DOI: 10.5061/dryad.dfn2z35bg
title: Impact of community size fraction on plants and greenhouse gases dataset

description: Soil communities are essential to ecosystem functioning, yet the impact of
 reducing soil biota on root-associated communities, tree performance, and
 greenhouse gas (GHG) fluxes remains unclear. This study examines how
 different size fractions of soil biota from young and mature forests
 influence Alnus glutinosa performance, root-associated community
 composition, and GHG fluxes. We conducted a mesocosm experiment using soil
 community fractions (wet sieving through 250, 20, 11, and 3 µm) from young
 and mature forest developmental stages as inocula. The results indicate
 that the root-associated community composition was shaped by forest
 developmental stage but not by the size of the community fractions.
 Inoculation with the largest size fraction from mature forests negatively
 affected tree growth, likely due to increased competition between the
 plants and soil biota. In addition, GHG fluxes were not significantly
 impacted by either size fraction or forest developmental stage despite the
 different community composition supplied. Overall, our research indicates
 that A. glutinosa strongly selects the composition of the root-associated
 community, despite differences in the initial inoculum, and this
 composition varies depending on the stage of ecosystem development,
 impacting the performance of the trees but not GHG fluxes.
description: To study the effect of a young and mature forest soil community,
 forests spanning two distinct developmental stages were selected. The
 forests were categorized based on their time of planting as either “young”
 (2010 – 2015) or “mature” (1880 – 1927). Within each developmental stage,
 six individual forests were sampled (6 young and 6 mature), each with
 their own soil community, characteristic of each developmental stage. Each
 individual forest acted as a replicate for its corresponding development
 stage. All sampled forests were located in the province of Drenthe in the
 Netherlands, had sandy soil and were initially planted with oak
 (<em>Quercus robur</em> and <em>Quercus
 petraea</em>).  In each forest stand, soil was collected 15 cm deep
 from multiple points around the stand (February 2022). Each point was at
 least 10 m apart from the previous one and the soil from all points was
 placed in one bag to make one homogenate per forest stand. Additionally,
 three soil cores (150 cm<sup>3</sup>, 12 cm deep) were
 collected from each stand to measure soil properties. In total, we
 collected 12 bags of soil (one from each forest) and 36 soil cores. The
 soil from each bag was initially sieved through a 2 cm mesh to further
 homogenize the soil and remove large debris and stones.
 The soil from the cores (150 cm<sup>3</sup>, 129.5 ±
 0.76 g) was oven dried at 40 ℃ for 96 hours to measure soil moisture, pH
 (1:2 soil:water ratio) and the bulk density of the soil (Schofield et al.,
 1955). The loss on ignition (LOI) method was used to measure the soil
 organic matter of the soil by first drying the soil at 105 ℃ for 48 hours
 and then heating at 550 ℃ inside a muffle furnace for four hours (Heiri et
 al., 2001). The soil heated at 550 ℃ was then used to determine the
 sand/silt fractions using a HAVER EML 200 Premium automated shaker (Oelde,
 Germany). Soil texture was classified into gravel (&gt;2 mm), Sand (2
 - 0.045 mm) and Silt and Clay (&lt;0.045 mm). Black alder
 (<em>Alnus glutinosa</em>, (L.) Gaertnr) was selected as the
 species to grow in the inoculated sterile soils of our experiments.
 <em>A. glutinosa</em> trees were present in all of the sampled
 sites despite a clear dominance of oak trees (<em>Quercus
 robur</em> and <em>Quercus petraea</em>). Seeds of
 native alder were acquired from the Nature Agency Staatsbosbeheer located
 in the Netherlands. The seeds were germinated in a nursery using
 commercial potting soil where they were grown for 3 weeks and subjected to
 a watering regime of three times a week. In order to
 assess the impact of soil community size on <em>A.
 glutinosa</em> tree performance, we prepared four community size
 fractions (250, 20, 11 and 3 µm) from each of the forest soil samples,
 under sterile conditions, following methods from studies that have
 reliably proven that with reducing size fraction, the soil community
 simplifies (e.g. Wagg et al., 2014; Wang et al., 2019; Li et al., 2020).
 We prepared phosphate buffer (1 g KH₂PO₄ in 1 L DI
 H<sub>2</sub>O, pH 6.5) and mixed it with soil (1:2, w/w) in
 four batches, each followed by sieving through 1 mm and 250 µm meshes.
 One-quarter of the filtrate was stored at 4°C (the 250 µm fraction), and
 the rest was further processed. To prevent clogging of the filter papers,
 the filtrate was passed through 63 and 45 µm sieves to remove more soil
 particles, and then through 20, 11 and 3 µm Whatman filter papers to
 generate the remaining fractions. These steps were done using a Buchner
 funnel and a LABOPORT N816.3 KT.18 vacuum pump (KNF, Utrecht, the
 Netherlands). After each step, portions of the recovered filtrate
 (1/3<sup>rd</sup> at 20 µm, 1/2 at 11 µm and the rest at 3 µm)
 were stored at 4°C, representing the respective size fractions.
 To prepare the inocula, each fraction stock was thawed and
 centrifuged to decant the supernatant (LB medium + glycerol). For each of
 the four fractions of each forest, the pellets were then resuspended in
 KH<sub>2</sub>PO<sub>4</sub> buffer and
 transferred to a flask that was filled up to 200 ml with
 KH<sub>2</sub>PO<sub>4</sub> buffer. The same
 procedure was followed for each of the four fractions of all twelve
 forests, resulting in a total of 48 liquid inocula with each individual
 forest acting as a replicate in its forest development stage (six
 replicates for young and six replicates for mature). Each of these inocula
 was randomly applied to 3.15 L (15x15x20cm) pots that were filled with
 gamma-sterilized grassland soil (Salonius et al., 1967). Finally, ten pots
 with gamma-sterilized grassland soil received just the
 KH<sub>2</sub>PO<sub>4</sub> buffer without any
 community fraction inocula and another ten pots with gamma-sterilized
 grassland soil were left completely untreated and received neither buffer
 nor inocula bringing the total number of pots to 68.Two additional
 experiments were performed using the same setup but supplemented with
 either bactericide or fungicide with the aim of disentangling the effects
 of fungi and bacteria from each fraction. After
 receiving the inocula, the pots were placed in a climate room with
 relative humidity 70%, light regime of 16h:8h (light:dark), air
 temperature of 20 ℃ (light) and 18 ℃ (dark). The pots were then left to
 settle for four days while the soil was kept moist. On
 the fifth day after receiving the inocula, one four-week-old <em>A.
 glutinosa</em> seedling, germinated in autoclaved potting soil, of
 similar stem height (~ 2 cm) and number of leaves (at least two) was
 planted in each pot. The seeds were initially surface-sterilized with
 bleach solution (14%) to minimize surface contaminants (vertically shaking
 at 200 rpm for 10 minutes) before being thoroughly rinsed with autoclaved
 MQ H<sub>2</sub>O to remove residual bleach. Seedlings were
 watered three times per week. Each pot was fully saturated with water
 during each watering event. During the first week, four seedlings died and
 these were immediately replaced. After 18 weeks, stem height (cm) and
 diameter (mm) were measured. Hereafter, CO<sub>2</sub>,
 N<sub>2</sub>O and CH<sub>4</sub> gas fluxes were
 measured for each tree, see below. When measuring GHG fluxes, the 11 µm
 size fraction was excluded from the measurements. Since access to the
 necessary equipment was limited, this size fraction was excluded as it was
 deemed to be the most redundant based on stem height measurements from the
 last week before the harvest. Two days prior to the
 harvest of the pots, CO<sub>2</sub>,
 N<sub>2</sub>O and CH<sub>4</sub> gas fluxes were
 measured for each of the trees (pot + tree system). The measurements were
 conducted using an UGGA GLA-915-0011 (Los Gatos Research, Inc., Mountain
 View, CA, USA) gas analyzer instrument for CO<sub>2</sub> and
 CH<sub>4</sub> fluxes and a LICOR LI-7820 (LI-COR
 Environmental., Lincoln, Nebraska USA) instrument for
 N<sub>2</sub>O fluxes. Each pot was placed in a 30 cm diameter
 PVC base on top of which we fitted a 100x30 cm (70 L) plexiglass chamber
 covered with double-layer black plastic bag to prevent photosynthesis and
 the fluxes were measured over a 5-minute enclosure time. A fan was placed
 at the top of the chamber, to ensure mixing during flux measurements.
 Between each flux measurement, the chamber was removed from the base and
 placed on its side so that it was flushed with atmospheric air.  After
 each flux measurement soil moisture and temperature were measured in each
 pot using a Delta T probe HH2 moisture meter (Delta-T Devices Ltd,
 Cambridge, United Kingdom) and a HANNA checktemp1 (HANNA instruments,
 Nieuwegein, Netherlands) instrument, respectively. These two instruments
 were thoroughly washed with ethanol in between samples to avoid cross
 contamination. Two days later, all plants were
 harvested. After removing the soil, roots were thoroughly rinsed with
 running tap water, and the root nodule density was recorded. Root
 subsamples were then collected for both morphological characterization
 (preserved in water at 4 ℃) and DNA analysis (preserved at -20 ℃). Root
 scanning was conducted using an Epson Perfection V850 Pro scanner at 1200
 dpi, and the WinRHIZO software (Regent Instruments, Quebec, QC, Canada)
 was employed to determine root length, specific root length, and the
 percentage of fine roots (defined as those with a diameter &lt; 0.3
 mm). The subsample was then oven-dried for 96 hours (40 ℃).  After
 removing the leaves, the stems were separated from the roots and all of
 them were oven-dried, similar to the root subsamples. The dry weight of
 the stems, leaves, roots and root subsamples was then recorded and this
 was used to calculate the above and belowground dry biomass of the
 trees. After the plants were unpotted, the soil from
 each pot was thoroughly mixed to homogenize the top and bottom layer and
 subsamples of ~ 100 g of fresh soil were kept from each pot and stored at
 4 ℃ for a week. The subsamples were sent to the university of Copenhagen
 to measure soil respiration. Roughly 50 g of each sample was placed in an
 incubator and all samples were normalized to 10% soil moisture. The
 temperature sensitivity of soil heterotrophic respiration (Q10) rate was
 measured for moisture-normalized samples (10% gravimetric water content)
 using a respirometer (Nordgren, 1988). Shortly, the soil sample was placed
 in a sealed cup and the respired CO<sub>2</sub> produced from
 the soil dissolved in a 0.1 M KOH-solution placed inside the cup. The
 change in electric conductivity of the KOH-solution is directly
 proportional to the CO<sub>2</sub> production. Data was
 recorded automatically on a computer.  Soil CO<sub>2</sub>
 respiration was measured at 9 temperature intervals from 5 ℃ to 25 ℃ with
 increments of +2.5 ℃.   
description: # Community fractions dataset
 [https://doi.org/10.5061/dryad.dfn2z35bg](https://doi.org/10.5061/dryad.dfn2z35bg) ## Description of the data and file structure The data was collected by visiting 12 forests (6 young and 6 mature) in the area of Drenthe in the Netherlands. Soils were brought back to the university in large bags and the processing of the samples started immediately.  The description of the materials and methods of the uploaded dataset has all the processes in very thorough detail. Important details: Most of the Metada can be found int he supplemented excel files. As a general rule, to understand the labels you need the following information: -Number: A unique number given to each pot. This is not essential for use of the data it is only there to order the dataset and to match the rest of the variables between datasets. -Forest: Forest identity. A unique name given to each forest that was visited to use as random factor -Age: the age of each forest. Either young (10yo) or mature (100yo) -Treatment: the experiment each pot belongs to. Buffer is the main experiment while the others (bactericide and fungicide) are supplementary experiments where the pots were additionally supplied with bactericide or fungicide. Compare each with its respective control. Fraction: The community size fraction that each pot was inoculated with. Ranges between 250, 20, 11 and 3um. The smaller the fraction, the more simplified the community. Please refer to the dataset description for a very detailed explanation of what the fractionation process entailed. ### Files and variables #### File: GHG\_raw\_data\_simplified.xlsx **Description:** A simplified version of the Greenhouse gas datafile, organized for ready use. Only the final variables after processing and calculations appear in this file. ##### Variables * Number: Unique number given to each pot so it can be matched between datasets * Label: label of the pot. Abbreviation for forest, treatment and fraction * Age: The age of the forest * Treatment: the experiment the pot belongs to * Fraction: The size of the community fraction that was inoculated to the pot * Forest: A unique name given to each forest to be used as a random factor * X flux per g: each flux expressed per gram soil per hour * X flux per kg: each flux expressed per kg soil per hour #### File: qPCR\_fractions\_raw\_data.xlsx **Description:** This file contains the raw data of the copy numbers for bacteria, fungi and soil animals obtained from a qPCR on the initial inoculum from each forest ##### Variables * Number: Unique number given to each pot so it can be matched between datasets * Age: The age of the forest * Fraction: The size of the community fraction that was inoculated to the pot * Forest: A unique name given to each forest to be used as a random factor * number of COI copies: soil animal copy numbers * number of ITS copies: fungi copy numbers * number of 16S copies: bacteria copy numbers #### File: GHG\_fluxes\_raw\_data\_part\_1.xlsx **Description:** This is the first part of the greenhouse gas raw data as it was measured from the instrument. The working data sheet shows how the calculations were done to reach the final variables. **Disclaimer: **Blank cells in sheet 1 and working data in column Flux_no, between flux numbers are intentionally left blank so that the reader knows where each flux begins and ends (eg. Flux 1 begins at row 2 and ends at row 309 all highlighted in yellow with blank cells in between. Flux 2 then starts at 310 with another highlight color and blank cells between beginning and end). ##### Variables * Flux number: refers to the number of the pot so it can be matched with the metadata #### File: Respirometer\_raw\_data.xlsx **Description:** Raw data from the respirometer experiment. The sheet "work data-simplified" has the data in ready to use format. ##### Variables * Label no: Unique number given to each pot so it can be matched between datasets * Label: label of the pot. Abbreviation for forest, treatment and fraction * Age: The age of the forest * Treatment: the experiment the pot belongs to * Fraction: The size of the community fraction that was inoculated to the pot * Forest: A unique name given to each forest to be used as a random factor * Q10: the temperature coefficient normalized to 10C  #### File: GHG\_fluxes\_raw\_data\_part\_2.xlsx **Description:** This is the second part of the greenhouse gas raw data as it was measured from the instrument. The working data sheet shows how the calculations were done to reach the final variables. **Disclaimer: **Blank cells in sheet 1 and working data in column Flux_no, between flux numbers are intentionally left blank so that the reader knows where each flux begins and ends (eg. Flux 1 begins at row 2 and ends at row 309 all highlighted in yellow with blank cells in between. Flux 2 then starts at 310 with another highlight color and blank cells between beginning and end). ##### Variables * Flux number: refers to the number of the pot so it can be matched with the metadata #### File: Microbial\_Fractions\_raw\_data.xlsx **Description:** The main dataset used for the experiments Contains data from the main and the supplementary experiments. ##### Variables * Label no: Unique number given to each pot so it can be matched between datasets * Label: label of the pot. Abbreviation for forest, treatment and fraction * Age: The age of the forest * Treatment: the experiment the pot belongs to * Fraction: The size of the community fraction that was inoculated to the pot * Forest: A unique name given to each forest to be used as a random factor * Aboveground biomass: the dry weight of the stem and leaves after the harvest expressed in grams * Belowground biomass: the dry weight of the root and scanned root subsample after the harvest expressed in grams * nodule number: the number of Frankia alni nodules counted on the roots * nodule density: the number of nodules per gram dry root * The rest of the data is exactly what is written in the cell.  #### File: Soil\_Properties\_Raw\_Data\_Microbial\_Fractions.xlsx **Description:** Raw data of the soil properties measured from the 12 visited forests ##### Variables * Forest: A unique name given to each forest to be used as a random factor * number: replicate number from each forest * the cup weight refers to the cups used to place the soil sample in * %OM: percentage of organic matter calculated after burning the soil at 550C * The rest of the data is exactly what is written in the cell.  #### File: Soil\_Texture\_Raw\_Data\_Microbial\_Fractions.xlsx **Description:** Raw data of the soil texture measured from the 12 visited forests ##### Variables * Forest: A unique name given to each forest to be used as a random factor * \&gt;2mm: the amount of soil that stayed on top of a 2mm sieve it is equal to the % gravel * 2-1mm: the amount of soil that stayed between the 2 and 1mm sieves * 1-0.25mm: the amount of soil that stayed between the 1 and 0.25mm sieves * 0.25-0.063mm: the amount of soil that stayed between the 0.25 and 0.063mm sieves * 0.063-0.045mm: the amount of soil that stayed between the 0.063-0.045mm sieves * &lt;0.045mm: the amount of soil that ended in the collection tray. It is equal to the % silt and clay * %sand: the added value of the soil that ended up between the 2 and 0.045mm sieves ## Code/software All the data can be viewed in excel and the "work data" sheet from all datasets except the GHG flux raw data part 1 and 2 can be used directly in R.