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Molecular approaches to diagnostics for plant parasitic nematodes of biosecurity concern

Tan, Matthew (2012) Molecular approaches to diagnostics for plant parasitic nematodes of biosecurity concern. PhD thesis, Murdoch University.

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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 (<2hr) approach to identify plant-parasitic nematodes. Methods were developed to extract and analyse protein spectra by MALDI-TOF MS to identify nematode species of local and biosecurity concern. Protein profiles were generated for H. glycines, G. rostochiensis, H. schachtii and H. avenae, and for the RLNs, P. neglectus, P. zeae, P. thornei and P. penetrans. Diagnostic species-specific protein peaks were identified in the profiles for each species. The results obtained show that protein profiling using MALDI-TOF MS is a valid and rapid method for identifying plant nematodes, and the database provided here represents the most comprehensive resource for protein-based diagnostics of plant nematodes.

Two dimensional protein gel electrophoresis (2DE) was also assessed as a tool to analyse RLN and CN proteins in more detail. Nematode proteins were extracted and separated in a protein extraction buffer first by isoelectric point (pH range 3-10, 16.5% polyacrylamide gel) , followed by size separation using SDS gel electrophoresis, and gels stained with silver nitrate. The protein spots in the second dimension were recorded using a high resolution gel scanner. Ninety three distinct protein spots were found for Pratylenchus spp. and 89 distinct spots for Heterodera spp. Differences in the protein profiles between populations of one species, and between different species, were identified readily using ‘Progenesis SameSpots’ software. Thirteen protein spots for RLNs and nine spots for CNs were further analysed and characterised. From these, two significant proteins were identified as useful biomarkers that were present in two different populations of P. neglectus and one significant biomarker was identified present in P. penetrans. Similarly, specific differences in protein profiles were found within populations of H. schachtii species which provided biomarkers that identified the different populations. For H. schachtii and H. avenae, two distinct protein spots were chosen as potential species specific biomarkers since they were present in three H. schachtii populations, and three species-specific biomarkers were chosen as specific to H. avenae.

Individual protein biomarkers were excised and sequenced after trypsin digestion to release peptides from the gel. The m/z ratio of the peptides fragments were then analyses by MALDI-TOF MS, and the pattern of fragmented peptides was then compared using blastP with those recorded on Mascot and NCBI databases to identify proteins from which the peptides were derived. Identified proteins included RutC family protein C23G10.2, major sperm protein, probable arginine kinase, ATP synthase subunit alpha-mitochondrial, glyceraldehyde-3-phosphate dehydrogenase 2 and vacuolar H atpase protein 8, protein C14C10.2b, Y20F4.3 transcript:Y20F4.3, protein 19C07, arabinogalactan endo-1,4- β -galactosidase 2, heat shock 70 kDa protein C, annexin 4F01, β-1, 4-endoglucanase 1 precursor, aldolase, cathepsin L and pectate lyase 1. Although it is not necessary to identify the function of diagnostic proteins, such additional information is useful. The value of identified species-specific proteins is their potential be use to develop antibody-based diagnostic tests, such as Lateral Flow Devices (LFDs), in which antibodies raised against specific biomarker proteins can be developed to provide a rapid field-based method for nematode identification.
Another nucleic acid based approach was also investigated, which also made use of ITS sequence data – termed anti-primer quantitative PCR (aQPCR) technology, using a qPCR equipment platform. In this approach an additional ‘anti-primer’ was added to fluorescent QPCR reactions (specific primers labeled with FAM, Cy5 or TET), which binds to and quenches unbound fluorescent label. This approach decreases background fluorescence and so increases accuracy of the diagnostic test, and was developed to provide a multiplex high-throughput assay for nematode diagnostics. With different fluorescence labels to tag different primers specific to different RLN species (P. neglectus, P. penetrans and P. thornei), the results obtained successfully differentiated these three species in a multiplex system, in a total reaction time of 2.5 hours. For this approach, the number of detectable species depends on the number of different fluorescent channels the qPCR machine can detect.

A further diagnostic procedure was also developed to try to increase the number of samples that could be analysed at one time. This procedure was termed ‘Multiplex anti-primer denaturation PCR’ (MAD PCR). It was derived by combining aQPCR technology and ‘auto-sticky’ PCR. This approach makes use of ‘C3 linkers’ (or ‘blocks’) in primers, which prevent further PCR copying of the strand in which they occur, resulting in an overhang. With C3 linkers inserted at different positions in different primers, the qPCR was developed into a multiplex high-throughput assay. Different C3 positioning on the primer results in different melt temperatures in a melt analysis after PCR. The difference in melt temperatures enabled differentiation between different C3 inserted primers. To extend this technology, different fluorescent labels (such as FAM) were combined with different C3 positions for primers specific to different species. After PCR, melt analysis was done to differentiate the species by the different temperatures at which melting of dsDNA occurred, as followed by changes in fluorescence with changing temperature. In the work undertaken C3 blocks were incorporated in different primers to detect three different RLNs, ie a triplex system. The analysis of up to 12 species per qPCR could be done with the system as developed here, but with further refinement, and incorporating additional fluorescence labels with the ‘triplex’ system, 18 species could be detected using 6 channels.

As for other soil-borne disease agents, nematodes are usually extracted from plant tissue or soil samples for detection. The time taken to extract them is usually several days. In the final Results chapter extraction of DNA from nematode infected soil samples was combined with molecular identification, and developed as a potential package for a rapid nematode diagnostics in a field situation. To do this, a rapid isolation method was developed and termed ‘DNA isolation rapid technique from soil’- ‘DIRT(s)’. This involved extraction of DNA from nematode-infected soil samples using a customised blender (time taken about 2 min) and DNA capture column, followed by elution from the column and aQPCR analysis: the whole procedure took only 4 hours. Using the DIRT(s) technique and aQPCR technology, three different RLN species (P. neglectus, P. thornei and P. penetrans) infecting wheat plants were successfully identified after DIRT(s) extraction from soil using aQPCR in a multiplex assay.

The results are discussed in relation to current techniques used for nematode diagnostics. It is suggested that both protein-based and the novel PCR-based technologies (aQPCR, MAD PCR), and the new soil extraction method (DIRT(s)), can be developed to provide useful new approaches to detect and diagnose plant nematodes in biosecurity applications.

Publication Type: Thesis (PhD)
Murdoch Affiliation: Western Australian State Agricultural Biotechnology Centre
School of Biological Sciences and Biotechnology
Supervisor: Jones, Michael
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