See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/226973136 Decomposition of spruce litter needles of different quality by Setulipes androsaceus and Thysanophora penicillioides ARTICLE in PLANT AND SOIL · OCTOBER 2008 Impact Factor: 2.95 · DOI: 10.1007/s11104-008-9666-5 CITATIONS READS 6 66 6 AUTHORS, INCLUDING: Ondřej Koukol Magda Vosmanská 31 PUBLICATIONS 207 CITATIONS 19 PUBLICATIONS 75 CITATIONS Charles University in Prague SEE PROFILE University of Chemistry and Technolog… SEE PROFILE Miroslav Vosatka Marcela Kovářová 111 PUBLICATIONS 1,913 CITATIONS 34 PUBLICATIONS 241 CITATIONS Institute of Botany of the ASCR SEE PROFILE All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immediately. Mendel University in Brno SEE PROFILE Available from: Ondřej Koukol Retrieved on: 04 February 2016 Plant Soil (2008) 311:151–159 DOI 10.1007/s11104-008-9666-5 REGULAR ARTICLE Decomposition of spruce litter needles of different quality by Setulipes androsaceus and Thysanophora penicillioides Ondřej Koukol & Blanka Beňová & Magda Vosmanská & Tomáš Frantík & Miroslav Vosátka & Marcela Kovářová Received: 11 March 2008 / Accepted: 21 May 2008 / Published online: 18 June 2008 # Springer Science + Business Media B.V. 2008 Abstract Various biotic and abiotic factors may change the quality of cast spruce needles or induce premature casting, subsequently altering the composition of needle litter. We tested the decomposition efficiency of Setulipes androsaceus, a key litter decomposer in spruce forests, on needles of the Norway spruce (Picea abies) that fell into three different categories of quality. We designed a cultivation experiment to test the decomposition rate of the Responsible Editor: David E. Crowley. O. Koukol (*) Department of Botany, Faculty of Science, Charles University in Prague, Benátská 2, 128 01 Prague 2, Czech Republic e-mail: o.koukol@seznam.cz O. Koukol : T. Frantík : M. Vosátka : M. Kovářová Department of Mycorrhizal Symbioses, Institute of Botany ASCR, 252 43 Průhonice, Czech Republic B. Beňová Department of Analytical Chemistry, Faculty of Chemical Technology, University of Pardubice, nám. Čs. legií 565, 532 10 Pardubice, Czech Republic M. Vosmanská Institute of Chemical Technology, 166 28 Prague 6, Czech Republic following needle categories: (1) naturally senesced brown needles, (2) intact, prematurely fallen green needles, and (3) frass pellets produced by caterpillars of the spruce web-spinning sawfly (Cephalcia spp.). Needles from each category were cultivated both independently and in combination. After a 4-month incubation, dry weight loss and the decrease of phydroxyacetophenone (p-HAP) and catechin were measured as markers of decomposition. Colonization of green needles by mycelia of S. androsaceus was initially inhibited. However, within the experimental period, those green needles successfully colonized by S. androsaceus lost more mass (22% of dry weight) than the brown needles (18% of dry weight). S. androsaceus also decreased the p-HAP and catechin contents of the green needles. Another fungal decomposer, Thysanophora penicillioides, was introduced only to the treatment that contained all three needle categories, and it induced less weight loss than S. androsaceus, but degraded the two phenolics to a similar extent. Neither the green nor the brown needles exhibited a more rapid rate of decomposition when cultivated in combination with another category of needles. We conclude that the increased proportions of green needles and frass pellets in the litter will be connected with temporarily increased decomposition activity of S. androsaceus. Keywords Setulipes androsaceus . Thysanophora penicillioides . p-hydroxyacetophenone . Catechin . Norway spruce . Litter . Cephalcia spp. . Bark beetle 152 Introduction The horse feather fungus Setulipes (Marasmius) androsaceus (L.) Antonín is a widespread spruce and pine litter decomposing saprotroph, which is common in both temperate and boreal regions. Vegetative mycelia of the fungus colonize needle interiors and produce numerous visible black rhizomorphs (multihyphal vegetative structures) and basidiocarps (Gourbière and Pépin 1987; Mitchell and Millar 1978; Ponge 1991). S. androsaceus is regarded as a key decomposer of litter needles, and produces both cellulolytic and lignolytic enzymes (Cox et al. 2001; Gourbière and Corman 1987). Though it is a relatively strong competitor (Koukol et al. 2006), its presence in the L horizon of the soil profile suggests a trade off with other saprotrophic basidiomycetes (e.g. Mycena galopus (Pers.) P. Kumm.), saprotrophic ascomycetes (e.g. Scleroconidioma sphagnicola Tsuneda, Currah & Thormann), ectomycorrhizal basidiomycetes, and invertebrates (e.g. Onychiurus latus Gisin) (Frankland et al. 1995; Lindahl and Boberg 2008; Koukol et al. 2006). Unlike saprotrophic fungi with endophytic phase, S. androsaceus does not occur in freshly fallen needles (Kowalski and Stańczykiewicz 2000; Przybył et al. 2007). It spreads from established mycelial networks to other needles in the litter via rhizomorphs. The term “needle litter” is often used to collectively describe the needles on the forest floor. However, the needle litter is heterogeneous, and different categories (or habitats) may be identified. Gremmen (1960) distinguished two types of Pinus sylvestris L. and P. nigra Arnold trash needles (needles still attached to fallen or dead twigs), which differed in their humidity and were occupied by different fungal communities. Minter and Millar (1980) distinguished four categories of P. sylvestris litter needles: (1) naturally senesced needles, (2) trash needles, (3) green needles mechanically detached from the tree by wind or animals, and (4) green needles cast prematurely after an attack by a parasitic fungus such as Lophodermium spp. These different needle categories were colonized by different communities of saprotrophic ascomycetes. We established similar categories as Minter and Millar (1980) for spruce needles (Picea abies (L.) Karst.). Classifying by quality, we differentiated between (1) fallen naturally senesced brown needles, Plant Soil (2008) 311:151–159 (2) prematurely fallen green needles with an elevated phenolic content, with no evidence of mechanical degradation, and (3) frass pellets. Frass pellets are finely crushed green needles that have passed through the gut of caterpillars of the web-spinning sawfly (spruce webworm) Cephalcia spp. (Hymenoptera). This material is enriched with bacteria and micromycetes, particularly cellulose-decomposing species (Grunda 1999). Though frass pellets represent a minor fraction of natural litter, their number may substantially rise when a forest experiences sawfly outbreaks, which occasionally induce total defoliation and spruce die-back (Battisti et al. 2000; Marchisio et al. 1994). Similarly, bark beetle (Ips spp., Coleoptera) outbreaks or other environmental stress factors (including wind, snow, frost, drought, and acid precipitation) may cause mass defoliation and premature shedding of otherwise healthy green needles. Outbreaks of sawflies and bark beetles in the Bohemian Forest, Czech Republic between 2001 and 2002 produced litter with dry weight composed of up to 3.9% frass pellets and up to 66% green needles (Kovářová, unpubl. data). The contributions of frass pellets and green needles rose significantly with respect to the major fraction during this period. Under normal circumstances, naturally senesced brown needles generally comprise 70% of total litter dry weight. Due to the high content of phenolic substances in green needles, a higher proportion of green needles in the needle litter may exert various allelopathic effects on the autochthonous fungi and other microorganisms (Kuiters 1990, Przybył et al. 2007). However, positive effect of phenolics on fungal growth was also reported (Lindeberg et al. 1980). It is unknown whether S. androsaceus avoids colonizing freshly fallen green needles. Nor is it known whether sudden changes in needle category proportions, such as bulk increases of green needles and frass pellets, affect growth of prominent fungal decomposers or the turnover rate of the substrate. The objective of this study was to determine the decomposition of three needle categories by S. androsaceus in a multiple substrate experiment. Dry weight loss and the degradation of phenolics (represented by phydroxyacetophenone and catechin) were used as markers of decomposition after a 4-month cultivation period. Further, the decomposition rate of the three needle categories in combination by Thysanophora penicillioides (Roum.) W.B. Kendr., another frequent Plant Soil (2008) 311:151–159 153 colonizer of spruce litter needles, was determined. We hypothesized that (1) the green needles would decompose more slowly than the brown needles due to the inhibitory effect of a high phenolic content and their higher integrity and intact cuticle, and that (2) the decomposition process in green needles would be increased by co-cultivation with brown needles and frass pellets. Material and methods Substrate and fungal strains Spruce (Picea abies (L.) Karst.) needles and frass pellets produced by Cephalcia spp. were obtained from litter traps (0.5×0.5 m) placed in four stands with 10 traps per stand in a high-mountain spruce forest (elevation between 1100 and 1200 m; stand 1: 48°59′10”N, 13°27′30”E; stand 2: 48°59′180”N, 13° 27′54”E; stand 3: 48°59′6”N, 13°26′19”E; stand 4: 48°59′14”N, 13°28′13”E) in the Bohemian Forest (Šumava National Park), Czech Republic. Fallen material was collected in two to five month intervals between 2000 and 2004. Material was transported to the laboratory, air dried, and hand-sorted into the following categories: (1) brown, matte needles (B) colonized by fungi (bearing fungal fruit bodies, stromata of conidiophores) or with visible mechanically eroded cuticles, (2) intact, green to grey-green prematurely cast shiny needles (G) with no obvious evidence of fungal colonization or grazing, (3) frass pellets (F), and (4) other material including cone scales, twigs, lichen thali (this category was not included in the experiment). Collections from individual traps were weighed, and the proportions of needle categories were determined. Data concerning the dynamics of various litter fractions will be published at a later date. The material was pooled either by stand (needles) or from all four stands (frass pellets). Frass pellet pooling was necessary because frass amounts varied greatly across stands. Setulipes androsaceus strain CCBAS 859/I was isolated from fruit bodies, and Thysanophora penicillioides strain SM12-3 was isolated from litter needles collected from stand 1. Fungal cultures were preserved on “spruce agar” prior to inoculation. Spruce agar was prepared as follows: 40 g of litter needles were extracted overnight in 1 l of distilled water and filtered. After that, 6 g of glucose and 15 g of agar were added to 1 l of the filtered extract and sterilised in an autoclave. Experimental systems The experiment was performed in 50 ml Falcon polypropylene conical tubes (BD Bioscience, NJ, USA) with cotton plugs. Material was inserted into tubes either as loose needles or as needles or frass pellets contained in a bag of unwoven cloth, enabling colonization of the inner substrate by fungal mycelia. Up to two bags containing different material were placed within the loose needles. Substrate combinations and the amount of material in each system are listed in Table 1. The ratio of brown to green needles (3:1) and the ratio of needles to frass pellets (30:1) in the different treatments was approximated based on 5-year monitoring of litter traps (Kovářová, unpubl. data). The design simulated needle category proportions in both an ordinary forest (treatments BB, BGF, BF, BG) and a highly stressed forest (treatments GG, GF). Tubes containing brown needles embedded in Table 1 Needle category combinations, initial amount of the needle category added, inoculation with fungi, and the water content of the tubes Treatment GG BB GF BF BG BGF Green needles (G) Brown needles (B) Frass pellets (F) Loose (g) In bag (g) Loose (g) In bag (g) In bag (g) 3 − 4 − − − 1 − − − 1 1 − 3 − 4 3 3 − 1 − − − − − − 0.12 0.12 − 0.12 “−“ This material/fungus was not present in the treatment Water (ml) 3.23 4.28 3.35 4.40 4.02 4.14 Inoculation T. penicillioides S. androsaceus − − − − − + + + + + + + 154 green needles and tubes consisting of frass pellets only were not considered, as they did not correspond to any naturally occurring situations. Filled tubes were sterilized using gamma radiation (25 kGy). After sterilization, tubes were moistened with autoclaved distilled water in an amount equal to the water absorbed by the respective needle category after soaking for 24 hours (Table 1). Agar discs (5 mm in diameter) were cut from the mycelia of S. androsaceus and T. penicillioides growing on spruce agar (see above), and inserted into each tube on the surface of loose needles. T. penicillioides was inserted only into the BGF treatment. Control treatments received only sterilised water. Each tube was weighed after inoculation. All treatments were performed in five replicates per stand (total n=260). The tubes were incubated at a 40° angle in plastic boxes, with the material from a given stand in the same box. These were kept in climaboxes with an ambient day/night temperature of 16/ 20°C for four months. During this period, fungal mycelia thoroughly colonized the tube contents and material in the bags. To maintain the initial moisture level, the tubes were weighed every 20 days. Moisture loss determined approximately by weight loss was replenished as necessary with a corresponding amount of sterilised distilled water. After four months, loose needles, frass pellets, and needles in bags were removed from all tubes and dried at 60°C. All material was separately weighed, ground, and analyzed for p-hydroxyacetophenone and catechin content. Chemical analyses were performed with five to seven replicates per treatment (i.e. one or two from each stand), and samples were chosen randomly. Plant Soil (2008) 311:151–159 Catechin and p-HAP were identified and quantified using two HPLC systems. First, samples were analysed using a liquid chromatograph equipped with a multi-channel electrochemical coulometric CoulArray detector (ESA Inc., Chelmsford, MA, USA). The eight-channel CoulArray detector allowed high selectivity and sensitivity with low range limits of detection (2.0 μg l−1 and 1.2 μg l−1, for catechin and p-HAP, respectively). This system was equipped with a binary pump, a thermostatted autosampler, a thermostatted column compartment, and an eightchannel electrochemical detector. The compounds were analyzed with a LiChrospher 100 RP C18 column (125 ×4.6 mm i.d. 5 μm). Elution was performed at 35°C. Gradient conditions were applied with a mobile phase consisting of acidified redistilled water (1 ml orthophosphoric acid to 1 l of mobile phase) and acetonitrile at flow rate 0.5 ml min−1, with an injection volume of 20 μl. Working potentials of 200, 300, 400, 500, 600, 700, 800 and 900 mV were applied to the eight electrochemical cells of the detector. Second, the extracts were analyzed using a liquid chromatograph Shimadzu LC 2010 (Shimadzu, Columbia, MD, USA) equipped with a UV-VIS detector with two variable wavelengths. The conditions of analysis were as above. The compounds were detected at 280 nm and 220 nm for p-HAP and catechin, respectively (detection limits 3.8 μg l−1 and 1.8 μg l−1, for catechin and p-HAP, respectively). Authentic standards (Sigma Aldrich, Steiheim, Germany) were used for identification of p-HAP and catechin. Both compounds were quantified by integrating the peak areas using an external standard method. Relative amounts of p-HAP and catechin are reported as percent of dry weight. Chemical analyses Statistical analyses Catechin and p-hydroxyacetophenone (p-HAP) were extracted following the methods of Vosmanská et al. (2005). We used a two-step extraction (water, methanol) in an ultrasonic bath. Approximately 40 mg powder of ground material was inserted into a test tube with 5 ml redistilled water and extracted for one hour in the ultrasonic bath. The extract was then filtered through a 0.2 μm filter and 5 ml of methanol was added to the pellet and re-extracted for one hour in the ultrasonic bath. Both extracts were analyzed using HPLC. Dry weight loss was calculated as a percent loss of initial weight. Data for the dry weight loss, content of p-HAP and catechin were transformed by rank. ANOVA with nested factor (“stand”) and Tukey’s test were performed using SPSS 15.0 for Windows (SPSS, Cary, NC, USA). The following factors were tested: needle category, the treatment (Table 1) and inoculation with fungi or fungal species (only the BGF treatment). For all analyses, the significance level was set at P≤0.05. Plant Soil (2008) 311:151–159 Results S. androsaceus mycelia failed to grow in 14 of the 40 (35%) tubes where loose green needles were inoculated with this species, and these samples were thus excluded from the experiment. Likewise, 16 of 80 (20%) tubes with loose brown needles inoculated did not yield S. androsaceus colonies and were also excluded. However, when S. androsaceus mycelia were successfully established, they grew rapidly and thoroughly colonized the tube and also the bag content. S. androsaceus frequently formed black rhizomorphs, regardless of the treatment. T. penicillioides was able to grow and sporulate extensively. Inoculation with either S. androsaceus or T. penicillioides resulted in significantly greater weight loss in both needle categories and frass pellets as compared to the uninoculated controls. The difference between the fungal species was also significant, as treatment with S. androsaceus caused significantly greater weight loss than T. penicillioides (Fig. 1). The relative weight loss of the green needles was significantly higher than that of the brown needles (ANOVA, P≤0.05). 155 The p-HAP content of uninoculated green needles was significantly higher (ANOVA, P≤0.05) than that of uninoculated brown needles: 0.26% vs. 0.05% of dry weight, respectively. S. androsaceus caused a significant decrease in pHAP content in green needles, regardless of needle category combination (Fig. 2). Similarly, T. penicillioides induced a significant decrease in p-HAP in the green needles in the BGF treatment. The activity of S. androsaceus resulted in a significant decrease of p-HAP content in the brown needles for treatments BG and BF but not for treatments BB and BGF (Fig. 3). The content of p-HAP in brown needle in the BGF treatment decreased significantly when inoculated with T. penicillioides. Frass pellets contained p-HAP in amounts comparable to those of the green needles. The passage through sawfly larvae did not affect p-HAP content. Incubation with S. androsaceus resulted in a significant decrease in p-HAP when the frass pellets were combined with other needle categories in the GF and BGF treatments, but not in the BF treatment (Fig. 4). The decrease noted in the BGF treatment caused by T. penicillioides was comparable to that of S. androsaceus. S. androsaceus caused significant catechin loss in the green needles in treatments GG, GF, and BGF but not in the BG treatment (Fig. 5). The inoculation with T. penicillioides in the BGF treatment caused no significant decrease in catechin content in the green needles. Inoculation with S. androsaceus and/or T. penicillioides resulted in no significant loss of catechin in the brown needles. Discussion Fig. 1 Weight loss of green needles, brown needles and frass pellets in the bag inoculated with S. androsaceus and T. penicillioides compared to the uninoculated control. Values represent mean ± S.E. (n=14). Different letters indicate significant differences within particular needle categories (P≤0.05) Our experiment simulating decomposition of litter in both ordinary and stressed forests showed that the two strains of saprotrophic fungi, S. androsaceus and T. penicillioides, colonized all needle categories to a similar extent, and caused significant weight loss in colonized needles compared to that of uninoculated controls. These two fungal strains were isolated from a boreal spruce forest exposed to several outbreaks of Cephalcia spp. and I. typographus in recent decades (Zemek and Heřman 2001). The outbreaks had catastrophic stress effects on the tree population, the ecological implications of which have been studied from zoological and botanical perspectives (Battisti et 156 Plant Soil (2008) 311:151–159 Fig. 2 p-HAP content in green needles after cultivation with S. androsaceus or T. penicillioides when combined with green needles (GG), brown needles (BG), frass pellets (GF) or both brown needles and frass pellets (BGF). Values represent mean ± S.E. (n=6). Different letters indicate significant differences within particular treatments (P≤0.05) Fig. 4 p-HAP content in frass pellets after cultivation with S. androsaceus or T. penicillioides when combined with brown needles (BF), green needles (GF) or both brown and green needles (BGF). Values represent mean ± S.E. (n=6). Different letters indicate significant differences within particular treatments (p≤0.05) Fig. 3 p-HAP content in brown needles after cultivation with S. androsaceus or T. penicillioides when combined with brown needles (BB), green needles (BG), frass pellets (BF) or both brown needles and frass pellets (BGF). Values represent mean ± S.E. (n=6). Different letters indicate significant differences within particular treatments (P≤0.05) Fig. 5 Catechin content in green needles after cultivation with S. androsaceus or T. penicillioides when combined with green needles (GG), brown needles (BG), frass pellets (GF) or both brown needles and frass pellets (BGF). Values represent mean ± S.E. (n=6). Different letters indicate significant differences within particular treatments (P≤0.05) Plant Soil (2008) 311:151–159 al. 2000; Jonášová 2001; Zemek and Heřman 2001; Háněl 2004). We documented novel observations of the effects of two saprotrophic decomposing fungal species on different needle litter categories. As expected, S. androsaceus, a litter-colonizing basidiomycete with enzymatic abilities similar to white rot fungi (Cox et al. 2001), was able to decompose needles more efficiently than T. penicillioides. Weight loss was similar to the decomposition of pine needles inoculated with another strain of S. androsaceus (Cox et al. 2001). Surprisingly, the green needles were degraded more rapidly than the brown needles (Fig. 2), although they did not support initial colonization by S. androsaceus. When the inoculum was placed on the green needles, it failed to grow in one third of the experimental tubes, even at ideal humidity and temperature. We suspect that the intact needle epidermis and cuticle cause this inhibition (Gourbière et al. 1988). The inhibition effect of surface wax cuticle on fungal pathogens was reported by Smith et al. (2006). The partially eroded brown needles bearing visual evidence of fungal colonization, namely protruding stromata and conidiophores of ascomycetes, were more easily colonized by S. androsaceus. However, 20% of the tubes where inoculum was placed on the brown needles also remained sterile, suggesting that vegetative mycelia have a limited ability to spread in the litter layer and that specialized mycelial structures play a crucial role in the colonization. Under natural conditions, the rhizomorphs that emerge from established mycelial nets in the needles of the litter layer colonize freshly fallen needles (Gourbière and Pépin 1987; Frankland et al. 1995). Incubation with T. penicillioides did not yield different results for green and brown needles. The decomposition ability of T. penicillioides for spruce needles (measured as dry weight loss) was higher than that for fir needles as reported by Osono and Takeda (2006). However, the measured decomposition rate was comparable with other spruce needle colonizing ascomycetes, including Sclerophoma pythiophila (Corda) Höhn. and Tiarosporella parca Berk & Broome as reported by Müller et al. (2001). These three ascomycetes may have been already present as endophytes in freshly fallen needles and played the role of primary, pioneer decomposers. They spread mostly through dispersion of asexually produced spores (conidia), but do not produce specific mycelial 157 structures (rhizomorphs or mycelial cords) enabling the extensive colonization of litter. Although S. androsaceus showed a limited ability to establish colonies in loose green needles, (partially verifying our first hypothesis), we documented rapid decomposition in successfully colonized substrates after the 4-month cultivation period. The rapid weight loss of green needles compared to brown needles illustrated degradation of simple organic and non-structural organic compounds. It also suggests the stimulatory effect of some compounds present in green needle tissue. These putative stimulatory chemicals may include various phenolic compounds. For example, the quantity of p-HAP in the green needles was significantly higher than that in the brown needles. Catechin was detected only occasionally, and mostly in the green needles. We suggest that the presence of p-HAP and catechin enhanced S. androsaceus colonization of the green needle substrates. This hypothesis is consistent with previous in vitro experiments using S. androsaceus and other litter decomposers cultivated on liquid media and treated with simple phenolics (Lindeberg et al. 1980). The combination of flavonoids and phenols extracted from fresh pine needles stimulated the growth of S. androsaceus. Taxifolin glycoside was shown to have a prominent positive effect on fungal growth (Lindeberg et al. 1980). Similarly, Black and Dix (1976) documented a stimulatory effect of ferrulic acid on germination and growth of T. penicillioides. Souto et al. (2000) revealed that spruce-derived phenolics added to humus were utilized by autochthonous microorganisms as a source of carbon within several days, and that the added phenolics were even stimulatory for some fungi. However, Souto et al. only considered changes in entire fungal communities, so it is unclear which fungi were stimulated by the treatment. The design of our experiment enabled us to isolate effects of single fungal species under semi-natural conditions. S. androsaceus and T. penicilloides had similar utilization activities, and both induced significant loss of pHAP from green needles. Phenolics present in soil show seasonal variation as well as different contents in distinct soil horizons. Litter needles represent the main source of p-HAP in the soil ecosystem (Vrchotová et al. 2004), although p-HAP may also be rinsed from canopy needles by rainwater, and thus be introduced by percolating 158 through the soil (Hongve et al. 2000; Vosmanská et al. 2005; Muscolo and Sidari 2006). In the winter, pHAP may also rise through pores in the soil and enrich the snow cover (Gallet and Pellisier 1997). Thus, the effect of p-HAP and other phenolics on autochthonous litter decomposers might also occur during later stages of decomposition. We found that needle category combination had only a negligible effect on the decomposition of the green needles, and thus, our second hypothesis was not supported. This result may be due to the limited ability of fungi to translocate nutrients horizontally between needle categories introduced to the forest floor at roughly the same time. This result contrasts with others that described the importance of vertical nutrient transport in the mycelium, from the F towards the L horizon, enabling decomposition of recently fallen needles with high C:N ratios (Lindahl and Boberg 2008). Similarly, the addition of frass pellets had no effect on decomposition of either needle category. The mechanical breakdown of healthy needles by caterpillars and the supposed enhanced accessibility to nutrients (Grunda 1999) did not appear to be an advantage for S. androsaceus in this experiment. Not the composition of the litter, but environmental factors, including microclimatic conditions (i.e. frequent desiccation of the uppermost litter layer in partly shaded stands) or lower consistency of loose needle litter on the ground (requiring a higher investment into mycelial biomass to reach the needles) determine the efficiency and rate of colonization of fresh substrate in nature. Conclusion Sudden changes in the ratio of naturally senesced and prematurely cast green needles due to bark-beetle outbreaks and forest clear-cuttings are connected with huge inputs of organic matter and nutrients into the soil ecosystem. In the Bohemian Forest, mountain Norway spruce forests contain 18–20 tons of needles per hectare (Kovářová and Vacek 2003). Similarly, the input of frass pellets (transformed green needles) into the litter is increased during sawfly outbreaks (Kovářová, unpubl. data). The impacts of these events on the key litter decomposing saprotrophic fungus S. androsaceus were assessed in a multiple substrate experiment. The data showed that S. androsaceus did Plant Soil (2008) 311:151–159 not specifically avoid colonization or utilization of the green needles, and did not discriminate between needle categories in the mixed treatments. Though needle categories differed in the mechanical integrity and nutrient content (particularly with respect to pHAP and catechin), they served as equivalent substrates for S. androsaceus. Our hypothesis predicting lower decomposability of green versus brown needles was not verified. We assume that S. androsaceus would temporarily increase the rate of decomposition after following various stressful events in the spruce forest which increase the amount of green needles in the litter. Acknowledgements The work was financed by the grant project of the Grant Agency of the Czech Republic, project no. 206/05/0269 and is part of the research project AV0Z60050516 of the Institute of Botany, ASCR. The assistance of Helena Koblihová and Kristýna Procházková is heartily acknowledged. We also thank two anonymous reviewers who provided valuable critiques and substantially improved the manuscript. 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