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This study is the second of two surveys designed to systematically screen extracts from marine plants for antimicrobial effects against ecologically relevant marine microorganisms, and to compare results on a geographical basis. In the preceding survey, extracts from tropical Atlantic marine algae and seagrasses were screened in growth inhibition assays against the pathogenic fungus Lindra thalassiae, the saprophytic fungus Dendryphiella salina, the saprophytic stramenopiles, Halophytophthora spinosa and Schizochytrium aggregatum, and the pathogenic bacterium Pseudoaltermonas bacteriolytica. In this study, the same assay microorganisms were used to examine the antimicrobial effects of lipophilic and hydrophilic extracts from 54 species of marine algae and two species of seagrasses collected from Indo-Pacific reef habitats. Overall, 95% of all species surveyed in this study yielded extracts that were active against one or more, and 77% yielded extracts that were active against two or more assay microorganisms. Broad-spectrum activity against three or four assay microbes was observed in the extracts from 50 to 21% of all species, respectively. Extracts from the green alga Bryopsis pennata and the red alga Portieria hornemannii inhibited the growth of all assay microorganisms. Given that antimicrobial activity was prevalent among extracts of Indo-Pacific marine plants, it is interesting to note that the inhibitory effects of each extract varied considerably between the assay microorganisms. Overall, H. spinosa and D. salina were the most susceptible while L. thalassiae, S. aggregatum, and P. bacteriolytica were the most resistant to the extracts tested. These results provide good evidence that antimicrobial chemical defenses are widespread among Indo-Pacific marine plants. Further, the activity profiles of plant extracts suggest that antimicrobial secondary metabolites can have pathogen-selective or broad-spectrum effects. To confirm these results, chemical studies will be needed to isolate and characterize the compounds responsible for the observed antimicrobial activities. [PUBLICATION ABSTRACT]

The saprobic, cosmopolitan, marine fungi Dendryphiella arenaria and Dendryphiella salina, isolated from various plant and algal substrates from different geographical locations and climatic zones, were studied for their adaptations to the abiotic and biotic parameters commonly found in their natural marine habitats. All the tested strains of D. arenaria and D. salina grew optimally on culture media with added marine salts, at pH values between 6.5 and 8.0 and at an incubation temperature of 25°C. The D. arenaria strains had faster mean colony extension rates under all conditions of culture. All strains exhibited an increased salt optimum with increasing incubation temperature. The TLC profiles of strains of the two species were similar. The culture extracts were antimicrobial, though production of the biologically active metabolites was strain-specific. There were no significant correlations between source of origin and responses to the investigated parameters. These results demonstrate phenotypic plasticity and the ability of each isolate to adapt to diverse biotopes.[PUBLICATION ABSTRACT]

Summary When Dendryphiella salina was grown on non‐saline media, mannitol and arabitol were the only polyols present in the mycelium. In media made up with sea‐water or salinized by high concentrations of salts of potassium, sodium and magnesium, glycerol was also present. When mycelium grown in sea‐water was transferred to distilled water, the glycerol content dropped very rapidly to zero. The amounts of mannitol and arabitol also declined. Transfer of similarly grown mycelium to sea‐water alone reduced the rate of decline of glycerol, caused little change in the amount of mannitol and induced a rise in that of arabitol. These observations help to explain the absence of glycerol in mycelium in previous studies. The concentration (mol l−1) of solutes within mycelium were determined after 48 h growth in the presence of either sodium chloride, magnesium chloride, sodium sulphate, inositol or betaine at either 0.4 or 0.8 osmol kg−1water. There was a very similar total polyol concentration at each osmolality in the saline media irrespective of the salt, even though there could be different concentrations of individual polyols. With inositol in the medium, the same held if the endogenous concentration of that compound was included in the total. The same did not hold for betaine, in which medium growth was poor. The total polyol concentration increased in all cases with the osmolality of the medium. With respect to the total concentration of solutes present (excluding data for mycelium grown in betaine), polyols were 19 to 33 % of the total, α‐amino nitrogen, 11 to 34% and organic acids, 0 to 8 %. These percentages take into account data obtained by 24Na‐flux studies, which indicate that, in media containing sodium salts, much of the mycelial content of the cation may be located in the wall.

Summary When mycelium of Dendryphiella salina Nicot & Pugh is exposed for 5 h to media containing various concentrations of the very slowly metabolized sugar, L‐sorbose, the total ethanol‐soluble carbohydrate content increases and there is also a stimulated synthesis of mannitol. Provided L‐sorbose is absorbed, the presence of glucose, fructose, mannitol or arabitol in the medium does not prevent these changes which are different from those brought about by the non‐metabolizable sugar, 3‐O‐methyl glucose. It is suggested that the apparent regulation of mycelial ethanol‐soluble carbohydrate concentration following uptake of the latter sugar cannot be controlled primarily by turgor.

The marine hyphomycete Dendryphiella salina (Suth.) Nicot & Pugh has a resting membrane potential of -250 mV (inside negative). The respiratory inhibitors sodium azide and FCCP induced a rapid but reversible depolarization of the membrane of at least 180 mV; sodium azide also caused alkalinization of the medium. Vanadate brought about significant depolarization but this was not always reversible. EDTA induced depolarization though to a lesser extent. DIDS and SITS caused a depolarization of around 30-70 mV which was readily reversible. N-ethylmaleimide irreversibly depolarized the membrane by 180-200 mV. Ouabain had no effect. When external concentrations of H+, K+, Na+ or Cl- were changed singly, only changes in H+ affected membrane potential, with shifts decreasing with increasing pH. Glucose and 3-O-methyl glucose depolarized the membrane in a concentration-dependent manner which was enhanced by starvation of the hyphae. Recovery occurred in the presence of the hexose. Glucose caused an alkalinization of the medium, with time characteristics similar to the membrane potential changes. It is concluded that there is an electrogenic proton pump and a proton-glucose symporter in D. salina. The retention of proton-based transport systems suggests a terrestrial origin for the fungus.

X-ray microanalysis was performed on hyphae of the filamentous marine fungus Dendryphiella salina growing at different salinities to give sodium, potassium and chloride concentrations in the cytoplasm, vacuole and cell wall. Sodium and chloride concentrations increased with salinity in all compartments. Cytoplasmic and vacuolar sodium and chloride concentration were broadly similar, and vacuolar contents represented, at most, 19% of the total protoplasmic content of an individual ion species. Potassium concentrations decreased to some extent with salinity, although concentrations were not severely affected by competition with sodium uptake. Results are discussed with regard to the role of ions in the overall osmotic adjustment in this species.

When Dendryphiella salina was grown on non-saline media, mannitol and arabitol were the only polyols present in the mycelium. In media made up with sea-water or salinized by high concentrations of salts of potassium, sodium and magnesium, glycerol was also present. When mycelium grown in sea-water was transferred to distilled water, the glycerol content dropped very rapidly to zero. The amounts of mannitol and arabitol also declined. Transfer of similarly grown mycelium to sea-water alone reduced the rate of decline of glycerol, caused little change in the amount of mannitol and induced a rise in that of arabitol. These observations help to explain the absence of glycerol in mycelium in previous studies. The concentration (mol l-1) of solutes within mycelium were determined after 48 h growth in the presence of either sodium chloride, magnesium chloride, sodium sulphate, inositol or betaine at either 0.4 or 0.8 osmol kg-1water. There was a very similar total polyol concentration at each osmolality in the saline media irrespective of the salt, even though there could be different concentrations of individual polyols. With inositol in the medium, the same held if the endogenous concentration of that compound was included in the total. The same did not hold for betaine, in which medium growth was poor. The total polyol concentration increased in all cases with the osmolality of the medium. With respect to the total concentration of solutes present (excluding data for mycelium grown in betaine), polyols were 19 to 33% of the total, α-amino nitrogen, 11 to 34% and organic acids, 0 to 8%. These percentages take into account data obtained by24Na-flux studies, which indicate that, in media containing sodium salts, much of the mycelial content of the cation may be located in the wall.

The deuteromycetes Dendryphiella arenaria and D. salina occur on macroalgae and plant debris in marine habitats. Strains from 17 locations in Canada, New England, the Southeast, Pacific Northwest and Europe were isolated from beaches and salt marshes or obtained from culture collections. The relationships between the species and the degree of genetic variability among different populations were studied. Starch-gel electrophoresis was used to analyze electromorph variation from 11 enzyme systems encoded by 14 presumptive structural loci. Strains of D. salina from different localities had identical electromorph patterns except for 3 strains isolated from Shelter Cove, California. All strains identified as D. arenaria differed from D. salina for four enzyme systems. Our data demonstrate that consistent genetic differences exist between the taxa and support the retention of two species. Some strains could not be placed with certainty in either species because of overlapping morphological characteristics; however, electrophoretic results indicate that all of these belonged to D. arenaria. The genetic variability observed was very low compared to other organisms including fungi. Our electrophoretic data indicate that both D. arenaria and D. salina are generalists and it is unlikely they have teleomorphs.

A stereological analysis of the response of cellular characteristics of hyphae of the marine hyphomycete Dendryphiella salina to salinity is presented. Analyses of volume fractions, compartmental volumes, membrane surface densities and membrane areas have been made. A major finding has been that the wall forms 40% of the compartment volume. Total hyphal compartment volume was maximal at 200 mM NaCl at 109 μm3with compartments increasing in width and decreasing in length with salinity. Maximal protoplasmic and cytoplasmic volumes were also found at 200 mM. Electron microscopy demonstrated three different vacuolar types differentiated by their staining characteristics. A light-staining vacuolar type increased in total volume from 4.5 to 12.2 μm3from 0 to 1 M NaCl. Data are considered not only in terms of hyphal anatomical characteristics but also in terms of those physiological processes occuring in D. salina which allow it to grow in saline media.

The effect of 3-O-methyl glucose on the carbohydrate metabolism of, and loss of14C-labelled compounds from, mycelium of Dendryphiella salina pretreated with [U-14C]mannitol was investigated. Though concentrations of mycelial glycerol were higher than previously reported, the results for other internal carbohydrates were generally similar. In particular, the sugar reduced the concentration of mannitol. Loss of radioactivity into the medium was increased by 3-O-methyl glucose. The pattern of loss cannot yet be explained but since there was no loss of labelled mannitol or arabitol, unspecific membrane damage seems unlikely