Study region and experimental design
This study was part of the long-term experiment BELongDead within the Biodiversity Exploratories project, which focuses on the interactions of tree species, microbial communities, and local conditions at three exploratories in Germany (Schwäbische Alb, Schorfheide-Chorin, and Hainich-Dün). In each exploratory, forest plots with different management intensities in threefold repetition were chosen to represent local management practices (Kahl et al. 2015).
The forests in the Hainich-Dün exploratory in Central Germany (50°56’14’’ − 51°22’43’’N/10°10’24’’–10°46’45’’E) were dominated by European beech (Fagus sylvatica). The study region was characterized by mean annual temperatures of 6.5 to 8°C, mean annual precipitation of 500 to 800 mm, and altitude from 285 to 550 m a.s.l.. Soils were characterized as Luvisols and Stagnosols, which developed on calcareous bedrock with a loess layer (Fischer et al. 2010; Kahl et al. 2017). N deposition was 10.1 ± 0.8 kg ha− 1 per vegetation period in the Hainich-Dün exploratory (Schwarz et al. 2014). Different forest management practices include selective timber forest, timber forest, and unmanaged forest (Fischer et al. 2010).
On each of the nine experimental plots in Hainich-Dün, the BELongDead experiment was established in the winter of 2008/09. Freshly cut logs (4 m length, ~ 31 cm diameter) of 13 tree species were placed on the soil in random order within 1 m distance to each other. The 13 tree species included four gymnosperms (Larix decidua, Picea abies, Pinus sylvestris, and P. menziesii), seven diffuse-porous angiosperms (Acer spp, Betula pendula, Carpinus betulus, F. sylvatica, Populus spp., Prunus avium, and Tilia spp.) and two ring-porous angiosperms (Fraxinus excelsior and Quercus spp.) (Kahl et al. 2017). All logs originated from the state forest of the Federal State of Thuringia in Central Germany and were thus grown under similar climatic conditions.
Sample collection
In June 2020, deadwood samples were cut laterally with an electric saw from the outer 7 cm of each log. Deadwood samples consisted almost exclusively of sapwood, but mixtures of sapwood and heartwood were taken from Quercus logs due to the decay and loss of sapwood. One part of the sample was stored in a plastic bag in the field and kept cool (~ 5°C) until further treatment. A second part of the sample (sawdust) was transferred into a 15 mL falcon tube in the field. After that, 10 mM bromodeoxyuridine (BrdU) was added sufficiently until the sawdust was completely soaked with solution. The falcon tubes were placed in an upright position and covered with aluminum foil, followed by incubation at room temperature for 48 h. The samples were then frozen at -80°C in the laboratory before the characterization of the abundance of metabolically active diazotrophs (see below).
BNF
At the laboratory, deadwood samples (~ 20 cm³) were saturated with water for 16 h to achieve maximum water holding capacity in all samples, then drained, and weighted into glass flasks (~ 131 mL, Media Bottle Rasotherm ISO, GL45). After five days of acclimatization in the dark at 20°C, the lids were equipped with a 6 mm thick chlorbutyl septum. After closure, the glass flasks were flushed for 10 min with a prepared gas mixture consisting of 8% oxygen (O2), 30% N2, and 62% helium (He) (Riessner-Gase GmbH, Lichtenfels, Germany). To determine the BNF rate (Weaver and Danso 1994), 25 mL of acid-washed 15N2 (99.1%, batch number MBBC7366, Sigma-Aldrich Inc., St. Louis, MI, USA) was added to each glass flask using a syringe. The gas pressure was measured before and after the addition of 15N2 to calculate the exact proportion of 15N2 in the gas phase in the glass flasks. After adding 15N2, the samples were incubated at 20° C for 72 h in the dark. The flasks were then opened, and the samples were immediately dried at 60°C for at least 72 h until mass constancy to terminate the incubation. The WC of the samples was determined by the wet and dry weight of the incubation samples. The dried samples were ground at low temperatures using a ball mill (MM400, Retsch GmbH, Haan, Germany). Incubated and respective non-incubated subsamples were analyzed for δ15N signatures and N concentration at the Centre for Stable Isotope Research and Analysis, Göttingen, using an element analyzer (Euro EA 3000, EuroVector S.p.A., Milano, Italy), coupled to a Delta C isotope mass spectrometer with a ConFlo III interface (Thermo Electron, Bremen, Germany) and an element analyzer (Euro MA 3000, EuroVector S.p.A., Milano, Italy), coupled to a Delta C isotope mass spectrometer with a ConFloII interface (Thermo Electron, Bremen, Germany) for natural abundance and 15N enriched samples, respectively.
As certified 15N2 gas (Sigma-Aldrich Inc., St. Louis, MI, USA) contained significant amounts of NH3 and N oxides (Dabundo et al. 2014), the 15N2 gas was acid-washed before adding to deadwood samples. For this purpose, an acid solution with a pH of 3 to 4 was produced by adding 100 mL deionized water and ~ 2 µL of 2 M H2SO4 (95%, Chemsolute, Th. Geyer GmbH & Co.KG, Renningen, Germany) to a 650 mL glass flask. The flask was closed with a screw cap and a gas-tight, 6 mm thick chlorbutyl septum. After that, the bottle was evacuated to 200 mbar, flushed with He 4.6 for 15 min, and then shaken overhead (6 rpm) for 1 h. This procedure of evacuating, flushing, and shaking was repeated three times. Afterwards, the bottle was evacuated to 200 mbar, and 15N2 gas was transferred directly from the 15N2 gas container to the flask up to a pressure of ~ 2200 mbar. The glass flask was shaken overhead for 1 h, left standing overnight, and shaken again for 1 h before use. Gas pressure measurements determined the proportion of 15N2 in the headspace before and after 15N2 addition. Finally, the purification amounted to a 15N2 concentration of 98 to 99% in the headspace. To determine potential contamination of the 15N2 gas, four wood samples were autoclaved at 121°C with 1 bar overpressure for 20 min. The samples were incubated and processed as described above. The enrichment in the δ15N signature in the autoclaved deadwood samples was neglectable (0.003 ± 0.0007 µg 15N per ml 15N2).
After the conversion of δ15N signatures of enriched and non-enriched (natural abundance, NA) deadwood samples into 15N atom fractions, BNF rates (µg N g − 1 d− 1) referring to the dry weight of deadwood was calculated using Eq. 1:
$$BNF= \frac{N \times \left({}_{ }{}^{15}{N}_{enriched}-{}_{ }{}^{15}{N}_{NA}\right)}{DW \times t \times HS \times 100 }$$
1
where N is the amount of N in deadwood (µg), 15Nenriched is 15N atom% in deadwood after incubation, 15NNA is the 15N atom% natural abundance in deadwood, DW is the dry weight of deadwood (g), t is the incubation time (d), and HS is the ratio of 15N2 to total N2 in the headspace of incubation flasks.
Respiration rate
The respiration rate of deadwood was determined by measuring the increase in carbon dioxide (CO2) concentration during the incubation. Gas samples of 100 µL were taken with a syringe from the glass jars after 21 h (t1) and 45 h (t2) after flushing with the CO2-free gas mixture at t0 (see above). The gas samples were directly injected into a gas chromatograph (SRI 8610C, SRI Instruments, Torrance, CA, USA) equipped with a flame ionization detector coupled to a methanizer catalyst. Certified gas standards (10,000 and 20,000 ppm CO2, Riessner Gase, Lichtenfels, Germany) were used to calibrate the gas chromatograph.
The respiration rate (µg C g− 1 h− 1) was calculated using Eq. 2:
$$respiration rate= \frac{{\varDelta CO}_{2}}{\varDelta t } \times \frac{{V}_{gas}}{DW} \times \frac{{{\rho }}_{air}}{R \times {T}_{air}} \times M$$
2
where ΔCO2 Δt− 1 (ppm h− 1) is the change in CO2 concentration within the incubation flasks during the incubation, assuming that the initial CO2 concentration was 0 ppm after flushing, Vgas (m³) is the gas volume of the incubation flasks, DW (g) is the dry weight of the deadwood sample, ρair (Pa) is the air pressure, R (8.314 J K− 1 mol− 1) is the gas constant, Tair (K) is the air temperature, and M (g mol− 1) is the molar mass of C.
NSC and elemental analyzes
The concentrations of ethanol-soluble NSC were measured at the Max Planck Institute of Biogeochemistry, Jena, as reported earlier (Landhäusser et al. 2018). Briefly, 1.5 mL of 80% ethanol was mixed with 30 mg of ground wood sample and heated to 90°C. After cooling down to room temperature, the mixture was centrifuged at 13,000 g for 1 min. The supernatant was filtered, and about 1 mL was injected into a High-Performance Anion Exchange Chromatograph with Pulsed Amperometric Detection (DIONEX ICS-3000 with CarboPac columns, Thermo Fisher Scientific Inc., Waltham, MA, USA) to analyze concentrations of glucose, arabinose, xylose, galactose, and total NSC. We assumed that starch was not present in deadwood after 12 years of decay.
Moreover, the concentrations of calcium (Ca), magnesium (Mg), potassium (K), phosphor (P), sulfur (S), manganese (Mn), and Mo were determined using pressure digestion. 1 g of dried wood sample was filled in vessels, and 12 mL of HNO3 (65%, Chemsolute, Th. Geyer GmbH & Co.KG, Renningen, Germany) was added. The mixture was microwaved (MARS6, CEM Corporation, Matthews, NC, USA) and filtered using sterile syringe filters (0.45 µm). Afterwards, the supernatant was analyzed at the Analytical Chemistry Lab, University Bayreuth, using an ICP-MS (Agilent 7500ce, Cetac ASX-510, Santa Clara, CA, USA) for Mo and an ICP-OES (Varian, Vista-Pro radial, Palo Alto, CA, USA) for Ca, Mg, K, P, S and Mn according to manufacture’s instructions. The C concentration was analyzed with a vario Max CN element analyzer (elementar Analysensysteme GmbH, Hanau, Germany) at the Department of Soil Ecology, University Bayreuth. With the concentrations of C and N (see above), the CN ratio was calculated.
In-situ deadwood BrdU labeling and molecular biological analysis
For in-situ labeling with BrdU, 1 mL of 10 mM BrdU solution (Sigma-Aldrich Inc., St. Louis, MI, USA) was added directly after sampling to each deadwood sample (~ 1 g) and incubated in sterile 50 mL tubes covered with aluminum foil with loose cap for 48 h at room temperature as explained earlier (Purahong et al. 2022). Only the metabolically active, replicating cells can incorporate the BrdU during DNA synthesis (McMahon et al. 2011). According to the manufacturer’s instructions, DNA was extracted from the BrdU-treated deadwood samples using Quick-DNA Fecal/Soil Microbe Miniprep Kit (Zymo, California, USA). We named this total DNA as it included all types of genomic DNA (dormant cells, dead cells, metabolically active, and replicating cells). As outlined earlier (McMahon et al. 2011; Purahong et al. 2022), this immunocapture approach was employed to separate the BrdU-labeled DNA from the total DNA. Briefly, for each sample, 2 µL monoclonal BrdU antibodies (1 mg µL− 1 mouse anti-BrdU, clone BU-33, Sigma-Aldrich Inc., St. Louis, MI, USA) were added to 18 mL denatured herring sperm DNA (1.25 mg mL− 1 in phosphate buffer saline (PBS), Promega, Walldorf, Germany), and then incubated for 45 min at 30°C to form antibody-herring sperm DNA complex. Denatured sample DNA (25 µL; ∼200 ng DNA + 10 µL PBS) was then mixed with antibody-herring sperm DNA complex and incubated for 30 min at 30°C to capture BrdU-labeled DNA. 6.26 µL washed Dynabeads ™ Goat Anti-Mouse IgG (Invitrogen, Life Technologies GmbH, Darmstadt, Germany) were then added to the incubated solution and rotated (66 rpm) for 30 min at room temperature on an RM-2 roll mixer (Carl Roth GmbH, Karlsruhe, Germany) to form the Dynabead complex (Dynabead-BrdU antibodies-BrdU-labeled DNA). After that, the Dynabead complex was treated eight times with 100 µL PBS–BSA solution (0.05 g bovine serum albumin in 50 mL phosphate-buffered saline) and washed with a DynaMagTM-2 (Invitrogen, Life Technologies GmbH, Darmstadt, Germany). BrdU-labeled DNA was isolated from the washed Dynabead complex by adding 20 µL BrdU solution (1.7 mM) followed by 35 min incubation under slow rotation. Finally, the BrdU-labeled DNA was separated from the Dynabeads using a DynaMag ™ -2 (Life Technologies GmbH, Darmstadt, Germany), thereafter named active DNA.
The abundance of nifH gene copy numbers in deadwood samples of the total and active DNA fraction was accessed by quantitative PCR (qPCR). Diazotrophic gene copy numbers were quantified by the primer set PolF and PolR (Poly et al. 2001). All reactions were performed in 96-well plates using the CFX96™ Real-Time System (Bio-Rad Laboratories GmbH, Feldkirchen, Germany), and nuclease-free master mix blanks were run as a negative control. Gene copy number was calculated by comparing PCR-cycle threshold values (CT) to a standard curve of triplicate 10-fold dilutions of genomic DNA. Genomic DNA extracted from a culture of Azotobacter vinelandii (DSM 2289) by employing the Quick-DNA Fecal/Soil Microbe Miniprep Kit was used to establish quantification standards for generating a standard curve. Petroff counting chamber (Paul Marienfeld GmbH, Germany) was used to determine the genomic DNA concentration (gene copies µL− 1). A five-point tenfold serial dilution of the A. vinelandii genomic DNA (10–100000 fg) was run in triplicate with each set of reactions to generate the standard curve. For nifH gene-based qPCR, the reactions were performed in 10-µL assays containing 5 µL SYBR® Green Supermix (Bio-Rad Laboratories GmbH, Feldkirchen, Germany), PolF and PolR primers (0.5 µL each of the primer (2.5 µM)), 3 µL sterile and nuclease-free water (Carl Roth GmbH, Karlsruhe, Germany) and 1 µL of either 1:10 diluted DNA-extract, ten-fold diluted standard DNA or 1 µL water as no template control. After an initial denaturation at 94°C for 5 min, 40 amplification cycles (regardless of the active or total fraction) were performed for 1 min at 95ºC (denaturation), 1 min at 55ºC (annealing), and 1 min 30 s at 72ºC (extension), followed by a final extension of 5 min at 72°C. Runs were completed with a melting analysis starting from 65°C to 95°C with temperature increments of 0.5°C and a transition rate of 5 s to check for product specificity and potential primer dimer formation. The purity of the amplified products was checked by electrophoresis on a 1.5% agarose gel. Multiple dilutions (non-dilution, with 1:10 and 1:100 dilution) were run simultaneously to check for inhibitors in qPCR assays. We observed no inhibition as the CT value shifted for each decimal dilution step in the same CT gap. CT and efficiency were calculated automatically by the Bio-Rad software CFX manager version 3.1.
Statistical analyses
Statistics were performed using R version 4.0.4 (R Core Team 2021) with additional packages. Data were screened visually for outliers with box plots and Cleveland’s dot plots. Normal distribution was checked with histograms, quantile-quantile plots, and the Shapiro-Wilk-Test. Significant differences (p < 0.05) were calculated using the Kruskal-Wallis-Test and pairwise Wilcoxon Rank Sum test for non-normal distributed data between tree species and phylogenetic clades (gymnosperms, diffuse-porous and ring-porous angiosperms). Due to the non-normal distribution of the data, correlations were assessed with Spearman’s rank correlation coefficient. Graphics were generated with the package ggplot2 (Whickham 2016).
To find direct and indirect effects of nifH gene abundance, BNF rate, respiration rate (as a proxy for fungal activity), WC, NSC, Mo, P, and N, a SEM was applied. Before analysis, a theory model with possible connections was built based on theoretical knowledge (Online Resource Fig. S1) and the significant correlation of BNF with all measured parameters. The package piecewiseSEM version 2.1.0 (Lefcheck 2016) was used for the analysis, as plot identity was included as a random factor. Before fitting the piecewise SEM, the single linear mixed-effect models within the SEM were checked for meeting model assumptions, using the package nlme version 3.1–152 (Pinheiro et al. 2021). For the piecewise SEM, the model fitting and the detection of missing paths were accomplished by Fisher’s C and Shipley’s test of d-separation, respectively (Shipley 2013). Models were considered valid when the model’s p-value was > 0.05 and the degrees of freedom (df) > 1, meaning that the piecewise SEM was not different from our data but displayed the data. For endogenous, response variables (BNF, nifH gene abundance, and respiration rate), a conditional Rc2 is given. Standardized path coefficients determine relationships between variables.