No description available.

<i>Ectocarpus siliculosus </i>is commonly challenged by the intracellular oomycete pathogen <i>Eurychasma dicksonii </i>and unlike most other pathogens affecting algae, it is available in a laboratory-controlled pathosystem.  In the context of this PhD project, the molecular processes of algal response to pathogen infection has for the first time been studied on a pathosystem using genome-enabled approaches. The proteomic investigation of the compatible (disease-causing) interaction between <i>E. siliculosus </i>and <i>Eu. dicksonii </i>via comparative two-dimensional electrophoresis elucidated 21 differentially expressed proteins. A number of proteins, identified in this course, have been associated with various stress responses (e.g. heat shock proteins, superoxide dismutases) including defence response in previous studies on macroalgae.  Most important, some  results of this study uncovered molecular aspects of the host response to biotic stress which have not been documented with elicitor-based studies so far, stressing the biological value of this pathosystem. The identification of a <i>Eurychasma-</i>resistant <i>Ectocarpus </i>strain via a qPCR assay, specifically developed for this pathosystem, will allow future applications of the proteomic approach in the investigation of the incompatible (resistant) interaction. Furthermore, <i>in-silico </i>analysis of the <i>E. siliculosus </i>genome identified homologues of plant defence-related genes, coding for so-called pathogenesis-related (PR) proteins.  Taken together, these results open new routes for the understanding of algal host pathogen interactions.

No description available.

Phytoplankton can exist in the water column, whole but non-functional, and the percentage of these dead cells in highly variable. These dead cells can contain chlorophyll and contribute to ocean colour, and hence estimates of oceanic primary productivity. The aim of this project was to assess indicators of phytoplankton physiological state, focusing on the formation of chlorophyll a oxidation products (allomers) and a chlorophyll precursor. Initially, to establish an appropriate method for the identification and quantification of chlorophyll allomers, a method selection and optimisation study was carried out. This assessment revealed that chlorophyll was prone to oxidation during sample analysis. Instrumentation, sample manipulation, method duration and HPLC solvent composition were all contributors to sample oxidation. The application of a method by Zapata et al. (2000) was found to produce minimal and consistent chlorophyll oxidation and was applied in subsequent studies. During a culture study of the picoeukaryote Ostreococcus tauri (Prasinophyceae), two chlorophyll allomers were formed solely during viral-infection, and not during environmental limitation of growth. Allomers began to increase 24 hours post viral-infection (hpi), simultaneously with decreases in population density and Fv/Fm, and an increase in membrane permeability. During viral-infection allomers reached a maximum level 48 hpi, which was 10-fold higher than the maximum level of allomers formed during environmental limitation. Chlorophyll a allomers were measured over an annual cycle for the first time, at the Western Channel Observatory (UK). Allomer occurrence (relative to chl-a) was maximal during April with a total allomer to chl-a ratio of 0.093 in surface water. Peaks in allomers were associated with blooms of Phaeocystis spp., Guinardia delicatula, Chaetoceros socialis and Emiliania huxleyi and associations were dependent on the cause of the taxas’ declines. In situ allomer measurements were also taken during a research cruise in the central and southern North Sea, where the maximum ratio of allomers to chl-a (0.15) was measured at the Flamborough Front.

A bacterial isolate and a microalga were identified to the genus level using 16S and 18S rRNA gene sequences respectively and· phylogenetic trees were constructed. The bacterial isolate belonged to the genus Halomonas and it was called Halomonas sp. NAH1, whereas the microalga was confirmed as belonging to the species Dunalie/la salina GGAP 19/30 which was the source of 18S rRNA gene. Glycerol production by three strains of the unicellular microalga Dunalie/la (D. parva 19/9, D. parva 19/10, and D. salina 19/30) was explored. The strains were grown in batch cultures in a range of salinities (0.1 4.0 M NaCI). Both intracellular and extracellular glycerol concentrations were measured. All three strains grew well over the range of salinities with optimum growth for all strains at 0.1 to 0.4 M NaC1. All strains leak significant amounts of glycerol in batch cultures. Significant leakage of glycerol into the growth medium was found to be an intrinsic property of the three strains tested. Two strains of Dunalie/la salina (GGAP 19/18 and 19/30) were grown in batch cultures and aerated with different concentrations of G02. Strain 19/18 accumulated large amounts of ~-carotene under nitrogen limitation whereas the strain 19/30 did not. Halomonas sp. NAH1 grew optimally at 1.0 M NaCI and utilised glucose, glycerol or betaine as the sole source of carbon. Glucose supported the most rapid growth rate. Sensitivity of NAH1 to antibiotics was determined and tetracycline had the most inhibitory effect on growth. The cyanobacteria Arthrospira fusiformis GGAP 1475/8 and Spirulina platensis UTEX LB 2340 were shown to be only slightly halotolerant with optimum growth for S. platensis at 0.1 M NaC1 and for A. fusiformis at 0.5 M NaC1. Phycobiliprotein content was very low in both strains, but very high protein content (92.9% of the dry weight biomass) was obtained for S. platensis. This strain looks very promising for mass cultivation for food and/or feed purposes. Glucosyl-glycerol was found to be the compatible solute in both A. fusiformis and S. platensis.

The macroalgae, or seaweeds, are ecologically and evolutionarily important but the progress of molecular research in seaweeds has been seriously hampered by the lack of transformation methodologies. One restriction is that tools which allow transformation in land plants such as biolistics and Agrobacterium have often not been tested or optimized in seaweeds. Accordingly, my thesis will use the recently sequenced genome of Chondrus crispus to develop transformation techniques in this seaweed. For biolistics, bombardment was optimised using the commonly used GUS expression cassette, pCAMBIA 1301; these bombardment parameters were subsequently used to test the expression of a native Chondrus promoter construct of my own design. Findings from biolistics were extended to test/present evidence that Chondrus thalli are also amenable to infection by Agrobacterium. Successful GUS localisation was viewed by histochemical staining within cortical and medullary cells.

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Twenty four strains of blue-green algae were screened for their ability to grow heterotrophically in complete darkness with sucrose. Eighteen of these strains proved capable of growth in the dark and all of these latter continued to grow on repeated subculture. Only six strains failed to grow in the dark and of these six, one was still viable after three months incubation in the dark. Among the organic substrates tested, sucrose (0.01M) was found to be the best substrate in allowing a considerable growth of the majority (ten out of eighteen) of the cultures in the dark. Chlorogloea fritschii and four other strains were selected for obtaining their growth rates under different environmental conditions. C. fritschii was further used in a comparison of the dark growth rates of three heterotrophic cultures: material first subcultured from light to dark, material subcultured from dark to dark and material after three years of subculturing in the dark. The growth rates (K) of all three heterotrophic cultures were found to be the same, thus suggesting that no physiological adaptation had taken place as a response to prolonged heterotrophic conditions. The addition of sucrose to cultures of C. fritschii of and of four selected strains in the light (500 lux and up to 4000 lux with C. fritschii) resulted in an increase of the growth rate (2). Growth of five strains incapable of growth in dark was significantly stimulated by sucrose at 500 and 1000 lux. Only Anabaena variabilis did not respond significantly to sucrose. Cultures grown in the dark were pigmented. Pigment analysis showed that the levels of phycocyanin and chlorophyll in dark-grown cells of selected strains are not appreciably lower than those in cells grown photoautotrophically at 500 lux- Unlike the cultures of C. fritschii which consisted of aseriate colonies only in the dark, all other strains consisted of filaments.

Seventy axenic microbial cultures were isolated from aquatic habitats, soil and air, according to their ability to lyse live blue-green algae. These isolates comprised 62 fungi, representing the genera, Acremonium, Emericellopsis and Verticillium, four bacteria of the genera Flexibacter and Pseudomonas, and four Streptomyces spp. In addition to these isolates, amoebae, identified as Acanthamoeba castellanii, were found predating algae in many of the initial plaques formed on Anabaena flos-aquae lawns. All isolates lysed A. flos-aquae, and in most cases, several other filamentous and unicellular blue-green algae also. The effects of varied experimental conditions on algal lysis by selected isolates were examined, as was the lytic mechanism involved. In general, the blue-green algae tested were most susceptible to microbial lysis when incubated at 25 to 30°C. Algal lysis also increased when the initial pH of the growth medium was raised. The fungi generally showed greater lytic activity than most of the other isolates towards a wider range of susceptible algae. Many of the fungal isolates retained their lytic activity after preservation under liquid nitrogen and freeze-drying. Emericellopsis and Acremonium isolates also inhibited the growth of blue-green algae and Gram positive bacteria, but did not lyse the latter. The exudates from these fungi also inhibited the Flexibacter flexilis isolate and Ac. castellanii. Acremonium and Emericellopsis spp. produced cephalosporin C and evidence is presented to suggest that the extracellular lytic and inhibitory activities of these fungi are due to the release of this ?-lactam antibiotic, Extracellular lytic activity was not detected with any of the other isolates, including the Verticillium fungi. The frequent isolation of predacious amoebae and lytic fungi from algal habitats suggests that these organisms may play an important algal-destroying role in natural ecosystems.

No description available.