Litter quality control of decomposition of leaves, twigs, and sapwood by the white-rot fungus Trametes versicolor

European Journal of Soil Biology 80 (2017) 1e8

Contents lists available at ScienceDirect

European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi

Original article

Litter quality control of decomposition of leaves, twigs, and sapwood by the white-rot fungus Trametes versicolor Takuya Hishinuma a, Jun-ichi Azuma b, Takashi Osono c, *, Hiroshi Takeda c a

Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan c Faculty of Science and Engineering, Doshisha University, Kyoto 610-0394, Japan b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 September 2016 Received in revised form 12 February 2017 Accepted 10 March 2017

Litter quality plays predominant roles in plant litter decomposition by modifying the activity of decomposer fungi, but little is known about the response of the decomposing activity of individual fungal strains to variations in litter quality. In the present study, the variability in the decomposing ability of a single fungal strain (Trametes versicolor IFO30340) was quantified under pure culture conditions to elucidate litter quality measures that control the decomposition. We used a total of 72 litters from 51 plant species, including leaves, twigs, sapwood, bark, heartwood, and petioles of broad-leaved trees, coniferous trees, and grass. Mass loss of litter caused by the fungus ranged from 0.9 to 59.8% of the original litter mass, was significantly higher in leaves, twigs, and petioles than in heartwood, and was significantly higher in broad-leaved than in coniferous litter. Tissue type (leaf, sapwood, or twig) and the relative amount of acid-unhydrolyzable residues to total nitrogen were selected as predictor variables of the mass loss of litters. Fourier transform infrared (FT-IR) spectroscopy showed that guaiacyl units of lignin negatively affected the fungal decomposition. © 2017 Elsevier Masson SAS. All rights reserved.

Handling Editor: Christoph Tebbe Keywords: Acid-unhydrolyzable residue FR-IR spectroscopy Leaf Ligninolytic fungi Nitrogen Wood decay

1. Introduction Decomposition processes of plant litter in soil are controlled by interactions of climatic conditions, litter quality, and decomposer organisms [1]. Under a given climatic condition, litter quality plays predominant roles in regulating the decomposition by modifying the activity of decomposer microbes, including fungi [2,3]. For example, plant tissues richer in recalcitrant organic compounds, such as lignin and polyphenols, decompose more slowly [4], whereas higher nitrogen (N) and phosphorus contents in litter are associated with faster decomposition [5,6]. Such variations in decomposition in relation to litter quality are attributed to either changes in the species composition of decomposer fungal assemblages or plasticity of individual fungal species (or individual strains) according to litter quality. Of these two processes, previous studies mostly focused on the shifts of fungal assemblages in relation to the litter quality [7e9]. In contrast, less is known about the response of the

* Corresponding author. E-mail address: [email protected] (T. Osono). http://dx.doi.org/10.1016/j.ejsobi.2017.03.002 1164-5563/© 2017 Elsevier Masson SAS. All rights reserved.

decomposing activity of individual fungi to variations in the quality of different litters, including leaves, twigs, and sapwood of broadleaved and coniferous trees with different levels of nitrogen and recalcitrant organic compounds. Only a few pure-culture experiments have documented the changes in decomposition in relation to litter quality. Mikola [10] showed that the mass loss of leaf litter varied when leaf litters of 25 plant species differing in chemical quality were inoculated with fungal isolates under constant conditions. Osono et al. [11] inoculated a total of 13 litter types with 10 Xylaria isolates under pure culture conditions and found that the fungal isolates caused greater mass loss of leaves than wood, and that the mass loss was related negatively to the lignin content and positively to the nitrogen content of the litters. Still, an integrative survey is lacking on the variability in the decomposing activity of individual fungal species or strains when inoculated to diverse litters from different plant species and tissues varied in chemical quality. The purposes of the present study were to quantify the response of the decomposing ability of a single fungal strain when inoculated to diverse plant litters under pure culture conditions and to elucidate litter quality measures that control the decomposition. Specifically, the effects of recalcitrant organic compounds and N in

2

T. Hishinuma et al. / European Journal of Soil Biology 80 (2017) 1e8

litter were investigated as potential limiting factors of the fungal decomposition. We used a total of 72 litters from 51 plant species, including leaves, twigs, sapwood, bark, heartwood, and petioles of broad-leaved trees, coniferous trees, and grass, which varied in the content of recalcitrant organic compounds, polymer carbohydrates, and N. Trametes versicolor (L.) Lloyd IFO30340 was chosen as a test strain to achieve our purposes. Trametes versicolor is a common white rot fungus of wood producing a suite of extracellular enzymes degrading recalcitrant organic compounds including lignin, such as laccase and manganese peroxidase [12e14]. Strain IFO30340 registered in a culture collection has been used as one of the model strains for extracellular enzymatic systems responsible for decomposition [15,16] and also as a standard for the decomposition test of timber under the Japanese Industrial Standard (JIS). Finally, we applied Fourier transform infrared (FT-IR) spectroscopy [17] to the analysis of chemical structure of lignin and carbohydrate components in twigs and sapwoods, two major substrata of T. versicolor in the field, to obtain further insights into the litter quality control on decomposition. 2. Materials and methods 2.1. Source of litter and fungal isolate A total of 72 litters from 51 plant species were used in the present study, including six tissues (27 leaves, 17 twigs, 19 sapwoods, 3 barks, 3 heartwoods, and 3 petioles) of three plant types (60 litters of broad-leaved trees, 11 of coniferous trees, and one grass) (detailed in Table A.1). Leaves in the present study denote lamina of newly shed leaves without obvious fungal or faunal attack, cut into pieces 1e1.5 cm in width. Twigs denote woody tissue of live trees less than 5 mm in diameter, cut into pieces 2e3 cm in length, and enclosed in thin bark. Sapwood and heartwood denote wood blocks of trees (approximately 10
10
5 mm) from live trees devoid of bark. Barks were derived from live trees and cut into blocks (approximately 10
10 mm, 5e10 mm in thickness). Of the 72 litters, leaves of 15 tree species were collected in a cool temperate forest located in northern Kyoto, Japan, in November 2000 [18]. Twigs, sapwood, and heartwood of 17, 13, and 3 tree species were collected in temperate forests near Kyoto city from January 2001 to March 2003. These materials were oven-dried at 40  C for one week and preserved in vinyl bags until the experiment was started. Data of the decomposition of 10 other litters were derived from previous pure culture tests conducted in the same laboratory and published by the same authors [19e25], and those of the decomposition of 14 litters were derived from an unpublished dataset related to papers published by the same authors [11,26] (detailed in Appendix A). The strain of Trametes versicolor IFO30340 deposited in the Institute of Fermentation, Osaka (IFO), Osaka, Japan, was used in all pure culture tests. 2.2. Pure culture decomposition test An individual pure culture decomposition test consisted of one fungal isolate inoculated to one of the 72 litters. Litters (0.2e1.0 g per dish, in most cases 0.2e0.4 g per dish) were sterilized by exposure to ethylene oxide gas at 60  C for 6 h and used in the tests according to the methods described in Osono [27]. The sterilized litters were placed on the surface of Petri dishes (9-cm diameter) containing 20 ml of 2% agar. Inocula for each assessment were cut out of the margin of previously inoculated Petri dishes on 1% MEA with a sterile cork borer (6 mm diameter) and placed on the agar adjacent to the litters, one plug per dish. The dishes were incubated for 12 weeks in the dark at 20  C. The dishes were sealed firmly

with laboratory film during incubation so that moisture did not limit decomposition on the agar. After incubation, the litters were retrieved, oven-dried at 40  C for 1 week, and weighed. The initial, undecomposed litters were also sterilized, oven-dried at 40  C for 1 week, and weighed to determine the original mass. These initial litters were used to determine the initial proportion of dry mass and the initial chemical property. Three to four plates were prepared for each test, and four uninoculated plates served as a control. Mass loss of litter was determined as a percentage of the original mass, taking the mass loss of litter in the uninoculated and incubated control treatment into account, and the mean values were calculated for each litter. Prior to these tests, the sterilized litters were placed on 1% MEA, and after 8 weeks of incubation at 20  C in darkness, no microbial colonies had developed on the plates. This assured the effectiveness of the sterilization method used in the present study. 2.3. Proximate chemical analyses Litter materials used in the pure culture tests were ground in a laboratory mill (0.5-mm screen) and used for chemical analyses according to the method described in Osono et al. [28]. The amount of acid-unhydrolyzable residue (AUR) in the samples was estimated by means of gravimetry as acid-insoluble residue, using hot sulfuric acid digestion [29]. Samples were extracted with alcohol-benzene at room temperature (15e20  C), and the residue was treated with 72% sulfuric acid (v/v) for 2 h at room temperature with occasional stirring. The mixture was diluted with distilled water to make a 2.5% sulfuric acid solution and autoclaved at 120  C for 60 min. After cooling, the residue was filtered and washed with water through a porous crucible (G4), dried at 105  C and weighed as AUR. This AUR fraction contains a mixture of organic compounds in various proportions, including condensed tannins, phenolic and carboxylic compounds, alkyl compounds such as cutins, and true lignin [30]. The filtrate (autoclaved sulfuric acid solution) was used for total carbohydrate (TCH) analysis. The amount of carbohydrates in the filtrate was measured by means of the phenol-sulfuric acid method [31]. One milliliter of 5% phenol (v/v) and 5 mL of 98% sulfuric acid (v/v) were added to the filtrate. The optical density of the solution was measured using a spectrophotometer at 490 nm, using known concentrations of D-glucose as standards. Total nitrogen content was measured by means of automatic gas chromatography (NC analyzer SUMIGRAPH NC-900, Sumitomo Chemical Co., Osaka, Japan). The relative amount of AUR and total N (AUR/N) is a useful index of the substrate quality and was calculated according to the equation: AUR/N ¼ AUR content (mg/g)/total N content (mg/g). 2.4. FT-IR analysis FT-IR spectroscopy measures the absorbance versus wavenumber (or equivalently, wavelength) of light to detect the vibration characteristics of chemical functional groups in a sample (Table 1) [32]. A total of 30 litters, including twigs of 17 tree species and sapwood of 13 tree species, were used for FT-IR analysis in the solid phase (Table B.1). Each ground sample was embedded in KBr and compacted into a disc using a bench press. FT-IR spectra were recorded in the absorbance mode at a resolution of 4 cm1 with wavenumber range of 400e4000 cm1, using FT/IR-4100 (JASCO Co., Tokyo, Japan). Each spectrum was composed of 100 scans. FT-IR spectra were baseline-corrected and normalized using JASCO Spectra Manager, Version 2 (JASCO Co., Tokyo, Japan). A total of 15 peaks that reflect functional groups associated with the lignocellulose matrix in litter were selected in the fingerprint region (800-2000 cm1) (Table 1). Peak heights were expressed as

T. Hishinuma et al. / European Journal of Soil Biology 80 (2017) 1e8

3

Table 1 Band origins of the 15 peaks detected in FT-IR spectra. The assignment followed Osono et al. [35]. G, guaiacylpropane; S, syringylpropane. Wavenumber (cm1)

Band origin

1735 1658 1593 1505 1462 1422 1367 1329 1266 1227 1126 1033 925 835 817

C¼O stretch in unconjugated ketones, carbonyls and in ester groups (frequently of carbohydrate origin); conjugated aldehydes and carboxylic acids C¼O stretch; in conjugated p-subst. aryl ketones; strong electronegative substituents lower the wavenumber aromatic skeletal vibrations plus C¼O stretch; S > G; G condensed > G etherified aromatic skeletal vibrations; G > S C-H deformations; asymmetry in -CH3 and -CH2aromatic skeletal vibrations combined with C-H in-plane deformations aliphatic C-H stretch in CH3, not in OCH3; phenolic OH S ring plus G ring condensed; (i.e., G ring substituted in position 5) G ring plus C¼O stretch C-C plus C-O plus C¼O stretch; G condensed > G etherified aromatic C-H in-plane deformation (typical for S units); plus secondary alcohols plus C¼O stretch aromatic C-H in-plane deformation, G > S; plus C-O deformation in primary alcohols; plus C¼O stretch (unconjugated) C-H out-of-plane; aromatic C-H out-of-plane in positions 2 and 6 of S, and in all positions of H units C-H out-of-plane in positions 2, 5, and 6 of G units

relative heights using the largest peak, a peak at 1028-1059 cm1 (Table 1). Original data of the relative height of 15 peaks in the spectra are given in Table B.1.

2.5. Data analysis In the present study, plant species and tissue types were not fully crossed, and the number and identity of plant species varied across tissue types, due to the limitation of sample preparation and data collection. However, these resulted in differences in compositional variation and could lead to potential biases in interpreting the statistical analyses. Therefore, we applied a series of statistical analysis to selected sub-datasets to extract possible tendencies. First, differences among the six tissues (leaf, twig, sapwood, bark, heartwood, petiole) in the contents of AUR, TCH, and total N, AUR/N and the mass loss of 71 litters (to exclude one grass leaf) caused by T. versicolor IFO30340 were examined with generalized linear models (GLMs) with a Gaussian distribution. The grass leaf was not included in the model because of no replication for this category. Secondly, GLMs were used with tissue (leaf, twig, sapwood) and litter type (broad-leaved, coniferous) as categorical predictors to evaluate the effect of tissue, litter type, and the interaction of tissue and type on the contents and the mass loss of 62 litters (26 leaves, 17 twigs, and 19 sapwoods). Thirdly, factors affecting the mass loss of 62 litters were analyzed with GLM with tissue (leaf, twig, sapwood), type (broad-leaved, coniferous) as categorical predictors, and the chemical properties (AUR, TCH, total N, AUR/N) as continuous predictors. Similarly, factors affecting the mass loss of leaves, twigs, and sapwood were analyzed with GLMs with type as a categorical predictor, and the chemical properties as continuous predictors. Using the results of FT-IR analysis, differences in the relative heights of 15 peaks in FT-IR spectra were examined with GLMs with a Gaussian distribution using tissue (twig, sapwood) and type (broad-leaved, coniferous) as categorical predictors. Factors affecting the mass loss of litter were analyzed with GLM with tissue and type as categorical predictors, and the relative heights of 15 peaks as continuous predictors. These GLMs were performed with the glm function of R version 3.0.2 for Mac (http://www.r-project. org) and with the glht function of the R multicomp package for multiple comparisons with Tukey's test. An automatic stepwise model selection with Akaike information criterion (AIC) was performed to find the most parsimonious model, using the minimum AIC as the best-fit estimator of GLMs.

3. Results 3.1. Proximate analysis of 72 litter The content of acid-unhydrolyzable residues (AUR) of 72 litter ranged from 88 to 454 mg/g, was not significantly different among the six tissues, and was significantly lower in broad-leaved than in coniferous litter (Table 2). The content of total carbohydrate (TCH) ranged from 235 to 712 mg/g, was significantly different among the tissues, and was significantly lower in broad-leaved than in coniferous litter (Table 2). Total N content ranged from 0.6 to 17.5 mg/g, was significantly higher in leaves than in other tissues, and higher in twigs, bark, and petioles than in sapwood or heartwood, and was significantly higher in broad-leaved than in coniferous litter (Table 2). The AUR/N ratio ranged from 8 to 484, was significantly higher in heartwood than in sapwood and higher in sapwood than in other tissues, and was not significantly different between broadleaved and coniferous litter (Table 2). A scatter plot of AUR versus total N content of 72 litters showed separate patterns for leaves, twigs, and sapwood (Fig. 1). That is, leaves showed a wider variation in AUR and higher total N content, whereas sapwood and heartwood showed less variation in AUR and lower total N content, and twigs, bark, and petioles showed intermediate AUR variation and total N content. 3.2. Mass loss of litter Mass loss of litter caused by T. versicolor IFO30340 ranged from 0.9 to 59.8% of the original litter mass, was significantly higher for leaves, twigs, and petioles than for heartwood, and was significantly higher for broad-leaved than for coniferous litter (Table 2). Overall, the mass loss of litter was positively correlated with nitrogen content and negatively correlated with AUR and TCH contents and AUR/N (Fig. 2). The tissue, AUR content, and AUR/N were selected as predictor variables of the mass loss of 62 litters and had significant regression coefficients ('All litter' column in Table 3). The litter type, TCH content, and AUR/N were selected as predictor variables of mass loss of broad-leaved and coniferous leaves, of which AUR/N had a significant coefficient ('Leaf' column in Table 3). Litter type and the contents of TCH and total N were selected as predictor variables of mass loss of twigs, of which type and total N showed significant coefficients ('Twig' column in Table 3). The content of TCH and AUR/N were selected as predictor variables of mass loss of sapwood and had significant coefficients ('Sapwood' column in Table 3). In all models, coniferous litter, AUR content, and AUR/N had negative coefficients, whereas total N content had

4

T. Hishinuma et al. / European Journal of Soil Biology 80 (2017) 1e8

Table 2 Contents (mg/g) of acid-unhydrolyzable residue (AUR), total carbohydrate (TCH), and total nitrogen (N) of litter, AUR/N ratio of litter, and mass loss of litter (% original mass) caused by Trametes versicolor IFO30340. Values indicate means ± standard deviations, with the minimum-maximum in parentheses. Generalized linear models (GLMs) were applied to examine the differences among six tissues and to examine the difference among three tissues (leaf, twig, sapwood) and between types (broad-leaved, coniferous). ***P < 0.001, **P < 0.01, *P < 0.05, ns not significant. Values marked with the same letters are not significantly different at the 5% level by Tukey's HSD test.

All data Tissue Leafa Twig Sapwood Heartwood Bark Petiole GLM (tissue) Typeb Broad-leaved Coniferous Grass GLM (tissuec) GLM (typea)

n

AUR

TCH

72

290 ± 71

26 17 19 3 3 3

309 292 268 294 314 249 ns

52 10 1

278 ± 67 361 ± 50 219 ns ***

± ± ± ± ± ±

95a 49a 35a 65a 101a 61a

Total N

AUR/N

Mass loss

(88454)

474 ± 144

(235e712)

6.0 ± 4.4

(0.6e17.5)

119 ± 130

(8e484)

25.0 ± 13.0

(0.9e59.8)

(88e454) (188e383) (204e335) (225e356) (198e377) (203e318)

312 528 637 596 455 390 ***

± ± ± ± ± ±

(235e467) (463e583) (581e712) (466e673) (416e518) (356e426)

10.7 ± 2.7a 5.0 ± 1.5b 1.3 ± 0.9c 0.7 ± 0.1c 5.3 ± 0.8b 5.6 ± 3.3b ***

(6.6e17.5) (2.2e8.1) (0.6e3.4) (0.6e0.8) (4.7e6.2) (3.5e9.5)

30 ± 12c 66 ± 30c 261 ± 109b 435 ± 55a 59 ± 19c 57 ± 35c ***

(8e59) (35e152) (80e441) (375e484) (40e77) (21e91)

31.1 ± 11.2a 22.1 ± 8.0a 20.6 ± 14.0ab 5.5 ± 1.6b 18.6 ± 17.3ab 36.2 ± 11.3a ***

(12.0e55.2) (11.7e44.5) (4.3e59.8) (3.6e6.5) (0.9e35.4) (23.5e45.2)

(88e427) (295e454)

466 ± 154 499 ± 119 628 *** *

(235e712) (262e659)

6.5 ± 4.7 5.0 ± 3.5 8.3 *** *

(0.6e17.5) (0.8e10.6)

105 ± 116 139 ± 135 26 *** ns

(8e441) (38e394)

27.6 ± 12.1 14.2 ± 5.4 43.1 ** ***

(7.6e59.8) (4.3e25.3)

53e 40bc 36a 113ab 55cd 35de

a

Leaves of broad-leaved and coniferous trees. Data of leaves, twigs, and sapwood were used. c The results of multiple comparisons were generally consistent with those of GLMs for the six tissues examined. The interaction of tissue and type was not statistically significant at 5% level in all models. b

Fig. 1. Scatter plot of total nitrogen (N) content against the content of acid unhydrolyzable residue (AUR) for 72 litters used in the pure culture decomposition tests. Litter types included broad-leaved trees (B), coniferous trees (C), and grass (G). Heartwood consisted of litter from two broad-leaved trees and one coniferous tree, and bark and petioles consisted of litter from broad-leaved trees.

positive coefficients (Table 2). 3.3. FT-IR analysis of 30 twigs and sapwoods The FT-IR spectra in the fingerprint region of 30 litters showed a degree of similarity in overall appearance, with major peaks at 1658 to 1735 cm1 and 1033 to 1126 cm1 (Fig. 3). The relative heights of the 15 peaks were significantly different between twigs and sapwoods, and those of 9 peaks were significantly different between broad-leaved and coniferous litters (Table 4). Mass loss of litter caused by T. versicolor IFO30340 was significantly and negatively

correlated with the relative height at two peaks at 1505 and 817 cm1 and was significantly and positively with the relative height of the peak at 1735 cm1 (Table 4). The tissue and relative heights of peaks at 1735, 1658, and 817 cm1 were selected as predictor variables of the mass loss of 30 litters, of which the tissue and the relative heights of peaks at 1735 and 817 cm1 had significant regression coefficients (Table 5). In this model, the relative height of peak at 817 cm1 had negative coefficient, whereas twigs and the relative height of peak at 1735 cm1 had positive coefficient (Table 5).

T. Hishinuma et al. / European Journal of Soil Biology 80 (2017) 1e8

5

Fig. 2. Mass loss of litter (% original mass) plotted against the content (mg/g) of acid-unhydrolyzable residue (AUR, upper left), total carbohydrate (TCH, upper right), and total nitrogen (N, lower left), and AUR/N ratio (lower right). Symbols are the same as in Fig. 1.

Table 3 Summary of generalized linear model for mass loss of 62 litters (leaves, twigs, and sapwood of broad-leaved and coniferous trees) caused by Trametes versicolor IFO30340 and selected predictor variables of tissue, type, and proximate analysis. Regression coefficients are shown. Dashes indicate that the variables were not selected in models. ***P < 0.001, **P < 0.01, *P < 0.05, þ P < 0.10, ns not significant. Tissue (sapwood, twig) indicates the regression coefficients for sapwood and twig. Type (C) indicates the regression coefficients for coniferous litter. AUR, acid-unhydrolyzable residue; TCH, total carbohydrate; Total N, total nitrogen; AUR/N, AUR/N ratio.

Tissue (sapwood, twig) Type (C) AUR TCH Total N AUR/N Intercept a

All litter

Leafa

Twig

Sapwood

10.545, 6.313*** e 0.044** e e 0.099*** 47.767

14.655ns e 0.066þ e 0.285*** 20.888

5.520*** e 0.119þ 3.359** e 55.766

e e 0.155** e 0.088*** 142.557

Data of broad-leaved and coniferous trees were used.

4. Discussion The present study clarifies comprehensively the plasticity of, and the litter quality control of, the decomposition of diverse plant tissues by a single fungal strain. Trametes versicolor IFO30340 had the ability to cause mass loss of diverse leaves, twigs, and sapwood under pure culture conditions (Table 2). This fungal species is generally known as a wood-decomposer but caused mass loss of not only twigs and sapwood but also leaves, petioles, and bark (Table 2), indicating its possession of the physiological capability of

decomposing diverse plant tissues despite the ecological constraints on its establishment and fruiting on non-woody tissues in the field. Abundance and functional roles of T. versicolor in the decomposition of leaves and barks remains unknown, but this fungus contributes significantly to the decomposition of wood; for example, its mycelia colonized 35%e59% of the total volume of fallen beech wood [33]. A suite of extracellular enzymes, such as laccase and manganese peroxidase, are considered responsible for the decomposition [12,13]. Trametes versicolor IFO30340 exhibited variability in its ability to

6

T. Hishinuma et al. / European Journal of Soil Biology 80 (2017) 1e8 Table 5 Summary of generalized linear model for mass loss of litter caused by Trametes versicolor IFO30340 and selected predictor variables of tissue, type, and relative height of FT-IR peaks. Regression coefficients are shown. ***P < 0.001, *P < 0.05, ns not significant. Tissue (T) indicates the regression coefficients for twig. Coefficient Tissue (T) Peak at 1735 cm1 Peak at 1658 cm1 Peak at 817 cm1 Intercept

Fig. 3. FT-IR spectra of 30 litters. Arrows indicate a total of 15 peaks in the fingerprint region (Table 1). Gray solid lines, twigs of broad-leaved trees; gray broken lines, twigs of coniferous trees; black solid lines, sapwood of broad-leaved trees; black broken lines, sapwood of coniferous trees.

decompose different plant tissues that was partly related to measures of litter quality examined in the present study (Tables 2, 3 and 5). Fruiting bodies of this fungal species are encountered on wood of both broad-leaved and coniferous trees, but more frequently on broad-leaved than on coniferous wood [34]. Trametes versicolor IFO30340 caused greater mass loss of broad-leaved litter than coniferous litter under our pure culture conditions, consistent with casual observations of its fruiting bodies. The greater content of lignin and other recalcitrant compounds and the lower N content in coniferous litter than in broad-leaved litter (Table 2) partly account for this difference of mass loss [2]. In fact, the GLM in the present study selected both litter type and AUR and N contents as significant predictors for the mass loss of all litters (Table 3). Other explanation included the difference in lignin structure, that is, conifer wood in general consists mainly of guaiacyl lignin, which is more recalcitrant than the guaiacyl-syringyl lignin present in angiosperms (Table 4) [35]. Alternatively, protective substances in

8.932* 34.015*** 16.613ns 289.726*** 14.372

conifers could inhibit fungal growth, such as terpenes, phenolic resins, and essential oils [36,37]. The lowest mass loss of heartwood (Table 2), which in general is rich in such antifungal compounds, may support the role of inhibitory substances. The GLM in the present study did not select tissue as a predictor variable for the mass loss of all litters (Table 3), indicating that the variation in mass loss was due to differences in chemical properties between the tissues tested, rather than to their structural properties. Moreover, chemical properties limiting the decomposition by T. versicolor IFO30340 were different between the tissues when examined separately. That is, N level limited the decomposition in twigs and sapwood, which had lower total N content, whereas AUR limited the decomposition of leaves, which were richer in N (Fig. 1, Table 3). Nitrogen is an essential element for hyphal growth, and its shortage obviously results in decreased amounts of extracellular enzymes responsible for the decomposition of plant tissues. The negative relationship between AUR content and mass loss caused by T. versicolor IFO30340 (Table 3, Fig. 2) was consistent with previous reports on fungal decomposition of litter [11,18], as well as decomposition in the field [38,39]. The AUR fraction contains such recalcitrant compounds as condensed tannins, phenolic and carboxylic compounds, cutins, and true lignin [30], which can retard the substrate utilization by T. versicolor IFO3040. Of the litter quality variables examined with proximate analysis, the present study demonstrated the usefulness of AUR/N as a predictor of decomposition (Fig. 2, Table 3), coincided with previous studies [40]. This is reasonable as AUR/N ratio incorporates both the positive effect of N as an essential nutrient for fungal growth and the negative effect of AUR as recalcitrant compounds for decomposition.

Table 4 Relative height (%) of peaks in FT-IR spectra for 30 litters. Values indicate means ± standard deviations. Generalized linear models (GLMs) were applied to examine the differences between tissues (twig, sapwood) and between types (broad-leaved, coniferous). Correlation coefficients are given for simple linear relationships between the relative heights and mass loss of litter caused by Trametes versicolor IFO30340. ***P < 0.001, **P < 0.01, *P < 0.05, ns not significant. Wavenumber (cm1)

Twig Broad-leaved (n ¼ 12)

Coniferous (n ¼ 5)

Broad-leaved (n ¼ 11)

Coniferous (n ¼ 2)

Tissue

Type

1735 1658 1593 1505 1462 1422 1367 1329 1266 1227 1126 1033 925 835 817

58 ± 9 56 ± 9 55 ± 7 43 ± 5 53 ± 6 55 ± 6 59 ± 5 59 ± 5 62 ± 4 65 ± 5 82 ± 3 98 ± 2 18 ± 4 5±1 3±1

40 ± 5 60 ± 14 53 ± 6 55 ± 7 52 ± 5 54 ± 5 58 ± 4 57 ± 5 65 ± 4 56 ± 4 76 ± 4 97 ± 3 15 ± 2 1±0 4±1

63 ± 5 39 ± 4 52 ± 5 56 ± 5 63 ± 5 63 ± 5 66 ± 4 64 ± 4 69 ± 4 73 ± 3 90 ± 2 99 ± 0 28 ± 3 8±1 4±1

31 ± 5 36 ± 10 34 ± 11 55 ± 16 51 ± 12 54 ± 11 56 ± 8 51 ± 8 66 ± 10 55 ± 8 80 ± 4 99 ± 0 20 ± 5 1±0 4±1

* *** * *** *** *** ** * *** *** *** * *** *** ***

*** ns ** ** ns ns ns ** ns *** *** ns *** *** *

Sapwood

GLM

Correlation with mass loss

0.42* 0.23ns 0.35ns 0.42* 0.01ns 0.02ns 0.05ns 0.15ns 0.30ns 0.23ns 0.04ns 0.01ns 0.11ns 0.12ns 0.57***

T. Hishinuma et al. / European Journal of Soil Biology 80 (2017) 1e8

Mass loss of litter caused by T. versicolor IFO30340 was negatively related to the relative height at two peaks at 1505 and 817 cm1 (Table 4) that result from guaiacyl unit of lignin (Table 1) and were greater in coniferous than in broad-leaved litters (Table 4). This result supports our suggestion above and indicated that the lower activity of T. versicolor IFO30340 to decompose coniferous litter than broad-leaved litter was attributed to guaiacyl units of lignin. In contrast, the mass loss was positively related to the relative height of the peak at 1735 cm1 (Table 4) that results from C¼O stretch of carbohydrate origin (Table 1) and was greater in broad-leaved than in coniferous litters (Table 4). This indicated that T. versicolor IFO3034 has an efficient ability to hydrolyze ester groups in broad-leaved litter, leading to the greater mass loss of broad-leaved litter than coniferous litter (Table 1). 5. Conclusion The present study showed that the litter quality controlled the decomposition by the single, representative fungal strain T. versicolor IFO30340 under pure culture conditions and explicitly demonstrated the importance of litter type (broad-leaved or coniferous), AUR, and N as regulatory factors of decomposition. These litter properties are also known to be limiting factors of plant litter decomposition by decomposer assemblages in the field [1], indicating that the plasticity of decomposing ability of an individual fungal species or strain in response to litter quality can contribute to the variability in litter decomposition observed in the field. Measures of litter quality potentially affecting the decomposition but not tested in the present study include the specific part of the leaf, gravimetric wood density, and phosphorus content [4,6,41]. Examining these properties, and more detailed evaluation of chemical properties of litter, using such tools as 13C nuclear magnetic resonance (MMR) analysis [42], will provide further insights into the litter quality control of decomposition by T. versicolor IFO30340. Future studies should also include some other measures of decomposition, such as respiration measurement and enzyme activities, and test whether the same trend in decomposition is found for other fungal species to test the general validity of the present findings. Acknowledgments We thank Dr. Elizabeth Nakajima for critical reading of the manuscript. This study received partial financial support from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT) (No.15K07480). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejsobi.2017.03.002. References [1] B. Berg, C. McClaugherty, Plant Litter, Decomposition, Humus Formation, Carbon Sequestration, Springer, Berlin, 2008. [2] T. Osono, Ecology of ligninolytic fungi associated with leaf litter decomposition, Ecol. Res. 22 (2007) 955e974. [3] A. Van der Wal, T.D. Geydan, T.W. Kuyper, W. de Boer, A thready affair: linking fungal diversity and community dynamics to terrestrial decomposition processes, FEMS Microbiol. Rev. 37 (2013) 477e494. [4] W.K. Cornwell, J.H.C. Cornelissen, K. Amatangelo, E. Dorrepaal, V.T. Eviner,
rez-Harguindeguy, O. Godoy, S.E. Hobbie, B. Hoorens, H. Kurokawa, N. Pe H.M. Quested, L.S. Santiago, D.A. Wardle, I.J. Wright, R. Aerts, S.D. Allison, P. van Bodegom, V. Brovkin, A. Chatain, T.V. Callaghan, S. Díaz, E. Garnier, D.E. Gurvich, E. Kazakou, J.A. Klein, J. Read, P.B. Reich, N.A. Soudzilovskaia, M.V. Vaieretti, M. Westoby, Plant species traits are the predominant control on litter decomposition rates within biomes worldwide, Ecol. Lett. 11 (2008)

7

1065e1071. [5] B. Berg, M. Berg, P. Bottner, E. Box, A. Breymeyer, R. Calvo de Anta, €lko €nen, M. Couteaux, A. Escudero, A. Gallardo, W. Kratz, M. Madeira, E. Ma ~ oz, P. Piussi, J. Remacle, A. Virzo de C. McClaugherty, V. Meentemeyer, F. Mun Santo, Litter mass loss rates in pine forests of Europe and eastern United States: some relationships with climate and litter quality, Biogeochemistry 20 (1993) 127e159. €nisch, J.M. Craine, [6] K.A. Pietsch, K. Ogle, J.H.C. Cornelissen, W.K. Cornwell, G. Bo B.G. Jackson, J. Kattge, D.A. Peltzer, J. Penuelas, P.B. Reich, D.A. Wardle, J.T. Weedon, I.J. Wright, A.E. Zanne, C. Wirth, Global relationship of wood and litter decomposability: the role of functional traits within and across plant organs, Glob. Ecol. Biogeogr. 23 (2014) 1046e1057. [7] C.H. Robinson, J. Dighton, J.C. Frankland, J.D. Roberts, Fungal communities on decaying wheat straw of different resource qualities, Soil Biol. Biochem. 26 (1994) 1053e1058. [8] M.K. Aneja, S. Sharm, F. Fleischmann, S. Stich, W. Heller, G. Bahnweg, J.C. Munch, M. Schloter, Microbial colonization of beech and spruce litter e influence of decomposition site and plant litter species on the diversity of microbial community, Microb. Ecol. 52 (2006) 127e135. [9] T. Mincheva, E. Barni, G.C. Varese, G. Brusa, B. Cerabolini, C. Siniscalco, Litter quality, decomposition rates and saprotrophic mycoflora in Fallopia japonica (Houtt.) Ronse Decraene and in adjacent native grassland vegetation, Acta Oecol 54 (2014) 29e35. [10] P. Mikola, Studies on the decomposition of forest litter by basidiomycetes, Commun. Inst. For. Fenn 48 (1956) 4e48. [11] T. Osono, C. To-Anun, Y. Hagiwara, D. Hirose, Decomposition of wood, petiole, and leaf litter by Xylaria species from northern Thailand, Fungal Ecol. 4 (2011) 210e218. [12] K. Addleman, T. Dumonceaux, M.G. Paice, R. Bourbonnais, F.S. Archibald, Production and characterization of Trametes versicolor mutants unable to bleach hardwood kraft pulp, Appl. Environ. Microb. 61 (1995) 3687e3694. [13] D. Schlosser, R. Grey, W. Fritsche, Patterns of ligninolytic enzymes in Trametes versicolor. Distribution of extra- and intracellular enzyme activities during cultivation on glucose, wheat straw and beech wood, Appl. Microbiol. Biot. 47 (1997) 412e418. [14] E. Bari, N. Nazarnezhad, S.M. Kazemi, M.A.T. Ghanbary, B. Mohebby, O. Schmidt, C.A. Clausen, Comparison between degradation capabilities of the white rot fungi Pleurotus ostreatus and Trametes versicolor in beech wood, Int. Biodeter. Biodegr 104 (2015) 231e237. [15] J. Mikuni, N. Morohoshi, Cloning and sequencing of a second laccase gene from the white-rot fungus Coriolus versicolor, FEMS Microbiol. Lett. 155 (1997) 79e84. [16] S. Kawai, M. Nakagawa, H. Ohashi, Aromatic ring cleavage of a non-phenolic b-O-4 lignin model dimer by laccase of Trametes versicolor in the presence of 1-hydroxybenzotriazole, FEBS Lett. 446 (1999) 355e358. [17] K.K. Pandey, A.J. Pitman, FTIR studies of the changes in wood chemistry following decay by brown-rot and white-rot fungi, Int. Biodeter. Biodegr 52 (2003) 151e160. [18] T. Osono, Effects of litter type, origin of isolate, and temperature on decomposition of leaf litter by macrofungi, J. For. Res. 20 (2015) 77e84. [19] Y. Fukasawa, T. Osono, H. Takeda, Decomposition of Japanese beech wood by diverse fungi isolated from a cool temperate deciduous forest, Mycoscience 46 (2005) 97e101. [20] T. Osono, Decomposition of grass leaves by ligninolytic litter-decomposing fungi, Grassl. Sci. 56 (2010) 31e36. [21] T. Osono, H. Takeda, Effect of malt extract addition on fungal decomposition of oak wood, Appl. For. Sci. 12 (2003) 177e180. [22] T. Osono, H. Takeda, Fungal decomposition of Abies needle and Betula leaf litter, Mycologia 98 (2006) 172e179. [23] T. Osono, Y. Fukasawa, H. Takeda, Roles of diverse fungi in larch needle litter decomposition, Mycologia 95 (2003) 820e826. [24] T. Osono, S. Iwamoto, J.A. Trofymow, Colonization and decomposition of salal (Gaultheria shallon) leaf litter by fungi in successional forests on coastal British Columbia, Can. J. Microbiol. 54 (2008) 427e434. [25] T. Osono, Y. Ishii, D. Hirose, Fungal colonization and decomposition of Castanopsis sieboldii leaf litter in a subtropical forest, Ecol. Res. 23 (2008) 909e917. [26] T. Osono, Y. Ishii, H. Takeda, T. Seramethakun, S. Khamyong, C. To-Anun, D. Hirose, S. Tokumasu, M. Kakishima, Fungal succession and lignin decomposition on Shorea obtusa leaves in a tropical seasonal forest in northern Thailand, Fungal Divers 36 (2009) 101e119. [27] T. Osono, Decomposing ability of diverse litter-decomposer macrofungi in subtropical, temperate, and subalpine forests, J. For. Res. 20 (2015) 272e280. [28] T. Osono, S. Hobara, K. Koba, K. Kameda, Reduction of fungal growth and lignin decomposition in needle litter by avian excreta, Soil Biol. Biochem. 38 (2006) 1623e1630. [29] H.G.C. King, G.W. Heath, The chemical analysis of small samples of leaf material and the relationship between the disappearance and composition of leaves, Pedobiologia 7 (1967) 192e197. [30] C.M. Preston, J.A. Trofymow, B.G. Sayer, J. Niu, 13C nuclear magnetic resonance spectroscopy with cross-polarization and magic-angle spinning investigation of the proximate-analysis fractions used to assess litter quality in decomposition studies, Can. J. Bot. 75 (1997) 1601e1613. [31] M. Dubois, K.A. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith, Colorimetric method for determination of sugars and related substances, Anal. Chem. 28

8

T. Hishinuma et al. / European Journal of Soil Biology 80 (2017) 1e8

(1956) 350e356. [32] T. Osono, S. Hobara, T. Hishinuma, J.I. Azuma, Selective lignin decomposition and nitrogen mineralization in forest litter colonized by Clitocybe sp, Eur. J. Soil. Biol. 47 (2011) 114e121. [33] Y. Fukasawa, T. Osono, H. Takeda, Beech log decomposition by woodinhabiting fungi in a cool temperate forest floor: a quantitative analysis focused on the decay activity of a dominant basidiomycete Omphalotus guepiniformis, Ecol. Res. 25 (2010) 959e966. [34] R. Imazeki, T. Hongo, Colored Illustrations of Mushrooms of Japan, vol. II, Hoikusha, Osaka, Japan, 1989. [35] K.E.L. Eriksson, R.A. Blanchette, P. Ander, Microbial and Enzymatic Degradation of Wood and Wood Components, Springer, Tokyo, 1990. ci, M. Dig rak, Antimicrobial activity of essential oils of some Abies (Fir) [36] E. Bag species from Turkey, Flavour Frag. J. 11 (1996) 251e256.

[37] N.J. Dix, J. Webster, Fungal Ecology, Chapman & Hall, London, 1995. [38] L.M. Stump, D. Binkley, Relationships between litter quality and nitrogen availability in Rocky Mountain forests, Can. J. For. Res. 23 (1993) 492e502. [39] K.L. Murphy, J.M. Klopatek, C.C. Klopatek, The effects of litter quality and climate on decomposition along an elevational gradient, Ecol. Appl. 8 (1998) 1061e1071. [40] J.M. Melillo, J.D. Aber, J.F. Muratore, Nitrogen and lignin control of hardwood leaf litter decomposition dynamics, Ecology 63 (1982) 621e626. [41] J.T. Weedon, W.K. Cornwell, J.H.C. Cornelissen, A.E. Zanne, C. Wirth, D.A. Coomes, Global meta-analysis of wood decomposition rates: a role for trait variation among tree species? Ecol. Lett. 12 (2009) 45e56. [42] T. Osono, H. Takeda, J.I. Azuma, Carbon isotope dynamics during decomposition with reference to lignin fractions, Ecol. Res. 23 (2008) 51e55.