Microb Ecol DOI 10.1007/s00248-015-0569-8
ENVIRONMENTAL MICROBIOLOGY
Resource Type and Availability Regulate Fungal Communities Along Arable Soil Profiles Julia Moll & Kezia Goldmann & Susanne Kramer & Stefan Hempel & Ellen Kandeler & Sven Marhan & Liliane Ruess & Dirk Krüger & Francois Buscot
Received: 14 April 2014 / Accepted: 13 January 2015 # Springer Science+Business Media New York 2015
Abstract Soil fungi play an essential role in the decomposition of plant-derived organic material entering soils. The quality and quantity of organic compounds vary seasonally as well as with soil depth. To elucidate how these resources affect fungal communities in an arable soil, a field experiment was set up with two plant species, maize and wheat. Resource availability was experimentally manipulated by maize litter
Electronic supplementary material The online version of this article (doi:10.1007/s00248-015-0569-8) contains supplementary material, which is available to authorized users. J. Moll (*) : K. Goldmann : D. Krüger : F. Buscot Department of Soil Ecology, UFZ – Helmholtz Centre for Environmental Research, Theodor-Lieser-Str. 4, 06120 Halle, Germany e-mail: [email protected] J. Moll : K. Goldmann : F. Buscot Institute of Biology, University of Leipzig, Johannisallee 21-23, 04103 Leipzig, Germany S. Kramer : E. Kandeler : S. Marhan Institute of Soil Science and Land Evaluation, Soil Biology Section, University of Hohenheim, 70593 Stuttgart, Germany S. Hempel Institute of Biology, Dahlem Center of Plant Sciences, Freie Universität Berlin, Altensteinstr. 6, 14195 Berlin, Germany
input on one half of these maize and wheat plots after harvest in autumn. Fungal biomass was determined by ergosterol quantification, and community structure was investigated by fungal automated ribosomal intergenic spacer analysis (FARISA). An annual cycle was assessed across a depth gradient, distinguishing three soil habitats: the plough layer, rooted soil below the plough layer, and deeper root-free soil. Fungal communities appeared highly dynamic and varied according to soil depth and plant resources. In the plough layer, the availability of litter played a dominant role in shaping fungal communities, whereas in the rooted layer below, community structure and biomass mainly differed between plant species. This plant effect was also extended into the root-free soil at a depth of 70 cm. In winter, the availability of litter also affected fungal communities in deeper soil layers, suggesting vertical transport processes under fallow conditions. These distinct resource effects indicate diverse ecological niches along the soil profile, comprising specific fungal metacommunities. The recorded responses to both living plants and litter point to a central role of fungi in connecting primary production and decomposition within the plant-soil system. Keywords Plant resources . Litter . Fungal community structure . Fungal biomass . Soil depth . Arable soil . Season . ARISA
S. Hempel Berlin-Brandenburg Institute of Advanced Biodiversity Research (BBIB), 14195 Berlin, Germany L. Ruess Institute of Biology, Ecology Group, Humboldt-Universität zu Berlin, Philippstr. 13, 10115 Berlin, Germany F. Buscot German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Deutscher Platz 5e, 04103 Leipzig, Germany
Introduction Due to their quantitatively dominant part of the microbial biomass, their diversity, and numerous life strategies, fungi are major drivers of key biogeochemical and ecological processes [1–3]. They rely on several main resources for their
J. Moll et al.
energy supply: litter entering the soil, plant roots and their exudates, as well as soil organic matter [4–6]. Hence, the major belowground fungal resources vary tremendously in chemical composition and physical structure [4, 7]. Moreover, these substrates are not homogeneously distributed in soils. While litter input from aboveground plant material dominates in upper soil horizons, soil in the rooting zone is characterized by root exudates and their residues. Below that, in the bulk soil, organic matter is probably the dominant nutrient source. As a consequence, the quantity of resources, e.g., carbon or nitrogen content, decreases with increasing depth [8, 9]. This general distribution of fungal resources along the soil profile is shaped spatially and temporally by two major processes: transport and seasonality. Organic substrates can be mobilized and translocated from the soil surface down to the subsoil predominantly by drying–wetting or freeze-thaw cycles after seepage events associated with heavy rain and snowmelt [10–13]. Thus, the highest translocation takes place in late autumn, winter, and spring. Besides this spatial variability in resources, arable systems show a distinct pattern in primary production and decomposition cycles related to seasons [14–16]. In particular, in temperate soils with annual plant cropping, root exudation is high in spring and summer, low in autumn, and lacking in winter after plant harvest [17]. In contrast, litter deposition is almost absent in the vegetation growing season, but high litter input occurs after harvest in autumn and winter when crop residues are returned to the field. These spatial- and temporal-based differences in input and accessibility of substrates strongly affect fungal diversity, biomass, and distribution in soil [18–22]. Different species exhibit a preference or specificity for distinct resource types, such as root exudates, related to single plant species or litter with a distinct composition [23, 24]. As fungal activity has a strong impact on soil nutrient cycling in arable soils [25], more knowledge about fungal community dynamics in relation to resource properties in this ecosystem is needed. Previous studies mainly focused on specific fungal groups such as arbuscular mycorrhizal fungi due to their importance as plant symbionts [26, 27]. Other studies investigated fungal communities in upper soil horizons, related to one plant type or on one sampling date [28–31]. A comprehensive assessment of fungal community dynamics in relation to resource availabilities considering seasonality and soil depth is still missing. The present study was performed as a part of a field experiment which was established in order to disentangle the respective impacts of litter input, root exudation, and bulk soil organic matter on microbial and faunal communities in an arable soil [32]. The experiment used two plant species, maize and wheat, which are characterized by different root biomass, distribution, and exudation [32, 33], thus enabling us to
distinguish plant-specific effects. Type and availability of plant resources were manipulated by the following treatments: fodder maize (plant removal at harvest), corn maize (litter amendment at harvest), wheat, and wheat plus maize litter. The plots were sampled at three soil depths: the plough layer, rooted soil below the plough layer, and deeper root-free soil. Sampling was undertaken at three resource phases: (1) during vegetation growing season - to examine root exudation; (2) shortly before harvest - to examine residue input from senescent plants; and (3) under fallow - to examine litter input and translocation processes. Fungal biomass was determined by ergosterol quantification and community structure by fungal automated ribosomal intergenic spacer analysis (F-ARISA) [34, 35]. F-ARISA is a frequently used, low-cost, high throughput DNA fingerprinting technique used to describe fungal communities in relation to environmental conditions [36–38]. It allows an assessment of the structure of fungal communities in large ecological studies such as in this comprehensive study in an arable system. Our objective for this study was to determine fungal biomass and community structure in relation to plant species, litter input, and soil depth over a time series in consecutive seasons. We hypothesized that (1) fungal communities in the topsoil (plough layer) are mostly affected by litter input, (2) while in the rooting zone, they are mainly shaped by root exudates which are related to plant species, and (3) the root-free soil layer below exhibits distinct fungal communities without resource effects.
Material and Methods Experimental Design and Sampling Campaign In April 2009, the experimental site was established in an arable field located at Göttingen-Holtensen (51° 33′ N, 9° 53′ E; 158 m NN, Lower Saxony, Germany). Detailed information about soil properties previously published in Kramer et al. [32] and Pausch and Kuzyakov [33] is given in Supplemental Table S1. Because of long-term agricultural management, two plough layers at 20 and 30 cm depth and severe soil compaction below the second layer were discovered at the field site. The A horizon reached an average depth of 34± 6.4 cm. More details about the study site, e.g., agricultural management (tillage and fertilizer practices), are given in Kramer et al. [32] and Scharroba et al. [39]. The field was cropped with maize (Zea mays L.) and wheat (Triticum aestivum L.) in a design with one strip of maize and one strip of wheat to allow standard agricultural management. Each strip included ten plots each of 24×24 m, with one half of the maize and wheat plots (5 plots each) randomly chosen and manipulated by litter amendment in autumn 2009 and
Resource Type and Availability Regulate Fungal Communities
2010. This resulted in the following four treatments with five replicated plots each: fodder maize (FM), corn maize (CM), wheat (W), and wheat with maize litter (WL). The CM and WL plots received litter input of aboveground hackled maize shoot material on the soil surface (without maize cobs; 0.8 kg m−2 dry weight equivalent to 0.35 kg C m−2, C/N ratio 16.8). In contrast, on the FM and W plots, all aboveground plant material was removed at harvest in autumn; so, there was no litter input. The amendment of maize derived litter on both, CM and WL plots, ensures that potential litter effects are based on the availability of similar resource and not biased by litter quality. After tillage with a chisel plough to a depth of 12 cm of all experimental plots in April 2010, wheat and maize were sown on the same plots as in the previous year. In addition, the amount of maize and wheat root biomass was determined by taking soil samples to a depth of 50 cm in a row and inter-row design in 2009. Roots were washed free from soil, dried, and weighed [32]. One plot of every treatment was set aside for 13C labeling experiments; four replicated plots of each treatment were sampled for this study. Within each plot, ten soil cores (diameter 2.5 cm) reaching 70 cm depth were randomly taken in December 2009 (high translocation of mobile organic particles), July 2010 (high root exudation), September 2010 (shortly before maize harvest), and January 2011 (high translocation of mobile organic particles). Soil cores were split into 10 cm layers, and three soil horizons were analyzed: samples from the 0–10 cm soil layer to investigate the topsoil within the plough layer, samples from 40 to 50 cm for the rooting zone below the plough layer and from 60 to 70 cm for soil below the rooting zone. Soil samples from the same depth within each plot were thoroughly homogenized by hand, transported to the lab on dry ice, and stored at −80 °C for later analyses. In total, 192 soil samples (three soil depths×four treatments× four replicates×four sampling dates) were analyzed for fungal biomass and community structure. Fungal Biomass (Ergosterol Quantification) Ergosterol as an indicator of fungal biomass was extracted from aliquots of 2 g (fresh weight) of topsoil samples and 4 g (fresh weight) of deeper soil layer samples with 25 ml ethanol using a modified version of the method of [40]. Details of modifications and values for the topsoil are published in Kramer et al. [32]. DNA Isolation and F-ARISA fingerprints Total DNA from 0.5 g bulk soil from each of the 192 samples was isolated using the PowerSoil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA) according to the manufacturer’s protocol with the following modifications: all 4 °C
steps were conducted at −20 °C and DNA was eluted in 80 μl elution buffer. Fungal communities were characterized by observing length heterogeneity of the fungal internal transcribed spacers (ITS) region. Fragments were amplified with the primer pair ITS1F-FAM/ITS4 in two technical replicates for each DNA extract. Detection was carried out by capillary electrophoresis on an ABI Prism 3700xl Genetic Analyzer (Applied Biosystems) using the internal size standard MapMarker 1000 ROX (BioVentures, Inc., Murfreesboro, TN, USA) as previously described [39]. Raw profiles were analyzed using the Gene Mapper software 4.0 (Applied Biosystems, Foster City, CA, USA) that converts fluorescence data into electropherograms with resulting peaks exported in a tabular form. All peaks above a threshold of 100 fluorescence units that were present in both technical replicates were considered for further analysis. Resulting peaks were binned into operational taxonomic units (OTUs) using an interactive binning script [41] in R version 2.12.2 [42]: to normalize the data set and to calculate relative peak areas, each peak area was divided by the total area of the whole profile, which was set to 1. The binning frame with the highest correlation values between samples and a window size of two base pairs was chosen. Peaks with a relative area less than 0.09 % were discarded as background noise [41]. Statistical Analyses To improve normality and variance homogeneity prior to statistical analyses, ergosterol data were log transformed, and OTU relative abundance data obtained by F-ARISA were arcsine square root transformed. An overview of treatment explanations, environmental factors, and statistical analyses performed in this study is given in Table S2. All analyses were performed with R version 3.0.1. [42]. Univariate statistical analyses on fungal biomass related to soil depth were done by one-way ANOVAs separately for each sampling date. To investigate treatment effects on fungal biomass, two-way ANOVAs related to plant species and litter input were undertaken separately for each soil depth and sampling date. All multivariate statistical analyses of fungal community structure were assessed using permutational multivariate analysis of variance (perMANOVA) [43] based on Bray–Curtis distance using the function Badonis^ in the R package Bvegan^ [44]. We investigated fungal community structure (a) related to soil depth within one treatment and (b) related to plant species and litter input (treatments) for each soil depth on all sampling dates. To avoid overinterpretation of the relative peak area abundances and to ensure that results were consistent, perMANOVAs were also calculated on the basis of Jaccard distance and using presence/absence data for the first sampling campaign in December 2009. Analyses revealed comparable results, although F values using Jaccard
J. Moll et al.
distance and presence/absence data were slight lower (Tables S5–8). Therefore, all remaining perMANOVAs were calculated based on Bray–Curtis distance using abundance data. Detailed perMANOVA results are given in Tables S9– 15. To its advantage, perMANOVA allows the inclusion of interactions between factors, but it does not provide a graphical output. Therefore, all multivariate results were visualized using nonmetric multidimensional scaling (NMDS) plots as previously described [45, 46]. For better clarity, P value (*P < 0.05; **P < 0.01; ***P < 0.001) and R 2 of each perMANOVA were directly included in the NMDS plots. R2 ranges between 0 and 1. Hence, the relative proportion of variation of each factor is directly visible.
Results Data pertaining to soil properties are available for the field site: organic carbon and total nitrogen, water content as well as extractable organic carbon (EOC) generally decreased with soil depth (Table S1). Soil water content and EOC showed strong seasonal variations, with higher water content in winter than in summer and autumn, while the highest EOC values were observed in the topsoil in July and in the deeper layers in September 2010. Litter application increased EOC in the topsoil especially in the winter samplings and in autumn 2010 [20]. The determination of root biomass revealed that wheat root biomass was higher than those of maize with about six times higher C contents [32].
Fungal Biomass Fungal biomass determined by ergosterol strongly decreased with increasing soil depth on all sampling dates (Fig. 1, Table S3). Plant type was the major significant driver of fungal biomass in the upper two layers (Fig. 1, Table S4). In the topsoil, wheat-cultivated plots revealed higher ergosterol values compared to maize plots during the growing season (July 2010: F1,12 =5.97, P=0.031; September 2010: F1,12 = 26.11, P
ENVIRONMENTAL MICROBIOLOGY
Resource Type and Availability Regulate Fungal Communities Along Arable Soil Profiles Julia Moll & Kezia Goldmann & Susanne Kramer & Stefan Hempel & Ellen Kandeler & Sven Marhan & Liliane Ruess & Dirk Krüger & Francois Buscot
Received: 14 April 2014 / Accepted: 13 January 2015 # Springer Science+Business Media New York 2015
Abstract Soil fungi play an essential role in the decomposition of plant-derived organic material entering soils. The quality and quantity of organic compounds vary seasonally as well as with soil depth. To elucidate how these resources affect fungal communities in an arable soil, a field experiment was set up with two plant species, maize and wheat. Resource availability was experimentally manipulated by maize litter
Electronic supplementary material The online version of this article (doi:10.1007/s00248-015-0569-8) contains supplementary material, which is available to authorized users. J. Moll (*) : K. Goldmann : D. Krüger : F. Buscot Department of Soil Ecology, UFZ – Helmholtz Centre for Environmental Research, Theodor-Lieser-Str. 4, 06120 Halle, Germany e-mail: [email protected] J. Moll : K. Goldmann : F. Buscot Institute of Biology, University of Leipzig, Johannisallee 21-23, 04103 Leipzig, Germany S. Kramer : E. Kandeler : S. Marhan Institute of Soil Science and Land Evaluation, Soil Biology Section, University of Hohenheim, 70593 Stuttgart, Germany S. Hempel Institute of Biology, Dahlem Center of Plant Sciences, Freie Universität Berlin, Altensteinstr. 6, 14195 Berlin, Germany
input on one half of these maize and wheat plots after harvest in autumn. Fungal biomass was determined by ergosterol quantification, and community structure was investigated by fungal automated ribosomal intergenic spacer analysis (FARISA). An annual cycle was assessed across a depth gradient, distinguishing three soil habitats: the plough layer, rooted soil below the plough layer, and deeper root-free soil. Fungal communities appeared highly dynamic and varied according to soil depth and plant resources. In the plough layer, the availability of litter played a dominant role in shaping fungal communities, whereas in the rooted layer below, community structure and biomass mainly differed between plant species. This plant effect was also extended into the root-free soil at a depth of 70 cm. In winter, the availability of litter also affected fungal communities in deeper soil layers, suggesting vertical transport processes under fallow conditions. These distinct resource effects indicate diverse ecological niches along the soil profile, comprising specific fungal metacommunities. The recorded responses to both living plants and litter point to a central role of fungi in connecting primary production and decomposition within the plant-soil system. Keywords Plant resources . Litter . Fungal community structure . Fungal biomass . Soil depth . Arable soil . Season . ARISA
S. Hempel Berlin-Brandenburg Institute of Advanced Biodiversity Research (BBIB), 14195 Berlin, Germany L. Ruess Institute of Biology, Ecology Group, Humboldt-Universität zu Berlin, Philippstr. 13, 10115 Berlin, Germany F. Buscot German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Deutscher Platz 5e, 04103 Leipzig, Germany
Introduction Due to their quantitatively dominant part of the microbial biomass, their diversity, and numerous life strategies, fungi are major drivers of key biogeochemical and ecological processes [1–3]. They rely on several main resources for their
J. Moll et al.
energy supply: litter entering the soil, plant roots and their exudates, as well as soil organic matter [4–6]. Hence, the major belowground fungal resources vary tremendously in chemical composition and physical structure [4, 7]. Moreover, these substrates are not homogeneously distributed in soils. While litter input from aboveground plant material dominates in upper soil horizons, soil in the rooting zone is characterized by root exudates and their residues. Below that, in the bulk soil, organic matter is probably the dominant nutrient source. As a consequence, the quantity of resources, e.g., carbon or nitrogen content, decreases with increasing depth [8, 9]. This general distribution of fungal resources along the soil profile is shaped spatially and temporally by two major processes: transport and seasonality. Organic substrates can be mobilized and translocated from the soil surface down to the subsoil predominantly by drying–wetting or freeze-thaw cycles after seepage events associated with heavy rain and snowmelt [10–13]. Thus, the highest translocation takes place in late autumn, winter, and spring. Besides this spatial variability in resources, arable systems show a distinct pattern in primary production and decomposition cycles related to seasons [14–16]. In particular, in temperate soils with annual plant cropping, root exudation is high in spring and summer, low in autumn, and lacking in winter after plant harvest [17]. In contrast, litter deposition is almost absent in the vegetation growing season, but high litter input occurs after harvest in autumn and winter when crop residues are returned to the field. These spatial- and temporal-based differences in input and accessibility of substrates strongly affect fungal diversity, biomass, and distribution in soil [18–22]. Different species exhibit a preference or specificity for distinct resource types, such as root exudates, related to single plant species or litter with a distinct composition [23, 24]. As fungal activity has a strong impact on soil nutrient cycling in arable soils [25], more knowledge about fungal community dynamics in relation to resource properties in this ecosystem is needed. Previous studies mainly focused on specific fungal groups such as arbuscular mycorrhizal fungi due to their importance as plant symbionts [26, 27]. Other studies investigated fungal communities in upper soil horizons, related to one plant type or on one sampling date [28–31]. A comprehensive assessment of fungal community dynamics in relation to resource availabilities considering seasonality and soil depth is still missing. The present study was performed as a part of a field experiment which was established in order to disentangle the respective impacts of litter input, root exudation, and bulk soil organic matter on microbial and faunal communities in an arable soil [32]. The experiment used two plant species, maize and wheat, which are characterized by different root biomass, distribution, and exudation [32, 33], thus enabling us to
distinguish plant-specific effects. Type and availability of plant resources were manipulated by the following treatments: fodder maize (plant removal at harvest), corn maize (litter amendment at harvest), wheat, and wheat plus maize litter. The plots were sampled at three soil depths: the plough layer, rooted soil below the plough layer, and deeper root-free soil. Sampling was undertaken at three resource phases: (1) during vegetation growing season - to examine root exudation; (2) shortly before harvest - to examine residue input from senescent plants; and (3) under fallow - to examine litter input and translocation processes. Fungal biomass was determined by ergosterol quantification and community structure by fungal automated ribosomal intergenic spacer analysis (F-ARISA) [34, 35]. F-ARISA is a frequently used, low-cost, high throughput DNA fingerprinting technique used to describe fungal communities in relation to environmental conditions [36–38]. It allows an assessment of the structure of fungal communities in large ecological studies such as in this comprehensive study in an arable system. Our objective for this study was to determine fungal biomass and community structure in relation to plant species, litter input, and soil depth over a time series in consecutive seasons. We hypothesized that (1) fungal communities in the topsoil (plough layer) are mostly affected by litter input, (2) while in the rooting zone, they are mainly shaped by root exudates which are related to plant species, and (3) the root-free soil layer below exhibits distinct fungal communities without resource effects.
Material and Methods Experimental Design and Sampling Campaign In April 2009, the experimental site was established in an arable field located at Göttingen-Holtensen (51° 33′ N, 9° 53′ E; 158 m NN, Lower Saxony, Germany). Detailed information about soil properties previously published in Kramer et al. [32] and Pausch and Kuzyakov [33] is given in Supplemental Table S1. Because of long-term agricultural management, two plough layers at 20 and 30 cm depth and severe soil compaction below the second layer were discovered at the field site. The A horizon reached an average depth of 34± 6.4 cm. More details about the study site, e.g., agricultural management (tillage and fertilizer practices), are given in Kramer et al. [32] and Scharroba et al. [39]. The field was cropped with maize (Zea mays L.) and wheat (Triticum aestivum L.) in a design with one strip of maize and one strip of wheat to allow standard agricultural management. Each strip included ten plots each of 24×24 m, with one half of the maize and wheat plots (5 plots each) randomly chosen and manipulated by litter amendment in autumn 2009 and
Resource Type and Availability Regulate Fungal Communities
2010. This resulted in the following four treatments with five replicated plots each: fodder maize (FM), corn maize (CM), wheat (W), and wheat with maize litter (WL). The CM and WL plots received litter input of aboveground hackled maize shoot material on the soil surface (without maize cobs; 0.8 kg m−2 dry weight equivalent to 0.35 kg C m−2, C/N ratio 16.8). In contrast, on the FM and W plots, all aboveground plant material was removed at harvest in autumn; so, there was no litter input. The amendment of maize derived litter on both, CM and WL plots, ensures that potential litter effects are based on the availability of similar resource and not biased by litter quality. After tillage with a chisel plough to a depth of 12 cm of all experimental plots in April 2010, wheat and maize were sown on the same plots as in the previous year. In addition, the amount of maize and wheat root biomass was determined by taking soil samples to a depth of 50 cm in a row and inter-row design in 2009. Roots were washed free from soil, dried, and weighed [32]. One plot of every treatment was set aside for 13C labeling experiments; four replicated plots of each treatment were sampled for this study. Within each plot, ten soil cores (diameter 2.5 cm) reaching 70 cm depth were randomly taken in December 2009 (high translocation of mobile organic particles), July 2010 (high root exudation), September 2010 (shortly before maize harvest), and January 2011 (high translocation of mobile organic particles). Soil cores were split into 10 cm layers, and three soil horizons were analyzed: samples from the 0–10 cm soil layer to investigate the topsoil within the plough layer, samples from 40 to 50 cm for the rooting zone below the plough layer and from 60 to 70 cm for soil below the rooting zone. Soil samples from the same depth within each plot were thoroughly homogenized by hand, transported to the lab on dry ice, and stored at −80 °C for later analyses. In total, 192 soil samples (three soil depths×four treatments× four replicates×four sampling dates) were analyzed for fungal biomass and community structure. Fungal Biomass (Ergosterol Quantification) Ergosterol as an indicator of fungal biomass was extracted from aliquots of 2 g (fresh weight) of topsoil samples and 4 g (fresh weight) of deeper soil layer samples with 25 ml ethanol using a modified version of the method of [40]. Details of modifications and values for the topsoil are published in Kramer et al. [32]. DNA Isolation and F-ARISA fingerprints Total DNA from 0.5 g bulk soil from each of the 192 samples was isolated using the PowerSoil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA) according to the manufacturer’s protocol with the following modifications: all 4 °C
steps were conducted at −20 °C and DNA was eluted in 80 μl elution buffer. Fungal communities were characterized by observing length heterogeneity of the fungal internal transcribed spacers (ITS) region. Fragments were amplified with the primer pair ITS1F-FAM/ITS4 in two technical replicates for each DNA extract. Detection was carried out by capillary electrophoresis on an ABI Prism 3700xl Genetic Analyzer (Applied Biosystems) using the internal size standard MapMarker 1000 ROX (BioVentures, Inc., Murfreesboro, TN, USA) as previously described [39]. Raw profiles were analyzed using the Gene Mapper software 4.0 (Applied Biosystems, Foster City, CA, USA) that converts fluorescence data into electropherograms with resulting peaks exported in a tabular form. All peaks above a threshold of 100 fluorescence units that were present in both technical replicates were considered for further analysis. Resulting peaks were binned into operational taxonomic units (OTUs) using an interactive binning script [41] in R version 2.12.2 [42]: to normalize the data set and to calculate relative peak areas, each peak area was divided by the total area of the whole profile, which was set to 1. The binning frame with the highest correlation values between samples and a window size of two base pairs was chosen. Peaks with a relative area less than 0.09 % were discarded as background noise [41]. Statistical Analyses To improve normality and variance homogeneity prior to statistical analyses, ergosterol data were log transformed, and OTU relative abundance data obtained by F-ARISA were arcsine square root transformed. An overview of treatment explanations, environmental factors, and statistical analyses performed in this study is given in Table S2. All analyses were performed with R version 3.0.1. [42]. Univariate statistical analyses on fungal biomass related to soil depth were done by one-way ANOVAs separately for each sampling date. To investigate treatment effects on fungal biomass, two-way ANOVAs related to plant species and litter input were undertaken separately for each soil depth and sampling date. All multivariate statistical analyses of fungal community structure were assessed using permutational multivariate analysis of variance (perMANOVA) [43] based on Bray–Curtis distance using the function Badonis^ in the R package Bvegan^ [44]. We investigated fungal community structure (a) related to soil depth within one treatment and (b) related to plant species and litter input (treatments) for each soil depth on all sampling dates. To avoid overinterpretation of the relative peak area abundances and to ensure that results were consistent, perMANOVAs were also calculated on the basis of Jaccard distance and using presence/absence data for the first sampling campaign in December 2009. Analyses revealed comparable results, although F values using Jaccard
J. Moll et al.
distance and presence/absence data were slight lower (Tables S5–8). Therefore, all remaining perMANOVAs were calculated based on Bray–Curtis distance using abundance data. Detailed perMANOVA results are given in Tables S9– 15. To its advantage, perMANOVA allows the inclusion of interactions between factors, but it does not provide a graphical output. Therefore, all multivariate results were visualized using nonmetric multidimensional scaling (NMDS) plots as previously described [45, 46]. For better clarity, P value (*P < 0.05; **P < 0.01; ***P < 0.001) and R 2 of each perMANOVA were directly included in the NMDS plots. R2 ranges between 0 and 1. Hence, the relative proportion of variation of each factor is directly visible.
Results Data pertaining to soil properties are available for the field site: organic carbon and total nitrogen, water content as well as extractable organic carbon (EOC) generally decreased with soil depth (Table S1). Soil water content and EOC showed strong seasonal variations, with higher water content in winter than in summer and autumn, while the highest EOC values were observed in the topsoil in July and in the deeper layers in September 2010. Litter application increased EOC in the topsoil especially in the winter samplings and in autumn 2010 [20]. The determination of root biomass revealed that wheat root biomass was higher than those of maize with about six times higher C contents [32].
Fungal Biomass Fungal biomass determined by ergosterol strongly decreased with increasing soil depth on all sampling dates (Fig. 1, Table S3). Plant type was the major significant driver of fungal biomass in the upper two layers (Fig. 1, Table S4). In the topsoil, wheat-cultivated plots revealed higher ergosterol values compared to maize plots during the growing season (July 2010: F1,12 =5.97, P=0.031; September 2010: F1,12 = 26.11, P
