A specific and sensitive PCR assay for the detection of <i>Phytophthora infestans</i>, the cause of late blight of potato, in soil and plant tissues was developed. A <i>P. infestans</i> specific primer pair (INF FW2 and INF REV) was designed by comparing the aligned sequences of internal transcribed spacer regions (ITS) of a range of <i>Phytophthora </i>species. Following PCR amplification with primers INF FW2 and INF REV, a 613 bp product was generated from DNA of an isolate of <i>P. infestans</i>. No product was amplified when DNA from isolates of various other <i>Phytophthora </i>species and potato blemish pathogens was tested, showing that the primers were species-specific. These primers can detect as little as 0.5 pg of pure <i>P. infestans</i> DNA. In a nested assay, sensitivity was increased by two fold, and as little as 5 fg <i>P. infestans</i> DNA was detected. The primer pair can also detect as few as two oospores, two sporangia and four zoospores of <i>P. infestans</i>. Using primer pair INF FW2 and INF REV, ten oospores of <i>P. infestans</i> could be detected in 0.5 g soil, besides detecting the pathogen in symptomatic and symptomless leaves, stems and tubers. The assay was validated on field soil samples and commercial potato seed stocks. Studies on the long term survival of sexual (oospores) and asexual (sporangia and mycelium) inoculum of <i>P. infestans</i> were undertaken under natural field conditions. PCR detection of oospores was possible up to 24 months (total length of the study) after burial in soil. The sporangia, although detectable up to 12 months, were less efficiently detected at the nine and twelve months sampling dates, presumably representing a degradation of inoculum not adapted to long term survival. In a baiting assay, sporangial inoculum proved non-viable whereas leaf material containing oospores remained viable up to 24 months after burial. Population studies are dependant on the development of co-dominant markers. Eight single nucleotide polymorphisms (SNPs) were identified in the <i>P. infestans</i> genes <i>2- phosphoglycerate dehydratase</i>, <i>transaldolase</i>, <i>glutamine synthetase</i>, <i>ubiquitin conjugating enzyme</i> and <i>ADP/ATP translocase</i> which represents a rate of ~ 2 SNP per kb. The majority of the SNPs were synonymous (i.e. did not code for a different amino acid). Screening of randomly selected fragments of non-coding DNA from a BAC (Bacterial Artificial Chromosome) library yielded an additional 28 SNPs which represents a rate of 2 SNP per kb. Allele specific PCR was used to develop SNPs into useful markers. Use of fluorescently labelled primers markedly increased the assay throughput. Segregation analysis revealed that with the exception of 56E14R and glutamine synthetase (linked in the coupling phase), the markers segregated independently. Seventeen genotypes were detected amongst 42 Scottish <i>P. infestans</i> isolates. Cluster analysis based on SNP markers grouped isolates into two clades and broadly supported previous studies in which AFLP markers were used. In the present studies, there was some evidence of cultivar-specific adaptation of <i>P. infestans</i> isolates. The isolates caused larger lesions on the cultivars on which they were maintained for seven successive weeks. The effect of cultivar adaptation was however lost upon removal of the selection pressure. The results were validated in an inter-isolate competition experiment. Generally, more sporangia were produced on a cultivar by the isolate adapted to that cultivar than by its non-adapted competitor isolate. The use of the above developed SNP markers allowed tracking of isolates in the inter-isolate competition within a single lesion. The availability of SNP markers will provide a powerful tool for epidemiological studies of <i>P. infestans</i> on a field scale.

Phytophthora cinnamomi is an important pathogen of many plant species in natural ecosystems and horticulture industries around the world. In Western Australia, a high proportion of native plant species are susceptible to P. cinnamomi attack. Acacia pulchella, a resistant legume species native to Western Australia has been considered as a potential biological control tool against P. cinnamomi. To develop effective control methods, it is important to understand the interactions between the control agent and the different life forms of the pathogen. In this thesis the interactions are investigated between P. cinnamomi and varieties of A. pulchella which occur in jarrah (Eucalyptus marginata) forest and sand plain ecosystems. The soil inoculum of P. cinnamomi was compared under the potted plants of the three common varieties of A. pulchella, var. pulchella, var. glaberrima and var. goadbyi. These were grown in infected jarrah forest soil in the glasshouse and in vitro in a sterilised soil-less mix aseptically. Acacia urophylla (a species non suppressive towards P. cinnamomi) was also included as a control. An isolate of the most commonly found clonal lineage of P. cinnamomi in the jarrah forest, A2 type 1 was selected for use in experiments after testing showed it reliably produced zoospores and chlamydospores both axenically and in non-sterile conditions, in comparison to several other isolates. The lowest survival of P. cinnamomi inoculum was found under A. pulchella var. goadbyi plants grown both in non sterile soil and in aseptic soil-less mix. All the life forms of P. cinnamomi were affected by A. pulchella (Chapters 2, 3, 4 and 5). The soil leachates from potted plants of A. pulchella var. goadbyi reduced sporangial production (Chapter 2) and caused cytoplasm collapse of chlamydospores (Chapter 3). The confirmation was obtained that soil under A. pulchella was inhibitory to sporangial stage of P. cinnamomi and new evidence was obtained on chlamydospore inactivation. Cytoplasm collapse in the chlamydospores was observed both for chlamydospores on mycelial discs on Mira cloth exposed to the soil leachate and within infected roots buried in soils under the three varieties of A. pulchella plants. The effect was strongest under the plants of A. pulchella var. goadbyi and indicated that the chlamydospores of P. cinnamomi are unlikely to act as persistent structures under A. pulchella var. goadbyi plants. In Chapter 4, bioassays were conducted with axenically produced mycelia, chlamydospores and zoospores to test the inhibitory effect of the root exudates collected from aseptically grown A. pulchella var. goadbyi plants. The zoospores of the same isolate used in the soil leachate tests were immobilised (became sluggish and encysted) within one to two minutes. When incubated for 24 h, zoospores predominantly clumped and germ tubes were observed only from the clumped ones. Chlamydospores produced by four isolates of the common A2 type 1 strain and the only one A2 type 2 strain available at the time were tested. A higher percentage of chlamydospores collapsed and a very low percentage germinated after 24 h. Chlamydospores of all the A2 type 1 isolates were inhibited by the root exudates whilst the A2 type 2 isolate remained viable. The findings showed that the suppressive effect must be due at least in part to substances exuded by the A. pulchella plants. However, it appeared that the A2 type 1 isolates were more vulnerable to this effect than the single A2 type 2 isolate. In Chapter 5, the effect of season on sporangial suppression of P. cinnamomi was shown using field soils collected from three jarrah forest soil vegetation types and a Banksia woodland on Bassendean sand, collected in winter and summer. The effect of age of A. pulchella plants was demonstrated using the soils collected from rehabilitated bauxite mine pits. In all the locations soils were collected under A. pulchella plants and 5 m away from the nearest A. pulchella. An effect of soil type was evident as whilst the soil leachates made from the three lateritic jarrah forest soil types where A. pulchella is common in the understorey were suppressive to the sporangial stage of P. cinnamomi, this effect was not evident in the Bassendean sand under A. pulchella. A. pulchella soils collected in winter were less suppressive towards sporangial production than soils collected in summer. An effect of plant age was demonstrated as soil leachates from four year-old A. pulchella stands in rehabilitated bauxite mine sites were more suppressive for sporangia than leachates from one year-old stands. Further information on the behaviour of the pathogen in soil and in potting mix with and without A. pulchella was obtained by infecting lupin radicles with an isolate of each A2 type, 1 and 2 strains of P. cinnamomi and burying them in the soil under the three varieties of A. pulchella plants. After a week, the chlamydospores were mostly collapsed and hyphae deteriorated. Oospores were observed and in significant numbers under the potted plants of A. pulchella var. glaberrima. Isolates of all three clonal lineages of P. cinnamomi found in Australian soil were tested for the ability to produce oospores. Two isolates of the A1 and A2 type 2 and three isolates of the common A2 type 1 were screened. The two isozyme types of the A2 clonal lineage isolated in Australia varied in ability to self and produce oospores in planta in several soils from the jarrah forest. The isozyme type 2 of the A2 clonal lineage of P. cinnamomi produced oospores under these experimental conditions. This stimulation was not effective for most of the tested isolates of the A2 type 1 and the A1 clonal lineage. The in planta oospores were viable but dormant and the oogonial-antheridial associations were amphigynous both in vitro and in vivo. For the first time it was established that, the stimulus for selfing and oospore formation in the A2 type 2 of P. cinnamomi is available in some jarrah forest soils, with and without A. pulchella and also in the potting mix used. This raises important questions for the management of the pathogen. Several factors were identified as potential stimuli for selfing. Among them, soil nutrient levels and essentially enhanced sulphur presence were found important. Temperature also played a key role. Oospores were produced abundantly at 21 – 25 ºC but not over 28 ºC. The biology of P. cinnamomi has been studied for several decades but some important aspects remain un-researched. This thesis pioneers research into the in planta selfing aspect of the pathogen in soil. It also improved the understanding of the interactions between P. cinnamomi and A. pulchella which to some extent supports use of A. pulchella as a biological control tool against P. cinnamomi. However, attention is drawn to the natural mechanisms of this complex pathogen to survive in planta by producing oospores, the most persistent form of its life cycle.

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.

Kransalger är en viktig nyckelart i Östersjön. De förökar sig med hjälp av oosporer och denna studie har syftat till att urskilja morfologisk differentiering mellan oosporer inom och mellan individer och populationer. Främst ställde jag mig frågan huruvida skillnader och likheter i morfologi kan associeras med skillnader mellan olika geografiska avstånd och habitat samt i vilken mån oosporer kan återföras till korrekt population och individ. Kransalger av arten Chara aspera har insamlats på lokaler i östra Svealand och elliptiska Fouriertransformationer har använts för att med hjälp av vågfunktioner beskriva oosporernas konturer. Parametrarna i vågfunktioner har sedan använts för statistiska analyser. Resultaten visade att de olika populationerna kunde separeras morfologiskt när oosporernas populationstillhörighet varit känd för analysen. Däremot var det svårare att separera individer från varandra men det fungerade bra i den population där flest oosporer insamlats. Då oosporernas identitet varit okänd för analysen återfördes endast hälften av dem till rätt population. Det gick inte att återföra okända oosporer till individer. De tre populationerna från bräckt vatten grupperades tillsammans i diskriminantanalysen, skilda från de två sötvattenpopulationerna som i sin tur var tydligt skilda från varandra. En spridning mellan de olika populationerna i brackvatten är trolig. Däremot är en spridning mellan populationerna i sött vatten inte sannolik. Det finns inte heller något som talar för en spridning mellan habitaten. Slutligen kan jag konstatera att det finns tillräckligt mycket information att hämta i oosporerna morfologi för att mäta relativa skillnader mellan individer och grupper liksom för att skatta variabilitet. / Charophytes are an important key species in the Baltic Sea. They reproduce by using oospores and this study aims at distinguish morphological differentiation between oospores within and between individuals and populations. Mainly I asked myself the question whether the differences and similarities in morphology could be associated with differences between geographic distance and habitat, and to what extent oospores could be reassigned to the correct population and individual. Charophytes of the genus Chara aspera were collected in eastern Svealand and harmonics from elliptic Fourier transforms have been used to describe the contours of the oospores. The parameters of the harmonics were then used for statistical analyses. The results showed that the different populations could be separated morphologically when the population affiliation of the oospores has been known to the analysis. It was difficult to separate individuals from each other, but it worked well in the population in which most oospores were collected. When the identity of the oospores was unknown to the analysis only half of them were returned to the correct population, and it did not work to reassign unknown oospores to individuals. The three populations from brackish water grouped together in the discriminant analysis, separated from the two freshwater populations, which, in turn, were clearly distinct from one another. Dispersal between the different populations in brackish water is likely. However, dispersal between the populations in fresh water is not likely. There is nothing to indicate dispersal between brackish and freshwater habitats. I can conclude that there is enough information in the morphology of the oospores to measure relative differences between individuals and groups, as well as to estimate variability.