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Fungal diversity in set-aide agricultural soil investigated using terminal-restriction fragment length polymorphism
Soil Biology & Biochemistry 36 (2004) 983–988 www.elsevier.com/locate/soilbio
Fungal diversity in set-aide agricultural soil investigated using terminal-restriction fragment length polymorphism Morten Klamer*, Katarina Hedlund Department of Ecology, Lund University, Ecology Building, S-223 62 Lund, Sweden. Received 15 August 2003; received in revised form 23 January 2004; accepted 29 January 2004
Abstract As part of the restoration of biodiversity on former agricultural land there has been focused on methods to enhance the rate of transition from agricultural land towards natural grasslands or forest ecosystems. Management practices such as sowing seed mixtures and inoculating soil of later successional stages have been used. The aim of this study was to determine the effects of a managed plant community on the diversity of soil fungi in a newly abandoned agricultural land. A field site was set up consisting of 20 plots where the plant diversity was managed by either sowing 15 plant species, or natural colonization was allowed to occur. The plant mixture contained five species each of grasses, legumes and forbs that all were expected to occur at the site. A subset of the plots (five from each treatment) was inoculated with soil cores from a late successional stage. The plant community composition was subject to a principal component analysis based on the coverage of each species. Five years after abandonment, soil samples were taken from the plots, DNA was extracted and the ITS region of the rDNA gene was amplified using fluorescently labelled fungal specific primers (ITS 1F/ITS 4). The PCR products were digested using Hinf I and Taq I and sequenced. Results from both restriction enzymes were combined and a principal component analysis performed on the presence/absence of fragments. Also the fungal diversity expressed as number of restriction fragments were analysed. There was significantly higher fungal species richness in the experimental plots compared to the forest and field soils, but no differences between sown and naturally colonized plots. The different plant treatments did not influence the below ground fungal community composition. Soil water content on the other hand had an impact on the fungal community composition. q 2004 Elsevier Ltd. All rights reserved. Keywords: Terminal-restriction fragment length polymorphism; Above and below ground interactions; Field experiment; Plant community management
1. Introduction Interactions between plants and belowground microbial communities is a topic of growing interest, mainly because restoration of biodiversity is an issue in conservation policies (Read, 1998). It is well known that plants interact with the soil community and recently it has been suggested that even closely related plant species may determine the composition and activity of the mycorrhizal community (Johnson et al., 1992), although this effect seems to differ between different plant species (Klironomos, 2002). Also the opposite view, that the composition of the mycorrhizal community may determine the composition of the plant * Corresponding author. Present address: Danish Technological Institute, P.O. Box 141, DK-2630 Taastrup, Denmark. Tel.: þ 45-7220-2330; fax: þ 45-7220-2330. E-mail address: [email protected] (M. Klamer). 0038-0717/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2004.01.017
community has been proposed (van der Heijden et al., 1998; Hartnett and Wilson, 1999; Marler et al., 1999). Within the framework of an EU research program the project Changing Land Usage and Enhancement of Ecosystem Processes (CLUE) field experiments were carried out in five countries with the aim of examining whether management practices used for manipulating above and belowground diversity would increase the rate of change from agricultural land into natural, and more diverse, ecosystems (van der Putten et al., 2000). In other parts of the CLUE project, the plant community interactions with arbuscular mycorrhiza, saprophytic fungi and bacterial communities were reported (Hedlund and Gormsen, 2002; Hedlund, 2002). In these studies an increased biomass of all three groups were reported in the experimental plots compared to the nearby field and forest soils by using phospholipid fatty acid analysis. Kowalchuk et al. (2002), on the other hand, studying the effects of different plant
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species on the microbial community, only found changes in the microbial community structure and diversity in the rhizosphere soil. In this paper we compared the belowground community composition of soil fungi obtained by using the molecular technique ‘terminal-restriction fragment length polymorphism’ (T-RFLP) to data of the plant community composition obtained by vegetation analysis. The T-RFLP technique has proved to be an efficient technique to detect changes in microbial soil communities, see e.g. (Osborn et al., 2000; Dollhopf et al., 2001; Klamer et al., 2002). T-RFLP is an automated and sensitive method for the characterization of complex microbial communities. T-RFLP uses a polymerase chain reaction (PCR) in which one or both primers are fluorescently labelled. After amplification, the PCR product is digested with one or more restriction enzymes, generating fragments with different lengths, depending on the DNA sequence of the organism analysed and on the specificity of the enzyme. Only the fluorescent-labelled terminal restriction fragment from each organism is detected. Thus, in principle each fragment represents a species and the data obtained can be used to study both community composition and richness (Kitts, 2001). We selected this technique because it allows sufficient sample throughput to enable replicate sample analysis, which is essential with the block design used in this study. Furthermore, the T-RFLP technique is at least as sensitive as other molecular techniques for microbial community analysis, including denaturing gradient gel electrophoresis and temperature gradient gel electrophoresis (Marsh et al., 1998; Moeseneder et al., 1999).
2. Materials and methods Field experiments were set up in spring 1996 on former agricultural land in order to test how management of the plant community will influence the development of the soil community during succession towards more diverse and low-productivity land (van der Putten et al., 2000; Hedlund, 2002). Briefly, out of a larger set of experimental treatments we here used 20 plots (4 £ 4 m2) that were established in 1996 on a former agricultural field, located at a slight slope, about 1 m in altitude (map from Lantma¨teriet 1976, 1:10000). After harrowing, the plots were either sown with 15 plant species common to the area of a mid successional stage (Table 1), called a high diversity treatment (HD), or natural colonization was allowed (NC). Half the number of the plots was inoculated with soil from a later successional stage (forest) one 25 £ 25 £ 25 cm3 soil core in each 1 m2. Thus, five replicate plots of each treatment were set up according to a randomised block design. In Table 1 the plant species selected for the sown plots are shown. They represent three functional groups: grasses, legumes and forbs. The development of the plant community was monitored by analysing plant composition in 1 m2 of each plot during
Table 1 Plant species sown in the plots at the field site at Trolleholm, S Sweden Grasses
Forbs
Legumes
Festuca rubra Phleum pratense Agrostis capillaris Cynocurus cristatus Anthoxantum odoratum
Plantago lanceolata Leontodon hispidus Galium verum Prunella vulgaris Campanula rotundifolia
Lotus corniculatus Anthyllis vulneraria Trifolium pratense Trifolium repens Medicago lupulina
August 2001. The cover of each species was determined on a scale from 0 to 6, where 0 means not present and 1 – 6 represent the following coverage (in %): 0 –1, 1– 4, 4– 10, 10– 25, 25– 50 and 50– 100, respectively. The data of plant cover was subjected to a principal component analysis using a Multi-Variate Statistical Package (Kovach Computing Services, version 3.01). Both above and belowground plant biomass was estimated yearly. Plant above ground biomass was cut just above the soil surface of a 25 £ 25 cm2 area adjacent to each of the 1 m2 plots. The plant material was collected and dried at 80 8C. Root biomass was determined by collecting soil cores of 3 cm diameter and 15 cm depth. The soil cores were collected at the same time as the clipping of the aboveground biomass and stored in a cold room at 5 8C until further processing. The roots were separated from the soil by washing with tap water, dried at 80 8C for 48 h and weighed. Soil samples for analyses of fungal composition were collected in September 2001. From every plot, two times five soil cores were taken and mixed, resulting in two composite samples from each plot. Five composite samples were also collected from the nearby forest and agricultural field, respectively. The samples were stored at 2 80 8C until analysed. Water content and organic matter was determined by drying soil at 80 8C for 24 h and heating at 600 8C for 6 h, respectively. The soil samples for fungal community analysis (about 20 g, w/w) were air dried at room temperature for 24– 48 h, ground and passed through a sieve (mesh size: 0.4 mm). DNA was extracted using FastDNA SPIN kit for soil (Bio101, Inc., Carlsbad, CA, USA) according to the manufacturers protocol. DNA yield was measured using a spectrometer reading at 260 nm. The extracts generated between 21.5 and 40.9 mg DNA g21 dw soil on average for the different treatments with no significant differences between treatments, while the forest samples yielded 119 mg g21 dw soil. Polymerase chain reaction (PCR) was performed on both composite soil samples in two dilutions of the template DNA (1:10 and 1:100), which gave a total of four sets of data from each plot. PCR was conducted in 50 ml reaction volumes using fluorescently-labeled forward (6-FAMe) and reverse (HEXe) oligonucleotide primers (synthesized by MWG Biotech AG, Germany) targeting the intergenic transcribed spacer (ITS) region of the ribosomal DNA operon. The used primer combination (ITS 1F/ITS 4) is supposed to be
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specific to higher fungi (Gardes and Bruns, 1993; Klamer et al., 2002). Final concentrations in the PCR reactions were: 2.0 mM MgCl2, 1 £ buffer (Applied Biosystems Buffer II), 200 mM of each dNTP, 1.0 mM of each primer, 0.4 mg/ml BSA, and 1.25 U AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA, USA). The thermocycler (Perkin Elmer 3600) reaction conditions were: 5 min initial denaturation at 94 8C followed by 35 cycles of 0.5 min at 94 8C, 2 min of annealing at 52 8C, and 3 min extension at 72 8C. The final extension was 5 min at 72 8C. Reaction yield was determined by agarose gel electrophoresis. The PCR products were digested with different restriction endonucleases (Hinf I, and Taq I at 5U per reaction according to manufacturer’s instructions), denatured at 95 8C for 10 min and separated by capillary electrophoresis on an ABI 377 Genetic Analyzer in GeneScan mode (Applied Biosystems, Foster City, CA, USA). Each restriction digest created four data sets, which were combined so that all terminal restriction fragments (TRFs) were represented in the composite data set, even though they only appeared in a single of the four subsets. In this way we have eliminated some of the within plot variation and bias from the PCR reaction. The TRFs were recorded as present or absent and the data from both restriction digests were combined and a principal component analysis was performed after normalizing the data using Genotyper (Applied BioSystems, version 2.0). In total 269 TRFs were included as variables.
3. Results 3.1. Plant community analysis Plant aboveground biomass was significantly higher in the high diversity sown plots (HD) than in the naturally colonized plots (ND) (two-way ANOVA, F ¼ 4:803; P , 0:05) (Table 2). The root biomass did not differ between treatments and there was no influence of the inoculated soil on any of the plant biomass parameters. Plant species richness varied between 6 and 14 m22 with no significant differences between treatments. Table 2 Average above and belowground plant biomass in natural colonized (NC) and high diversity sown plots (HD) (data from 2001) Treatment
Soil inoculation
Shoot (g/m2)
Root (g/m2)
Shoot:root ratio
NC NC HD HD
2 þ 2 þ
460a 675b 905b 792b
532 730 671 645
1.0 1.1 1.5 1.4
Similar letter or no letter in a column indicates that these treatments are not significantly different, in a multi-comparison of a one-way ANOVA.
Fig. 1. (A) Principal component analysis (PCA) of the vegetation composition, summer 2001. Only plant species present in at least two plots are included. (W) (NC 2 ) Natural colonisation without soil inoculation. (B) (NC þ ) Natural colonisation with soil inoculation of forest. (K) (HD 2 ) Plots sown with high diversity of plant species without soil inoculation. (V) (HD þ ) Plots sown with high diversity of plant species with soil inoculation of forest soil. (B) Loading plot of the PCA showed in (A) showing loading values for individual plant species. Elytrigia repens, E.r.; Cirsium arvense, C.a.; Lapsana communis, L.c.; Lotus corniculatus, Lo.c.; Festuca rubra, F.r.; Poa trivialis, P.t.; Agrostis capillaris, A.c.; Galium aparine, G.a.; Taraxacum sect. Ruderalia, T.s.; Epilobium lamyi, E.l.; Fraxinus excelsior, F.e.; Poa pratensis, P.p.; Rubus idaeus, R.i.; Trifolium repens, T.r.; Trifolium pratense, T.p.; Galeopsis bifida, G.b.; Plantago lanceolata, P.l.; Stellaria holostea, S.h.; Myosotis arvensis, M.a.; Cirsium vulgare, C.v.; Galium verum, G.v.; Anthoxanthum odoratum, A.o.; Artemisia vulgaris, A.v.; Ranunculus repens, R.r.; Deschampsia caespitosa, D.c.; Geum urbanum, G.u.; Carpinus betulus, C.b.
In a PCA of the cover of the plant species the first principal component (PC 1) separated sown plots (HD) from plots that were naturally colonized (NC) (Fig. 1A). HD plots were grouped more closely together than the NC plots as they were sown with the same mixture of plant species while NC plots had a more random set of plants originating from the seed bank and from naturally invading species. The second principal component separated the inoculated plots (þ ) from the un-inoculated (2 ), mainly caused by
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the introduction of plant species from the forest ecosystem to both treatments. For example Stellaria holostea, was a typical plant species present in the forest and is rarely seen in fields (Weimark 1977), while Galium aparine was only found in un-inoculated plots (Fig. 1B). These two species colonised the plots naturally and were not included in the plant species used in the seed mixtures. 3.2. Fungal species richness When evaluated with the two restriction enzymes, the species richness was significantly higher in the abandoned land than in the agricultural field and the forest (Table 3). The level of richness evaluated with Hinf I was lower than the richness evaluated with Taq I for all samples. There were no effects of the soil inoculations on species richness analysed with any of the restriction enzymes. 3.3. Fungal community composition In a principal component analysis (PCA) of all the samples a clear separation could be seen between the forest samples and those from the experimental and field samples (Fig. 2A). There was also a separation of the five replicate samples of NC and HD plots where the samples of the treatments from each block cluster together, while no effect of the aboveground plant treatment was detected (Fig. 2B). However, there was a pattern in the PCA that could be related to the positioning of the plots in the field where block A was placed at the top of a small slope and the site ended at block E further down (about 1 m of altitude). The water content of the soil could partly explain these results. Thus, a relation of soil water content to the case scores of the samples in the PCA was illustrated in Fig. 2 with a linear regression between case scores of samples of PC 1 and water content of the soil (r 2 ¼ 0:304; P , 0:05; n ¼ 20). There was no relation to organic matter content, which varied between 3.4 and 9.2% of the dry weight of the soil. Table 3 Species richness evaluated by the number of terminal restriction fragments from each restriction enzyme Treatment
Hinf forward
Hinf reverse
Taq forward
Taq reverse
HD 2 HD þ NC 2 NC þ Field Forest
26.2ab 30.4a 30.0a 30.2a 17.6b 22.2ab
27.4a 29.8a 28.6a 29.8a 14.2b 17.8ab
46.0a 46.8a 49.8a 51.2a 27.4b 30.8b
30.2a 31.2a 34.8a 33.8a 20.4b 19.0b
ANOVA
P , 0:05
P , 0:05
P , 0:001
P , 0:01
Similar letter in a column indicates that these treatments are not significantly different, in a multi-comparison of a one-way ANOVA. HD 2 : High diversity sown plots without inoculation of forest soil. HD þ : High diversity sown plots with inoculation of forest soil. NC 2 : Natural colonization of plants without inoculation of forest soil. NC þ : Natural colonization with inoculation of forest soil.
Fig. 2. (A) Principal component analysis of the fungal community composition in all samples. Only species present in at least two samples were included. (W) (NC 2 ) Natural colonisation without soil inoculation. (B) (NC þ ) Natural colonisation with soil inoculation of forest. (K) (HD 2 ) Plots sown with high diversity of plant species without soil inoculation. (V) (HD þ ) Plots sown with high diversity of plant species with soil inoculation of forest soil. Fields A– E and forest A–E represent the five replicate soil samples from the agricultural field and the forest, respectively, next to the experimental site. The letters A and E represent the top and bottom of the slope at the site, respectively. (B) Linear correlation between principal component 1 from (A) and water content in the plots. r 2 ¼ 0:304; P , 0:05: For legends see Fig. 1.
4. Discussion The amount of aboveground plant biomass had increased between 27 and 40% in 2001 compared to earlier determinations at the field site of both treatments in 1998. On the other hand, the belowground biomass had also increased between 8 and 37% (Hedlund et al., 2003). In both treatments the ratio between shoot and root biomass decreased during the five year period, though the naturally colonised plots reaching almost equal above and below biomass earlier than the sown plots. This indicates that through succession the plants allocate more resources to their roots.
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Sowing plant species on set aside land will decrease the variation of the plant community composition when compared to a naturally colonised land since some of the sown species can suppress the naturally colonising species and species from the seed bank (Leps et al., 2001). This can be seen in the results where the HD plots were more homogeneous (closer together in the PCA), than those that were naturally colonised. In the vegetation analyses it was also possible to detect an effect of the soil inoculations; not only as an increase in the number of woody species as previously described by Hedlund and Gormsen (2002). The introduction of soil, with root fragments and a seed bank of plant species from the forest community contributed with several plant species to the inoculated plots in both treatments. Why is the fungal diversity higher in the experimental plots compared to both field and forest soils? The species composition in the set-aside land has changed from that of the ongoing agricultural practice, we can say that there has been a shift in species composition due to abandonment of agriculture. An increased number of species in the set aside plots could be explained by species present in the forest and the field soils entering the experimental plots. However, if this was the case, it should be detected in the PCA, mixing the coordinates of the experimental plots with forest and the field. A more plausible explanation is that the set-aside land has not reached an equilibrium state regarding competition between species and can thus contain more species than that of a forest, which is considered the climax community in this region. Part of the reason why the results from Hinf I differed from Taq I may be that different enzymes have different recognition sites, but it emphasises the importance of using more than one restriction enzyme when evaluating microbial community profiles from environmental samples (Klamer et al., 2002). Klironomos (2002) gave evidence to the hypothesis that feedback between plants and soil communities may determine the ability of a plant to establish, invade and persist in a local habitat. Especially the ability of the plants to generate high levels of pathogens seems to play a key role in the development of plant diversity. Thus, he showed that rare species tend to accumulate pathogens quicker than invasive plants, which could explain why they are rare. If this hypothesis is valid for the system studied here, then it should be possible to detect changes in the fungal community caused by changes in plant cover. However, the data in this study did not support the hypothesis, since the plant cover data and data from the belowground fungal community did not show the same pattern. However, there are some limitations to this conclusion. The plant cover data are based on all species found in the plots, while the fungal community is based on samples from the soil. This renders the possibility that some fungal species are not included because of the heterogeneity of the distribution of fungi in the soil. We have tried to eliminate the heterogeneity by taking 10 soil cores from each plot and mix these into two
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composite samples. To further improve the amplification of DNA, also from rare species, PCR was performed on two different dilutions of the DNA templates. But still we are only including a subset of the species actually present in the soil. Another problem is the specificity of the used primer set. ITS 1F-ITS 4 is known to amplify a large range of Ascomycetes and Basidomycetes, while Zygomycetes and Glomeromycetes will not be amplified (Gardes and Bruns, 1993; Klamer et al., 2002). Thus, in the grassland system studied, we have mainly amplified saprophytic or pathogenic fungi, while the arbuscular mycorrhizal fungi are not included. There are some indications that AM fungi may constitute a large fraction of the fungal biomass in a field ecosystem (Olsson et al., 1995; Olsson et al., 1999). Hedlund (2002) found a higher amount of AM fungal biomass in the NC plots compared to HD, field and forest samples. She also showed that there was higher microbial biomass and activity in the HD plots as opposed to the NC and field plots, based on PLFA and respiration measurements. Wardle and Nicholson (1996) investigated the synergistic effects of grassland plants on soil microbial biomass and activity in a potting system using both plant monocultures and two species per pot. They found that the microbial biomass was determined by plant primary production and by which and how many plants species were present. Bever (1994) and Bever et al. (1997) demonstrated a negative feedback mechanism on growth rates in a pair wise comparison of different plant species. Finally, Zak et al. (2003) showed that microbial biomass, fungal abundance, and N mineralization rate increased with increasing plant diversity. By excluding the effects of abiotic factors these papers provided evidence for the potential importance of the soil community in maintaining diversity within plant communities. These results suggest that the influence of plant community composition on below-ground fungal communities seems to be confined to fungal species related to plant roots or rhizosphere, e.g. arbuscular mycorrhizal (Johnson et al., 1992), while the saprophytic community composition is unaffected by the changes in plant community composition. Fungal species richness was affected by the experimental treatments, clearly showing that the richness was higher in the set-aside land than in the nearby agricultural field and forest soils. The increase of species in the abandoned land shows that the restoration of former agricultural land does also increase the level of fungal diversity in the soil.
Acknowledgements This project was funded by the Swedish environmental protection agency and the Swedish Research council for environment, agricultural sciences and spatial planning.
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References Bever, J.D., 1994. Feedback between plants and their soil communities in an old field community. Ecology 75, 1965–1977. Bever, J.D., Westover, K.M., Antonovics, J., 1997. Incorporating the soil community into plant population dynamics: the utility of the feedback approach. Journal of Ecology 85, 561 –573. Dollhopf, S.L., Hashsham, S.A., Tiedje, J.M., 2001. Interpreting 16S rDNA T-RFLP data: application of self-organizing maps and principal component analysis to describe community dynamics and convergence. Microbial Ecology 42, 495 –505. Gardes, M., Bruns, T.D., 1993. ITS primers with enhanced specificity for basidiomycetes - application to the identification of mycorrhizae and rusts. Molecular Ecology 2, 113 –118. Hartnett, D.C., Wilson, G.W.T., 1999. Mycorrhizae influence plant community structure and diversity in tallgrass prairie. Ecology 80, 1187–1195. Hedlund, K., 2002. Soil microbial community structure in relation to plant diversity management on former agricultural land. Soil Biology & Biochemistry 34, 1299–1307. Hedlund, K., Gormsen, D., 2002. Mycorrhizal colonization of plants in setaside agricultural land. Applied Soil Ecology 19, 71–78. Hedlund, K., Santa Regina, I., van der Putten, W.H., Lepsˇ, J., Diaz, T., Korthals, G.W., Lavorel, S., Brown, V.K., Gormsen, D., Mortimer, S.R., Rodrigues Barrueco, C., Roy, J., Smilauer, P., Smilauerova, M., van Dijk, C., 2003. Plant species diversity, plant biomass and responses of the soil community on abandoned land across Europe: Idiosyncracy or above-belowground time lags. Oikos 103, 45–58. van der Heijden, M.G.A., Klironomos, J.N., Ursic, M., Moutoglis, P., Streitwolf-Engel, R., Boller, T., Wiemken, A., Sanders, I.R., 1998. Mycorrhizal fungal diversity determines plant biodiversity, ecosystem variability and productivity. Nature 396, 69 –72. Johnson, N.C., Tilman, D., Wedin, D.A., 1992. Plant and soil controls on mycorrhizal fungal communities. Ecology 73, 2034–2042. Kitts, C.L., 2001. Terminal restriction fragment patterns: a tool for comparing microbial communities and assessing community dynamics. Current Issues in Intestinal Microbiology 2, 17 –25. Klamer, M., Roberts, M.S., Levine, L.H., Drake, B.G., Garland, J.L., 2002. Influence of elevated CO2 on the fungal community in a coastal scrub oak forest soil investigated using terminal-restriction fragment length polymorphism (T-RFLP). Applied and Environmental Microbiology 68, 4370–4376. Klironomos, J.N., 2002. Feedback with soil biota contributes to plant rarity and invasiveness in communities. Nature 417, 67 –70.
Kowalchuk, G.A., Buma, D.S., De Boer, W., Klinkhamer, P.G.L., Van Veen, J.A., 2002. Effects of above-ground plant species composition and diversity on the diversity of soil-borne microorganisms. Antonie van Leeuwenhoek 81, 509–520. Leps, J., Brown, V.K., Len, T.A.D., Gormsen, D., Hedlund, K., Kailova, J., Korthals, G.W., Mortimer, S.R., Rodriguez-Barrueco, C., Roy, J., Regina, I.S., van Dijk, C., van der Putten, W.H., 2001. Separating the chance effect from other diversity effects in the functioning of plant communities. Oikos 92, 123–134. Marler, M.J., Zabinski, C.A., Callaway, R.M., 1999. Mycorrhizae indirectly enhance competitive effects of an invasive forb on native bunchgrass. Ecology 80, 1180–1186. Marsh, T.L., Liu, W.T., Forney, L.J., Hans, C., 1998. Beginning a molecular analysis of the eukaryal community in activated sludge. Water Science and Technology 37, 455–460. Moeseneder, M.M., Arrieta, J.M., Muyzer, G., Winter, C., Herndl, G.J., 1999. Optimization of terminal-restriction fragment length polymorphism analysis for complex marine bacterioplankton communities and comparison with denaturing gradient gel electrophoresis. Applied and Environmental Microbiology 65, 3518–3525. Olsson, P.A., Ba˚a˚th, E., Jakobsen, I., So¨derstro¨m, B.E., 1995. The use of phospholipid and neutral lipid fatty acids to estimate biomass of arbuscular mycorrhizal fungi in soil. Mycological Research 99, 623 –629. Olsson, P.A., Thingstrup, I., Jakobsen, I., Ba˚a˚th, E., 1999. Estimation of the biomass of arbuscular mycorrhizal fungi in a linseed field. Soil Biology & Biochemistry 31, 1879–1887. Osborn, A.M., Moore, E.R.B., Timmis, K.N., 2000. An evaluation of thermal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure and dynamics. Environmental Microbiology 2, 39– 50. van der Putten, W.H., Mortimer, S.R., Hedlund, K., van Dijk, C., Brown, V.K., LepTˇ, J., Rodriguez-Barrueco, C., Roy, J., Len, T.A.D., Gormsen, D., Korthals, G.W., Lavorel, S., Regina, I.S., Smilauer, P., 2000. Plant species diversity as a driver of early succession in abandoned fields: a multi-site approach. Oecologia 124, 91 –99. Read, D., 1998. Biodiversity: plants on the web. Nature 396, 22–23. Wardle, D.A., Nicholson, S., 1996. Synergistic effects of grassland plant species on soil microbial biomass and activity: implications for ecosystem-level effects of enriched plant diversity. Functional Ecology 10, 410–416. Zak, D.R., Holmes, W.E., White, D.C., Peacock, A.D., Tilman, D., 2003. Plant diversity, soil microbial communities, and ecosystem function: are there links? Ecology 84, 2042–2050.
Fungal diversity in set-aide agricultural soil investigated using terminal-restriction fragment length polymorphism Morten Klamer*, Katarina Hedlund Department of Ecology, Lund University, Ecology Building, S-223 62 Lund, Sweden. Received 15 August 2003; received in revised form 23 January 2004; accepted 29 January 2004
Abstract As part of the restoration of biodiversity on former agricultural land there has been focused on methods to enhance the rate of transition from agricultural land towards natural grasslands or forest ecosystems. Management practices such as sowing seed mixtures and inoculating soil of later successional stages have been used. The aim of this study was to determine the effects of a managed plant community on the diversity of soil fungi in a newly abandoned agricultural land. A field site was set up consisting of 20 plots where the plant diversity was managed by either sowing 15 plant species, or natural colonization was allowed to occur. The plant mixture contained five species each of grasses, legumes and forbs that all were expected to occur at the site. A subset of the plots (five from each treatment) was inoculated with soil cores from a late successional stage. The plant community composition was subject to a principal component analysis based on the coverage of each species. Five years after abandonment, soil samples were taken from the plots, DNA was extracted and the ITS region of the rDNA gene was amplified using fluorescently labelled fungal specific primers (ITS 1F/ITS 4). The PCR products were digested using Hinf I and Taq I and sequenced. Results from both restriction enzymes were combined and a principal component analysis performed on the presence/absence of fragments. Also the fungal diversity expressed as number of restriction fragments were analysed. There was significantly higher fungal species richness in the experimental plots compared to the forest and field soils, but no differences between sown and naturally colonized plots. The different plant treatments did not influence the below ground fungal community composition. Soil water content on the other hand had an impact on the fungal community composition. q 2004 Elsevier Ltd. All rights reserved. Keywords: Terminal-restriction fragment length polymorphism; Above and below ground interactions; Field experiment; Plant community management
1. Introduction Interactions between plants and belowground microbial communities is a topic of growing interest, mainly because restoration of biodiversity is an issue in conservation policies (Read, 1998). It is well known that plants interact with the soil community and recently it has been suggested that even closely related plant species may determine the composition and activity of the mycorrhizal community (Johnson et al., 1992), although this effect seems to differ between different plant species (Klironomos, 2002). Also the opposite view, that the composition of the mycorrhizal community may determine the composition of the plant * Corresponding author. Present address: Danish Technological Institute, P.O. Box 141, DK-2630 Taastrup, Denmark. Tel.: þ 45-7220-2330; fax: þ 45-7220-2330. E-mail address: [email protected] (M. Klamer). 0038-0717/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2004.01.017
community has been proposed (van der Heijden et al., 1998; Hartnett and Wilson, 1999; Marler et al., 1999). Within the framework of an EU research program the project Changing Land Usage and Enhancement of Ecosystem Processes (CLUE) field experiments were carried out in five countries with the aim of examining whether management practices used for manipulating above and belowground diversity would increase the rate of change from agricultural land into natural, and more diverse, ecosystems (van der Putten et al., 2000). In other parts of the CLUE project, the plant community interactions with arbuscular mycorrhiza, saprophytic fungi and bacterial communities were reported (Hedlund and Gormsen, 2002; Hedlund, 2002). In these studies an increased biomass of all three groups were reported in the experimental plots compared to the nearby field and forest soils by using phospholipid fatty acid analysis. Kowalchuk et al. (2002), on the other hand, studying the effects of different plant
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species on the microbial community, only found changes in the microbial community structure and diversity in the rhizosphere soil. In this paper we compared the belowground community composition of soil fungi obtained by using the molecular technique ‘terminal-restriction fragment length polymorphism’ (T-RFLP) to data of the plant community composition obtained by vegetation analysis. The T-RFLP technique has proved to be an efficient technique to detect changes in microbial soil communities, see e.g. (Osborn et al., 2000; Dollhopf et al., 2001; Klamer et al., 2002). T-RFLP is an automated and sensitive method for the characterization of complex microbial communities. T-RFLP uses a polymerase chain reaction (PCR) in which one or both primers are fluorescently labelled. After amplification, the PCR product is digested with one or more restriction enzymes, generating fragments with different lengths, depending on the DNA sequence of the organism analysed and on the specificity of the enzyme. Only the fluorescent-labelled terminal restriction fragment from each organism is detected. Thus, in principle each fragment represents a species and the data obtained can be used to study both community composition and richness (Kitts, 2001). We selected this technique because it allows sufficient sample throughput to enable replicate sample analysis, which is essential with the block design used in this study. Furthermore, the T-RFLP technique is at least as sensitive as other molecular techniques for microbial community analysis, including denaturing gradient gel electrophoresis and temperature gradient gel electrophoresis (Marsh et al., 1998; Moeseneder et al., 1999).
2. Materials and methods Field experiments were set up in spring 1996 on former agricultural land in order to test how management of the plant community will influence the development of the soil community during succession towards more diverse and low-productivity land (van der Putten et al., 2000; Hedlund, 2002). Briefly, out of a larger set of experimental treatments we here used 20 plots (4 £ 4 m2) that were established in 1996 on a former agricultural field, located at a slight slope, about 1 m in altitude (map from Lantma¨teriet 1976, 1:10000). After harrowing, the plots were either sown with 15 plant species common to the area of a mid successional stage (Table 1), called a high diversity treatment (HD), or natural colonization was allowed (NC). Half the number of the plots was inoculated with soil from a later successional stage (forest) one 25 £ 25 £ 25 cm3 soil core in each 1 m2. Thus, five replicate plots of each treatment were set up according to a randomised block design. In Table 1 the plant species selected for the sown plots are shown. They represent three functional groups: grasses, legumes and forbs. The development of the plant community was monitored by analysing plant composition in 1 m2 of each plot during
Table 1 Plant species sown in the plots at the field site at Trolleholm, S Sweden Grasses
Forbs
Legumes
Festuca rubra Phleum pratense Agrostis capillaris Cynocurus cristatus Anthoxantum odoratum
Plantago lanceolata Leontodon hispidus Galium verum Prunella vulgaris Campanula rotundifolia
Lotus corniculatus Anthyllis vulneraria Trifolium pratense Trifolium repens Medicago lupulina
August 2001. The cover of each species was determined on a scale from 0 to 6, where 0 means not present and 1 – 6 represent the following coverage (in %): 0 –1, 1– 4, 4– 10, 10– 25, 25– 50 and 50– 100, respectively. The data of plant cover was subjected to a principal component analysis using a Multi-Variate Statistical Package (Kovach Computing Services, version 3.01). Both above and belowground plant biomass was estimated yearly. Plant above ground biomass was cut just above the soil surface of a 25 £ 25 cm2 area adjacent to each of the 1 m2 plots. The plant material was collected and dried at 80 8C. Root biomass was determined by collecting soil cores of 3 cm diameter and 15 cm depth. The soil cores were collected at the same time as the clipping of the aboveground biomass and stored in a cold room at 5 8C until further processing. The roots were separated from the soil by washing with tap water, dried at 80 8C for 48 h and weighed. Soil samples for analyses of fungal composition were collected in September 2001. From every plot, two times five soil cores were taken and mixed, resulting in two composite samples from each plot. Five composite samples were also collected from the nearby forest and agricultural field, respectively. The samples were stored at 2 80 8C until analysed. Water content and organic matter was determined by drying soil at 80 8C for 24 h and heating at 600 8C for 6 h, respectively. The soil samples for fungal community analysis (about 20 g, w/w) were air dried at room temperature for 24– 48 h, ground and passed through a sieve (mesh size: 0.4 mm). DNA was extracted using FastDNA SPIN kit for soil (Bio101, Inc., Carlsbad, CA, USA) according to the manufacturers protocol. DNA yield was measured using a spectrometer reading at 260 nm. The extracts generated between 21.5 and 40.9 mg DNA g21 dw soil on average for the different treatments with no significant differences between treatments, while the forest samples yielded 119 mg g21 dw soil. Polymerase chain reaction (PCR) was performed on both composite soil samples in two dilutions of the template DNA (1:10 and 1:100), which gave a total of four sets of data from each plot. PCR was conducted in 50 ml reaction volumes using fluorescently-labeled forward (6-FAMe) and reverse (HEXe) oligonucleotide primers (synthesized by MWG Biotech AG, Germany) targeting the intergenic transcribed spacer (ITS) region of the ribosomal DNA operon. The used primer combination (ITS 1F/ITS 4) is supposed to be
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specific to higher fungi (Gardes and Bruns, 1993; Klamer et al., 2002). Final concentrations in the PCR reactions were: 2.0 mM MgCl2, 1 £ buffer (Applied Biosystems Buffer II), 200 mM of each dNTP, 1.0 mM of each primer, 0.4 mg/ml BSA, and 1.25 U AmpliTaq DNA polymerase (Applied Biosystems, Foster City, CA, USA). The thermocycler (Perkin Elmer 3600) reaction conditions were: 5 min initial denaturation at 94 8C followed by 35 cycles of 0.5 min at 94 8C, 2 min of annealing at 52 8C, and 3 min extension at 72 8C. The final extension was 5 min at 72 8C. Reaction yield was determined by agarose gel electrophoresis. The PCR products were digested with different restriction endonucleases (Hinf I, and Taq I at 5U per reaction according to manufacturer’s instructions), denatured at 95 8C for 10 min and separated by capillary electrophoresis on an ABI 377 Genetic Analyzer in GeneScan mode (Applied Biosystems, Foster City, CA, USA). Each restriction digest created four data sets, which were combined so that all terminal restriction fragments (TRFs) were represented in the composite data set, even though they only appeared in a single of the four subsets. In this way we have eliminated some of the within plot variation and bias from the PCR reaction. The TRFs were recorded as present or absent and the data from both restriction digests were combined and a principal component analysis was performed after normalizing the data using Genotyper (Applied BioSystems, version 2.0). In total 269 TRFs were included as variables.
3. Results 3.1. Plant community analysis Plant aboveground biomass was significantly higher in the high diversity sown plots (HD) than in the naturally colonized plots (ND) (two-way ANOVA, F ¼ 4:803; P , 0:05) (Table 2). The root biomass did not differ between treatments and there was no influence of the inoculated soil on any of the plant biomass parameters. Plant species richness varied between 6 and 14 m22 with no significant differences between treatments. Table 2 Average above and belowground plant biomass in natural colonized (NC) and high diversity sown plots (HD) (data from 2001) Treatment
Soil inoculation
Shoot (g/m2)
Root (g/m2)
Shoot:root ratio
NC NC HD HD
2 þ 2 þ
460a 675b 905b 792b
532 730 671 645
1.0 1.1 1.5 1.4
Similar letter or no letter in a column indicates that these treatments are not significantly different, in a multi-comparison of a one-way ANOVA.
Fig. 1. (A) Principal component analysis (PCA) of the vegetation composition, summer 2001. Only plant species present in at least two plots are included. (W) (NC 2 ) Natural colonisation without soil inoculation. (B) (NC þ ) Natural colonisation with soil inoculation of forest. (K) (HD 2 ) Plots sown with high diversity of plant species without soil inoculation. (V) (HD þ ) Plots sown with high diversity of plant species with soil inoculation of forest soil. (B) Loading plot of the PCA showed in (A) showing loading values for individual plant species. Elytrigia repens, E.r.; Cirsium arvense, C.a.; Lapsana communis, L.c.; Lotus corniculatus, Lo.c.; Festuca rubra, F.r.; Poa trivialis, P.t.; Agrostis capillaris, A.c.; Galium aparine, G.a.; Taraxacum sect. Ruderalia, T.s.; Epilobium lamyi, E.l.; Fraxinus excelsior, F.e.; Poa pratensis, P.p.; Rubus idaeus, R.i.; Trifolium repens, T.r.; Trifolium pratense, T.p.; Galeopsis bifida, G.b.; Plantago lanceolata, P.l.; Stellaria holostea, S.h.; Myosotis arvensis, M.a.; Cirsium vulgare, C.v.; Galium verum, G.v.; Anthoxanthum odoratum, A.o.; Artemisia vulgaris, A.v.; Ranunculus repens, R.r.; Deschampsia caespitosa, D.c.; Geum urbanum, G.u.; Carpinus betulus, C.b.
In a PCA of the cover of the plant species the first principal component (PC 1) separated sown plots (HD) from plots that were naturally colonized (NC) (Fig. 1A). HD plots were grouped more closely together than the NC plots as they were sown with the same mixture of plant species while NC plots had a more random set of plants originating from the seed bank and from naturally invading species. The second principal component separated the inoculated plots (þ ) from the un-inoculated (2 ), mainly caused by
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the introduction of plant species from the forest ecosystem to both treatments. For example Stellaria holostea, was a typical plant species present in the forest and is rarely seen in fields (Weimark 1977), while Galium aparine was only found in un-inoculated plots (Fig. 1B). These two species colonised the plots naturally and were not included in the plant species used in the seed mixtures. 3.2. Fungal species richness When evaluated with the two restriction enzymes, the species richness was significantly higher in the abandoned land than in the agricultural field and the forest (Table 3). The level of richness evaluated with Hinf I was lower than the richness evaluated with Taq I for all samples. There were no effects of the soil inoculations on species richness analysed with any of the restriction enzymes. 3.3. Fungal community composition In a principal component analysis (PCA) of all the samples a clear separation could be seen between the forest samples and those from the experimental and field samples (Fig. 2A). There was also a separation of the five replicate samples of NC and HD plots where the samples of the treatments from each block cluster together, while no effect of the aboveground plant treatment was detected (Fig. 2B). However, there was a pattern in the PCA that could be related to the positioning of the plots in the field where block A was placed at the top of a small slope and the site ended at block E further down (about 1 m of altitude). The water content of the soil could partly explain these results. Thus, a relation of soil water content to the case scores of the samples in the PCA was illustrated in Fig. 2 with a linear regression between case scores of samples of PC 1 and water content of the soil (r 2 ¼ 0:304; P , 0:05; n ¼ 20). There was no relation to organic matter content, which varied between 3.4 and 9.2% of the dry weight of the soil. Table 3 Species richness evaluated by the number of terminal restriction fragments from each restriction enzyme Treatment
Hinf forward
Hinf reverse
Taq forward
Taq reverse
HD 2 HD þ NC 2 NC þ Field Forest
26.2ab 30.4a 30.0a 30.2a 17.6b 22.2ab
27.4a 29.8a 28.6a 29.8a 14.2b 17.8ab
46.0a 46.8a 49.8a 51.2a 27.4b 30.8b
30.2a 31.2a 34.8a 33.8a 20.4b 19.0b
ANOVA
P , 0:05
P , 0:05
P , 0:001
P , 0:01
Similar letter in a column indicates that these treatments are not significantly different, in a multi-comparison of a one-way ANOVA. HD 2 : High diversity sown plots without inoculation of forest soil. HD þ : High diversity sown plots with inoculation of forest soil. NC 2 : Natural colonization of plants without inoculation of forest soil. NC þ : Natural colonization with inoculation of forest soil.
Fig. 2. (A) Principal component analysis of the fungal community composition in all samples. Only species present in at least two samples were included. (W) (NC 2 ) Natural colonisation without soil inoculation. (B) (NC þ ) Natural colonisation with soil inoculation of forest. (K) (HD 2 ) Plots sown with high diversity of plant species without soil inoculation. (V) (HD þ ) Plots sown with high diversity of plant species with soil inoculation of forest soil. Fields A– E and forest A–E represent the five replicate soil samples from the agricultural field and the forest, respectively, next to the experimental site. The letters A and E represent the top and bottom of the slope at the site, respectively. (B) Linear correlation between principal component 1 from (A) and water content in the plots. r 2 ¼ 0:304; P , 0:05: For legends see Fig. 1.
4. Discussion The amount of aboveground plant biomass had increased between 27 and 40% in 2001 compared to earlier determinations at the field site of both treatments in 1998. On the other hand, the belowground biomass had also increased between 8 and 37% (Hedlund et al., 2003). In both treatments the ratio between shoot and root biomass decreased during the five year period, though the naturally colonised plots reaching almost equal above and below biomass earlier than the sown plots. This indicates that through succession the plants allocate more resources to their roots.
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Sowing plant species on set aside land will decrease the variation of the plant community composition when compared to a naturally colonised land since some of the sown species can suppress the naturally colonising species and species from the seed bank (Leps et al., 2001). This can be seen in the results where the HD plots were more homogeneous (closer together in the PCA), than those that were naturally colonised. In the vegetation analyses it was also possible to detect an effect of the soil inoculations; not only as an increase in the number of woody species as previously described by Hedlund and Gormsen (2002). The introduction of soil, with root fragments and a seed bank of plant species from the forest community contributed with several plant species to the inoculated plots in both treatments. Why is the fungal diversity higher in the experimental plots compared to both field and forest soils? The species composition in the set-aside land has changed from that of the ongoing agricultural practice, we can say that there has been a shift in species composition due to abandonment of agriculture. An increased number of species in the set aside plots could be explained by species present in the forest and the field soils entering the experimental plots. However, if this was the case, it should be detected in the PCA, mixing the coordinates of the experimental plots with forest and the field. A more plausible explanation is that the set-aside land has not reached an equilibrium state regarding competition between species and can thus contain more species than that of a forest, which is considered the climax community in this region. Part of the reason why the results from Hinf I differed from Taq I may be that different enzymes have different recognition sites, but it emphasises the importance of using more than one restriction enzyme when evaluating microbial community profiles from environmental samples (Klamer et al., 2002). Klironomos (2002) gave evidence to the hypothesis that feedback between plants and soil communities may determine the ability of a plant to establish, invade and persist in a local habitat. Especially the ability of the plants to generate high levels of pathogens seems to play a key role in the development of plant diversity. Thus, he showed that rare species tend to accumulate pathogens quicker than invasive plants, which could explain why they are rare. If this hypothesis is valid for the system studied here, then it should be possible to detect changes in the fungal community caused by changes in plant cover. However, the data in this study did not support the hypothesis, since the plant cover data and data from the belowground fungal community did not show the same pattern. However, there are some limitations to this conclusion. The plant cover data are based on all species found in the plots, while the fungal community is based on samples from the soil. This renders the possibility that some fungal species are not included because of the heterogeneity of the distribution of fungi in the soil. We have tried to eliminate the heterogeneity by taking 10 soil cores from each plot and mix these into two
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composite samples. To further improve the amplification of DNA, also from rare species, PCR was performed on two different dilutions of the DNA templates. But still we are only including a subset of the species actually present in the soil. Another problem is the specificity of the used primer set. ITS 1F-ITS 4 is known to amplify a large range of Ascomycetes and Basidomycetes, while Zygomycetes and Glomeromycetes will not be amplified (Gardes and Bruns, 1993; Klamer et al., 2002). Thus, in the grassland system studied, we have mainly amplified saprophytic or pathogenic fungi, while the arbuscular mycorrhizal fungi are not included. There are some indications that AM fungi may constitute a large fraction of the fungal biomass in a field ecosystem (Olsson et al., 1995; Olsson et al., 1999). Hedlund (2002) found a higher amount of AM fungal biomass in the NC plots compared to HD, field and forest samples. She also showed that there was higher microbial biomass and activity in the HD plots as opposed to the NC and field plots, based on PLFA and respiration measurements. Wardle and Nicholson (1996) investigated the synergistic effects of grassland plants on soil microbial biomass and activity in a potting system using both plant monocultures and two species per pot. They found that the microbial biomass was determined by plant primary production and by which and how many plants species were present. Bever (1994) and Bever et al. (1997) demonstrated a negative feedback mechanism on growth rates in a pair wise comparison of different plant species. Finally, Zak et al. (2003) showed that microbial biomass, fungal abundance, and N mineralization rate increased with increasing plant diversity. By excluding the effects of abiotic factors these papers provided evidence for the potential importance of the soil community in maintaining diversity within plant communities. These results suggest that the influence of plant community composition on below-ground fungal communities seems to be confined to fungal species related to plant roots or rhizosphere, e.g. arbuscular mycorrhizal (Johnson et al., 1992), while the saprophytic community composition is unaffected by the changes in plant community composition. Fungal species richness was affected by the experimental treatments, clearly showing that the richness was higher in the set-aside land than in the nearby agricultural field and forest soils. The increase of species in the abandoned land shows that the restoration of former agricultural land does also increase the level of fungal diversity in the soil.
Acknowledgements This project was funded by the Swedish environmental protection agency and the Swedish Research council for environment, agricultural sciences and spatial planning.
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