ESI-MS and MS/MS identification of the first ceramide analogues of fumonisin B1 mycotoxin from a Fusarium verticillioides culture following RP-HPLC separation.

Following the earlier detection of six new esterified fumonisin B1 (EFB1) isomers containing three acyl groups in a Fusarium verticillioides-inoculated rice culture, it was assumed that linoleic, palmitic or oleic acid esterifies one of the free OH groups on the fumonisin backbone. On the basis of the results of our recent investigations we now propose that these EFB1 isomers are actually 3-O- and 5-O-acyl derivatives of FB1 (3-O-linoleoyl-FB1, 5-O-linoleoyl-FB1, 3-O-palmitoyl-FB1, 5-O-palmitoyl-FB1, 3-O-oleoyl-FB1 and 5-O-oleoyl-FB1). A F. verticillioides strain was identified that produced not only O-acyl-FB1 isomers, but also low amounts of three N-acyl derivatives (N-linoleoyl-FB1, N-palmitoyl-FB1 and N-oleoyl-FB1), which eluted from the HPLC column after the six O-acyl compounds and in the same sequence as for the O-acyl compounds. The characteristic positive and negative ESI-MS/MS spectra obtained after solid-phase extraction of the culture extract facilitated identification of these N-acyl-FB1 derivatives. The biosynthesis of N-palmitoyl-FB1 by F. verticillioides was verified by spiking the culture extract with synthetic N-palmitoyl-FB1. This is the first report of the separation and mass spectrometric identification of the six O-acyl- and three N-acyl-FB1 derivatives extracted from a F. verticillioides culture.

Rapid purification method for fumonisin B1 using centrifugal partition chromatography.

Fumonisin B1 (FB1) is a highly toxic mycotoxin produced by fungal strains belonging to the Fusarium genus, which can be found mainly in maize products, and is gaining interest in food safety. To produce large amounts of pure FB1, a novel purifying method was developed by using centrifugal partition chromatography, which is a prominent member of the liquid-liquid chromatographic techniques. Rice cultured with Fusarium verticillioides was extracted with a mixture of methanol/water and found to contain 0.87 mg of FB1 per gram. The crude extracts were purified on a strong anion-exchange column and then separated by using a biphasic solvent system consisting of methyl-tert-butyl-ether-acetonitrile-0.1% formic acid in water. The collected fractions were analysed by flow injection-mass spectrometry and high-performance liquid chromatography coupled with Corona-charged aerosol detector and identified by congruent retention time on high-performance liquid chromatography and mass spectrometric data. This method produced approximately 120 mg of FB1 with a purity of more than 98% from 200 g of the rice culture. The whole purification process is able to produce a large amount of pure FB1 for analytical applications or for toxicological studies.

Identification of the first fumonisin mycotoxins with three acyl groups by ESI-ITMS and ESI-TOFMS following RP-HPLC separation: palmitoyl, linoleoyl and oleoyl EFB1 fumonisin isomers from a solid culture of Fusarium verticillioides.

The aim of this study was to apply RP-HPLC/ESI-ITMS and RP-HPLC/ESI-TOFMS to investigate and characterise six new higher molecular weight fumonisins (three pairs of isomers) extracted from a Fusarium verticillioides-infected solid rice culture. The ITMS and ITMS² spectra clearly indicated the m/z values (960, 984 and 986) of the protonated molecules and the FB1 toxin-like structures of these compounds, respectively. Moreover, the data evaluation software of the TOFMS equipment unambiguously demonstrated the exact masses of the protonated molecules and the suggested empirical formulae (C50H89NO16, C52H89NO16 and C52H91NO16) of the new fumonisins, with mass accuracy in the range between 0.1 and -1.1 ppm. Subtraction of the empirical formula of FB1 toxin (C34H59NO15) from these formulae and correction for the mass of water split-off from the fumonisin molecule during ester formation resulted in the empirical formulae of the fumonisin backbone esterifying agents (fatty acids): C16H32O2 (palmitic acid, PA), C18H32O2 (linoleic acid, LA) and C18H34O2 (oleic acid, OA). We denoted the new compounds as esterified FB1 (EFB1) toxins, with the suggested names EFB1 PA, iso-EFB1 PA, EFB1 LA, iso-EFB1 LA, EFB1 OA and iso-EFB1 OA. The total amount of these new compounds comprised 0.1% of the FB1 concentration, which may be rated as significant when it is considered that these new components are significantly more apolar than earlier-described fumonisins, and their uptake into and toxicity elicited in the various tissues of living organisms may therefore also be significantly different from those of other fumonisins.

Identification of Fusarium species by isozyme analysis.

Cellulose-acetate electrophoresis (CAE) was used to investigate isozyme polymorphisms among different isolates of Fusarium cerealis, F. culmorum, F. graminearum and F. pseudograminearum. After initial testing of 18 enzymes in three buffer systems for activity and resolution of bands, 12 proved to be appropriate for analysis of the full sample set. Comparing the different electrophoretic types (ETs), adenylate kinase (AK), NADP dependent glutamate dehydrogenase (NADP GDH), peptidase B (PEP B), peptidase D (PEP D) and phosphoglucomutase (PGM) proved to be diagnostic for at least one species examined. However, only PEP D was useful alone as a marker to distinguish the four taxa studied providing a rapid and simple CAE based diagnostic protocol.

Distinct electrophoretic isozyme profiles of Fusarium graminearum and closely related species.

Cellulose-acetate electrophoresis (CAE) was used to investigate isozyme polymorphisms among different isolates of Fusarium cerealis, F. culmorum, F. graminearum and F. pseudograminearum from around the world. After initial testing of 22 enzymes in three buffer systems for activity and resolution of bands, 12 proved to be appropriate for analysis of the full sample set. Remarkably uniform isozyme patterns were obtained intraspecifically, irrespective of the geographical origin of the isolates or the host/substratum from which they were isolated. This result indicated that isolates within a given species are descendant from a same ancestral population. Comparing the different electrophoretic types (ETs), adenylate kinase (AK), NADP dependent glutamate dehydrogenase (NADP GDH), peptidase B (PEP B), peptidase D (PEP ID) and phosphoglucomutase (PGM) proved to be diagnostic for at least one species examined. However, only PEP D was useful alone as a marker to distinguish the four taxa studied providing a rapid and simple CAE based diagnostic protocol. Cluster analysis of band sharing coefficients grouped the isolates into four distinct groups corresponding to the 4 species studied. Isolates of F. cerealis were clustered between those of F. culmorum and F. graminearum corroborating their known close relationship to both species. For common ETs, the similarity values between F. cerealis and F. culmorum and between F. cerealis and F. graminearum were the same. Furthermore, the similarity values and the resulting phenogram indicated that F. graminearum is more closely related to F. cerealis and F. culmorum than to F. pseudograminearum, thus the morphological similarity of F. graminearum and F. pseudograminearum does not reflect their generic relationship. This fact supports the species status of F. pseudograminearum.

Analysis of esterase zymograms of Fusarium sambucinum and related species.

Esterase zymograms were obtained following polyacrylamide slab gel electrophoresis of protein extracts Fusarium sambucinum and related species originating from different geographic locations and different matrices. The sites of esterase activity were recorded, and the Rfs were calculated. The data were used for the construction of phenograms by cluster analysis and nonlinear mapping by computerized classification techniques. The fifteen isolates of F. sambucinum, the eight isolates of F. torulosum and the six isolates of F. spec. nov. each had identical profiles, and are therefore electrophoretically distinct species. The isolates of F. sarcochroum, one of F. sambucinum sensu lato (BBA 64280) and fifteen isolates of F. sambucinum were electrophoretically indistinguishable from each other. We assume they are synonymous. The isolate of F. bacteridiodes, one of F. sambucinum sensu lato (BBA 64993) and eight isolates of F. torulosum had uniform EST patterns, therefore the two species are electrophoretically identical. We assume they are also synonymous. The remaining three isolates of F. sambucinum sensu lato are somewhat closely related to F. sambucinum isolates on the basis of our investigations.

Trichothecene chemotypes ofFusarium graminearum isolated from corn in Hungary.

Twenty three strains ofFusarium graminearum isolated from corn were screened for their ability to produce type A and B trichothecenes and zearalenone (ZEA) on the solid substrate rice grains in the dark at 28 °C for 21 days. Toxin analyses were made with HPLC technique. Of 23 total isolates, 10 produced deoxynivalenol (DON), 4 produced DON and nivalenol (NIV), 1 produced DON and 15 acetyl-DON (15-ADON), 1 produced NIV and 4-acetyl-NIV (4-ANIV) and 1 produced NIV. Of 23 totalF. graminearum isolates, 20 produced ZEA. These results suggest that strains ofF. graminearum, prevailing in Hungarian corn growing regions, might belong to DON-and NIV-chemotypes. This is the first report demonstrating that DON-, DON-NIV-, DON-15-ADON-, NIV-4-ANIV and NIV-producingF. graminearum isolates are distributed in Hungary.

Isoelectric focusing isozyme profiles and taxonomic distances among Fusarium species of the sections Arthrosporiella and Sporotrichiella.

Isozymes from 18 isolates representing seven species of the Fusarium sections Arthrosporiella and Sporotrichiella were compared by isoelectric focusing in polyacrylamide gels. Of the six enzyme systems tested esterase and malate dehydrogenase showed the largest variation. A numerical analysis of the pI values determined for acid phosphatase, esterase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, phosphoglucose isomerase and phosphoglucomutase resulted in a dendrogam demonstrating the taxonomical relationships of the seven species. Fusarium avenaceum and Fusarium pallidoroseum were the two most closely related species. The high degree of isoenzyme dissimilarity among Fusarium chlamydosporum, Fusarium poae, Fusarium tricinctum, the fungi that produce pyriform or citriform microconidia, suggests that they are distinct species and their reduction to a variety level is not reasonable. The taxonomical distinctness of Fusarium camptoceras, a lesser known and rarely occurring fungus was also proven.

Genetic distance in fungus genus Fusarium measured by comparative computer analysis of DNA thermal denaturation profiles.

High resolution thermal denaturation profiles of different members of fungus genus Fusarium were compared with respect to the shape of their DNA melting curves. Quantitative comparison of the shape (areas under differential curves) of all thermal denaturation profiles was made. Thermal denaturation profiles can be used to derive the quantitative parameter, genetic distance, defined by Soumpasis (12). Based on such data of genetic distance a dendrogram and a genetic distance tree was constructed.

Phylogenetic relationships among Fusarium species measured by DNA reassociation.

Deoxyribonucleic acid of 11 Fusarium species (F. acuminatum, F. arthrosporioides, F. avenaceum, F. culmorum, F. graminearum, F. heterosporum, F. moniliforme, F. oxysporum, F. sambucinum, F. semitectum, F. solani) have been compared with respect to their physical characteristics (Tm, % G + C), homology values and nucleotide sequence divergence. A phylogenetic tree based on physical characteristics, homology values and differences in percentage divergence of Fusarium species DNAs has been constructed.

Melting fine structure of filamentous fungus nuclear DNA.

Melting fine structure of the nuclear DNA isolated from the filamentous fungus Fusarium graminearum Schwabe is presented. Optical melting profiles of nuclear DNA were analyzed by using a combination of curve fitting and derivative techniques. The "melting components" were obtained from the derivative curve by a simple decomposition technique. Differential optical melting curves of unsheared nuclear DNA indicate the presence of 15 "melting components" in filamentous fungus nuclear genome. It should be emphasized that the "melting components" observed here are different from the "thermalites" which can be observed in bacteriophage DNA. The "melting components" reported here represent the separately melting of large "blocks" of fungus nuclear DNA.