UV-induced DNA damage promotes resistance to the biotrophic pathogen Hyaloperonospora parasitica in Arabidopsis

Plant innate immunity to pathogenic microorganisms is activated in response to recognition of extracellular or intracellular pathogen molecules by transmembrane receptors or resistance proteins, respectively. The defense signaling pathways share components with those involved in plant responses to UV radiation, which can induce expression of plant genes important for pathogen resistance. Such intriguing links suggest that UV treatment might activate resistance to pathogens in normally susceptible host plants. Here, we demonstrate that pre-inoculative UV (254 nm) irradiation of Arabidopsis (Arabidopsis thaliana) susceptible to infection by the biotrophic oomycete Hyaloperonospora parasitica, the causative agent of downy mildew, induces dose- and time-dependent resistance to the pathogen detectable up to 7 d after UV exposure. Limiting repair of UV photoproducts by postirradiation incubation in the dark, or mutational inactivation of cyclobutane pyrimidine dimer photolyase, (6-4) photoproduct photolyase, or nucleotide excision repair increased the magnitude of UV-induced pathogen resistance. In the absence of treatment with 254-nm UV, plant nucleotide excision repair mutants also defective for cyclobutane pyrimidine dimer or (6-4) photoproduct photolyase displayed resistance to H. parasitica, partially attributable to short wavelength UV-B (280–320 nm) radiation emitted by incubator lights. These results indicate UV irradiation can initiate the development of resistance to H. parasitica in plants normally susceptible to the pathogen and point to a key role for UV-induced DNA damage. They also suggest UV treatment can circumvent the requirement for recognition of H. parasitica molecules by Arabidopsis proteins to activate an immune response.

Effects of phytophthora cinnamomi on heathland flora and fauna of the Eastern Otway Ranges

The plant pathogen, Phytophthora dnnamomi, is a cause of dieback disease observed in sclerophyll vegetation in Australia, The effects of P. dnnamomi on flora and fauna were studied at two locations in heathland vegetation near the coastal town of Anglesea, Victoria. The pathogen was isolated from soils beneath diseased heathland plants. The extent of diseased vegetation was assessed by the presence and absence of highly sensitive indicator species, Xanthorrhoea australis and hopogon ceratophyllus. The characteristics of heathland vegetation exhibiting dieback disease associated with the presence of P. dnnamomi were investigated. Plant species richness was similar between diseased and non-diseased areas however diseased areas were characterised by significant declines in the cover and frequency of susceptible species, increases in resistant species and increases in percent cover of open ground. Compared to non-diseased areas, diseased areas exhibited fewer shrub species and decreased shrub cover. The percentage cover and number of species of sedges, lilies and grasses were higher in diseased areas. Structural differences were significant between 0-0.6 m with decreased cover of vegetation in diseased areas. Differences in structure between diseased and non-diseased areas were not as great as expected due to increases in the cover of resistant species. A number of regenerating X australis were observed in post-disease areas. Cluster analysis of floristic data could clearly separate diseased and non-diseased trap stations. The population dynamics and habitat use of eight small mammal species present were compared in diseased and non-diseased areas using trapping and radio-tracking techniques. The number of small mammal species captured in post-disease areas was significantly lower than non-diseased areas. Mean captures of Antechinus stuartii and Rattus fiisdpes were significantly lower in diseased areas on Grid B. Mean captures of Rattus lutreolus were significantly lower in diseased areas on both study grids. Significant differences were not observed in every season over the two year study period. Radio tracking revealed more observations of Sminthopsis leucopus in non-diseased vegetation than in diseased. Cercartetus nanus was frequently observed to utilise the disease susceptible X. australis for nesting. At one location, the recovery of vegetation and small mammal communities in non-diseased and diseased vegetation after fuel reduction burning was monitored for three years post-fire. Return of plant species after fire in both disease classes were similar, reaching 75% of pre-fire richness after three years. Vegetation cover was slower to return after fire in diseased areas. Of the seven small mammal species captured pre-fire, five were regularly captured in the three years after fire. General linear model analysis revealed a significant influence of disease on capture rates for total small mammals before fire and a significant influence of fire on capture rates for total small mammals after fire. After three years, the influence of fire on capture rates was reduced no significant difference was detected between disease classes. Measurements of microclimate indicate that diseased, burnt heathland was likely to experience greater extremes of temperature and wind speed. Seeding of diseased heathland with X. australis resulted in the establishment of seedlings of this sensitive species. The reported distributions of the mamma] species in Victoria were analysed to determine which species were associated with the reported distribution of dieback disease. Twenty-two species have more than 20% of their known distribution in diseased areas. Five of these species, Pseudomys novaehollandiae, Pseudomys fumeust Pseudomys shortridgei, Potorous longipes and Petrogale pencillata are rare or endangered in Victoria. Four of the twenty-two species, Sminthopsis leucopus, Isoodon obesulus, Cercartetus nanus and Rottus lutreolus am observed in Victorian heathlands. Phytophthora cinnamomi changes both the structure and floristics of heathland vegetation in the eastern Qtway Ranges. Small mammals respond to these changes through decreased utilisation of diseased heathland. The pathogen threatens the diversity of species present and future research efforts should be directed towards limiting its spread and rehabilitating diseased areas.

Use of phosphite as a control for Phytophthora cinnamomi in southeastern Victorian vegetation communities

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.

Aspects of the interaction between Xanthorrhoea australis and Phytophthora cinnamomi in south-western Victoria, Australia

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.

Effects of Phytophthora cinnamomi on heathland fauna and ecosystem function

Progression of disease caused by the plant pathogen Phytophthora cinnamomi was correlated to rainfall events and resulted in a loss of plant species diversity in heathland vegetation at Anglesea, Victoria. Lower captures of small mammals were recorded in diseased areas. Management of disease using the chemical phosphite was also evaluated.

Profiling secondary metabolites of plant roots inoculated with Phytophthora cinnamomi

The soil-borne pathogen, Phytophthora cinnamomi, continues to cause severe dieback in Australian native forest species and is of great international significance due to its global distribution. This research established a protocol to successfully identify phyto-chemicals associated with the defense response of plants challenged by the disease caused by Phytophthora cinnamomi.

The antimicrobial efficacy of elaeis guineensis : characterization, in Vitro and in Vivo studies

The urgent need to treat multi-drug resistant pathogenic microorganisms in chronically infected patients has given rise to the development of new antimicrobials from natural resources. We have tested Elaeis guineensis Jacq (Arecaceae) methanol extract against a variety of bacterial, fungal and yeast strains associated with infections. Our studies have demonstrated that E. guineensis exhibits excellent antimicrobial activity in vitro and in vivo against the bacterial and fungal strains tested. A marked inhibitory effect of the E. guineensis extracts was observed against C. albicans whereby E. guineensis extract at =, 1, or 2 times the MIC significantly inhibited C. albicans growth with a noticeable drop in optical density (OD) of the bacterial culture. This finding confirmed the anticandidal activity of the extract on C. albicans. Imaging using scanning (SEM) and transmission (TEM) electron microscopy was done to determine the major alterations in the microstructure of the extract-treated C. albicans. The main abnormalities noted via SEM and TEM studies were the alteration in morphology of the yeast cells. In vivo antimicrobial activity was studied in mice that had been inoculated with C. albicans and exhibited good anticandidal activity. The authors conclude that the extract may be used as a candidate for the development of anticandidal agent.

Optimisation and analysis of three polymerase chain reaction (PCR) systems for the detection of environmentally significant microorganisms

Conventional assessments of water quality are based on the determination of bacterial indicator numbers. The detection of an indicator organism only provides presumptive evidence of the presence of harmful organisms By using polymerase chain reaction (PCR) to monitor water quality, indicators and pathogenic organisms can be specifically detected with a high sensitivity. The research illustrates the importance of optimisation and the maintenance of reaction efficiency in the accuracy and precision of the PCR.

Eukaryotic microorganisms for producing lipids and antioxidants

Disclosed are compositions and methods related to eukaryotic microorganisms that can produce unsaturated fatty acids which can be purified and used.

Multi-envelope HIV-1 vaccine devoid of SIV components controls disease in macaques challenged with heterologous pathogenic SHIV

A central obstacle to the design of a global HIV-1 vaccine is virus diversity. Pathogen diversity is not unique to HIV-1, and has been successfully conquered in other fields by the creation of vaccine cocktails. Here we describe the testing of an HIV-1 envelope cocktail vaccine. Six macaques received the vaccine, delivered by successive immunizations with recombinant DNA, recombinant vaccinia virus and recombinant envelope proteins. Following vaccination, animals developed a diversity of anti-envelope antibody binding and neutralizing activities toward proteins and viruses that were not represented by sequence in the vaccine. T-cells were also elicited, as measured by gamma-interferon production assays with envelope-derived peptide pools. Vaccinated and control animals were then challenged with the heterologous pathogenic SHIV, 89.6P. Vaccinated monkeys experienced significantly lower virus titers and better maintenance of CD4+ T-cells than unvaccinated controls. The B- and T-cell immune responses were far superior post-challenge in the vaccinated group. Four of six vaccinated animals and only one of six control animals survived a 44-week observation period post-challenge. The present report is the first to describe pathogenic SHIV disease control mediated by a heterologous HIV-1 vaccine, devoid of 89.6 or SIV derivatives.

Hampered foraging and migratory performance in swans infected with low-pathogenic avian influenza A virus

It is increasingly acknowledged that migratory birds, notably waterfowl, play a critical role in the maintenance and spread of influenza A viruses. In order to elucidate the epidemiology of influenza A viruses in their natural hosts, a better understanding of the pathological effects in these hosts is required. Here we report on the feeding and migratory performance of wild migratory Bewick's swans (Cygnus columbianus bewickii Yarrell) naturally infected with low-pathogenic avian influenza (LPAI) A viruses of subtypes H6N2 and H6N8. Using information on geolocation data collected from Global Positioning Systems fitted to neck-collars, we show that infected swans experienced delayed migration, leaving their wintering site more than a month after uninfected animals. This was correlated with infected birds travelling shorter distances and fuelling and feeding at reduced rates. The data suggest that LPAI virus infections in wild migratory birds may have higher clinical and ecological impacts than previously recognised.

Antibacterial activity of stevioside towards food-borne pathogenic bacteria

This study determines the inhibitory effect of Stevia rebaudiana leaf extracts and its purified bioactive compound ‘stevioside’ against food-related pathogens. The S. rebaudiana solvent extracts (1000 μg/mL) displayed antibacterial activity to Serratia marcescens, Klebsiella pneumoniae, Bacillus cereus, Pseudomonas aeruginosa, B. subtilis, Alcaligenes denitrificans and Salmonella typhimurium. Of the six solvents, ethanol and acetone extracts displayed the highest zone of inhibition. The bioactive compound from S. rebaudiana was purified by solvent extraction, thin-layer chromatography followed by structural characterization by spectroscopy evidence. Purified stevioside prevented the growth of tested bacterial species, i.e. B. subtilis, K. pneumoniae and S. typhimurium. Significant zone of inhibition (12 mm) was observed against B. cereus which proposes potential application of stevioside in foods to increase their shelf life.