P.R. Wallnöfer

Determination of ergosterol as a measure of fungal growth using Si 60 HPLC

ZusammenfassungZur Erfassung des Pilzwachstums vonFusarium graminearum 480 wurde eine Methode der Extraktion und Messung des spezifischen Pilzsterins Ergosterin beschrieben. Diese Methode beruht auf einem direkten Verseifen des gebundenen Ergosterins im Pilzmycel, anschließender Extraktion mit n-Hexan und Quantifizierung mittels Si 60 HPLC und UV-Detektion. Verglichen mit den bisher bekannten Methoden liegen ihre Vorteile in der über 40% höheren Ergosterinausbeute, der geringeren die UV-Detektion störenden Verunreinigungen der n-Hexanextrakte und einer Halbierung der Retentionszeit der Verbindung durch Si 60 HPLC. Die Protein- und Ergosteringehalte von wachsendenF. graminearum-Kulturen waren über einen Zeitraum von 3 Wochen proportional. Die Bildung des Mykotoxins Zearalenon setztebei einer Ergosterinmenge von ca. 50 mg/kg ein und stieg bei Beginn der stationären Wachstumsphase, die durch eine geringere Ergosterinbildungsrate gekennzeichnet war, steil an.SummaryIn order to determine to fungal growth ofFusarium graminearum 480, a method was developed for the extraction and estimation of ergosterol, a sterol specific for fungi. This method includes the direct saponification of bound ergosterol to fungal mycelia followed by n-hexane extraction and quantification using. High performance liquid chromatography (HPLC) with UV-detection. This procedure proved to be superior compared with other methods, since the yield of ergosterol yields was higher (up to 40%). n-Hexane extracts contained minor impurities which interfered with the UV-detection and the retention time of the compound was halved using Si 60 HPLC. The protein and ergosterol contents inF. graminearum cultures increased proportionally over a 3-week incubation period. The fungal formation of the mycotoxin zearalenone started at a level of 50 mg/kg ergosterol and increased rapidly in the stationary phase of growth, which was characterized by decreasing rates of ergosterol formation.

Oxidative dealkylation of five phenylurea herbicides by the fungus Cunninghamella echinulata Thaxter

Microorganisms isolated from soil degrade phenylurea herbicides via two major pathways: (i) direct hydrolysis by an amidase leading to N,O-dimethylhydroxylamine, CO2 and aniline1 and (ii) N-dealkylation, which has been described as the first step in urea herbicide degradation by a variety of organisms including mammals, plants and microbial systems (for a review see reference 2). Until now no attempts have been made to investigate the mechanism of N-demethylation of substituted ureas in soil microorganisms, due to the instability of the N-hydroxymethyl intermediates. This reaction mechanism has only been described in detail in green plants3–5. As among soil fungi Phycomycetes are known to demethylate phenylurea herbicides6,7 this study has been made to identify intermediate hydroxymethyl compounds from urea herbicides, when incubated with the fungus Cunninghamella echinulata Thaxter.

Metabolism of o-phthalic acid by different gram-negative and gram-positive soil bacteria

Different bacteria, isolated from soil by the enrichment method, were able to grow on phthalic acid as carbon source. Protocatechuate was identified as intermediate in phthalate metabolism. All phthalategrown bacteria oxidized phthalate and protocatechuate rapidly without having a lag-period. Benzoic acid, terephthalic acid, protocatechuic acid, salicylic acid, di- and mono-butyl phthalate were also metabolized by some of the organisms, benzoic acid being degraded via catechol and terephthalic acid via protocatechuate as intermediate. All organisms tested cleaved protocatechuate or catechol, respectively, by the “ortho” fission, when grown on phthalate, terephthalate, or benzoate as carbon source. A characterization and tentative identification of the organisms is given.

Influence of soil properties on the degradation of the nematocide fenamiphos

Abstract The decline and mineralization to 14CO2 of [phenyl-1-14C]fenamiphos (f) together with the main metabolite fenamiphos sulfoxide (ox) was investigated over a period of 90 days, at two temperatures using 16 soils originating from different geographic areas (9 soils from a cool to temperate climate at 16–22°C and 7 soils from a subtropical to tropical climate at 22–28°C, resp.). The mineralization rates at 22°C, as determined by curve fitting, were in good correlation with the % Cmic/Corg-ratio (percentage of the microbial biomass in the total organic carbon content in the soil). The degradation rates for (f) plus (ox) could be described by first-order kinetics. The half-lives at 22°C ranged from 12 to 87 days. By means of a multiple computation, using regression analysis, between soil properties and decline rates it was possible to explain 70% of the total variance by differences in the pH (H2O)-values and in the % Cmic/Corg-ratio.

Transformation of the mycotoxin ochratoxin A in plants: 1. Isolation and identification of metabolites formed in cell suspension cultures of wheat and maize

The metabolism of the mycotoxin ochratoxin A in plant cells was investigated using cell suspension cultures of wheat and maize. A number of metabolites were detected by HPLC-chromatography with fluorescence detection. The main metabolites were ochratoxin alpha, ochratoxin A methyl ester, two isomers of hydroxyochratoxin A, and the glucosides and methyl esters of both hydroxyochratoxin A isomers. The compounds were isolated by TLC and preparative HPLC and identified by mass spectrometry and specific enzymic reactions.

Transformation of the mycotoxin ochratoxin A in plants. 2. Time course and rates of degradation and metabolite production in cell-suspension cultures of different crop plants

Ochratoxin A, one of the most toxic mycotoxins, can be metabolized nearly completely by suspension cultures of various plant cells. The transformation products identified in this study were almost the same in the cell-suspension cultures of maize, carrot, tomato, potato, soybean, wheat and barley, but the quantitative distribution differed strongly depending on incubation time and species of plant-cell culture. The compounds were extracted with ethyl acetate and detected by reversed-phase HPLC with gradient elution. From the result it is supposed that besides ochratoxin A also ochratoxin derivatives may occur in food and feedstuff of plant origin.

Chemically and biologically synthesized zearalenone-4-β-d-glucopyranoside: Comparison and convenient determination by gradient HPLC

Abstract Zearalenone-4-β- d -glucopyranoside was synthesized via a chemical reaction between zearalenone and 2,3,4,6-tetra-O-acetyl-α- d -glucopyranosylbromide, followed by deacetylation. By this way the exact molecular structure of the glucose conjugate, formerly isolated from corn tissue cultures was confirmed. For the simultaneous estimation of zearalenone, β-zearalenol and zearalenone-4-β- d -glucopyranoside a HPLC method was developed using fluorescence detection and a gradient solvent system.

Production of mycotoxins byFusarium species isolated in Germany

ZusammenfassungKulturen vonFusarium tricinctum 434 bildeten auf feuchtem, autoklaviertem Mais, Reis bzw. Hafer relativ hohe Mengen der Trichothecen-Mykotoxine Deoxynivalenol (DON) und 3-Acetyldeoxynivalenol (AcDON) sowie das zu den makrocyclischen Lactonen zählende Mykotoxin Zearalenon. Die Zearalenonbildung war mit Werten von 15 bis 72 mg/kg gegenüber der Trichothecenproduktion mit maximal 917 mg/kg AcDON auf Reis und 750 mg/kg DON auf Hafer deutlich niedriger. In den Kulturen vonF. graminearum 183 wurden insgesamt geringere Toxinmengen gefunden mit maximalen Zearalenon-konzentrationen bis zu 150 mg/kg sowie AcDON Mengen bis zu 160 mg/kg auf Reis. Dagegen erreichte die DON-Bildung auf Reis 740 mg/kg.SummaryCultures ofFusarium tricinctum 434 formed large amounts of the trichothecene mycotoxins deoxynivalenol (DON) and 3-acetyldeoxynivalenol (AcDON), as well as the macrocyclic secondary metabolite zearalenone on moistened, autoclaved maize, rice and oats. The formation of zearalenone was low, with levels from 15 to 72 mg/kg as compared to the trichothecene production with maximum quantities of 917 mg/kg of AcDON on rice and 750 mg/kg DON on oats. In the cultures ofF. graminearum 183, total mycotoxin amounts found were lower, with maximum levels of zearalenone up to 150 mg/kg and AcDON up to 160 mg/kg on rice. DON, however, was produced in quantities of about 740 mg/kg on rice.

Verhal ten von 3,4- Dichloranilin in wachsenden Pilzkul turen

Abstract 3,4- Dichloraniline is being transformed by the white rot fungi Pleurotus ostreatus and Phanerochaete chrysosporium in subinhibitory concentrations up to 90%. Detailled tests with P . chrysosporium are indicating in contrast to previous reports that the fungus is not able to mineralize this aniline. The corresponding acylanilides from acetic-,succinic-,and hydroxyglutaric acid are being observed as main transformation products.

Formation, distribution and bioavailability of cell wall bound residues of 4- chloroaniline and 2,4-dichlorophenol

Maize cells associated respectively 30–40% more of the 14C-activity with the residue fraction than tomato cells. A major portion of the bound residues was deposited in the starch, protein, lignin, and pectine cell wall polymers; the distribution depended on plant species and the test compound applied. Enzymatic hydrolysis of the starch fraction liberated the parent compounds and their β-D-glucosides. Mineralization rates of bound residues in soils were ranging from 5.5–8% by 4-chloroaniline (4-CA), 12–16% by 2,4-dichlorophenol (2,4-DCPh). They were reduced by a factor of two after separating the readily degradable cell wall polymers. The uptake of released bound residues by Lolium multiflorum varied between 2% and 3% and did not cause phytotoxic damages.