Effect of four chlorophytes on larval survival, development and adult body size of the mosquito Aedes aegypti

The effect of four microalgal chlorophytes, Chlorella vulgaris,Scenedesmus quadricauda, Chlorococcum sp. and Ankistrodesmus convolutus, on the survival, larval development and adultbody size of the mosquito Aedes aegypti was investigated. The percentage mortality of larvae fed with C. vulgaris, Chlorococcum sp.or S. quadricauda after six days was 100%, 84% and 88%,respectively. Delayed pupation and body size reduction of the mosquitoesfed with C. vulgaris, Chlorococcum sp. and S. quadricaudawere observed. In contrast, larvae fed with A. convolutus werebigger than those fed with normal insectory feed. The study showed thatC. vulgaris, S. quadricauda and Chlorococcum sp. have potentialto be used as larvicidal agents.     

Comparative analysis of astaxanthin and its esters in the mutant E1 of Haematococcus pluvialis and other green algae by HPLC with a C30 column

A gradient reversed-phase high-performance liquid chromatography (HPLC) method using a C30 column was developed for the simultaneous determination of astaxanthin, astaxanthin monoesters and astaxanthin diesters in the green algae Chlorococcum sp., Chlorella zofingiensis, Haematococcus pluvialis and the mutant E1, which was obtained from the mutagenesis of H. pluvialis by exposure to UV-irradiation and ethyl methanesulphonate (EMS) with subsequent screening using nicotine. The results showed that the contents of total astaxanthins including free astaxanthin and astaxanthin esters ranged from 1.4 to 30.9 mg/g dry biomass in these green algae. The lower total astaxanthin levels (< 2 mg/g dry biomass) were detected in the green algae Chlorococcum sp. and C. zofingiensis. The higher total astaxanthin levels (>16 mg/g dry biomass) were found in the green alga H. pluvialis and its mutant E1. It is notable that the mutant E1 is found to have considerably higher amounts of total astaxanthin (30.9 mg/g) as compared to the wild strain of H. pluvialis (16.1 mg/g). This indicates that UV-irradiation and EMS compound mutagenesis with subsequent screening using nicotine is an effective method for breeding of a high-producing astaxanthin strain of H. pluvialis. In addition, the green alga C. zofingiensis had a remarkably higher percentage of astaxanthin diesters (76.3% of total astaxanthins) and a remarkably lower percentage of astaxanthin monoesters (18.0% of total astaxanthins) in comparison with H. pluvialis (35.5% for diesters and 60.9% for monoesters), the mutant E1 (49.1% and 48.1%) and Chlorococcum sp. (18.0% and 58.6%). Supported by the Frontier Research Grant of the SCSIO, the Hundred Talents program of Chinese Academy of Sciences, and National Natural Sciences of China projects (Grant No. 40776087)     

Changes in chlorophyll fluorescence quenching and pigment composition in the green alga Chlorococcum sp. grown under nitrogen deficiency and salinity stress

Changes in the in vivo chlorophyll fluorescencequenching, photosynthesis and pigment composition werefollowed in the green alga Chlorococcum sp.during exposure of the culture to nitrogen deficiencyand salinity stress with the aims to study theinterrelations between changes in physiological andphotochemical parameters and xanthophyll-cyclepigments content during adaptation to stress, and toevaluate the capacity of this green alga to producesecondary carotenoids in tubular photobioreactors.Exposure of Chlorococcum to nitrogendeficiency, 0.2 M NaCl and high irradiance outdoorscaused a strong depression of the photosyntheticactivity and of photochemical quantum yield ofPSII (F_v/F_m). These changes wereaccompanied by an increase of the non-photochemicalquenching coefficient (NPQ), of the amount ofxanthophyll-cycle pigments and of thecarotenoid/chlorophyll ratio. As a result of exposureto stress conditions, cell division completelystopped, although an increase in the biomass dryweight could be detected due to an increase in thecell size. These processes were followed, with acertain delay (15–20 h), by massive appearance ofsecondary carotenoids that reached the maximum (about50% total carotenoids) after 2–3 days of cultivation.The results show that despite of the lower carotenoidcontent (2 mg g^-1 dry wt) as compared with Haematococcus, Chlorococcum can be apotentially interesting strain for secondarycarotenoid production because of its higher growthrate.     

Ultrastructural characterization of eubacteria residing within leaf cavities of symbiotic and cyanobiont-freeAzolla mexicana

The occurrence and ultrastructure of eubacteria within leaf cavities of symbiotic and cyanobiont-freeAzolla mexicana were examined with transmission electron microscopy. Bacteria were observed in all leaf cavities of bothAzolla cultures, showing increasing numbers concomitant with leaf age. In symbioticAzolla mexicana two ultrastructurally distinct types of bacteria were found; a helical bacterium and a slightly curved, rod-shaped bacterium. Both had typical Gram-negative cell wall ultrastructure. The helical bacteria comprised 60% of the total population in young leaf cavities (1–5) and 74–80% of the population in older cavities. The mode of cell division for the rod-shaped bacterium was binary fission. In cyanobiont-freeAzolla mexicana at least three distinct types of bacteria were observed; two were ultrastructurally similar to, if not identical, with the types present in symbioticAzolla mexicana.     

Interactions Between Hyphosphere-Associated Bacteria and the Fungus <Emphasis Type="Italic">Cladosporium herbarum</Emphasis> on Aquatic Leaf Litter

We investigated microbial interactions of aquatic bacteria associated with hyphae (the hyphosphere) of freshwater fungi on leaf litter. Bacteria were isolated directly from the hyphae of fungi from sedimented leaves of a small stream in the National Park “Lower Oder,” Germany. To investigate interactions, bacteria and fungi were pairwise co-cultivated on leaf-extract medium and in microcosms loaded with leaves. The performance of fungi and bacteria was monitored by measuring growth, enzyme production, and respiration of mono- and co-cultures. Growth inhibition of the fungus Cladosporium herbarum by Ralstonia pickettii was detected on leaf extract agar plates. In microcosms, the presence of Chryseobacterium sp. lowered the exocellulase, endocellulase, and cellobiase activity of the fungus. Additionally, the conversion of leaf material into microbial biomass was retarded in co-cultures. The respiration of the fungus was uninfluenced by the presence of the bacterium.     

The Role of a Green Alga in the Precipitation of Calcite and the Coprecipitation of Phosphate in Freshwater

The precipitation of calcite from a calcium bicarbonate solution, similar in ionic strength to natural hardwaters, was observed in a series of experiments utilizing an automated culture apparatus. Seeded growth experiments, using calcite seed crystals, were performed at a range of phosphate concentrations to observe inhibitory effects. These experiments demonstrated a linear relationship of increasing inhibition with increasing initial phosphate concentration. A further series of experiments was performed in which an actively photosynthesizing culture of a unicellular green alga (Chlorococcum sp.) was added to the culture vessel in order to initiate precipitation. Experiments to observe spontaneous precipitation, occurring in the absence of both seed and alga additions, were carried out to compare with precipitation rates in the algal experiments. A control experiment was also performed to investigate whether precipitation occurrred in algal cultures maintained in darkness. The carbonate site mechanistic model, developed for calcite precipitation in abiotic conditions, was used to analyse the results of the algal experiments and found to be applicable.     

The occurrence of coryneform bacteria in the leaf cavity of Azolla

Coryneform bacteria were found associated with the nitrogen fixing blue-green alga, Anabaena azollae in the leaf cavity of Azolla caroliniana. Plate counts indicated ca. 7,400±1,900 bacterial cells per mature leaf cavity or approximately 1 bacterial cell for every algal cell. No other type of bacterium was found in these cavities.     

Degradation of Cinnamate via β-Oxidation to Benzoate by a Defined,Syntrophic Consortium of Anaerobic Bacteria

A syntrophic consortium was enriched in a basal medium containing cinnamate as the carbon and energy source. It was found to consist of three morphologically distinct microbes, viz., a short, rod-shaped, non-motile bacterium with distinctly pointed ends, Papillibacter cinnamivorans; a rod-shaped, motile bacterium with rounded ends, Syntrophus sp.; and a methanoarchaeon, Methanobacterium sp. This methanogen was then replaced by a collection strain of Methanobacterium formicicum. A syntrophic interdependency of the three partners of the consortium was observed during growth on cinnamate. In the presence of bromoethanesulfonic acid (BESA), cinnamate was transformed to benzoate, whereas under methanogenic conditions without BESA, cinnamate was first transformed to benzoate via β-oxidation and subsequently completely degraded into acetate, CH₄, and CO₂. Papillibacter cinnamivorans was responsible for benzoate production from cinnamate, whereas a syntrophic association between Syntrophus sp. and the methanogen degraded benzoate to acetate, CH₄, and CO₂. A new anaerobic degradation pathway of cinnamate into benzoate via β-oxidation by a pure culture of P. cinnamivorans is proposed. Received: 27 December 2001 / Accepted: 28 March 2002     

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.     

Proteome analysis of fungal and bacterial involvement in leaf litter decomposition

Fungi and bacteria are key players in the decomposition of leaf litter, but their individual contributions to the process and their interactions are still poorly known. We combined semi‐quantitative proteome analyses (1‐D PAGE‐LC‐MS/MS) with qualitative and quantitative analyses of extracellular degradative enzyme activities to unravel the respective roles of a fungus and a bacterium during litter decomposition. Two model organisms, a mesophilic Gram‐negative bacterium (Pectobacterium carotovorum) and an ascomycete (Aspergillus nidulans), were grown in both, pure culture and co‐culture on minimal medium containing either glucose or beech leaf litter as sole carbon source. P. carotovorum grew best in co‐culture with the fungus, whereas growth of A. nidulans was significantly reduced when the bacterium was present. This observation suggests that P. carotovorum has only limited capabilities to degrade leaf litter and profits from the degradation products of A. nidulans at the expense of fungal growth. In accordance with this interpretation, our proteome analysis revealed that most of the extracellular biodegradative enzymes (i.e. proteases, pectinases, and cellulases) in the cultures with beech litter were expressed by the fungus, the bacterium producing only low levels of pectinases.     

Carbohydrates Oxidation by Pseudomonas albosesamae nov. sp.

Taxonomic behaviors of a newly isolated bacterium which produces 2,5-diketogluconic acid in high yield were examined in this paper. The bacterium was isolated from sesame seed. It is aerobic, rod-shaped bacilli (0.6~0.8 × 1.0~3.0 microns) with rounded ends. It occurs singly or as a small mass and shows motility with a polar flagellum. Gram staining and acid-fast staining are both negative. Endospore and capsule are not observed. It does not possess photosynthetic and usual pigments the cell. Glucose and other sugars are attacked with acid production, but no gas formation. Polysaccharides and sucrose are not attacked. This bacterium does not produce acetic acid from ethanol. The above behaviors and other physiological properties which are described in the text lead to the conclusion that the bacterium is a new species situated in the genus Pseudomonas. So, it was named Pseudomonas albosesamae, nov. sp. (Wakisaka) in relation with its isolated origin.     

Coexisting <Emphasis Type="Italic">Curtobacterium</Emphasis> Bacterium Promotes Growth of White-Rot Fungus <Emphasis Type="Italic">Stereum</Emphasis> sp.

White-rot basidiomycetes are the main decomposers of woody biomass in forest ecosystems. Little is known, however, about the interactions between white-rot fungi and other microorganisms in decayed wood. A wood-rotting fungus, Stereum sp. strain TN4F, was isolated from a fruit body, and its coexisting cultivable bacteria were isolated from its substrate; natural white-rot decayed wood. The effects of bacteria on fungal growth were examined by confrontational assay in vitro. A growth-promoting bacterium for this Stereum strain was identified as Curtobacterium sp. TN4W-19, using 16SrRNA sequencing. A confrontational assay revealed that Curtobacterium sp. TN4W-19 significantly promoted the mycelial growth of Stereum sp. TN4F in the direction of the bacterial colony, without direct contact between the mycelium and bacterial cells. This is the first report of a positive interaction between a white-rot fungus and a coexisting bacterial strain in vitro.     

Experimental Studies on the Physiology of the Phycobiont and Mycobiont Ramalina ecklonii By

The lichenized fungus and alga of the fruticose lichen Ramalini ecklonii were isolated into pure cultures. The ascospores of the fungus failed to germinate in less than five weeks incubation in spite of the use of a variety of cultural conditions. The fungus showed a considerable increase in growth on malt extract agar. Both organisms showed a marked tolerance for high concentrations of glucose although growth was quantitatively reduced. The fungus was able to use a variety of carbon and nitrogen sources as well as an extract of algal cells. Cultivation in the absence of biotin and thiamine failed to yield significant amounts of growth. The alga yielded 27 mg of dry weight after three weeks in a synthetic medium under low light intensities. The alga could be grown in satisfactory amounts on CO₂ and inorganic salts with moderate light intensities. Experiments using ¹⁴CO₂ showed the fungus able to incorporate the extra-cellular and intra-cellular products of algal metabolism. The rate of incorporation of extra-cellular products was inhibited by high concentrations of biotin and thiamine. The alga assimilated ^l4CO₂ which was retained by the cells over a period of 14 days, at which time 78 per cent of the activity was insoluble in 80 per cent ethanol. An extract of the fungus labelled with ¹⁴C glucose was partially taken up by the alga and 50 per cent of the label was insoluble in 80 per cent after three days incubation in the light. No lichen acids were found in either the fungal cultures or the algal cultures although large amounts (e.g. 2 liters) of material were extracted and chromatographed. Usnic acid was produced by the intact lichen thallus.     

Isolation and alga-inhibiting characterization of Vibrio sp. BS02 against Alexandrium tamarense

The bacterium BS02 which is closely related to the genus Vibrio sp. and capable of inhibiting the toxic dinoflagellate Alexandrium tamarense was isolated from a mangrove area in Zhangjiangkou, Fujian Province, China. The bacterium was not species-specific since it displayed varying degrees of lysing activities against eight of the eighteen algae tested. There was a close interaction between initial bacterial and A. tamarense cell densities, indicating that algal growth was prompted at low bacterial concentrations, while the number of the alga cells was reduced at high concentrations. Alga-lysing characterization of Vibrio sp. BS02 suggested that the alga-lysing substance was extracellularly produced, less than 500 in molecular weight, as well as non proteinaceous, stable under wide range of temperature and pH conditions, UV radiation, repeated freezing and thawing and heavy metal treatments. These findings suggested that BS02 could play an important role in controlling harmful algal blooms.     

A bacterial leaf spot of Protea cynaroides (king protea)

The etiology of a new leaf spot disease on king protea was investigated. The causal organism was a Gram negative rod-shaped bacterium with between two and seven flagella. On the basis of cultural, biochemical and physiological characters it was identified as a pathovar of Pseudomonas syringae.     

ASCOPHYLLUM NODOSUM (PHAEOPHYTA) IN AXENIC CULTURE AND ITS RESPONSE TO THE ENDOPHYTIC FUNGUS MYCOSPHAERELLA ASCOPHYLLI AND EPIPHYTIC BACTERIA1

Ascophyllum nodosum (L.) Le Jol. was freed from bacteria and the endophytic fungus Mycosphaerella ascophylli Cotton by repeated treatment with chlorine solutions and grown in artificial seawater. Two types of axenic culture of different origin were obtained. Type 1 was developed from apices of A. nodosum collected in the sea. Type 2 was from plants which developed from adventitious embryos on rhizoids formed by type 1. This is the first time A. nodosum has been cultivated axenically. Growth of the axenic alga was increased by IAA, 21P and zeatin. Without external growth regulators some strains of the axenic alga deteriorated within a year; others developed a filamentous habit. Sulfur in a reduced state also stimulated growth. Addition of either glucose, mannose or mannitol to the medium caused the formation of calluslike layers of loosely packed colorless cells under the epidermis of the thalli and the epidermis was sloughed off. No increase in thallus length was noticed. Mycosphaerella ascophylli in axenic culture did not excude any substances stimulating growth of the alga, but that does not exclude an influence of the fungus on the alga in vivo. The fungus, when growing within the alga, seemed to have little influence on algal morphology. A bacterized but fungus-free A. nodosum was cultivated in an artificial seawater for 8 years. In the bacteria-free alga, the fungus protruded from the epidermis and evidently utilized the alga as a carbon source. The bacteria thus seem much more important than the fungus for normal growth of the Ascophyllum plant.     

Symbiosis constraints: Strong mycobiont control limits nutrient response in lichens

Symbioses such as lichens are potentially threatened by drastic environmental changes. We used the lichen Peltigera aphthosa—a symbiosis between a fungus (mycobiont), a green alga (Coccomyxa sp.), and N₂‐fixing cyanobacteria (Nostoc sp.)—as a model organism to assess the effects of environmental perturbations in nitrogen (N) or phosphorus (P). Growth, carbon (C) and N stable isotopes, CNP concentrations, and specific markers were analyzed in whole thalli and the partners after 4 months of daily nutrient additions in the field. Thallus N was 40% higher in N‐fertilized thalli, amino acid concentrations were twice as high, while fungal chitin but not ergosterol was lower. Nitrogen also resulted in a thicker algal layer and density, and a higher δ¹³C abundance in all three partners. Photosynthesis was not affected by either N or P. Thallus growth increased with light dose independent of fertilization regime. We conclude that faster algal growth compared to fungal lead to increased competition for light and CO₂ among the Coccomyxa cells, and for C between alga and fungus, resulting in neither photosynthesis nor thallus growth responded to N fertilization. This suggests that the symbiotic lifestyle of lichens may prevent them from utilizing nutrient abundance to increase C assimilation and growth.     

Rates of photosynthesis of two lichens and of their isolated algal components

Abstract

The rate of photosynthesis of two lichen species (Peltigera leucophlebia and Ramalina farinacea) was found to be 30 to 40% that of spinach leaf dises and 20% that of the free-living alga Chlorella when the results were expressed on a per mg chlorophyll basis. When the algae were isolated from the thalli, the rate of photosynthesis per mg chlorophyll increased for Ramalina farinacea and decreased for Peltigera leucophlebia. Product analysis indicated that the products of photosynthesis depended on the association of the alga with the fungus: algae isolated from the thalli showed a «shift» in products from sugars and sugar alcohols. to compounds such as organic acids. The results suggest that a symbiotic relationship with a fungus alters both the rate and products of algal photosynthesis.     

Artificial tripartite symbiosis involving a green alga (Chlamydomonas), a bacterium (Azotobacter) and a fungus (Alternaria): Morphological and physiological characterization

A long-living artificial tripartite symbiosis involving a green alga (Chlamydomonas), a bacterium (Azotobacter) and a fungus (Alternaria) was established on carbon- and nitrogen-free medium. The basis of the interdependence is the complementation of photosynthetic CO₂ assimilation and atmospheric nitrogen fixation. Green color of the colonies indicated that the algal cells had enough nitrogen to synthesize chlorophylls. The chlorophyll content was nearly 40 % of the control cells. The relatively high rate of photosynthetic oxygen evolution proved that nitrogen was effectively used for building up a well functioning photosynthetic apparatus. This was supported by the analysis of photosystems and ultrastructural investigations. In comparison with degreened algae cultured on nitrogen-free medium, the chloroplasts in the symbiont algal cells contained a well-developed, stacked thylakoid membrane system without extreme starch or lipid accumulation. The occurrence of the fungus in the association greatly increased the chlorophyll content. Far fewer types of amino acids were excreted by the tripartite cultures than by pure cultures. Cystathionine, which is a common intermediate in the sulfur-containing amino acid metabolism, was produced in high quantities by the tripartite symbiosis. This can mostly be attributed to the activity of the fungus.     

Degradation of phenol via carboxylation to benzoate by a defined,obligate syntrophic consortium of anaerobic bacteria

Summary An obligate syntrophic culture was selected in mineral medium with phenol as the only carbon and energy source. The consortium consisted of a short and a long rod-shaped bacterium and of low numbers of Desulfovibrio cells, and grew only in syntrophy with methanogens, e. g. Methanospirillum hungatei. Under N₂/CO₂, phenol was degraded via benzoate to acetate, CH₄ and CO₂, while in the presence of H₂/CO₂ benzoate was formed, but not further degraded. When 4-hydroxybenzoate was fed to the mixed culture, it was decarboxylated to phenol prior to benzoate formation and subsequent ring cleavage. Isolation of pure cultures of the two rod-shaped bacteria failed. Microscopic observations during feeding of either 4-hydroxybenzoate, phenol or benzoate implied an obligate syntrophic interdependence of the two different rod-shaped bacteria and of the methanogen. The non-motile rods formed phenol from 4-hydroxybenzoate and benzoate from phenol, requiring an as yet unknown co-substrate or co-factor, probably cross-fed by the short, motile rod. The short, motile rodshaped bacterium grew only in syntrophy with methanogens and degraded benzoate to acetate, CO₂ and methane. Desulfovibrio sp., present in low numbers, apparently could not contribute to the degradation of phenol or 4-hydroxybenzoate.