The influence of the chemical phosphite on Phytophthora species was investigated by studying the morphological and molecular changes induced by phosphite. In vitro experiments were conducted to study the effects of phosphite on five isolates of each of five species of Phytophthora grown in low phosphate defined medium. Sensitivity to phosphite varied greatly among the five isolates of each species and resulted in a significant interaction between isolate and phosphite effect. The EC50 values ranged from less than 5 to 10 ìg/ml for P. cinnamomi, to 13 ìg/ml for P. nicotianae, to 27 ìg/ml for P. citricola, to 24 ìg/ml for P. palmivora and to 49 ìg/ml for P. capsici. Phosphite concentrations from 5 to 100 ìg/ml caused different degrees of morphological changes. Mycelial growth of all species was significantly suppressed by phosphite at 5 ìg/ml while at 100 ìg/ml there was hyphal lysis. Swelling of hyphae with stunted sidebranches and shrinking of cytoplasm from hyphal tips and hyphal walls were characteristic changes observed. Phosphite also retarded the development and caused distortion and lysis of chlamydospores, sporangia and zoospores. Zoosporogenesis was also adversely affected. Differential display reverse transcription-PCR was used to study changes in gene expression in P. cinnamomi induced in response to phosphite stress. The differential conditions were simulated by growth on a defined medium with and without phosphite amendment. This technique resulted in the isolation of 34 putative differentially expressed cDNA fragments which were cloned and sequenced. Nucleotide sequences of 26 of these cDNA clones were generated. BLASTX analysis of these nucleotide sequences against the NCBI database revealed that 18 exhibited homology to gene sequences encoding known proteins involved in various biological processes. The remaining eight showed homology to either hypothetical or unknown or unnamed proteins. The expression level of four of these cDNA clones were further analysed by real-time quantitative RT-PCR using SYBR Green 1 assay. Three candidate endogenous reference genes namely, tubulin, cyclophilin and actin were evaluated to determine their expression level under the influence of phosphite. None of these genes were significantly regulated by phosphite. As tubulin had the highest expression among the three, it was chosen as the endogenous reference gene. Amplification efficiencies between the reference gene and each of the target genes were validated and found to be approximately equal or within 5% of each other. The relative gene expression between the phosphite-treated and untreated samples can thus be determined using the comparative CT (ÄÄCT) method. One of the cDNA clones, CP6 which showed differential expression of three-fold was up-regulated. The remaining three were constitutively expressed. CP6 which encodes 1564 nucleotides showed sequence homology, at the amino acid level with proteophosphoglycans from Leishmania major. This study demonstrated the growth inhibition and morphological deformities caused by phosphite in Phytophthora species. It also illustrated the use of a modified DDRT-PCR method to study genes expressed in phosphite stress regulation. The application of real-time quantitative RT-PCR with SYBR Green I assay facilitated the quantification of the expression level of some of these genes.

Thesis (M.Phil.)--Murdoch University, 2006. / Thesis submitted to the Division of Science and Engineering. Bibliography: leaves 103-116.

Wasting and death of deciduous trees, including oaks, is among the most serious problems today. Since 2003, the decline of oak trees in the Czech republic, among others, has been associated with the pathogenic fungus Phytophtora plurivora causing serious damage to root systems, which can lead to insufficient transport of water to transpiring leaves. The thesis is partly built on my Bachelor's thesis and includes two experiments. The objective of the first experiment,based on anatomical investigations, was to determine a theoretical maximum flow of water in the sapwood in oak seedlings. The goal of the second experiment,based on the temperature of the leaves, was to find out the functionality of the root system infected by the fungus Phytophthora plurivora. If the root system works, the transpiration is not disrupted and the leaf is cooler. The results of the first experiment give us evidence that the seedlings infected by the fungus Phytophthora plurivora are of the lower hydraulic conductivity and the lower theoretical maximum flow of water in the sapwood. The oak seedlings inoculated by the fungus Phytophthora plurivora compared with the control seedlings had smaller area of the vessels lumina, maximum theoretical flow of water in the sapwood, theoretical hydraulic conductivity, number of the vessel elements, maximum and minimum diameter of the vessel elements. In contrast, the inoculated seedlings had greater specific hydraulic conductivity and specific flow of water in the sapwood. The disruption of the root system by the fungus Phytophthora was demonstrated by 1°C higher temperature in the inoculated seedlings leaves.

No description available.

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.