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Genetic variation, taxonomy and pathogenicity of rust pathogen isolates (Puccinia boroniae) from south Western Australian Boronia cultivars

Driessen, Susanna (2001) Genetic variation, taxonomy and pathogenicity of rust pathogen isolates (Puccinia boroniae) from south Western Australian Boronia cultivars. Honours thesis, Murdoch University.


Puccinia boroniae is a rust fungus that infects some species and cultivars of the Australian native shrub, Boronia. Due to its commercial viability as a cut flower, many species and varieties of Boronia are now cultivated throughout south Western Australia. This increase in genetically uniform plantings of Boronia has led to high levels of rust infection being reported and is now considered the most prominent problem faced by Boronia growers in Western Australia. As current control methods are inadequate for eradicating the fungus, research into the host specificity and diversity of P. boroniae was initiated. The extent of variation among 27 isolates of Puccinia boroniae collected from cultivated Boronia within the south west of Western Australia was investigated using PCR-RFLPs and a comparison of teliospore dimensions. Several infection methods were also assessed such that the host specialization of isolates could be confirmed.

Genetic variation among the isolates was determined by looking at the variation in restriction sites within the internal transcribed spacers (ITS) of the nuclear ribosomal RNA gene. Amplification of the rust ITS regions in samples composed predominantly of host plant DNA was achieved using the polymerase chain reaction with primers Rustl (rust specific) and ITS5 (universal specificity). 21 out of the 27 isolates screened (78%) were successfully amplified using this primer pair, resulting in a single band of approximately 1290bp. These samples were digested with nine restriction enzymes, of which only one isolate was distinguished from the remaining population with a different restriction pattern generated using HinFI.

All infection trials conducted in temperature-controlled cabinets were unsuccessful. Uniform germination of the teliospores was a particular problem, with a germination rate of 5% and less occurring among the trials. Therefore rust biotypes based on host preferences could not be determined.

Morphological variation among Puccinia boroniae teliospores collected from two of the most economically important Boronia species was investigated. Six isolates collected from cultivated Boronia heterophylla and Boronia megastigma were measured at XlOOO. A highly significant (p<O.OOl) relationship between the two groups of rust fungi (based on host plant species) was determined using multivariate analysis of variance. On average, B. heterophylla teliospores had wider apical and basal cell widths, thinner cell walls and thinner apical papilla than teliospores isolated from B. megastigma. Discriminant function analysis of the two rust groups indicated that the combination of apical papillium thickness, apical and basal cell width and apical cell length provided the greatest level of discrimination between the two groups. Despite a highly significant relationship existing between the two groups based on these teliospore dimensions (p<0.001), the discriminant analysis indicated that teliospore dimensions alone would be insufficient to categorize unknown isolates (Wilks lambda= 0.656) and therefore could not be used as a taxonomic tool at this stage.

Overall, the data would suggest that a certain level of diversity exists among the Puccinia boroniae isolates screened. However, the level of variation seen within the ITS regions using PCR-RFLP indicates that subspecies of Puccinia boroniae are not present. Investigations into alternative rDNA and other DNA regions would be warranted. Furthermore, as the host specialization of the rust isolates analysed by teliospore morphology could not be confirmed, it would be inappropriate to further define these two rust groups as separate varieties of Puccinia boroniae.

Item Type: Thesis (Honours)
Murdoch Affiliation(s): School of Biological Sciences and Biotechnology
Notes: A digital copy of this thesis is not available. Your library can request a copy from Murdoch University Library via Document Delivery. A fee applies to this service.
Supervisor(s): Hardy, Giles and O'Brien, Philip
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