Assessment of root endophytic aquatic hyphomycetous fungi on plant growth

The role of root endophytic aquatic hyphomycetous fungi in plant health was studied by employing pot experiments. Three aquatic hyphomycetous fungi Heliscus lugdunensis, Tetrachaetum elegans and Tetracladium nainitalense isolated as root endophytes of riparian plants were artificially inoculated into two test plants viz., Solanum melongena and Hibiscus esculentus. T. elegans and H. lugdunensis showed significant effects (fresh weight, dry weight and length of shoots and roots) on both test plants, whereas T. nainitalense had no effect.     

Metabolism of aromatic hydrocarbons by yeasts

Six yeasts were examined for their ability to metabolize naphthalene, biphenyl and benzo(a)pyrene. All of the organisms tested oxidized these aromatic hydrocarbons. Candida lipolytica oxidized naphthalene to 1-naphthol, 2-naphthol, 4-hydroxy-1-tetralone and trans-1,2-dihydroxy-1,2-dihydronaphthalene. The major metabolite was 1-naphthol. C. lipolytica oxidized biphenyl to produce 2-, 3-, and 4-hydroxybiphenyl, 4,4′-dihydroxybiphenyl and 3-methoxy-4-hydroxybiphenyl. 4-Hydroxybiphenyl was the predominant metabolite formed. C. lipolytica oxidized benzo(a)pyrene to 3-hydroxybenzo(a)pyrene and 9-hydroxybenzo(a)pyrene. Metabolites were isolated and identified by absorption spectrophotometry, mass spectrometry and thin-layer, gasliquid and high-pressure liquid chromatography. Where possible the structures of these metabolites were confirmed by comparison with authenic compounds.     

Specific rates of substrate oxidation and product formation in autotrophically growingChromatium vinosum cultures

Eighty-six species of fungi belonging to sixty-four genera were examined for their ability to metabolize naphthalene. Analysis by thin-layer and high pressure liquid chromatography revealed that naphthalene metabolism occurred in forty-seven species belonging to thirty-four genera from the major fungal taxa. All organisms tested from the order Mucorales oxidized naphthalene with species of Cunninghamella, Syncephalastrum and Mucor showing the greatest activity. Significant metabolism was also observed with Neurospora crassa, Claviceps paspali and four species of Psilocybe. The predominant metabolite formed by most organisms was 1-naphthol. Other products identified were, 4-hydroxy-1-tetralone, trans-1,2-dihydroxy-1,2-dihydronaphthalene, 2-naphthol, 1,2-and 1,4-naphthoquinone.     

Evidence for an arene oxide-NIH shift pathway in the transformation of naphthalene to 1-naphthol by Bacillus cereus

Bacillus cereus ATCC 14579 transformed naphthalene predominately to 1-naphthol. Experiments with [¹⁴C]naphthalene showed that over a 24 h period, B. cereus oxidized 5.2% of the added naphthalene. 1-Naphthol accounted for approximately 80% of the total metabolites. B. cereus incubated with naphthalene under the presence of ¹⁸O₂ led to the isolation of 1-naphthol that contained 94% ¹⁸O. The metabolism of [1-²H]-and [2-²H]-naphthalene by B. cereus yielded 1-naphthol which retained 95% and 94% deuterium, respectively, as determined by mass spectral analysis. NMR spectroscopic analysis of the deuterated 1-naphthol formed from [1-²H]-naphthalene indicated an NIH shift mechanism in which 19% of the deuterium migrated from the C-1 to the C-2 position. The ¹⁸O₂ and NIH shift experiments implicate naphthalene-1,2-oxide as an intermediate in the formation of 1-naphthol from naphthalene by B. cereus.Abbreviations HPLC High performance liquid chromatography - NMR nuclear magnetic resonance     

Microbial models of soil metabolism: biotransformations of danofloxacin

Danofloxacin is a new synthetic fluoroquinolone antibacterial agent under development for exclusive use in veterinary medicine. Such use could lead to deposition of low levels of danofloxacin residues in the environment in manure from treated livestock. This study was conducted to evaluate the potential for indigenous soil microorganisms to metabolize danofloxacin. Cultures of 72 soil microorganisms representing a diverse panel of bacteria, fungi and yeast were incubated with danofloxacin mesylate substrate and samples analyzed periodically by high performance liquid chromatography for loss of danofloxacin and formation of metabolites. Some samples were further analyzed by liquid chromatography-mass spectrometry and mass spectrometry to confirm metabolite identification. Twelve organisms, representing eight different genera, biotransformed danofloxacin to metabolites detectable by the chromatographic methods employed. Two Mycobacterium species, two Pseudomonas species, and isolates of Nocardia sp, Rhizopus arrhizus and Streptomyces griseus all formed N-desmethyldanofloxacin. The formation of the 7-amino danofloxacin derivative, 1-cyclopropyl-6-fluoro-7-amino-4-oxo-1,4-dihydroquinoline-3-carboxylic acid by cultures of Candida lipopytica, Pseudomonas fluorescens, two Mycobacterium species and three Penicillium species demonstrates the propensities of these cultures to completely degrade the piperazine ring. At least two additional and unidentified metabolite peaks were observed in chromatograms of Aspergillus nidulans and Penicillium sp cultures. Radiolabled [2-¹⁴C]danofloxacin added to cultures of the fungus Curvularia lunata was apparently mineralized, with approximately 31% of the radiolabel recovered as volatile metabolites after 24 h of incubation, indicating the susceptibility of the quinolone ring to microbial metabolic degradation. Received 09 December 1996/ Accepted in revised form 09 April 1997     

The identification of 5β,7α-dihydroxy-11-oxotetranor-prostane-1, 16-dioic acid as a urinary metabolite of prostaglandin F2α in the rat

5β,7α-Dihydroxy-11-oxotetranor-prostane-1,16-dioic acid has been identified by gas chromatography-mass spectrometry as a urinary metabolite of [9β-³H]prostaglandin F_2α in the rat. This tetranor prostaglandin F derivative, which is the 5β epimer of the major urinary metabolite of prostaglandin F_2α, accounted for at least 2% of the total dose. Absence from the metabolite of tritium label at the C-5 position indicated the existence of a minor, previously unknown metabolic pathway by which prostaglandin F_α derivatives may be converted by oxido-reduction into prostaglandins of F_β stereochemistry.     

UHPLC-Q-Orbitrap HR-MS-Based Metabolomics for Profiling the Sida rhombifolia Metabolites with Different Plant Organs and Cultivation Ages

Plant organs and cultivation ages can result in different compositions and concentration levels of plant metabolites. The metabolite profile of plants can be determined using liquid chromatography. This study determined the metabolite profiles of leaves, stems, and roots of Sida rhombifolia at different cultivation ages at 3, 4, and 5 months post-planting (MPP) using liquid chromatography-mass spectrometry/mass spectrometry (LC/MS/MS). The results identified that 41 metabolites in S. rhombifolia extract for all plant organs and cultivation ages. We successfully identified approximately 36 (leaves), 22 (stems), and 18 (roots) compounds in all extract. Using principal component analysis (PCA) with peak area as the variable, we clustered all sample extracts based on plant organs and cultivation ages. As a result of PCA, S. rhombifolia extracts were grouped according to plant organs and cultivation ages. In conclusion, a clear difference in the composition and concentration levels of metabolites was observed in the leaves, stems, and roots of S. rhombifolia harvested at 3-, 4-, and 5-MPP.     

Isolation and Characterization of a Novel Atrazine Metabolite Produced by the Fungus Pleurotus pulmonarius, 2-Chloro-4-Ethylamino-6-(1-Hydroxyisopropyl)Amino-1,3,5-Triazine

The white rot fungus Pleurotus pulmonarius exhibited metabolism of atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) in liquid culture, producing the dealkylated metabolites desethylatrazine, desisopropylatrazine, and desethyl-desisopropylatrazine. A fourth, unknown metabolite was also produced. It was isolated and was identified as 2-chloro-4-ethylamino-6-(1-hydroxyisopropyl)amino-1,3,5-triazine by gas chromatography-mass spectrometry, Fourier transformed infrared spectroscopy, and ¹H nuclear magnetic resonance analysis. The structure of this metabolite was confirmed by chemical synthesis of the compound and comparison with the fungally produced metabolite.     

Endogenous indoles and the biosynthesis and metabolism of indole-3-acetic acid in cultures of Rhizobium phaseoli

Gas chromatography-mass spectrometric analyses of purified extracts from cultures of Rhizobium phaseoli wild-type strain 8002, grown in a non-tryptophan-supplemented liquid medium, demonstrated the presence of indole-3-acetic acid (IAA), indole-3-ethanol (IEt), indole-3-aldehyde and indole-3-methanol (IM). In metabolism studies with ³H-, ¹⁴C- and ²H-labelled substrates the bacterium was shown to convert tryptophan to IEt, IAA and IM; IEt to IAA and IM; and IAA to IM. Indole-3-acetamide (IAAm) could not be detected as either an endogenous constituent or a metabolite of [³H]tryptophan nor did cultures convert [¹⁴C]IAAm to IAA. Biosynthesis of IAA in R. phaseoli, thus, involves a different pathway from that operating in Pseudomonas savastanio and Agrobacterium tumefaciens-induced crown-gall tumours.Abbreviations IAA indole-3-acetic acid - IAld indole-3-aldehyde - IAAm indole-3-acetamide - IEt indole-3-ethanol - IM indole-3-methanol - HPLC-RC high-performance liquid chromatography-radio counting - GC-MS gas chromatography-mass spectrometry     

Glucuronide and sulfate conjugation in the fungal metabolism of aromatic hydrocarbons.

Cunninghamella elegans oxidized naphthalene to ethyl acetate-soluble and water-soluble metabolites. Experiments with [14C]-naphthalene indicated that 21% of the substrate was converted into metabolites. The ratio of organic-soluble metabolites to water-soluble metabolites was 76:24. The major ethyl acetate-soluble naphthalene metabolites were trans-1,2-dihydroxy-1,2-dihydro-naphthalene, 4-hydroxy-1-tetralone, and 1-naphthol. Enzymatic treatment of the aqueous phase with either arylsulfatase or beta-glucuronidase released metabolites of naphthalene that were extractable with ethyl acetate. In both cases, the major metabolite was 1-naphthol. The ratio of water-soluble sulfate conjugates to water-soluble glucuronide conjugates was 1:1. Direct analysis of the aqueous phase by high-pressure liquid and thin-layer chromatographic and mass spectrometric techniques indicated that 1-naphthyl sulfate and 1-naphthyl glucuronic acid were major water-soluble metabolites formed from the fungal metabolism of naphthalene. C. elegans oxidized biphenyl primarily to 4-hydroxy biphenyl. Deconjugation experiments with biphenyl water-soluble metabolites indicated that the glucuronide and sulfate ester of 4-hydroxy biphenyl were metabolites. The data demonstrate that sulfation and glucuronidation are major pathways in the metabolism of aromatic hydrocarbons by fungi.     

Molecular Species of Free Ceramides in Aspergillus oryzae

Free ceramides isolated from A. oryzae were fractionationed into three groups by thin-layer chromatography on silica gel G according to degree of hydroxylation of the molecules. Each group was converted to trimethylsilyl ether derivatives, which were analyzed by gas-liquid chromatography-mass spectrometry for the structure of the molecular species. As a result, the representative molecular species of ceramides were characterized as N-lignoceroyl-phytosphingosine (1%), N-2-hydroxylignoceroyl-phytosphingosine (31%) and N-2,3-dihydroxylignoceroyl-phytosphingosine (31%).     

The identification of 5beta,7alpha-dihydroxy-11-oxotetranor-prostane-1,16-dioic acid as a urinary metabolite of prostaglandin F2alpha in the rat.

5beta,7alpha-Dihydroxy-11-oxotetranor-prostane-1,16-dioic acid has been identified by gas chromatography-mass spectrometry as a urinary metabolite of [9beta-3H]prostaglandin F2alpha in the rat. This tetranor prostaglandin F derivative, which is the 5beta epimer of the major urinary metabolite of prostaglandin F2alpha, accounted for at least 2% of the total dose. Absence from the metabolite of tritium label at the C-5 position indicated the existence of a minor, previously unknown metabolic pathway by which prostaglandin Falpha derivatives may be converted by oxido-reduction into prostaglandins of Fbeta stereochemistry.     

Spectrophotometric assay for detection of aromatic hydroxylation catalyzed by fungal haloperoxidase–peroxygenase

Agrocybe aegerita peroxidase (AaP) is a versatile heme-thiolate protein that can act as a peroxygenase and catalyzes, among other reactions, the hydroxylation of aromatic rings. This paper reports a rapid and selective spectrophotometric method for directly detecting aromatic hydroxylation by AaP. The weakly activated aromatic compound naphthalene served as the substrate that was regioselectively converted into 1-naphthol in the presence of the co-substrate hydrogen peroxide. Formation of 1-naphthol was followed at 303 nm (ɛ ₃₀₃ = 2,010 M⁻¹ cm⁻¹), and the apparent Michaelis–Menten (K _m) and catalytic (k _cat) constants for the reaction were estimated to be 320 μM and 166 s⁻¹, respectively. This method will be useful in screening of fungi and other microorganisms for extracellular peroxygenase activities and in comparing and assessing different catalytic activities of haloperoxidase–peroxygenases.     

Identification of 1-(malonylamino)cyclopropane-1-carboxylic acid as a major conjugate of 1-aminocyclopropane-1-carboxylic acid,an ethylene precursor in higher plants

When labeled 1-aminocyclopropane-1-carboxylic acid, an ethylene precursor, was administered to light-grown wheat leaves, it was primarily converted into a nonvolatile metabolite, which was identified as 1-(malonylamino)cyclopropane-1-carboxylic acid. The natural occurrence of this conjugate in the wilted wheat leaves was confirmed by gas chromatography-mass spectrometry.     

Phagotrophic protists are a key component of microbial communities processing leaf litter under contrasting oxic conditions

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The major product of PGG2 metabolism by aortic microsomes is 6, 15-dioxo-PGF1α

Incubations of PGG₂ with aortic microsomes yielded two products which were not formed in boiled enzyme control, one of which was 6-oxo-PGF_1α. The major metabolite was identified by gas-liquid chromatography-mass spectrometry as 6,15-dioxo-PGF_1α. Thus, unlike PGH₂, PGG₂ is probably converted to 15-hydroperoxy PGI₂ which subsequently decomposes to 6,15-dioxo-PGF_1α.     

Metabolic fate of endogenously synthesized prostaglandin D2 in a human female with mastocytosis

Increased production of prostaglandin D₂ was recently demonstrated in patients with systemic mastocytosis. One female patient investigated with mastocytosis was found to have overproduction of prostaglandin D₂ of such magnitude (150-fold above normal) that it provided the unique opportunity to delineate the metabolic fate of endogenously synthesized prostaglandin D₂. A five percent aliquot of a twenty-four hour urine collection from the patient was extracted, purified by silicic acid chromatography, methylated, and finally subjected to high pressure liquid chromatography. Column fractions collected were derivatized and analyzed by gas chromatography-mass spectrometry. Increased quantities of sixteen urinary metabolites were identified and included a series of metabolites retaining the PGD-ring as well as series of metabolites with a PGF-ring. PGF-ring metabolites were excreted in approximately 4-fold greater relative abundance than PGD-ring metabolites. More than one apparent isomeric form of some PGF-ring metabolites were found. The predominant urinary metabolite was 2,3-dinor-prostaglandin F₂. This study provides evidence that endogenously synthesized prostaglandin D₂ is converted in substantial part to prostaglandin F₂ metabolites in humans.     

Transformation of arachidonic acid in leukocytes. isolation and structural analysis of a novel dihydroxy derivative

A suspension of mixed peripheral blood leukocytes was incubated with arachidonic acid. After ether extraction and silicic acid fractionation of the products, the fraction containing the mono- and dihydroxy derivatives of arachidonic acid was further analyzed by high pressure liquid chromatography on silica gel and octadecyl silica (reversed-phase) columns. A previously undescribed metabolite was detected and isolated in pure form. The compound co-chromatographed with leukotriene B₄ on octadecyl silica but was eluted earlier than leukotriene B₄ from silica gel columns. Ultraviolet spectrophotometry, gas chromatography-mass spectrometry, ozonolysis and steric analysis indicated that the new metabolite was the 5S,12S-dihydroxy-6,8,10,14-icosatetraenoic acid. The yield of the novel dihydroxy acid was 1 to 4% of the added substrate. The new metabolite showed less than 1% of the myotropic activity of leukotriene B4 on the guinea pig lung parenchymal strip.     

Studies of Aquatic Fungi. XIII. Mycoflora of the River Czarna Hańcza and its Tributary,the River Marycha

The authors investigated the mycoflora and the environmental factors in the River Czarna Hańcza (10 stations) and its tributary River Marycha (1 station) as well as Lakes Hańcza (2 stations) and Wigry (2 stations) on the occurrence of various aquatic fungi. At the stations investigated the presence of 45 aquatic fungi species was noted. The following fungi, up to now unknown in Poland, were found: Monoblepharis hypogyna, Rozellopsis inflata, Cladolegnia eccentrica, Apostemidium guernisaci, Anguillospora gigantea, Geniculospora grandis, Clavariopsis aquatica, Lemonniera aquatica and Lemonniera terrestris.     

The Effect of the Herbicide Mecoprop on Heliscus lugdunensis and Its Influence on the Preferential Feeding of Gammarus Pseudolimnaeus

Abstract Exposure of the aquatic hyphomycete Heliscus lugdunensis to the herbicide Mecoprop did not significantly affect production of the antigen recognized by the specific monoclonal antibody NG-CF10. Therefore, an ELISA method, developed in a previous study, could be used to quantify the biomass of H. lugdunensis colonizing leaves exposed to this herbicide. Exposure to Mecoprop significantly reduced the mycelial biomass associated with alder leaves. This was shown to be a threshold response rather than a dose response, with higher biomass recorded on control leaves. No significant differences were found over the range of Mecoprop concentrations used. In laboratory experiments, Gammarus pseudolimnaeus was offered a choice of alder leaves exposed to a range of Mecoprop concentrations. The animals were able to discriminate between the exposed and control leaves, and between inoculated and sterile leaves. Presence of the fungus resulted in increased leaf consumption, but no interaction between the Mecoprop concentrations and fungal colonization was observed. The major factor affecting food choice was the concentration of Mecoprop that the leaves were exposed to—not the Mecoprop-mediated effects on fungal biomass. Received: 10 February 1997; Accepted: 8 May 1997