Condensed tannins mediate the effect of long-term nitrogen addition on soil nematodes in a boreal spruce forest

Condensed tannins mediate the effect of long-term nitrogen addition on soil nematodes in a boreal spruce forest | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Condensed tannins mediate the effect of long-term nitrogen addition on soil nematodes in a boreal spruce forest Lea-Rebekka Tonjer, Line Nybakken, Tone Birkemoe, Marek Renčo, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2446831/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Fertilization and combustion have increased nitrogen (N) deposition over the last decades. Most boreal forests are N limited; thus, increased N availability may have considerable implications for boreal forest food web structure and functioning. Soil biota are important drivers of ecosystem processes through their effect on carbon (C) and nutrient cycling. Among these, nematodes are ubiquitous in soil and respond quickly to environmental changes. They are thus useful in determining changes in ecosystem status. Boreal forest plants produce large quantities of condensed tannins (hereafter tannins), and these may decrease following N addition. As defence compounds, tannins may alter soil community structure by reducing microbial activity and deter microfauna. Using nematodes as indicators, we here investigated the linkages between N-induced changes in soil food web and soil tannin concentration. We utilized a fertilization experiment where plots have been fertilized since 2003 at a rate of 150 kg ha − 1 yr − 1 in the form of granulated pellets containing 24.6% N, 2% P, 6% K and trace elements. This have resulted in a shift in the understory, and consequently a decrease in soil tannin concentration. Fertilization led to a less structured nematode community dominated by bacterivores. The abundance of bacterivores relative to fungivores increased, indicating a more bacteria-dominated energy channel. Furthermore, the dominance of bacteria appears to have been inhibited by soil tannin concentrations in the fertilized plots. Overall, our study demonstrate that soil community structure strongly changes upon increased N availability, and lower soil tannin concentrations further facilitate the dominance of bacteria. Fertilization Plant secondary metabolites Energy channel Food webs Interactions Figures Figure 1 Figure 2 Figure 3 Figure 4 1 Introduction The availability of nitrogen (N) is a critical factor controlling plant productivity and soil processes within all ecosystems. Most boreal forests are N limited but are subject to N enrichment both through intentional fertilization and pollution. However, N addition to a primarily N limited forest will not only impact aboveground production; it will also have important implications for belowground structure and functioning (Meunier et al., 2016 ). Soil microorganisms are the primary decomposers of organic matter, thus essential in carbon (C) and nutrient cycling, with fungi commonly dominating the decomposition pathway in boreal forests. However, increased N availability favours bacteria over fungi, causing a shift towards a more bacteria-dominated energy channel (Demoling et al., 2008 ; Högberg et al., 2017 ; Maaroufi et al., 2018 ; Shaw et al., 2019 ). Such shifts in the microbial community may further affect the soil food web at higher trophic levels (Moore et al., 2003 ; Wardle et al., 2004 ), possibly leading to a less structured and complex community after fertilization (Maaroufi et al., 2018 ; Shaw et al., 2019 ). Moreover, increased N availability may lead to reduced soil microbial activity and respiration rates, and subsequently increased C sequestration (Treseder, 2008 ; Janssens et al., 2010 ; Maaroufi et al., 2015 ). Accordingly, the structure and functioning of soil food webs may be directly or indirectly affected by increased N availability. Directly through increased soil nutrient availability influencing soil conditions, or more indirectly through changes in plant community composition as well as litter production and quality (Fig. 1 ). In boreal Norway spruce ( Picea abies ) forests, bilberry ( Vaccinium myrtillus ) and other ericaceous dwarf shrubs (e.g. V. vitis-idaea and Empetrum nigrum ) commonly dominate the understory vegetation. Norway spruce and ericaceous dwarf shrubs all produce large quantities of condensed tannins (hereafter tannins), one of the main classes of phenolic defence compounds (Hättenschwiler & Vitousek, 2000 ). Boreal forest soils are thus rich in tannins. As defence compounds, tannins are known to reduce microbial activity and deter microfauna, thereby altering soil community structure. (Hättenschwiler & Vitousek, 2000 ). Additionally, some studies report a higher inhibitory effect on bacteria compared to fungi (Kanerva et al., 2006 ; Mutabaruka et al., 2007 ; Ushio et al., 2013 ). Tannins in soil also play a role in the N cycle by forming recalcitrant complexes with N-rich organic compounds, thereby inhibiting N mineralization processes (Kraus et al., 2003 ; Chomel et al., 2016 ). The afterlife effects of tannins in soil are thus considerable. However, as plant-derived compounds, their concentration in the soil is controlled by the aboveground plant community. Increased N may affect tannin concentrations through changes both within- and between plant species (e.g., species turnover). According to the protein competition model, within species tannin concentrations are likely to decrease with increased nutrient availability (Wright et al., 2010 ). This is because growth proteins and phenolic compounds compete for the same amino acid precursor phenylalanine. At higher N availability, the incorporation of phenylalanine into proteins will increase, and the production of phenolic compounds will decrease (Wright et al., 2010 ). Further, an increase in soil fertility commonly leads to a shift from a community dominated by slow-growing species investing more C to structural and chemical defence to one dominated by species that allocate more C to growth and thus have higher foliage N and specific leaf area, and lower defence levels (Aerts, 1999 ; Wardle et al., 2004 ; Meunier et al., 2016 ). The concentration of tannins in soil may thus vary with N availability, with further implications for soil processes. Soil biota is an essential part of the ecosystem, and play important roles in nutrient and C cycling and by modifying soil community structure (Ferris, 2010 ; Nielsen et al., 2011 ; Bardgett & Van Der Putten, 2014 ; Jesús & Briones, 2014 ). Among these, nematodes are ubiquitous in soil, occupy several trophic niches, and respond quickly to environmental changes (Ferris et al., 2001 ). They are thus useful in determining changes in ecosystem status (Neher, 2001 ; Du Preez et al., 2022 ). In 1990 the first nematode-based index, the Maturity Index, was established to assess level of disturbance (Bongers, 1990 ). Later, a complex toolset of indices has been developed (Table 1 ), and several studies have used nematode communities and nematode-based indices to assess the effect of disturbances on soil food web (Čerevková et al., 2020 ; Renčo et al., 2021 ; Du Preez et al., 2022 ; Renčo et al., 2022b ). The nematode indices are calculated by assigning taxa into functional guilds (Bongers, 1990 ; Yeates et al., 1993 ; Ferris et al., 2001 ) with similar feeding habit and life history strategy, i.e. if they are colonizers (r-selected) or persisters (K-selected). Colonizers are more tolerant to disturbances due to their short generation time, large population fluctuations and high fecundities. Persisters on the other hand, have long life cycles, low reproductive rates, low metabolic activities, and slow movement; they are thus sensitive to disturbances. Table 1 An overview of the indices used in this study and what they indicate (Du Preez et al., 2022 ). Index Indicates Maturity Index Successional maturity Structure Index Food web complexity and disturbances Channel Index Decomposition dominance by bacteria vs. fungi Enrichment Index Nutrient availability Basal Index Food web complexity Plant-Parasitic Index Compilation of plant-parasitic nematodes Metabolic Footprints Magnitude of ecosystem services fulfilled by nematode community Here, we used nematodes as indicators to study the linkages between N-induced changes in soil tannin concentration and the soil food web. We utilized a long-term Norway spruce fertilization experiment where plots have been fertilized annually since 2003 resulting in a shift in the understory from a community dominated by bilberry, producing high levels of tannins, and feathermosses to a system dominated by grasses and forbs, with no or low amounts of tannins (Lorentzen, 2017 ). Our overall aim was to investigate the long-term effect of fertilization on the soil ecosystem defined by nematode communities and indices, and we specifically wanted to test the hypotheses that fertilization will (i) favour bacteria over fungi leading to a lower Channel Index; (ii) decrease soil tannin concentrations, which will ease bacteria and thus further enhance the effect on the Channel Index; and (iii) lead to a less structured nematode community that are more dominated by short-lived stress-tolerant species. By testing these three hypotheses, we will advance the understanding of how high fertilization rates affect soil food web. 2 Materials And Methods 2.1 Study site and sampling The study was conducted in an > 200 year old sub-alpine boreal Norway spruce ( Picea abies ) dominated forest east of Dokkfløyvatn, Gausdal Vestfjell, SE Norway (61°10’N, 09°90’E, 800 m a.s.l.). Bilberry ( Vaccinium myrtillus ) and feathermosses dominated the understory. A fertilization experiment was established in 2003 with 10 fertilized 15×15 m plots, and 10 control plots of the same size (Davey et al., 2017 ). Granulated pellets containing 24.6% N, 2% P, 6% K and trace elements (YaraMila Fullgjødsel) have been added yearly since 2003 at a rate of 150 kg ha − 1 year − 1 in fertilized plots, locally placed to avoid runoff to control plots. The N addition (ca. 37 kg N ha − 1 year − 1 ) was significantly higher than the upper rates of N deposition in the southern boreal zone of northern Europe (12 kg ha − 1 year − 1 ; Gundale et al., 2011 ). As such, it represents an extreme change, but is comparable with many long-term forest fertilization experiments in boreal forests (e.g. Sponseller et al., 2016 ). In June 2020, we sampled the entire organic soil and approximately 4 cm of the mineral soil using a ø10 cm soil corer at five sub-plots per plot. Sub-plots were established as followed: A grid of 5×5 m squares within the 15×15 m plots were defined. Four sub-plots were located at each crossing point, in addition to one sub-plot in the centre of the 15×15 m plot. Soil cores were divided vertically in the field, where one half was saved for nematode analyses (both organic and mineral layer combined). From the other half, the organic layer was used for chemical analyses. Fresh-weight of soil samples was measured immediately after sampling. Further, in September 2020, senesced V . myrtillus leaves and P . abies needles still attached to the plant (for tannin analyses) were sampled randomly at plot-level. Only leaves and needles that detached easily were collected. All samples were stored at room temperature until further processing. 2.2 Sample preparation and analyses Organic soil samples for chemical analyses were dried at 30°C until stable weight (at least 48 h). Gravimetric soil water content (hereafter soil moisture) was determined after drying ((wet weight – dry weight)/dry weight). Green parts, roots and twigs larger than approx. 2 mm in diameter were removed. The samples were then homogenized by grounding into fine powder using a Retsch MM 400 ball mill (Retsch, Haan, Germany) at 30 rpm. Senesced V . myrtillus leaves and P . abies needles were air-dried in paper bags for 15 days and then powdered the same way as the soil samples. We determined soil C and N concentrations in 5–7 mg sub-samples by combustion at 950°C using a vario MICRO cube analyser (Elementar Analysensysteme GmbH, Hanau, Germany) with a thermal conductivity detector. For soil pH analysis, 5 ml of soil was diluted in 12.5 ml of dH 2 O for 24 h. The pH value was then measured using an inoLab pH 720 precision pH meter (WTW GmbH, Weilheim, Germany), following the manufacturers protocol. For tannin extraction, 4 ml 70% acetone was added to glass tubes containing 100 mg of powdered soil or 10 mg of powdered leaves or needles, respectively. The samples were then mixed on a planar shaker (KS 501 digital, IKA-WERKE, Germany) at 200 rpm for 1 hour, followed by centrifugation (10 min, 1,500 x g; Hettich Universal 16 centrifuge [Hettich Zentrifugen, Kirchlengern Germany]). The supernatant was transferred to a separate glass tube, and the extraction process was repeated twice. The mixed supernatants were then evaporated in a vacuum centrifuge (Eppendorf concentrator plus; Eppendorf, Hamburg, Germany) and stored in a freezer (-18°C) until further analysis. Concentration of tannins in both soil and leaves and needles were determined following the acid butanol assay for proanthocyanidins (condensed tannins) described in Hagerman ( 2002 ). The extracts were redissolved in 0.5 ml MeOH and mixed properly. Further, 3 ml acid butanol (95% butanol, 5% HCl) and 0.1 ml iron reagent (2% ferric ammonium sulphate in 2N HCl) were added before placing the samples in boiling water for 1 hour. After cooling, the absorbance at 550 nm was determined using a spectrophotometer (UV-1800; Shimadzu Corp., Kyoto, Japan). 2.3 Nematode extraction and analyses Soil samples were homogenized by gentle hand mixing and soaked in 1 L of tap water for 30–60 min. A combination of Cobb sieving and decanting (Cobb, 1918 ) and a modified Baermann technique (Baermann, 1917 ) was used for nematode extraction. Firstly, an aqueous soil suspension was sieved in 1 mm (16 mesh) and 0.05 mm (300 mesh) sieves. The obtained suspension with nematodes without coarse soil was then placed on a funnel contained a sieve 0.5 mm (30 mesh) and a set of two cotton–propylene filters. Sub-samples were removed after 48 h of extraction at room temperature. The aqueous suspensions were examined under a stereomicroscope (40 and 60× magnification), excessive water was removed and the nematodes were fixed with a hot 99:1 solution of 4% formaldehyde:pure glycerol (Seinhorst, 1962 ). The nematodes were microscopically (100, 200, 400, 600 and 1000× magnification) identified to the genus level using an Eclipse 90i Nikon light microscope (Nikon, Tokyo, Japan) from temporary slides using keys, including those of Brzeski ( 1998 ), Andrássy ( 2005 ; 2007 ; 2009 ), Geraert ( 2008 ; 2010 ) Loof ( 1999 ) and several others. Nematodes in each sample were assigned into trophic groups based on their feeding habits, according to Yeates et al. ( 1993 ) and Wasilewska ( 1997 ), adjusted and supplemented following Sieriebriennikov et al. ( 2014 ): bacterivores, fungivores, root-fungal feeders (facultative plant parasites), obligatory plant parasites, predators and omnivores. We calculated basic ecological indices, commonly used to assess the status of the soil ecosystems using nematode communities. Plant-Parasitic Index and Maturity Index were calculated following Bongers ( 1990 ). Enrichment, Structure, Channel and Basal Index were calculated following Ferris et al. ( 2001 ). Metabolic footprints, a metric of the magnitudes of ecosystem functioning and services, were calculated following Ferris ( 2010 ) for each trophic group. The footprint incorporates both the lifetime amount of C partitioned into growth and egg production, and the C use in metabolic activity (respiration). All indices and footprints were calculated using the NINJA automated calculation system (Sieriebriennikov et al., 2014 ). 2.4 Data analysis The Shannon index of species diversity (Shannon, 1948 ) was calculated as: H´ = −∑ P i × ln P i where P i is the proportion of individuals of the i th taxon. All statistical analyses were conducted in R version 4.2.2 (R Core Team, 2021 ). To visualize the nematode community composition, we performed a global nonmetric multidimensional scaling (GNMDS), based on a Bray-Curtis distance matrix with the “metaMDS” function in the vegan package (Oksanen et al., 2013 ), using settings as recommended by Liu et al. ( 2008 ). The “envfit” (permutations = 999) function were used to test if soil variables (i.e. soil N, C and tannin concentration, C:N ratio, moisture and pH) and nematode indices correlated with the nematode community structure. Significant (P < 0.05) correlated variables were fitted onto the diagram. The “ordiellipse” function was used to plot 95% confidence intervals of the treatment centroids. For modelling the effect of treatment on soil variables, linear mixed effects models were used using the “lme” function of the nlme package (Pinheiro et al., 2022 ), with plots as a random effect. The same function was used when modelling the interaction between Channel Index and soil tannin concentration, in addition to “ggpredict” in the ggeffect package (Lüdecke, 2018 ) as well as ggplot2 (Wickham, 2016 ) for graphical plotting. The effect of treatment on tannin concentration in bilberry leaves and spruce needles was modelled in a linear model using the “lm” function in the stats package (R Core Team, 2021 ). 3 Results Fertilization had a significant effect on all measured soil variables and nematode indices (Table 2 ). Soil N and C concentration were on average 26% and 6% higher in fertilized plots, respectively. Thus, the C:N ratio decreased. Fertilization further led to a lower soil pH. Moreover, soil tannin concentration was 30% higher in control plots, whereas fertilization had no effect on tannin concentration in senesced V. myrtillus leaves and P . abies needles (Table 2 ). Table 2 Mean values ± standard error (SE) of soil variables, condensed tannin concentrations of senesced V. myrtillus leaves and P . abies needles and nematode indices. CT = condensed tannins, N = nitrogen, C = carbon. Bold numbers indicate statistical significance (P < 0.05). *Samples collected at plot-level (i.e., not subplot-level). Mean control ± SE Mean fertilized ± SE t-value p-value Soil pH 3.81 ± 0.04 3.66 ± 0.03 -2.61 0.018 Soil CT (mg g − 1 ) 6.61 ± 0.44 5.07 ± 0.34 -2.23 0.039 Soil N (%) 1.25 ± 0.03 1.58 ± 0.03 6.96 0.000 Soil C (%) 38.89 ± 0.82 41.38 ± 0.54 2.55 0.020 Soil C:N ratio 31.53 ± 0.62 26.63 ± 0.46 -4.9 0.000 Soil moisture 2.16 ± 0.12 2.14 ± 0.09 -0.07 0.946 V. myrtillus CT* 176.7 ± 5.52 168.3 ± 6.8 -0.96 0.349 P. abies CT* 112.6 ± 10.9 105.5 ± 14.3 -0.40 0.694 Nematode indices Maturity Index 2.66 ± 0.03 2.08 ± 0.01 -18.27 0.000 Structure Index 74.79 ± 1.04 44.02 ± 1.51 -13.78 0.000 Basal Index 20.17 ± 0.76 32.54 ± 1.07 6.8 0.000 Plant-Parasitic Index 2.41 ± 0.03 2.04 ± 0.01 -10.07 0.000 Channel Index 41.44 ± 2.11 26.95 ± 1.60 -3.94 0.001 Enrichment Index 48.70 ± 1.09 56.17 ± 1.28 3.02 0.007 Shannon Diversity 2.96 ± 0.02 2.62 ± 0.02 -8.45 0.000 3.1 Soil community structure The nematode community composition was clearly influenced by fertilization, as shown by the clear separation in the GNMDS ordination (Fig. 2 ). Except soil C concentration and moisture, all measured soil variables (i.e., soil tannin and N concentration, C:N ratio and pH) and nematode indices significantly correlated with nematode community structure. Fertilized plots inhabited a more disturbed and less-structured nematode community with lower diversity in contrast to more mature and complex communities in the controls. Both Maturity Index and Structure Index, related to soil food web disturbance and community structure (see Table 1 ), had significantly lower values after fertilization (22% and 41% respectively; Table 2 ). At the same time, a higher Basal Index value indicated a more depleted food web in fertilized plots. Moreover, the Enrichment Index, based on the expected responsiveness of the opportunistic guilds to organic-resource enrichment, was 15% higher in fertilized plots. We observed a distinct difference in the relative abundance of trophic groups between treatments (Figs. 2 and 3 a). Fertilization led to a nematode community dominated by bacterivores. The relative abundance of omnivores, predators and herbivores were negatively affected by fertilization, whereas fungivores had a proportionally slight increase (Fig. 3 a). Furthermore, fertilization led to a considerable decline in plant parasites (Fig. 3 a), resulting in a lower Plant-Parasitic Index (Table 2 ). The relative distribution of trophic groups in terms of their metabolic footprint – i.e., how much they contribute to C utilization, and thus to soil processes (see Table 1 ) – gave a slightly different picture. The bacterivore footprint dominated in fertilized plots (Fig. 3 b). Fungivore footprint was unaffected by N addition, whereas the footprint of omnivores decreased considerably. Predator and herbivore footprints turned in to negligible after fertilization. 3.2 Effect of condensed tannins on Channel Index Channel Index was 54% higher in control plots, indicating a more fungi dominated energy channel here (Table 2 ). This is also demonstrated by the Channel Index vector fitted onto the ordination diagram (Fig. 2 ). Furthermore, Channel Index was negatively affected by soil tannin concentration in the fertilized plots (Fig. 4 ). This suggests that fertilized plots with higher tannin concentrations approaches a more fungi dominated energy channel, whereas fertilized plots with lower tannin concentrations have more bacteria dominated energy channel. This response was, however, not significant in the control plots. 4 Discussion We found support for our first hypothesis that fertilization favour bacteria over fungi resulting in a more bacteria-based Channel Index (i.e., low Channel Index). Fertilization decreased soil tannin concentrations, in line with our second hypothesis. We also found that the lower soil tannin concentrations in fertilized plots further facilitated the bacteria-based energy channel. Our last hypothesis, that fertilization would lead to a more disturbed soil food web, was also supported, demonstrated by the change in nematode community and nematode-based indices. 4.1 Direct and indirect effects of fertilization on dominating energy channel Our study indicate that the soil food web changed from fungi-dominated to bacteria-dominated in response to N addition. In general, soils with high nutrient availability are associated with a bacteria-dominated energy channel and lower fungi:bacteria ratio (Moore et al., 2005 ; Van Der Heijden et al., 2008 ; Högberg et al., 2017 ). Fertilization thus had a direct bottom-up effect on the bacterial advantage relative to fungi (indicated through Channel Index and by nematode feeding groups). This led to a lower fungi:bacteria ratio by increasing the amounts of easily available N, in line with other studies (De Vries et al., 2006 ; Treseder, 2008 ; Maaroufi et al., 2018 ). The N-induced shift in the understory may also have enhanced the effect on the energy channel through positive plant-soil-feedbacks. Slow growing plant species in the control plots like ericaceous shrubs and feathermosses produce low quality litter which promotes fungi (ectomycorrhizal and ericoid mycorrhizal) due to their ability to decompose recalcitrant organic matter and exploit organic bound N (Bending & Read, 1997 ; Bödeker et al., 2014 ; Lindahl & Tunlid, 2015 ). On the other hand, fast-growing plant species like grasses and forbs in the fertilized plots produce high quality litter that are easily available for bacteria. Thus, different plant functional groups may be associated with either a fungal or bacterial dominated soil community (Wardle et al., 2004 ; Van Der Heijden et al., 2008 ; Eskelinen et al., 2009 ). Further, our data suggest that the decline in soil tannins amplified the fertilization and vegetation effect on the bacterial community. However, this effect was only significant in fertilized plots. An explanation for this could be that the low N availability is a limiting factor for bacteria in the control plots, and thus overrides the negative effect of tannins here. The lower soil tannin concentration in fertilized plots compared to control plots is in line with another boreal forest fertilization study (Smolander et al., 2022 ). Since N addition did not have any effect on the tannin concentration of leaves and needles, the lower soil tannin levels likely resulted from the decline in V. myrtillus abundance. Tannins negatively influence microbes, both through enzyme inhibition and direct toxic effects, but it is discussed whether fungi or bacteria are more sensitive to tannins (Scalbert, 1991 ; Kraus et al., 2003 ; Smolander et al., 2012 ). However, both Mutabaruka et al. ( 2007 ) and Ushio et al. ( 2013 ) explained higher fungi:bacteria ratio in their studies from tropical forests by higher concentrations of soil tannins. Further, the ability of fungi, especially ericoid mycorrhiza, to exploit N from tannin-protein complexes (Bending & Read, 1996 ; Joanisse et al., 2007 ; Wurzburger & Hendrick, 2009 ), may additionally have promoted fungal advantage over bacteria the in controls in our study. Fungivorous and bacterivorous nematodes have long been recognized as useful indicators of the abundances of fungi and bacteria in soil (Ferris et al., 2001 ; Neher, 2001 ). However, in the studies by De Long et al. ( 2016 ) and Maaroufi et al. ( 2018 ), the nematode fungivore and bacterivore ratios did not clearly mirror the respective microbe ratios (based on microbial phospholipid fatty acids method (PLFA)). Further, Cesarz et al. ( 2015 ) found that functional guilds (i.e. feeding habits combined with life history strategies) within one trophic group, had different responses to the treatment in their study. They thus suggested that functional guilds, which are used to calculate the indices (Table 1 ), are better in reflecting shifts in soil communities rather than trophic groups. Several studies report a reduction in total microbial abundance, but also that fungi are particularly sensitive to N addition (Högberg et al., 2007 ; Demoling et al., 2008 ; Treseder, 2008 ; Maaroufi et al., 2018 ). We observed no major change in the relative abundance of fungivores and a large relative increase in bacterivores in response to N addition. However, the explanation that the nematode-based indices reflect the microbial community more correctly than the feeding groups may also be applied to our study. 4.2 Effect of fertilization on nematode communities and soil food web Adding heavy loads of N to a forest developed under N-limitation had large impacts on the soil food web leading to lower diversity and maturity. This indicates a disturbed system with fewer trophic links. Overall, the fertilized plots inhabited less omnivores and predators and a relatively high abundance of bacterivores. Omnivores and predators, i.e. persisters, are more susceptible to N addition and other disturbances (Bongers, 1990 ; Ferris et al., 2001 ). On the contrary, most colonizers are bacterivores responding rapidly to environmental changes. When the food web is dominated by bacterivorous colonizers with short lifecycles and high respiratory rates, less amounts of C are assimilated to higher trophic levels (Ferris & Bongers, 2006 ). Additionally, as mentioned above, several studies have shown that fertilization reduce microbial activity. As microbes are an important C source for nematodes, this combination may have caused the lower abundance of nematodes at higher trophic levels in the fertilized plots in this study. Furthermore, the lower pH after fertilization may have affected omnivores and predators negatively (reviewed in Xing et al. ( 2022 )). Both Maturity Index and Plant-Parasitic Index had lower values in fertilized plots compared to the untreated controls in this study. It has been claimed that these indices usually act in the opposite direction of each other (Bongers et al., 1997 ). A low Maturity Index value indicate low soil food web maturity because N enrichment result in a stimulation of opportunistic species, whereas the Plant-Parasitic Index tend to increase with increasing nutrients due to the consequently better quality in the associated plant root cells. However, the fertilizers in this study contain 13.8% ammonia, which is reported to be toxic to plant parasites (Rodríguez-Kábana, 1986 ; Wei et al., 2012 ; Xing et al., 2022 ), and this toxicity may override the positive effect of higher root quality. Furthermore, our finding that fertilization exclude plant parasitic nematodes corresponds with the decline in V. myrtillus cover in favour of a more graminoid dominated community (Lorentzen, 2017 ). This pattern was also found in Renčo et al. ( 2022a ). We cannot assure a causal relationship, but some of the nematode genera that had a substantial decline in abundance in fertilized plots (i.e. Helicotylenchus and Pratylenchus ) are known parasites on V. corrymbosum , a species closely related to V. myrtillus . However, these reports are from agriculture and might be of low relevance to our system (Jagdale et al., 2013 ; Rivera et al., 2015 ). Another possible explanation could be that fertilization lead to higher abundances of bacteria which suppress plant-parasitic nematodes by colonizing roots (Siddiqui & Mahmood, 1999 ). 4.3 Conclusions This study has shown that both direct and indirect effects must be considered when examining the consequences of increased N availability in boreal forests. The soil community structure strongly changed upon N addition. Such transitions may consequently impact soil functions. Under ambient conditions, nutrient rich soils typically have a bacteria dominated energy channel associated with faster decomposition, higher N mineralization rates and C fluxes. However, high N addition leads to soil acidification through oxidation of ammonia to nitrate. This inhibits microbial activity and organic matter accumulates, even with a bacteria dominated energy channel. Accordingly, the amount of N added determines whether the ecosystem will have a fast or slow decomposition pathway, which subsequently have consequences for soil C sequestration. Furthermore, the changes in soil nutrient availability, community structure and function influence the plant community, litter quality and production. The likely effect of tannins on bacteria in fertilized plots corroborate that plant-derived compounds are important players in soil processes. However, it also demonstrates that when N is no longer a limiting nutrient, other factors affecting soil biota may emerge. Thus, assessing the interaction between members of soil food web and the elements involved in nutrient cycling, including plant secondary metabolites, should be a subject for future studies. This could generate valuable insight into how N enrichment through fertilization or pollution affect ecosystem processes in boreal forests. Declarations Acknowledgement s We acknowledge Mikael Ohlson for initiating the fertilization experiment in 2003. We thank Annie Aasen, Claus Kreibich and Puspa Subedi for help with sampling and chemical analyses. Funding: Not applicable. Conflicts of interest/Competing interests: The authors declare that they have no conflict of interest. Ethics approval: Not applicable. Consent to participate: Not applicable. Consent for publication: Not applicable. Availability of data and material: Data associated with this manuscript are deposited in the NMBU Open Research Data database (doi will be given if the manuscript is accepted). Code availability: Not applicable. Authors' contributions: JA and LN designed the study. Field work was conducted by LRT, ZF, JA and LN. ZF performed laboratory analyses under supervision of LN and JA. MR extracted and identified nematodes. Data analyses were done by LRT. LRT led the writing under supervision of JA, LN and TB. 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Feeding Habits in Soil Nematode Families and Genera-An Outline for Soil Ecologists. Journal of Nematology , 25 (3), 315–331. pmid: 19279775; PMCID: PMC2619405. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2446831","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":167626596,"identity":"39860f5e-2ae9-4698-ae51-343a79e617a1","order_by":0,"name":"Lea-Rebekka Tonjer","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEElEQVRIie2Qv0oDMRjAEwLJkjPrlUD7CpGCRQR9lZMOXTzomEHwDuGm4O7SZ9BFO0YC7fI9gMINh8O53iS4SM+2DoV46iaYH4F8ge/3/QlCgcAfZG9zWRTzTUTaYxHOvlbojmLXCk1+p7Rw1a2wy4poXfZ7xlWk0SUTEl6fn+YOjZjfo3yhMEA9lFGhkIWa9K7S+2EKDh0a61fiZPSWF+50JpBCD4UjCqI7mRYOqcfErwxeGpy/u4uZYM1aOQFedysxVzjPXCIjs+3COe1W+NkUZ4t6/9rA1AI4EgM9kClMuAL/LoItb3B2Xg5imNxWWruxMKQdbH7UV0v/j+3wUXX8+eDf5285/nFmIBAI/BtWIZtf0Yw6LUgAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-7704-7382","institution":"Norwegian University of Life Sciences Department of Ecology and Natural Resource Management: Norges miljo- og biovitenskapelige universitet Institutt for naturforvaltning","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Lea-Rebekka","middleName":"","lastName":"Tonjer","suffix":""},{"id":167626597,"identity":"a4e40780-3ef0-4c02-9bf9-2cabe880d816","order_by":1,"name":"Line Nybakken","email":"","orcid":"","institution":"Norwegian University of Life Sciences Faculty of Environmental Sciences and Natural Resource Management: Norges miljo- og biovitenskapelige universitet Fakultet for miljovitenskap og naturforvaltning","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Line","middleName":"","lastName":"Nybakken","suffix":""},{"id":167626598,"identity":"e8a17f11-3cc3-4647-a06f-273041c6e416","order_by":2,"name":"Tone Birkemoe","email":"","orcid":"","institution":"Norwegian University of Life Sciences Faculty of Environmental Sciences and Natural Resource Management: Norges miljo- og biovitenskapelige universitet Fakultet for miljovitenskap og naturforvaltning","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tone","middleName":"","lastName":"Birkemoe","suffix":""},{"id":167626599,"identity":"168359f9-6211-4708-8fc6-f120611cc7ee","order_by":3,"name":"Marek Renčo","email":"","orcid":"","institution":"Institute of Parasitology of the Slovak Academy of Sciences: Parazitologicky ustav Slovenskej akademie vied","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marek","middleName":"","lastName":"Renčo","suffix":""},{"id":167626600,"identity":"87910b1d-c3ae-4be9-9016-f712f026d15a","order_by":4,"name":"Zannatul Ferdous","email":"","orcid":"","institution":"Norwegian University of Life Sciences Faculty of Environmental Sciences and Natural Resource Management: Norges miljo- og biovitenskapelige universitet Fakultet for miljovitenskap og naturforvaltning","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zannatul","middleName":"","lastName":"Ferdous","suffix":""},{"id":167626601,"identity":"27d1e41b-4c5e-4d5e-a837-33bb9b2b5306","order_by":5,"name":"Johan Asplund","email":"","orcid":"","institution":"Norwegian University of Life Sciences Faculty of Environmental Sciences and Natural Resource Management: Norges miljo- og biovitenskapelige universitet Fakultet for miljovitenskap og naturforvaltning","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Johan","middleName":"","lastName":"Asplund","suffix":""}],"badges":[],"createdAt":"2023-01-05 13:30:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2446831/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2446831/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":31693265,"identity":"99eb5941-ee3f-4d30-baaa-34616c2e1f4e","added_by":"auto","created_at":"2023-01-17 15:56:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":286934,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic view of the potential effects of nitrogen (N) addition on soil structure in our study system. The microbial community composition, consisting of fungi and bacteria, as well as the nematode community composition, may be directly affected through higher N availability from fertilization. Further, the soil community may be indirectly affected through N-induced changes in the plant community. A shift in the plant community may lead to a concomitant shift in the microbial and nematode communities. Soil condensed tannin concentration may decrease, which in turn influence the composition of microbes. Additionally, the fungi:bacteria ratio of the microbial community, further structure the nematode community composition.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-2446831/v1/1c773ad90820cfc2f1e429ae.png"},{"id":31690547,"identity":"0021e3e3-26b2-4eed-b84d-7032df090b0a","added_by":"auto","created_at":"2023-01-17 15:40:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":345385,"visible":true,"origin":"","legend":"\u003cp\u003eGlobal\u003cstrong\u003e \u003c/strong\u003enon-metric multidimensional scaling (GNMDS) of the nematode community based on species scores. Indices and soil variables fitted onto the diagram significantly correlated with the community structure (pH = soil pH, N = soil nitrogen concentration, C:N = soil carbon to nitrogen ratio, CT = soil condensed tannin concentration, MI = Maturity Index, SI = Structure Index, CI = Channel Index, PPI = Plant-Parasitic Index, BI = Basal Index, EI = Enrichment Index). The two centroids represent treatment (green = control, purple = fertilized). Sizes of points are proportional to abundance, each point representing one genus: (1)\u003cem\u003eAcrobeloides\u003c/em\u003e, (2) \u003cem\u003ePlectus\u003c/em\u003e, (3) \u003cem\u003eAphelenchoides\u003c/em\u003e, (4) \u003cem\u003eEudorylaimus\u003c/em\u003e, (5) \u003cem\u003ePrismatolaimus\u003c/em\u003e, (6) \u003cem\u003eMalenchus\u003c/em\u003e, (7) \u003cem\u003eDitylenchus\u003c/em\u003e, (8) \u003cem\u003eRhabditis\u003c/em\u003e, (9) \u003cem\u003eCephalobus\u003c/em\u003e, (10) \u003cem\u003eFilenchus\u003c/em\u003e, (11) \u003cem\u003eTeratocephalus\u003c/em\u003e, (12) \u003cem\u003eHelicotylenchus\u003c/em\u003e, (13) \u003cem\u003eEuteratocephalus\u003c/em\u003e, (14) \u003cem\u003eTylencholaimus\u003c/em\u003e, (15) \u003cem\u003eLelenchus\u003c/em\u003e, (16) \u003cem\u003ePanagrolaimus\u003c/em\u003e, (17) \u003cem\u003eWilsonema\u003c/em\u003e, (18) \u003cem\u003eEucephalobus\u003c/em\u003e, (19) \u003cem\u003eMesorhabditis\u003c/em\u003e, (20) \u003cem\u003eAporcelaimellus\u003c/em\u003e, (21) \u003cem\u003eBunonema\u003c/em\u003e, (22) \u003cem\u003eRhabdolaimus\u003c/em\u003e, (23) \u003cem\u003eAlaimus\u003c/em\u003e, (24) \u003cem\u003eDeladenus\u003c/em\u003e, (25) \u003cem\u003eDiploscapter\u003c/em\u003e, (26) \u003cem\u003eEumonhystera\u003c/em\u003e, (27) \u003cem\u003eCervidellus\u003c/em\u003e, (28) \u003cem\u003eSeinura\u003c/em\u003e, (29) \u003cem\u003eAcrobeles\u003c/em\u003e, (30) \u003cem\u003eClarkus\u003c/em\u003e, (31) \u003cem\u003ePungentus\u003c/em\u003e, (32) \u003cem\u003eRotylenchus\u003c/em\u003e, (33) \u003cem\u003eGeocenamus\u003c/em\u003e, (34) \u003cem\u003eDiphtherophora\u003c/em\u003e, (35) \u003cem\u003eCoslenchus\u003c/em\u003e, (36) \u003cem\u003eChiloplacus\u003c/em\u003e, (37) \u003cem\u003eEpidorylaimus\u003c/em\u003e, (38) \u003cem\u003eBoleodorus\u003c/em\u003e, (39) \u003cem\u003eParatylenchus\u003c/em\u003e, (40) \u003cem\u003eMicrodorylaimus\u003c/em\u003e, (41) \u003cem\u003eAllodorylaimus\u003c/em\u003e, (42) \u003cem\u003eTripyla\u003c/em\u003e, (43) \u003cem\u003eNygolaimus\u003c/em\u003e, (44) \u003cem\u003eSteinernema\u003c/em\u003e, (45) \u003cem\u003eMesodorylaimus\u003c/em\u003e, (46) \u003cem\u003eEudiplogaster\u003c/em\u003e, (47) \u003cem\u003eTylenchus\u003c/em\u003e, (48) \u003cem\u003eBastiania\u003c/em\u003e, (49) \u003cem\u003eProdorylaimus\u003c/em\u003e, (50) \u003cem\u003eNothotylenchus\u003c/em\u003e, (51) \u003cem\u003eParaphelenchus\u003c/em\u003e, (52) \u003cem\u003eAmphidelus\u003c/em\u003e, (53) \u003cem\u003eMylonchulus\u003c/em\u003e, (54) \u003cem\u003eEcphyadophora\u003c/em\u003e, (55) \u003cem\u003eDorylaimoides\u003c/em\u003e, (56) \u003cem\u003eCephalenchus\u003c/em\u003e, (57) \u003cem\u003ePratylenchus\u003c/em\u003e, (58) \u003cem\u003eDiscolaimus\u003c/em\u003e, (59) \u003cem\u003eCriconema\u003c/em\u003e, (60) \u003cem\u003eBasiria\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-2446831/v1/6f4dea8fea98128ab66f4cd2.png"},{"id":31692274,"identity":"3eb974b9-c344-48ff-9de7-ae4bae12ee28","added_by":"auto","created_at":"2023-01-17 15:48:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":107709,"visible":true,"origin":"","legend":"\u003cp\u003eBar plots illustrating the relative abundances of (a) nematode trophic groups and (b) the metabolic footprints of the trophic groups. Herbi = herbivore. Note that herbivore root feeders and parasites are merged into “herbivore footprint” in (b).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-2446831/v1/7f306066599c30ec9d490ad4.png"},{"id":31690545,"identity":"4535a5c2-e9b5-4a68-ad6a-00b0911b5f69","added_by":"auto","created_at":"2023-01-17 15:40:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":54950,"visible":true,"origin":"","legend":"\u003cp\u003eIllustration of the interaction effect between Channel Index and soil condensed tannin concentration (P = 0.006). Higher Channel Index value indicates increasing fungi dominated energy channel. In fertilized plots, Channel Index value increased with higher condensed tannin concentration (significant, indicated by the solid line). In control plots, Channel Index value decreased with higher tannin concentration (non-significant, indicated by the dashed line).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-2446831/v1/77e1f85c9744863fe86b7261.png"},{"id":34481187,"identity":"2769ef58-06a0-46ec-8647-271934ec1dd0","added_by":"auto","created_at":"2023-03-19 21:17:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1135015,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2446831/v1/c0e7ea2a-b0e2-4288-a2d7-6346e5c89f64.pdf"}],"financialInterests":"","formattedTitle":"Condensed tannins mediate the effect of long-term nitrogen addition on soil nematodes in a boreal spruce forest","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eThe availability of nitrogen (N) is a critical factor controlling plant productivity and soil processes within all ecosystems. Most boreal forests are N limited but are subject to N enrichment both through intentional fertilization and pollution. However, N addition to a primarily N limited forest will not only impact aboveground production; it will also have important implications for belowground structure and functioning (Meunier et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Soil microorganisms are the primary decomposers of organic matter, thus essential in carbon (C) and nutrient cycling, with fungi commonly dominating the decomposition pathway in boreal forests. However, increased N availability favours bacteria over fungi, causing a shift towards a more bacteria-dominated energy channel (Demoling et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; H\u0026ouml;gberg et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Maaroufi et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Shaw et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Such shifts in the microbial community may further affect the soil food web at higher trophic levels (Moore et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Wardle et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), possibly leading to a less structured and complex community after fertilization (Maaroufi et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Shaw et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Moreover, increased N availability may lead to reduced soil microbial activity and respiration rates, and subsequently increased C sequestration (Treseder, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Janssens et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Maaroufi et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Accordingly, the structure and functioning of soil food webs may be directly or indirectly affected by increased N availability. Directly through increased soil nutrient availability influencing soil conditions, or more indirectly through changes in plant community composition as well as litter production and quality (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn boreal Norway spruce (\u003cem\u003ePicea abies\u003c/em\u003e) forests, bilberry (\u003cem\u003eVaccinium myrtillus\u003c/em\u003e) and other ericaceous dwarf shrubs (e.g. \u003cem\u003eV. vitis-idaea\u003c/em\u003e and \u003cem\u003eEmpetrum nigrum\u003c/em\u003e) commonly dominate the understory vegetation. Norway spruce and ericaceous dwarf shrubs all produce large quantities of condensed tannins (hereafter tannins), one of the main classes of phenolic defence compounds (H\u0026auml;ttenschwiler \u0026amp; Vitousek, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Boreal forest soils are thus rich in tannins. As defence compounds, tannins are known to reduce microbial activity and deter microfauna, thereby altering soil community structure. (H\u0026auml;ttenschwiler \u0026amp; Vitousek, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Additionally, some studies report a higher inhibitory effect on bacteria compared to fungi (Kanerva et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Mutabaruka et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Ushio et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Tannins in soil also play a role in the N cycle by forming recalcitrant complexes with N-rich organic compounds, thereby inhibiting N mineralization processes (Kraus et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Chomel et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The afterlife effects of tannins in soil are thus considerable. However, as plant-derived compounds, their concentration in the soil is controlled by the aboveground plant community.\u003c/p\u003e \u003cp\u003eIncreased N may affect tannin concentrations through changes both within- and between plant species (e.g., species turnover). According to the protein competition model, within species tannin concentrations are likely to decrease with increased nutrient availability (Wright et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). This is because growth proteins and phenolic compounds compete for the same amino acid precursor phenylalanine. At higher N availability, the incorporation of phenylalanine into proteins will increase, and the production of phenolic compounds will decrease (Wright et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Further, an increase in soil fertility commonly leads to a shift from a community dominated by slow-growing species investing more C to structural and chemical defence to one dominated by species that allocate more C to growth and thus have higher foliage N and specific leaf area, and lower defence levels (Aerts, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Wardle et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Meunier et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The concentration of tannins in soil may thus vary with N availability, with further implications for soil processes.\u003c/p\u003e \u003cp\u003eSoil biota is an essential part of the ecosystem, and play important roles in nutrient and C cycling and by modifying soil community structure (Ferris, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Nielsen et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Bardgett \u0026amp; Van Der Putten, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Jes\u0026uacute;s \u0026amp; Briones, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Among these, nematodes are ubiquitous in soil, occupy several trophic niches, and respond quickly to environmental changes (Ferris et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). They are thus useful in determining changes in ecosystem status (Neher, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Du Preez et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In 1990 the first nematode-based index, the Maturity Index, was established to assess level of disturbance (Bongers, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). Later, a complex toolset of indices has been developed (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), and several studies have used nematode communities and nematode-based indices to assess the effect of disturbances on soil food web (Čerevkov\u0026aacute; et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Renčo et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Du Preez et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Renčo et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). The nematode indices are calculated by assigning taxa into functional guilds (Bongers, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Yeates et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Ferris et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) with similar feeding habit and life history strategy, i.e. if they are colonizers (r-selected) or persisters (K-selected). Colonizers are more tolerant to disturbances due to their short generation time, large population fluctuations and high fecundities. Persisters on the other hand, have long life cycles, low reproductive rates, low metabolic activities, and slow movement; they are thus sensitive to disturbances.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAn overview of the indices used in this study and what they indicate (Du Preez et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIndex\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIndicates\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaturity Index\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSuccessional maturity\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStructure Index\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFood web complexity and disturbances\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChannel Index\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDecomposition dominance by bacteria vs. fungi\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnrichment Index\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNutrient availability\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBasal Index\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFood web complexity\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlant-Parasitic Index\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCompilation of plant-parasitic nematodes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMetabolic Footprints\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMagnitude of ecosystem services fulfilled by nematode community\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eHere, we used nematodes as indicators to study the linkages between N-induced changes in soil tannin concentration and the soil food web. We utilized a long-term Norway spruce fertilization experiment where plots have been fertilized annually since 2003 resulting in a shift in the understory from a community dominated by bilberry, producing high levels of tannins, and feathermosses to a system dominated by grasses and forbs, with no or low amounts of tannins (Lorentzen, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Our overall aim was to investigate the long-term effect of fertilization on the soil ecosystem defined by nematode communities and indices, and we specifically wanted to test the hypotheses that fertilization will (i) favour bacteria over fungi leading to a lower Channel Index; (ii) decrease soil tannin concentrations, which will ease bacteria and thus further enhance the effect on the Channel Index; and (iii) lead to a less structured nematode community that are more dominated by short-lived stress-tolerant species. By testing these three hypotheses, we will advance the understanding of how high fertilization rates affect soil food web.\u003c/p\u003e"},{"header":"2 Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Study site and sampling\u003c/h2\u003e \u003cp\u003eThe study was conducted in an \u0026gt;\u0026thinsp;200 year old sub-alpine boreal Norway spruce (\u003cem\u003ePicea abies\u003c/em\u003e) dominated forest east of Dokkfl\u0026oslash;yvatn, Gausdal Vestfjell, SE Norway (61\u0026deg;10\u0026rsquo;N, 09\u0026deg;90\u0026rsquo;E, 800 m a.s.l.). Bilberry (\u003cem\u003eVaccinium myrtillus\u003c/em\u003e) and feathermosses dominated the understory. A fertilization experiment was established in 2003 with 10 fertilized 15\u0026times;15 m plots, and 10 control plots of the same size (Davey et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Granulated pellets containing 24.6% N, 2% P, 6% K and trace elements (YaraMila Fullgj\u0026oslash;dsel) have been added yearly since 2003 at a rate of 150 kg ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e year\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in fertilized plots, locally placed to avoid runoff to control plots. The N addition (ca. 37 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e year\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was significantly higher than the upper rates of N deposition in the southern boreal zone of northern Europe (12 kg ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e year\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; Gundale et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). As such, it represents an extreme change, but is comparable with many long-term forest fertilization experiments in boreal forests (e.g. Sponseller et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn June 2020, we sampled the entire organic soil and approximately 4 cm of the mineral soil using a \u0026oslash;10 cm soil corer at five sub-plots per plot. Sub-plots were established as followed: A grid of 5\u0026times;5 m squares within the 15\u0026times;15 m plots were defined. Four sub-plots were located at each crossing point, in addition to one sub-plot in the centre of the 15\u0026times;15 m plot. Soil cores were divided vertically in the field, where one half was saved for nematode analyses (both organic and mineral layer combined). From the other half, the organic layer was used for chemical analyses. Fresh-weight of soil samples was measured immediately after sampling. Further, in September 2020, senesced \u003cem\u003eV\u003c/em\u003e. \u003cem\u003emyrtillus\u003c/em\u003e leaves and \u003cem\u003eP\u003c/em\u003e. \u003cem\u003eabies\u003c/em\u003e needles still attached to the plant (for tannin analyses) were sampled randomly at plot-level. Only leaves and needles that detached easily were collected. All samples were stored at room temperature until further processing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Sample preparation and analyses\u003c/h2\u003e \u003cp\u003eOrganic soil samples for chemical analyses were dried at 30\u0026deg;C until stable weight (at least 48 h). Gravimetric soil water content (hereafter soil moisture) was determined after drying ((wet weight \u0026ndash; dry weight)/dry weight). Green parts, roots and twigs larger than approx. 2 mm in diameter were removed. The samples were then homogenized by grounding into fine powder using a Retsch MM 400 ball mill (Retsch, Haan, Germany) at 30 rpm. Senesced \u003cem\u003eV\u003c/em\u003e. \u003cem\u003emyrtillus\u003c/em\u003e leaves and \u003cem\u003eP\u003c/em\u003e. \u003cem\u003eabies\u003c/em\u003e needles were air-dried in paper bags for 15 days and then powdered the same way as the soil samples.\u003c/p\u003e \u003cp\u003eWe determined soil C and N concentrations in 5\u0026ndash;7 mg sub-samples by combustion at 950\u0026deg;C using a vario MICRO cube analyser (Elementar Analysensysteme GmbH, Hanau, Germany) with a thermal conductivity detector. For soil pH analysis, 5 ml of soil was diluted in 12.5 ml of dH\u003csub\u003e2\u003c/sub\u003eO for 24 h. The pH value was then measured using an inoLab pH 720 precision pH meter (WTW GmbH, Weilheim, Germany), following the manufacturers protocol.\u003c/p\u003e \u003cp\u003eFor tannin extraction, 4 ml 70% acetone was added to glass tubes containing 100 mg of powdered soil or 10 mg of powdered leaves or needles, respectively. The samples were then mixed on a planar shaker (KS 501 digital, IKA-WERKE, Germany) at 200 rpm for 1 hour, followed by centrifugation (10 min, 1,500 x g; Hettich Universal 16 centrifuge [Hettich Zentrifugen, Kirchlengern Germany]). The supernatant was transferred to a separate glass tube, and the extraction process was repeated twice. The mixed supernatants were then evaporated in a vacuum centrifuge (Eppendorf concentrator plus; Eppendorf, Hamburg, Germany) and stored in a freezer (-18\u0026deg;C) until further analysis.\u003c/p\u003e \u003cp\u003eConcentration of tannins in both soil and leaves and needles were determined following the acid butanol assay for proanthocyanidins (condensed tannins) described in Hagerman (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The extracts were redissolved in 0.5 ml MeOH and mixed properly. Further, 3 ml acid butanol (95% butanol, 5% HCl) and 0.1 ml iron reagent (2% ferric ammonium sulphate in 2N HCl) were added before placing the samples in boiling water for 1 hour. After cooling, the absorbance at 550 nm was determined using a spectrophotometer (UV-1800; Shimadzu Corp., Kyoto, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Nematode extraction and analyses\u003c/h2\u003e \u003cp\u003eSoil samples were homogenized by gentle hand mixing and soaked in 1 L of tap water for 30\u0026ndash;60 min. A combination of Cobb sieving and decanting (Cobb, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1918\u003c/span\u003e) and a modified Baermann technique (Baermann, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1917\u003c/span\u003e) was used for nematode extraction. Firstly, an aqueous soil suspension was sieved in 1 mm (16 mesh) and 0.05 mm (300 mesh) sieves. The obtained suspension with nematodes without coarse soil was then placed on a funnel contained a sieve 0.5 mm (30 mesh) and a set of two cotton\u0026ndash;propylene filters. Sub-samples were removed after 48 h of extraction at room temperature. The aqueous suspensions were examined under a stereomicroscope (40 and 60\u0026times; magnification), excessive water was removed and the nematodes were fixed with a hot 99:1 solution of 4% formaldehyde:pure glycerol (Seinhorst, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e1962\u003c/span\u003e). The nematodes were microscopically (100, 200, 400, 600 and 1000\u0026times; magnification) identified to the genus level using an Eclipse 90i Nikon light microscope (Nikon, Tokyo, Japan) from temporary slides using keys, including those of Brzeski (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1998\u003c/span\u003e), Andr\u0026aacute;ssy (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), Geraert (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) Loof (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) and several others.\u003c/p\u003e \u003cp\u003eNematodes in each sample were assigned into trophic groups based on their feeding habits, according to Yeates et al. (\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) and Wasilewska (\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), adjusted and supplemented following Sieriebriennikov et al. (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2014\u003c/span\u003e): bacterivores, fungivores, root-fungal feeders (facultative plant parasites), obligatory plant parasites, predators and omnivores. We calculated basic ecological indices, commonly used to assess the status of the soil ecosystems using nematode communities. Plant-Parasitic Index and Maturity Index were calculated following Bongers (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). Enrichment, Structure, Channel and Basal Index were calculated following Ferris et al. (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Metabolic footprints, a metric of the magnitudes of ecosystem functioning and services, were calculated following Ferris (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) for each trophic group. The footprint incorporates both the lifetime amount of C partitioned into growth and egg production, and the C use in metabolic activity (respiration). All indices and footprints were calculated using the NINJA automated calculation system (Sieriebriennikov et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Data analysis\u003c/h2\u003e \u003cp\u003eThe Shannon index of species diversity (Shannon, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e1948\u003c/span\u003e) was calculated as:\u003c/p\u003e \u003cp\u003eH\u0026acute; = \u0026minus;\u0026sum;\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e\u0026times; ln\u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e\u003c/p\u003e \u003cp\u003ewhere \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003ei\u003c/em\u003e\u003c/sub\u003e is the proportion of individuals of the \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003ei\u003c/span\u003eth taxon.\u003c/p\u003e \u003cp\u003eAll statistical analyses were conducted in R version 4.2.2 (R Core Team, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo visualize the nematode community composition, we performed a global nonmetric multidimensional scaling (GNMDS), based on a Bray-Curtis distance matrix with the \u0026ldquo;metaMDS\u0026rdquo; function in the \u003cem\u003evegan\u003c/em\u003e package (Oksanen et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), using settings as recommended by Liu et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The \u0026ldquo;envfit\u0026rdquo; (permutations\u0026thinsp;=\u0026thinsp;999) function were used to test if soil variables (i.e. soil N, C and tannin concentration, C:N ratio, moisture and pH) and nematode indices correlated with the nematode community structure. Significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) correlated variables were fitted onto the diagram. The \u0026ldquo;ordiellipse\u0026rdquo; function was used to plot 95% confidence intervals of the treatment centroids.\u003c/p\u003e \u003cp\u003eFor modelling the effect of treatment on soil variables, linear mixed effects models were used using the \u0026ldquo;lme\u0026rdquo; function of the \u003cem\u003enlme\u003c/em\u003e package (Pinheiro et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), with plots as a random effect. The same function was used when modelling the interaction between Channel Index and soil tannin concentration, in addition to \u0026ldquo;ggpredict\u0026rdquo; in the \u003cem\u003eggeffect\u003c/em\u003e package (L\u0026uuml;decke, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) as well as \u003cem\u003eggplot2\u003c/em\u003e (Wickham, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) for graphical plotting. The effect of treatment on tannin concentration in bilberry leaves and spruce needles was modelled in a linear model using the \u0026ldquo;lm\u0026rdquo; function in the \u003cem\u003estats\u003c/em\u003e package (R Core Team, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cp\u003eFertilization had a significant effect on all measured soil variables and nematode indices (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Soil N and C concentration were on average 26% and 6% higher in fertilized plots, respectively. Thus, the C:N ratio decreased. Fertilization further led to a lower soil pH. Moreover, soil tannin concentration was 30% higher in control plots, whereas fertilization had no effect on tannin concentration in senesced \u003cem\u003eV. myrtillus\u003c/em\u003e leaves and \u003cem\u003eP\u003c/em\u003e. \u003cem\u003eabies\u003c/em\u003e needles (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean values\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE) of soil variables, condensed tannin concentrations of senesced \u003cem\u003eV. myrtillus\u003c/em\u003e leaves and \u003cem\u003eP\u003c/em\u003e. \u003cem\u003eabies\u003c/em\u003e needles and nematode indices. CT\u0026thinsp;=\u0026thinsp;condensed tannins, N\u0026thinsp;=\u0026thinsp;nitrogen, C\u0026thinsp;=\u0026thinsp;carbon. Bold numbers indicate statistical significance (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). *Samples collected at plot-level (i.e., not subplot-level).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean control\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean fertilized\u0026thinsp;\u0026plusmn;\u0026thinsp;SE\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003et-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoil pH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e3.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-2.61\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.018\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoil CT (mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e6.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e5.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-2.23\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.039\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoil N (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e1.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e1.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e6.96\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoil C (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e38.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e41.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e2.55\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.020\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoil C:N ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e31.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e26.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-4.9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoil moisture\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.946\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eV. myrtillus\u003c/em\u003e CT*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e176.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e168.3\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.349\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. abies\u003c/em\u003e CT*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e112.6\u0026thinsp;\u0026plusmn;\u0026thinsp;10.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e105.5\u0026thinsp;\u0026plusmn;\u0026thinsp;14.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e-0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.694\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eNematode indices\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaturity Index\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e2.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e2.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-18.27\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStructure Index\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e74.79\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e44.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.51\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-13.78\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBasal Index\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e20.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e32.54\u0026thinsp;\u0026plusmn;\u0026thinsp;1.07\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e6.8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlant-Parasitic Index\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e2.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e2.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-10.07\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eChannel Index\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e41.44\u0026thinsp;\u0026plusmn;\u0026thinsp;2.11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e26.95\u0026thinsp;\u0026plusmn;\u0026thinsp;1.60\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-3.94\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnrichment Index\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e48.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e56.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.28\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e3.02\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.007\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShannon Diversity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e2.96\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e2.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-8.45\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e0.000\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Soil community structure\u003c/h2\u003e \u003cp\u003eThe nematode community composition was clearly influenced by fertilization, as shown by the clear separation in the GNMDS ordination (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Except soil C concentration and moisture, all measured soil variables (i.e., soil tannin and N concentration, C:N ratio and pH) and nematode indices significantly correlated with nematode community structure. Fertilized plots inhabited a more disturbed and less-structured nematode community with lower diversity in contrast to more mature and complex communities in the controls. Both Maturity Index and Structure Index, related to soil food web disturbance and community structure (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), had significantly lower values after fertilization (22% and 41% respectively; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). At the same time, a higher Basal Index value indicated a more depleted food web in fertilized plots. Moreover, the Enrichment Index, based on the expected responsiveness of the opportunistic guilds to organic-resource enrichment, was 15% higher in fertilized plots.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe observed a distinct difference in the relative abundance of trophic groups between treatments (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Fertilization led to a nematode community dominated by bacterivores. The relative abundance of omnivores, predators and herbivores were negatively affected by fertilization, whereas fungivores had a proportionally slight increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Furthermore, fertilization led to a considerable decline in plant parasites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), resulting in a lower Plant-Parasitic Index (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The relative distribution of trophic groups in terms of their metabolic footprint \u0026ndash; i.e., how much they contribute to C utilization, and thus to soil processes (see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) \u0026ndash; gave a slightly different picture. The bacterivore footprint dominated in fertilized plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Fungivore footprint was unaffected by N addition, whereas the footprint of omnivores decreased considerably. Predator and herbivore footprints turned in to negligible after fertilization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effect of condensed tannins on Channel Index\u003c/h2\u003e \u003cp\u003eChannel Index was 54% higher in control plots, indicating a more fungi dominated energy channel here (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This is also demonstrated by the Channel Index vector fitted onto the ordination diagram (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Furthermore, Channel Index was negatively affected by soil tannin concentration in the fertilized plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This suggests that fertilized plots with higher tannin concentrations approaches a more fungi dominated energy channel, whereas fertilized plots with lower tannin concentrations have more bacteria dominated energy channel. This response was, however, not significant in the control plots.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eWe found support for our first hypothesis that fertilization favour bacteria over fungi resulting in a more bacteria-based Channel Index (i.e., low Channel Index). Fertilization decreased soil tannin concentrations, in line with our second hypothesis. We also found that the lower soil tannin concentrations in fertilized plots further facilitated the bacteria-based energy channel. Our last hypothesis, that fertilization would lead to a more disturbed soil food web, was also supported, demonstrated by the change in nematode community and nematode-based indices.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Direct and indirect effects of fertilization on dominating energy channel\u003c/h2\u003e \u003cp\u003eOur study indicate that the soil food web changed from fungi-dominated to bacteria-dominated in response to N addition. In general, soils with high nutrient availability are associated with a bacteria-dominated energy channel and lower fungi:bacteria ratio (Moore et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Van Der Heijden et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; H\u0026ouml;gberg et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Fertilization thus had a direct bottom-up effect on the bacterial advantage relative to fungi (indicated through Channel Index and by nematode feeding groups). This led to a lower fungi:bacteria ratio by increasing the amounts of easily available N, in line with other studies (De Vries et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Treseder, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Maaroufi et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe N-induced shift in the understory may also have enhanced the effect on the energy channel through positive plant-soil-feedbacks. Slow growing plant species in the control plots like ericaceous shrubs and feathermosses produce low quality litter which promotes fungi (ectomycorrhizal and ericoid mycorrhizal) due to their ability to decompose recalcitrant organic matter and exploit organic bound N (Bending \u0026amp; Read, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; B\u0026ouml;deker et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Lindahl \u0026amp; Tunlid, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). On the other hand, fast-growing plant species like grasses and forbs in the fertilized plots produce high quality litter that are easily available for bacteria. Thus, different plant functional groups may be associated with either a fungal or bacterial dominated soil community (Wardle et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Van Der Heijden et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Eskelinen et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurther, our data suggest that the decline in soil tannins amplified the fertilization and vegetation effect on the bacterial community. However, this effect was only significant in fertilized plots. An explanation for this could be that the low N availability is a limiting factor for bacteria in the control plots, and thus overrides the negative effect of tannins here. The lower soil tannin concentration in fertilized plots compared to control plots is in line with another boreal forest fertilization study (Smolander et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Since N addition did not have any effect on the tannin concentration of leaves and needles, the lower soil tannin levels likely resulted from the decline in \u003cem\u003eV. myrtillus\u003c/em\u003e abundance. Tannins negatively influence microbes, both through enzyme inhibition and direct toxic effects, but it is discussed whether fungi or bacteria are more sensitive to tannins (Scalbert, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Kraus et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Smolander et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). However, both Mutabaruka et al. (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) and Ushio et al. (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) explained higher fungi:bacteria ratio in their studies from tropical forests by higher concentrations of soil tannins. Further, the ability of fungi, especially ericoid mycorrhiza, to exploit N from tannin-protein complexes (Bending \u0026amp; Read, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Joanisse et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Wurzburger \u0026amp; Hendrick, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), may additionally have promoted fungal advantage over bacteria the in controls in our study.\u003c/p\u003e \u003cp\u003eFungivorous and bacterivorous nematodes have long been recognized as useful indicators of the abundances of fungi and bacteria in soil (Ferris et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Neher, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). However, in the studies by De Long et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and Maaroufi et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), the nematode fungivore and bacterivore ratios did not clearly mirror the respective microbe ratios (based on microbial phospholipid fatty acids method (PLFA)). Further, Cesarz et al. (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) found that functional guilds (i.e. feeding habits combined with life history strategies) within one trophic group, had different responses to the treatment in their study. They thus suggested that functional guilds, which are used to calculate the indices (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), are better in reflecting shifts in soil communities rather than trophic groups. Several studies report a reduction in total microbial abundance, but also that fungi are particularly sensitive to N addition (H\u0026ouml;gberg et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Demoling et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Treseder, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Maaroufi et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). We observed no major change in the relative abundance of fungivores and a large relative increase in bacterivores in response to N addition. However, the explanation that the nematode-based indices reflect the microbial community more correctly than the feeding groups may also be applied to our study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Effect of fertilization on nematode communities and soil food web\u003c/h2\u003e \u003cp\u003eAdding heavy loads of N to a forest developed under N-limitation had large impacts on the soil food web leading to lower diversity and maturity. This indicates a disturbed system with fewer trophic links. Overall, the fertilized plots inhabited less omnivores and predators and a relatively high abundance of bacterivores. Omnivores and predators, i.e. persisters, are more susceptible to N addition and other disturbances (Bongers, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1990\u003c/span\u003e; Ferris et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). On the contrary, most colonizers are bacterivores responding rapidly to environmental changes. When the food web is dominated by bacterivorous colonizers with short lifecycles and high respiratory rates, less amounts of C are assimilated to higher trophic levels (Ferris \u0026amp; Bongers, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Additionally, as mentioned above, several studies have shown that fertilization reduce microbial activity. As microbes are an important C source for nematodes, this combination may have caused the lower abundance of nematodes at higher trophic levels in the fertilized plots in this study. Furthermore, the lower pH after fertilization may have affected omnivores and predators negatively (reviewed in Xing et al. (\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)).\u003c/p\u003e \u003cp\u003eBoth Maturity Index and Plant-Parasitic Index had lower values in fertilized plots compared to the untreated controls in this study. It has been claimed that these indices usually act in the opposite direction of each other (Bongers et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). A low Maturity Index value indicate low soil food web maturity because N enrichment result in a stimulation of opportunistic species, whereas the Plant-Parasitic Index tend to increase with increasing nutrients due to the consequently better quality in the associated plant root cells. However, the fertilizers in this study contain 13.8% ammonia, which is reported to be toxic to plant parasites (Rodr\u0026iacute;guez-K\u0026aacute;bana, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e1986\u003c/span\u003e; Wei et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Xing et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and this toxicity may override the positive effect of higher root quality. Furthermore, our finding that fertilization exclude plant parasitic nematodes corresponds with the decline in \u003cem\u003eV. myrtillus\u003c/em\u003e cover in favour of a more graminoid dominated community (Lorentzen, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This pattern was also found in Renčo et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). We cannot assure a causal relationship, but some of the nematode genera that had a substantial decline in abundance in fertilized plots (i.e. \u003cem\u003eHelicotylenchus\u003c/em\u003e and \u003cem\u003ePratylenchus\u003c/em\u003e) are known parasites on \u003cem\u003eV. corrymbosum\u003c/em\u003e, a species closely related to \u003cem\u003eV. myrtillus\u003c/em\u003e. However, these reports are from agriculture and might be of low relevance to our system (Jagdale et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Rivera et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Another possible explanation could be that fertilization lead to higher abundances of bacteria which suppress plant-parasitic nematodes by colonizing roots (Siddiqui \u0026amp; Mahmood, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1999\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Conclusions\u003c/h2\u003e \u003cp\u003eThis study has shown that both direct and indirect effects must be considered when examining the consequences of increased N availability in boreal forests. The soil community structure strongly changed upon N addition. Such transitions may consequently impact soil functions. Under ambient conditions, nutrient rich soils typically have a bacteria dominated energy channel associated with faster decomposition, higher N mineralization rates and C fluxes. However, high N addition leads to soil acidification through oxidation of ammonia to nitrate. This inhibits microbial activity and organic matter accumulates, even with a bacteria dominated energy channel. Accordingly, the amount of N added determines whether the ecosystem will have a fast or slow decomposition pathway, which subsequently have consequences for soil C sequestration.\u003c/p\u003e \u003cp\u003eFurthermore, the changes in soil nutrient availability, community structure and function influence the plant community, litter quality and production. The likely effect of tannins on bacteria in fertilized plots corroborate that plant-derived compounds are important players in soil processes. However, it also demonstrates that when N is no longer a limiting nutrient, other factors affecting soil biota may emerge. Thus, assessing the interaction between members of soil food web and the elements involved in nutrient cycling, including plant secondary metabolites, should be a subject for future studies. This could generate valuable insight into how N enrichment through fertilization or pollution affect ecosystem processes in boreal forests.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003cstrong\u003es\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge Mikael Ohlson for initiating the fertilization experiment in 2003. We thank Annie Aasen, Claus Kreibich and Puspa Subedi for help with sampling and chemical analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding: \u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest/Competing interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material:\u003c/strong\u003e Data associated with this manuscript are deposited in the NMBU Open Research Data database (doi will be given if the manuscript is accepted).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions:\u003c/strong\u003e JA and LN designed the study. Field work was conducted by LRT, ZF, JA and LN. ZF performed laboratory analyses under supervision of LN and JA. MR extracted and identified nematodes. Data analyses were done by LRT. LRT led the writing under supervision of JA, LN and TB. All authors contributed to revisions and discussions and approved the final version.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAerts, R. (1999). Interspecific competition in natural plant communities: Mechanisms, trade-offs and plant-soil feedbacks. \u003cem\u003eJournal of Experimental Botany\u003c/em\u003e, \u003cem\u003e50\u003c/em\u003e(330), 29\u0026ndash;37. https://doi.org/10.1093/jxb/50.330.29\u003c/li\u003e\n\u003cli\u003eAndr\u0026aacute;ssy, I. (2005). Free-living nematodes of Hungary (Nematoda errantia), Volume I. Budapest, Hungary, Hungarian Natural History Museum and Systematic Zoology Research Group of the Hungarian Academy of Sciences.\u003c/li\u003e\n\u003cli\u003eAndr\u0026aacute;ssy, I. 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Feeding Habits in Soil Nematode Families and Genera-An Outline for Soil Ecologists. \u003cem\u003eJournal of Nematology\u003c/em\u003e, \u003cem\u003e25\u003c/em\u003e(3), 315\u0026ndash;331. pmid: 19279775; PMCID: PMC2619405.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Fertilization, Plant secondary metabolites, Energy channel, Food webs, Interactions","lastPublishedDoi":"10.21203/rs.3.rs-2446831/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2446831/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFertilization and combustion have increased nitrogen (N) deposition over the last decades. Most boreal forests are N limited; thus, increased N availability may have considerable implications for boreal forest food web structure and functioning. Soil biota are important drivers of ecosystem processes through their effect on carbon (C) and nutrient cycling. Among these, nematodes are ubiquitous in soil and respond quickly to environmental changes. They are thus useful in determining changes in ecosystem status. Boreal forest plants produce large quantities of condensed tannins (hereafter tannins), and these may decrease following N addition. As defence compounds, tannins may alter soil community structure by reducing microbial activity and deter microfauna. Using nematodes as indicators, we here investigated the linkages between N-induced changes in soil food web and soil tannin concentration. We utilized a fertilization experiment where plots have been fertilized since 2003 at a rate of 150 kg ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e yr\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in the form of granulated pellets containing 24.6% N, 2% P, 6% K and trace elements. This have resulted in a shift in the understory, and consequently a decrease in soil tannin concentration. Fertilization led to a less structured nematode community dominated by bacterivores. The abundance of bacterivores relative to fungivores increased, indicating a more bacteria-dominated energy channel. Furthermore, the dominance of bacteria appears to have been inhibited by soil tannin concentrations in the fertilized plots. Overall, our study demonstrate that soil community structure strongly changes upon increased N availability, and lower soil tannin concentrations further facilitate the dominance of bacteria.\u003c/p\u003e","manuscriptTitle":"Condensed tannins mediate the effect of long-term nitrogen addition on soil nematodes in a boreal spruce forest","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-01-17 15:40:41","doi":"10.21203/rs.3.rs-2446831/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f46b5e31-1956-473e-9499-4acc186b16ef","owner":[],"postedDate":"January 17th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-03-19T21:16:48+00:00","versionOfRecord":[],"versionCreatedAt":"2023-01-17 15:40:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2446831","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2446831","identity":"rs-2446831","version":["v1"]},"buildId":"omnImTCwR2MFx8CMYfrG7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}