2 Methodology
2.1 Site Selection
Four perennial ryegrass trial sites across New Zealand were chosen for microbiome comparison. These trial sites were chosen to represent the common dairy farming locations across New Zealand and to gain a geographical contrast in soil types and environmental conditions. One South Island, and three North Island sites, were selected due to pasture persistence being a more significant problem for North Island farmers. The trial sites were governed by the NZ Plant Breeding and Research Association (NZPBRA) as a part of the network of National Forage Variety Trials (NFVT). The four trial sites were: Cambridge (Waikato), Havelock North (Hawkes Bay), Palmerston North (Manawatu), and Burnham (Canterbury) (Figure 2.1).
Figure 2.1 Map of New Zealand showing the four NFVT trial locations (Cambridge, Havelock North, Palmerston North, and Burnham) sampled for the current project.
36 2.2 Experimental design
The NFVT plots were set out in randomised blocks containing 10 different cultivar- Epichloë combinations. The current project only sampled the four replicates of the One50 AR37 ryegrass combinations from these plot sites. By randomly allocating the sowing positions of the One50 AR37 ryegrass, it catered for spatial differences in the soil type, pests, elevation, and moisture retention ability along the plot blocks.
2.2.1 Ryegrass plot management
The NZPBRA managed the perennial ryegrass plots at each sampling site. Defoliation of the plots at each site was required after yield sampling, which occurred on average ten times per year (refer to yield sampling details in section 2.8.2). All three North-Island sites were defoliated through livestock grazing: Cambridge and Palmerston North by cows and Havelock North by sheep. The Burnham site ryegrass was manually defoliated with a mower instead of being grazed by livestock throughout the trial. Mowing and grazing achieved the same purpose for defoliating the ryegrass; however, grazingadds nutrient inputs from dung and urine, and livestock treading during grazing can alter the soil structure. The Burnham site was the only site to receive irrigation over the summer months of the three-year trial, replicating the typical Canterbury irrigated dairy system.
Both mowing and irrigation became highly important to consider when exploring the microbiome. The plots at all four sites received nitrogen fertilisers during the trial, with the Cambridge site receiving the highest – 446 kgs/ha/year (Table 2.1).
Table 2.1 Defoliation, irrigation, and nitrogen inputs for the four NZPBRA trial sites in the current study.
NZPBRA Trial Site
Summer irrigation
Defoliation Quantity of Nitrogen applied per year (kgs/ha)
Type of Nitrogen
applied
Cambridge No Grazed by cows 446 SustaiN
Havelock North
No Grazed by sheep 154 SustaiN and
CropZeal20 Palmerston
North
No Grazed by cows 360 SustaiN
Burnham Yes Mown 344 Urea,
ammonium sulphate, nitrophoska,
cropmaster 2.3 Field sample collection
The field sampling followed the same protocol for each site, with a few minor adjustments described. Each plot replicate was marked with a flag to avoid confusion from neighbouring plots. All equipment, including metal rings, scissors, spades, and soil coring devices, were sprayed and cleaned thoroughly with 70 % ethanol between each plot replicate. Gloves were worn and replaced between each replicate to limit cross- contamination between each plot and human microorganisms.
37 Figure 2.2 Annotation of the field sampling method utilised to obtain the ryegrass stem and soil samples from the four sampling sites across New Zealand. Diagram made using Biorender.com.
For the four One50 AR37 replicates at each sampling site, four sterilised metal rings (7.5 cm diameter) were randomly tossed onto the plot area to limit sampling bias (Figure 2.2).
At the location the metal rings landed, the rings were fitted around the base of the ryegrass plant/s, and sterilised scissors were used to carefully cut off the shoots at around 2 cm above the soil to avoid contamination with soil particles. The four ryegrass shoot cut-offs from each plot replicate were pooled and placed into a sterile plastic zip lock bag.
Once shoots were removed from each marked-out core area, cores (7.5 cm diameter x 10 cm deep) were taken from each ring using one of two methods.
A) The first method involved using a soil corer to extract the soil cores. This method was used when the soils at the sites were relatively moist and not compacted. The cores were removed by physically forcing and twisting the corer into the ground to a 10 cm depth (marked by a red line on the corer). Once lodged into the ground, the corer was pulled out, and the core was pushed straight into a sterile plastic Ziplock bag.
B) The second method was the backup method utilised for the Burnham and Havelock North sites when the soil was hard, compacted, or stony. This method involved a spade marked with a 10 cm line to dig out a square of soil around the marked soil core area (Figure 2.3). The soil square was dug out and placed onto a sterile tray. A sterilised knife was used to cut around the metal sore core area to produce a circular core, which was then placed into a sterile Ziplock bag.
38 Figure 2.3: Photo of myself (Bernadette) and Mark McNeil (AgResearch) demonstrating the spade, tray and knife method (B) for extracting 10cm cores at the NFVT Burnham site.
Once four core samples were extracted from each replicate, the cores were combined as one bulk field sample. The bulk field sample bag was stored in a chilly bin with icepacks, for no longer than half a day, before being relocated into a cool store (4℃).