Methods

Study area

This field experiment was carried out in the Town of Hinton (Alberta), that is located within the ancestral and traditional territory of the Indigenous peoples of this area.

Ten healthy Lodgepole pine trees were selected 22 km North-East from Hinton town (Alberta) for the experiment (53°30'50.7"N 117°17'31.2"W).

This forest area was selected because not attacked by MPB and so resulting in no infection by their symbiotic fungi.

Figure 3. Picture of the project's area using software QGIS. The red dot on the left indicate where is the area in Canada; the red square on the right side indicate where samples were collected in the lodgpepole pine forest close to Hinton Town.

Field experiment design

Figure 4. Professor Nadir Erbilgin creating a wound in Lodgepole pine tree with an arch punch and axe.

For each tree selected, we decided to inoculate the 3 symbiotic fungi in the phloem and leave one positive treatment as control (wound without inoculation). We decided to create 4 wounds for each treatment and for each tree (for a total of 16 wounds per tree), following the four cardinal points (North, South, East, West).

Each wound have been taken at the DBH level, at upper level compare to the previous one and every two weeks (starting to count from the inoculation's day).

All process in the field had three steps:

1) Wound, using a hammer and an arch punch to extract a rounded piece of phloem that was immediately stored in dry ice (to preserve samples and compare before and after treatment). The tools were disinfected each time with ethanol 70% in concentration.

2) Inoculation of one fungus inside the wound. Using a forcep, a small plug or the fungus was inoculated, and the wound closed with saran wrap (control had just a wound without inoculation).

3) Sampling the trees every two weeks for a total of four times (counting wound and inoculation as starting point). Samples have been collected above the previous one, stored in dry ice and brought to the laboratory to analyze.

Figure 5. This figure represent the experimental design protocol, simplified in pictures. Starting from the left side, there is schematic image of lodgepole pine and where the samples were collected at the DBH level. Going to the right side, the red dot at Week 0 represent visually how one of the four wounds (one for each cardinal point), was created on the bark of the tree in order to collect a rounded sample of phloem and to inoculate the treatments, starting the same day (Week 0). After two weeks (W 2) a new sample of phloem was collected above the previous collection (Week 0). The same procedure was applied after two weeks for Week 4 and Week 6. Samples of phloem were processed in order to extract the chemical compounds through the Gas chromatography–mass spectrometry (GC-MS).

Laboratory procedure for samples

All samples collected were stored in freezer at -40°C and proceeded to extract the chemical from the phloem.

Each phloem tissue was ground with liquid nitrogen and crumbled using a mortar-pestel.

After the ponder was transferred into a vial and mixed with 1ml of specific solvent. The vial was sonicated for 10 minutes and centrifuge for 30 minutes for 18000 rpm.

The upper layer was transferred 1ml GC vial.

Extraction of data

The eluted volatile samples were subjected to Gas chromatography–mass spectrometry (GC-MS).

From this machine, we get a graph with different pics. For each sample GC-MS produce one chromatograph.

The final stage is the quantification of the chemical’s constituents in the sample, comparing the cromatograph with standard chemical (bought from specific companies).

The result is an excel file with all the chemicals present in the sample of phloem tissue.

Figure 6. This is a picture of the Gas chromatography–mass spectrometry (GC-MS), used to create the chromatograph.

Figure 7. Above, an example of chromatograph took from one of the samples. On the X axes there is chronological time of extraction. On the Y axes, the Abundance in unit of ug/ml, that is a response variable. Each pick on the graph, represent the abundance of one compound.

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