VALORACIÓN DE CRITERIOS DE EVALUACIÓN PARA SCA

4.2.1 Lysimeter study

4.2.1.1 Design of the lysimeter study

Twenty soil monolith lysimeters, measuring 50 cm in diameter and 70 cm deep, were collected from the Lincoln University Ashley Dene Farm (43o 39’ 2” S; 172o 19’ 45” E) following the protocol outlined in Section 3.2.2 and transported back to Lincoln University for installation (see Section 3.2.2) into the field trench lysimeter facility (43o 38’ 52” S; 172o 28’ 7” E).

In February 2011, kale seeds were sown into each of the lysimeters (see Section 3.2.4). After germination, the kale plants were thinned to represent the standard kale density on a winter feed system (4.5 kg ha-1). The kale was then left to grow until early winter when the trial commenced.

4.2.1.2 Treatment application

Five different treatments (Table 4.1), each with four replicates were applied onto the lysimeters in a random design following the protocols outlined in Section 3.2.4.1. Each treatment was applied between June 23rd and 29th 2011 immediately after simulated grazing of the kale. The water input was maintained after treatment application following the protocols outlined in

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Section 3.2.5. A second rotation crop was planted November 3rd 2011 following the protocols outlined in Section 3.2.7.

Table 4.1 Treatments applied to the soil.

Treatment Urine (kg N ha-1) DCD (kg DCD ha-1) Biochar (tonnes biochar ha-1) Control 0 0 0 Urine 700 0 0 Urine + DCD 700 20 0 Urine + biochar 700 0 5 Urine + DCD + biochar 700 20 5

4.2.1.3 Leachate collection and sample analysis

Leachate collection began six weeks prior to treatment application to determine the background levels of NO3--N and NH4+-N present in the soil and ended in early May the following year.

Leachate was collected either when the drainage volume was greater than 200 ml, or weekly. All leachate samples were analysed for NO3--N and NH4+-N through FIA (Tecator Inc., Sweden) and

the samples that had DCD as a treatment were analysed using HPLC (Shimadzu, Japan) (see Section 3.2.9).

4.2.2 Companion soil blocks

4.2.2.1 Design of the soil block

For each lysimeter there was a corresponding soil block, measuring 50 cm in diameter and 7.5 cm deep, for destructive soil sampling. The blocks were collected from the Lincoln University Ashley Dene Farm following the protocols outlined in Section 3.2.3 and placed next to the lysimeters at Lincoln University. In February 2011, kale seeds were sown into each of the soil blocks (see Section 3.2.4). After germination, the number kale plants were thinned to represent the standard kale density on the winter feed system (4.5 kg ha-1). The kale was then left to grow until early winter when the trial commenced.

4.2.2.2 Treatment application

The treatments (Table 4.1) were the same as that of the lysimeter study and were applied following the protocols outlined in Section 3.2.4.1. Each treatment was applied between June 23rd and 29th 2011 immediately after simulated grazing of the kale.

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4.2.2.3 Soil sample collection and sample analysis

Soil cores were collected on day 0, 1, 7, 21, 52, and 90 after treatment. At each sampling occasion, three soil cores were removed from each soil block and bulked into a single sample for analysis. Following the protocols in Section 3.2.8, each soil sample was analysed to determine the NO3--N and NH4+-N concentrations while the samples that had DCD as part of the treatment

were also analysed for DCD concentration. The soil extracts were analysed for NO3--N and

NH4+-N using FIA (Tecator Inc., Sweden) and for DCD using HPLC (Shimadzu, Japan) ) (see Section

3.2.9).

4.2.2.4 Soil AOB and AOA assays

From the soil samples collected on day 0, 1, 7, 21, 52, and 90 after treatment, the population abundance of AOB and AOA present within the soil was determined using real-time qPCR by targeting the functional amoA gene (Di et al. 2009b). All fresh soil samples (subsampled from the bulk soil sample) were stored at -80oC before DNA extraction and analysis.

DNA was extracted from 0.4 g frozen soil using the MO BIOTM PowerSoil® DNA Isolation Kit (MO BIO Laboratories, GeneWorks Pty Ltd, South Australia, Australia) according to the manufacturer’s instructions outlined in Section 3.2.10.1. After the extraction process, the samples of DNA were stored at -20oC before analysis.

On day 52 after treatment application, a 1 g sample of soil was collected for RNA analysis, to determine microbial activity. The soil was stored at -80oC until use. RNA was extracted using the MO BIOTM RNA PowerSoil® Total RNA Isolation Kit (MO BIO Laboratories, GeneWorks Pty Ltd, South Australia, Australia), following the manufacturer’s instructions outlined in Section 3.2.11.1. To remove residual amounts of genomic DNA, the extracted RNA samples were treated with the TURBO DNA-free Kit (Ambion®, Life Technologies, Auckland, New Zealand) (Section 3.2.11.2). cDNA was produced using Superscript III reverse transcriptase (RT) (Life Technologies, Auckland, New Zealand) (Section 3.2.11.3). RNA analysis was only done on samples from a single sampling date and not on other samples due to resource constraints.

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4.2.3 Statistical analysis

Annual NO3--N leaching losses were calculated using the NO3--N concentrations in, and volume

of, the leachate collected from each lysimeter. Average annual NO3--N leaching losses were then

calculated using the four replicates. Mean values and standard errors of the means for NO3--N

leaching losses, NO3--N concentrations, DCD concentrations, and ammonia oxidiser populations

were calculated based on the four replicates for each treatment using Microsoft Excel 2010 (Microsoft Corporation, USA). For both the leachate and the soil block data a 2 x 2 + 1 factorial design was used. P-values, main effects, and interactions were calculated following analysis of variance (ANOVA) using Genstat© (Version 15.1, VSN International Ltd, U.K.).