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
Session 1: Fungi and the Environment (Chairperson Monika Olsen)
The production of ochratoxin A/B and citrinin increases competitiveness of Penicillium under certain environmental influences
Rolf Geisen*, Julia Batzler, Peter Schütz and Markus Schmidt-Heydt
Max Rubner Institut, Haid-und-Neu-Str. 9, 76131 Karlsruhe, Germany
*Presenter: rolf.geisen@mri.bund.de
Ochratoxin A is a nephrotoxic mycotoxin which is produced by several species of the genus Aspergillus as well as species of the genus Penicillium. P. verrucosum occur predominately in wheat and is solely responsible for the occurrence of ochratoxin A in this commodity, whereas P. nordicum is especially adapted to sodium chloride rich fermented foods. The influence of external parameters on the activation of ochratoxin A biosynthesis genes has been extensively investigated by transcriptional analysis. During that analysis it became obvious, that the concentration of NaCl plays an essential role for the production of ochratoxin A. At a certain concentration of NaCl, which is in the range of the NaCl concentration of the fermented products, a shift in the metabolism of P. nordicum could be observed. This could be correlated at the molecular level to an activation of the hog signal cascade. The hog MAP kinase cascade is responsible for gene regulation in relation to osmotic changes in the environment. Moreover the inactivation of the main ochratoxin A polyketide synthase by gene disruption in P. nordicum revealed a second polyketide synthase which obviously can substitute for the former, but only at low NaCl concentrations. A comparison of the competitiveness of the mutant strain which only produce ochratoxin A at lower NaCl concentrations with the wild type revealed that the growth rate of the wild type at higher NaCl concentrations is much less affected than that of the mutant. Moreover the mutant accumulated chloride salts to a much higher concentration than the wild type. These results suggest, that the biosynthesis of ochratoxin A increase the capacity of a strain to be compatible under high NaCl concentrations. Also in P. verrucosum two differentially regulated pks genes have been identified. One seems to be responsible for citrinin biosynthesis, whereas the other is involved in ochratoxin A biosynthesis. Also in case of P. verrucosum high NaCl concentrations shifts production from citrinin towards ochratoxin A. Interestingly other ecological parameters, like the activity of light lead to the opposite effect and shifts production towards citrinin instead of ochratoxin A. Under certain light conditions ochratoxin A biosynthesis could be completely stopped. In case of the non-citrinin producing P. nordicum OTB is produced instead.
The influence of light on ochratoxin A producing Penicillia
Markus Schmidt-Heydt1* and Rolf Geisen1
1Max Rubner Institut Karlsruhe, Haid-Neu-Str.09, 76131 Karlsruhe, Germany
Presenter: Markus.Schmidt-Heydt@mri.bund.de
Ochratoxin A is a nephrotoxic mycotoxin produced predominantly by several Aspergillus species, like A. ochraceus, A. carbonarius, A. niger and two Penicillium species, in particular P. verrucosum and P. nordicum. It is known that the production of mycotoxins strongly dependents on environmental parameters like temperature, water activity or pH. The influence of these parameters on the regulation of the ochratoxin A biosynthesis has been well analysed. Much less information exists about the influence of the physical parameter LIGHT on mycotoxin biosynthesis in fungi.
Growth and ochratoxin biosynthesis of P. nordicum and various other mycotoxin producing fungi could be influenced with light of different wavelengths. Interestingly wavelengths from both sides of the spectrum, e. g. red (long wavelength) and blue (short wavelength) had the strongest inhibitory effects on growth and ochratoxin A biosynthesis. Blue light generally had a stronger effect. Light of moderate wavelength, (yellow to green) had no negative but rather a positive influence on growth and induces ochratoxin A biosynthesis compared to incubation in the dark or under white light. The light effect on growth and ochratoxin A biosynthesis was also dependent on the growth medium. In contrast to malt extract medium (MEA), YES medium as an especially rich nutrient medium, had an attenuating effect on the reactivity against light. However the tendency of the response in both media was the same. Moreover the light intensity is an important factor for the reactive action of the fungus which either results in complete cessation of growth and/or inhibition of ochratoxin A biosynthesis. Light has a growth stalling but not inactivating effect on aerial mycelia. If a non-growing colony under light is shifted to the dark it immediately grows normally. However, blue light has a deactivating effect on spores. After incubation of spores of P. nordicum for 24 h under blue light up to 73 % of the spores were no longer able to germinate. This effect, which has been demonstrated in vitro before can also be exerted in a wheat system. Wheat was inoculated with 107 conidio spores/g of P. verrucosum and incubated for 5 days at 25 °C in the dark (control) or under various light conditions. After that incubation growth was determined by colony counting and the ochratoxin A produced by TLC and HPLC. Again at certain light wavelengths only trace amounts of ochratoxin A were produced and in parallel drastically reduced cfu values were found in contrast to the control. These results indicate the capacity of light to control the growth of ochratoxin A producing Penicillia on food.
Modelling the relationship between environmental factors, transcriptional genes and deoxynivalenol mycotoxin production by strains of two
Fusarium species
M. Schmidt-Heydt2, R. Parra3, R. Geisen2, N. Magan1*
1Applied Mycology Group, Cranfield Health, Cranfield University, Bedford MK43 0AL, U.K.
2Max-Rubner Institute, Karlsruhe, Germany
3Centro del Agua para America Latina y el Caribe (CAALCA), Escuela de Ingeniería y Tecnologías de Información, ITESM Campus Monterrey, NL 64849, Mexico
*Presenter: n.magan@cranfield.ac.uk
The effect of changes in temperature/water activity (aw) on growth, deoxynivalenol (DON) production and trichothecene gene cluster expression (18 genes) for strains of Fusarium culmorum and Fusarium graminearum was studied. The expression data for 6 key transcription genes (TRI4, TRI5, TRI6, TRI10, TRI12 and TRI13) were analysed using multiple regression analyses to model the relationship between these various factors for the first time. Changes in aw and temperature significantly (P=0.05) affected growth and DON. Microarray data on expression of these genes were significantly related to DON production for both strains. Multi-regression analysis was done and polynomial models found to best fit the relationship between actual/predicted DON production relative to the expression of these TRI genes and environmental factors. This allowed prediction of the amounts of DON produced in two dimensional contour maps to relate expression of these genes to either aw or temperature. These results suggest complex interactions between gene expression (TRI genes), environmental factors and mycotoxin production. This is a powerful tool for understanding the role of these genes in relation to environmental factors and enables more effective targeted control strategies to be developed.
Differential response to environmental factors of the Fusarium species associated to cereals (F. verticillioides, F. proliferatum and F. graminearum) and effects on their distribution pattern and toxin risk
Marín, P. 1, Magan, N. 2, Jurado, M. 1, Vázquez, C. 3, González-Jaén, M.T. 1*
1Dpt. Genetics, Faculty of Biology, University Complutense of Madrid, Spain
2Applied Mycology Group, Cranfield Health, Cranfield University, UK
3Dpt. Microbiology III, Faculty of Biology, University Complutense of Madrid, Spain
*Presenter: tegonja@bio.ucm.es
Fusarium verticillioides, F. proliferatum and F. graminearum are important pathogens and toxin producers on cereals. Environmental factors, especially water availability and temperature, significantly influence fungal growth and toxin production. We have performed several studies to know the effects of interacting conditions of temperature and water activity on growth rate in Spanish field isolates of those three species in a standardized in vitro system. Similarly, the effect of these conditions on toxin production was analyzed using an approach based on the quantification of the expression of key toxin biosynthetic genes by real-time RT-PCR species specific assays. TRI5 gene expression was quantified for the trichothecene-producer F. graminearum and FUM1 gene for the fumonisin producers F. verticillioides and F. graminearum. The two-dimensional maps produced in these studies showing the optimum and marginal rates of growth rates were compared and discussed in relation to the distribution of these species in different cereals and climatic regions from Spain. Similarly, the different toxin biosynthetic gene expression patterns obtained for these species are described and the implications for prediction of risk discussed.
The influence of water activity and temperature on the germination, growth and sporulation of Stachybotrys chartarum isolates
S. Frazer1, N. Magan1* and D. Aldred1
1Applied Mycology Group, Cranfield Health, Cranfield University, Cranfield, Bedford MK43 OAL, UK
Presenter: n.magan@cranfield.ac.uk
There is little detailed knowledge of the response of S.chartarum to interacting environmental factors. This may be particularly important when examining the potential for spores to be dispersed in semi-damp or previously flooded buildings. The objectives of this study were to examine the effect of interacting conditions of water activity (0.99-0.90), temperature (15-37oC) on (a) conidial germination, (b) germ tube extension, (c) growth and (d) sporulation in vitro on a potato dextrose agar medium.
Germination of the conidia of the two tested isolates was significantly influenced by aW and temperature and was most rapid at 0.99 and 0.97 aW between 15 and 30°C with all conidia germinating within 24 hours. These factors also played a significant role in the rate of germ tube extension of both isolates and were found to be most rapid at 25-30°C for all tested aw levels. Mycelial growth rates of both isolates were optimal at 0.99 aW and 25-30°C with very little growth at 37°C. No growth was observed at 0.91 aw. The numbers of spores produced by colonies was quantified and compared. The optimum spore production rates were at 0.99 aw and 30oC (1030-1745 conidia mm2) for two strains. At 0.95 aw maximum conidia were produced at 25-30oC with none produced at 15-20 or at 37oC. These types of data are now being compared with that on building materials and will be related to mycotoxin production.
National emergency planning for mould and mycotoxins in response to climate change
Monica Olsen1*, Elisabeth Fredlund1, Mats Lindblad1, Gunnar Andersson2, Cecilia Lerenius3, Michael Sulyok4 and Thomas Börjesson5
1Microbiology Division, Research and Development Department, National Food Administration, SE-751 26 Uppsala, Sweden
2National Veterinary Institute, SE-751 89 Uppsala, Sweden
3 Swedish Board of Agriculture, SE-551 82 Jönköping, Sweden
4Center for Analytical Chemistry Department of Agrobiotechnology (IFA-Tulln)
Konrad Lorenzstr. 20, A-3430 Tulln, Austria
5Lantmännen Business Development, Grain, SE-531 87 Lidköping, Sweden
*Presenter: monica.olsen@slv.se
Climate change may very likely lead to increased levels of mycotoxins in raw food material. There is already an apparent shift in prevalence of Fusarium species on infected heads in Northern Europe toward F. graminearum. It has been suggested that F. graminearum is displacing other species such as F. culmorum, presumably due, in part, to short-term climate variations and ecological differences among fungi, but also perhaps due to differences in aggressiveness and pathogenicity of the various species. Furthermore, climate change will lead to the introduction of new plants to regions where climate used to be an obstacle for successful production. One example is the increased production of maize in Northern Europe. Increased production of maize and the dramatic increase in maize and other residues that remain on the soil surface may provide an increased reservoir of fungal spores, especially of F. graminearum.
The only real way that we can determine whether climate change is having an effect on fungal and mycotoxin levels ‘in the real world’, is if we monitor for them. This new project is focused to develop a national coordination system between different authorities and agricultural industry for future emergency planning and decisions in response to climate change. As tools we are using both predictive models for mycotoxins in grain, fast multi methods for myco-toxins and real-time PCR methods for fungi. Furthermore, international collaboration with other institutes and projects are important components of the project.
