Discussion of Results
Discussion of Results
Rapid Detection of Rhizoctonia solani
When the initial primer test was conducted on the Perkin-Elmer PCR machine, it showed that the designed primer, PITS2RSc, was indeed annealing to the R. solani DNA, but not to the other fungi or grasses. The size of the bands for the positive control for R. solani was around 700 base pairs. The size of the bands for the DNA that was amplified using the specific primer was about 500 base pairs. The fact that the designed primer and the ITS-1 primer frame a sequence smaller than the ITS-1 and ITS-4 primers accounts for this difference. Next, the same tests were run using the Rapid Cycler PCR machine. This test also showed that the specific primers were annealing to Rhizoctonia specifically.
Consequent tests were run on fungal cultures of different species of Rhizoctonia and the results showed that the designed primer was genus-specific, not species-specific. This does not turn out to be a problem because there are several species of Rhizoctonia that cause brown patch and control measures do not vary among these species.
Using the rapid extraction method, the Rapid Cycler PCR machine, and gel electrophoresis, a diagnosis of a fungal culture could be made in 2 1/2 hours. Problems arose however, when turfgrass samples were to be diagnosed. The rapid extraction method was not able to extract the fungal DNA along with the plant DNA. Hence, a diagnosis could not be made.
A possible experiment that could result in an accurate diagnosis of an infected turfgrass sample would involve extracting the plant and fungal DNA using the Dellaporta extraction method, and then running the sample through the Rapid Cycler PCR machine. Since the Dellaporta extraction takes 1 1/2 hours, the time for diagnosis would still be under four hours.
Rapid Detection of Sclerotinia homoeocarpa
In the initial primer test run in the Perkin-Elmer PCR machine at an annealing temperature of 55ºC, the designed primer, PITS2SHc, did not amplify the DNA of any of the samples tested. The positive controls (ITS-1 and ITS-4) for the two Sclerotinia samples, K1 and KN, did not show bands. It was therefore concluded, that the DNA in the K1 and KN samples had degraded. New DNA extractions were made from fresh Sclerotinia sample and the experiment was repeated. The results from this experiment (see gel #2) showed that the designed primer is not specific to Sclerotinia homoeocarpa at an annealing temperature of 55ºC. Also, a new stock of ITS-4 primer needed to be obtained as none of the positive controls showed bands.
Other annealing temperatures were tried in subsequent experiments (see table in Results section), but the results were not encouraging. At 55ºC, there was no specificity with the designed primer, and at 56ºC, the primer did not amplify DNA from any of the samples tested. In conclusion, the designed primer was not specific to Sclerotinia homoeocarpa in the experiments conducted during this research project. Further studies could be attempted with new fungal cultures as many of them were heavily utilized during the experiment and may have become contaminated. If the designed primer is not specific when used with new cultures, then another primer could be designed in the ITS 1 region to be used with PITS2SHc.