DOI: 10.5061/dryad.g79cnp5rs
title: Cross-kingdom interactions and functional patterns of active microbiota matter in governing deadwood decay

description: 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.
description: <strong>1. Study sites, plot factors, experimental design,
 sample preparation, wood-physicochemical properties and enzyme
 analysis.</strong> 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.
 <strong>2. Analyses of
 metabolically active wood microbiota using BrdU labelling technique and Illumina
 sequencing. </strong>
 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<sup>-1</sup> mouse anti-BrdU, clone BU-33, Sigma-Aldrich)
 was mixed with 18 mL denatured herring sperm DNA (1.25 mg mL<sup>-1
 </sup>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.