Single-kernel analysis of fumonisins and other fungal metabolites in maize from South African subsistence farmers.

Fumonisins are important Fusarium mycotoxins mainly found in maize and derived products. This study analysed maize from five subsistence farmers in the former Transkei region of South Africa. Farmers had sorted kernels into good and mouldy quality. A total of 400 kernels from 10 batches were analysed; of these 100 were visually characterised as uninfected and 300 as infected. Of the 400 kernels, 15% were contaminated with 1.84-1428 mg kg(-1) fumonisins, and 4% (n=15) had a fumonisin content above 100 mg kg(-1). None of the visually uninfected maize had detectable amounts of fumonisins. The total fumonisin concentration was 0.28-1.1 mg kg(-1) for good-quality batches and 0.03-6.2 mg kg(-1) for mouldy-quality batches. The high fumonisin content in the batches was apparently caused by a small number (4%) of highly contaminated kernels, and removal of these reduced the average fumonisin content by 71%. Of the 400 kernels, 80 were screened for 186 microbial metabolites by liquid chromatography-tandem mass spectrometry, detecting 17 other fungal metabolites, including fusaric acid, equisetin, fusaproliferin, beauvericin, cyclosporins, agroclavine, chanoclavine, rugulosin and emodin. Fusaric acid in samples without fumonisins indicated the possibility of using non-toxinogenic Fusaria as biocontrol agents to reduce fumonisin exposure, as done for Aspergillus flavus. This is the first report of mycotoxin profiling in single naturally infected maize kernels.

Dynamics in the microbiology of maize silage during whole-season storage.

AIMS: To monitor seasonal variations in the microbiology of maize silage and to determine whether the risk of fungal spoilage varies during whole-year storage. METHODS AND RESULTS: A continuous survey of 20 maize silage stacks was conducted over a period from three to 11 months after ensiling. Filamentous fungi, yeasts and lactic acid bacteria (LAB) were enumerated at five time-points, and cultivable species of filamentous fungi were identified. Significant differences in the numbers of filamentous fungi, yeast and LAB were detected. The highest numbers of fungi were five to seven and the lowest 11 months after ensiling, while the LAB decreased in numbers during the study. Filamentous fungi were isolated from all stacks at all time-points. The most abundant toxigenic mould species were Penicillium roqueforti, Penicillium paneum and Aspergillus fumigatus. CONCLUSIONS: There are significant variations in the microbiology of maize silage over a whole storage season. The risk of fungal spoilage was highest 5-7 months after ensiling and lowest after 11 months. SIGNIFICANCE AND IMPACT OF THE STUDY: This information is valuable in the assessment of health risks connected with spoiled maize silage and may be useful in the management of maize silage stacks, when whole-season storage is applied.

Aspergillus acidus from Puerh tea and black tea does not produce ochratoxin A and fumonisin B2.

Puerh tea is a unique Chinese fermented tea. Unlike other teas it is stored for a long period of time. Aspergillus niger is claimed to be the dominant microorganism in the Puerh tea manufacturing process and also to be common on tea in general. A. niger sensu stricto is known to produce the mycotoxins ochratoxin A, fumonisins B(2) and B(4). With this in mind, we performed a preliminary study to determine if production of these mycotoxins by black Aspergilli isolated from Puerh and black tea can occur. An examination of 47 isolates from Puerh tea and black tea showed that none of these was A. niger. A part of the calmodulin gene in 17 isolates were sequenced, and these 17 isolates were all identified as Aspergillus acidus (=A. foetidus var. acidus). The rest of the 47 isolates were also identified as A. acidus from their metabolite profile. Neither production of ochratoxin A nor fumonisins B(2) and B(4) by any of the 47 isolates were observed.

Host-derived media used as a predictor for low abundant, in planta metabolite production from necrotrophic fungi.

AIMS: Penicillium ser. Corymbifera strains were assayed on a variety of media and from infected Allium cepa tissues to evaluate the stimulation and in planta prediction of low abundance metabolites. METHODS AND RESULTS: Stimulated production of corymbiferones and the corymbiferan lactones were observed for Penicillium albocoremium, Penicillium allii, Penicillium hirsutum, Penicillium hordei and Penicillium venetum strains cultured on tissue media. Target metabolites were sporadically detected from strains cultured on common laboratory media (CYA, MEA and YES). Up to a 376 times increase in corymbiferone and corymbiferan lactone production was observed when culture extracts from CYA and A. cepa agar were compared by high pressure liquid chromatography with ultraviolet and mass spectrometry (LC-UV-MS). The novel metabolite corymbiferone B was purified and structure elucidated from a P. allii/A. cepa tissue medium extract. In planta expression of low abundance, target metabolites were confirmed from infected A. cepa tissue extracts by LC-UV-MS. CONCLUSIONS: Secondary metabolite production was directly dependent and influenced by media conditions, resulting in the stimulated production of low abundance metabolites on host-derived media. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of macerated host tissue media can be applied in vitro to predict in planta expression of low abundance metabolites and aid in metabolite origin annotation during in planta metabolomic investigations at the host/pathogen interface.

An integrated taxonomic study of Fusarium langsethiae, Fusarium poae and Fusarium sporotrichioides based on the use of composite datasets.

An integrated systematic study was carried out to clarify the taxonomical position and relationship of Fusarium langsethiae to other taxa within the Fusarium section Sporotrichiella. Strains of this species were compared with strains of the closely related species Fusarium poae and Fusarium sporotrichioides using a composite dataset. This set consisted of DNA sequences derived from the ribosomal internal transcribed spacer (ITS) regions, partial sequences of the ribosomal intergenic spacer (IGS) region, the beta-tubulin and translation elongation factor-1 alpha (EF-1alpha) genes, AFLP fingerprints, chromatographic data on secondary metabolites and morphological data and growth characteristics. From these combined data, a consensus matrix was calculated by taking the mean of all pairwise distances between single isolates over all separate datasets. The consensus matrix was used as the basis for the construction of a UPGMA dendrogram and a multidimensional scaling, both of which revealed a clear separation of the three taxa. Partial IGS, EF-1alpha and beta-tubulin sequence-as well as chromatography-and AFLP-derived similarities turned out to be comparably consistent, while ITS sequence- and morphology-derived similarity matrices were rather divergent.

Determination of fungal spore release from wet building materials.

The release and transport of fungal spores from water-damaged building materials is a key factor for understanding the exposure to particles of fungal origin as a possible cause of adverse health effects associated to growth of fungi indoors. In this study, the release of spores from nine species of typical indoor fungi has been measured under controlled conditions. The fungi were cultivated for a period of 4-6 weeks on sterilized wet wallpapered gypsum boards at a relative humidity (RH) of approximately 97%. A specially designed small chamber (P-FLEC) was placed on the gypsum board. The release of fungal spores was induced by well-defined jets of air impacting from rotating nozzles. The spores and other particles released from the surface were transported by the air flowing from the chamber through a top outlet to a particle counter and sizer. For two of the fungi (Penicillium chrysogenum and Trichoderma harzianum), the number of spores produced on the gypsum board and subsequently released was quantified. Also the relationship between air velocities from 0.3 to 3 m/s over the surface and spore release has been measured. The method was found to give very reproducible results for each fungal isolate, whereas the spore release is very different for different fungi under identical conditions. Also, the relationship between air velocity and spore release depends on the fungus. For some fungi a significant number of particles smaller than the spore size were released. The method applied in the study may also be useful for field studies and for generation of spores for exposure studies.

Fast methods for screening of trichothecenes in fungal cultures using gas chromatography-tandem mass spectrometry.

The paper presents a fast method for trichothecene profiling and chemotaxonomic studies in species of Fusarium, Stachybotrys. Trichoderma and Memnoniella. Micro scale extracted crude Fusarium extracts were derivatised using pentafluoropropionic anhydride and analysed by gas chromatography with simultaneous full scan and tandem mass spectrometric detection. It was possible to monitor for up to four compounds simultaneous, making detection of acetyl T-2 toxin, T-2 toxin, HT-2 toxin, T-2 triol. T-2 tetraol, neosolaniol, iso-neosolaniol, scirpentriol, 4,15-diacetoxyscirpenol, 15-acetoxyscirpenol, 4-acetoxyscirpentriol, nivalenol, fusarenon-X, deoxynivalenol, 15-acetyl-deoxynivalenol and 3-acetyldeoxynivalenol possible during a 23-min GC run. A slightly modified method could detect trichothecenes produced by Stachybotrys, Memnoniella and Trichoderma, by hydrolysing crude extracts prior to derivatisation with heptafluorobuturyl imidazole. All types of derivatised extracts could be reanalysed using negative ion chemical ionisation (NICI) GC-MS for molecular mass determination and verification purposes. A retention time index could be used for correction in retention time drifts between sequences and worked both in EI+ and NICI mode.

Identification of Trichoderma strains by image analysis of HPLC chromatograms.

Forty-four Trichoderma strains from water-damaged building materials or indoor dust were classified with chromatographic image analysis on full chromatographic matrices obtained by high performance liquid chromatography with UV detection of culture extracts. The classes were compared with morphological identification and rDNA sequence data, and for each class all strains were of the same identity. With all three techniques each strain--except one--was identified as the same species. These strains belonged to Trichoderma atroviride (nine strains), Trichoderma viride (three strains), Trichoderma harzianum (10 strains), Trichoderma citrinoviride (12 strains), and Trichoderma longibrachiatum (nine strains). The odd strain was identified as Trichoderma hamatum by morphology and rDNA sequencing, but not by image analysis as no reference strains of this species were included. It is concluded that the secondary metabolite profile contains sufficient information for classification and species identification.

Fusaria and fumonisins in maize from Ghana and their co-occurrence with aflatoxins.

Fifteen maize samples from four markets and processing sites in Accra, Ghana were analysed for fumonisins B1, B2, and B3. All samples contained fumonisins. Total fumonisin levels for 14 samples ranged from 70 to 4222 microg kg(-1). One sample of visibly mouldy kernels contained 52 670 microg kg(-1) total fumonisins. Mycological examination of the samples showed Aspergillus spp. as the most dominant fungi (76.4%) followed by Penicillium spp. (19.9%). Fusarium formed 2.6% with Fusarium verticillioides as the predominant Fusarium species. Thirty-two Fusarium strains representing five species isolated from the maize samples were tested for the production of fumonisins in maize substrates. From 95% (21 of 22) of the F. verticillioides strains tested, all three types of fumonisins were produced. Total fumonisin levels ranged from 127 to 11 052 microg g(-1). Additional studies on maize samples from 15 processing sites in Accra revealed a co-occurrence of both fumonisins and aflatoxins in 53% (8 of 15) of the samples.

Identification of Trichoderma strains from building materials by ITS1 ribotyping, UP-PCR fingerprinting and UP-PCR cross hybridization.

To study the role of Trichoderma in sick building syndrome, it is essential to be able to accurately identify species. Forty-four strains of Trichoderma spp. isolated from Danish buildings damaged by water leaks were identified using ITS1 ribotyping and universally primed PCR, UP-PCR. Ribotyping allowed the assignment of the strains into three distinct groups. High similarity of UP-PCR banding profiles of the strains allowed species designation for almost all strains (43 out of 44) when compared with the UP-PCR banding profiles obtained from reference strains of T. atroviride, T. citrinoviride, T. harzianum, T. longibrachiatum and T. viride. However, cross hybridization of UP-PCR products showed that the latter strain had high DNA homology to the ex-type strain of T. hamatum. The combined approach is a convenient way for reliable identification of Trichoderma strains.

Fast methods for screening of trichothecenes in fungal cultures using GC-MS/MS.

Fast methods for screening for production of simple and macrocyclic trichothecenes, produced on solid substrates were developed. Crude extracts fromFusarium cultures grown on yeast extract sucrose agar (approx. 0.3 cm(2) culture) were derivatised using pentafluoropropionic anhydride and analysed by GC - tandem mass spectrometry using a Finnigan GCQ(+) ion trap. The MS was operated in the EI(+) multiscan mode allowing simultaneous full scan and MS/MS of 3-4 parent ions. Production of acetyl T-2 toxin (AT-2), T-2 toxin, HT-2 toxin (HT-2), T-2 triol (T-2TR), T-2 tetraol (T-2TE), neosolaniol (NEO), iso-neosolaniol (I-NEO), scripentriol (SCR), 4,15-diacetoxyscirpenol (DAS), 15-acetoxyscripentriol (15MAS), 4-acetoxyscripentriol (4MAS), nivalenol (NIV), fusarenon-X (F-X), deoxynivalenol (DON), 15-acetoxy-DON (15DON) and 3-acetoxy-DON (3DON) was studied for severalFusarium species. In hydrolysed crude extracts ofStachybotrys albipes, Trichoderma harzianum, andMemnoniella echinata trichodermol was detected, in cultures ofS. chartarum both verrucarol and trichodermol were detected as the heptafluorobuturyl esters after derivatisation with a imidazole based reagent.

Production of mycotoxins on artificially and naturally infested building materials.

In this study, the ability to produce mycotoxins during growth on artificially infested building materials was investigated for Penicillium chrysogenum, Pen. polonicum, Pen. brevicompactum, Chaetomium spp., Aspergillus ustus, Asp. niger, Ulocladium spp., Alternaria spp., and Paecilomyces spp., all isolated from water-damaged building materials. Spores from the different isolates of the above mentioned species were inoculated on gypsum board with and without wallpaper and on chipboard with and without wallpaper. Fungal material was scraped off the materials, extracted, and analyzed using high performance liquid chromatography-diode array detection and thin layer chromatography. All six isolates of C. globosum produced the toxic chaetoglobosins A and C, at levels of up to 50 and 7 microg/cm2 respectively. The quantities of secondary metabolites produced by Penicillia were generally low, and no toxin production was detected from any of the five isolates of Pen. chrysogenum. Both isolates of Pen. polonicum produced 3-methoxy-viridicatin, verrucosidin, and verrucofortine. Two of five isolates of Pen. brevicompactum produced mycophenolic acid. From five out of six isolates of Alternaria spp., altenariol and alternariol monomethyl ether were detected. From Ulocladium spp., Paecilomyces spp., and Asp. ustus no known mycotoxins were detected, although the latter two are known mycotoxin producers. Asp. niger produced several naphtho-gamma-pyrones and tetra-cyclic compounds. All investigated species, especially Asp. ustus and Asp. niger produced many unknown secondary metabolites on the building materials. Analyses of wallpaper and glass-fibre wallpaper naturally infested with Asp. versicolor revealed sterigmatocystin and 5-methoxysterigmatocystin. Analyses of naturally infested wallpaper showed that C. globosum produced the chaetoglobosins A and C, and Pen. chrysogenum produced the antibiotic meleagrin.

Moulds in food spoilage.

There is an increasing knowledge and understanding of the role played by moulds in food spoilage. Especially the discovery of mycotoxin production in foods has highlighted the importance of moulds in food quality. It is, however, only within the last 5-10 years that major progresses have been made towards the prevention of spoilage caused by moulds. This is due to recent international agreements on taxonomy and analytical methods for foodborne moulds, which has led to the discovery, that a specific, very limited funga (= mycobiota) is responsible for the spoilage of each kind of food. This is called the associated or critical funga and has been shown to consist of less than ten species. In this paper the associated funga is described for the following foods: citrus and pomaceous fruit, potato and yam tubers, onions, rye, wheat, rye bread, cheese and fermented sausage and whenever possible the selective principle of the food is discussed. In the description only references which are using the new taxonomy and mycological methods have been used. The individual fungas are very different from each other, which again means that the potential appearance of a specific mycotoxin is restricted to a limited number of foods. The important mycotoxin pattern of each food is described including toxins which originate from 'carry over'. For some foods examples are also given on spoilage of sensoric properties due to moulds. Finally, preventive action against the growth of the associated funga is described for some of the foods and it is concluded that optimization of the prevention and control of moulds in foods must be based on knowledge of the associated funga.

Comparison of three selective media for detecting Fusarium species in foods: a collaborative study.

At the Second International Workshop on Standardisation of Methods for the Mycological Examination of Foods in Baarn, 1990, three selective media for food-borne Fusarium species were recommended: Czapek-Dox Iprodione Dichloran Agar (CZID), Dichloran Chloramphenicol Peptone agar (DCPA) and Pentachloronitrobenzene Peptone agar (PPA). Lack of sufficient data made it impossible to recommend one Fusarium selective medium. In the present study nine laboratories from seven countries compared CZID, DCPA, and PPA by analysing 10 samples of flour spiked with conidia of Fusarium avenaceum and F. verticillioides (= F. moniliforme). The colony forming units were counted. The total counts on each of the three media were within a similar range for each participant, and no significant differences between the three media were evident. However, the development of Fusarium colonies was quite different on the three media, and most collaborators found it possible to differentiate these Fusarium species by pigmentation on CZID. Pigmentation was much less conspicuous on PPA, and inconspicuous in DCPA cultures. CZID is recommended as the best currently available selective medium for Fusarium isolates from foods.

Secondary metabolites produced byAltemaria infectoria and their use as chemotaxonomic markers.

Twenty-one isolates ofAlternaria infectoria have been screened for their production of secondary metabolites for chemotaxonomic characterization. For comparison isolates ofA. tenuissima andStemphylium sarciniforme were also screened.A. infectoria andA. tenuissima had two unknown metabolites and none known metabolites in common.A. infectoria had two other unknown metabolites in common withS. sarciniforme. The positions of six unknown metabolites in the metabolite profile ofA. infectoria have been determined. UV-spectra and retention time indices of these six metabolites are given.

Chemical and physiological characterization of taxa in the Fusarium sambucinum complex.

Forty-one isolates of Fusarium sambucinum sensu lato were screened for production of secondary metabolites in agar cultures. Of 16 strains of F. sambucinum sensu stricto all but two strains produced diacetoxyscirpenol and two unidentified metabolites, TB1 and TB2 respectively. The two remaining F. sambucinum strains produced T-2 toxin, TB1 and TB2. Fusarium venenotum (6 strains) produced diacetoxyscirpenol and an unidentified metabolite BB. Fusarium torulosum (8 strains) produced wortmannin and antibiotic Y. The three species could be differentiated by their pattern of identified and unidentified metabolites detected by agar plug TLC combined with chemical data from HPLC-diode array detection of fungal extracts, and data on growth rates on potato sucrose agar and tannin sucrose agar.

FUSKEY, an interactive computer key to commonFusarium species.

An interactive computer key to 17 commonFusarium species has been developed. The key, FUSKEY, uses morphological, physiological and chemical criteria and is running on a PC (MS-DOS structured) using easy obtainable software.

Natural occurrence of the mycotoxin fusarochromanone, a metabolite of Fusarium equiseti, in cereal feed associated with tibial dyschondroplasia.

The mycotoxin fusarochromanone, a metabolite of Fusarium fungi, is able to induce tibial dyschondroplasia (TD) in chickens under experimental conditions. On the basis of health surveillance data on TD, two broiler farms with TD prevalence rates of up to 56% were identified. In the corresponding pelleted feed samples, fusarochromanone was detected in all 12 samples analyzed by column purification and TLC, with concentrations 4 to 59 micrograms/kg. No Fusarium fungi were available from the feed because of the pelleting process, but seven Fusarium equiseti strains previously isolated from Danish cereals were checked for fusarochromanone production, and all produced fusarochromanone at 57 to 1,435 mg/kg. Thus, the potential for fusarochromanone production by F. equiseti is considerable. The identification of fusarochromanone from feed and F. equiseti was confirmed by mass, infrared, and nuclear magnetic resonance spectral analyses. This is the first report of fusarochromanone as a naturally occurring contaminant.

Analysis and screening for mycotoxins and other secondary metabolites in fungal cultures by thin-layer chromatography and high-performance liquid chromatography.

Methods for the screening of fungal cultures for toxic secondary metabolites are reviewed. Thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) are good general analytical methods for secondary metabolites in unpurified extracts. The combination of normal phase TLC using different chemical spray reagents with reversed phase HPLC, using alkylphenone retention indices and diode array detection, is a powerful technique for identifying the individual mycotoxins detected. The results of the screening methods are very dependent of the growth media and incubation conditions and a general method for the detection of Penicillium, Aspergillus, Fusarium, Alternaria and Cladosporium toxins is suggested.

Screening for Fusarin C production by European isolates of Fusarium species.

A total of 137Fusarium isolates were screened forin vitro production of the mutagenic metabolite fusarin C, using a simple thin layer chromatographic method. It has been proven thatFusarium species (F. culmorum, F. graminearum, F. crookwellense, F. sporotrichioides, F. poae, F. tricinctum, andF. Avenaceum) isolated from European agricultural crops and soils are able to produce fusarin C. No fusarin C production was detected among isolates ofF. arthrosporioides, F. acuminatum, or F. equiseti.Results obtained by High-Performance Liquid Chromatography (HPLC) analyses of fungal extracts show that up to 26 chromatographic peaks having UV spectra similar to that of fusarin C are produced. It is not known if any of these metabolites are as mutagenic as fusarin C.