International Commission on Food Mycology
International Commision on Food Mycology
Workshop 2010 -“Fungi in food and beverages: new research on spoilage, mycotoxins and prevention”
Freising, Germany,
7 – 9 June, 2010
Abstracts
Posters
Stress regulation of ochratoxin biosynthesis in Penicillium
Julia Batzler1*, Markus Schmidt-Heydt1 and Rolf Geisen1
1Max Rubner Institut, Haid-und-Neu-Str. 9, 76131 Karlsruhe, Germany
*Presenter: julia.batzler@mri.bund.de
MAP-Kinase cascades mediate different cellular progresses in response to extracellular signals such as different stresses. One of these cascades is the high osmolarity glycerol pathway of Saccharomyces cerevisiae where the MAP-Kinase HOG1 is involved in osmotic stress induced signal transduction. Homologues of hog1 in different fungi have been shown to be involved in regulation of secondary metabolism, stress responses, expression of mycotoxin biosynthesis genes and synthesis of mycotoxins.
In Penicillium nordicum and Penicillium verrucosum a hog1 homologue could be identified. Both fungi produce the mycotoxin ochratoxin A in dependence of different environmental parameters like temperature, water activity, salt concentration and pH. Growth of P. verrucosum at low temperatures (15°C and 20°C) showed that HOG1 is activated and, with a time lag, the expression of otapks, a key gene in ochratoxin synthesis, increases. This increase in otapks expression results in an increased ochratoxin A synthesis.
hog1 tranformants of P. verrucosum showed strongly reduced ochratoxin A synthesis. At high NaCl concentrations, HOG1 is activated in the wild type, followed by an increase in ochratoxin A synthesis. In the transformants, HOG1 phosphorylation only occurred at the highest NaCl concentration or was even completely absent. otapks expression was strongly reduced compared to wild type. hog1 transformants showed higher growth rates at high NaCl concentration as compard to the wild type.
These results indicate that HOG1 activation is correlated to ochratoxin A synthesis in response to different stresses in Penicillium.
Quantification of Fusarium and other dominating species in Swedish wheat and correlation to dominating mycotoxins
Elisabeth Fredlund1*, Ann Gidlund1, Ronnie Eriksson1, Thomas Börjesson2, Michael Sulyok3, and Monica Olsen1
1Microbiology Division, Research and Development Department, National Food Administration, Box 622, 751 26 Uppsala, Sweden
2Lantmännen Business Development, Grain, SE-531 87 Lidköping, Sweden
3Center for Analytical Chemistry Department of Agrobiotechnology (IFA-Tulln)
University of Natural Resources and Applied Life Sciences, Vienna
Konrad Lorenzstr. 20, A-3430 Tulln, Austria
*Presenter: elisabeth.fredlund@slv.se
Fusarium moulds are common contaminants in cereal grain such as wheat, which is the most important cereal crop cultivated in Sweden. Several Fusarium species are able to produce one or more mycotoxins. Some of these mycotoxins are regulated within the European legislation (deoxynivalenol, fumonisins, zearalenon) and some are not (T-2, HT-2, enniatins etc.). Growth of Fusarium species and subsequent production of mycotoxins depend on climatic conditions and cultivation techniques or systems. Climatic changes will therefore have great impact on growth of Fusarium and other mould species and their potential to produce mycotoxins. In this base-line study, 30 samples of winter wheat from three different regions in the middle and south parts of Sweden were screened for moulds and mycotoxins. A broad approach was taken and moulds were detected with both traditional isolation techniques, i.e cultivation on DG18 and CZID, and real-time PCR. Isolates were further identified by morphological characters, chemical analysis and sequence analysis of elongation factor 1. Species detected and quantified with real-time PCR were F. graminearum, F. culmorum, F. poae, F. langsethiae, and F. sporotrichioides. With traditional methods, various species of Fusarium such as F. poae, F. tricinctum and F. culmorum were isolated but also species of Cladosporium, Penicillium, Scopulariopsis, and Arthrinium. Of the specific species detected with real-time PCR, F. poae was by far the most dominating, followed by F. graminearum. F. langsethiae and F. culmorum were present in many samples in low amounts but F. sporotrichioides was not present at all. In addition, samples were also analyzed for mycotoxins and the Fusarium toxins identified were deoxynivalenol, nivalenol, monoliformin, enniatins and beauvericin. Correlation was seen between the level of F. graminearum and DON and F. poae and enniatins. This study will be extended during 2010 and 2011 in order to follow changes in mould and mycotoxin patterns and correlate these to changes in weather conditions.
Comparison of efficacy of the fungicides Fenpropimorph, Prochloraz and Tebuconazole on growth of Fusarium langsethiae strains on an oat-based medium
E.M. Mateo1*, F.M.Valle-Algarra1, A. Medina2, M. Jiménez1, and N. Magan2
1 Dep. de Microbiología y Ecología. Universitat de València. Dr. Moliner 50, E-46100, Burjassot, Valencia, Spain.
2 Applied Mycology Group, Cranfield Health, Cranfield University, Cranfield, Bedforshire, MK43 0AL, U.K.
*Presenter: eva.mateo@uv.es
Fusarium langsethiae is an important mycotoxigenic species which has been isolated from oats in different countries of northern Europe. This species is responsible for the production of type-B trichothecenes, such as T-2 and HT-2 toxins. The EU is considering legislative limits on the combined total of these two toxins in the very near future. The objective of this study was to examine the influence of three fungicides, namely fenpropimorph, prochloraz and tebuconazole on the growth of two strains of Fusarium langsethiae on a milled oat-based medium at three temperatures (15, 20 and 25ºC) and 0.96 water activity. Tebuconoazole and prochloraz were added directly to autoclaved oat medium to provide doses in the range 0 - 0.5 mg/l. For fenpropimorph treatments ranged from 0 to 50 mg/l. Mycelial growth rates (mm/day) were calculated by linear regression of the colony radius against time. The ED50 values were calculated and compared. In general, for the three antifungal agents, growth rates decreased with increasing fungicide dose and with decreasing temperature. Prochloraz was the most active antifungal agent regardless of the temperature with growth being significantly inhibited up to day 10 at 15 ºC (ED50 values were 0.03-0.1 mg/l). This fungicide was followed by tebuconazole with ED50 values ranging from 0.29 to 0.9 mg/l while fenpropimorph, the least effective fungicide, allowed F. langsethiae to grow, even at the highest concentration used at 15 ºC. The ED50 values were 21-25 mg/l at 15ºC and more than 30 mg/l at 20 and 25 ºC. These studies suggest that there are big differences in efficacy of different fungicides against F. langsethiae strains. Studies are now in progress to determine what effect these fungicides have on T-2 and HT-2 production over these time scales. Subsequent work will examine their effects in controlling F. langsethiae and mycotoxin production in oats, pre- and post-harvest.
Acknowledgments:
The authors wish to thank for financial support from FEDER and Spanish Government “Ministerio de Ciencia e Innovación” (Project AGL2007–66416–C05–01/ALI, and two research grants).
Efficacy of mancozeb, copper oxychloride and sulfur on the production of ochratoxin A by Aspergillus ochraceus and Aspergillus carbonarius in barley-based medium
E. M. Mateo1*, F.M. Valle-Algarra1, R. Mateo1, and M. Jiménez1
1Dep. de Microbiología y Ecología. Universitad de Valencia. Dr. Moliner 50, E-46100, Burjassot, Valencia, Spain
*Presenter: eva.mateo@uv.es
Barley is contaminated worldwide by various fungal species and some of them not only produce plant diseases but are also capable of producing mycotoxins. Among toxigenic species Aspergillus ochraceus has been shown to be able to produce ochratoxin A (OTA). A. carbonarius, which usually grows on grape, has also been isolated from Spanish barley being thus another species that can be of concern regarding OTA accumulation in this cereal. The aim of this study was to assess the capacity of A. ochraceus and A. carbonarius isolated from Spanish barley for producing OTA in cultures performed on barley-based medium containing three widely used fungicides (mancozeb, copper oxychloride and sulfur). Cultures were performed by inoculation of a strain of each of the two species on barley meal extract agar containing different doses of the above mentioned fungicides. Then, inoculated Petri dishes were incubated at 15 °C and 25 ºC and at 0.97 water activity for 60 days (15 ºC) and 20 days (25 ºC). Negative controls were run in parallel. OTA was determined by liquid chromatography with fluorescence detection over time in cultures where growth was apparent. OTA accumulation in cultures was significantly affected by temperature, fungal species, incubation time and type of fungicide. There were also differences concerning the concentration of the fungicide. Toxin levels were generally higher at 25 ºC than at 15ºC. The A. ochraceus strain produced more OTA than the A. carbonarius strain. Mancozeb was the most active antifungal product; it inhibited fungal growth at a dose of 40 mg/l thus preventing OTA production. OTA production was unaffected with regard to controls at doses of 10 mg/l at 25 ºC but the toxin was not detected at 15 ºC. Using copper oxychloride the toxin was not detected in cultures of A. carbonarius or in any culture of A. ochraceus at high doses. However, low doses of this fungicide did not arrest OTA accumulation in cultures of A. ochraceus, particularly at 25ºC. Sulfur, the less active fungicide, favoured OTA production in A. ochraceus cultures at 3 g/l at 25 ºC and 0.2 g/l at 15 ºC, but not at low (<0.1 g/l) or high doses (>5 g/l). It did not promote OTA production in A. carbonarius cultures.
Acknowledgments: the authors wish to thank financial support from FEDER and Spanish Government “Ministerio de Ciencia e Innovación” (Project AGL2007–66416–C05–01/ALI, and two research grants).
Fungicidal effects of food preservatives: a comparison between lactic acid bacteria metabolites and traditionally used weak organic acids
Åsa Svanström1*, Emma Boström1 and Petter Melin1
1Department of Microbiology, Swedish University of Agricultural Sciences (SLU), Box 7025, SE-750 07 Uppsala
*Presenter: asa.svanstrom@mikrob.slu.se
To protect food from spoilage organisms is a growing need. Two substances frequently used to prevent growth of filamentous fungi in food are the weak acid preservatives sorbic and benzoic acid. These compounds are effective against mould but with drawbacks; some are degradable by various spices and high dosage can cause allergic reactions and off flavors. An alternative preservation technique is to ferment the food by lactic acid bacteria that continuously produce inhibiting substances. In this study we are evaluating the food preservation potential of three such metabolites; lactic acid, phenyllactic acid and 3-hydroxydodecanoic acid, in comparison with sorbic and benzoic acids. The common food spoilage mould Aspergillus niger is the model organism. Many previous studies have been performed in liquid media with relatively high concentration of fungal spores. We wanted an experimental setup that was more comparable to the actual conditions when food is contaminated with mould. Therefore we are using solid media with different acid concentrations and spread a very small spore quantity. This enables us to evaluate the minimal inhibitory concentrations on individual spores. In addition, any change in radial growth and sporulation induction or deficiency can be monitored. Besides measuring the inhibiting activities of the lactic acid metabolites we also investigate their mode of action. The first question to ask is whether the metabolites inhibit fungi with similar mechanisms as weak acid preservatives, i.e. similar pH dependency and as inhibitors of nutrient uptake. Preliminary results indicate that, in contrast to weak acid preservatives, phenyl lactic acid strongly reduces radial growth and sporulation even at sub-inhibiting concentrations.
Ochratoxin A and fumonisin production by Aspergillus section Nigri in food from different origins
Marta H. Taniwaki1, Beatriz T. Iamanaka1, Larissa Souza Ferranti1, Marina V. Copetti2, Luciana M. R. Esper1*
1 Food Technology Institute – ITAL, Campinas, SP, Brazil
2 Federal University of Pampa – Itaqui, RS, Brazil
*Presenter: lumaesper@gmail.com
Knowledge of toxigenic fungi distribution in food is important because it gives parameters to control and prevent mycotoxin production. Ochratoxin A and fumonisin are two mycotoxins produced by Aspergillus section Nigri which are of concern to human health. After a lot of research on coffee, cocoa and dried fruits, several species of Aspergillus section Nigri have been isolated from different origins. The objective of this study was to verify the ability of these isolates to produce ochratoxin A and fumonisins. The isolates were grown in yeast extract 20% Sucrose agar at 25ºC for 7 days. Ochratoxin A was tested from agar plug technique using thin layer chromatography plates under UV light. A total of 408 samples of coffee, 226 of cocoa and 117 dried fruits from all over the world were analysed. From these samples, 1,246 species of Aspergillus section Nigri, were isolated with 88.4% identified as Aspergillus section Nigri, and 11.6% as Aspergillus carbonarius. In spite of this high incidence of Aspergillus section Nigri, only 4.1% were ochratoxin A producers, while 91% of Aspergillus carbonarius produced ochratoxin A. In relation to fumonisin production, so far few isolates from cocoa samples were tested and preliminary results have shown that 87.5% produced fumonisin B2, 25% produced fumonisin B2 and ochratoxin A and 12.5% did not produce fumonisin or ochratoxin A.
Growth of Aspergillus carbonarius and production of ochratoxin A in various culture media
F. M. Valle–Algarra1, E. M. Mateo2*, R. Mateo1, J. V. Gimeno–Adelantado1, and M. Jiménez2
1 Departamento de Química Analítica, Universidad de Valencia, Dr. Moliner 50, 46100 Burjassot, Valencia, Spain
2 Departamento de Microbiología y Ecología, Universidad de Valencia, Dr. Moliner 50, 46100 Burjassot, Valencia, Spain
Presenter: eva.mateo@uv.es
Ochratoxin A (OTA) is a mycotoxin produced by various species of Aspergillus and Penicillium, such as A. ochraceus, A. carbonarius or P. verrucosum. OTA is nephrotoxic, hepatotoxic, teratogenic and immunotoxic to animals. It has been associated to fatal endemic human nephropathies and has been classified as possible carcinogen to humans (group 2B). This mycotoxin is widely distributed and has been reported to occur mainly in cereals, coffee, wine and beer, but also in bee pollen. It is usually accepted that mycotoxin production is dependent on various factors, such as the strain, the environmental conditions and the substrate. Ochratoxin-producing species are widely distributed but only a percentage of the strains belonging to reported producing species are OTA producers. This percentage may increase when studies in progress can establish the ideal conditions for OTA biosynthesis. Bees use pollen as their nutritional source of proteins, carbohydrates, lipids, vitamins and minerals. Beekeepers catch bee pollen in traps put at the hive entry where pollen remains for some time. Afterwards, bee pollen is taken out and carried to stores where it is cleaned, dried, sometimes fumigated, stored and marketed. Before harvest or during these stages pollen may be contaminated with various fungal species like A. carbonarius. The aim of the present study was to assess the capacity of culture media with or without bee pollen to serve as substrates for OTA production by A. carbonarius. The solid media assayed were yeast-extract sucrose medium (YES), YES supplemented with 0.5, 1 and 3 % bee pollen, potato-glucose medium, Wickerham medium, aflatoxin production medium and bee pollen medium containing 0.5, 1, 2 and 3% bee pollen. Media inoculated with A. carbonarius were kept at 25 °C for 2 weeks at 0.98 water activity. Lag phases and radial growth rates were determined. Cultures were analysed for OTA over time by liquid chromatography with fluorescence detection. Preliminary results indicate that there were not significant differences concerning lag phases among the culture media assayed, but there were differences concerning growth rates among the culture media. Growth rates were lower in culture media containing bee pollen. OTA production in media containing bee pollen as a substrate was significantly higher than production in other culture media regardless of incubation time. A correlation between the proportion of pollen added to the medium and OTA level was observed. On the basis of the results obtained in this study it can be assumed that bee pollen is a suitable substrate for OTA production and therefore it may be a risk factor for consumers despite its usually accepted healthy properties.
Acknowledgments: the authors wish to thank financial support from FEDER and Spanish Government “Ministerio de Ciencia e Innovación” (Project AGL2007–66416–C05–01/ALI, and two research grants).
