Microbial community members are the primary microbial colonizers and active decomposers of deadwood. This study placed sterilized standardized beech and spruce sapwood specimens on the forest ground of 8 beech- and 8 spruce-dominated forest sites. After 370 days, specimens were assessed for mass loss, nitrogen (N) content and ¹⁵N isotopic signature, hydrolytic and lignin-modifying enzyme activities. Each specimen was incubated with bromodeoxyuridine (BrdU) to label metabolically active fungal and bacterial community members, which were assessed using an amplicon sequencing. Fungal saprotrophs colonized the deadwood accompanied by a distinct bacterial community that was capable of cellulose degradation, aromatic depolymerisation, and N₂ fixation. The latter were governed by the genus Sphingomonas, which was co-present with the majority of saprotrophic fungi regardless of whether beech or spruce specimens were decayed. Moreover, the richness of the diazotrophic Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium group were significantly correlated with mass loss, N content and ¹⁵N isotopic signature. In contrast, presence of obligate predator Bdellovibrio spp. shifted bacterial community composition and were linked to decreased beech deadwood decay rates. Our study provides the first account of the composition and function of metabolically active wood-colonising bacterial and fungal communities, highlighting cross-kingdom interactions during the early and intermediate stages of wood decay.

1. Study sites, plot factors, experimental design, sample preparation, wood-physicochemical properties and enzyme analysis.

Investigated forest plots were part of the German Biodiversity Exploratories and located in the UNESCO Biosphere Reserve Swabian Alb in the southwest of Germany. The field experiment was conducted on 16 forest plots (each one hectare) of the exploratory of which 8 plots are located in a Fagus sylvatica and 8 plots in a Picea abies dominated forest site. Furthermore, investigated plots differed in forest management intensity (ForMI). Soil type, soil pH, humidity (%), soil moisture (%), air temperature (°C), soil temperature (°C), elevation (m), Slope (°), Aspect (°) were measured/analyzed and reported. We excluded the results from Norway spruce and European beech specimens in some plots due to low DNA quality and quantity. The final experimental design consisted of specimens of 2 tree species x 2 forest types x 6 independent replicate plots.

Sapwood deadwood specimens of one F. sylvatica and one P. abies tree trunk were cut in 50 x 25 x 15 mm. Each specimen was gamma sterilized at 70 kGy by Synergy Health Radeberg GmbH (Radeberg, Germany) to inactivate endophytes. Afterwards specimens were dried at 103 ± 2 °C for 48 h, and dry weight was determined precisely under kiln-dried conditions. In May 2017 both two F. sylvatica and two P. abies deadwood specimens were placed in mesh bags on top of the forest ground of each forest plot with 10 cm distance between each specimen to obtain the same environmental conditions, and after 370 days of exposure specimens were collected. One specimen was directly treated with 0.1 ml of 10 mM BrdU on top for 48 h at room temperature, while the other specimen was shock frozen with liquid N at the field site to measure mass loss, wood pH, enzyme activities, C and N content and its isotopic signatures. After BrdU treatment, specimens were shock frozen with liquid nitrogen and stored at -80 °C until further use.

Mass loss and wood pH measurement was carried out. Enzyme activities such as acid phosphatase, β-glucosidase, cellobiohydrolase, manganese peroxidase, N-acetylglucosaminidase, laccase, peroxidase and xylosidase were assessed to differentiate C, N and P acquisition. Content of C and N and its isotopic signatures was measured from milled sub-samples by a vario EL III element analyser combined with an Isoprime 100 stable isotope ratio mass spectrometer (EA-IRMS) (Elementar Analysensysteme GmbH, Langenselbold, Germany). Specimens were ground into powder by adding liquid N2 using a swing mill with zirconium balls (MM2 Retsch, Haan, Germany) according to manufacturer`s instructions and thereafter stored at -80°C until further use. 20 mg powder of each specimen was burned with an Oxygen metering of 120 s and the generated CO2 and N2 were detected with a thermal conductivity detector. Downstream, the isotope signatures of 13C and 15N were determined with the IRMS with separated maps for each isotope. CO2 and N2 were used as reference gases and Helium as carrier gas. 5 mg of birch leaf dust (Elemental Microanalysis Ltd, Okehampton, United Kingdom) was used as EA standard and 10 mg wheat flour as certified reference material (Elemental Microanalysis Ltd, Okehampton, United Kingdom) for IRMS. For comparison, wood specimens without field exposure were analyzed in the same way.

2. Analyses of metabolically active wood microbiota using BrdU labelling technique and Illumina sequencing.

Bromodeoxyuridine (BrdU) labelling was done at the field site by adding 0.1 ml of 10 mM BrdU on top of each specimen. Each specimen was incubated in sterile 50 ml tubes covered with aluminium with loose cap for 48 h at room temperature. Only the metabolically active proliferating microbial cells were able to incorporate the BrdU during DNA synthesis. DNA was extracted from the BrdU-treated specimens using Quick-DNA Fecal/Soil Microbe Miniprep Kit (Zymo, California, USA) according to the manufacturer’s instructions. DNA samples from this step contained all types of genomic DNA including those from active, dead, and dormant cells. BrdU-labeled DNA from the metabolically active microbial cells was isolated from each total DNA extract by a BrdU immune-capture approach. Briefly, for each sample, 2 µL monoclonal BrdU antibodies (1 mg µL^-1 mouse anti-BrdU, clone BU-33, Sigma-Aldrich) was mixed with 18 mL denatured herring sperm DNA (1.25 mg mL^-1 in phosphate buffer saline (PBS), Promega) and incubated for 45 min at 30°C to form antibody-herring sperm DNA complex. Denatured DNA sample (25 µL ~200 ng DNA + 10 µL PBS) was then added to antibody-herring sperm DNA complex and incubated for 30 min at 30°C to capture BrdU-labeled DNA. After incubation, the mixture was added to 6.26 µL aliquots of washed DynabeadsTM goat anti-mouse IgG (Invitrogen) in PBS–bovine serum albumin solution (PBS–BSA) and incubated under slow rotation for 35 min. The Dynabead complex (Dynabead-BrdU antibodies-BrdU-labeled DNA) was washed with 100 ml PBS–BSA eight times by adding the wash solution, and trapping the complex with a magnetic particle concentrator (Dynal). BrdU-labeled DNA was released from the washed Dynabeads by adding 25 µl BrdU solution (1.7mM in PBS–BSA) then incubated under slow rotation for 35 min. The obtained immocaptured DNA is now referred as active DNA isolated from metabolically microbial active cells.

The active DNA from each specimen were subjected to PCR. For construction of the bacterial amplicon libraries, the 16S rRNA gene V4 region was amplified using the universal bacterial primer pair 515F (5-GTGCCAGCMGCCGCGGTAA-3) and 806R (5-GGACTACHVGGGTWTCTAAT-3) with Illumina adapter sequences. For establishing fungal amplicon libraries, the fungal internal transcribed spacer region sequences (ITS)2 was amplified using the fungal primer pair fITS7 [5-GTGARTCATCGAATCTTTG-3] and ITS4 primer [5-TCCTCCGCTTATTGATATGC-3] with Illumina adapter sequences. Amplifications were performed using 20 µL reaction volumes with 5× hot fire pol blend master mix (Solis BioDyne, Tartu, Estonia). The amplified products were visualised by gel electrophoresis and purified using an Agencourt AMPure XP kit (Beckman Coulter, Krefeld, Germany). Illumina Nextera XT indices were added to both ends of the bacterial and fungal amplicons. The products from three technical replicates were then pooled in equimolar concentrations. Paired-end sequencing (2 × 300 bp) was performed on the pooled PCR products using a MiSeq Reagent kit v3 on an Illumina MiSeq system (Illumina Inc., San Diego, CA, United States) at the Department of Soil Ecology, Helmholtz Centre of Environmental Research, Germany.