Phosphite has been used to effectively control the soil borne plant pathogen Phytophthora cinnamomi in many horticultural crops, forest trees and natural ecosystems. However, the molecular mechanisms behind phosphite action on this pathogen are poorly understood. Several studies have shown that phosphite inhibits growth and zoospore production of P. cinnamomi and in addition induces significant physiological and metabolic changes in the mycelium. As an approach to understanding the mechanisms and relevance of these changes in the pathogen, the effect of phosphite on gene expression was investigated using microarray analysis. To construct the microarray, RNA was extracted from phosphite-treated (40 ug/ml) mycelium of P. cinnamomi isolate MP 80. The chosen phosphite concentration inhibited the mycelial growth by 70% but provided sufficient mycelium for RNA extractions after 4 days growth at 25C. The mRNA was reverse transcribed into cDNA and cloned into lambda to construct a library consisting of 2 million pfu of which 80 % were recombinant phage. The inserts were sequenced for a random selection of clones from the library. The nucleotide sequences generated revealed a range of different P. cinnamomi genes being expressed and demonstrated that the cDNA library provided a good representation of the transcripts expressed in P. cinnamomi. The types of genes found to be expressed in the mycelium of P. cinnamomi included genes encoding GTP binding proteins involved in vesicle transport, structural proteins involved in maintaining cell membrane integrity,elicitors, phosphatases and ribosomal proteins. Over nine thousand cDNA transcripts were randomly selected from the cDNA library and prepared by PCR amplification and purification for microarray construction. Custom made cDNA arrays containing 9216 cDNA transcripts were constructed and probed with RNA from untreated mycelium and mycelium grown in medium with 40 ug/ml phosphite. Two genes, EF-1 alpha and cinnamomin gene, identified by qRT-PCR as being constitutively expressed were also positioned on the arrays as positive controls. In the process of identifying constitutively expressed genes, qRT PCR revealed that phosphite down-regulated a gene encoding ubiquitin-conjugating enzyme, a component of the ubiquitin/proteasome pathway involved in the removal of abnormal and short lived-regulatory proteins and rate limiting enzymes. From the arrays a further seventy-two transcripts with altered patterns in gene expression (fold change > 2) were identified. The majority of the cDNA transcripts spotted on the array were down-regulated with changes in gene expression ranging from 2- to 3.5-fold. Thirty-two cDNA transcripts were up-regulated with changes in gene expression ranging from 2- to 16-fold. Characterisation by sequencing revealed that the most highly induced transcripts coded for ADP-ribosylation factors, an ABC cassette transporter and a glycosyl transferase. A transcript encoding a vitamin B6 biosynthesis protein was also identified as up-regulated by 2.9-fold. In contrast, the down-regulated transcripts coded for cellulose synthase I, annexin, glutamine synthetase, metallothionein and an alternative oxidase. The results are discussed in terms of possible roles and mechanism(s) of phosphite action within the mycelium of P.cinnamomi. This work is the first comprehensive screen for phosphite regulated-gene expression in P. cinnamomi and represents a significant step towards an understanding of the mode of action of phosphite on this organism. This thesis provides valuable information on the molecular interaction between phosphite and P. cinnamomi, which in future studies may stimulate the discovery of novel methods and cellular targets for the control of plant pathogenic Oomycetes.

One of the major aims of the research presented in this thesis was to assist managers of native vegetation communities in southeastern Australia in understanding the dynamics of P. cinnamomi with an important ecological species, Xanthorrhoea australis. It trialed the use of phosphite in large-scale field applications to establish the usefulness of this management option for the first time on Victorian flora. This thesis describes the process of disease development within mature X. Australia plants. For the first time it was shown that within X. australis plants, secondary disease symptoms are related to the percentage of stem that has been infested by the disease. It was evident that after initial invasion the pathogen moves via root xylem and throughout the plant within vascular to the stem, especially within the desmium. The research shows that the pathogen could not be isolated consistently even though it was considered to be responsible for disease symptoms. Trials of a control fungicide (Foli-R-fos 200) shows that protection occurs in many susceptible plants when 2 and 6g a.i./L phosphite is applied. Phytotoxicity occurred in native plants at Anglesea and within controlled environment trials when using ≥ 6g a.i./L. It will be shown that 2g a.i./L phosphite controls disease in sprayed plots within heathlands at Anglesea and a recently burnt coastal woodland community at Wilson’s Promontory. The proportion of healthy X. australis plants treated with phosphite was significantly higher than the proportion in control plots without phosphite. The research shows that phosphite was recovered from leaves of three species treated with Foli-R-fos 200 in the field. For the first time it has been shown that seed germination was reduced in two species when high concentrations of phosphite were applied. The first documentation of the effect that phosphite has on soil properties showed that nitrogen and oxidised organic carbon were the only parameters to alter significantly. This thesis provides answers to some important questions, answers that can now be used by managers in formulating better policies and actions at an operational level. There has been a dire need in Victoria to address many issues regarding P. cinnamomi and this thesis provides relevant and informative approaches to disease control, and a better understanding of the disease progress.

Thesis (Ph.D.)--Murdoch University, 2003. / Thesis submitted to the Division of Science. Bibliography: leaves 151-176.

Thesis (Ph.D.)--Murdoch University, 2006. / Thesis submitted to the Division of Science and Engineering. Bibliography: leaves 183-201.

Thesis (M. Sc.)--University of Canterbury, 2007. / Typescript (photocopy). "June 2007." Includes bibliographical references (leaves 104-110). Also available via the World Wide Web.

This study focuses on the soil- and water-borne plant pathogen Phytophthora cinnamomi Rands and the phenomenon of P. cinnamomi suppressive soil. In particular, this thesis reports on the outcome of field surveys and glasshouse assays undertaken to locate P. cinnamomi suppressive soils and to confirm the involvement of biological processes in suppression. The potential role of cellulase and laminarinase in suppression was investigated and a molecular technique known as length heterogeneity PCR (LH-PCR) was used to analyse the structure and diversity of bacterial and fungal communities in avocado orchard soils that were suppressive and conducive to P. cinnamomi. Four avocado orchards with P. cinnamomi suppressive soils were identified and soils were ã-irradiated to destroy their suppressive capacity, thus confirming biological suppression. Suppression was also partially transferred to ã-irradiated and conducive soils by mixing with 10% suppressive avocado soils. Cellulase and laminarinase activities measured in avocado orchard soils inoculated with P. cinnamomi were not associated with disease severity in lupin seedlings during glasshouse assays involving the same soil samples. Minor shifts in bacterial and fungal community structure were observed in response to mixing conducive and irradiated soils with suppressive soils. This was associated with decreased disease severity in avocado seedlings in these treatments. The shift in bacterial community structure was partially determined by the appearance and increased abundance of several bacterial 16S rDNA sequences, which were unique to the suppressive soils, in the mixed soil treatments. It is suggested that the bacteria and fungi from which these sequences originated may be involved in suppression and further work should be undertaken to determine their identity and confirm their potential role in the development and maintenance of P. cinnamomi suppressive soils. / Doctor of Philosophy (PhD)

Diseases in natural ecosystems are often assumed to be less severe than those observed in domestic cropping systems due to the extensive biodiversity exhibited in wild vegetation communities. In Australia, it is this natural biodiversity that is now under threat from Phytophthora cinnamomi. The soilborne Oomycete causes severe decline of native vegetation communities in south-western Victoria, Australia, disrupting the ecological balance of native forest and heathland communities. While the effect of disease caused by P. cinnamomi on native vegetation communities in Victoria has been extensively investigated, little work has focused on the Anglesea healthlands in south-western Victoria. Nothing is known about the population structure of P. cinnamomi at Anglesea. This project was divided into two main components to investigate fundamental issues affecting the management of P. cinnamomi in the Anglesea heathlands. The first component examined the phenotypic characteristics of P. cinnamomi isolates sampled from the population at Anglesea, and compared these with isolates from other regions in Victoria, and also from Western Australia. The second component of the project investigated the effect of the fungicide phosphonate on the host response following infection by P. cinnamomi. Following soil sampling in the Anglesea heathlands, a collection of P, cinnamomi isolates was established. Morphological and physiological traits of each isolate were examined. All isolates were found to be of the A2 mating type. Variation was demonstrated among isolates in the following characteristics: radial growth rate on various nutrient media, sporangial production, and sporangial dimensions. Oogonial dimensions did not differ significantly between isolates. Morphological and physiological variation was rarely dependant on isolate origin. To examine the genetic diversity among isolates and to determine whether phenotypic variation observed was genetically based, Random Amplified Polymorphic DNA (RAPD) analyses were conducted. No significant variation was observed among isolates based on an analysis of molecular variance (AMQVA). The results are discussed in relation to population biology, and the effect of genetic variation on population structure and population dynamics. X australis, an arborescent monocotyledon indigenous to Australia, is highly susceptible to infection by P. cinnamomi. It forms an important component of the heathland vegetation community, providing habitat for native flora and fauna, A cell suspension culture system was developed to investigate the effect of the fungicide phosphonate on the host-pathogen interaction between X. australis and P. cinnamomi. This allowed the interaction between the host and the pathogen to be examined at a cellular level. Subsequently, histological studies using X. australis seedlings were undertaken to support the cellular study. Observations in the cell culture system correlated well with those in the plant. The anatomical structure of X australis roots was examined to assist in the interpretation of results of histopathological studies. The infection of single cells and roots of X. australis, and the effect of phosphonate on the interaction are described. Phosphonate application prior to inoculation with P. cinnamomi reduced the infection of cells in culture and of cells in planta. In particular, phosphonate was found to stimulate the production of phenolic material in roots of X australis seedlings and in cells in suspension cultures. In phosphonate-treated roots of X australis seedlings, the deposition of electron dense material, possibly lignin or cellulose, was observed following infection with P. cinnamomi. It is proposed that this is a significant consequence of the stimulation of plant defence pathways by the fungicide. Results of the study are discussed in terms of the implications of the findings on management of the Anglesea heathlands in Victoria, taking into account variation in pathogen morphology, pathogenicity and genotype. The mode of action of phosphonate in the plant is discussed in relation to plant physiology and biochemistry.

Phytophthora cinnamomi has been recognised as a key threatening process to Australia’s biodiversity by the Commonwealth’s Environment Protection and Biodiversity Conservation Act 1999. Despite over 80 years of extensive research, its exact mode of survival is still poorly understood. It is widely accepted that thin- and thick-walled chlamydospores are the main survival propagules while oospores are assumed to play no role in the survival of the pathogen in the Australian environment, yet evidence is limited. The saprophytic ability of the pathogen is still unresolved despite the important role this could play in the ability of the pathogen to survive in the absence of susceptible hosts. This thesis aimed to investigate chlamydospores, oospores and the saprophytic ability of P. cinnamomi to determine their contribution to survival. Phytophthora cinnamomi did not show saprophytic ability in non-sterile soils. The production of thick-walled chlamydospores and selfed oospores of P. cinnamomi in vitro was documented. Thick-walled chlamydospores were sporadically formed under sterile and non-sterile conditions in vitro but exact conditions for stimulating their formation could not be determined. The formation of thick-walled chlamydospores emerging from mycelium of similar wall thickness was observed, challenging the current knowledge of chlamydospore formation. Selfed oospores were abundant in vitro on modified Ribeiro’s minimal medium in one isolate. Three other isolates tested also produced oospores but not in large numbers. Although the selfed oospores did not germinate on a range of media, at least 16 % were found to be viable using Thiozolyl Blue Tetrazolium Bromide staining and staining of the nuclei with 4´, 6-diamidino-2-phenylindole.2HCl (DAPI). This indicated the potential of selfed oospores as survival structures and their ability to exist dormantly. The ability of phosphite to kill chlamydospores and selfed oospores was studied in vitro. Results challenged the efficacy of this chemical and revealed the necessity for further study of its effect on survival propagules of P. cinnamomi in the natural environment. Phosphite was shown to induce dormancy in thin-walled chlamydospores if present during their formation in vitro. Interestingly, dormancy was only induced by phosphite in isolates previously reported as sensitive to phosphite and not those reported as tolerant. Chlamydospores were produced uniformly across the radius of the colony on control modified Ribeiro’s minimal medium but on medium containing phosphite (40 or 100 µg ml-1), chlamydospore production was initially inhibited before being stimulated during the log phase of growth. This corresponded to a point in the colony morphology where mycelial density changed from tightly packed mycelium to sparse on medium containing phosphite. This change in morphology did not occur when the pathogen was grown on liquid media refreshed every four days, and chlamydospores were evenly distributed across the radius of these colonies. This trend was not observed in selfed oospores produced in the presence of phosphite. Selfed oospore production was found to be inhibited by phosphite at the same concentrations that stimulated chlamydospore production. Isolates of P. cinnamomi were transformed using a protoplast/ polyethylene glycol method to contain the Green Fluorescent Protein and geneticin resistance genes to aid in future studies on survival properties of the organism. Although time constraints meant the stability of the transgene could not be determined, it was effective in differentiating propagules of the transformed P. cinnamomi from spores of other microrganisms in a non-sterile environment. Two different sized chlamydospores (approximately 30 µm diameter and < 20 µm diameter) were observed in preliminary trials of transformed P. cinnamomi inoculated lupin roots floated in non-sterile soil extracts and these were easily distinguished from microbial propagules of other species. The growth and pathogenicity was reduced in two putative transformants and their ability to fluoresce declined over ten subcultures but they still remained resistant to geneticin. This study has improved our knowledge on the survival abilities of P. cinnamomi in vitro and has provided a useful tool for studying these abilities under more natural glasshouse conditions. Important implications of phosphite as a control have been raised.

This project assesses two aspects of DNA detection of Phytophthora species from soil samples. Firstly, a nested PCR protocol was established with both primary and nested PCR specific for P. cinnamomi detection. PCR amplification of P. cinnamomi DNA isolated from soil was optimised with the addition of bovine serum albumin and formamide. This was found to improve both the specificity and sensitivity of PCR amplification of DNA in the presence of inhibitors co-extracted along with the target DNA from soil samples. The application of diagnostic nested PCR with the addition of BSA and formamide was verified by comparison with routine culture based detection methods. In all cases, nested PCR detection incorporating BSA and formamide was found to be considerably more sensitive than the culture based detection methods. The second component of this thesis investigates the simultaneous detection of multiple species of Phytophthora using microarray analysis. Microarray based detection has been previously limited by variable and inconsistent hybridisation intensities across the diversity of probes used in each array. In this study a novel concept for the differentiation of detection targets using duplex melting kinetics is introduced. A microarray assay was developed on a PamChip „¥ microarray enabling the differentiation of target Phytophthora species using the melting kinetics of probe-target duplexes. In the majority of cases the hybridization kinetics of target and non-target duplexes differed significantly. Analysis of the melting kinetics of duplexes formed by probes with target and non-target DNA was found to be an effective method for determining specific hybridization and was independent of fluctuations in hybridization signal intensity. This form of analysis was more robust than the traditional approach based on hybridisation intensity, and allowed the detection of individual Phytophthora species and mixtures there of.

Thesis (Ph.D)--Murdoch University, 2003. / Thesis submitted to the Division of Science and Engineering. Bibliography: leaves 219-235.