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Comparative single-strand conformation polymorphism (SSCP) and microscopy-based analysis of nitrogen cultivation interactive effects on the fungal community of a semiarid steppe soil
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Comparative single-strand conformation polymorphism (SSCP) and microscopy-based analysis of nitrogen cultivation interactive e¡ects on the fungal community of a semiarid steppe soil Jennifer L. Lowell *, Donald A. Klein Department of Microbiology, Colorado State University, Fort Collins, CO 80523-1677, USA Received 13 October 2000; received in revised form 2 April 2001; accepted 2 April 2001 First published online 27 April 2001
Abstract The effects of nitrogen accretion on fungal diversity and community structure in early-seral (cultivated) and native (uncultivated) shortgrass steppe soils were evaluated using single-strand conformation polymorphism (SSCP) and microscopy in a comparative experiment. Selected haplotypes generated from fungal 18S gene fragments were also sequenced for species identification. Microscopy-based analyses showed significantly shorter fungal hyphal lengths in the early-seral control plots in comparison with the native control plots (P 6 0.0003), independent of nitrogen addition. Although diversity indices did not show significant differences between the plots, SSCP analyses indicated that fungal community structure differed in the native and early-seral control sites. In nitrogen-amended sites, gene sequences from dominant haplotypes indicated a shift to a more common nitrogen-impacted fungal community. While nitrogen amendments appear to be more important than cultivation in influencing these soil fungal communities, hyphal lengths were only decreased due to cultivation. The use of microscopic and molecular techniques, as carried out in this study, provided integrative information concerning fungal community responses to wide spread stresses being imposed globally on terrestrial ecosystems, that is not provided by the individual techniques. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Diversity; Fungus; Microscopy ; Nitrogen; Semiarid steppe ; SSCP
1. Introduction The fungal community is an important and extremely diverse component of the soil biota, making up the largest percentage of biomass of the soil microbial community [1], with documented values ranging from 500 to 5000 wet kg ha31 [2]. This important part of the microbial community is often ignored when examining processes that are important in succession, including assessing possible impacts of successional development after disturbances such as cultivation and nitrogen accretion [3]. Understanding the possible role of fungi as a part of the soil microbial community structure in disturbed ecosystems may be very important for predicting rates of plant community succession and recovery. Currently, more than 64 000 fungal species are recognized [4], however, Cannon [5] estimated,
* Corresponding author. Tel. : +1 (970) 491-2091; Fax: +1 (970) 491-1815; E-mail: [email protected]
based on the numbers of known ascomycetes, that as many as 9.9 million fungal species exist worldwide. The fungi include the only decomposers of lignin found in nature [4], and these often neglected microbes are important in the biogeochemical cycling of nitrogen, carbon and other elements. Nitrogen availability plays a major role in the development of the microbial community structure as plant^soil systems develop in succession [6]. The role of nitrogen cycling during the recovery of disturbed ecosystems and the rate at which secondary succession occurs are of particular interest. Lands disturbed by either natural or anthropogenic processes tend to have disrupted nitrogen cycles due to the removal of plants and resulting increased mineralization [7^9], which have multiple e¡ects on fungi, including a trend towards reduced hyphal lengths [6]. The ¢lamentous fungi, in general, are particularly sensitive to disturbance and high levels of mineral nitrogen [10^12] as they extend their hyphae to exploit environments with spatially separated resources [13].
0168-6496 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 0 1 ) 0 0 1 2 1 - 0
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Fungal succession, especially in the shortgrass steppe ecosystem, has been largely ignored due to the di¤culty in characterizing the fungal communities present in these soils. Historically, many techniques have been used to measure fungal activity, diversity, and community structure [14^23] and microscopic approaches have been very useful in the assessment of the fungal hyphal structure. Klein et al. [6] used microscopy-based active and total hyphal length measurements, and conversion factors, to determine fungal biovolumes and to examine how the active fungal community responds to disturbances such as N accretion and cultivation with the recognition that the ¢lamentous fungi are non-discrete microbes [24]. These considerations have been used to develop a ratio approach to characterize fungal^bacterial community development in soils [25] to estimate total and functional biovolume changes. Such microscopy-based measurements, however, do not provide information on fungal species diversity or community structure, which can be provided by molecular techniques. Jonsson et al. [23] compared mycorrhizal fungal species that were identi¢ed by counting fruiting bodies to those that were identi¢ed using restriction fragment length polymorphism (RFLP) techniques. Five taxa identi¢ed by RFLP colonized over 60% of the mycorrhizal roots investigated, however none of these taxa were found among the fungi that formed fruiting bodies. The use of a combination of techniques [23], may make it possible to provide additional information on fungal diversity, community structure, and dynamics of fungi in disturbed soils. Although fungal succession has been extensively studied in the laboratory [26^28], few studies have been completed where soil fungal community development in the ¢eld has been followed during seral succession, and these studies have been based primarily on cultural techniques and fungal biomass estimates [29]. The objectives of this study were to use combined microscopic and 18S rDNA gene molecular markers and sequence analyses to assess fungal community structure, and to identify dominant fungal species within and between shortgrass steppe sites for the evaluation of successional nitrogen interactive e¡ects on fungal community development in the ¢eld. 2. Materials and methods 2.1. Experimental site Soil samples were taken from sites used in a larger investigation of nitrogen availability in secondary succession [3] and for studying fungal^bacterial structure in the shortgrass steppe [25]. The sites are located on and near the Central Plains Experimental Range (CPER), approximately 50 km northeast of Ft. Collins, CO, USA. One
site is a native control site (uncultivated), and the second is an early-seral site, last cultivated in 1989. The sites were divided into four blocks with each block containing two 10U10 m plots that were separated by 2 m wide bu¡er zones. One plot in each block was amended with nitrogen, with the others left as controls. This yielded a total of four replicates of each treatment at each site [6]. The fungal diversity and fungal hyphal lengths within and among the control plots and the nitrogen-amended plots were compared. 2.2. Soil sampling Within each plot, seven soil samples of approximately 20 cm in depth and 3 cm in diameter were taken during the spring of 1998, using randomly generated coordinates, and the individual samples were composited. Larger pieces of root and plant matter were removed, samples were sieved through 8-mesh sieves, and any remaining smaller plant fragments were removed. Soil samples of 45 g were shipped overnight at 5³C to Soil Foodweb, Corvallis, OR, USA, where microscopic evaluations were completed within 24 h of sampling. The remainder of the soil was immediately frozen at 380³C for use in molecular analyses. 2.3. Microscopic analysis Total fungal hyphal lengths were determined by preparing agar ¢lm soil suspensions and measuring the diameters and lengths of hyphae in the suspensions [30^32]. Means of total fungal hyphal lengths in cm per g of dry soil were calculated for each site and t-test comparisons were made using SAS for Windows version 7.0 (SAS Institute, Cary, NC, USA), to identify signi¢cant di¡erences. 2.4. Molecular analysis Community DNA was extracted from soils of the nitrogen-amended and control plots within the early-seral and the uncultivated sites. Extraction was carried out using 0.5 g soil and the BIO 101 FastDNA0 SPIN0 Kit for soil. The extracted DNA was loaded on a 1.2% agarose gel, containing 0.2% ethidium bromide, and run at 100 V for 1 h at room temperature. The DNA was viewed and photographed on an Eagle Eye1 II system (Stratagene, La Jolla, CA, USA). The remaining extract was stored at 380³C. Approximately 1839 bp of the fungal 18S rDNA gene was PCR ampli¢ed from soil community DNA using primers NS1 and NS8 [33]. Each 50 Wl reaction contained 25 Wl of 2UGC bu¡er (PanVera, Madison, WI, USA), 2.5 mM of each dNTP, 50 pmol of each primer, 100 ng of template DNA, and 2.5 U LA Taq polymerase (PanVera, Madison, WI, USA). Cycle parameters were 4 min at 94³C, load Taq at 80³C, 30 cycles of 1 min at 94³C, 30 s
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at 55³C, 1 min 30 s at 72³C, and a ¢nal elongation of 8 min at 72³C. The PCR products were puri¢ed using a QIAquick PCR puri¢cation column (Qiagen, Valencia, CA, USA), quanti¢ed by UV spectrophotometry, digested with T7 endonuclease I, and cloned according to [34]. Two hundred recombinants from each of the four sites were detected by K-complementation [35] and were screened according to [34]. The desired product was 310 bp long. 2.5. Single-strand conformation polymorphism analyses Polyacrylamide gel plates and solutions were prepared and poured according to [36]. The PCR products described above were prepared for SSCP by adding 1 Wl of product to 9 Wl of denaturing loading mixture (DLM) (0.02 mM NaOH, 0.05% xylene cyanol blue, 0.05% bromophenol blue, and 90% formamide). A total of 589 samples were prepared in 96-well plates, denatured in a PTC1001 programmable thermal controller (MJ Research, Incline Village, NY, USA), at 95³C for 5 min, and immediately plunged into ice. Each PCR product was loaded into the (12.5 ml) non-denaturing 8.1% polyacrylamide gels and run at 4³C at a constant current of 20 mAmp for approximately 5 h. The upper and lower bu¡er chambers contained 1/2UTBE (0.045 M Tris^borate, 0.001 M EDTA). Following electrophoresis, silver staining was carried out according to [36]. The SSCP gels were scored by comparing sample banding patterns. Each pattern, or haplotype, was assigned a number, beginning with 1. Unique haplotypes were numbered sequentially while identical haplotypes were assigned the same numbers. Haplotype SSCP patterns appearing similar to each other, but run on di¡erent gels, were rerun side by side on the same gel to verify similarity and pattern reproducibility. Each haplotype number was then entered into a Microsoft Excel spreadsheet, and the haplotype frequencies between and within sites were determined.
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2.7. Sequence analysis All haplotypes shared by sites, and haplotypes dominant but unique to one site, were chosen for sequencing. The fungal 1839 bp 18S insert was ampli¢ed from the 11 chosen haplotypes using primers NS1 and NS8 as described previously. The PCR product was puri¢ed using a QIAquick PCR puri¢cation column (Qiagen, Valencia, CA, USA), and quanti¢ed by UV spectrophotometry. Each product was transferred to two new 0.5 Wl microfuge tubes at a concentration of 37 ng Wl31 to yield two sets of tubes for each product. One set of tubes received 3 mM of primer NS1 and the other set received 3 mM of NS2 [33]. Product and primer were mixed by centrifugation and then dried in a speed vacuum (Savant SC 110, Holbrook, NY, USA). The NS1 and NS2 primer sets yielded a sequenced fragment of approximately 550 bp. Sequences were aligned using SeqMan1 II, version 4.03. The consensus sequences were compared to known sequences in GenBank using the BLAST program at http://www.ncbi.nlm.nih.gov/BLAST. Known sequences yielding the greatest percent similarity to submitted sequences were chosen for haplotype identi¢cation. 3. Results 3.1. Microscopic measurement of hyphal lengths Microscopic analyses of fungal hyphal lengths showed signi¢cantly shorter fungal hyphal lengths in the early-seral control site than in the native control site (P 6 0.0003). The average hyphal length in the early-seral control site was 565 cm mg dry weight soil31 , with a standard error of þ 159, while the average hyphal length in the native control site was 2136 cm mg dry weight soil31 , with a stan-
2.6. TU diversity index and analysis of variance comparisons After scoring the gels, frequency spreadsheets were used to derive an estimation of species diversity using Keefe and Bergersen's TU diversity index [37]. ( ) k X 2 TU 13 n= n31 pi 31=n ; 06TU61 i1
TU values approaching 1 represent a more diverse community. The diversity index was calculated for each treatment site, and then compared with mean hyphal length changes in each treatment site. An ANOVA test was carried out using PROC GLM in SAS for Windows version 7.0 (SAS Institute, Cary, NC, USA).
Fig. 1. Microscopic evaluation of fungal hyphal lengths from soils collected in the spring of 1998, at native and early-seral shortgrass steppe sites, with and without N amendment. Letters denote signi¢cant di¡erences seen between early-seral and native plots with and without N amendments, however, no signi¢cant e¡ect was seen between plots of the same age with the addition of N. Error bars represent one standard deviation for seven replicate determinations of fungal hyphal length.
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termine the haplotype frequency between and within sites (Fig. 3). Of these 589 samples, 312 di¡erent haplotype patterns were produced. Based on these results, each site exhibited dominant haplotypes that were unique, with few haplotypes shared between sites. In addition, many unique haplotypes existed within each site that were not considered dominant because they appeared very infrequently ( 6 5%). Haplotypes that were shared between sites were primarily shared between the early-seral N-amended and the native N-amended sites. For example, Phaeosphaeria nodorum, fungi from the order Pleosporales, and fungi from the domain Basidiomycota, were shared by and dominant in the N-amended sites, while only one control site shared a dominant haplotype with an N-amended site. All other dominant haplotypes seen in this study were only unique to one site, suggesting a shift to a more common dominant nitrogen-impacted community. Fig. 2. Polyacrylamide gel showing fungal haplotype SSCP analysis of 17 individuals from the native control plots. Lanes 1 and 18 contain a 1 kb standard ladder (Promega, Madison, WI, USA), and lanes 2^16 contain individual haplotypes generated from the 310 bp 18S native control plot DNA fragments. Lane 17 contains control DNA from a Spizzelomyces dolichospermus.
3.3. Diversity analyses Keefe and Bergersen's TU diversity index yielded similar values of mean diversity for each site, with the native control site = 0.947, the native N-amended site = 0.965, the early-seral control site = 0.981, and the early-seral Namended site = 0.972. ANOVA tests determined that there were no signi¢cant di¡erences in diversity between sites (P = 0.779, F = 0.08, n = 16), or between treatments within sites (P = 0.596, F = 0.30, n = 16). Although all of the sites were equally diverse, the community composition as described by SSCP analyses was markedly di¡erent in each site, with most of the dominant and shared haplotypes appearing in the N-amended sites.
dard error of þ 335, with n = 4 for both sites. No nitrogen e¡ect was evident (Fig. 1). 3.2. SSCP analysis A total of 589 samples generated from the 310 bp 18S gene fragment were compared using SSCP (Fig. 2) to de-
Table 1 Unique but dominant haplotype sequences from each of the native control, native N-amended, early-seral control, and early-seral N-amended sites, and shared haplotype sequences between sites, submitted to GenBank for identi¢cation Dominant but unique haplotypes found in the four study sites Native control
Native N-amended
Early-seral control
Early-seral N-amended
x x x x x x x
% Similarity 96 94 98 93 99 99 99
Organism (taxonomic level)
GenBank accession numbers
Dothideales (O) Spizellomycetaceae (F) Lophiostomataceae (F) Glomus sp. Ascomycota (D) Asconzonus sp. Pleosporales (O)
U42477/U42476 AF164251/M59759 AF164364/AF164362 Z14008 U04236/U04238 AF010590 U04236/U04238
Organism
GenBank accession numbers
P. nodorum Pleosporales (O) Basidiomycota (D) Dothideales (O)
U04236 U00975/U42481 D13460 U77668/L25429
Dominant but shared haplotypes found between sites Native control
Native N-amended
x
x x x x
Early-seral control
Early-seral N-amended x x x
% Similarity 100 99 92 99
Haplotypes showing highest homology to several di¡erent species at varied taxonomic levels were noted at the domain level (D), if all similar species were in the same domain, at the family level (F), if all similar species were in the same family, and at the order level (O), if all similar species were in the same order.
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Fig. 3. Upper panel: Haplotype frequency as determined by SSCP analysis, demonstrating the di¡erence in species composition in the native control plots as compared with the native N-amended plots. The haplotype frequency from the native control plots is represented on the positive y-axis and the frequency from the N-amended plots is represented on the negative y-axis. Lower panel: Haplotype frequency as determined by SSCP analysis, demonstrating the di¡erence in species composition in the early-seral control plots as compared with the early-seral N-amended plots. The haplotype frequency from the early-seral control plots is represented on the positive y-axis and the frequency from the early-seral N-amended plots is represented on the negative y-axis.
3.4. Sequence analysis The haplotypes that were dominant but unique to each site, and the haplotypes that were dominant but shared between sites were chosen for sequencing. BLAST searches
returned known sequences with 92^100% similarity to the sequences submitted (Table 1). Submitted sequences that matched more than one returned sequence to the same percentage of similarity were identi¢ed at the taxonomic level to which all known sequences belonged. In some
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cases, this method of identi¢cation yielded broad groups of fungi, however, this result was expected due to the use of universal primers that are designed to amplify a broad array of fungal 18S genes. In addition, the use of universal 18S primers to assess large fungal groups met the objective of this study by enabling us to examine the fungal community as a whole rather than looking at speci¢c species changes. 4. Discussion 4.1. Microscopy vs. molecular techniques These results indicate that fungal communities in a shortgrass steppe respond to disturbances of cultivation and N amendment in di¡erent ways, and that the combined use of microscopic and molecular approaches clearly provides more information concerning these fungal responses to disturbance than either technique used individually. The microscopy-based analyses revealed that the fungal hyphal lengths decreased in response to disturbance by cultivation but not in response to added nitrogen. This negative e¡ect of cultivation on hyphal lengths in soils has been observed previously [6], and may be the result of homogenized resources created by physical disturbance, which allow fungi to use nutrients in their immediate environments. In comparison, a more heterogeneous environment, typically found in late-seral or climax communities, may stimulate fungal hyphal extension to bridge the nutrient poor zones [13]. In contrast, molecular techniques revealed that cultivation and the addition of N might have stimulated shifts in fungal community structure that could not be detected using microscopy. Furthermore, SSCP and sequencing determined that these shifts resulted in a more common dominant fungal community in N-amended sites. 4.2. Sequence analysis Gene sequences identi¢ed from dominant haplotypes showed an evident shift in dominant fungal groups present in each site (Table 1). However, sequencing of dominant haplotypes revealed that those shared by more than one site were largely found in N-amended environments, indicating that added N results in a shift to a more common nitrogen-impacted community (Table 1). Although the levels to which many haplotypes were identi¢ed were rather broad, the universal 18S primers used in this study were chosen in order to amplify as much of the ¢lamentous fungal community as possible, and to examine the community as a whole rather than focusing on single species. Furthermore, shifts in large groups of dominant ¢lamentous fungi may provide an indication as to what is happening at the species level. Speci¢c genera or species with-
in these larger groups may be examined by selecting primers for organisms of interest. 4.3. Community diversity The TU diversity index indicated that only a slight increase in fungal diversity had occurred in the early-seral control plots as compared with the native control plots, while increased N resulted in a slight decrease in diversity. By comparing the hyphal length values with the TU diversity data, it appears, that while the hyphal lengths decreased in response to disturbance, the fungal diversity may have increased. In addition, based on the SSCP analyses and TU diversity data, the fungal diversity may decrease in response to increased N, however, the species diversity did not vary statistically between the di¡erent sites. Although no signi¢cant change in diversity occurred, there was an apparent shift in fungal species composition. Based on SSCP analyses, the fungal species composition di¡ered dramatically in the sites with added N in comparison with adjacent non-N-amended plots, in both earlyseral and uncultivated sites. The dominant haplotypes present among and between sites were identi¢ed to a variety of levels and showed a clear shift in dominant fungal groups. One haplotype showing 93% similarity to Glomus spp., a member of the Zygomycota, and one haplotype with high homology to the Ascomycota, were dominant only in the early-seral control plots. Many members of the Zygomycota cannot utilize cellulose as a C source, and therefore may colonize disturbed soils forming mutualistic relationships with cellulose degraders, such as members of the Ascomycota [38]. The presence of Zygomycota in the early-seral control site thus may indicate that fungal^fungal interactions are occurring in an environment high in resources with minimal fungal competition. The native control sites were dominated by fungi in the family Spizellomycetaceae, and by fungi in the order Dothideales, both of which contain parasitic and saprophytic species, and known lichen-forming fungi, capable of utilizing woody stems from plants [39]. These characteristics suggest that fungi in these groups may be highly competitive in a late-seral environment such as the native control sites, which contain more ligni¢ed organic matter in the soil and copious amounts of litter, as compared with an early-seral environment. Fungi from the family Lophiostomataceae are also capable of utilizing ligni¢ed organic matter as a substrate [39], however, they were present only in the native N-amended plots. This result suggests that members of this family are not sensitive to relatively high levels of N, perhaps enabling them to out-compete groups with the same substrate utilization capability, but di¡erent N sensitivity, explaining the increase in Lophiostomataceae and the decrease in Dothideales in the native N-amended plots. The haplotype showing 100% similarity to P. nodorum
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was present only in the native and early-seral N-amended plots, suggesting that this species may also be important in environments that are high in N. P. nodorum is the causal agent of glume blotch on wheat, and forms ascoscarps enabling this species to survive di¤cult environmental conditions. In a recent study, Halama et al. [40] showed that several wild-type isolates of P. nodorum are capable of 3 growing on media containing NO3 2 , NO3 , or NH4 . The ability to utilize several forms of N, and to develop resistant spores, may provide a competitive advantage for this species over others in high N environments and in succession. In addition to P. nodorum, fungi in the order Pleosporales and Asconzonus sp. were found only in the N-amended sites. Although the submitted sequences were similar to more than one known sequence in some cases, thereby associating our haplotypes with large groups of fungi, the goal of this study was to examine the ¢lamentous fungal community as a whole, and to provide a broad view of community changes in response to disturbances on a fairly large scale. The use of SSCP and haplotype sequencing provided information on fungal community changes that may be important throughout succession and in response to high N levels. 5. Conclusions The use of a combined microscopic and molecular approach to evaluating fungal community responses to nitrogen and successional processes indicated that major changes in physical structure and fungal community composition had occurred. The combined use of microscopic and molecular techniques provided unique integrative information, as also shown by [23], which is not provided by either technique when used individually. Microscopy provided information concerning hyphal length changes while the molecular techniques provided information on fungal community composition changes that may be important throughout succession and in response to high N levels. In addition, these results indicate that a molecular technique-based measure of fungal diversity, using SSCP combined with the TU diversity index, did not provide information on hyphal length changes that were occurring in the fungal community. By using these complementary molecular and microscopic methods, it is possible to provide a better understanding of fungal community responses to disturbances such as cultivation and N accretion in a shortgrass steppe ecosystem. These results emphasize that the combined use of molecular and microscopic techniques provides unique information not provided when either technique is used individually. This integrated approach may be critical to more fully assess possible impacts of cultivation and nitrogen accretion on fungi in soils and in other environments where ¢lamentous fungi play important but often poorly understood roles.
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Acknowledgements This research was carried out with support from the USDA-NRICGP under awards 93-37101-8601 and 9735101-4317. The assistance of M.W. Paschke and E.F. Redente with maintenance of the ¢eld sites used in these sites is deeply appreciated. References [1] Wollum, A.G. (1998) Introduction and historical perspective. In: Principles and Applications of Soil Microbiology (Sylvia, D.M., Fuhrmann, J.J., Hartel, P.G. and Zuberer, D.A., Eds.), pp. 3^20. Prentice-Hall, Englewood Cli¡s, NJ. [2] Metting, F.B. (1992) Soil Microbial Ecology. Marcel Dekker, New York. [3] Paschke, M.W., McLendon, T. and Redente, E.R. (2000) Nitrogen availability and old-¢eld succession in a shortgrass steppe. Ecosystems 3, 144^158. [4] Carlisle, M.J. (1995) The success of the hypha and mycelium. In: The Growing Fungus (Gow, N.A.R. and Gadd, G.M., Eds.), pp. 1^18. ChapmanpHall, London. [5] Cannon, P. (1997) Diversity of the Phyllachoraceae with special reference to the tropics. In: The Biodiversity of Microorganisms and Invertebrates: Its Role in Sustainable Agriculture (Hyde, K.D., Ed.), pp. 255^278. Hong Kong University Press, Hong Kong. [6] Klein, D.A., McLendon, T., Paschke, M.W. and Redente, E.F. (1996) Nitrogen availability and fungal-bacterial responses in successional semiarid steppe soils. Arid Soil Res. Rehabil. 10, 321^332. [7] Marrs, R.H., Roberts, R.D., She¤ngton, R.A. and Bradshaw, A.D. (1983) Nitrogen and the Development of Ecosystems in Nitrogen as an Ecological Factor. Blackwell Scienti¢c Publications, Boston, MA. [8] Carpenter, A.T. and Allen, M.F. (1988) Responses of Hydysarum boreale Nut. to mycorrhizas and rhizobium : plant and soil nutrient changes in a disturbed shrub-steppe. New Phytol. 109, 125^132. [9] George, T.J.K., Ladha, R.J.B. and Garrity, D.P. (1993) Nitrate dynamics during aerobic soil phase in lowland rice-based cropping systems. Soil Sci. Soc. Am. J. 57, 1526^1532. [10] So«derstro«m, B., Ba®a®th, E. and Lundgren, B. (1983) Decrease in soil microbial activity and biomasses owing to nitrogen amendments. Can. J. Microbiol. 29, 1500^1506. [11] Klein, D.A., Frederick, B.A. and Redente, E.F. (1989) Fertilizer effects on microbial communities and organic matter in the rhizosphere of Sitanion hystrix and Agropyron smithii. Arid Soil Res. Rehabil. 3, 397^404. [12] Arnolds, E. (1991) Decline of Ectomycorrhizal fungi in Europe. Agricult. Ecosyst. Environ. 35, 209^244. [13] Holland, E.A. and Coleman, D.C. (1987) Litter placement e¡ects on microbial and organic matter dynamics in an agroecosystem. Ecology 68, 425^433. [14] Bligh, E.G. and Dyer, W.J. (1959) A rapid method of total lipid extraction and puri¢cation. Can. Biochem. Physiol. 37, 911^917. [15] Trappe, J.M. (1962) Fungus associates of ectotrophic mycorrhizae. Bot. Rev. 28, 538^606. [16] Witkamp, M. and van der Drift, J. (1961) Breakdown of forest litter in relation to environmental factors. Plant Soil 15, 295^311. [17] White, D.C., Davis, W.M., Nickels, J.S., King, J.C. and Bobbie, R.J. (1979) Determination of the sedimentary microbial biomass by extractable lipid phosphate. Oecologia 40, 51^62. [18] Kreisel, H. and Schauer, F. (1983) Methoden des Mykologischen Laboratoriums. Gustav Fischer, Stuttgart. [19] Kaczmarek, W. (1984) A comparison of bacterial and fungal biomass in several early-seral soils. Acta Microbiol. Pol. 33, 239^247.
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[20] Vance, E.D., Brooks, P.C. and Jenkinson, D.S. (1987) Microbial biomass measurements in forest soils: the use of the chloroform fumigation method in strongly acid soils. Soil Biol. Biochem. 19, 697^702. [21] Sarquis, M.I.D. and De Oliveira, P.C. (1996) Diversity of microfungi in the sandy soil of Ipanema Beach, Rio de Janeiro, Brazil. J. Basic Microbiol. 36, 51^58. [22] Frankland, J.C. (1998) Fungal Succession - unraveling the unpredictable. Mycol. Res. 102, 1^15. [23] Jonsson, T., Kokalj, S., Finlay, R. and Erland, S. (1999) Ectomycorrhizal community structure in a limed spruce forest. Mycol. Res. 103, 501^508. [24] Rayner, A.D.M., Beeching, J.R., Crowe, J.D. and Watkins, Z.R. (1999) De¢ning individual fungal boundaries. In: Structure and Dynamics of Fungal Populations (Worrall, J.J., Ed.), pp. 19^42. Kluwer Academic Publishers, Dordrecht. [25] Klein, D.A. and Paschke, M.W. (2000) A soil microbial community structural index: the microscopy-based total/active active fungal/bacterial biovolumes ratio. Appl. Soil Ecol. 14, 257^268. [26] Park, D. (1968) The ecology of terrestrial fungi. In: The Fungi (Ainsworth, G.C. and Sussman, A.S., Eds.), pp. 5^39. Academic Press, London. [27] Frankland, J.C. (1981) Mechanisms in fungal successions. In: The Fungal Community : Its Organization and Role in the Ecosystem (Wicklow, D.T. and Carroll, G.C., Eds.), pp. 403^426. Marcel Dekker, New York. [28] Sampo¨, S., Bergero, R., Bu¡a, G. and Luppi-Mosca, A.M. (1997) Soil fungal communities in a young and an old Alnus viridis coenosis. Mycologia 89, 837^845. [29] KjÖller, A. and Struwe, S. (1982) Microfungi in ecosystems - fungal occurrence and activity in litter and soil. Oikos 39, 391^422. [30] Ingham, E.R. and Klein, D.A. (1984) Soil fungi: measurement of hyphal length. Soil Biol. Biochem. 16, 279^280.
[31] Lodge, D.J. and Ingham, E.R. (1991) A comparison of agar ¢lm techniques for estimating fungal biovolumes in litter and soil. Agricult. Ecosyst. Environ. 34, 131^144. [32] Stamatiadis, S., Doran, J.W. and Ingham, E.R. (1990) Use of staining and inhibitors to separate fungal and bacterial activity in soil. Soil Biol. Biochem. 22, 81^88. [33] White, T.J., Bruns, T., Lee, S. and Taylor, J. (1990) Ampli¢cation and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols : A Guide to Methods and Applications (Innis, M.A., Gelfand, D.H., Sninsky, J.J. and White, T.J., Eds.), pp. 315^322. Academic Press, San Diego, CA. [34] Lowell, J.L. and Klein, D.A. (2000) Heteroduplex resolution using T7 endonuclease I in microbial community analyses. BioTechniques 28, 676^681. [35] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [36] Black, W.C. and Duteau, N.M. (1997) RAPD-PCR and SSCP analysis for insect population genetic studies. In: The Molecular Biology of Insect Disease Vectors. A Methods Manual (Crampton, J.M., Beard, C.B. and Louis, C., Eds.), pp. 361^371. ChapmanpHall, New York. [37] Keefe, T.J. and Bergersen, E.P. (1977) A simple diversity index based on the theory of runs. Water Res. 11, 689^691. [38] Dix, N.J. and Webster, J. (1995) Structure of fungal communities. In: Fungal Ecology, (Dix, N.J. and Webster, J., Eds.) pp. 39^84. ChapmanpHall, London. [39] Hawksworth, D.L. (1991) The fungal dimension of biodiversity magnitude, signi¢cance, and conservation. Mycol. Res. 95, 641^655. [40] Halama, P., Skajenniko¡, M. and Dehorter, B. (1999) Tetrad analysis of mating type, mutations, esterase and aggressiveness in Phaeosphaeria nodorum. Mycol. Res. 103, 43^49.
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Comparative single-strand conformation polymorphism (SSCP) and microscopy-based analysis of nitrogen cultivation interactive e¡ects on the fungal community of a semiarid steppe soil Jennifer L. Lowell *, Donald A. Klein Department of Microbiology, Colorado State University, Fort Collins, CO 80523-1677, USA Received 13 October 2000; received in revised form 2 April 2001; accepted 2 April 2001 First published online 27 April 2001
Abstract The effects of nitrogen accretion on fungal diversity and community structure in early-seral (cultivated) and native (uncultivated) shortgrass steppe soils were evaluated using single-strand conformation polymorphism (SSCP) and microscopy in a comparative experiment. Selected haplotypes generated from fungal 18S gene fragments were also sequenced for species identification. Microscopy-based analyses showed significantly shorter fungal hyphal lengths in the early-seral control plots in comparison with the native control plots (P 6 0.0003), independent of nitrogen addition. Although diversity indices did not show significant differences between the plots, SSCP analyses indicated that fungal community structure differed in the native and early-seral control sites. In nitrogen-amended sites, gene sequences from dominant haplotypes indicated a shift to a more common nitrogen-impacted fungal community. While nitrogen amendments appear to be more important than cultivation in influencing these soil fungal communities, hyphal lengths were only decreased due to cultivation. The use of microscopic and molecular techniques, as carried out in this study, provided integrative information concerning fungal community responses to wide spread stresses being imposed globally on terrestrial ecosystems, that is not provided by the individual techniques. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Diversity; Fungus; Microscopy ; Nitrogen; Semiarid steppe ; SSCP
1. Introduction The fungal community is an important and extremely diverse component of the soil biota, making up the largest percentage of biomass of the soil microbial community [1], with documented values ranging from 500 to 5000 wet kg ha31 [2]. This important part of the microbial community is often ignored when examining processes that are important in succession, including assessing possible impacts of successional development after disturbances such as cultivation and nitrogen accretion [3]. Understanding the possible role of fungi as a part of the soil microbial community structure in disturbed ecosystems may be very important for predicting rates of plant community succession and recovery. Currently, more than 64 000 fungal species are recognized [4], however, Cannon [5] estimated,
* Corresponding author. Tel. : +1 (970) 491-2091; Fax: +1 (970) 491-1815; E-mail: [email protected]
based on the numbers of known ascomycetes, that as many as 9.9 million fungal species exist worldwide. The fungi include the only decomposers of lignin found in nature [4], and these often neglected microbes are important in the biogeochemical cycling of nitrogen, carbon and other elements. Nitrogen availability plays a major role in the development of the microbial community structure as plant^soil systems develop in succession [6]. The role of nitrogen cycling during the recovery of disturbed ecosystems and the rate at which secondary succession occurs are of particular interest. Lands disturbed by either natural or anthropogenic processes tend to have disrupted nitrogen cycles due to the removal of plants and resulting increased mineralization [7^9], which have multiple e¡ects on fungi, including a trend towards reduced hyphal lengths [6]. The ¢lamentous fungi, in general, are particularly sensitive to disturbance and high levels of mineral nitrogen [10^12] as they extend their hyphae to exploit environments with spatially separated resources [13].
0168-6496 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 6 4 9 6 ( 0 1 ) 0 0 1 2 1 - 0
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Fungal succession, especially in the shortgrass steppe ecosystem, has been largely ignored due to the di¤culty in characterizing the fungal communities present in these soils. Historically, many techniques have been used to measure fungal activity, diversity, and community structure [14^23] and microscopic approaches have been very useful in the assessment of the fungal hyphal structure. Klein et al. [6] used microscopy-based active and total hyphal length measurements, and conversion factors, to determine fungal biovolumes and to examine how the active fungal community responds to disturbances such as N accretion and cultivation with the recognition that the ¢lamentous fungi are non-discrete microbes [24]. These considerations have been used to develop a ratio approach to characterize fungal^bacterial community development in soils [25] to estimate total and functional biovolume changes. Such microscopy-based measurements, however, do not provide information on fungal species diversity or community structure, which can be provided by molecular techniques. Jonsson et al. [23] compared mycorrhizal fungal species that were identi¢ed by counting fruiting bodies to those that were identi¢ed using restriction fragment length polymorphism (RFLP) techniques. Five taxa identi¢ed by RFLP colonized over 60% of the mycorrhizal roots investigated, however none of these taxa were found among the fungi that formed fruiting bodies. The use of a combination of techniques [23], may make it possible to provide additional information on fungal diversity, community structure, and dynamics of fungi in disturbed soils. Although fungal succession has been extensively studied in the laboratory [26^28], few studies have been completed where soil fungal community development in the ¢eld has been followed during seral succession, and these studies have been based primarily on cultural techniques and fungal biomass estimates [29]. The objectives of this study were to use combined microscopic and 18S rDNA gene molecular markers and sequence analyses to assess fungal community structure, and to identify dominant fungal species within and between shortgrass steppe sites for the evaluation of successional nitrogen interactive e¡ects on fungal community development in the ¢eld. 2. Materials and methods 2.1. Experimental site Soil samples were taken from sites used in a larger investigation of nitrogen availability in secondary succession [3] and for studying fungal^bacterial structure in the shortgrass steppe [25]. The sites are located on and near the Central Plains Experimental Range (CPER), approximately 50 km northeast of Ft. Collins, CO, USA. One
site is a native control site (uncultivated), and the second is an early-seral site, last cultivated in 1989. The sites were divided into four blocks with each block containing two 10U10 m plots that were separated by 2 m wide bu¡er zones. One plot in each block was amended with nitrogen, with the others left as controls. This yielded a total of four replicates of each treatment at each site [6]. The fungal diversity and fungal hyphal lengths within and among the control plots and the nitrogen-amended plots were compared. 2.2. Soil sampling Within each plot, seven soil samples of approximately 20 cm in depth and 3 cm in diameter were taken during the spring of 1998, using randomly generated coordinates, and the individual samples were composited. Larger pieces of root and plant matter were removed, samples were sieved through 8-mesh sieves, and any remaining smaller plant fragments were removed. Soil samples of 45 g were shipped overnight at 5³C to Soil Foodweb, Corvallis, OR, USA, where microscopic evaluations were completed within 24 h of sampling. The remainder of the soil was immediately frozen at 380³C for use in molecular analyses. 2.3. Microscopic analysis Total fungal hyphal lengths were determined by preparing agar ¢lm soil suspensions and measuring the diameters and lengths of hyphae in the suspensions [30^32]. Means of total fungal hyphal lengths in cm per g of dry soil were calculated for each site and t-test comparisons were made using SAS for Windows version 7.0 (SAS Institute, Cary, NC, USA), to identify signi¢cant di¡erences. 2.4. Molecular analysis Community DNA was extracted from soils of the nitrogen-amended and control plots within the early-seral and the uncultivated sites. Extraction was carried out using 0.5 g soil and the BIO 101 FastDNA0 SPIN0 Kit for soil. The extracted DNA was loaded on a 1.2% agarose gel, containing 0.2% ethidium bromide, and run at 100 V for 1 h at room temperature. The DNA was viewed and photographed on an Eagle Eye1 II system (Stratagene, La Jolla, CA, USA). The remaining extract was stored at 380³C. Approximately 1839 bp of the fungal 18S rDNA gene was PCR ampli¢ed from soil community DNA using primers NS1 and NS8 [33]. Each 50 Wl reaction contained 25 Wl of 2UGC bu¡er (PanVera, Madison, WI, USA), 2.5 mM of each dNTP, 50 pmol of each primer, 100 ng of template DNA, and 2.5 U LA Taq polymerase (PanVera, Madison, WI, USA). Cycle parameters were 4 min at 94³C, load Taq at 80³C, 30 cycles of 1 min at 94³C, 30 s
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at 55³C, 1 min 30 s at 72³C, and a ¢nal elongation of 8 min at 72³C. The PCR products were puri¢ed using a QIAquick PCR puri¢cation column (Qiagen, Valencia, CA, USA), quanti¢ed by UV spectrophotometry, digested with T7 endonuclease I, and cloned according to [34]. Two hundred recombinants from each of the four sites were detected by K-complementation [35] and were screened according to [34]. The desired product was 310 bp long. 2.5. Single-strand conformation polymorphism analyses Polyacrylamide gel plates and solutions were prepared and poured according to [36]. The PCR products described above were prepared for SSCP by adding 1 Wl of product to 9 Wl of denaturing loading mixture (DLM) (0.02 mM NaOH, 0.05% xylene cyanol blue, 0.05% bromophenol blue, and 90% formamide). A total of 589 samples were prepared in 96-well plates, denatured in a PTC1001 programmable thermal controller (MJ Research, Incline Village, NY, USA), at 95³C for 5 min, and immediately plunged into ice. Each PCR product was loaded into the (12.5 ml) non-denaturing 8.1% polyacrylamide gels and run at 4³C at a constant current of 20 mAmp for approximately 5 h. The upper and lower bu¡er chambers contained 1/2UTBE (0.045 M Tris^borate, 0.001 M EDTA). Following electrophoresis, silver staining was carried out according to [36]. The SSCP gels were scored by comparing sample banding patterns. Each pattern, or haplotype, was assigned a number, beginning with 1. Unique haplotypes were numbered sequentially while identical haplotypes were assigned the same numbers. Haplotype SSCP patterns appearing similar to each other, but run on di¡erent gels, were rerun side by side on the same gel to verify similarity and pattern reproducibility. Each haplotype number was then entered into a Microsoft Excel spreadsheet, and the haplotype frequencies between and within sites were determined.
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2.7. Sequence analysis All haplotypes shared by sites, and haplotypes dominant but unique to one site, were chosen for sequencing. The fungal 1839 bp 18S insert was ampli¢ed from the 11 chosen haplotypes using primers NS1 and NS8 as described previously. The PCR product was puri¢ed using a QIAquick PCR puri¢cation column (Qiagen, Valencia, CA, USA), and quanti¢ed by UV spectrophotometry. Each product was transferred to two new 0.5 Wl microfuge tubes at a concentration of 37 ng Wl31 to yield two sets of tubes for each product. One set of tubes received 3 mM of primer NS1 and the other set received 3 mM of NS2 [33]. Product and primer were mixed by centrifugation and then dried in a speed vacuum (Savant SC 110, Holbrook, NY, USA). The NS1 and NS2 primer sets yielded a sequenced fragment of approximately 550 bp. Sequences were aligned using SeqMan1 II, version 4.03. The consensus sequences were compared to known sequences in GenBank using the BLAST program at http://www.ncbi.nlm.nih.gov/BLAST. Known sequences yielding the greatest percent similarity to submitted sequences were chosen for haplotype identi¢cation. 3. Results 3.1. Microscopic measurement of hyphal lengths Microscopic analyses of fungal hyphal lengths showed signi¢cantly shorter fungal hyphal lengths in the early-seral control site than in the native control site (P 6 0.0003). The average hyphal length in the early-seral control site was 565 cm mg dry weight soil31 , with a standard error of þ 159, while the average hyphal length in the native control site was 2136 cm mg dry weight soil31 , with a stan-
2.6. TU diversity index and analysis of variance comparisons After scoring the gels, frequency spreadsheets were used to derive an estimation of species diversity using Keefe and Bergersen's TU diversity index [37]. ( ) k X 2 TU 13 n= n31 pi 31=n ; 06TU61 i1
TU values approaching 1 represent a more diverse community. The diversity index was calculated for each treatment site, and then compared with mean hyphal length changes in each treatment site. An ANOVA test was carried out using PROC GLM in SAS for Windows version 7.0 (SAS Institute, Cary, NC, USA).
Fig. 1. Microscopic evaluation of fungal hyphal lengths from soils collected in the spring of 1998, at native and early-seral shortgrass steppe sites, with and without N amendment. Letters denote signi¢cant di¡erences seen between early-seral and native plots with and without N amendments, however, no signi¢cant e¡ect was seen between plots of the same age with the addition of N. Error bars represent one standard deviation for seven replicate determinations of fungal hyphal length.
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termine the haplotype frequency between and within sites (Fig. 3). Of these 589 samples, 312 di¡erent haplotype patterns were produced. Based on these results, each site exhibited dominant haplotypes that were unique, with few haplotypes shared between sites. In addition, many unique haplotypes existed within each site that were not considered dominant because they appeared very infrequently ( 6 5%). Haplotypes that were shared between sites were primarily shared between the early-seral N-amended and the native N-amended sites. For example, Phaeosphaeria nodorum, fungi from the order Pleosporales, and fungi from the domain Basidiomycota, were shared by and dominant in the N-amended sites, while only one control site shared a dominant haplotype with an N-amended site. All other dominant haplotypes seen in this study were only unique to one site, suggesting a shift to a more common dominant nitrogen-impacted community. Fig. 2. Polyacrylamide gel showing fungal haplotype SSCP analysis of 17 individuals from the native control plots. Lanes 1 and 18 contain a 1 kb standard ladder (Promega, Madison, WI, USA), and lanes 2^16 contain individual haplotypes generated from the 310 bp 18S native control plot DNA fragments. Lane 17 contains control DNA from a Spizzelomyces dolichospermus.
3.3. Diversity analyses Keefe and Bergersen's TU diversity index yielded similar values of mean diversity for each site, with the native control site = 0.947, the native N-amended site = 0.965, the early-seral control site = 0.981, and the early-seral Namended site = 0.972. ANOVA tests determined that there were no signi¢cant di¡erences in diversity between sites (P = 0.779, F = 0.08, n = 16), or between treatments within sites (P = 0.596, F = 0.30, n = 16). Although all of the sites were equally diverse, the community composition as described by SSCP analyses was markedly di¡erent in each site, with most of the dominant and shared haplotypes appearing in the N-amended sites.
dard error of þ 335, with n = 4 for both sites. No nitrogen e¡ect was evident (Fig. 1). 3.2. SSCP analysis A total of 589 samples generated from the 310 bp 18S gene fragment were compared using SSCP (Fig. 2) to de-
Table 1 Unique but dominant haplotype sequences from each of the native control, native N-amended, early-seral control, and early-seral N-amended sites, and shared haplotype sequences between sites, submitted to GenBank for identi¢cation Dominant but unique haplotypes found in the four study sites Native control
Native N-amended
Early-seral control
Early-seral N-amended
x x x x x x x
% Similarity 96 94 98 93 99 99 99
Organism (taxonomic level)
GenBank accession numbers
Dothideales (O) Spizellomycetaceae (F) Lophiostomataceae (F) Glomus sp. Ascomycota (D) Asconzonus sp. Pleosporales (O)
U42477/U42476 AF164251/M59759 AF164364/AF164362 Z14008 U04236/U04238 AF010590 U04236/U04238
Organism
GenBank accession numbers
P. nodorum Pleosporales (O) Basidiomycota (D) Dothideales (O)
U04236 U00975/U42481 D13460 U77668/L25429
Dominant but shared haplotypes found between sites Native control
Native N-amended
x
x x x x
Early-seral control
Early-seral N-amended x x x
% Similarity 100 99 92 99
Haplotypes showing highest homology to several di¡erent species at varied taxonomic levels were noted at the domain level (D), if all similar species were in the same domain, at the family level (F), if all similar species were in the same family, and at the order level (O), if all similar species were in the same order.
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Fig. 3. Upper panel: Haplotype frequency as determined by SSCP analysis, demonstrating the di¡erence in species composition in the native control plots as compared with the native N-amended plots. The haplotype frequency from the native control plots is represented on the positive y-axis and the frequency from the N-amended plots is represented on the negative y-axis. Lower panel: Haplotype frequency as determined by SSCP analysis, demonstrating the di¡erence in species composition in the early-seral control plots as compared with the early-seral N-amended plots. The haplotype frequency from the early-seral control plots is represented on the positive y-axis and the frequency from the early-seral N-amended plots is represented on the negative y-axis.
3.4. Sequence analysis The haplotypes that were dominant but unique to each site, and the haplotypes that were dominant but shared between sites were chosen for sequencing. BLAST searches
returned known sequences with 92^100% similarity to the sequences submitted (Table 1). Submitted sequences that matched more than one returned sequence to the same percentage of similarity were identi¢ed at the taxonomic level to which all known sequences belonged. In some
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cases, this method of identi¢cation yielded broad groups of fungi, however, this result was expected due to the use of universal primers that are designed to amplify a broad array of fungal 18S genes. In addition, the use of universal 18S primers to assess large fungal groups met the objective of this study by enabling us to examine the fungal community as a whole rather than looking at speci¢c species changes. 4. Discussion 4.1. Microscopy vs. molecular techniques These results indicate that fungal communities in a shortgrass steppe respond to disturbances of cultivation and N amendment in di¡erent ways, and that the combined use of microscopic and molecular approaches clearly provides more information concerning these fungal responses to disturbance than either technique used individually. The microscopy-based analyses revealed that the fungal hyphal lengths decreased in response to disturbance by cultivation but not in response to added nitrogen. This negative e¡ect of cultivation on hyphal lengths in soils has been observed previously [6], and may be the result of homogenized resources created by physical disturbance, which allow fungi to use nutrients in their immediate environments. In comparison, a more heterogeneous environment, typically found in late-seral or climax communities, may stimulate fungal hyphal extension to bridge the nutrient poor zones [13]. In contrast, molecular techniques revealed that cultivation and the addition of N might have stimulated shifts in fungal community structure that could not be detected using microscopy. Furthermore, SSCP and sequencing determined that these shifts resulted in a more common dominant fungal community in N-amended sites. 4.2. Sequence analysis Gene sequences identi¢ed from dominant haplotypes showed an evident shift in dominant fungal groups present in each site (Table 1). However, sequencing of dominant haplotypes revealed that those shared by more than one site were largely found in N-amended environments, indicating that added N results in a shift to a more common nitrogen-impacted community (Table 1). Although the levels to which many haplotypes were identi¢ed were rather broad, the universal 18S primers used in this study were chosen in order to amplify as much of the ¢lamentous fungal community as possible, and to examine the community as a whole rather than focusing on single species. Furthermore, shifts in large groups of dominant ¢lamentous fungi may provide an indication as to what is happening at the species level. Speci¢c genera or species with-
in these larger groups may be examined by selecting primers for organisms of interest. 4.3. Community diversity The TU diversity index indicated that only a slight increase in fungal diversity had occurred in the early-seral control plots as compared with the native control plots, while increased N resulted in a slight decrease in diversity. By comparing the hyphal length values with the TU diversity data, it appears, that while the hyphal lengths decreased in response to disturbance, the fungal diversity may have increased. In addition, based on the SSCP analyses and TU diversity data, the fungal diversity may decrease in response to increased N, however, the species diversity did not vary statistically between the di¡erent sites. Although no signi¢cant change in diversity occurred, there was an apparent shift in fungal species composition. Based on SSCP analyses, the fungal species composition di¡ered dramatically in the sites with added N in comparison with adjacent non-N-amended plots, in both earlyseral and uncultivated sites. The dominant haplotypes present among and between sites were identi¢ed to a variety of levels and showed a clear shift in dominant fungal groups. One haplotype showing 93% similarity to Glomus spp., a member of the Zygomycota, and one haplotype with high homology to the Ascomycota, were dominant only in the early-seral control plots. Many members of the Zygomycota cannot utilize cellulose as a C source, and therefore may colonize disturbed soils forming mutualistic relationships with cellulose degraders, such as members of the Ascomycota [38]. The presence of Zygomycota in the early-seral control site thus may indicate that fungal^fungal interactions are occurring in an environment high in resources with minimal fungal competition. The native control sites were dominated by fungi in the family Spizellomycetaceae, and by fungi in the order Dothideales, both of which contain parasitic and saprophytic species, and known lichen-forming fungi, capable of utilizing woody stems from plants [39]. These characteristics suggest that fungi in these groups may be highly competitive in a late-seral environment such as the native control sites, which contain more ligni¢ed organic matter in the soil and copious amounts of litter, as compared with an early-seral environment. Fungi from the family Lophiostomataceae are also capable of utilizing ligni¢ed organic matter as a substrate [39], however, they were present only in the native N-amended plots. This result suggests that members of this family are not sensitive to relatively high levels of N, perhaps enabling them to out-compete groups with the same substrate utilization capability, but di¡erent N sensitivity, explaining the increase in Lophiostomataceae and the decrease in Dothideales in the native N-amended plots. The haplotype showing 100% similarity to P. nodorum
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was present only in the native and early-seral N-amended plots, suggesting that this species may also be important in environments that are high in N. P. nodorum is the causal agent of glume blotch on wheat, and forms ascoscarps enabling this species to survive di¤cult environmental conditions. In a recent study, Halama et al. [40] showed that several wild-type isolates of P. nodorum are capable of 3 growing on media containing NO3 2 , NO3 , or NH4 . The ability to utilize several forms of N, and to develop resistant spores, may provide a competitive advantage for this species over others in high N environments and in succession. In addition to P. nodorum, fungi in the order Pleosporales and Asconzonus sp. were found only in the N-amended sites. Although the submitted sequences were similar to more than one known sequence in some cases, thereby associating our haplotypes with large groups of fungi, the goal of this study was to examine the ¢lamentous fungal community as a whole, and to provide a broad view of community changes in response to disturbances on a fairly large scale. The use of SSCP and haplotype sequencing provided information on fungal community changes that may be important throughout succession and in response to high N levels. 5. Conclusions The use of a combined microscopic and molecular approach to evaluating fungal community responses to nitrogen and successional processes indicated that major changes in physical structure and fungal community composition had occurred. The combined use of microscopic and molecular techniques provided unique integrative information, as also shown by [23], which is not provided by either technique when used individually. Microscopy provided information concerning hyphal length changes while the molecular techniques provided information on fungal community composition changes that may be important throughout succession and in response to high N levels. In addition, these results indicate that a molecular technique-based measure of fungal diversity, using SSCP combined with the TU diversity index, did not provide information on hyphal length changes that were occurring in the fungal community. By using these complementary molecular and microscopic methods, it is possible to provide a better understanding of fungal community responses to disturbances such as cultivation and N accretion in a shortgrass steppe ecosystem. These results emphasize that the combined use of molecular and microscopic techniques provides unique information not provided when either technique is used individually. This integrated approach may be critical to more fully assess possible impacts of cultivation and nitrogen accretion on fungi in soils and in other environments where ¢lamentous fungi play important but often poorly understood roles.
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