Phytophthora diseases have caused worldwide economic, social and environmental
impacts for decades. Once their presence is confirmed, they are difficult to eradicate. To
reduce and manage the damage inflicted by the pathogen, fast and reliable disease
management protocols are required. Tests that enable the rapid and reliable
identification of the pathogen assist greatly in disease management.
Phytophthora species are traditionally not only detected by baiting but also by plating of
symptomatic tissue on selective media. Species can be identified by the characteristics
of the mycelium growing out of the bait. However, the method is low throughput,
labour intensive, and prone to false negatives. An alternative approach would be to
detect the pathogen by the presence of its DNA. This involves amplification of the
pathogen DNA using Polymerase Chain Reaction (PCR) and detection of the
amplification product. Detection is usually by agarose gel electrophoresis. However,
this is also a labour intensive process involving pouring, loading, running, and staining
of the gels. The aim of this thesis is to explore the use of Matrix Assisted Laser
Desorption/ Ionisation Time-of-Flight (MALDI-TOF) mass spectrometry for detection
of PCR products. This procedure enables the analysis of large numbers of samples
within a very short time-frame as the average time for analysis of each sample is in the
order of milliseconds.
The assay involves annealing an extension (genotyping) primer to the PCR product and
its extension by a single nucleotide. The nature of the nucleotide added differentiates
species as does the site to which the primer anneals. Multiple extension (genotyping)
primers can be used together in a single reaction for detection of multiple species. In
this project four genotyping primers (GPs) were designed from the ITS regions of
Phytophthora palmivora, Phytophthora cinnamomi, Phytophthora citricola, and
Phytophthora cambivora.
The extension primers were tested for their specificity on the DNA of the target species.
The four primers designed were specific for their intended targets except for GPpalm3
which in addition to being extended by ddT when tested with DNA from P. palmivora,
was also extended by ddC when tested with DNA from other species of Phytophthora
or Pythium.
These primers were also tested for their ability to detect multiple Phytophthora species
in a single reaction (multiplexing). Mixtures of primers were added to mixed DNA
templates and the primer extension reaction carried out. The primers were designed so
that their masses were sufficiently different for them to be identified from a mixture.
Six replicates were analysed for each reaction. In general only about 1-3 of the six
replicates gave a positive reaction. This indicates that there may be some interference
between primers, or that the presence of all four nucleotides interfered with the primer
extension reaction. Increasing either the amount of enzyme, the amount of nucleotides
or both did not improve the results.
The sensitivity of detection was tested by the addition of different amounts of mycelium
to soil. The detection sensitivity depended on the primer pair used for PCR
amplification. The ITS1/2 primer pair was more sensitive than the ITS1/4 pair. The
limit of detection was 1 ìg mycelium g soil-1. However using nested PCR, levels of
sensitivity comparable to those obtained using the ITS1/2 primer pair could be
achieved. Primers to other regions of the genome such as the beta cinnamomin elicitin
gene gave very low levels of sensitivity compared to the ITS primers.
In comparison with DNA detection we found that the limit of detection using baiting
was 4 ìg mycelium g soil-1. Results below this limit were unreliable. The method
suffered from the additional disadvantage that it took a long time in comparison to DNA
detection.
DNA detection methods do not distinguish between living and dead organisms in the
soil. However it can be hypothesised that DNA is unlikely to persist for any significant
length of time in soil. To test this, we added plasmid DNA to soil and tested the
persistence of this DNA using a variety of methods such as precipitation of labelled
DNA, southern blotting and PCR amplification. It was found that in general, in soils
from different ecosystems, the bulk of the DNA was undetectable after 24 hours. The
rate of DNA breakdown differed with the soil type. In some soils, the added DNA was
not detected even after 2 hours, whereas in others it could be observed after 10 hours.
The detection depended on the method. Southern blotting showed that although DNA
could be observed at 10 hours, by 24 hours it was completely degraded. In contrast a
PCR product could be obtained from the soil extracts up to 24 hours. In a separate
experiment, plasmid DNA was detectable over a 24 hour incubation period in 5 soil
samples from 5 different sites. The results suggest that DNA is degraded rapidly in soil
and is unlikely to persist longer than 24 hours.
The results in this thesis demonstrate that MALDI-TOF MS is a suitable alternative to
agarose gel electrophoresis for analysis of PCR products. The technique is rapid,
differentiates species from mixtures, is high-throughput and amenable to automation.
Implementation will require further research to automate the primer extension assay to
reduce the sensitivity to impurities in the DNA and to design parameters for sampling
asymptomatic material.
| Identifer | oai:union.ndltd.org:ADTP/221854 |
| Date | January 2005 |
| Creators | siricordcc@yahoo.co.uk, Cornelia Charito Siricord |
| Publisher | Murdoch University |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://www.murdoch.edu.au/goto/CopyrightNotice, Copyright Cornelia Charito Siricord |
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