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Roles of diverse fungi in larch needle-litter decomposition

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

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

Functional biodiversity of fungi in larch (Larix leptolepis) forests needle-litter decomposition was examined by a pure-culture test. Weight loss of larch-needle litter, utilization pattern of lignocellulose and chemical composition of remaining litter were investigated and compared for 31 isolates in 27 species of basidiomycetes and ascomycetes. Weight loss (% original weight) of litter ranged from -2.0% to 14.2%. Mean weight loss of litter caused by the basidiomycetes was not significantly different from that caused by the ascomycetes. Basidiomycetes caused loss of lignin and carbohydrates in variable proportions, while ascomycetes exclusively attacked carbohydrates without delignification. The content of lignin and nitrogen in remaining litter was not significantly correlated when both basidiomycetes and ascomycetes were included. However, the correlation coefficient was significant when the relationship was examined separately for basidiomycetes, indicating that the degree of selective delignification determined the final nitrogen content in litter. Possible effects of fungal colonization on needle-litter decomposition in larch forests are discussed.

Key words: carbohydrates, ecology, Larix leptolepis, lignin, selective delignification, simultaneous decay

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Fungi play an important role in plant litter decomposition in forest ecosystems through nutrient recycling and humus formation in soil (Swift 1979) because they attack the lignocellulose matrix in litter that other organisms are unable to assimilate (Kjøller and Struwe 1982, Cooke and Rayner 1984). Litter decomposing abilities of fungi have been examined by pure-culture tests (Lindeberg 1944, 1946, Mikola 1956, Saito 1960, Hering 1967, De-Boois 1976, Dix and Simpson 1984, Kuyper and Bokeloh 1994, Miyamoto 2000, Osono and Takeda 2002). These studies showed that fungi were divided into three functional groups according to their substrate utilization patterns: lignocellulose decomposers that attack both lignin and cellulose in various proportions, cellulose decomposers that preferentially attack carbohydrates and sugar fungi that rely on soluble sugar for growth. Most previous studies were based on deciduous broadleaf litters, and further studies are necessary to evaluate the functional biodiversity of decomposer fungal communities on coniferous needle litters that have different structural and chemical properties from broadleaf litters.

Japanese larch (Larix leptolepis) is one of the most important trees subject to silvicultural practices in Japan and Korea. Diverse fungi, including sexual and asexual basidiomycetes and ascomycetes occur in larch forests (McBride and Hayes 1977, Imazeki 1987, Hosoya and Otani 1997). However, few studies have evaluated the role of diverse fungi in larch-needle decomposition (McBride 1972). A study of functional diversity of fungi is needed for the understanding of biological aspects of needle-litter decomposition in larch forests (Kawahara 1981, Son 1999).

The purpose of this study is to assess by a pure-culture test the functional biodiversity of fungi encountered in larch forests in needle-litter decomposition. Weight loss of needle litter, utilization patterns of lignocellulose and chemical composition of remaining litter were investigated and compared for 31 isolates in 27 species of basidiomycetes and ascomycetes. Possible effects of fungal colonization on needle-litter decomposition in larch forests are discussed.

	MATERIALS AND METHODS

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Source of fungi and litter – Thirty-one isolates in 27 species were used in this test, including 15 basidiomycetes, 14 ascomycetes and two isolates of white, sterile mycelium, coded LLS12 (Table I). Some basidiomycetes were obtained from basidiocarps collected in a Larix plantation at Nagasaka, Yamanashi, Japan, in Sep 2001, and others that are common in Larix forests (Imazeki 1987, Takahashi 1991) from the culture collection (IFO, Osaka, Japan). Ascomycetes and LLS12 were obtained from Larix litter by surface sterilization and washing, according to the procedures described in Osono and Takeda (2001). The needles were collected from litter (L) and fermentation (F) layers in a Larix plantation at Sanada, Nagano, Japan, in Jun 2000. Frequencies of these fungi on needle litter are shown in Table II. Lambertella advenula was isolated from multiple ascospores discharged from an ascocarp on a larch needle.

Decomposition test – Needle litter (1.0 g) was placed in a nylon mesh bag (6 x 6 cm, 0.1 mm mesh) and sterilized with ethylene oxide gas at 60 C for 3 h. The bags were placed on the surface of Petri dishes (9 cm diam) containing 20 mL 2% agar. Inocula for each assessment were cut out of the margin of the previously inoculated Petri dishes on 2% malt-extract agar (malt extract 2% and agar 2% (w/v)) with a sterile cork borer (6 mm diam) and placed on the agar adjacent to the bag, one plug per plate. The plates were incubated for 3 mo at 20 C in the dark. After incubation, the needles were collected, oven-dried at 40 C for 4 d and weighed. The initial litter also was sterilized, oven-dried at 40 C for 4 d and weighed to determine the original weight. Weight loss of the needles was determined as a percentage of the original weight. Three plates were prepared for each isolate, and three uninoculated plates served as a control. The needles then were combined and used for chemical analyses as described below.

Chemical analyses – Needle samples were ground in a laboratory mill (0.5 mm screen). The amount of lignin in the 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 was treated with 72% sulfuric acid (v/v) for 2 h at room temperature (15–20 C) 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 insoluble acid residue. The filtrate (autoclaved sulfuric acid solution) was used for total carbohydrate analysis. The amount of carbohydrates in the filtrate was estimated by the phenol-sulfuric acid method (Dubois 1956). One mL 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 then was measured by a spectrophotometer at 490 nm, using known concentrations of D-glucose as standards. Total nitrogen contents were measured by automatic gas chromatography (NC analyzer SUMIGRAPH NC-900, Sumitomo Chemical Co., Osaka, Japan).

Lignin/weight loss ratio (L/W) and lignin/carbohydrate loss ratio (L/C) are useful indices of substrate utilization patterns of each fungal species (Osono and Takeda 2002, Osono 2003). L/W and L/C of each fungal species were calculated according to these equations:

	RESULTS

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Litter decomposition – Weight loss of larch-needle litter ranged from -2.0% to 14.2% (Table I). Trametes versicolor caused the highest weight loss, followed by Collybia dryophila, Cyathus striatus, Clitocybe gibba, Collybia peronata, LLS12, Pestalotiopsis neglecta, Micromphale sp. and Mycena polygramma. Other species caused weight loss of less than 5.0%. Preliminary DNA analysis indicated that LLS12 belonged to the ascomycetes, therefore the decomposition results of LLS12 are subsumed under the ascomycetes in this data analysis: Mean weight loss of litter caused by basidiomycetes was 4.8% ± 1.3% (mean ± SE, n = 15) and was not significantly different (t-test, P = 0.22) from that caused by the ascomycetes (2.9% ± 0.7%, n = 16).

Substrate utilization – Weight losses of lignin and carbohydrates were measured for the litters decomposed by 11 isolates (seven basidiomycetes and four ascomycetes) that caused weight loss of more than 5.0% (Table III). Weight loss of lignin ranged from -3.7 to 39.6%. Mean weight loss of lignin caused by basidiomycetes was 18.4% ± 4.9% (mean ± SE, n = 7) and was significantly higher (t-test, P < 0.01) than that by the ascomycetes (-2.0% ± 0.8%, n = 4). Weight loss of carbohydrates ranged from 4.8 to 27.2%. Mean weight loss of carbohydrates caused by basidiomycetes was 14.5% ± 3.5% (mean ± SE, n = 7) and significantly was lower (t-test, P < 0.05) than that by the ascomycetes (23.4% ± 1.3%, n = 4).

Chemical composition of remaining litter – Initial contents of lignin, carbohydrates and nitrogen in larch litter were 45.4%, 26.2% and 0.77%, respectively. Lignin, carbohydrate and nitrogen contents of litter decomposed by 11 fungi are shown in Table III. Lignin content ranged from 31.9 to 51.5%. Mean lignin content of litter decomposed by basidiomycetes was 41.6% ± 3.0% (mean ± SE, n = 7) and was significantly lower (t-test, t = -3.4, P < 0.05) than those by the ascomycetes (50.2% ± 0.4%, n = 4). The carbohydrate content range was 21.2–29.0%. Mean carbohydrate content of litter decomposed by basidiomycetes was 25.2% ± 1.2% (mean ± SE, n = 7) and was significantly higher (t-test, P < 0.01) than that by the ascomycetes (21.7% ± 0.2%, n = 4). Nitrogen content range was 0.77–0.89%. Mean nitrogen content of litter decomposed by the basidiomycetes was 0.84% ± 0.01% (mean ± SE, n = 7) and was not significantly different (t-test, P = 0.11) from that by the ascomycetes (0.81% ± 0.02%, n = 4).

Lignin and nitrogen content were not significantly correlated when all 11 basidiomycete and ascomycete isolates were included (r = 0.07, n = 11; Fig. 1). However, the correlation coefficient was significant but only marginally so (P = 0.07) when the relationship was examined separately for the basidiomycetes (r = 0.72, n = 7).

View larger version (17K): [in this window] [in a new window]   FIG. 1. Relationship between lignin and nitrogen contents for seven basidiomycetes (black box) and four ascomycetes (open circle). The line is fitted only to the basidiomycetes

	DISCUSSION

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On the other hand, the other eight basidiomycetes caused negligible weight loss in larch litter. Among these fungi, however, Clitocybe odora, C. clavipes, Collybia butyracea and Stropharia aeruginasa have been reported as lignin decomposers (Lindeberg 1946, Steffen 2000). Thus, these eight might have a limited ability to attack larch-needle litter or the cultural conditions adopted in this study might have been unsuitable for their growth.

Pestalotiopsis neglecta and LLS12 are regarded as cellulose decomposers that exclusively attacked carbohydrates without delignification. Xylaria species, Geniculosporium species and Phialophora lignicola have ligninolytic activity (see references in Domsch 1980, Osono and Takeda 2002) but showed a limited ability to decompose larch litter in the present study. The other seven ascomycetes functionally were regarded as "sugar fungi", and their growth may depend on readily available energy sources such as soluble carbohydrates (Hudson 1968).

In the present study, it is difficult to differentiate between loss of needle mass and gain in fungal biomass. The biomass produced by the fungus relative to that consumed—defined as the economic coefficient by Mikola (1956)—was variable among species and nutrient conditions, usually ranging from 20 to 30%. Mikola (1956) reported forest soil basidiomycetes synthesized biomass corresponding to 16–49% (28% on mean, n = 19) of the glucose consumed under pure-culture conditions.

Substrate utilization pattern – Basidiomycetes and ascomycetes differed in their ability to use lignin and carbohydrates in larch litter. This result is consistent with Osono and Takeda (2002), who reported that ascomycetes decompose holocellulose in preference to lignin more so than do basidiomycetes on beech litter. The seven basidiomycetes that caused higher litter weight loss attacked both lignin and carbohydrates in variable proportions: Collybia dryophila, C. peronata, Micromphale sp. and Clitocybe gibba caused selective delignification, while Trametes versicolor, Cyathus striatus and Mycena polygramma attacked carbohydrates in preference to lignin. The results are consistent with previous reports. Lignin/weight loss ratio (L/W) of C. dryophila previously was determined as 2.1 and 2.0 for beech wood and leaf litter, respectively, while values of L/W of M. polygramma for beech-leaf litter have been reported as 0.9 and 1.4 (Lindeberg 1946, Tanesaka 1993, Osono and Takeda 2002). Trametes versicolor has caused simultaneous decay of lignin and cellulose in beech wood and leaf litter (Enoki 1988, Osono and Takeda 2002), and Cyathus striatus has caused simultaneous decompisition of lignin and cellulose in wheat straw and maple wood (Wicklow 1984). White rot in wood caused by basidiomycetes has been divided into two functional types—selective delignification and simultaneous decomposition—based on the relative utilization pattern of lignin and cellulose (Otjen and Blanchette 1986). The same functional types are recognized in basidiomycete white rot on larch-needle litter.

Chemical composition of decomposition products – Lignin and carbohydrate content was different in the remaining litter, depending on the selectivity of lignin and carbohydrate use by basidiomycetes and ascomycetes. The positive relationship between lignin and nitrogen contents for seven basidiomycetes indicated that the degree of selective delignification determined the final concentration of nitrogen in litter. However, lignin and nitrogen content did not correlate when P. neglecta and LLS12 were included, suggesting that these fungi caused nitrogen loss without delignification, in contrast to the basidiomycetes.

In forest ecosystems, litter decomposition is carried out by a succession of multiple fungi that is regulated by the availability of substrate, such as organic matter and nutrients, and by environmental factors, such as temperature and moisture (Hudson 1968, Swift 1979). The current study demonstrated the role of diverse fungi in larch needle-litter decomposition. The relative rate of lignin and carbohydrate loss and the chemical composition of decomposition products were dependent on the selectivity of lignin and carbohydrate decomposition by basidiomycetes or ascomycetes. Ascomycetes are deemed component restricted in that individual colonies are limited by the physical boundaries of the space they occupy (Cooke and Rayner 1984), such as a leaf. These communities may attack structural and soluble carbohydrates in preference to lignin in each leaf.

On the other hand, needle litter decomposes differently when colonized by basidiomycetes. In this case, both lignin and carbohydrates are attacked in various proportions depending on the species and nitrogen content is reduced in relation to the selectivity of delignification. It should be noted that the majority of basidiomycetous fungi are not component restricted, i.e., their habitat included the entire litter system (Cooke and Rayner 1984). Their mycelia often are extensive but unevenly distributed because of their tendency to develop as fairy rings and to form a mosaic of individuals in forest floors. Hence, a portion of needle litter may undergo delignifying decomposition typical of the basidiomycetes. This situation has been described as "bleached litter" by Harris (1945), Saito (1960) and Hintikka (1970). The proportion of litter decomposed by basidiomycetes or ascomycetes may vary among forest stands, depending on the biodiversity of fungal community within the forests. Further studies are required to evaluate the effect of functional biodiversity of fungal communities on nutrient cycling and the development of soil organic matter in forest ecosystems.

	ACKNOWLEDGMENTS

 
We thank Dr. H. Barclay for his critical reading of the manuscript; Drs. S. Tokumasu, T. Hosoya, I. Tanaka and A. Nakagiri for their helpful identification of fungi; Drs. S. Inaba and S. Iwamoto for their help with the fieldwork; Mr. D. Hirose and Ms. K. Koide for their useful discussion; Dr. G. J. Samuels and two anonymous reviewers for their critical reading of the manuscript. We especially thank Dr. S. Iwamoto for his kindness in providing preliminary data on DNA analysis. This study received partial financial support from the Japanese Ministry of Education, Culture and Sports (No. 14760099).

	FOOTNOTES

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

Accepted for publication April 18, 2003.

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