An experimental association between Chlorella xanthella and a streptomyces

An association in the form of a lichen-like crust (“actinolichen”) was obtained by seeding Chlorella xanthella cells and spores of a Streptomyces sp. isolated from soil on the surface of potato sections partially immersed in water at temperatures with a diurnal fluctuation of 9–19 C or 14–25 C, and with illumination continuous or during the hours of the higher temperatures. Sections of the crust showed a lichen-like organized tissue with layers of Streptomyces sp. spores, hyphae alone and associated hyphae and algae.     

PRELIMINARY INVESTIGATION OF THE DOUBLE-DIFFUSION TECHNIQUE AS A TOOL IN DETERMINING RELATIONSHIPS AMONG SOME MYXOMYCETES,ORDER PHYSARALES

Double-diffusion technique was used to investigate myxomycete relationships within the order Physarales. Extracts of plasmodia of 22 slime mold isolates were reacted with five antisera produced to Plasmodia of Didymium nigripes, Physarella oblonga, Physarum polycephalum, Physarum gyrosum and Fuligo septica. Two isolates of Fuligo septica tested alike. Four isolates of Physarum pusillum did not test alike, and no valid conclusion of the relationship of this species was possible. These isolates showed strong serological affinity: (1) Physarum gyrosum, Physarella oblonga, two isolates of Fuligo septica, and possibly two isolates of Physarum pusillum, and Physarum tenerum; (2) Physarum polycephalum and Physarum flavicomum; (3) Fuligo septica and many of the species tested; (4) Didymium nigripes and at least one isolate of Didymium iridis. In most cases serologial relationships among species tested did not coincide with current taxonomy based on morphology of fructification.     

Extracellular Carbohydrates and Polysaccharides of the Alga Chlorella pyrenoidosa Chick S-39

In the growing culture of the thermophilic alga Chlorella pyrenoidosa Chick S-39, the amount of extracellular carbohydrates in the medium reached 5–17% of their content in the cells and 20–40% of the total content of extracellular organic matter. Experiments with the enrichment and synchronous algal cultures showed that the accumulation of extracellular carbohydrates and polysaccharides in the media occurred due to their release from the cells, rather than to cell lysis, and depended on cell photosynthetic activity and reproduction. Chromatographic determination of free sugars revealed the presence of saccharose, glucose, and fructose in the culture medium. Extracellular carbohydrates in C. pyrenoidosa cultures were represented mainly by water-soluble polysaccharides containing galactose, mannose, arabinose, xylose, ribose, fucose, and rhamnose.     

HETEROTHALLISM AND HOMOTHALLISM IN TWO MYXOMYCETES

Collins , O'Neil Ray . (Queens Coll., New York City.) Heterothallism and homothallism in two Myxomycetes. Amer. Jour. Bot. 48(8): 674–683. Illus. 1961.—Single-spore studies of 2 Myxomycetes, Didymium iridis and Fuligo cinerea, revealed that the former is heterothallic and the latter is homothallic. In D. iridis, 256 single-spore isolations were made from sporangia which developed in mass-spore cultures. Of these, 101 germinated and 22 yielded plasmodia that later fructified in most cases. The remaining 79 single-spore cultures produced clones of myxamoebae and swarm cells only. When 18 of the 79 clones were mated in all possible combinations, plasmodia developed in a pattern which showed that the clones were either (+) or (–) with regard to mating type. Fructifications were readily obtained from these plasmodia. Fifty-three single spores of the F₁ generation were isolated. Of the 44 that germinated, 9 yielded plasmodia in monospore cultures, and 35 produced clones of myxamoebae and swarm cells only. Twenty-five of the F₁ clones were back-crossed with their parents. Results of the back crosses show that each F₁ clone is capable of yielding plasmodia with either the (+) or the (–) parent, never with both. When 14 of the F₁ clones were mated among themselves, a (+) and (–) mating type system was again revealed. Most of the 22 original single-spore cultures which produced plasmodia, later formed sporangia. From these sporangia, 88 spores were isolated. Seventy-two of these germinated and yielded large populations of swarm cells and myxamoebae, but none produced plasmodia. Twenty of the 72 clones were then mated among themselves. Some matings resulted in plasmodial formation, but the pattern was difficult to interpret. However, when these 20 clones were mated with known (+) and (–) clones, the results appear to be in keeping with a (+) and (–) mating type system. In F. cinerea, 219 single spores were isolated from aethalia derived from mass-spore cultures. Of these, 144 germinated and the same number yielded plasmodia. Fructifications were easily obtained from such plasmodia. Thirty-five second-generation single spores were isolated, of which 15 germinated and 15 yielded plasmodia. These results indicate that F. cinerea is homothallic.     

EXCRETION OF GLYCOLATE BY A SPECIES OF CHLORELLA (CHLOROPHYCEAE)1

The claim that Chlorella sp. (CCAP 211/8p), sometimes referred to as C. fusca, Shihira and Krauss, does not excrete glycolate has been reexamined. Chlorella sp. grown on 5% CO₂in air, excreted glycolate when incubated in light in 10 mM bicarbonate. Excretion ceased 30–60 min after transfer of the cells to air and no excretion could be detected with air-grown cells or with cells grown on 5% CO₂in media buffered at pH 8.0. Incubation with 10 mM isonicotinyl hydrazide, a glycolate pathway inhibitor, caused excretion in air-grown cells and stimulated excretion in CO₂-grown cells indicating that both the rate of glycolate synthesis and metabolism is higher in CO₂grown cells than in air-grown cells. Enhanced glycolate synthesis and excretion in CO₂-grown cells is correlated with law photosynthetic rate in 10 mM bicarbonate, and the photosynthetic rate of these cells doubles over a period of 2–2.5 h after initial transfer from high CO₂to bicarbonate. This correlation of photosynthetic induction with cessation of glycolate excretion is similar to that reported in a bluegreen alga and thought to occur in other green algae. These results indicate that glycolate excretion and its regulation in this species of Chlorella is not different from that in other algae.     

On the Properties of the Starch Granules from Unicellular Green Algae

Starch granules from Chlorella, Chlamydomonas and Scenedesmus, grown heterotro-phically in a medium containing organic carbon sources, were isolated by means of the toluol treatment of the sonicate of alga. The toluol treatment separated the starch granules in the water layer from the cells and cell debris coagulated in the upper toluol layer.

The starch granules of Chlorella vulgaris and Chlamydomonas sp. were composed of amylose (12 to 3%) and amylopectin. The amylose content of the starch granules of Scenedesmus basilensis was 22 %. All the X-ray diffraction patterns of algal starch obtained in this investigation were of the A-type, identical to that of corn starch.     

Inorganic carbon acquisition in the acid‐tolerant alga Chlorella kessleri

The ability of the freshwater alga, Chlorella kessleri, to maintain a carbon concentrating mechanism when grown at acid pH was investigated. The alga grows over the pH range 4.0–9.0 and was found to take up bicarbonate and CO₂ actively when grown at pH 6.0. However, when grown at acid pH (below 5.5), it does not have active CO₂ uptake. The acidotolerant species maintained an internal pH of 6.1–7.5 over the external pH range 4.5–7.5, thus the pH difference between the cell interior and the external medium was large enough to allow for the diffusive uptake of CO₂ at acid external pH. Mass spectrometric monitoring of O₂ and CO₂ fluxes by suspensions of C. kessleri, grown at acid pH, and maintained at pH 7.5 showed that the rates of O₂ evolution did not exceed those of CO₂ uptake. The final CO₂ compensation concentrations of 14.0–17.7 µM reached by photosynthetic cells were above the CO₂ equilibrium concentration in the external medium, indicating a lack of active CO₂ uptake at acid pH. Chlorella kessleri accumulated CO₂ with internal concentrations that were 9.9, 18.7 and 22.7‐fold that of the external medium for cells grown, respectively, at pH 4.5, 5.0 and 5.5. The ability of C. kessleri cells to accumulate high intracellular concentrations of inorganic carbon at acid pH would provide a sufficiently high concentration of CO₂ at the active site of Rubisco thus allowing the alga to maintain growth rates similar to those at alkaline pH.     

Physiologische Untersuchungen an einer ungewöhnlich säureresistenten Chlorella

Summary The influence of the hydrogen-ion concentration on the growth and metabolism of a highly acid-resistant green alga, Chlorella ellipsoidea (strain Marburg St), was studied. Chlorella pyrenoidosa (Emerson strain) served as a normal control organism. Growth of Chlorella ellipsoidea occurs in the entire range from Ph 2.0 to Ph 10, whereas for Chlorella pyrenoidosa the limits were found to be Ph 3.5 and Ph 10. Respiration is much less sensitive to hydrogen-ion concentration in the acid-resistant as compared to the normal strain. Thus an increase in acidity from Ph 4.0 to Ph 2.0 increases the respiratory oxygen uptake by 120% in Chlorella pyrenoidosa and by 25% in Chlorella ellipsoidea. In addition, only the less resistant Chlorella pyrenoidosa shows an accumulation of nitrite in the dark in acid culture media, indicating a disturbance of the normal course of nitrate reduction under these conditions. On the other hand, the rate of photosynthesis of both organisms was found to be almost independent of acidity between Ph 4.0 and Ph 2.0. At the acid and alkaline limits of growth in both algae, an inhibition of cell division leads to an increase of cell size and dry weight per cell, frequently connected with the occurrence of bizarre giant cells. — In addition, adaptation phenomena were found to play a role in determining the acid limit of growth. Cells of Chlorella ellipsoidea, after inoculation from normal medium (Ph about 6) into a solution of Ph 2.0, begin growth at a high rate only after a lag of about two weeks. Cells grown previously in an acid medium, however, immediately resume growth upon inoculation into a medium of Ph 2.0. This adaptation involves a considerable reduction of cell size.     

TAXONOMIC SIGNIFICANCE OF INTRASPECIFIC ISOZYME PATTERNS OF THE SLIME MOLD FULIGO SEPTICA PRODUCED BY DISC ELECTROPHORESIS

Plasmodial extracts of 45 isolates of Fuligo septica (L.) Wiggers selected from diverse geographical areas were subjected to acrylamide disc electrophoresis. Profiles for ten enzymes and general protein were determined. Profile variability among isolates occurred. Indices of similarity calculated for each isolate indicated that the variations of the yellow and white plasmodial isolates fell within different ranges. Determination of the total range of variation for the species must wait on the analysis of more white isolates. The extreme sensitivity of disc electrophoresis in detecting color forms suggests it can be useful as a tool in myxomycete taxonomy.     

Brief in vitro study on Botrytis cinerea and Aspergillus carbonarius regarding growth and ochratoxin A

Aims: To evaluate the effect of Botrytis cinerea growth on ochratoxin A (OTA) production by Aspergillus carbonarius and degradation. Methods and Results: OTA‐producing A. carbonarius and B. cinerea were grown on grape‐like medium at 20°C for 7 days. Radii of colonies were daily recorded and OTA was analysed. In addition, each B. cinerea isolate was inoculated on grape‐like synthetic nutrient medium (SNM) paired with each A. carbonarius isolate at a distance of 45 mm. Botrytis cinerea isolates were also grown in OTA‐spiked SNM. Growth rates of B. cinerea and A. carbonarius were 20 and 7·5 mm day^?1, respectively. The growth of the colonies of each species stopped when they contacted each other in paired cultures. OTA production by A. carbonarius in the contact area was affected by B. cinerea, but no clear trend was observed. All B. cinerea isolates showed to degrade between 24·2% and 26·7% of OTA from spiked SNM. Conclusions: The ecological advantage of B. cinerea, in terms of growth rate, vs. OTA‐producing Aspergillus in some wine‐growing regions and its ability to degrade OTA may explain the low levels of this toxin in noble wines. Significance and Impact of the Study: At determinate conditions, the presence of B. cinerea in grapes with A. carbonarius may help in reducing OTA accumulation.     

BIOTIC RELATIONSHIPS BETWEEN SOIL ALGAE AND OTHER MICROORGANISMS

Parker , Bruce C., and Harold C. Bold . (U. Texas, Austin.) Biotic relationships between soil algae and other microorganisms. Amer. Jour. Bot. 48(2): 185–197. Illus. 1961.—A study was conducted of biotic relationships between various algae and other microorganisms isolated from a sample of Texas soil. From 143 two-membered combinations of organisms tested in soil-water cultures, 3 were selected for detailed studies of the nature of the causal mechanisms of the associative effects. These were: (1) an association between a species of Bracteacoccus (Br. A-20) and a heterotrophic bacterium (B-6); (2) an association between a species of Chlamydomonas (Ch. 10) and a species of Streptomyces (Act-1); and (3) an association between a blue-green alga, Phormidium sp. (Ph. 14), and an as-yet-unidentified fungus (F-2). In the first association, the heterotrophic bacterium increased growth of Bracteacoccus up to 20 times in soil-water culture; the chief cause of stimulation was shown by a series of experiments to be the decomposition by the bacterium of complex nitrogenous substrates in the soil resulting in the release of simplified products which were available to the alga as a nitrogen source. In soil-water culture, Streptomyces (Act-1) enhanced the growth and motility of Chlamydomonas (Ch. 10) and induced akinetogenesis in the alga, while the actinomycete itself was stimulated in growth and production of conidia. The mutual stimulation was shown to be caused, in part, by carbon dioxide-oxygen interchange between the organisms. Motility of Ch. 10 was enhanced by a decrease in nitrogen as a result of the growth of Act-1 and by the ability of Act-1 to decompose and assimilate the extracellular polysaccharide of the alga. The assimilation of the extracellular polysaccharide by the actinomycete promoted its growth and conidia production. Initiation of akinetogenesis in Chlamydomonas occurred exclusively in close association with the filaments of Streptomyces in soil-water cultures, and only when the concentration of available nitrogen dropped below a critical level. The akinetogenic factor has not yielded to isolation and identification, but there is some suggestion that it may be an antibiotic substance. Phormidium (Ph. 14) was frequently antagonized and annihilated by fungus 2 in the soil-water medium. Attempts to extract growth inhibitors from filtrates of the fungal medium were unsuccessful. Indirect evidence suggested that perhaps the consumption of extracellular polysaccharide with concomitant release of organic acid by the fungus might be the factor inhibiting growth of Phormidium. Attempts to confirm experimentally the ecological significance for these biotic relationships are reported.     

Cellular response of <Emphasis Type="Italic">Chlorella zofingiensis</Emphasis> to exogenous selenium

An investigation of the cellular response of the freshwater microalga Chlorella zofingiensis to exogenous selenium showed that Chlorella cells can tolerate sodium selenite up to a concentration of 100 mg l⁻¹. Cells grown in such a selenium-supplemented medium accumulated boiling-stable proteins in a concentration-dependant manner. Western blot analysis revealed that three of these boiling-stable proteins cross-reacted with anti-dehydrin antibody. Selenium was also found to exert an effect on antioxidative enzymes: superoxide dismutase (Fe-SOD and Mn-SOD isoforms) accumulated in response to selenium stress of 100 mg l⁻¹ sodium selenite, as did a new form of selenium-dependent glutathione peroxidase. Upon transfer of the cells to a selenium-free medium, the boiling-stable proteins, the superoxide dismutase isoforms and the selenium-dependent glutathione peroxidase were all down regulated. The accumulation of boiling-stable proteins and the increased activities of the antioxidant enzymes in selenium-treated Chlorella cells suggest that these compounds are probably involved in the mechanism(s) of selenium tolerance of this alga.     

Myxomycete biodiversity of the Colorado Plateau

A rapid biodiversity assessment for myxomycetes (plasmodial slime moulds) was carried out during a two-week field trip to the Colorado Plateau (western U.S.A.). Due to the very arid climate of the region, the moist chamber culture technique formed a major part of the survey. A total of 1165 records belonging to 93 species and 1 variety from 27 genera was collected from the 433 moist chamber cultures prepared with the bark surface of living plants, litter and weathered dung of herbivorous animals. Only 31 specimens of 16 wood-inhabiting species were collected in the field, mainly at higher elevations. Subsequent moist chamber cultures produced numerous bark-inhabiting species that are usually considered to be rare, including Echinostelium coelocephalum, Protophysarum phloiogenum, and Macbrideola declinata. The most common litter-inhabiting species were Didymium mexicanum and Badhamia melanospora (mainly on decaying parts of Opuntia spp. and Agave spp.); the most abundant coprophilous species were Badhamia cf. apiculospora, Fuligo cinerea, Perichaena liceoides, and Licea tenera. Both, species richness and diversity, increase from sagebrush desert to pine-juniper open woodland and pine-oak woodland. The myxomycete biota of the Colorado Plateau displays a high level of similarity to those of other arid regions of the world (mean coefficient of community C_s = 0.67), differing considerably from temperate (mean C_s = 0.34), boreal (mean C_s = 0.49) and tropical biotas (mean C_s = 0.42).     

The effect of conditioned medium obtained from <Emphasis Type="Italic">Scenedesmus subspicatus</Emphasis> on suspension culture of <Emphasis Type="Italic">Silene vulgaris</Emphasis> (<Emphasis Type="Italic">Caryophyllaceae</Emphasis>)

The effect of culture filtrate (conditioned medium, CM) containing cell exudates obtained from green alga, Scenedesmus subspicatus, on cell suspension of dicotyledonous plant Silene vulgaris was examined. The addition of diluted CM to the modified MS medium, supplemented with dicamba and BAP, stimulates cell biomass production. The biomass was composed of association of single non-dividing cells, cells during mitosis stage and cellular aggregates. Silene cells began mitotic divisions earlier in the presence of CM in medium when compared to control treatments. Results of performed bioassay showed that some factor or factors released by green alga to the culture medium could be responsible for sustained proliferation of phylogenetically distant species cells. Although it is still unclear which culture constituent influenced most the mitotic response of Silene suspension, results point at versatile stimulatory character of green alga exudates in higher plant cell culture.     

The toxicity of phenolic compounds to soil algal population and toChlorella vulgaris andNostoc linckia

Summary p-Nitrophenol (PNP),m-nitrophenol (MNP), 2,4-dinitrophenol (DNP) and catechol were tested for their effects on algal population in a soil and on pure cultures of two algae isolated from soil. Both PNP and MNP, even at 0.5 kg ha⁻¹ level were toxic to the soil algae; high doses effected increase in toxicity. Inhibition of algae was relatively more with PNP compared to the other two nitrophenols. Catechol treatment up to 1.0 kg ha⁻¹ led to a significant initial enhancement of algae with a subsequent far less toxic effect. The toxicity of the phenolic compounds towardChlorella vulgaris, a green alga andNostoc linckia, a blue-green alga, decreased in the order: MNP≧PNP>DNP>Catechol. However, algicidal or algistatic effect of the test chemicals was fairly more againstC. vulgaris, suggesting that the eukaryotic alga is highly sensitive to such soil pollutants compared to the prokaryotic alga.     

The effect of host Chlorella NC64A carbon : phosphorus ratio on the production of Paramecium bursaria Chlorella Virus-1

1. We used the freshwater alga Chlorella NC64A (Division Chlorophyta) and its virus Paramecium bursaria Chlorella virus‐1 (PBCV‐1) as a model system to test for potential stoichiometric constraints on a virus–host interaction. 2. Media phosphorus concentrations were manipulated to create Chlorella NC64A host cells with low (91 ± 23) or high (453 ± 246) C : P ratio. In contrast, the C : P ratio of PBCV‐1, calculated from its biochemical composition, was 17 : 1. 3. Stoichiometric theory predicts that infection success and postinfection viral production should be depressed in high C : P cultures due to insufficient intracellular P for production of P‐rich viral particles. 4. Consistent with this hypothesis, viral production was strongly affected by host C : P ratio. While host C : P ratio did not affect viral attachment or the percentage of new viral particles that were infectious, in the low C : P Chlorella NC64A treatment, nine times more viruses were produced per infected cell than in the high C : P treatment (158 ± 138 versus 18 ± 18), indicating that the low C : P cells were higher quality for PBCV‐1 proliferation. 5. This result implies that the stoichiometric quality of algal cells can have a major effect on host–virus population dynamics.     

NUCLEOTIDE SEQUENCE OF THE GENES FOR RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE LARGE SUBUNIT AND RIBOSOMAL PROTEIN S14 FROM A CHLORELLA-LIKE GREEN ALGA1

Two chloroplast genes were sequenced from an exsymbiotic strain of a eukaryotic, Chlorella-like green alga. The genes for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) and the ribosomal protein S14 (rps14) were oriented in the same direction and were separated by 402 bp. The rbcLs of the exsymbiont and a free living Chlorella ellipsoidea were compared with other reported rbcL sequences. The rbcL gene of the exsymbiont is closely related to that of free-living Chlorella ellipsoidea. This is the first published report of an rps 14 gene sequence from an alga.     

Fatty acid production by heterotrophic Chlorella saccharophila

The fatty acid composition of the alga Chlorella saccharophila was investigated under different growth conditions. Using glucose as the sole carbon source, heterotrophically-grown Chlorella saccharophila produced a greater proportion of the polyunsaturated fatty acids (C18: 2 and C18: 3) than photosynthetic cultures, with linoleic acid (C18: 2) predominating. An unexpected discovery was the observation that at the lowest glucose concentration (2.5 gl^–1) the lipid content of the algae increased to between 36–47% of the cell weight, depending on the temperature. At glucose concentrations of 5 g l^–1 or more, the lipid content fell to 10–12% of the cell, although total fatty acid yield was higher due to higher biomass concentrations. Aeration of heterotrophic cultures promoted the production of unsaturated fatty acids compared to non-aerated cultures.     

A cryptic intracellular green alga in <Emphasis Type="Italic">Ginkgo biloba</Emphasis>: ribosomal DNA markers reveal worldwide distribution

Intracellular symbioses involving eukaryotic microalgae and a variety of heterotrophic protists and invertebrates are widespread, but are unknown in higher plants. Recently, we reported the isolation and molecular identification of a Coccomyxa-like green alga from in vitro cell cultures of Ginkgo biloba L. This alga resides intracellularly in an immature “precursor” form with a nonfunctional chloroplast, implying that algal photosynthetic activity has no role in this endosymbiosis. In necrotizing Ginkgo cells, precursors evolved into mature algae, proliferated, and were liberated into the culture medium after host cell bursting. In the present paper we demonstrate by molecular methods a worldwide distribution of the alga in planta. Endosymbiont-specific sequences of ribosomal DNA could be traced in Ginkgo tissues of each specimen examined from different geographic locations in Europe, North America, and Asia. The Ginkgo/Coccomyca association represents a new kind of intracellular, vertically inherited symbiosis. Storage bodies, probably of lipid nature, present in the cytoplasm of each partner suggest a possible involvement of the endosymbiont in metabolic pathways of its host.     

The Relationship between Inorganic Nitrogen Metabolism and Proline Accumulation in Osmoregulatory Responses of Two Euryhaline Microalgae

Chlorella autotrophica, a euryhaline marine alga, and Stichococcus bacillaris, a salt-tolerant soil alga, grow in the presence of methionine sulfoximine (MSX), an inhibitor of glutamine synthetase, by maintaining high levels of NADPH-glutamate dehydrogenase. Nitrate reductase showed no change in MSX-adapted cells. For both species, MSX-adapted cells retained their capacity to accumulate proline in response to salinity, and in S. bacillaris no major shift was observed in the presence of MSX toward the accumulation of sorbitol. Following transfer from 33 to 150% artificial seawater (ASW), both algae exhibited increases in organic solute levels without a lag. Within 6 h of this sudden increase in salinity, the levels of proline in C. autotrophica and of proline and sorbitol in S. bacillaris were similar to those found in steady state 150% ASW cultures. Following transfer from 33 to 150% ASW, S. bacillaris continued [¹⁴C] bicarbonate photoassimilation at a normal rate and maintained active enzymes of nitrogen assimilation. The incorporation of [¹⁴C]phenylalanine into proteins was inhibited for about 30 minutes in MSX-free cells and 90 minutes in MSX-adapted cells following transfer from 33 to 150% ASW; the recovery after these lag periods was almost complete.