Decomposition nitrogen and phosphorus

New Phytol. (19%), 134, 123-132

Decomposition, nitrogen and phosphorus mineralization from beech leaf litter colonized by ectomycorrhizal or litterdecomposing basidiomycetes BY

JAN V. COLPAERTi* AND K A T I A K. VAN TICHELEN^

^Laboratory of Developmental Biology, Institute of Botany, Katholieke Universiteit Leuven, K. Mercierlaan, 92, B-3001 Leuven, Belgium ^Laboratory of Plant Ecology, Institute of Botany, Katholieke Universiteit Leuven, K. Mercierlaan, 92, B-3001 Leuven, Belgium (Received 12 October 1995; accepted 12 March 1996)

SUMMARY T h e decomposition a n d t h e nitrogen a n d phosphorus mineralization of fresh beech (Fagus sylvatica L . ) leaf litter are described. Leaves were b u r i e d for u p to 6 m o n t h s in plant containers m which Scots pine (Pxnus sylvestris L . ) seedlings were cultivated at a low rate of nutrient addition. T h e saprotrophic abilities of three ectomycorrhizal fungi, Thelephora terrestris E h r h . : F r . , Suillus bovinus ( L . : F t . ) O. K u n t z e and Paxillus involutus (Batsch: F r ) F t . , were c o m p a r e d with t h e degradation caused by t h e iittet-decomposing basidiomycete, Lepista nuda (Bull.: F t . ) Cooke. U n i n o c u l a t e d leaves were included as controls. T h e investigation was performed at two different p H values since substrate p H is supposed to have an effect on the activities of extracellular enzymes of ectomycorrhizal fungi. T h e enzyme expression m i g h t also be largely influenced by t h e substrate they colonised. T h e mycorrhizal fungi caused only a low decomposition rate of t h e litter compared with that of L . nuda, and nitrogen was released only by L nuda. Leaves colonized by mycorrhizal fungi showed no net release of nitrogen; on t h e contrary, a small accumulation of N in the litter was observed. It therefore seems likely that the ectomyccrrhizal fungi studied do not have t h e ability to d e c o m p o s e efficiently t h e ligDOcellulose matrix of the relatively recalcitrant beech Jeaf litter. T h e degradation of this matrix seems to be essential for the fungi to gain access to the leaf nitrogen pool of fresh beech litter A direct release of nitrogen from organic compounds by ectomycorrhizal fungi seems therefore to be confined to t h e older litter layers. T h e beech leaf litter contained an i m p o r t a n t fraction of easily mineralizable p h o s p h o r u s . P was n o t a g r o w t h limiting factor m t h e cultivation system, and could therefore accumulate m the leaf litter colonized by t h e ectomycorrhizal mycelium. Key w o r d s : Basidiomycetes, ectomycorrhiza, litter decomposition, leaf litter mineralization, nutrient cycling.

INTRODUCTION The ectomycorrhizal mycehum of many basidiomvcetes which colonize the organic bonzons of forest soils is thought to be tnvolved tn direct mobilization of nutrients from organic substrates (Abuzinadah, Fmlay & Read, 1986; Read, 1991). There ts clear evidence that mycorrhizal fung. can produce enzymes which m.ght allow them to derive Lmeral nutrknts and carbon from organic resources (Dtghton 1991). However, Dighton (1991) concludes that the whole st^bject is far from completely ,

* To whom correspondence should be addressed.

understood, as many studies on this topic have been made under fairly artificial conditions. Very httle work has been done w.th intact mycorrbizal systems colonizing organic substrates that occur in forest ecosystems (Bending & Read. 1995). The importance of the saprotrophic abihties o the ectomycorrhizal fungi for a particular host and for nutrient cycling m forest ecosystems ,s still unclear. Comparisons artd .nteract,on studies with true sapro roph.c fung a e .nd.spensable as they will allo^w us to °btain a be r understanding of the decomposition and m.nerahzation processes m forest '^'^^A'f ^ " f . In forests both nitrogen (Iv) and phosphorus (f) accumulate in a wide array of organic compounds

124

y. V. Colpaert and K. K. Van Tichelen

and the availability of these elements is inherently fungi might be much larger in the natural enlinked to the turnover of organic N and P compounds vironment. The investigation was performed at two which can have very long half-lives (Attiwill & different pH values since substrate pH is also Adams, 1993). Free inorganic N and P can hardly be supposed to have an important effect on the detected, because of their fast turnover in the soil. extracellular enzyme activities of ectomycorrhizal Competition for uptake of ammonium between the fungi (Read, 1991). different soil-inhabiting organisms is high, nonmycorrhizal roots being poor competitors (Smith & MATERI.'iLS .IND METHODS Smith, 1990). In temperate forests, saprotrophic and mycorPlant and fungus material rhizal basidiomycetes colonize intensively the F litter layers of the mor and moder humus (Hering, Two successive experiments were performed at two 1982). Vegetation growing on these soils produces a different pH conditions: pH 4 and pH 6. In the pH 4 litter that is not especially palatable to soil animals treatment 86 Scots pine {Pinus sylvestris L.) seedlings since it contains a lot of phenolic compounds. It is were grown in pairs in 4 1 plant containers with a also poor in N which, in addition, is not readily transparent wall (Colpaert,, Van Assche & Luijtens, accessible as it is retained within refractory com- 1992). Twenty seedlings were inoculated with Thelepounds such as protein-polyphenol complexes. Leaf phora terrestris Ehrh.; Fr., another 20 with Suillus litter falling on the soil surface is normally already bovinus (L.: Er.) O. Kuntze and six plants were colonized by microfungi, which originally become infected with Paxillus involutus (Batsch: Er) Fr.. established as biotrophs or necrotrophs. Once the The remaining 40 plants were not inoculated. In the easily degradable carbohydrates from the fresh leaf pH 6 treatment, P. involutus was omitted. T. terreslitter have been metabolized by the microfungi, tris was isolated from a carpophore collected in a saprotrophic basidiomycetes take over and the young pine stand planted on shifting inland dunes. decomposition and mineralization of more recal- S. bovinus and P. involutus, two 'protein fungi' citrant compounds can start. The decomposition and (Abuzinadah & Read, 1986), were collected from a mineralization of recalcitrant litters requires a well mixed semi-natural forest on a podzolic soil with a balanced production and activity of a whole set of mor humus (Zolder). The tree vegetation consisted extracellular enzymes, in an enzyme-hostile en- mainly of several deciduous tree species intermingled \'ironment. The purely litter-decomposing basidio- with some old pines. The litter white-rot fungus mycetes are probably best-equipped to perform this Lepista nuda (Bull.: Er.) Cooke was cultivated from basidiospores of a carpophore growing in the mor task (Cooke & Rayner, 1984). Although specific ectomycorrhizal fungi seem to humus of the same forest. This species can degrade have the enzymatic potential for the degradation of both cellulose and lignin (Norkrans, 1950) and causes organic N and P compounds, our knowledge about a severe bleaching of several types of leaf litter the naturally occurring substrates which are most (beech, pine, Taxus) (Watling, 1982), but does not susceptible to enzymatic attack is sparse. Bending & appear to degrade wood (Tanesaka, Masuda & Read (1995) studied the nutrient export from organic Kinugawa, 1993), and does not form mycorrhizas matter of the fermentation horizon (FHOM) by (Norkrans, 1950). ectomycorrhizal fungi. They demonstrated that Suillus bovinus was able to export 23, 22 and 30 % of the initial N, P and K present in this organic matter. The quantity of N exported from the FHOM exceeded that mineralized in uncolonized FHOM, suggesting that the additional N was mobilized directly from the organic matter. In the present study, we compared the decomposition and mineralization of fresh beech leaf litter by an important leaf litter decomposing basidiomycete, Lepista nuda (syn. Tricholoma nudum) and three ectomycorrhizal fungi, growing in an intact plantfungus system: Thelepkora terrestris Ehrh.: Fr., Paxillus involutus (Batsch: Fr) Fr. and Suillus bovinus (L.: Fr.) O. Kuntze. The mass loss and the release of nutrients from the litter were followed over a period of 6 months. Howe\'er, the expression of the enzymes which cause the degradation and mineralization of a particular substrate are affected by the qualitj' of that substrate, so that the degradative abilities of the

Growth conditions The perlite growth substratum (1—2 mm particles) was acid-washed then thoroughly rinsed with water until the conductivity of the eluted solution was < 50 /iS cm"^ Thereafter the perlite was dried again and 1 d before planting it was re-wetted with nutrient solution up to 80% of its maxima! water holding capacitv'. The plant containers were weighed at regular intervals to control the moisture level of the substratum. AU seedlings received daily equal additions of a dilute nutrient-solution containing increasing amounts of nutrients (Ingestad, Arveby & Kahr, 1986), sometimes supplemented with some extra distilled water to compensate for the differences in transpiration rates between the plants. In this nutrient solution for Pinus sylvestris, nutrients are present in well balanced proportions so that a steady state nutrition of the seedlings is obtained at a

Mineralization of beech leaves constant relative-nutrient addition. The N concentration in Ingestads' stock solution was decreased by 10%, in order to be sure that N was the growth linniting factor. The two different pH conditions were brought about by changing the NO^"; NH^^ ratio (60:40 and 50:50). Every month the pH and nutrient status of the perlite solution were controlled. Plants were maintained under non-sterile conditions in a growth chamber under a 16:8 h light: dark cycle, which was gradually changed to 20:4 h. The day: night temperature cycle was 23:15 °C, relative air humidity was at least 70 % and photosynthetically active radiation (PAR) was 400//mol m"' s"\ Organic material For each pH condition senescent beech (Fagus sylvatica L.) leaves were collected from a single tree at the University campus. Loosely attached leaves were picked by hand from twigs in October, just before abscission. The leaves were washed with plenty of tap water, put on grilles and 8 h later quickly dried under a stream of warm air (35 °C). This treatment was repeated three times. Before a fourth washing with distilled water, the leaves were cut into strips with a maximum width of 5 mm, and 2-00 g of the dried material was put in nylon bags with a mesh size of 100/(m to prevent root growth. When the plant containers were filled with perlite, three plastic tubes (diameter, 2-5 cm) had been introduced in al! pots. One month after the pine seedlings had been planted, the closed nylon bags were placed in the pots after removal of the plastic tubes. The bags fitted perfectly in the prepared holes in the growth medium, so that the penetration of the mycorrhizal mycelium, that was already growing around the tubes,, would not be hampered by large air gaps. The top of the bags was covered with 2 cm of perlite.

125

saprotrophic basidiomycetes (mostly Mycena sp.) so that it was difficult to study a single species. The presentation of beech leaf litter to mycorrhizal fungi of pine is not ecologically irrelevant since Pinus sylvestris often persists in mixed forests of deciduous trees; typical pine beech communities can be found on acid soils in central Europe (Ellenberg, 1978). Analysis of the litters from the preliminary experiment did not reveal important differences in the decomposition and mineralization pattern between the beech and pine litter. The inoculation of the OM with L. nuda was performed in vitro. The fungus was cultured in abundantly inoculated large Petri dishes (diameter, 18 cm) on Fries agar medium (Fries, 1978). Two weeks later when most of the agar surface was colonized with mycelium, 35 bags with re-wetted organic matter were put in the dishes. After 1 wk the bags were removed from the Petri dishes and buried in half of the plant containers which were left nonmycorrhizal. After 6 wk, a second inoculation was performed. For this treatment, spores of a L. nuda carpophore that were kept m a refrigerator for 3 months were suspended in water. Ten ml of this suspension was injected with a syringe in each bag of the L. nuda treatment. Atialyses Harvest. From each fungal treatment, three plant containers were harvested on three subsequent days after 13, 18 and 26 wk (pH 4 condition) or after 18, 22 and 26 «'k (pH 6 condition). Two bags from each container were used for an enzyme study (not reported here) and one bag was used to determine CO.,-release. Plants were partitioned into roots and shoots which were oven-dried at 80' °C.

Microhial activity. CO, release was measured with an IRGA system at 22 °C (Soderstrom & Read, 1987). The colonization and the condition of the organic The nylon net was removed from the sample, the material (OM) was inspected during the experiment: leaves were lightly sprayed with distilled water and 2 g of leaves were introduced in square holes put in an open Petri dish (diameter, 9 cm), that was (2-5 X 1-5 cm) just behind the transparent walls of placed in a flow-through cell (300 cm'') supplied with two pots in each treatment. This technique was also a constant air stream (r.h. = +80°o, 360 ppm COJ. used in a preUminary experiment in which we COo concentrations were measured after a standard compared the colonization of fresh leaf or needle equilibrium time of 15 min. Four months after litter of the following species: Betula pendula Roth, inoculation the density of the vegetative mycelium of F. sylvatica and P. sylvestris. Fresh birch leaves were the mycorrhizal fungi in the perlite was estimated by colonized within a few^ days by a whole array of the ergosterol assay (Nylund & Wallander, 1992). saprotrophic fungi exhibiting antagonism towards Fresh perlite samples (5 g) were crushed in a mortar the species we wanted to study. A more restrained in 5 ml of MeOH and then transferred to test tubes growth of microfungi was found on the OM from the with another 10 ml of MeOH. Two ml of 100"., other species. The final choice for beech OM was KOH (ww) were added to the sample and heated to influenced by the fact that the mycorrhizal coloniz- 100 °C for 30 min. The sample was neutralized with ation of dead pine needles was considerably slower 4 ml of 18 " 0 HCl. The alcoholic solution was washed and consequently more irregular when compared three times with 5 nr»l of pentane. The pentane layer with the colonization of the beech leaves. The was pipetted ofl' and collected in a glass vial. The needles also became more easily infected with pooled fractions were evaporated to dryness below

126

J. V. Colpaert and K. K. Van Tichelen

40 °C. The extract was dissolved in 2 ml of MeOH. Ergosterol was quantified with HPLC, using a reverse-phase Cjg column and pure MeOH as the mobile phase. Standard concentrations of pure ergosterol were added to clean perlite and treated in the same way as the samples to determine the efficiency of the extraction procedure. Trials to measure ergosterol in the organic matter were unsuccessful with this method because of dirty samples and irregular extractability of ergosterol.

These data confirm that N was the growth limiting factor in our cultivation system. The differences in plant growth between the two pH conditions were negligible and only in the pH 4 condition did the N O g ' i N H / ratio have to be corrected slightly to avoid excessive acidification of the perlite (lowest value pH 3-5)., Colonization of the leaves

Under both pH regimes, the vegetative mycelium of Litter decomposition. The organic matter of all bags, Thelephora terrestris colonized the whole perlite including those of the enzyme study, was dried at substratum within 4 wk. Paxillus involutus and 50 °C for 3 d. Litter decomposition {k) was calcu- Suillus bovinus did the same in 5 wk and 6 wk lated using an e.x;ponential decay model (Olson, respectively. The ergosterol measurements indicated 1963): X, = Xj e-", where X^ = initial weight of the that the mycelial densities were highest in the leaves and X, — weight of the leaves after time t in substratum of the two latter species {Table 1). yr. This model however could explain only the Compared with the fast vegetative growth of the weight loss of the leaves inoculated with L. nuda ectomycorrhizal mycelium in perlite, the coloniz(r^ > 0-95). For the other fungal treatments a better ation of the beech leaves took much longer. In the prediction of the data could be obtained when this first 2 wk the mycelial fronts of all mycorrhizal fungi equation was split into two distinct exponential did not penetrate the OM placed behind the curves (Paul & Clark, 1989): X, = C, e-^-' + C^ e-'V, transparent walls. It was only during the third and Cj is the easily degradable and leachable fraction the fourth week that the rate of colonization of the (19 %) of the organic matter, Cg is the fraction which leaves increased, T. terrestris being the first to exploit is only slowly degradable and k.^ represents the the material. At the end of the third month, all leaves decomposition rate of this fraction. were surface colonized with mycorrhizal mycelium. The density of this mycelium was soon equal to or Mineralization. Nitrogen in the leaves was extracted even larger than that of the perlite. From the first with the Kjeldahl method, NH^ was steam distilled month, scattered colonies with conidia could be into H,SOj (0-05 M) and determined colorimetrically observed on the surface of several beech leaves in all with Nessler's reagent. The P concentration in the treatments. The growth of L. nuda mycelium could leaves was measured after ashing two subsamples of scarcely be evaluated macroscopically and we do not three bags in a mufHe-furnace (500 °C, 3 h), then know whether the double inoculation was necessary. assayed by the molybdate-blue method (Murphy & However, a microscopic analysis of the OM after 2 Riley, 1962). The ash-free dry mass was measured months revealed the presence of a diffuse hasidioby the loss on ignition of 200 mg suhsamples in a mycete mycelium with clamp connections, which muffle furnace (600 °C, 8 h). To calculate the C:N was penetrating leaf cells. A few weeks later the ratio, a conversion factor of 0'48 was used to leaves started to show bleached patches and at the determ.ine carbon from ash-free dry mass (Anderson, last harvest most leaves had lost their initial strength. 1973). Cations were measured with AAS, after They became strongly bleached and some were ashing a subsample at 600 °C for 8 h. The pine reduced to skeletons of veins after 6 months. During seedlings were dried at 80 °C for 3 d, N and P the later stages of colonization this species formed concentrations in the needles were determined by mycelial cords which grow from the bags in search for new substrates. the same methods used for the OM.

RESULTS Plant growth The low nutrient addition rate resulted in a low relative growth rate (KGR) of the seedlings (Table 1), far below the maximal growth rate for Scots pine (Ingestad et al., 1986). Plants inoculated with mycorrhizal fungi grew slightly more slowly than the non-mycorrhizal ones. The shoots of the seedlings had a low N concentration and young needles were yellow-green. The tissue P concentrations and P : N ratio were relatively high (Ingestad et al., 1986).

Weight loss of organic matter The treatment of the lea\'es resulted in effective removal or elimination of saprotrophic basidiomycetes (spores) which could have been present on the leaves when collected. Only in six bags buried in the non-mycorrhizal substrate was an undesired infection with basidiomycete mycelium apparent. In one of these the invader could be identified as Mycena galopus (Pers.: Er.) Kummer. In the other fungal treatments invaders probably had more difficulty in becoming established in the bags owing to inhibitors or competition effects. Attempts to

Mineralization of beech leaves

127

Table 1. Growth parameters of the pine seedlings and mycorrhizal fungi, grown at a low N addition rate (% d-')

N {mg g-' d.wt)

P (mg g"^ d. wt)

P/N

1-90 1-76 1-70 1-92

8-3 (0-1) 7-5 (0-2) 7-4 (0-2) 8-5 (0-2)

1-44 1-73 1-68 1-35

(0-04) (0-04) (0-03) (0-03)

0-18 (O'Ol) 0-23 (O-OI) 0-23 (O'Ol) 0-16 (O'Ol)

0

1-78 1-69 1-59 1-58 176

8-2 (0-2) 7-1 (0-1) 7-0 (0-2) 6-9 (0-3) 8-1 (0-2)

1-55 1-85 1-65 1-65 1-57

(0-04) (0-03) (0-03) (0-05) (0-02)

0-19 0-26 0-25 0-24 0-18

0

EGR

Condition

Ergosterol (/*g g"' perlite)

pH6

Control Thelephora terrestris Suilhts bovinus Lepista nuda pH4 Control Thelephora terrestris Suillus bovimts Paxillus involutus Lepista nuda

(0-01) (O'Ol) (0-01) (O'Ol) (O'Ol)

7-6 (0-8) 15-1 (1-2) 0

8-1 (0-3) 22-5 (1-7) 33-1 (1-6) 0

Values in parentheses are standard errors of the mean. BGR was calculated from the tnean dry weights of the plants at the start of the experiment and at the different harvests. Nand P concentrations in the shoots are means of all plants (m = 20, except for Paxillus: n = 6). Ergosterol was measured 18 wk after inoculation (n = 3).

0-8

15 20 Time (wk)

25

Figure 1. Dry weight of beech leaf litter not inoculated with a basidiomycete (A, co) or colonized by mycelium of Thelephora terrestris ( • , T.t.), Suillus bovinus ( • , S.b.), Paxillus involutus {D) or Lepista nuda (A, L.n.). Curves follow mass loss according to the two-phase decomposition model.

sterilize the beech leaves with more rigorous treatments resulted in a greater palatability of the OM which finally resulted in more infections with other fungi, often compost species. In Figure 1 the rate of weight loss, according to the two-phase decomposition model, is plotted together with the remaining weights of the beech leaves. The decotnposition rate of the easily de-

composable fraction, has been set equal for all treatments. This phase of the decay process was finished before we started our measurements and was at least partly performed by the non-basidiomycete fungi in the system. In the second phase of the decay process the OM in the mycorrhizal treatment showed a very low decay rate, A^, indicating that beech leaves were very resistant to breakdown even at a relatively high temperature and high moisture contents. After 6 months the buried leaves showed no visible changes except for a slight discoloration to a darker brown. However, when L. nuda was inoculated on the OM, the decay rate was stimulated significantly (Table 2), similar to results obtained in vitro with other litter decomposing white-rot fungi (Hering, 1982). Taylor, Parkinson & Parsons (1989) reported similar decomposition rates for litter with a relatively low substrate quality in microcosms. Leaves colonized with S. bovinus and T. terrestris always lost more weight than did the control leaves. This small difference was significant for the OM colonized with S. bovinus (Table 2). By contrast the leaves colonized with P. involutus showed a smaller mass loss than did the controls. The microbial biomass in the mycorrhizal treatments was higher, however, than in the control treatment since carbon from the host plant was imported into the bags. This implies that the true decomposition is somewhat higher in the mycorrhizal treatments than can be deduced from the data for mass loss. Nevertheless, it is clear that the decomposition rate, mediated by the mycorrhizal fungi, is much lower than that of L. nuda. The mycelium of the mycorrhizal fungi can be stripped off easily from the leaf material with forceps, unlike Lepista mycelium. Microscopic analysis reveals that hyphae from the latter species penetrate and grow inside the leaf tissues. After removal of the mycelium, the leaf surfaces in the S. bovinus treatment showed a slight discoloration to yellow-brown.

128

J. V. Colpaert and K. K. Van. Tichekn

Table 2. Dry weight of beech leaf litter buried in pots with Pinus sylvestris seedlings C:N

Dry weight (g) 18 wk

22 wk

26 wk

26 wk

1-53 a 1-51 a 1-47 a 1-28 b

1-51 1-50 1-44 1-02

1-51 X

l'39v 0-92 z

38 a 37 a 34 b 30 c

13 wk

18 wk

26 wk

26 wk

1-60 a 1-54 a 1-51 a

1-53 148 143 1-63 1-20

1-50 X

1-45 xy 1-38 y

34 a 32 ab 30 b

1-09 z

30 b

k (yr-')Olson

k, (yr-')

*n.a. n.a. n.a. 1-55

0-12 0-15 0-22 1-00

3-92 3-13 2-14 0-47

n.a. n.a. n.a. n.a. 1-25

0-12 0-19 0-29

3-92 247 1-62

0-80

0-59

pH 6

Control Thelephora terrestris Suillus bovinus Lepista nuda

m mn n p

1 4 8 XV

pH4

Control Thelephora terrestris Suillus bovinus Paxillus involutus Lepista nuda

l'33b

mp mn n p q

The litter was colonized by a mycorrhizal or a saprotrophie basidiomycete (n = 6). Initial weight of the lea^'es was 2-00 g, C:N = 52 (pH 6) and 47 (pH 4). A is the decomposition rate according to the Olson (1963) model, A^ is the weight loss rate during the second phase of decomposition in a two-phase model with /jj = 15, <,,,, is the time required to obtain a 50 % weight loss. Within each pH treatment one-way .4NOVA was used to separate the means from one harvest. Means in a column not followed by the same letter are significantly different (Tukey's test, x = 0-01). * n.a. = not applicable.

Table 3. CO.^ release from beech leaf litter buried in pots with Pinus sylvestris seedlings Total CO, release (mg cr') 18 wk

22 wk

2-4 a 6-3 b 17-1 c 13-1 c

2-9 m 6-2 n 13-5 p 15-8 p

13 wk

18 wk

4-5 a 10-1 ac 18-6 h

3-5 m 8-8 n 22-3 p 17-2 p 8-5 n

CO2 release per g material left (mg d ' g ') 18 wk

22 wk

26 wk

15-2 z 12-8 yz

1-6 (0-3) 4-2 (0-8) 11-5(1-9) 10-4 (2-0)

1-9(0-3) 4-2 (0-6) 9-6(1-0) 154(1-7)

2-0 (0-3) 4-5 (0-3) 11-0(1-3) 14-0(1-5)

26 wk

13 wk

18 wk

26 wk

7-5 y 21-5 z

2-8 (0-5) 6-5 (0-6) 12-3 ( M )

2-0 (0-4) 5-2 (0-4) 154 (17)

7-5 y

7-6 (0-4)

2-2 (0-3) 6-0 (0-3) 15-6 (0-7) 10-6(1-3) 7-3 (0-6)

26 wk

pH6

Control Thelephora terrestris Suillus bovinus Lepista nuda

pH4 Control Thelephora terrestris Suillus bovinus Paxillus involutus Lepista nuda

10-2 c

3-1 X 7-0 V

3-2 X

7-0 (0-6)

The litter was colonized with niycelium from mycorrhizal or saprotrophic hasidiomycetes. Initial %veight of the leaves w-as 2-00 g (K = 3). Within each pH treatment one-way AXOVA was used to separate the means from one harvest. Means in a column not folio-wed by the same letter are significantly different (Tukey's test, a = 0.05). Values in parentheses are standard errors of the mean.

The respiration measurements indicated that the microbial activity was lowest in the control treatment (Tahle 3). At pH 4, decaying leaves colonized hy L. nuda released less CO^ than did the leaves colonized hy S. bovinus. This observation demonstrates that the microhial activity in the mycorrhizal treatments could be higher than in the saprotrophic treatment. Soderstrom & Read (1987) found comparable respiratory activities in unsterile peat colonized hy ectomycorrhizal mycelium.

Release of minerals

After 3 months the N concentration in the leaf litter was higher in all treatments (not shown). Figure 2 shows the release or accumulation of total N in the leaf litter. In the control leaves, there was no net release or accumulation of total N, even after 6 months. This confirms that virtually all of the N in this fresh tree leaf litter is in organic form, insoluble in water and is retained within refractory compounds

Mineralization of beech leaves

129

pH 6, week 18; Paxillus, pH 4, week 18) which showed a significant accumulation of N in the litter compared to the control leaves (P < O-OS). A significant release of N was only observed in the litter colonized with L. nuda. At the last harvest the C: N ratio had decreased significantly in all treatments. However, the differences between the different fungal treatments remained small (Table 2) and the C: N ratio was poorly correlated with the decay rate. The P concentrations in the litter varied considerably during the experiment and were more irregular than the N concentrations (Figure 3). After 3 months, P was released from the beech leaves in all treatments. A substantial part of the P in leaf litter of deciduous trees is often released easily (Attiwill & Adams, 1993). However, the P concentrations at the later harvests increased again in the mycorrhizal treatments. Staaf & Berg (1982) also found increasing fluctuations in the P release from litter in the later stages of decomposition. 10

15 Tirriie (wk)

20 DISCUSSION

Figure 2. Changes in the nitrogen content of beech leaf litter not inoculated with a basidiomycete (A) or colonized In natural conditions fresh beech leaf litter is invaded by mycelium of Thelephora terrestris ( • ) , Suillus bovinussuccessively by a number of different micro-organ(#), Paxillus involutus (Q) or Lepista nuda (A). isms. Fallen leaves are initially colonized by a group of microfungi which have predominantly stresstolerant ruderal strategies, and a significant proportion of them might be present before litter fall (Cooke & Rayner, 1984). In England the colonization of beech leaves with basidiomycete mycelia only starts from the second year after leaf-fall (Hogg & Hudson, 1966). Although we recognize that the natural fungal succession was not fully reproduced in the present study, the pre-treatment of the beech leaves did not prevent a rapid colonization by several species of microfungi. Cooke & Rayner (1984) argue that the invasion of leaf litter hy hasidiomycete myceiia (saprotrophs and mycorrhizal species) is delayed mainly because of the environmental extremes operating in the loose surface litter. Freshly fallen leaf litter can be as susceptible to colonization by the most active litter-degrading fungi as older litter in which microfungi are eliminated once the litter becomes sufHciently compacted. Knowledge about the spatial distribution and abundance of ectomycorrhizal mycelia in the different soil horizons is sparse, because of problems in tracing the mycelia. 10 15 20 Time (wkl At least some ectomycorrhizas can he found in Figure 3. Changes in the phosphorus content of beech leaf unstable layers of surface litter (Harley, 1989), and litter not inoculated with a hasidiom3'cete (A) or colonized we observed that even Suillus mycelia can intensively by mycelium of Thelephora terrestris ( • ) , Suillus bovinuspenetrate the needle litter of the L- and Fj-layer (•), Paxillus involutus (D) or Lepista nuda (A)during periods of high humidity. Several orders of the Basidiomycotina, for instance or complexes (Staaf & Berg, 1982; Cooke & Rayner, the Boletales and Tricholomatales, contain both 1984; Attiwill & Adams, 1993). In the leaves mycorrhizal species and obligate litter decomposers. colonized with mycorrhizal mycelium the total N Although some of the mycorrhizal representatives content in the bags tended to increase. A one-way seem to be able to grow slowly on complex organic ANOVA test revealed two data points {Suillus, resources (e.g. cellulose) in axenic conditions, it is

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J. V. Colpaert and K. K. Van Tichelen

evident that ectomycorrhizal fungi have only a reduced ability to decompose complex organic compounds of carbon (Norkrans, 1950; Kirk & Farrell, 1987; Haselwandter, Bobleter & Read, 1990; Dighton, 1991). Experiments of Dighton, Thomas & Latter (1987) and of Durall, Todd & Trappe (1994) indicate that ectomycorrhizal species in association with a host can degrade cellulose in sterile conditions. The latter authors found that the decay rate of Douglas fir needles was very small, though significant, for some mycorrhizal fungi. Our results confirm the observations that ectomycorrhizal fungi can slowly degrade leaf litter, at least where litter decomposers have been eliminated (Table 2, Figure 1).

However, the ecological significance of this decomposition remains unclear. The fungi were not carbon-limited since they obtained carbohydrates from a host which was N-limited. The effect on nutrient release appears also to be negligible. Norkrans (1950) suggested that this low decomposition capacity' might sustain an independent saprotrophic existence of the mycorrhizal fungi, and Linkins & Antibus (1982) thought that the utilization of organic compounds should reduce the carbon drain imposed on the host plant by the mycobiont. If we assume that the CO^ release from the leaves is relevant for the period between two subsequent harvests, then we can calculate that hardly 10-15 % of the released C could originate from the OM colonized with S. bovinus. This balance might however change under other climatic conditions, e.g. low temperatures. In the treatment with mycorrhizal fungi, an increase in total N was observed. An initial input of ' exogenous' labile N in the upper litter layers is one of the factors that control decay rate (Melillo et al., 1989). This accumulation of N is explained by an input of N from precipitation and from hyphae importing N from surrounding older litter richer in N (Berg & Staaf, 1981). Pine needle litter deposited in the L-layer of a mature Scots pine stand contained about 4-8 mg fungal biomass g"' litter, which corresponds to an increase of total N of 4-9 % (Berg & SoderstrSm, 1979). At that time the weight loss of the needles was already greater than 30 %. Anderson (1973), in his study of beech leaves, found that N accumulation occurred at an initial N content of 0-6%, but not at 0-8%. In our study the initial N content of the beech leaves was 0-9 and 1-0%, suggesting that an input of ' exogenous' N was not necessary which might also explain the lack of a N accumulation in the L. nuda treatment. However, the hypothesis that the nitrogen concentration is the main limiting factor for the microbial decomposition of organic substrates with a high C:N ratio is debated by some authors. Limiting P concentrations or the accessibility and availability of a decomposable C or N resource might be more important factors

that control the decomposition process (Attiwill & Adams, 1993; Sinsabaugh et al., 1993; Prescott, 1995). The increase of N in the micro-organisms of the litter is then a "luxury uptake' (Fog, 1988). The role of N in the regulation of litter decomposition is too complex to be generalized by measures of total N (Sinsabaugh et al., 1993). In the present study N was only exported from the OM inoculated with L. nuda. However, the N release was slower than the rate of weight loss, resulting in an increasing N concentration end a decreasing C: N ratio in the litter, which might finally result in the formation of a N-rich material with a very long halflife (Melillo et al., 1989). In all other treatments there was no net release of N from the OM, indicating that N in the leaves was not leachable and probably unavailable to the mycorrhizal fungi. The proteases from ectomycorrhizal fungi often have a sharp pH optimum between 3 and 4-5 (Read, 1991). Nevertheless, we found no difference in N mineralization between the pH treatments. Although it is possible that part of the litter N was transformed into fungal N, it would have been a very minor fraction. As a consequence, we could not demonstrate a net transport of N from the leaf litter to the host. Such transport would have taken place if the fungi had access to the N pool of the beech leaves. Bending & Read (1995) demonstrated that mycorrhizal fungi can readily transport N from pine litter to a host plant. However the litter used in their experiment was collected from a fermentation horizon where mycorrhizal fungi experience organic substrates of a different quality, including dead microbial hiomass. The availability and the composition of organic nutrients changes considerably during the degradative succession of leaf and needle litter. It seems likely that in older litter specific N compounds are produced or become more available which can be broken down directly by the mycobionts. Northup et al. (1995) have shown that decomposing pine litter releases both mineral and dissolved organic N. The fraction released as organic N increases considerably with a decreasing litter quality (high polyphenol content, high C:N ratio), in contrast to the mineral N release. Soluble organic N compounds might be important target molecules for the extracellular peptidases of mycorrhizal fungi. Kalisz, Wood & Moore (1987) found that basidiomycete proteases were largely immobilized to wail or ' sheath' layers of the hyphae. Close contact between the hypha producing the extracellular protease and the proteinaceous substrate are necessary to ensure eiBcient utilization of the substrate. Our results suggest that the mycorrhizal fungi we have tested do not have access to the organic N present in fresh leaf litter of beech. Apparently N in this kind of material can only be released by fungi which are able to degrade efficiently the lignoceliulose matrix of the leaves. A direct N release by mycorrhizal fungi

Mineralisation of beech leaves during the initial stages of the degradation of recalcitrant leaf litter is not very likely. Such a direct N release seems to be confined to the older litter layers. The increase of the P concentration in the litter with mycorrhizal mycelium suggests that the vegetative mycelium accumulates a considerable amount of P. None of the organisms in our cultivation system is P-limited and therefore an accumulation of P is conceivable. Ectomycorrhizal fungi are known to accumulate large quantities of polyphosphate, when this element is present in excess (Harley, 1989). This was not observed with L. nuda. The P data do not allow us to draw any conclusions on the P mineralization abilities of the mycorrhizal fungi. A large proportion of the labile P in litter is in ester forms, which are more mohile than inorganic P and can be minerahzed by the action of plant and microbial phosphatases. Competition for this P-pool might be great since several groups of soil-inhabiting organisms might have access to this organic P. Despite the low decomposition rate of the litter and the separation of the mycobiont from its carbohydrate suppHer, the COj measurements confirm that mycorrhizal fungi can release more CO, than a saprotrophic fungus. It demonstrates that the energy drain to the vegetative mycelium of the mycobiont is considerable (Rygiewicz & Andersen, 1994) and it also confirms that a significant amount of the carbon released in soils is directly converted from photosynthetic carbon through the mycorrhizal fungi (Harley, 1989).

ACKNOWLEDGEMENTS Jan Colpaert thanks the Belgian National Fund for Scientific Research (N.F.W.O.) for providing a postdoctoral fellowship.

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