BINDINGS

The methodology for the soil AOB and AOA assays followed Di et al. (2009b). Soil samples were collected on day 0, 1, 7, 21, 52, and 90 after treatment application. DNA was extracted from 0.4 g of soil using a MO BIOTM PowerSoil® DNA Isolation Kit (MO BIO Laboratories, GeneWorks Pty Ltd, South Australia, Australia) according to the manufacturer’s instructions (Section 3.2.10.1). After the extraction process, the samples of DNA were stored at -20oC before analysis. The population abundance of both AOB and AOA present within the soil was determined using real- time quantitative PCR (qPCR) (Section 3.2.10.2) by targeting the functional amoA gene (Di et al. 2009b).

3.2.10.1 PowerSoil DNA Isolation Kit Protocol

A 0.4 g sample of frozen soil was weighed and added to the power bead tubes provided. The tubes were briefly vortexed and 60 µl of solution C1 was added to each tube. The samples were inverted several times before securing them onto a form pad tube holder for vortexing. The samples were then vortexed at maximum speed for 10 minutes. Following this, the tubes were centrifuged at 10,000 x g for 1 minute at room temperature. The supernatant of each sample was transferred into clean 2 ml collection tube and 250 µl of solution C2 was added to each sample before vortexing for 5 seconds and incubating at 4oC for 5 minutes. The samples were then centrifuged at room temperature for 1 minute at 10,000 x g. Avoiding the pellet that had formed, 600 µl of the supernatant was transferred into clean 2 ml collection tubes and 200 µl of solution C3 was added to each sample. The samples were vortexed briefly and incubated at 4oC for 5 minutes. The samples were then centrifuged at room temperature for 1 minute at 10,000 x g. Avoiding the pellet that had formed, 750 µl of supernatant was transferred to clean 2 ml

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collection tubes. Solution C4 was shaken before use and 1.2 ml was added to each sample. Each sample was vortexed for 5 seconds before 675 µl of sample was loaded into a spin filter tube. This was then centrifuged at 10,000 x g for 1 minute at room temperature before pouring off the flow-through. Another 675 µl of sample was loaded onto the spin filter and centrifuged again at 10,000 x g for 1 minute at room temperature. The flow through was poured off again before loading the remainder of the sample onto the spin filter and centrifuging at 10,000 x g for 1 minute at room temperature. After the flow through was poured off, 500 µl of solution C5 was added onto the spin filter and centrifuged at 10,000 x g for 30 seconds at room temperature. Once again, the flow through was poured off and centrifuged again at 10,000 x g for 1 minute at room temperature. The spin filter was then carefully removed and placed into a clean 2 ml collection tube. To elute the DNA, 100 µl of solution C6 was added onto the centre of the white filter membrane of the spin filter and centrifuged at 10,000 x g for 30 seconds at room temperature. Once the spin filter had been discarded, the DNA samples were stored at -20oC.

3.2.10.2 Real-time qPCR

All qPCR reactions were prepared using the CAS-1200 Robotic liquid handling system (Corbett Life Science, BioStrategy, Auckland, New Zealand), and the real-time qPCR analysis was performed on a Rotor-GeneTM 6000 (Corbett Life Science, BioStrategy, Auckland, New Zealand) (Tables 3.5 and 3.6). Both bacterial and archaeal amoA genes were quantified using the following PCR primer pairs: amoA1F/amoA2R primers (Rotthauwe et al. 1997) and Arch- amoAF/Arch-amoAR primers (Francis et al. 2005). The 16 µl reaction mixture was prepared using the CAS-1200 and consisted of 8.0 µl of 2x SYBR Premix Ex TaqTM (TaKaRa, Nori Biotech, Auckland, New Zealand), 0.4 µl of each primer at 10 µM concentration, and 1.5 µl of 1:10 diluted soil genomic DNA. A melting curve analysis was performed to confirm PCR product specificity after amplification by measuring fluorescence continuously as the temperature increased from 50oC to 99oC. Data analysis was then carried out using Rotor-GeneTM 6000 series software 1.7.

Table 3.5 AOB cycling conditions.

Step Temperature Time # of cycles

Initial denaturation 94oC 2 minutes 1 Denaturation 94oC 20 seconds

Primer annealing 57oC 30 seconds Extension 72oC 50 seconds

Hold 4oC HOLD 1

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Table 3.6 AOA cycling conditions.

Step Temperature Time # of cycles

Initial denaturation 94oC 2 minutes 1 Denaturation 94oC 20 seconds

Primer annealing 55oC 30 seconds Extension 72oC 30 seconds

Hold 4oC HOLD 1

For each gene, a standard curve was produced as follows. Bacterial and archaeal amoA genes were PCR amplified from extracted DNA with the primers amoA1F/amoA2R and Arch- amoAF/Arch-amoAR respectively. Using the AxyPrepTM PCR Cleanup Kit (Axygen, Total Lab Systems, New Zealand) the PCR products were then purified and cloned into the pGEM-T Easy Vector (Promega, In Vitro Technologies, Auckland, New Zealand). Following the manufacturer’s instructions, the resulting clones were transformed in Escherichia coli (E. coli) JM109 competent cells (Promega, In Vitro Technologies, Auckland, New Zealand). The transformed E. coli cells were grown on solid LB plates at 37oC overnight. Ten to fifteen bacterial colonies from the plate were then individually inoculated into a 3 ml LB broth medium and incubated overnight in an orbital incubator-shaker at 37oC and 250 rpm. The plasmids carrying correct amoA gene inserts were then extracted from bacterial cultures using QIA Prep Spin Miniprep Kit (Qiagen, BioStrategy, Auckland, New Zealand) and sent for sequencing. The plasmid DNA concentration was determined on a QubitTM fluorometer using the Quant-iTTM ds DNA BR Assay Kit (Life Technologies, Auckland, New Zealand). The copy numbers of the target genes were then calculated directly from the concentration of the extracted plasmid DNA. Tenfold serial dilutions of a known copy number of the plasmid DNA were included in each real-time qPCR run (Table 3.7). Triplicates were used to generate a standard curve and estimate amplification efficiency for each assay.

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Table 3.7 Serial dilutions and concentrations of the AOB and AOA standards.

AOB AOA Dilution Concentration (copies µL-1) Dilution Concentration (copies µL-1) -4 4,960,000 -4 596,000 -5 496,000 -5 59,600 -6 49,600 -6 5,960 -7 4,960 -7 596 -8 496 -8 59.6 -9 49.6 -9 5.96