC098-L. chrysorheus) the rate of decomposition was higher for tissues grown in standard N medium. This is difficult to explain at present because we have no evidence that N concentrations were significantly different between tissues produced in high and standard N media. Thus, variation in decomposition may be controlled to some extent by factors other than tissue N concentration. However, such factors do not appear to be as important as strain variation because far more variability in decomposition was ascribable to variation among strains than variation due to differences between nutrient solutions. Some authors have hypothesized that fungal tissues containing high concentrations of pigmented substances, such as melanins, are more resistant to decomposition than tissues without them (Butler & Day 1998). Strain SC052 (C. geophilum) is dark and contains melanins (Paris et al. 1993), and it was among the most slowly decomposing strains in the present study. However, strain SC098 (L. chrysorheus) is light colored and slowly decomposing, while strain SC099 (R. caperata) is dark and rapidly decomposing. Moreover, decomposition of each of these strains appears to be well explained by tissue N concentration without invoking any effect of melanins. Strain SC038 (S. citrinum) appeared to be exceptional as it had an unexpectedly low decomposition rate given its rather high N concentration. The reason for this is unclear but many factors may have contributed, e.g. its hydrophobicity. Hyphae of S. citrinum can be hydrophobic (personal observations, see also color photos in Agerer 1997–2002) and this may reduce wettability and retard decomposition (Lu¨tzow et al. 2006). While the chemical composition of individual hyphae and rhizomorphs of a given strain are likely to be similar, rhizomorphs may be more resistant to decomposition than individual hyphae because of differences in the relationship between effective surface area and volume. Thus, variation among species in rhizomorph production could lead to variation in decomposition rate. In the present study, the tissues

Decomposition of ectomycorrhizal fungal tissues

used in the decomposition experiments were not rhizomorphic. The liquid cultures produced only individual hyphae. In future studies it would be valuable to compare the decomposition rate of individual hyphae and cords/rhizomorphs. In addition to containing C, the tissues of ectomycorrhizal fungi also contain substantial quantities of N. According to Wallander et al. (2004), the standing crop of ectomycorrhizal fungi in some ecosystems may contain between 121 and 187 kg N ha1. In a microcosm system, mycelium of ectomycorrhizal fungi retained a significant fraction (up to 32 %) of the available N in the system (Colpaert et al. 1996). Therefore, variation among strains in the rate of decomposition suggests that the structure of the ectomycorrhizal fungal community may influence ecosystem N dynamics. The C:N ratios of individual strains of ectomycorrhizal fungi in the present study varied from approximately 8 to 22, with a mean value of approximately 13.3. Wallander et al. (2003) found that the mean C:N ratio of mycelia from communities of ectomycorrhizal fungi in ingrowth bags from forest soils in southern Sweden was higher (approximately 20.2), with a range among individual replicate bags of 14–29. The reason for this difference is unknown, but it could be related to the fact that in the current study C:N ratios were determined on individual strains without regard to their natural abundance, while in the Wallander et al. (2003) study, C:N ratios were determined on communities of ectomycorrhizal fungi, the tissue qualities of which may differ from any individual species. Moreover, in the present experiment the fungi were grown in liquid cultures that contained relatively high concentrations of N, that may have led to a different C:N ratio compared to fungi growing in nature. In any case, significant differences among either species or communities of ectomycorrhizal fungi in C:N ratio suggest that they may decompose at different rates. Some authors have assumed that fungal cell walls are recalcitrant to decomposition relative to other common sources of litter (Treseder & Allen 2000; Langley & Hungate 2003; Godbold et al. 2006). However, we are aware of no direct comparisons of the decomposition of fungi and plant litter. Koide & Wu (2003) showed that the average initial rate of decomposition of forest floor litter (mostly pine needles) was about 10 % during 1 month in the red pine plantation in which the current study was performed (17 Jul.–16 Aug. 2000, see Koide & Wu 2003). The average initial rate of decomposition for the fungal tissues in the current study was approximately 50 % in 1 month (7 May–11 Jun. 2008, Fig 2). Some of this variation may have been due to different microclimatic conditions. Rainfall for 17 Jul.–16 Aug. 2000 was 5.7 cm, while rainfall for 7 May–11 Jun. 2008 was 10.2 cm, so it is quite possible that decomposition of any tissue would have been more rapid in the current study than in 17 Jul.–16 Aug. 2000. Nevertheless, our limited evidence suggests that fungal tissues, if anything, are less recalcitrant to decomposition than plant tissues, at least in the early phases of decomposition. Whether fungal tissues are more or less resistant to decomposition than plant tissues is ecologically consequential. For example, some have speculated that the relative recalcitrance of ectomycorrhizal fungal tissues should result in a reduction in the rate of decomposition of colonized fine roots (Langley & Hungate 2003). Langley et al. (2006) did, in fact,

201

show clearly that in a pinyon pine (Pinus edulis) system, ectomycorrhizal fungal colonization of roots substantially decreased the rate of fine root decomposition despite increasing N concentration. However, whether ectomycorrhizal fungal tissues are more recalcitrant than plant tissues remains to be demonstrated. The fungi colonizing the roots of the pinyon pines in the study by Langley et al. (2006) may frequently be ascomycetes (Gehring et al. 1998). It is possible that some peculiar trait(s) of ascomycetes makes them more recalcitrant to decomposition than the basidiomycetes of the current study. More experimentation will obviously be necessary in order to determine general effects of ectomycorrhizal fungi on fine root decomposition.

Acknowledgements The authors acknowledge the National Science Foundation and the Northeast SunGrant Initiative for funding.

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