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The effects of nitrogen fertilization and soil properties on mycorrhizal formation of Salix viminalis
Forest Ecology and Management 160 (2002) 35–43
The effects of nitrogen fertilization and soil properties on mycorrhizal formation of Salix viminalis C. Bauma,*, M. Weihb, T. Verwijstb, F. Makeschinc a
b
University of Rostock, Institute of Soil Science, Justus-von-Liebig-Weg 6, D-18059 Rostock, Germany Department of Short Rotation Forestry, Swedish University of Agricultural Sciences, P.O. Box 7016, S-750 07 Uppsala, Sweden c Dresden University of Technology, Institute of Soil Science, Pienner Street 7, D-01735 Tharandt, Germany Received 7 March 2000; received in revised form 12 December 2000; accepted 12 December 2000
Abstract The effects of nitrogen fertilization (100, 200 kg N ha1 per year) and soil properties on mycorrhizal formation on Salix viminalis were investigated at three short rotation plantations on Gleysols and Cambisols (Abbachhof (ABB) and Wildeshausen (WIL) in Germany, Ultuna (ULT) in Sweden). During 3 years the ectomycorrhizal colonization, the composition of ectomycorrhizal morphotypes and the VAM spore density in the soil were analyzed. The ectomycorrhizal colonization was significantly altered due to N-fertilization at all sites. The quality and magnitude of the fertilization effects on mycorrhizal formation on Salix viminalis varied due to the soil properties, i.e. soil texture, soil N content and pH. The WIL site was characterized by sandy soil (low pH, high soil N content), whereas the ABB site was characterized by clayey soil (high pH, low N content). The ULT site was characterized by clayey soil (high pH, high N content). In the unfertilized control plots (C), ectomycorrhizal colonization was higher at WIL than at ABB. Fertilization reduced the ectomycorrhizal colonization at WIL but increased it at ABB. The distribution of the ectomycorrhizal morphotypes was very heterogeneous within the treatments, therefore significant differences were rare. Sporocarps were collected at ABB during one growing period. The sporocarps were mostly from saprophytic species, with exception of the ectomycorrhizal species Inocybe glabripes. Significant effects of N-fertilization on VAM spore density were observed at two of the three plantations. The pattern in VAM spore density was similar to the pattern seen for ectomycorrhizal colonization. Thus, VAM spore density was increased by fertilization at ABB (low soil N) and decreased at ULT (high soil N). The soil properties have been shown to modify the effects of fertilization on ectomycorrhizal colonization and VAM spore density. Therefore, in management practice of short rotation plantations, the benefit of N-fertilization should be evaluated keeping secondary effects caused by changed mycorrhizal formations in mind. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Ectomycorrhiza; Vesicular arbuscular mycorrhiza; Willow; Short rotation forestry
1. Introduction Salix viminalis is frequently used in short rotation forestry because of its high biomass production of up *
Corresponding author. Tel.: þ49-381-498-2136; fax: þ49-381-498-2159. E-mail address: [email protected] (C. Baum).
to 30 t DW ha1 after a 5-year growing period (Jug et al., 1999). The yields are affected by the intraspecific variation of the genotype, the site conditions and management practices (Ro¨nnberg-Wa¨stljung and Thorse´n, 1988; Verwijst, 1996a,b). Nitrogen fertilization and irrigation are common management practices to increase willow growth rates (Larsson et al., 1998). For example, biomass production of Salix viminalis at
0378-1127/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 2 7 ( 0 1 ) 0 0 4 7 0 - 4
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the German short rotation plantation Abbachhof (ABB) was strongly increased by fertilization with 100 kg N ha1 per year (Jug, 1997; Hofmann-Schielle et al., 1999). Salix spp. are able to form vesicular-arbuscular mycorrhizas (VAM) and ectomycorrhizas (ECM) (Khan, 1993). On short rotation plantations on former arable soils in Germany, average ectomycorrhizal colonization of 2–20% of the fine roots of Salix viminalis was observed at 0–10 cm soil depth (Baum and Makeschin, 1997). Mycorrhizal colonization on Salix viminalis is known to increase biomass production (Backhaus et al., 1986). It can cause a two-fold increase in growth due to substantially higher P uptake (Jones et al., 1991). Positive effects of colonization by mycorrhizal fungi on growth of different plant species are improved nutrition (Cooper and Tinker, 1978; Barea et al., 1991) and enhanced protection against root pathogen fungi (Hooker et al., 1994). Together with plant roots, hyphae of mycorrhizal fungi are important factors for the aggregation of soils (Bethlenfalvay et al., 1997). The low rooting intensity of many tree species in agroforestry systems suggests that growth rates of trees may strongly respond to management practices that increase the populations of indigenous mycorrhizal fungi, in particular where these are highly effective but low in number (Haselwandter and Bowen, 1996). N-fertilization has
been found to cause changes both in mycorrhizal formation on fine roots and production of sporocarps of ectomycorrhizal fungi (Ritter and To¨ lle, 1978; Termorshuizen, 1993; Wiklund et al., 1995). Fertilization can reduce the positive effect that mycorrhiza can have on plant growth (Bethlenfalvay et al., 1997). Reduced mycorrhizal colonization caused by N-fertilization might reduce the benefit of N-fertilization on biomass production of Salix viminalis. The aims of our investigation were (a) to quantify the effects of N-fertilization on ectomycorrhizal colonization of Salix viminalis and VAM spore density in the soil, as one source of endomycorrhizal colonization, at plantations exposed to different soil conditions, (b) to determine the fungal sporocarps and the composition of ectomycorrhizal morphotypes.
2. Material and methods 2.1. Experimental sites The site descriptions of the two German experimental plantations Abbachhof (ABB), Wildeshausen (WIL) and the Swedish plantation Ultuna (ULT) and the fertilization treatments (N1) are summarized in Table 1. The treatments (N þ I1 and N þ I2) at ULT were fertilized and irrigated according to different
Table 1 Site description of the three experimental plantations (soil conditions before fertilization treatments)a Feature
Experimental plantation Abbachhof
Wildeshausen
Ultuna
Region South Germany Situation 49850 N, 128100 E Annual average temperature (8C) 8.0 Annual precipitation (mm) 650 Bed rock Sandy-clayey tertiary sediments Soil type (FAO-Unesco, 1988) Stagnic Gleysols 1 N addition on N plots (kg ha per year) 100 Fertilization period 1985–1987, 1989–1992, 1994–1995 Plot size (m2) 100 Plant spacing (m) 2.0 0.3
North Germany 528530 N, 88280 E 8.6 760 Fluvio-glacial sandy sediments Gleyic Cambisols 100 1991–1993, 1995 270 2.0 0.3
Middle Sweden 608490 N, 178400 E 5.8 544 Fluvial-glacial clayey sediments Vertic Cambisols 100 respectively 200 1985–1999 150 0.7 0.7
Soil parameter (0–10 cm) pH (CaCl2) Corg (mg g1) Nt (mg g1)
4.5 17 1.6
7.3 31 2.2
a
5.5 14 1.0
References: Olsson and Samils, 1984, SMHI, 1961–1990, Hofmann-Schielle et al., 1999.
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Table 2 The additional inputs of nitrogen and water at the experimental site Ultuna near Uppsala, Sweden, during the periods from 1985 to 1996 and 1997–1999, respectively, and for three different treatmentsa 1985–1996 Treatment
C N þ I1 N þ I2 a
1997–1999
Nitrogen (kg ha1 per annum)
Irrigation (mm per annum)
Nitrogen (kg ha1 per annum)
Irrigation (mm per annum)
0 100 100
0 100 350
0 100 200
0 650 650
C: control; N þ I1, N þ I2: nitrogen fertilization and irrigation.
nitrogen and irrigation regimes (Table 2). Irrigation and fertilization were supplied according to Ingestad et al. (1981) and Ingestad (1987). The control plots (C) at the three plantations did not receive any fertilizer since their establishment. ABB was established in 1983, ULT in 1984 and WIL in 1989. At ULT weed control was maintained chemically during the first 3 years. At the German plantations chemical weed control was not applied. The Salix viminalis clones planted at the experimental sites were the clone 72251 in Germany and the clone 77-683 in Sweden. 2.2. Sampling of fine roots, soil and sporocarps Fine root samples were taken from 0 to 10 cm soil depth from 1996 to 1998 at the German sites and from 1997 to 1999 at the Swedish site. At the German plantations nine replicate samples from each treatment (C and N1) and at ULT six replicate samples from the treatments C, N þ I1 and N þ I2 were investigated. The samples were taken and prepared according to Agerer (1991). At least 200 fine roots were investigated from each sample. The soil surrounding the fine roots was shaken off carefully for VAM spore quantification. Sporocarps were collected within the 1997 growing season every third week at ABB. 2.3. Ectomycorrhizal analyses The ectomycorrhizae were separated into morphotypes on the basis of characters described by Agerer (1987–1993), (a) the texture of the outer layer of the fungal mantle covering the fine root, (b) the presence and appearance of rhizomorphs, (c) the presence and appearance of cystidia. Each morphotype includes different fungal species.
2.4. Quantification of VAM spores The soil was analyzed by wet-sieving and decanting to measure the numbers of VAM spores following the method of Jenkinson (1964) as modified by Smith and Skipper (1979). A sample of each soil was dried at 110 8C for 24 h, so that the results could be expressed on a dry weight basis. The spore density is only one parameter, which can affect the endomycorrhizal colonization. The direct quantification of endomycorrhizal colonization of fine roots would be preferable, but the ectomycorrhizal investigation was too time consuming, and it is impossible to use the same fine roots for investigation of endo- and ecto-mycorrhizal colonization. 2.5. Statistical analysis All data were analyzed statistically by analysis of variance (ANOVA) for each site separately. We used one-way ANOVA with fixed factor fertilization for each sampling date and two-way ANOVA, with fixed factors sampling date and fertilization and the interaction between them. Ectomycorrhizal data expressed as percentages and VAM spore density were log (n þ 1) transformed prior to analysis to obtain normality. All statistics were computed using Statistica (StatSoft Inc., 1995).
3. Results 3.1. Ectomycorrhizal colonization Ectomycorrhizal colonization on roots was significantly affected by sampling date at all locations
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Table 3 Analysis of variance for effects of sampling date, N-fertilization and sampling date N-fertilization interaction on ectomycorrhizal colonization (ECM) and VAM spore density at the short rotation plantations Abbachhof (ABB), Wildeshausen (WIL) and Ultuna (ULT)a Site and parameter
Source of variation
df
ABB-ECM
Within þ residual Sampling date Fertilization Sampling date fertilization
80 4 1 4
8.24 20.55 0.04 62.99
0.049 0.942 <0.001
Within þ residual Sampling date Fertilization Sampling date fertilization
80 4 1 4
2.13 123.35 0.68 5.51
<0.001 0.573 0.042
Within þ residual Sampling date Fertilization Sampling date fertilization
64 3 1 3
6.31 34.17 69.39 70.22
0.002 0.001 <0.001
Within þ residual Sampling date Fertilization Sampling date fertilization
64 3 1 3
1.70 35.13 0.07 0.41
<0.001 0.836 0.867
Within þ residual Sampling date Fertilization þ irrigation Sampling date (fertilization þ irrigation)
28 2 1 2
4.40 22.59 136.18 6.20
0.013 <0.001 0.262
Within þ residual Sampling date Fertilization þ irrigation Sampling date (fertilization þ irrigation)
28 2 1 2
1.70 16.01 1.93 3.91
<0.001 0.296 0.120
Within þ residual Sampling date Fertilization þ irrigation Sampling date (fertilization þ irrigation)
28 2 1 2
4.97 31.67 87.29 11.74
0.006 <0.001 0.115
Within þ residual Sampling date Fertilization þ irrigation Sampling date (fertilization þ irrigation)
28 2 1 2
1.34 16.07 8.25 1.62
<0.001 0.020 0.317
ABB-VAM
WIL-ECM
WIL-VAM
ULT N þ I1-ECM
ULT N þ I1-VAM
ULT N þI2-ECM
ULT N þ I2-VAM
a
MS
P
df: degrees of freedom; MS: mean squares; P: probability of effect caused by random chance; : significant effect.
(Table 3). N-fertilization significantly influenced ectomycorrhizal colonization at all locations, at least at certain sampling dates (Table 3 fertilization and sampling date fertilization). At the two German plantations the fertilizer effect significantly varied depending on the sampling date (Table 3 sampling date fertilization). At ABB with a low soil N content (Table 1), the ectomycorrhizal colonization was increased by fertilization in autumn 1996, 1 year after the N-application (Fig. 1). In spring 1997 the ectomycorrhizal
colonization was similar in control and fertilized plots. However, in autumn 1997 and spring 1998, higher ectomycorrhizal colonization was observed at the control plots. Mycorrhizal colonization decreased continuously throughout the observation period from 1996 to 1998, (Fig. 1) following the last N-application in 1995. At WIL the ectomycorrhizal colonization was significantly reduced until 2 years after the Nfertilization compared to the non-fertilized control plots, whereas in the third year the ectomycorrhizal colonization was significantly higher at the formerly
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Three morphological types of ectomycorrhiza were distinguished on the basis of features of the outer mantle, Type A: plectenchymatic outer mantle, no rhizomorphs, no cystidia; Type B: plectenchymatic outer mantle, interconnected or ramified rhizomorphs, no cystidia; Type C: parenchymatic outer mantle, no rhizomorphs, awl-shaped, bristle like cystidia. Each morphological ECM type included different fungal species. The distribution of the morphotypes within the treatments was very variable. The proportion of species which form rhizomorphs (Type B) was always low (Table 4). At ABB in spring 1998 colonization by Type C was significantly reduced at the former fertilized plots, whereas Type B was only detected at these plots and never at control plots. In the previous year there were no differences between control and fertilized plots in the distribution of morphotypes. At WIL the colonization with Type A was significantly reduced due to the Table 4 Compositions of ectomycorrhizal morphotypes (% colonized fine roots) in spring 1997 and 1998 at Abbachhof (ABB), Wildeshausen (WIL) and Ultuna (ULT), Type A: plectenchymatic outer mantle, no rhizomorphs, no cystidia; Type B: plectenchymatic outer mantle, interconnected or ramified rhizomorphs, no cystidia; Type C: parenchymatic outer mantle, no rhizomorphs, awl-shaped, bristle like cystidia; C: control; N1 ¼ 100 kg N ha1 per annum; N þ I1 and N þ I2 ¼ 100, 200 kg N ha1 per annum with irrigationa Treatment
Fig. 1. Percentage of fine roots of Salix viminalis colonized with ectomycorrhizal fungi in 0–10 cm soil depth under various fertilizer regimes at the short rotation plantations Abbachhof, Wildeshausen and Ultuna during the years 1996–1999. C: control; N1: fertilization with 100 kg N ha1 per annum; N þ I1 and N þ I2: different fertilizer and irrigation treatments (see Table 2); S.E.: standard error; : significant effect of fertilization.
fertilized treatments. At ULT N-fertilization also reduced the ectomycorrhizal colonization. No differences were found between the two different fertilization and irrigation regimes, i.e. N þ I1 and N þ I2.
Morphotypes A
B
C
Mean S.D.
Mean S.D.
Mean S.D.
1997 ABB—C ABB—N1 WIL—C WIL—N1 ULT—C ULT—N þI1 ULT—N þ I2
5.1 4.2 15.6 4.4 9.8 4.4 9.8
2.9 7.2 3.1 3.5 6.2 2.9 4.1
– – 0.7 1.3 – – 0.4 0.8 0.2 0.5
2.8 7.2 0.5 2.4 5.1 2.0 –
4.9 9.8 0.4 4.1 8.7 5.2
1998 ABB—C ABB—N1 WIL—C WIL—N1 ULT—C ULT—N þI1 ULT—N þI2
5.2 1.9 4.0 4.9 2.5 1.8 3.1
7.5 1.8 2.8 3.9 2.2 1.4 3.2
– 1.6 2.8 2.4 4.2 8.1 7.0 – – –
14.4 2.9 4.4 6.1 6.1 0.4 –
6.4 1.7 7.6 6.3 9.3 0.7
a –: Absent; S.D.: standard deviation; : significant effect of N fertilization.
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fertilization in 1997. Type B was found only at control plots. In 1998 there were no longer effects of fertilization on the composition of morphotypes. At ULT Type B was observed only at fertilized plots, whereas Type C was not present at the N þ I2 plots. Only one ectomycorrhizal fungus (Inocybe glabripes) produced sporocarps at ABB under Salix viminalis within the growing season 1997. The sporocarps were detected only on plots without N-fertilization, but in low numbers. The saprophytic species Coprinus micaceus and Mycena rosella were observed at both treatments. Sporocarps of Coprinus disseminatus and Polyporus brumalis were observed only on the control plots and Agrocybe praecox only at the N1 plots. 3.2. VAM spore density A general fertilizer effect on the VAM spore density was found only at the highly fertilized and irrigated Swedish site (ULT-N2, Table 3). The density of VAM spores in the rhizosphere soil was affected mainly by the location of the sites (Fig. 2) and the sampling date (Table 3). Similar to the ectomycorrhizal colonization, the VAM spore density at ABB was generally high and was increased by fertilization in autumn in 1996, 1 year after the end of the N treatment (Fig. 2, Table 3 sampling date and sampling date fertilization). At WIL the VAM spore density in the rhizosphere soil was generally low, but remained unaffected by the Nfertilization. At ULT the VAM spore density was intermediate between the two German sites and Napplication reduced the density of VAM spores significantly in May 1997 and June 1999 (Fig. 2).
4. Discussion N-fertilization at three short rotation plantations in Germany and Sweden with different site conditions and management affected the ectomycorrhizal colonization, the composition of morphotypes and the VAM spore density in the soil surrounding the fine roots of Salix viminalis. It is known that mycorrhizal formation in general depends on the soil conditions (Baar, 1995). The soil P content, for example, affected the production of extramatrical mycelium of ectomycorrhizal fungi on Salix viminalis (Jones et al., 1990). Soil texture, the organic matter content and the pH
Fig. 2. Number of VAM spores g1 dry rhizosphere soil of Salix viminalis (0–10 cm depth) under various fertilizer regimes at the short rotation plantations Abbachhof, Wildeshausen and Ultuna during the years 1996–1999. C: control; N1: fertilization with 100 kg N ha1 per annum; N þ I1 and N þ I2: different fertilizer and irrigation treatments (see Table 2); S.E.: standard error; : significant effect of fertilization.
level were important differences between the soils of our experimental plantations (Table 1). At the clayey sites ABB and ULT (soil pH of 5.5 and 7.3) the average level of ectomycorrhizal colonization on Salix
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viminalis was lower, whereas the VAM spore density was higher than at the sandy site WIL (soil pH 4.5). Differences in the composition of morphotypes between the plantations may be caused partly by the management of the neighbouring sites. The German plantations ABB and WIL include plots with poplar (Populus tremula tremuloides and P. trichocarpa), whereas the Swedish plantation is neighboured only by arable soils and other Salix viminalis stands. The poplar clones in general had higher ectomycorrhizal colonization indices than willows (Baum and Makeschin, 1997) and belong to the same plant family (Salicaceae). Therefore, similarity in the selection of mycorrhizal partners can be suspected and introduction of mycorrhizal fungi from these plots could be possible. Altered composition of morphotypes due to N-fertilization suggests selective inhibition or promotion of fungal species as observed on pine (Termorshuizen, 1993). The low sporocarp production at ABB within the growing season 1997 with only one mycorrhizal species does not allow any conclusions to be drawn on fertilization effects on this parameter. The application of nutrients, e.g. through wastewater sludge is a frequently used management practice in short rotation forestry. The effects of N-fertilization on growth of Salix viminalis depend not only on the absolute quantity of N-application but on fertilization regime and site characters (soil type, climate, number of applications, irrigation) (Alriksson et al., 1997). Altered mycorrhizal formation due to fertilization may sometimes be one reason for these observations. Our investigations at the German plantations have shown that similar amounts of fertilizer (100 kg N ha1 per annum) caused different responses in mycorrhizal formation by Salix viminalis under different site conditions. Pot experiments have already shown the existence of an optimum N concentration for the formation of extraradical mycelium and mycorrhizas (Wallenda and Kottke, 1998; Baum et al., 2000). We suggest that the soil N content could be used as a parameter for the prediction of fertilization effects on mycorrhizal responses in willow plantations. This hypothesis should be tested with similar boundary conditions. At the Swedish plantation we found similar effects on the mycorrhizal formation of Salix viminalis with the two different fertilization and irrigation treatments using 100 or 200 kg N ha1 per annum. The influence
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of irrigation should be tested in further investigations without fertilization. Since it was impossible to separate fertilization and irrigation effects at ULT in our investigation, our results document an integrated response of mycorrhizal formation to the management practice at this plantation.
5. Conclusions Soil properties, e.g. soil organic matter content, soil texture and soil pH affect mycorrhiza formation. The two German plantations ABB and WIL had similar organic matter content and soil pH but different soil texture. The clayey plantation ABB had lower ectomycorrhizal colonization than the sandy plantation WIL. The soil properties have been shown to modify the effects of N-fertilization on mycorrhizal formation of Salix viminalis. Therefore, in management practice of short rotation plantations, the suspected increase of biomass production after Nfertilization should be estimated keeping secondary effects caused by changed mycorrhizal formations in mind. It was found that the response to N-application could be increased and decreased ectomycorrhizal colonization. The quantitative effects of fertilization on ectomycorrhizal colonization might be caused in part by changes in species composition as was observed on pine and spruce by Baar (1995), Ka˚ ren and Nylund (1997) and Fransson et al. (2000). The changes in the morphotype composition at ABB and WIL after N-fertilization support this assumption. Undoubtedly, it is necessary to further test this hypothesis for willows, possibly in pot experiments with similar boundary conditions.
Acknowledgements The analyses of mycorrhization on the German experimental sites were founded by the Deutsche Forschungsgemeinschaft (MA 1257/3). We thank Mrs. Ch. Fenger for technical assistance. We are ¨ Z, Freiberg, grateful to Prof. Dr. H. Heilmeier (IO Germany), Mr. J. Schumacher (Institute for Forest Botany, Tharandt, Germany) and Dr. S. Thiele (Institute of Soil Science, Rostock, Germany) for their critical reviewing of the manuscript.
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C. Baum et al. / Forest Ecology and Management 160 (2002) 35–43 Verwijst, T., 1996a. Cyclic and progressive changes in shortrotation willow coppice systems. Biomass Bioenergy 11, 161– 165. Verwijst, T., 1996b. Stool mortality and development of a competitive hierachy in a Salix viminalis coppice system. Biomass Bioenergy 11, 245–250.
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Wallenda, T., Kottke, I., 1998. Nitrogen deposition and ectomycorrhizas. New Phytol. 139, 169–187. Wiklund, K., Nilsson, L.O., Jacobsson, S., 1995. Effect of irrigation, fertilization, and artificial drought on basidioma production in a Norway spruce stand. Can. J. Bot. 73, 200– 208.
The effects of nitrogen fertilization and soil properties on mycorrhizal formation of Salix viminalis C. Bauma,*, M. Weihb, T. Verwijstb, F. Makeschinc a
b
University of Rostock, Institute of Soil Science, Justus-von-Liebig-Weg 6, D-18059 Rostock, Germany Department of Short Rotation Forestry, Swedish University of Agricultural Sciences, P.O. Box 7016, S-750 07 Uppsala, Sweden c Dresden University of Technology, Institute of Soil Science, Pienner Street 7, D-01735 Tharandt, Germany Received 7 March 2000; received in revised form 12 December 2000; accepted 12 December 2000
Abstract The effects of nitrogen fertilization (100, 200 kg N ha1 per year) and soil properties on mycorrhizal formation on Salix viminalis were investigated at three short rotation plantations on Gleysols and Cambisols (Abbachhof (ABB) and Wildeshausen (WIL) in Germany, Ultuna (ULT) in Sweden). During 3 years the ectomycorrhizal colonization, the composition of ectomycorrhizal morphotypes and the VAM spore density in the soil were analyzed. The ectomycorrhizal colonization was significantly altered due to N-fertilization at all sites. The quality and magnitude of the fertilization effects on mycorrhizal formation on Salix viminalis varied due to the soil properties, i.e. soil texture, soil N content and pH. The WIL site was characterized by sandy soil (low pH, high soil N content), whereas the ABB site was characterized by clayey soil (high pH, low N content). The ULT site was characterized by clayey soil (high pH, high N content). In the unfertilized control plots (C), ectomycorrhizal colonization was higher at WIL than at ABB. Fertilization reduced the ectomycorrhizal colonization at WIL but increased it at ABB. The distribution of the ectomycorrhizal morphotypes was very heterogeneous within the treatments, therefore significant differences were rare. Sporocarps were collected at ABB during one growing period. The sporocarps were mostly from saprophytic species, with exception of the ectomycorrhizal species Inocybe glabripes. Significant effects of N-fertilization on VAM spore density were observed at two of the three plantations. The pattern in VAM spore density was similar to the pattern seen for ectomycorrhizal colonization. Thus, VAM spore density was increased by fertilization at ABB (low soil N) and decreased at ULT (high soil N). The soil properties have been shown to modify the effects of fertilization on ectomycorrhizal colonization and VAM spore density. Therefore, in management practice of short rotation plantations, the benefit of N-fertilization should be evaluated keeping secondary effects caused by changed mycorrhizal formations in mind. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Ectomycorrhiza; Vesicular arbuscular mycorrhiza; Willow; Short rotation forestry
1. Introduction Salix viminalis is frequently used in short rotation forestry because of its high biomass production of up *
Corresponding author. Tel.: þ49-381-498-2136; fax: þ49-381-498-2159. E-mail address: [email protected] (C. Baum).
to 30 t DW ha1 after a 5-year growing period (Jug et al., 1999). The yields are affected by the intraspecific variation of the genotype, the site conditions and management practices (Ro¨nnberg-Wa¨stljung and Thorse´n, 1988; Verwijst, 1996a,b). Nitrogen fertilization and irrigation are common management practices to increase willow growth rates (Larsson et al., 1998). For example, biomass production of Salix viminalis at
0378-1127/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 1 2 7 ( 0 1 ) 0 0 4 7 0 - 4
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the German short rotation plantation Abbachhof (ABB) was strongly increased by fertilization with 100 kg N ha1 per year (Jug, 1997; Hofmann-Schielle et al., 1999). Salix spp. are able to form vesicular-arbuscular mycorrhizas (VAM) and ectomycorrhizas (ECM) (Khan, 1993). On short rotation plantations on former arable soils in Germany, average ectomycorrhizal colonization of 2–20% of the fine roots of Salix viminalis was observed at 0–10 cm soil depth (Baum and Makeschin, 1997). Mycorrhizal colonization on Salix viminalis is known to increase biomass production (Backhaus et al., 1986). It can cause a two-fold increase in growth due to substantially higher P uptake (Jones et al., 1991). Positive effects of colonization by mycorrhizal fungi on growth of different plant species are improved nutrition (Cooper and Tinker, 1978; Barea et al., 1991) and enhanced protection against root pathogen fungi (Hooker et al., 1994). Together with plant roots, hyphae of mycorrhizal fungi are important factors for the aggregation of soils (Bethlenfalvay et al., 1997). The low rooting intensity of many tree species in agroforestry systems suggests that growth rates of trees may strongly respond to management practices that increase the populations of indigenous mycorrhizal fungi, in particular where these are highly effective but low in number (Haselwandter and Bowen, 1996). N-fertilization has
been found to cause changes both in mycorrhizal formation on fine roots and production of sporocarps of ectomycorrhizal fungi (Ritter and To¨ lle, 1978; Termorshuizen, 1993; Wiklund et al., 1995). Fertilization can reduce the positive effect that mycorrhiza can have on plant growth (Bethlenfalvay et al., 1997). Reduced mycorrhizal colonization caused by N-fertilization might reduce the benefit of N-fertilization on biomass production of Salix viminalis. The aims of our investigation were (a) to quantify the effects of N-fertilization on ectomycorrhizal colonization of Salix viminalis and VAM spore density in the soil, as one source of endomycorrhizal colonization, at plantations exposed to different soil conditions, (b) to determine the fungal sporocarps and the composition of ectomycorrhizal morphotypes.
2. Material and methods 2.1. Experimental sites The site descriptions of the two German experimental plantations Abbachhof (ABB), Wildeshausen (WIL) and the Swedish plantation Ultuna (ULT) and the fertilization treatments (N1) are summarized in Table 1. The treatments (N þ I1 and N þ I2) at ULT were fertilized and irrigated according to different
Table 1 Site description of the three experimental plantations (soil conditions before fertilization treatments)a Feature
Experimental plantation Abbachhof
Wildeshausen
Ultuna
Region South Germany Situation 49850 N, 128100 E Annual average temperature (8C) 8.0 Annual precipitation (mm) 650 Bed rock Sandy-clayey tertiary sediments Soil type (FAO-Unesco, 1988) Stagnic Gleysols 1 N addition on N plots (kg ha per year) 100 Fertilization period 1985–1987, 1989–1992, 1994–1995 Plot size (m2) 100 Plant spacing (m) 2.0 0.3
North Germany 528530 N, 88280 E 8.6 760 Fluvio-glacial sandy sediments Gleyic Cambisols 100 1991–1993, 1995 270 2.0 0.3
Middle Sweden 608490 N, 178400 E 5.8 544 Fluvial-glacial clayey sediments Vertic Cambisols 100 respectively 200 1985–1999 150 0.7 0.7
Soil parameter (0–10 cm) pH (CaCl2) Corg (mg g1) Nt (mg g1)
4.5 17 1.6
7.3 31 2.2
a
5.5 14 1.0
References: Olsson and Samils, 1984, SMHI, 1961–1990, Hofmann-Schielle et al., 1999.
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Table 2 The additional inputs of nitrogen and water at the experimental site Ultuna near Uppsala, Sweden, during the periods from 1985 to 1996 and 1997–1999, respectively, and for three different treatmentsa 1985–1996 Treatment
C N þ I1 N þ I2 a
1997–1999
Nitrogen (kg ha1 per annum)
Irrigation (mm per annum)
Nitrogen (kg ha1 per annum)
Irrigation (mm per annum)
0 100 100
0 100 350
0 100 200
0 650 650
C: control; N þ I1, N þ I2: nitrogen fertilization and irrigation.
nitrogen and irrigation regimes (Table 2). Irrigation and fertilization were supplied according to Ingestad et al. (1981) and Ingestad (1987). The control plots (C) at the three plantations did not receive any fertilizer since their establishment. ABB was established in 1983, ULT in 1984 and WIL in 1989. At ULT weed control was maintained chemically during the first 3 years. At the German plantations chemical weed control was not applied. The Salix viminalis clones planted at the experimental sites were the clone 72251 in Germany and the clone 77-683 in Sweden. 2.2. Sampling of fine roots, soil and sporocarps Fine root samples were taken from 0 to 10 cm soil depth from 1996 to 1998 at the German sites and from 1997 to 1999 at the Swedish site. At the German plantations nine replicate samples from each treatment (C and N1) and at ULT six replicate samples from the treatments C, N þ I1 and N þ I2 were investigated. The samples were taken and prepared according to Agerer (1991). At least 200 fine roots were investigated from each sample. The soil surrounding the fine roots was shaken off carefully for VAM spore quantification. Sporocarps were collected within the 1997 growing season every third week at ABB. 2.3. Ectomycorrhizal analyses The ectomycorrhizae were separated into morphotypes on the basis of characters described by Agerer (1987–1993), (a) the texture of the outer layer of the fungal mantle covering the fine root, (b) the presence and appearance of rhizomorphs, (c) the presence and appearance of cystidia. Each morphotype includes different fungal species.
2.4. Quantification of VAM spores The soil was analyzed by wet-sieving and decanting to measure the numbers of VAM spores following the method of Jenkinson (1964) as modified by Smith and Skipper (1979). A sample of each soil was dried at 110 8C for 24 h, so that the results could be expressed on a dry weight basis. The spore density is only one parameter, which can affect the endomycorrhizal colonization. The direct quantification of endomycorrhizal colonization of fine roots would be preferable, but the ectomycorrhizal investigation was too time consuming, and it is impossible to use the same fine roots for investigation of endo- and ecto-mycorrhizal colonization. 2.5. Statistical analysis All data were analyzed statistically by analysis of variance (ANOVA) for each site separately. We used one-way ANOVA with fixed factor fertilization for each sampling date and two-way ANOVA, with fixed factors sampling date and fertilization and the interaction between them. Ectomycorrhizal data expressed as percentages and VAM spore density were log (n þ 1) transformed prior to analysis to obtain normality. All statistics were computed using Statistica (StatSoft Inc., 1995).
3. Results 3.1. Ectomycorrhizal colonization Ectomycorrhizal colonization on roots was significantly affected by sampling date at all locations
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Table 3 Analysis of variance for effects of sampling date, N-fertilization and sampling date N-fertilization interaction on ectomycorrhizal colonization (ECM) and VAM spore density at the short rotation plantations Abbachhof (ABB), Wildeshausen (WIL) and Ultuna (ULT)a Site and parameter
Source of variation
df
ABB-ECM
Within þ residual Sampling date Fertilization Sampling date fertilization
80 4 1 4
8.24 20.55 0.04 62.99
0.049 0.942 <0.001
Within þ residual Sampling date Fertilization Sampling date fertilization
80 4 1 4
2.13 123.35 0.68 5.51
<0.001 0.573 0.042
Within þ residual Sampling date Fertilization Sampling date fertilization
64 3 1 3
6.31 34.17 69.39 70.22
0.002 0.001 <0.001
Within þ residual Sampling date Fertilization Sampling date fertilization
64 3 1 3
1.70 35.13 0.07 0.41
<0.001 0.836 0.867
Within þ residual Sampling date Fertilization þ irrigation Sampling date (fertilization þ irrigation)
28 2 1 2
4.40 22.59 136.18 6.20
0.013 <0.001 0.262
Within þ residual Sampling date Fertilization þ irrigation Sampling date (fertilization þ irrigation)
28 2 1 2
1.70 16.01 1.93 3.91
<0.001 0.296 0.120
Within þ residual Sampling date Fertilization þ irrigation Sampling date (fertilization þ irrigation)
28 2 1 2
4.97 31.67 87.29 11.74
0.006 <0.001 0.115
Within þ residual Sampling date Fertilization þ irrigation Sampling date (fertilization þ irrigation)
28 2 1 2
1.34 16.07 8.25 1.62
<0.001 0.020 0.317
ABB-VAM
WIL-ECM
WIL-VAM
ULT N þ I1-ECM
ULT N þ I1-VAM
ULT N þI2-ECM
ULT N þ I2-VAM
a
MS
P
df: degrees of freedom; MS: mean squares; P: probability of effect caused by random chance; : significant effect.
(Table 3). N-fertilization significantly influenced ectomycorrhizal colonization at all locations, at least at certain sampling dates (Table 3 fertilization and sampling date fertilization). At the two German plantations the fertilizer effect significantly varied depending on the sampling date (Table 3 sampling date fertilization). At ABB with a low soil N content (Table 1), the ectomycorrhizal colonization was increased by fertilization in autumn 1996, 1 year after the N-application (Fig. 1). In spring 1997 the ectomycorrhizal
colonization was similar in control and fertilized plots. However, in autumn 1997 and spring 1998, higher ectomycorrhizal colonization was observed at the control plots. Mycorrhizal colonization decreased continuously throughout the observation period from 1996 to 1998, (Fig. 1) following the last N-application in 1995. At WIL the ectomycorrhizal colonization was significantly reduced until 2 years after the Nfertilization compared to the non-fertilized control plots, whereas in the third year the ectomycorrhizal colonization was significantly higher at the formerly
C. Baum et al. / Forest Ecology and Management 160 (2002) 35–43
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Three morphological types of ectomycorrhiza were distinguished on the basis of features of the outer mantle, Type A: plectenchymatic outer mantle, no rhizomorphs, no cystidia; Type B: plectenchymatic outer mantle, interconnected or ramified rhizomorphs, no cystidia; Type C: parenchymatic outer mantle, no rhizomorphs, awl-shaped, bristle like cystidia. Each morphological ECM type included different fungal species. The distribution of the morphotypes within the treatments was very variable. The proportion of species which form rhizomorphs (Type B) was always low (Table 4). At ABB in spring 1998 colonization by Type C was significantly reduced at the former fertilized plots, whereas Type B was only detected at these plots and never at control plots. In the previous year there were no differences between control and fertilized plots in the distribution of morphotypes. At WIL the colonization with Type A was significantly reduced due to the Table 4 Compositions of ectomycorrhizal morphotypes (% colonized fine roots) in spring 1997 and 1998 at Abbachhof (ABB), Wildeshausen (WIL) and Ultuna (ULT), Type A: plectenchymatic outer mantle, no rhizomorphs, no cystidia; Type B: plectenchymatic outer mantle, interconnected or ramified rhizomorphs, no cystidia; Type C: parenchymatic outer mantle, no rhizomorphs, awl-shaped, bristle like cystidia; C: control; N1 ¼ 100 kg N ha1 per annum; N þ I1 and N þ I2 ¼ 100, 200 kg N ha1 per annum with irrigationa Treatment
Fig. 1. Percentage of fine roots of Salix viminalis colonized with ectomycorrhizal fungi in 0–10 cm soil depth under various fertilizer regimes at the short rotation plantations Abbachhof, Wildeshausen and Ultuna during the years 1996–1999. C: control; N1: fertilization with 100 kg N ha1 per annum; N þ I1 and N þ I2: different fertilizer and irrigation treatments (see Table 2); S.E.: standard error; : significant effect of fertilization.
fertilized treatments. At ULT N-fertilization also reduced the ectomycorrhizal colonization. No differences were found between the two different fertilization and irrigation regimes, i.e. N þ I1 and N þ I2.
Morphotypes A
B
C
Mean S.D.
Mean S.D.
Mean S.D.
1997 ABB—C ABB—N1 WIL—C WIL—N1 ULT—C ULT—N þI1 ULT—N þ I2
5.1 4.2 15.6 4.4 9.8 4.4 9.8
2.9 7.2 3.1 3.5 6.2 2.9 4.1
– – 0.7 1.3 – – 0.4 0.8 0.2 0.5
2.8 7.2 0.5 2.4 5.1 2.0 –
4.9 9.8 0.4 4.1 8.7 5.2
1998 ABB—C ABB—N1 WIL—C WIL—N1 ULT—C ULT—N þI1 ULT—N þI2
5.2 1.9 4.0 4.9 2.5 1.8 3.1
7.5 1.8 2.8 3.9 2.2 1.4 3.2
– 1.6 2.8 2.4 4.2 8.1 7.0 – – –
14.4 2.9 4.4 6.1 6.1 0.4 –
6.4 1.7 7.6 6.3 9.3 0.7
a –: Absent; S.D.: standard deviation; : significant effect of N fertilization.
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fertilization in 1997. Type B was found only at control plots. In 1998 there were no longer effects of fertilization on the composition of morphotypes. At ULT Type B was observed only at fertilized plots, whereas Type C was not present at the N þ I2 plots. Only one ectomycorrhizal fungus (Inocybe glabripes) produced sporocarps at ABB under Salix viminalis within the growing season 1997. The sporocarps were detected only on plots without N-fertilization, but in low numbers. The saprophytic species Coprinus micaceus and Mycena rosella were observed at both treatments. Sporocarps of Coprinus disseminatus and Polyporus brumalis were observed only on the control plots and Agrocybe praecox only at the N1 plots. 3.2. VAM spore density A general fertilizer effect on the VAM spore density was found only at the highly fertilized and irrigated Swedish site (ULT-N2, Table 3). The density of VAM spores in the rhizosphere soil was affected mainly by the location of the sites (Fig. 2) and the sampling date (Table 3). Similar to the ectomycorrhizal colonization, the VAM spore density at ABB was generally high and was increased by fertilization in autumn in 1996, 1 year after the end of the N treatment (Fig. 2, Table 3 sampling date and sampling date fertilization). At WIL the VAM spore density in the rhizosphere soil was generally low, but remained unaffected by the Nfertilization. At ULT the VAM spore density was intermediate between the two German sites and Napplication reduced the density of VAM spores significantly in May 1997 and June 1999 (Fig. 2).
4. Discussion N-fertilization at three short rotation plantations in Germany and Sweden with different site conditions and management affected the ectomycorrhizal colonization, the composition of morphotypes and the VAM spore density in the soil surrounding the fine roots of Salix viminalis. It is known that mycorrhizal formation in general depends on the soil conditions (Baar, 1995). The soil P content, for example, affected the production of extramatrical mycelium of ectomycorrhizal fungi on Salix viminalis (Jones et al., 1990). Soil texture, the organic matter content and the pH
Fig. 2. Number of VAM spores g1 dry rhizosphere soil of Salix viminalis (0–10 cm depth) under various fertilizer regimes at the short rotation plantations Abbachhof, Wildeshausen and Ultuna during the years 1996–1999. C: control; N1: fertilization with 100 kg N ha1 per annum; N þ I1 and N þ I2: different fertilizer and irrigation treatments (see Table 2); S.E.: standard error; : significant effect of fertilization.
level were important differences between the soils of our experimental plantations (Table 1). At the clayey sites ABB and ULT (soil pH of 5.5 and 7.3) the average level of ectomycorrhizal colonization on Salix
C. Baum et al. / Forest Ecology and Management 160 (2002) 35–43
viminalis was lower, whereas the VAM spore density was higher than at the sandy site WIL (soil pH 4.5). Differences in the composition of morphotypes between the plantations may be caused partly by the management of the neighbouring sites. The German plantations ABB and WIL include plots with poplar (Populus tremula tremuloides and P. trichocarpa), whereas the Swedish plantation is neighboured only by arable soils and other Salix viminalis stands. The poplar clones in general had higher ectomycorrhizal colonization indices than willows (Baum and Makeschin, 1997) and belong to the same plant family (Salicaceae). Therefore, similarity in the selection of mycorrhizal partners can be suspected and introduction of mycorrhizal fungi from these plots could be possible. Altered composition of morphotypes due to N-fertilization suggests selective inhibition or promotion of fungal species as observed on pine (Termorshuizen, 1993). The low sporocarp production at ABB within the growing season 1997 with only one mycorrhizal species does not allow any conclusions to be drawn on fertilization effects on this parameter. The application of nutrients, e.g. through wastewater sludge is a frequently used management practice in short rotation forestry. The effects of N-fertilization on growth of Salix viminalis depend not only on the absolute quantity of N-application but on fertilization regime and site characters (soil type, climate, number of applications, irrigation) (Alriksson et al., 1997). Altered mycorrhizal formation due to fertilization may sometimes be one reason for these observations. Our investigations at the German plantations have shown that similar amounts of fertilizer (100 kg N ha1 per annum) caused different responses in mycorrhizal formation by Salix viminalis under different site conditions. Pot experiments have already shown the existence of an optimum N concentration for the formation of extraradical mycelium and mycorrhizas (Wallenda and Kottke, 1998; Baum et al., 2000). We suggest that the soil N content could be used as a parameter for the prediction of fertilization effects on mycorrhizal responses in willow plantations. This hypothesis should be tested with similar boundary conditions. At the Swedish plantation we found similar effects on the mycorrhizal formation of Salix viminalis with the two different fertilization and irrigation treatments using 100 or 200 kg N ha1 per annum. The influence
41
of irrigation should be tested in further investigations without fertilization. Since it was impossible to separate fertilization and irrigation effects at ULT in our investigation, our results document an integrated response of mycorrhizal formation to the management practice at this plantation.
5. Conclusions Soil properties, e.g. soil organic matter content, soil texture and soil pH affect mycorrhiza formation. The two German plantations ABB and WIL had similar organic matter content and soil pH but different soil texture. The clayey plantation ABB had lower ectomycorrhizal colonization than the sandy plantation WIL. The soil properties have been shown to modify the effects of N-fertilization on mycorrhizal formation of Salix viminalis. Therefore, in management practice of short rotation plantations, the suspected increase of biomass production after Nfertilization should be estimated keeping secondary effects caused by changed mycorrhizal formations in mind. It was found that the response to N-application could be increased and decreased ectomycorrhizal colonization. The quantitative effects of fertilization on ectomycorrhizal colonization might be caused in part by changes in species composition as was observed on pine and spruce by Baar (1995), Ka˚ ren and Nylund (1997) and Fransson et al. (2000). The changes in the morphotype composition at ABB and WIL after N-fertilization support this assumption. Undoubtedly, it is necessary to further test this hypothesis for willows, possibly in pot experiments with similar boundary conditions.
Acknowledgements The analyses of mycorrhization on the German experimental sites were founded by the Deutsche Forschungsgemeinschaft (MA 1257/3). We thank Mrs. Ch. Fenger for technical assistance. We are ¨ Z, Freiberg, grateful to Prof. Dr. H. Heilmeier (IO Germany), Mr. J. Schumacher (Institute for Forest Botany, Tharandt, Germany) and Dr. S. Thiele (Institute of Soil Science, Rostock, Germany) for their critical reviewing of the manuscript.
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