Accumulation of free amino acids and humic substances in a freshwater modular system and their ecological significance

1. Methods for measuring concentrations of total and individual free amino acids (TFAA, FAA), other amino compounds and humic substances (HS) in media containing the following three treatments and the control are described: (i) the non-axenic aquatic macrophyte Ceratophyllum demersum alone; (ii) the pulmonate snail, Biomphalaria glabrata alone; (iii) the snail, plant and the epiphytic bacteria and algae together as a four-component modular system; (iv) control without organisms. 2. TFAA accumulated to give asymptotic values of 2.5 μm , 10 μm and 15 μm in treatments (i), (ii) and (iii), respectively, by the end of the 30-day incubation period. The mass-specific accumulation rate in the treatment with the snail alone [89.4 nmol g^–1 (wet weight minus shell) day^–1] was approximately ten times that with the plant alone [8.4 nmol g^–1 (wet mass) day^–1]. No FAA or HS could be detected at any time in (iv). 3. On the second day of incubation the concentrations of TFAA and of some individual amino acids were significantly higher in the treatment containing the snail and plant together than the sum of the concentrations in the treatments with the plant and snail alone, presumably due to an increase in the snails’ metabolic rate and ‘sloppy feeding’. 4. The differences in the relative abundance of amino compounds accumulating in media conditioned by snails alone (e.g. much larger proportions of ammonia, ethanolamine and phosphoserine than in plant-conditioned media) and plants alone (e.g. larger proportions of asparagine and glutamine than in snail-conditioned media) suggest that snails and plants may derive mutual benefits by exchanging amino compounds. 5. The accumulation patterns of the more recalcitrant HS differ from those of the amino acids in two respects. First, the HS concentrations continued to increase throughout the 30-day incubation period in all three treatments. Second, most of the HS in the module originates from the plant as both the concentrations and mass-specific accumulation rates were significantly higher in the treatment with the plant alone [2.6 mg l^–1, 9.09 μg g^–1 (wet mass) day^–1, respectively] than in the treatment with the snail alone [0.75 mg l^–1 and 7.7 μg g^–1 (wet mass) day^–1, respectively]. 6. The possible reasons for the differences in the accumulation patterns of FAA and HS in the three treatments are discussed. Evidence is also given in support of the testable hypothesis that the four components of the module derive several mutual benefits, including those arising from the release of dissolved organic matter, such as FAA and HS, by living organisms.     

Fruit-body production of an ectomycorrhizal fungus in genus <Emphasis Type="Italic">Boletus</Emphasis> in pure culture

Mycelial growth and fruit-body production of an ectomycorrhizal Boletus sp. were examined in pure culture. Mycelia of the strain Bo1 grew well on a medium consisting of sawdust and barley grains. Mature fruit bodies bearing basidiospores were produced after incubation at 22°C for 90 days in the dark, followed by incubation at 26°C for 30–46 days under conditions of high humidity and illumination. The addition of porous stone as a casing on the medium increased fruit-body yield. Deposited spores germinated well on an agar medium and formed mycelial colonies, thus completing the life cycle of Bo1 without a host plant and under axenic conditions. The ability of Bo1 to form ectomycorrhizas was confirmed by axenic resynthesis of mycorrhizas on Quercus serrata. Cultured fruit bodies of Bo1 resembled Gyroporus castaneus and Boletus subcinnamomeus, but its taxonomic position was not elucidated at the species level.     

Isolation and purification of Australian isolates of the toxic cyanobacteriumMicrocystis aeruginosa Kütz

Isolation and laboratory culture ofMicrocystis aeruginosa Kütz. using a growth medium (MLA medium) suitable for both non-axenic and axenic cultures is described. Seventeen established strains ofM. aeruginosa were subjected to one or more of three purification methods: centrifugation cleaning, sulphide gradient selection, and antibiotic treatment (Imipenem^®). While each method purified only about half of the strains attempted, the selective application of each method, based on the morphological characteristics of the strains, succeeded in purifying 12 of the 17 strains. Three of the 5 strains not purified were contaminated with a sulphide-tolerant, Imipenem-resistant spirochaete,Spirochaeta cf.aurantia, which could not be detected on normal, broad spectrum bacterial test media. The presence of this bacterial species was detected only by phase contrast and DAPI (4,6-diamidino-2-phenylindole) stained fluorescence microscopy.Author for correspondence     

Amylase production by Mucor pusillus and Humicola lanuginosa as related to mycelial growth

Mycelial dry weights of Mucor pusillus and Humicola lanuginosa reached maxima after two and eight days of incubation, respectively, in starch-yeast media. Maximum levels of extracellular amylase activities measured in the growth media were recorded after three to 11 days of incubation of M. pusillus, and after 9–25 days of incubation of H. lanuginosa, periods corresponding to observed reductions in mycelial dry weights. In both cases, abnormally high concentrations of reducing sugars were measured in the growth media prior to the attainment of maximum mycelial dry weight. It is suggested that a membrane-bound form of amylase might be principally responsible for providing reducing sugars necessary for growth, and extracellular amylase might be due principally to autolysis.     

Production of microsclerotia of the fungal entomopathogen Metarhizium anisopliae and their potential for use as a biocontrol agent for soil-inhabiting insects

Microsclerotia (MS), overwintering structures produced by many plant pathogenic fungi, have not been described for Metarhizium anisopliae. Three strains of M. anisopliae – F52, TM109, and MA1200 – formed MS in shake flask cultures using media with varying carbon concentrations and carbon-to-nitrogen (C:N) ratios. Under the conditions of this study, all strains produced MS, compact hyphal aggregates that become pigmented with culture age, in addition to more typical blastospores and mycelia. While all strains formed desiccation tolerant MS, highest concentrations (2.7–2.9 × 10⁸ L⁻¹ liquid medium) were produced in rich media with C:N ratios of 30:1 and 50:1 by strain F52. All three strains of M. anisopliae produced similar biomass concentrations when media and growth time were compared. Strain MA1200 produced higher concentrations of blastospores than the other two strains of M. anisopliae with highest blastospore concentrations (1.6 and 4.2 × 10⁸ blastospores ml⁻¹ on days 4 and 8, respectively) in media with the highest carbon and nitrogen concentrations. Microsclerotial preparations of M. anisopliae containing diatomaceous earth survived air-drying (to <5 % moisture) with no significant loss in viability. Rehydration and incubation of air-dried MS granules on water agar plates resulted in hyphal germination and sporogenic germination to produce high concentrations of conidia. Bioassays using soil-incorporated, air-dried MS preparations resulted in significant infection and mortality in larvae of the sugar beet root maggot, Tetanops myopaeformis. This is the first report of the production of sclerotial bodies by M. anisopliae and provides a novel approach for the control of soil-dwelling insects with this entomopathogenic fungus.     

Decolourisation of reactive textile dyes Drimarene Blue X3LR and Remazol Brilliant Blue R by <Emphasis Type="Italic">Funalia trogii</Emphasis> ATCC 200800

Decolourisation of reactive dyes Drimarene Blue X3LR and Remazol Brilliant Blue R by white rot fungi Funalia trogii was studied under static conditions. The effect of various conditions such as mycelial age, initial dye and glucose concentrations on decolourisation were also investigated. Decolourisation activity of F. trogii was compared with Phanerochaete chrysosporium known as test microorganism. It was found that 7-day-old cultures were more effective than 5-day-old cultures of F. trogii for decolourisation of these dyes. Decolourisations by F. trogii of both dyes were increased with glucose concentration decreasing. In contrast, decolourisations by P. chrysosporium were decreased. F. trogii decolourised 92–98% of both dyes within 4–10 h. However, P. chrysosporium partially decolourised (11–20%) these dyes during 10days incubation period under the same conditions.     

Decomposition of duckweed (Lemna gibba) under axenic and microbial [-2pt] conditions: flux of nutrients between litter water and sediment, the impact of leaching and microbial degradation

The decomposition of axenic Lemna gibba has been studied over a 200 day period under laboratory conditions in the presence and absence of wastewater micro-organisms. The residual mass of plant litter in the decomposition vessels decreased three times more rapidly under biotic than abiotic conditions. The organic matter in the duckweed litter lost about half its weight within 67.9 days in the presence of micro-organisms while more than 200 days were required in axenic vessels. In the former case, AFDW loss followed an exponential pattern of decay. The rate constant was 0.0102 day ^–1 and the decay was virtually complete after 200 days. The C and K concentration of the remaining duckweed litter decreased; the N, Ca, Fe and B concentration increased in both treatments. The concentration of total N, P, K, Mg, and Mo increased in the receiving water in both treatments but was much higher under biotic than abiotic conditions. Mass balances of nutrients in the vessels and flux of these nutrients between compartments in the vessels (duckweed litter, water and sediment) have been determined. Under axenic conditions the release of elements was very slow. Only notably potassium leaching had occurred. Leaching of potassium, magnesium and organic carbon took place mainly during the first term of incubation and then slowed down. Under biotic decomposition the elemental content of the litter decreased by more than 50% over 43 days for K, 53 days for Mo, 64 days for C, 81 days for Mg, 101 days for S, 104 days for P, 108 days for Na, 111 days for N, 140 days for B. Calcium and iron immobilised in the litter. Most of the released N, S, P, K, Mg and Mo remained in the water, but B and Mn settled into the sediment. The result of the investigation demonstrated that the nutrient flux from decomposing duckweed litter is mainly a microbially mediated process.     

Culture and taxonomic status of Chlorochytrium lemnae a green algal endophyte

Pure (axenic) strains of an intercellular endophyte identified as Chlorochytrium lemnae Cohn (= Chlorosphaeropsis lemnae Moewus) were isolated from infected duckweed (Lemna spp.). Pure cultures of the host were also obtained. Both grew well in mineral media, requiring no organic growth factors. In suitably dilute media, the algae could be induced to infect dead leaves of several different Lemna clones. Since its cells divide vegetatively by the formation of common cross walls (“desmoschisis”), Chlorochytrium lemnae should be transferred from the Chlorococcales to the Chaetophorales. The taxonomic status of other algae identified as Chlorochytrium—some of them demonstrated to be stages in the life-cycle of filamentous algae—should be re-evaluated.     

Assignment of <Emphasis Type="Italic">Lemna gibba</Emphasis> L. (duckweed) bioassay for <Emphasis Type="Italic">in situ</Emphasis> ecotoxicity assessment

To narrow the differences between the results obtained from radionuclides and heavy metal ecotoxicity investigations in the laboratory and in the abandoned uranium mines, a few standardised plant bioassay procedures were selected from the literature for testing with Lemna gibba L. The bioassay procedures were tested in situ and ex situ. The laboratory culturing was performed in batch and semicontinuous modes. The results revealed that most of the standardised plant bioassay procedures require modification for the L. gibba bioassay to predict the actual effects under field conditions. L. gibba performed relatively better in the field than laboratory batch cultures despite that the batch cultures had many-fold higher nutrient concentrations than in the field. For instance, the phosphorus concentration of the mine tailing water was 0.13 ± 0.09 μg l⁻¹ in the field, while the literature range for phosphorus in the laboratory culture media is 13.6–40 mg l⁻¹. L. gibba growth in the laboratory batch culture was influenced by speciation changes due to consumption of nutrients, CO₂ and O₂ phase exchanges, and excretion of organic substances by the test plants. Semicontinuous culture modes performed significantly better than batch cultivation even after 10× dilution of the nutrient solution. The growth behaviour revealed that L. gibba exhibited intrapopulation and probiotic interaction for best performance. Growth performance of L. gibba was influenced by the anions that balanced essential cations despite equal cation concentration in the culture media; e.g., the best growth was observed in culture media that had more SO₄²⁻ than Cl⁻. Water samples from the field had higher SO₄²⁻ concentrations than Cl⁻. The test vessel material, sterilisation and axenic culturing procedures also influenced the sensitivity of the bioassay. These, for instance, and a few others are neither described nor reported in most standard Lemna tests or the literature. Thus, this work presents results of a series of tests conducted on the selected methods. Common and possible errors and corrective measures in assigning L. gibba bioassay from laboratory population levels to field community levels are discussed.     

Phenology and photosynthetic activity in sterile and fertile sporophytes of Dryopteris filix-mas (L.) Schott

Summary This study describes the phenology of sporophytes of the fern Dryopteris filix-mas in relation to whole plant development. Sterile and fertile potted sporophytes were set out at an exposed site and the seasonal development of the fronds was measured from the commencement of unfolding, through the phase of increasing length, up to discoloration. The physiological activity of the fronds was determined by measuring photosynthetic gas exchange. The fronds of sterile sporophytes unfolded in April, about a week earlier than those of fertile plants, but the colour had already begun to turn in September and their life span was 1–2 months shorter. However, between mid-June and the end of August the sterile sporophytes put out several sets of new fronds: these overwintered without changing color and were still photosynthetically active in the following spring. All types of fronds were fully expanded 1–2 months from the beginning of unfolding and, with a natural supply of CO₂, had similar maximum net photosynthetic rates of 8–9 mol/m² · s. The decline in photosynthetic performance began before symptoms of senescence were visible and was due to decreased efficiency of the mesophyll. It is concluded that the phenology of D. filix-mas changes with transition from the sterile to the fertile phase. Whereas fertile sporophytes are genuinely summergreen, the sterile sporophytes with their summer fronds remain green throughout the winter and should therefore be termed semi-evergreen. The formation of overwintering summer shoots clearly extends the period of photosynthetic productivity of sterile sporophytes.     

Dual axenic culture of sheared-root inocula of vesicular-arbuscular mycorrhizal fungi associated with tomato roots

Surface-sterilized sheared-root inocula of two vesicular-arbuscular mycorrhizal (VAM) fungi (Glomus intraradices and G. versiforme) from pot cultures associated with excised tomato roots showed significant sporulation and the production of an extensive hyphal biomass. As many as 10²–10³ axenic mature spores were recovered in Petri dishes during 3 months incubation in the dark. Propagules of both species were able to complete their vegetative life cycle in vitro and efficiently colonize Acacia albida roots after 1 month under greenhouse conditions. The effectiveness of 0.5 cm pieces of VAM roots as starter inocula indicates the high inoculum potential of intravesicle propagules.     

Modelling on isoamyl isovalerate synthesis from Rhizomucor miehei lipasein organic media: optimization studies

Immobilized lipase from Rhizomucor miehei (Lipozyme IM-20) was employed in the esterification of isovaleric acid and isoamyl alcohol to synthesize isoamyl isovalerate in n-heptane. Response surface methodology (RSM) based on a five-level, five-variable central composite rotatable design (CCRD) was used to evaluate the effects of important variables: enzyme concentration (20–40% w/w of acid), acid concentration (0.2–1.0 M), incubation period (24–120 h), alcohol concentration (0.25–1.25 M) and temperature (30–70 °C) on the esterification yield of isoamyl isovalerate. Extent of conversion was found to be excellent at all acid and alcohol concentrations employed in the range of 0.2–1.25 M, even at low enzyme concentration (20% w/w). The optimum conditions arrived at are as follows: 35% (w/w) enzyme concentration, 1.0 M acid concentration, 1.25 M alcohol concentration and 120 h incubation period, at 35 °C. Under these conditions, the predicted value was 680 mM ester matched very well with an experimental value of 678 mM.     

Fecundity,spore recruitment and size in Gelidium sesquipedale (Gelidiales,Rhodophyta)

Gelidium sesquipedale fecundity was quantified by counting tetrasporangial sori and cystocarps per meter squared and by estimating the number of spores contained inside them. These were obtained by regression on a size metric of reproductive structures. Tetrasporangial sori length and cystocarp thickness were the best estimators of spore number. To assess spore recruitment, 12 pottery tiles were fixed to the bottom, and the appearance of small fronds was monitored.No clear seasonal pattern of reproduction was found. Tetraspore production peaked in March 1990 with 10.4 × 10⁶ spores m^–2, whereas the carpospore peak was lower, 4.9 × 10⁵ spores m^–2 in July 1989. Recruitment followed tetraspore peaks. The probability of a G. sesquipedale tetraspore making the transition to a recruit was 4.7 × 10^–5. Frond length was significantly related to tetrasporangial sori number, while cystocarp number was only related to frond branching order. Minimum size for reproduction was 6.9 cm for gametophytes and 5.4 cm for tetrasporophytes; very rarely were cystocarpic fronds smaller than 9 cm, while tetrasporic fronds were often longer than 15 cm. Cystocarpic fronds were significantly shorter and had more branches than tetrasporic fronds.     

Mycelial elongation and sporulation of two fungi on amended media in light or dark

Botrytis allii andCollectotrichum dematium are onion pathogens which can infect in the field and cause decay in storage. Some phenolics can hinder development of these fungi, but the effect of cytokinins is not clear. Cytokinins (kinetin or 6-benzyladenine) or phenolics (caffeic or chlorogenic acids) were added to agar at concentrations of 0 to 10^–3 M. Cultures were continuously irradiated with fluorescent light or maintained in the dark for 6 days. On unamended media, final mycelial elongation was 45 or 17.8 mm and sporulation was 28 or 10.6 × 10⁴ spores/ml forBotrytis andColletotrichum, respectively. ForBotrytis, mycelial elongation was slightly (5%) but significantly increased and sporulation increased by 21% by incubation on phenolics as compared to cytokinins. Mycelial extension ofColletotrichum was not affected by amendment. Sporulation ofColletotrichum on kinetin was 16 to 28% greater than on the other amendments. As amendments concentration increased elongation of mycelia of both fungi decreased. Sporulation ofBotrytis increased by 60% as amendment concentration increased from 0 to 10^–5 M and then decreased 25% at 10^–3 M. As amendment concentration increased from 0 to 10^–3 M, sporulation ofColletotrichum increased by 45%. Incubation in light increased mycelial extension 3 to 17% forBotrytis andColletotrichum respectively, and sporulation was increased approximately 78% for both fungi. These compounds do not appear to inhibit development of theseBotrytis orColletotrichum species in culture.     

The role of bacteria in the metal tolerance of the fouling diatom Amphora coffeaeformis Ag.

Components of “spent” axenic and non-axenic Amphora coffeaeformis Ag. culture media were tested for their effect on copper and tributyltin tolerance in axenic A. coffeaeformis. Growth of such algal cultures in the presence of either toxin was enhanced by the addition of enriched “spent” medium from exponentially growing non-axenic cultures. This response was seen whether or not the bacteria had been removed. It appears that bacteria or bacteria-algal interactions produce some soluble (<0.2 μm) factor which either decreases the toxicity of copper and tributyltinfluoride (TBTF) or increases the tolerance of A. coffeaeformis to these toxins.     

The dose of 1-naphthaleneacetic acid determines flower-bud regeneration in tobacco explants at a large range of concentrations

Short-term applications of very high concentrations of 1-naphthaleneacetic acid (NAA) to expiants from flower stalks of tobacco (Nicotiana tabacum L. cv. Samsun) induced flower-bud regeneration to the same extent as longer or continuous incubation on lower concentrations. The maximum number of flower buds per explant after 15 d of culture was obtained not only by continuous culturing at 1 mol·l^–1 NAA but also by 12 h of culturing at 22 mol·l^–1 or 0.5 h at 220 mol· l^–1, followed by incubation on medium without auxin for the remaining period. Continuous application of such high concentrations resulted in callus formation or caused the death of the explanted tissue. In all experiments in which auxin concentration and time of application were independently varied, the product of concentration and time determined the number of buds formed. Most, but not all, of the NAA taken up by the tissues was converted into conjugates. In expiants which had received a dose which was optimal for regeneration, the internal concentration of free NAA remaining beyond the pulse period was between 1.7 and 6.2 mol·l^–1. Suboptimal applications led to lower values, supraoptimal treatments to much higher internal concentrations. The physiological effect, which depends on the internal hormone concentration, thus manifested itself as dose-dependent with regard to applied hormone.Abbreviations BAP N⁶-benzylaminopurine - NAA 1-naphthaleneacetic acid     

Increase in activity of acetylcholinesterase by 20-OH-ecdysone in a Chironomus tentans cell line

Summary An epithelial cell line from Chironomus tentans exhibits acetylcholinesterase activity (specific activity 0.05–0.2 nkat/mg protein), which rises 30– to 40-fold after addition of 10^–6 M 20-OH-ecdysone. The first visible increase occurs after 4 days of incubation with hormone. The enzyme has an apparent K _m of 2.3±0.2×10^–4 M for acetylthiocholine iodide as substrate and is inhibited by eserine and BW284 C51 (50% inhibition at 5×10^–7 M for both inhibitiors) as well as by high concentrations of substrate, but not by tetraisopropylpyrophosphamide. The sensitivity against inhibitors is the same in extracts from hormone-treated cells and from controls. The cholinesterase activity correlates with morphological changes (shape and cell arrangement) and is indepenent of neuronal differentiation. We therefore propose a function for this activity during morphogenesis.     

Tris not only controls the pH in microalgal cultures,but also feeds bacteria

Tris (Tris(hydroxymethyl)amino methane), a compound often used as a buffer in microalgal culture media, sustains active bacterial growth in non-axenic microalgal cultures when sodium phosphate is present. The low pH levels caused by bacterial growth and probably the depletion of phosphorus in the medium caused the collapse ofPhaeodactylum tricornutum cultures resulting in a reduction of microalgal growth from 32 x 10⁶ to 1.1 x 10⁶ cells ml^–1. This emphasizes the need for care when interpreting the results of non-axenic microalgae cultures in which Tris or other organic buffer is added.     

Effects of kinetin,paclobutrazol and their interactions on the microtuberization of potato stem segments cultured in vitro in the light

Single-nodal cuttings of Solanum tuberosum (four cultivars) and Solanum chacoense were induced to produce in vitro microtubers on Murashige & Skoog (MS) medium supplemented with 8 g l^–1 sucrose and various concentrations of kinetin and paclobutrazol. The cultures were kept 10 days in darkness and then transferred to a 14 h daylength with 100 µE m^–2 sec^–1 light intensity at 21 °C. Kinetin (2.5 mg l^–1) had no significant influence on tuber formation. However, its addition together with paclobutrazol (0.001 mg l^–1) significantly enhanced tuberization. Paclobutrazol alone stimulated early tuber initiation and inhibited stem growth. Despite some genotype × treatment interactions, all genotypes (from very early to late and wild type) formed the maximum proportion of explants bearing microtubers on the media containing both plant growth regulators.     

Conversion of ribulose-1,5-bisphosphate carboxylase to an acidic and catalytically inactive form by extracts of osmotically stressed Lemna minor fronds

The fronds of Lemna minor L. respond to a number of stresses, and in particular to an osmotic stress, by producing an enzyme system which catalyzes the oxidation of ribulose-1,5-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39) to an acidic and catalytically inactive form. During the first 24 h of osmotic stress the induced oxidase system does not seem to exert a significant in-vivo effect on RuBPCase, presumably because of compartmentation. Subsequently, the oxidase system gains access to the enzyme and converts it to the acid and catalytically inactive form and eventually the oxidase system declines in activity.A number of partially acidified forms of RuBPCase are formed during oxidation, and this process appears to be correlated with the disappearance of varying numbers of SH residues. The number of-SH residues in RuBPCase from Lemna has been estimated at 89. However, RuBPCase isolated from 24-h osmotically stressed fronds showed a reduction in the number of-SH residues per molecule from 89 to 54. It seems likely that the oxidation of-SH groups is causally related to the acidification of RuBPCase which occurs during osmotic stress.Abbreviations DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - FPLC fast protein liquid chromatography - PMSF phenylmethylsulphonyl fluoride - RuBPCase ribulose-1,5-bisphosphate carboxylase - SDS sodium dodecyl sulfate