Molecular approaches to diagnostics for plant parasitic nematodes of biosecurity concern

Nematode identification by classical methods is a highly skilled undertaking, in which trained taxonomists examine samples microscopically and identify nematodes using keys based on morphological details. The accuracy of diagnosis depends considerably on the skill of the taxonomist. More recently, molecular diagnostic techniques have been developed to identify such nematodes, and the focus of this thesis is the development and application of new approaches to nematode diagnostics. The species studied included root lesion nematodes (RLNs, Pratylenchus spp.), cyst nematodes (CNs Heterodera and Globodera spp.), and the pine wood nematode (PWN, Bursaphelenchus xylophilus). Six populations of four species of RLNs isolated from wheat and sorghum plants were maintained on carrot pieces in vitro for the work. Similarly, seventeen populations of five species of CNs were also studied: in some cases for biosecurity species (eg for soybean and potato cyst nematodes, and PWN) the materials had either to be studied overseas or extracts obtained from overseas for analysis. For nucleic acid based diagnostics, an ITS-based PCR approach was used to identify different species of RLNs and study sequence differences within and between different species and populations. Phylogenetic trees were constructed to compare the data generated in this thesis with those of published sequences for the nematodes studied. The results obtained showed that there were relatively small differences in sequence between different populations of a individual species, but significantly greater differences between species. Comparing ITS sequences of different RLN species, that of P. neglectus was 58% similar to that of P. thornei, and compared to that of P. penetrans and P. zeae, the similarity was 60% and 53% respectively. Similarly, comparing ITS sequences, P. penetrans was more closely related to P. neglectus (60%) than to P. thornei (59%) and P. zeae (58-59%). For CNs, for the genus Heterodera, the similarity in ITS sequence of H. schachtii and H. glycines was 97-98%. When comparing H. schachtii and cereal cyst nematodes (CCNs), the similarity in ITS sequences was less, at 77-78% for H. avenae. H. glycines, a biosecurity listed pathogen, is not present in Australia and DNA from a Japanese population was obtained and sequenced. Based on the close sequence similarity of ITS regions (97-98%) between H. schachtii and H. glycines, it is suggested that H. schachtii can be use as a model for detecting future incursions of H. glycines. Of the three species of CCNs studied (H. avenae, H. latipons and H. filipjevi), the two species, H. latipons and H. filipjevi, are also not present in Australia, and so published sequence data for these species was used for comparisons. ITS sequences of Australian H. avenae populations were compared to those of H. latipons and H. filipjevi: which were 86-88% and 94-96% similar to those of H. avenae. In the absence of the two biosecurity pathogens, H. avenae can be used as a suitable model to develop methods to detect the other two CCN species. For the genus Globodera, a comparison of ITS sequences of G. rostochiensis (potato cyst nematode) populations from New Zealand and Japan differed by 2% and 3% from the consensus world collection of G. rostochiensis ITS sequences. These differences might reflect different routes of introduction of potato into Japan and New Zealand. It was also noted that, in some cases, PCR analysis can lead to mis-identification. For example, in this study, a RLN from Queensland was identified by classical taxonomy as P. zeae, but when DNA sequencing was undertaken for this population, the resulting ITS sequence obtained was more similar to database sequences identified as P. bolivianus than to P. zeae. Such differences may result from initial mis-identification of the original sample, or from sequencing error. Protein profiling was used as an alternative approach to ITS-based PCR identification of plant nematodes. This involves separating nematode proteins using Matrix Assisted Laser Desorption Ionisation Time-of-Flight Mass Spectrometry (MALDI-TOF MS) to generate diagnostic protein profiles. Protein profiling by MALDI-TOF MS was developed as a novel, rapid (