DOI: 10.5285/59f81d41-a789-4c5c-8ab8-36baa7ac2c55
title: Soil biological properties from an inorganic fertiliser (grass trial) experiment at North Wyke, Henfaes Farm and Easter Bush, UK (2016)

description: The data nitrogen gene data, soil biodiversity indices and microbial community composition, for three soil depths (0-15, 15-30 and 30-60 cm) from a three-cut silage plot trial located at three grassland sites within the UK collected between April 2016 and October 2016. The sites were Rothamsted Research at North Wyke in Devon, Bangor University at Henfaes Research Station in North Wales and Easter Bush in Scotland.

At each site measurements were taken from 16 plots, organised within a randomised complete block design where 4 plots did not receive fertilizers (controls), 4 plots received urea only, 4 plots received urea and urea-inhibitors, and 4 plots received ammonium-nitrate (Nitram). Fertiliser was applied three times and three cuts were performed, all parameters measured were following a fertiliser application. Samples were taken before the fertilizer additions, at peak growth and before the last silage cut.

Soil chemical parameters were: soil nitrate, ammonium, dissolved organic carbon and nitrogen, amino acids and peptides, soil organic matter content as loss-on-ignition, pH, sodium, potassium, calcium, magnesium, permanganate oxdisable carbon citric acid extractable phosphorous, Olsen-P and total carbon, nitrogen and phosphorus. Soil biological measure were: microbial biomass carbon and nitrogen. Soil physico-chemical parameters were measured on the same samples and are available in a related dataset measured on the same soil samples and are presented in a separate dataset in the project data series (https://catalogue.ceh.ac.uk/id/7a87dde4-b54e-49b0-8751-1d59e8aebb90).

Measurements were undertaken by members of staff from the Centre of Ecology & Hydrology (Bangor, Edinburgh, Lancaster, Wallingford), Bangor University, School of Environment, Natural Resources & Geography and Rothamsted Research, Sustainable Agricultural Sciences, North Wyke.

Data was collected for the Newton Fund project "UK-China Virtual Joint Centre for Improved Nitrogen Agronomy". Funded by Biotechnology and Biological Sciences Research Council (BBSRC) and NERC -  Ref BB/N013468/1
description: Soil samples were taken by members of the sites. At North Wyke and Henfaes farm, staff from CEH Bangor helped taking the samples. Two sets of soil cores were taken. One was sent in cooling boxes on ice to CEH Bangor for measuring soil physicochemical properties. The second set was sent to Rothamsted Research Harpenden for the analysis of nitrogen genes and microbial community composition (related dataset). The topsoil 0-15 cm were taken with the same type of corer across the sites, samples below 15 cm soil depth were sampled with corers available at the sites. Samples were wet sieved to 5 mm by staff from Bangor University and CEH Bangor. Subsamples of these were analysed for physicochemical and biological parameters.

DNA extractions were performed by Rothamsted Research Harpenden. Extracts were analyzed for nitrogen genes at Rothamsted Research in Harpenden. Extracts were also sent to CEH Wallingford for analyses of Operational Taxonomic Units (OTU's).

Soil community DNA was extracted from 0.25 gams soil using the MoBio DNA PowerSoil DNA isolation kit (Mo Bio Laboratories, Inc. Carlsbad, CA), following manufacturer’s protocol. Extracted DNA was quantified by fluorometer Qubit® 2.0 dsDNA BR Assay Kit (Thermo Fisher Scientific) and quality checked by nanodrop (Thermo Fisher Scientific).

Quantitative-polymerase chain reaction (qPCR) protocols were as detailed in De Sosa et al. 2018.

Microbial nitrogen cycling gene abundance was investigated by qPCR targeting speciﬁc genes or genetic regions. Bacterial and archaeal communities were targeted via the 16S rRNA genes, while the fungal community abundance was targeted by the ITS region. The diﬀerent genes involved in soil nitrogen cycling were investigated: 
•	Nitrogen ﬁxation (nifH gene)
•	Nitriﬁcation by targeting the ammonia oxidising bacteria (AOB) and archaea (AOA) (amoA gene)
•	Denitriﬁers via the nitrite reductase (nirK and nirS genes) and the nitrous oxide reductase (nosZ genes clade I and II)
•	Degradation of urea was also assessed by targeting the ureC gene

qPCR ampliﬁcations were performed in 10 μL volumes containing 5 μL of QuantiFast (Qiagen, Manchester, UK), 2.8 μL of nuclease-free water (Severn Biotech, Kidderminster, UK), 0.1 μL of each primer (1 μM) and 2 μL of template DNA at 5 ng μL−1, using a CFX384 Touch® Real-Time PCR Detection System (Bio-Rad, Hemel Hempstead, UK). 

Standards for each molecular target were obtained using a 10 fold serial dilution of PCR products ampliﬁed from an environmental reference DNA (also used as positive control) and puriﬁed by gel extraction using the Wizard® SV Gel and PCR Clean Up System (Promega, Southampton, UK) following the manufacturer's instruction and quantiﬁed by ﬂuorometer Qubit® 2.0 dsDNA BR Assay Kit (Thermo FisherScientiﬁc). 

Standard curve template DNA and the negative/positive controls were ampliﬁed in triplicate. Ampliﬁcation conditions for all qPCR assays consisted in 2 steps: ﬁrst denaturation at 95 degrees Celsius for 5 minutes followed by 40 cycles at 95 degrees Celsius for 10 seconds and 60 degrees Celsius for 30 seconds that included annealing, elongation and reading. Each ampliﬁcation was followed by melting curve (increase in temperature from 60 degrees Celsius to 95 degrees Celsius, with a reading every 0.5degrees Celsius) to assess the speciﬁcity of each assay.

The eﬃciency of the qPCR varied between 85.2 percent and 95.9 percent (except nozII 74.4 percent, amplicon 746 bp long), and R2 between 0.987 and 0.999. The melting curves showed speciﬁcity for all the genes, except as expected for the fungal Internal Transcribed Spacer (ITS), that showed the ampliﬁcation of products of diﬀerent lengths, due to the variability in length of the ITS region between diﬀerent fungal taxa (Manter and Vivanco, 2007).

Microbial diversity indices:
Extracted DNA was supplied from Rothamsted Research in Harpenden (see section 5.4.13 in main supporting document) for amplicon sequencing at the facilities of the Molecular Ecology Group, Centre for Ecology & Hydrology, Wallingford. <50 μL volumes of DNA extract were supplied in 96-well plate format, these were subject to:
•	16S rRNA amplicon sequencing as per Kozich 2013 (Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform), utilising V3-V4 region primers
•	ITS rRNA amplicon sequencing employing the Kozich 2013 strategy, utilizing ITS primers by Ihrmark 2012 (New primers to amplify the fungal ITS2 region--evaluation by 454-sequencing of artificial and natural communities)

Amplicons were sequenced on Illumina MiSeq platform with V3 chemistry. Resulting sequences were demultiplexed using Illumina Basespace. 16S amplicons were processed using in-house developed pipelines for quality filtering, merging and taxonomic assignment (GreenGenes database) of operational taxonomic units (OTUs). ITS amplicons were processed using the R package Dada2 for quality filtering, merging. 
R package Vegan was used to analyse amplicon sequences. Bacterial and fungal Shannon’s Index diversity scores for each sample were produced from rarefied data using diversity function. Species richness for each sample were similarly produced using ‘specnumber’ function.

Bacterial phylum level frequencies were produced from taxonomically identified OTU sequences (using Greengenes database), these values are a percentage of the rarefied total.

All results were entered into CSV spreadsheet.