Physiological aspects of fungi isolated from root nodules of faba bean (Vicia faba L.)

The present study was made to isolate and assess some physiological characteristics of root nodule-colonizing fungi. During this study, 17 fungal species were isolated from root nodule samples taken from faba bean plants (Vicia faba L.) collected from different sites at Assiut area (Egypt). The growth of faba bean plants in pots was significantly promoted by soil inoculation with most fungi. Growth was checked in pots with inocula of Cladosporium cladosporioides, Fusarium moniliforme, F: oxysporium, F solani, Macrophominia phaseolina and Rhizoctonia solani which were added separately. All growth-promoting fungi were capable of producing cellulase, pectin lyase, polygalacturonase, protease, urease, amidase, acid phosphatase, alkaline phosphatase and arylsulfatase in growth medium supplemented with the corresponding substrates. Four fungal species, Aspergillus awamori, A. flavus, Penicillium chrysogenum and Trichoderma koningii showed the highest rates of enzyme formation. The effect of the addition of six trace elements to the growth media at 30 micromol/ml on enzyme production revealed some dependency on species, enzyme and metal ion. Cd2+, Hg2+ and Zn2+ generally inhibited enzyme activity. Cu(1+), Fe3+ and Al3+ showed a stimulatory effect. Fungicides (afugan and tilt) and herbicides (brominal and fusilade) at 50 ppm generally promoted enzyme activity, but insecticides (kelthane and fenvalerate) caused some inhibition to enzyme activities. Salinization of the growth media with NaCl strongly inhibited the enzymatic activity of all fungi at concentrations between 0.5 and 1.5%.     

Preparation of cellodextrins and isolation of oligomeric side components and their characterization

Cellodextrin (beta-1,4-glucose oligomer) mixtures are prepared by precipitation of oligomers with 1-propanol and ethanol after partial hydrolysis of cellulose with hydrochloric acid or by acetolysis of cellulose. Cellooligomers (DP3-DP8) can be isolated by high-resolution size-exclusion chromatography on Bio-Gel P 4 using water as eluent. Recycle operation of the columns allows the separation of oligomers up to a degree of polymerization of 12. However, ion-exchange chromatography of their borate complexes demonstrates the heterogeneity of cellodextrins, homogeneous according to size-exclusion chromatography. At least four secondary oligomeric components are observed in the different samples. By preparative affinity chromatography on phenyl-boronate-agarose two of these components could be purified and subsequently characterized. In one series of oligosaccharides the glucose unit at the reducing end of the beta-1,4-glucose oligomers is derivatized to fructose. This enolization reaction occurs during size-exclusion chromatography. The precipitation step with alkanols during preparation of oligomer mixtures generates oligomeric glycosides. Additionally, the formation of amines from respective beta-1,4-glucose oligomers is observed with the ammonium carbonate eluent used in affinity chromatography. Analysis methods combined to assess for the homogeneity of cellodextrins include enzyme- and acid-catalyzed (partial) hydrolysis of the different oligomers and subsequent analysis of degradation products by sugar borate chromatography; 13C and 1H NMR spectroscopy; and fast atom bombardment mass spectroscopy.     

Coenzyme Q10 supplementation and recovery from ischemia in senescent rat myocardium

Many studies have suggested that parenteral administration of coenzyme Q10 (Q10) protects the myocardium of young experimental animals from post-ischemic reperfusion injury. Although parenteral administration, in contrast to per os supplementation, seems to elevate coenzyme Q concentrations in heart tissue, it is not suitable for prophylactic use. In addition, the incidence of ischemic events is greatest in older age. We studied the effect of Q10 supplementation on myocardial postischemic recovery in 18-month-old Wistar rats. The treated group (n=9) received 10 mg/kg/day of Q10 for 8 weeks in their chow while the normal chow of the control group (n=9) contained less than 0.5 mg/kg/day of Q10. The treatment clearly elevated plasma Q10 concentration (286 +/- 25 micromol/l and 48 +/- 30 micromol/l, treated and controls, respectively, p<0.0001) but neither Q9 nor Q10 concentrations in heart tissue were affected by the supplementation. The isolated perfused hearts were subjected to 20 minutes of ischemia and 30 minutes of reperfusion. The preischemic values of developed pressure (DP) but not contractility (+DP/delta t) and relaxation (-DP/delta t) were improved by Q10 supplementation (p=0.034, p=0.057 and p=0.13, respectively) while in postischemic recovery no differences were observed between the groups (p>0.05 at all time points). Also, in myocardial flow, myocardial oxygen consumption (MVO2) and myocardial aerobic efficiency (DP/MVO2) the groups did not differ at any time points. Although dietary Q10 supplementation clearly elevated plasma Q10 concentrations in senescent rats, the coenzyme Q contents in heart tissue and myocardial recovery from ischemia were not affected. However, it is possible that the site of action for the reported beneficial effects of Q10 is in the coronary endothelium rather than myocardium itself.     

Effects of sabinene and γ-terpinene from coastal redwood leaves acting singly or in mixtures on the growth of some of their fungus endophytes

Sabinene and γ-terpinene were assayed in vitro acting singly or in mixtures in the gaseous state on the following redwood endophytes: Botrytis cinerea, Cryptosporiopsis abietina, Pestalotiopsis funerea, Phomopsis occulta, Pleuroplaconema sp. and Seiridium juniperi. The hypothesis that these redwood monoterpenes were acting additively or synergistically in inhibiting the growth on leaf endophytes was tested. Dose-response curves were obtained for each endophyte growing under five concentrations of both sabinene and γ-terpinene. Three mixtures of different ratios were assayed keeping constant 0.25 mg of monoterpenes per ml air. Either acting singly or in mixtures sabinene and γ-terpinene were inhibitory to all fungi, but their effect varied according to species. Both compounds had similar effects acting singly on each endophyte with doses from 0.0625 to 0.25 mg ml air⁻¹. With doses of 1.6667 mg ml air⁻¹ of either monoterpene, S. juniperi was mildly inhibited by sabinene and strongly inhibited by γ-terpinene, but other species were strongly and equally inhibited by both compounds. Sabinene: γ-terpinene mixtures of 1:3, 1:1 and 3:1 ratios were equally inhibitory for each species. Results suggest that when these compounds co-occur, they act additively on leaf endophytic fungi.     

Bifidogenic effect and in vitro immunomodulatory roles of melibiose-derived oligosaccharides produced by the acceptor reaction of glucansucrase E81

Glucansucrases are responsible for the production of α-glucans using sucrose as the substrate. Glucansucrases may also produce different oligosaccharides by transferring the glucose moiety from sucrose to a variety carbohydrates acting as acceptor nucleophile. In this study, melibiose-derived oligosaccharides were produced by the glucansucrase from Lactobacillus reuteri E81 expressed without the N-terminal region (GtfA-ΔN). The reaction products were characterized by TLC, LC-MS and NMR analysis and it was found that GtfA-ΔN synthesized melibiose-derived oligosaccharides (DP3 and DP4) by adding glucose units through alpha 1->3 or 1->6 glycosidic bond. The functional characteristics of these melibiose-derived oligosaccharides were determined by testing the immune-modulatory functions in HT-29 cells and testing their growth promoting effects for important probiotic and pathogenic strains. The melibiose-derived oligosaccharides triggered the production of anti-inflammatory cytokine IL-10 and pro-inflammatory cytokine TNF-α depending on their concentrations. Finally, melibiose-derived oligosaccharides showed bifidogenic effect as potential prebiotics.     

Regulation of the Chlamydomonas cell cycle by a stable, chromatin-associated retinoblastoma tumor suppressor complex

We examined the cell cycle dynamics of the retinoblastoma (RB) protein complex in the unicellular alga Chlamydomonas reinhardtii that has single homologs for each subunit-RB, E2F, and DP. We found that Chlamydomonas RB (encoded by MAT3) is a cell cycle-regulated phosphoprotein, that E2F1-DP1 can bind to a consensus E2F site, and that all three proteins interact in vivo to form a complex that can be quantitatively immunopurified. Yeast two-hybrid assays revealed the formation of a ternary complex between MAT3, DP1, and E2F1 that requires a C-terminal motif in E2F1 analogous to the RB binding domain of plant and animal E2Fs. We examined the abundance of MAT3/RB and E2F1-DP1 in highly synchronous cultures and found that they are synthesized and remain stably associated throughout the cell cycle with no detectable fraction of free E2F1-DP1. Consistent with their stable association, MAT3/RB and DP1 are constitutively nuclear, and MAT3/RB does not require DP1-E2F1 for nuclear localization. In the nucleus, MAT3/RB remains bound to chromatin throughout the cell cycle, and its chromatin binding is mediated through E2F1-DP1. Together, our data show that E2F-DP complexes can regulate the cell cycle without dissociation of their RB-related subunit and that other changes may be sufficient to convert RB-E2F-DP from a cell cycle repressor to an activator.     

The effects of syntheticdl-dormin (Abscisin II) on the growth of the oat mesocotyl

Summary The growth-inhibitory properties of syntheticdl-dormin (Abscisin II) and itstrans, trans isomer were assayed using the oat mesocotyl section. Growth of this tissue was promoted by IAA and GA; dormin inhibited the elongation caused by both compounds and also the small amount of growth that occurred in blank buffer. A given concentration of dormin inhibited growth to about the same proportion in all these cases. IAA and GA mixtures stimulated growth in the presence of 2.0 mg/l dormin slightly more than the sum of growth with IAA+2.0 mg/l and GA+2.0 mg/l dormin. At lower concentrations of dormin (0.02 mg/l and below) the relationship was reversed. Dormin inhibited growth slightly at 0.02 mg/l but below that concentration it had no observable effect.Kinetin did not affect mesocotyl elongation, nor did it interact with the inhibition of IAA- or GA-stimulated growth.Thetrans, trans isomer had 1/30th the activity of the natural 2-cis-4-trans compount; when they were assayed together in mixtures of different proportions the inhibitions were additive.Most experiments were carried out with GA₃ but dormin also inhibited the action of gibberellins 1, 4, 5, 6, 7 and 9. The action of dormin was not competitive with that of IAA or GA.The effect of dormin as an inhibitor of plant growth is discussed.     

Improved chemical analysis of cellulose ethers using dialkylamine derivatization and mass spectrometry

Oligosaccharides of hydroxypropylmethyl cellulose, hydroxypropyl cellulose, and methyl cellulose were investigated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The cellulose ether oligosaccharides were produced either by enzymatic depolymerization utilizing the purified family 5 endoglucanase from Bacillus agaradhaerens or by partial acidic depolymerization. To lower the limit of detection in MALDI-MS three dilakylamines, dimethyl-, diethyl-, and dipropylamine were studied as reagents for reductive amination of the oligosaccharides. All three amines contributed to a significant increase in sensitivity in MALDI-MS, especially for oligosaccharides with a degree of polymerization (DP) < 3. These reagents were also attractive due to their high volatility, which facilitated the purification of the reaction mixtures. It was established that low-mass discrimination in MALDI-MS in the DP range 1-7 was substantially reduced with dialkylamine derivatization. Hence, dialkylamine derivatization of cellulose ether oligosaccharides obtained by endoglucanase depolymerization increased the number of detected analyte components. Dimethylamine was concluded to be the preferred reagent of those evaluated.     

Complement C5a exhibits anxiolytic-like activity via the prostaglandin D2-DP1 receptor system coupled to adenosine A2A and GABAA receptors

We have recently found that central PGD(2) exhibits anxiolytic-like activity. Here we show that complement C5a exhibits anxiolytic-like activity via the PGD(2) system. Centrally administered C5a had anxiolytic-like activity at a dose of 0.3 pmol/mouse in the elevated plus-maze test in mice. C5a-induced anxiolytic-like activity was inhibited by indomethacin, a cyclooxygenase inhibitor, or BWA868C, an antagonist of DP(1) receptor for PGD(2), respectively. The anxiolytic effect of C5a was also blocked by SCH58261 or bicuculline, antagonists of adenosine A(2A) and GABA(A) receptors, respectively, which were activated downstream of PGD(2)-DP(1) receptor. These results suggest that C5a exhibits anxiolytic-like activity via the PGD(2)-DP(1) receptor system coupled to the activation of adenosine A(2A) and GABA(A) receptors.     

Naturally occurring prebiotic oligosaccharides in poultry feed mixtures

The presence of raffinose series oligosaccharides (RSO) was determined by an enzymic method in three commercially available chicken feed mixtures. All feed mixtures contained RSO at a concentration of 2.1–2.2 %. Soya meal was identified as the exclusive source of RSO. Subsequently, the bifidogenic effect of stachyose (main soya bean RSO) was also assigned on the growth of poultry intestinal bifidobacteria. Bifidobacteria were counted in chicken intestinal tract using cultivation and FISH methods. Four out of 6 bifidobacterial strains tested grew significantly better on stachyose than on glucose. It can be thus concluded that chicken feed mixtures naturally contain prebiotic oligosaccharides in the form of RSO in higher levels (>2 %) compared with the concentration (usually up to 1 %) recommended for artificially added prebiotics. Our results therefore indicate that there is no reason for the supplementation of chicken feed mixtures with prebiotics with bifidogenic properties.     

Characterization of water-soluble hemicelluloses from spruce and aspen employing SEC/MALDI mass spectroscopy

Partly depolymerized hemicelluloses isolated from wood chips of spruce and aspen employing microwave treatment were resolved using size-exclusion chromatography (SEC) into oligo- and polysaccharide fractions containing components with a narrow range of sizes, as determined by MALDI mass spectroscopy. The degree of substitution with acetyl moieties (DS) was also calculated on the basis of the MALDI-MS spectra obtained prior to and following deacetylation. For spruce hemicelluloses, the low molecular mass fraction contained small arabino-4-O-methylglucuronoxylan oligosaccharides, with DP values ranging from 4 to approximately 20, separated primarily on the basis of their charge density. The fraction eluted last consisted of an O-acetyl-(galacto)glucomannan polysaccharide of peak-average DP value (DP(p)) 14. The degree of substitution with acetyl groups (DS) decreased with decreasing DP, a value DS of 0.39 being obtained for the fraction with DP(p) 12. For the aspen hemicelluloses, the SEC fractions eluted first contained an acidic O-acetyl-4-O-methylglucuronoxylan polysaccharide with DP ranging from 10 to approximately 28 and an average DS of approximately 0.75. The fractions eluted last consisted of oligosaccharide mixtures composed primarily of small neutral O-acetyl-xylooligosaccharides (DP(p) 6, DS 0.41), together with minor quantities of an O-acetyl-glucomannan.     

Studies on the polyphenol metabolism of tissue cultures derived from the tea plant (Camellia sinensis L.)

1. The growth characteristics on various media of solid and liquid suspension cultures derived from the stem of the tea plant are described; chlorophyll and anthocyanin synthesis occurred in the light. 2. Only the simplest catechins and leucoanthocyanins were present in callus tissue, although oligomeric and polymeric leucoanthocyanin fractions were also represented. Light caused an increase in all monomeric components analysed, but inhibited polymerization of the leucoanthocyanins. 3. The polyphenol oxidase activity of cultures was comparable with that of the apical regions of the intact plant, and was inversely correlated with growth rate. 4. Growth was stimulated by hormonal variation, and inhibited by high concentrations of sucrose and by high light-intensity; polyphenol concentrations were generally inversely correlated with growth rate. 5. From the inability of callus tissue and of cultured root apices to synthesize complex catechins, it is inferred that complex catechin formation in intact plants is associated with the process of cell vacuolation.     

Studies on the polyphenol metabolism of tissue cultures derived from the tea pant (Camellia sinensis L.)

1. The growth characteristics on various media of solid and liquid suspension cultures derived from the stem of the tea plant are described; chlorophyll and anthocyanin synthesis occurred in the light. 2. Only the simplest catechins and leucoanthocyanins were present in callus tissue, although oligomeric and polymeric leucoanthocyanin fractions were also represented. Light caused an increase in all monomeric components analysed, but inhibited polymerization of the leucoanthocyanins. 3. The polyphenol oxidase activity of cultures was comparable with that of the apical regions of the intact plant, and was inversely correlated with growth rate. 4. Growth was stimulated by hormonal variation, and inhibited by high concentrations of sucrose and by high light-intensity; polyphenol concentrations were generally inversely correlated with growth rate. 5. From the inability of callus tissue and of cultured root apices to synthesize complex catechins, it is inferred that complex catechin formation in intact plants is associated with the process of cell vacuolation.     

Studies on the nutrition of Arthrobotrys oligospora Fres. and A. robusta Dudd: The saprophytic phase

Growth requirements of two predaceous hyphomycetes in the absence of eelworm prey were determined in specified culture media, in an investigation of the interrelationship of eelworm and fungi. Both fungi were able to utilize a wide range of energy sources and gave the highest yields of mycelium when the carbon sources supplied had the same configuration as glucose around carbon atoms, 3, 4, 5 and 6, or were oligosaccharides yielding glucose on hydrolysis. The species differed in their ability to utilize nitrogen sources, A. robusta producing very little growth when nitrate was the sole source of nitrogen, and comparatively little on aspartate nitrogen. Both responded to thiamin, biotin and p-aminobenzoic acid, but neither species showed absolute dependence on an external source of vitamin.     

Antifungal Activity of the Essential Oil of Hyssop (Hyssopus officinalis)

The antifungal and fungicidal effects of hyssop ( Hyssopus officinalis ) oil and its individual components were studied in a series of in vitro and in vivo experiments. Mycelial growth of the plant pathogenic fungi Pyrenophora avenae and Pyricularia oryzae was completely inhibited by 0.4% hyssop oil. Volatile components diffusing from agar medium containing 0.4% hyssop oil also completely inhibited the growth of these two fungi. Various components of hyssop oil ( L -bornyl acetate, isopinocampheol and pinocamphone), used individually, reduced growth of P. avenae and, where combinations of individual components were used, any mixture containing isopinocampheol completely inhibited fungal growth. Growth of P. oryzae was less affected by individual components of the oil. Hyssop oil reduced germination of Botrytis fabae conidia and uredospores of Uromyces viciae-fabae , but in contrast to the data from in vitro experiments, its effects on pathogen infection were less clear cut. Thus, although 0.05% hyssop oil reduced rust infection of broad bean when applied 1, 2 or 3 days before, or 1 or 2 days after inoculation, its effects against barley powdery mildew and apple powdery mildew were variable. It is suggested that this variability might be the result of the volatile components of the oil diffusing away from leaf surfaces, thus reducing the concentration of active components on the leaf surface.     

On-farm production of AM fungus inoculum in mixtures of compost and vermiculite

On-farm production of arbuscular mycorrhizal (AM) fungus inoculum can reduce the cost of the inoculum and increase utilization of this symbiosis in plant production. Bahiagrass (Paspalum notatum Flugge) seedlings, colonized by AM fungi, were transplanted into raised bed enclosures. Media within the enclosures was vermiculite mixed with either field soil or yard clippings compost in Experiment I and vermiculite mixed with yard clippings compost or dairy manure/leaf compost in Experiment II. Compost and vermiculite mixtures yielded more propagules of AM fungi than soil-based mixtures in Experiment I. Growth of plants in a 1:4 (v/v) mixture of yard clippings compost and vermiculite produced more inoculum (503 propagules cm(-3)) than growth in 1:9 and 1:99 (v/v) mixtures (240 and 42 propagules cm(-3), respectively). Water, inorganic nutrient solution minus P, and fish protein digest were added to inoculum production enclosures in Experiment II. Results indicated that supplemental nutrient addition was unnecessary. This method produces a concentrated inoculum of AM fungi in a form readily used as an amendment to horticultural potting media for the production of vegetable seedlings.     

Asparagine-linked oligosaccharides of BHK cells treated with inhibitors of oligosaccharide processing.

Baby-hamster kidney (BHK) cells were labelled with [2-3H]mannose for 1-2 days in media containing 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin or 1-deoxymannojirimycin. Glycopeptides obtained by Pronase digestion of disrupted cells were analysed by lectin affinity chromatography, by Bio-Gel P4 gel filtration and by paper chromatography of oligosaccharides released by endo-beta-N-acetylglucosaminidase H. Biosynthesis of complex-type oligosaccharides was diminished but not abolished, the greatest effect being obtained by continuous culture of cells with 1-deoxymannojirimycin. Under these conditions cells contained only 20-30% of the concentration of complex-type chains found in control cells and correspondingly increased amounts of oligomannose-type chains. Similar concentrations of asparagine-linked Man6-GlcNAc2 and Man5GlcNAc2 were present in 1-deoxymannojirimycin-treated cells and control cells, indicating that the inhibition of complex-type chain formation was not related simply to an inability of inhibitor-treated cells to carry out extensive mannosidase-catalysed processing. N-Methyl-1-deoxynojirimycin induced accumulation of oligomannose-type chains containing three glucose residues, and cells treated with 1-deoxynojirimycin contained oligosaccharides with one to three glucose residues. Cells cultured in the presence of the inhibitors retained sensitivity towards the galactose-binding lectins ricin and modeccin.     

Ectomycorrhizal fungi: A new source of atmospheric methyl halides?

Incomplete source budgets for methyl halides – compounds that release inorganic chlorine and bromine radicals which, in turn, catalyze atmospheric ozone depletion – limit our ability to predict the fate of the stratospheric ozone layer. We report here the first measured emissions of methyl chloride, methyl bromide, and methyl iodide from ectomycorrhizal fungi. We grew nine fungal isolates on growth media containing halide concentrations similar to those found in soils and plant tissues. The observed range of emissions was 0.003–65 μg methyl chloride, 0.001–3 μg methyl bromide, and 0.02–12 μg methyl iodide g^?1 dry weight fungi day^?1. Species varied in production rates of methyl chloride vs. methyl bromide vs. methyl iodide. Cenococcum geophilum, a widespread ectomycorrhizal fungus, was further tested to investigate the effects of halide substrate concentration in growth media. Emissions from this species increased linearly with increasing concentrations of both bromide and iodide. In addition, a subset of four fungi was studied with two media concentrations each of chloride, bromide, and iodide (0.2 or 20 mm ). These fungi had similar responses to halide concentration, despite 1000‐fold differences in baseline emission rates between isolates. Finally, high chloride concentrations (20 mm ) in media did not appear to inhibit emissions of methyl bromide or methyl iodide. Overall, ectomycorrhizal fungi might be an important source of methyl halides to the atmosphere, and substrate concentrations and community composition may influence production levels in ecosystems.     

Utilization of Carbohydrates by Rhynchosporium secalis. I. Growth and Sporulation on Glucose, Galactose, and Galacturonic acid

The fungus Rhynchosporium secalis (Oud.) Davis grew and speculated in liquid nutrient media that contained glucose, galactose or galacturonic acid, or a pair of those substances, as the sole carbon source. Sporulation was inhibited by high concentrations of glucose and galacturonic acid. Growth and sporulation were greatest on glucose, and least on galactose. Growth was increased when glucose and galacturonic acid were mixed. Nitrogen concentration affected sporulation but not growth.     

Pure culture growth of ectomycorrhizal fungi on inorganic nitrogen sources

Four ectomycorrhizal fungi were tested for their ability to grow (i.e., mycelial mat radial extension and fungal biomass) on nutrient media either supplemented with ammonium-nitrogen or nitrate-nitrogen or in the absence of an inorganic nitrogen source.Pisolithus tinctorius, Cenococcum geophilum andThelephora terrestris exhibited greater growth on ammonium-nitrogen.Suillus granulatus grew better on the nitrate-nitrogen nutrient medium. Regardless of inorganic nitrogen form preference (i.e., ammonium-nitrogen or nitrate-nitrogen), all 4 species showed some growth on each of the 3 nutrient media. Growth rate maxima varied by fungal species as well as by inorganic nitrogen source. Maximum growth rate forT. terrestris exceeded rates exhibited by the other 3 fungi by 2–5 times.