2 Methodology
2.4 Sample processing
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℃).
39 Figure 2.4: Annotated diagram displaying the sample processing overview used in the current project.
2.4.1 Bulk soil
Once arriving at the laboratory, the bulk field samples contained ryegrass cores comprised of shoots, roots, and soil. These bulk samples were taken out from their bags and emptied onto a sterilised tray, where they were processed into the three microbiome habitats: bulk soil, rhizosphere, and root endosphere.
To obtain the bulk soil samples, soil that was not in direct contact with the roots was scooped into a labelled 50 ml Falcon tube until the tube was roughly ¾ full. The Falcon tubes containing the bulk soil were rinsed with 70% ethanol and agitated for 60 seconds before rinsing with distilled water three times, giving the finished ryegrass bulk soil samples.
2.4.2 Root endosphere and rhizosphere
Roots were separated from the bulk field samples to obtain the root endosphere and rhizosphere samples. These root samples (with some soil still attached) were placed into labelled 50 ml Falcon tubes (Tube 1), filling the tubes to just over halfway (loosely packed) (Figure 2.5).
40 Figure 2.5 Annotated diagram of the sample processing method used to obtain the root endosphere and rhizosphere sample. This process was repeated for the four-bulk sample One50 AR37 reps at each ryegrass plot location. Diagram made using Biorender.com.
Twenty-five mls of phosphate-buffered saline (PBS) was added to the falcon tube (Tube 1) containing the roots and some rhizosphere soil before vortexing for 15 seconds. The liquid supernatant from this tube was poured into another sterile 50 ml Falcon tube (Tube 2). Another 20 mls of PBS was added to Tube 1 before vortexing for another 15 seconds (acting as a second rinse to remove the soil attached to the roots). The liquid supernatant was added to Tube 2, containing the previous supernatant.
Tube 2 containing the supernatant was centrifuged for 5 minutes at 8000 rpm. This produced a soil pellet at the bottom of the tube with liquid floating above. The supernatant liquid could then be poured off carefully, leaving the remaining soil pellet in the Falcon tube. Tube 2, containing the soil pellet, represented the finished ryegrass rhizosphere sample ready for DNA extraction.
The roots remaining in Tube 1 were rinsed using 70% ethanol. This involved adding ethanol to the tube until the roots were fully submerged and then lightly shaking the tube for 60 seconds. The ethanol was then drained off, and the roots were rinsed three times with distilled (sterilised) water. After the rinsing, the processed ryegrass root samples were ready for DNA extraction.
41 2.4.3 Shoot endosphere and phyllosphere
The ryegrass shoots endosphere, and phyllosphere sub-samples were extracted from the field shoot samples (see section 2.3). The shoot samples were processed in a PC2 laboratory within two days of being collected from the field.
Five-gram sub-samples of ryegrass shoots from the field shoot sample were added to a ziplock bag with 15 mls of PBS and lightly shaken for 1 minute to remove any loosely attached microorganisms (likely contaminants) and soil particles (Figure 2.7). The rinsed shoots were added to 50 ml glass tubes (30 mm x 114 mm) containing 30 mls of PBS. It was essential to ensure the ryegrass shoots were placed into the tube with the cut ends facing upwards and the shoot tips facing downwards to avoid contaminating the PBS medium with endosphere microorganisms (Figure 2.6).
Figure 2.6: An image of the glass tube set up before sonication. Note that the ryegrass cut ends are facing upwards, with the uncut ends of the shoot facing down.
The shoots were transferred to the glass tubes using sterilised metal tweezers. Between each sample, the tweezers were sterilised by dipping the tips into 70 % ethanol and heating under a Bunsen burner blue flame for 5 seconds (Bykowski & Stevenson, 2020).
The glass tube samples were secured in a plastic holding rack and placed into a pre-set- up sonicator bath - two-thirds full of room temperature water (Figure 2.7). In each cycle, no more than 20 glass tubes were placed into the sonicator. The sonicator was turned on for 5 minutes. This vibrated the ryegrass tubes and the water, allowing any microorganisms attached to the outer surface of the ryegrass leaves to become dislodged into the liquid PBS medium.
42 After sonication, the ryegrass shoots were removed from the glass tubes using sterilised metal tweezers and placed into clear plastic bags. The shoots were rinsed with 70 % ethanol, agitated for 60 seconds, and rinsed three times again with distilled water. This resulted in the finished shoot endosphere samples before DNA extraction.
The liquid remaining in the glass tubes was filtered using filter lids into 50ml Falcon tubes. This was to remove any free-floating plant debris or soil. The Falcon tube containing the liquid was centrifuged for 30 mins at 3200 rpm (Figure 2.7). The tube was then centrifuged for a subsequent 30 seconds at 1000 rpm. The supernatant was drained off and discarded, leaving a white pellet on the side of the centrifuge tube. 270 µL of PBS was added, and the Falcon tube was vortexed to resuspend the pellet. 270 µL of the remaining solution was then pipetted into a clean, sterile 1ml tube. This resulted in the finished phyllosphere samples before DNA extraction.
Figure 2.7: Annotated diagram of the sample processing method used to obtain the shoot endosphere and phyllosphere samples. Diagram made using Biorender.com.