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Comparison of litter decomposing ability among diverse fungi in a cool temperate deciduous forest in Japan

     Laboratory of Forest Ecology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502 Japan

	ABSTRACT

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The litter decomposing ability of 79 fungal isolates (41 genera, 60 species) was assessed with the pure culture decomposition test. The isolates were collected qualitatively in a cool temperate deciduous forest in Japan during a 21-mo period. Loss of original weight of sterilized litter ranged from 0.1% to 57.6%. Six isolates in the Basidiomycota caused high weight losses ranging from 15.1% to 57.6%. Fourteen isolates in Xylaria and Geniculosporium (the Xylariaceae and its anamorph) also caused high weight losses ranging from 4.0% to 14.4%. Other isolates in the Ascomycota and associated anamorphs and in the Zygomycota caused low weight losses on mean. Six fungi in the Basidiomycota, and all in the Xylariaceae showed a bleaching activity of the litter and caused lignin and carbohydrate decomposition. Mean lignin/weight loss ratios (L/W) and lignin/carbohydrate loss ratios (L/C), were 0.9 and 0.7 for the Basidiomycota and 0.7 and 0.4 for the Xylariaceae, respectively. Significant differences were found in L/W and L/C between the two groups when the result of Xylaria sp. that showed marked delignification was excluded. These differences in lignin and carbohydrate utilization patterns are discussed in relation to the structural and the chemical properties of the decomposed litter and to the implications for organic chemical changes during litter decomposition processes.

Key words: Basidiomycota, beech leaf, carbohydrate, lignin, Xylariaceae

	INTRODUCTION

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Fungi play fundamental roles in decomposition processes of leaf litter within forest ecosystems (Swift et al 1979 , Cooke and Rayner 1984 ). Fungal species composition and successional changes during the decomposition process have been qualitatively investigated on several litter types (Hudson 1968 ), but the frequency of occurrence of individual species is a poor guide to their importance in the decomposition processes. Pure culture decomposition tests have been therefore carried out to assess the decomposing abilities and the substrate utilization patterns of fungi (Lindeberg 1944, 1946 , Mikola 1956 , Saito 1960 , Hering 1967 , De-Boois 1976 , Dix and Simpson 1984 , Kuyper and Bokeloh 1994 , Osono and Takeda 1999 ).

Lignin and holocellulose are structural components that constitute 70–80% of fresh organic material and are major energy sources in plant tissues that are available to fungi (Swift et al 1979 ). Many studies have reported that the Basidiomycota account for most of the lignocellulose decomposition in leaf litter (Lindeberg 1944, 1947 , Hering 1967 , Dix and Simpson 1984 , Miyamoto et al 2000 ). In addition, Osono and Takeda (1999) reported that fungi in the Xylariaceae (Ascomycota) that colonize the interior of leaf tissues have the ability to decompose lignin and carbohydrate in beech litter. Because decomposition of lignin and holocellulose are key factors controlling litter decomposition rates (Aber et al 1990 ), it is important to evaluate the lignin and cellulose decomposing ability of fungi occurring on litter in order to understand their roles in decomposition processes (Lindeberg 1946 , Saito 1960 , Hering 1967 , Kuyper and Bokeloh 1994 , Osono and Takeda 1999 , Miyamoto et al 2000 ). However, in contrast to wood decomposition (e.g., Otjen et al 1987 , Rayner and Boddy 1988 , Nilsson et al 1989 , Tanesaka et al 1993 , Worrall et al 1997 ), few surveys have been carried out comparing the leaf litter decomposing ability of diverse fungi.

In this study, we investigated and compared the ability of 79 fungal isolates (41 genera, 60 species) in the Basidiomycota, the Ascomycota (Xylariaceae and others), and the Zygomycota to decompose beech leaf litter under laboratory conditions. Fungi were isolated either from sporocarps on leaf litter, twigs, cupules, or wood; from green leaves, leaf litter, or twigs of beech; or from mineral soils. Isolates from green leaves were used to examine litter decomposing ability because some of these fungi persisted after litter fall and occurred as litter decomposers (Osono in press ).

	MATERIALS AND METHODS

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Fungi – The 79 fungal isolates used in the decomposition test, date, source, and method of isolation are listed in Table I . The isolates were qualitatively collected on several occasions during a 21-month period from Oct 1996 to Jun 1998 from a cool temperate deciduous forest dominated by Fagus crenata B1. in the Ashiu Experimental Forest of Kyoto University (35° 18'N and 135° 43'E), Kyoto, Japan. Isolations were made either from mass basidiospores, a single ascospore, or mass ascospores discharged from sporocarps on leaf litter, twigs, cupules, or wood; from green leaves, leaf litter, or twigs of beech, with the surface sterilization or the washing method (Osono and Takeda 1999 ); or from mineral soils with the dilution plating method (Osono and Takeda 2000). All fungal isolates were maintained on slants of a modified malt-yeast-soytone agar (Kinugawa 1988 ) at a room temperature (ca 15–20 C) in darkness. Malt-yeast-soytone agar contains malt extract 0.35%, soytone 0.05%, yeast extract 0.025%, and agar 1.5% (w/v).

Leaf disks were pressed in moistened paper towels between the base and lid of a Petri dish, then autoclaved at 120 C for 20 min. The sterilized disks were placed on surfaces of Petri dishes containing 20 mL 2% plain agar. Inocula for each assessment were cut out of the margin of the growing colonies on 2% malt extract agar with a sterile cork borer (5.5 mm diam) and placed on the center of the plates including a subsample of seven disks around the inoculum. Plates were incubated for 8 wk at 20 C in darkness.

After 8 wk leaf disks were collected, oven dried for 4 d at 40 C, and weighed. Observations were made of the external appearance of the decomposed litter, and bleaching (i.e., change in color from brown to white) was observed under a binocular microscope with a magnification of 20x. Weight loss of the leaf disks was determined as a percentage of the original weight. Ten plates were prepared for each strain. A portion of the sample leaves were used for chemical analyses as described below.

A part of the results has already been presented in Osono and Takeda (1999) , and, in the present study, sterile mycelium CHL and unidentified GSH1 in Osono and Takeda (1999) were reclassified as Geniculosporium sp. 1 and white sterile 5LS12, respectively.

Chemical analyses – Leaf samples for chemical analyses were ground in a laboratory mill (0.5 mm screen). The amount of lignin in samples was estimated by gravimetry using hot sulfuric acid digestion (King and Heath 1967 ). Samples were extracted with alcohol-benzene at room temperature and the residue treated with 72% sulfuric acid (v/v) for 2 h at room temperature with occasional stirring. The mixture was then 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 acid-insoluble residue. The filtrate (autoclaved sulfuric acid solution) was used for total carbohydrate analysis as described below.

Total carbohydrate amount in the filtrate was estimated by the phenol–sulfuric acid method (Dubois et al 1956 ). The filtrate was added with 5% phenol (v/v) and 98% sulfuric acid (v/v). The optical density of the solution was then measured by a spectrophotometer at 490 nm using the known concentrations of D-glucose as standards.

Mean concentrations of lignin and carbohydrate in the initial litter were 39.6% and 34.2%, respectively. Weight losses of lignin and carbohydrate were expressed as percentage of the original weights.

Lignin/weight loss ratio (L/W) and lignin/carbohydrate loss ratio (L/C) are useful indices of the substrate utilization pattern of each fungal isolate (Worrall et al 1997 ). L/W and L/C of an isolate are calculated according to the following equations:

	RESULTS

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Weight loss – Weight loss of beech leaf litter ranged from 0.1% to 57.6% (Table I ). High weight losses were caused by six isolates in the Basidiomycota (Lentinula edodes, Microporus vernicipes, Mycena polygramma, Mycena sp., Naematoloma sublateritium, and Panellus serotinus) and ranged from 15.1% to 57.6%, whereas Guepiniopsis sp. and Xylobolus frustulatus caused weight losses of 4.2% and 4.3%, respectively. The mean weight loss of the Basidiomycota was 25.5%. Xylaria spp. and Geniculosporium spp. also caused high weight losses ranging from 4.8% to 14.4% and a mean weight loss for this group was 7.6%. Other Ascomycota (including anamorphs) caused weight losses ranging from 0.1% to 6.5% and a mean weight loss of 2.6%. Fungi in the Zygomycota caused weight losses ranging from 1.1% to 4.9% and a mean weight loss of 2.7%. Sterile mycelia caused weight losses ranging from 1.4% to 8.8% and a mean weight loss of 5.0%.

Bleaching was noticeable in leaf litter decomposed by six isolates in the Basidiomycota, all 14 isolates in the Xylariaceae, and two isolates of white sterile 5LS12 (Table I ). Mean weight loss of only bleached litter was 14.4% ± 2.9% (mean ± SE) and was significantly (P < 0.01, t-test) higher than that of the non-bleached litter (2.6% ± 0.2%).

Chemical changes and substrate utilization – Weight losses of lignin and carbohydrate were measured for the litters decomposed by 13 isolates (six in the Basidiomycota, five in the Xylariaceae, and two white sterile 5LS12) that were representative of 22 isolates with the bleaching activity. Preliminary DNA analysis indicated the white sterile 5LS12 isolate belonged to the Xylariaceae, therefore decomposition results are subsumed under the Xylariaceae in the analysis.

Weight loss of lignin ranged from 6.5% to 59.8% and from 2.0% to 12.2%, in the Basidiomycota and in the Xylariaceae, respectively (Table II ). Weight loss of carbohydrate ranged from 30.0% to 66.6% and from 7.6% to 29.1%, respectively. L/W ranged from 0.4 to 1.1 and from 0.2 to 1.5, and L/C ranged from 0.2 to 0.9 and from 0.1 to 1.6, respectively. Variation in L/W and L/C was higher in the Xylariaceae than in the Basidiomycota, because a strain of Xylaria sp. showed marked delignification and high L/W and L/C.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Weight loss and chemical changes – Our results indicate that some litter decomposing ability occurs in many groups of fungi. However, a marked decomposing ability was limited to members in the Basidiomycota and in the Xylariaceae. These fungi also had bleaching activity that was associated with lignin and carbohydrate decomposition, and were regarded as lignocellulose decomposers.

High weight losses of the litter with lignin and carbohydrate decomposition were caused by six isolates in the Basidiomycota, i.e., Mycena polygramma, Mycena sp., Lentinula edodes, Microporus vernicipes, Naematoloma sublateritium, and Panellus serotinus. Mycena spp. were known as vigorous decomposers of lignin and cellulose in leaf litter (Lindeberg 1946 ), and the others as white-rotters of wood (Imazeki and Hongo 1987, 1989 , Tanesaka et al 1993 ). Mycena polygramma and Mycena sp. decomposed lignin and carbohydrate in beech leaf litter as intensively as the wood fungi.

On the other hand, Guipiniopsis sp. and Xylobolus frustulatus caused lower weight losses and showed no bleaching of the litter, suggesting that some fungi in the Basidiomycota are not vigorous decomposers of beech leaf litter. Mikola (1956) also reported that decomposing abilities of basidiomycetous fungi were highly variable among species.

Fungi in the Xylariaceae caused the second highest weight loss of beech litter and lignin and carbohydrate decomposition. Xylariaceous species are known to be lignin and cellulose decomposers of wood (Merrill et al 1964 , Rogers 1979 , Sutherland and Crawford 1981 , Nilsson et al 1989 , Whalley 1996 , Worrall et al 1997 ). There have been a few studies on leaf litter decomposition by xylariaceous fungi (Osono and Takeda 1999 ), but the present study showed that they caused substantial decomposition of lignin and carbohydrate. A field evidence of their bleaching activity associated with lignocellulose decomposition has already presented on the beech litter (Osono and Takeda 2001a ). These thus indicate that Xylariaceous species are major decomposers of lignin and cellulose in beech leaf litter.

Other species within the Ascomycota and in the Zygomycota had low weight losses on mean and had no bleaching activity. Among them, Discosia sp. and Trichoderma hamatum caused 22.7% and 9.3% loss of carbohydrate in the litter, respectively, and were regarded as cellulose decomposers (Osono and Takeda 1999 ). Hence, some isolates that caused litter weight loss of 3.0% to 6.0%, such as Pestalotiopsis sp. and the Nigrospora state of Khuskia oryzae, may be considered as cellulose decomposing fungi. The growth of other fungi that caused weight losses below 3.0% in the test may rely mainly on readily available energy sources and be regarded as sugar fungi (Hudson 1968 ), as beech litter contained about 3.0% of soluble carbohydrate that fungi might consume without destroying cell wall polymers.

Substrate utilization patterns – Lignin/weight loss ratio (L/W) and lignin/carbohydrate loss ratio (L/C) are useful indices of the substrate utilization pattern of fungi. In this study, mean L/W were 0.9 and 0.7, and mean L/C were 0.7 and 0.4 for the Basidiomycota and the Xylariaceae, respectively. There has been only one comparable study of L/W and L/C of litter decomposers. In the decomposition of Fagus sylvatica litter by 26 basidiomycetous fungi, Lindeberg (1946) reported mean values of L/W (1.8 ± 0.5, mean ± SD) and L/C (1.4 ± 1.2) that were significantly (P < 0.01, t-test) higher than those recorded in the present study. This difference is probably due to the difference in the fungal strains used and/or in the incubation method utilized.

Mean L/W and L/C of fungi in the Basidiomycota were significantly higher than in the Xylariaceae when the result of Xylaria sp. that caused marked delignification was excluded in the analysis. The difference in L/W and L/C between these two groups was also found in studies on decomposition of birch wood block (Otjen et al 1987 , Nilsson et al 1989 , Worrall et al 1997 ). Thus, it is suggested that L/W and L/C were higher in the Basidiomycota than in the Xylariaceae in the litter decomposition and that the Xylariaceae decomposed holocellulose in preference to lignin more so than in the Basidiomycota. A strain of Xylaria sp. caused marked delignification. As Xylaria sp. occurred most frequently on bleached portions of beech litter with frequency of 60%, some strains in this species may be important in lignin decomposition of beech litter in the study site (Osono and Takeda 2001a ).

This difference may reflect the anatomical and chemical characteristics of plant cell wall decomposition by these fungi (Rayner and Boddy 1988 ). In the white-rot process by basidiomycetous fungi, all cell wall constituents were decomposed in the secondary wall and middle lamella (Blanchette 1995 ) and/or lignin was selectively removed (Otjen and Blanchette 1986 ). On the other hand, xylariaceous fungi caused soft-rot type decomposition by formation of cavities within the secondary wall along the microfibrillar axis or of cell wall erosion towards the middle lamella (Blanchette 1995 ), in which carbohydrates were preferentially attacked (Nilsson et al 1989 , Worrall et al 1997 ).

The difference in L/W and L/C between the Basidiomycota and the Xylariaceae provides the implication to their roles in lignocellulose decomposition on several deciduous and coniferous litter types in temperate regions (Berg et al 1984 , Aber et al 1990 , Osono and Takeda 2001b ). Osono and Takeda (2001b) suggested in a three year decomposition study of beech litter that selective holocellulose decomposition in the first 21 mo may be ascribed to the mycelial growth of Xylaria sp. and Geniculosporium sp. 1. Consequently content of lignin and related humic substances increased in the decomposing litter that was less available to these fungi than freshly fallen litter. The disappearance of lignin and holocellulose then proceeded at a similar rate during the 21th to the 35th month. In this phase the Basidiomycota increased its mycelial abundance, indicating they replaced the Xylariaceae and attacked these residual compounds. Therefore, the difference in the substrate utilization between the Xylariaceae and the Basidiomycota is related to the fungal succession and organic chemical changes during beech litter decomposition. Other species within the Ascomycota and in the Zygomycota were, on the other hand, regarded as cellulose decomposers or sugar fungi that depended their growth on holocellulose or on soluble carbohydrate of plant origin or sugars released from holocellulose fraction by ligninolytic activity of the lignocellulose decomposers (Osono and Takeda 2001b ).

	ACKNOWLEDGMENTS

 
We thank Dr. K. Kikuzawa for providing leaf litters used in the decomposition test; Dr. C. Tanaka for providing preliminary data on DNA analysis and for his valuable comments on fungal taxonomy; Drs. S. Tokumasu, E. Nagasawa, T. Hattori, T. Hosoya, and I. Tanaka, for their helpful identification of fungi; Dr. F. Hyodo for his critical reading of the manuscript. We acknowledge the helpful discussions of the members of the Laboratory of Forest Ecology, Kyoto University. This study was partly financially supported by a grant from the Japanese Ministry of Education, Culture and Sports relating to a JSPS Fellowship for Japanese Young Scientists (No. 4546).

	FOOTNOTES

 
¹ Corresponding author, fujijun{at}kais.kyoto-u.ac.jp

Accepted for publication September 25, 2001.

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