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 2: Xerophilic and Heat Resistant Fungi (Chairperson Rob Samson)
The extreme xerophile Xeromyces bisporus: our current state of
knowledge
Olga Vinnere Pettersson1*, Su-lin L. Leong1, Therese Rice1, Jan Dijksterhuis2, Jos Houbraken2, Robert A. Samson2, Johan Schnürer1
1Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, SE-750 07 Uppsala, Sweden
2Applied and Industrial Mycology, Centraalbureau voor Schimmelcultures, Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands
*Presenter: olga.vinnere.pettersson@mikrob.slu.se
The filamentous ascomycete Xeromyces bisporus Fraser is, arguably, the most xerophilic organism known on Earth, able to grow at as low as 0.62 aw. The fungus is most commonly isolated from dried fruit and other sugary substrates with low water activity. The genus Xeromyces is monotypic and its taxonomic position has long been debated, however it is clear that it is closely related to the genus Monascus, based on morphology of the fruiting bodies as well as DNA sequences. Von Arx (1970) united the genera Monascus and Xeromyces, introducing the new name, Monascus bisporus, which was later supported by phylogenetic studies (Stchigel et al. 2004). At the same time Hawksworth & Pitt (1983), did not accept this unification and proposed to retain Xeromyces as a separate genus.
Our results of multilocus sequence typing as well as electron microscopy data show clearly that Xeromyces is indeed closely related to Monascus, however it should be retained as a separate genus. Moreover, our data suggest that the closest relatives of Xeromyces are two xerophilic food-borne species of Chrysosporium – C. inops and C. xerophilum. For X. bisporus we have: established the minimal, optimal and maximal temperature and water activity requirements for the worldwide population; discovered extremely low level of genetic variation within this species; and have investigated the physiological aspects of its response to water stress (accumulation of glycerol as compatible solute). The fungus stores trehalose in its ascospores. We discovered that X. bisporus regulates membrane permeability by changing saturation of the membrane fatty acids to cope with hyperosmosis. We are currently investigating this phenomenon in closely related fungal xerophiles (collaboration with Dr H. Heipieper, Helmholtz Centre for Environmental Research, Leipzig). According to our data, the ascospores of X. bisporus are moderately heat-resistant and can be activated by temperatures around 50-65°C. The latter result is valuable for the bakery industry: X. bisporus is an emerging spoilage agent in Europe, significantly reducing the shelf life of bakery products (Samson, CBS, pers. com.).
The genome sequence of the holotype strain of X. bisporus has been recently obtained and is currently in the process of annotation. The genome size is in the range of 19 Mbp (PFGE data) – 22 Mbp (draft assembly data), consisting of presumably five chromosomes.
References:
von Arx, J.A. (1970) The genera of fungi sporulating in pure culture. J. Cramer, Lehre.
Hawksworth D. L and Pitt J. I. (1983) A new taxonomy for Monascus species based on cultural and microscopic characteristics. Australian Journal of Botany, 31, 51-61.
Stchigel A. M., Cano J. F. Abdullah S. K. and Guarro J. (2004) New and interesting species of Monascus from soil, with a key to the known species. Studies in Mycology 50, 299-306.
Xeromyces bisporus: Growth and Competition at Low Water Activities
Su-lin L. Leong1*, Olga Vinnere Pettersson1, Therese Rice1, Ailsa D. Hocking2, Johan Schnürer1
1Department of Microbiology, Swedish University of Agricultural Sciences, Box 7025, SE-750 07 Uppsala, Sweden
2CSIRO Food and Nutritional Sciences, PO Box 52, North Ryde, NSW 1670, Australia
*Presenter: Su-lin.Leong@mikrob.slu.se
Xeromyces bisporus is unique in its ability to grow at water activity (aw) 0.62, which is lower than for any other known organism. The effect of temperature and water activity on the radial growth rates of a fast and slow growing strain of X. bisporus were examined. Optimal conditions for growth of both strains were approx. 0.84 aw and 30°C, despite FRR 2347 growing two- to five-fold faster than CBS 185.75. Strain FRR 2347 also grew well at 37°C, whereas strain CBS 185.75 did not grow. The growth response of FRR 2347 was more typical for the majority of 19 strains of X. bisporus examined. Of 19 strains, 14 grew rapidly at 37°C and 11 were able to grow at relatively high aw, 0.98.
The slow-growing CBS 185.75 and fast-growing FRR 2347 had identical ITS sequences, whereas other strains which differed at one or two loci displayed similar growth responses. RAPD analysis did not suggest extensive genetic variation among strains.
The ability of FRR 2347 and CBS 185.75 to compete with other xerophilic moulds at low water activity seems to depend primarily on growth rate. At aw ≤ 0.80, the fast growing strain FRR 2347 was highly competitive against other xerophiles, although E. chevalieri also grew well. Excretion of inhibitory substances acting over a long range was not observed by any species; inhibitors acting over a short range that temporarily slowed the growth of X. bisporus or produced a protective zone around the colony were occasionally observed for Aspergillus penicillioides, Chrysosporium inops and C. xerophilum.
Preliminary data on radial growth rates of X. bisporus on different solutes at aw ~0.88–0.90 suggested a strong preference for glucose. Moderate growth was observed on sucrose and sorbitol, whereas glycerol and xylitol supported weaker growth. Unlike other xerophiles, X. bisporus failed to grow when NaCl was the controlling solute. As for X. bisporus, the xerophilic species Eurotium chevalieri, C. inops, C. fastidium and C. xerophilum displayed faster radial growth on glucose than on glycerol; however, growth of the xerotolerant A. niger appeared to be equivalent or even improved on glycerol and xylitol compared to glucose.
The ever changing spore: surprises of early germination
Richard van Leeuwen1*, Timon Wyatt1, Robert Jan Bleichrodt2, Elena Golovina3, Hildegard Menke4, Han Wösten2, Hein Stam4, Jan Dijksterhuis1.
1Applied and Industrial Mycology, CBS-KNAW/Fungal Biodiversity Centre, Utrecht. 2Molecular Microbiology and Kluyver Centre for Genomics of Industrial Fermentation, Utrecht. 3Wageningen NMR Centre, Wageningen University,Wageningen. 4DSM Food Specialties, Delft. All affiliations in The Netherlands.
*Presenter: r.vanleeuwen@cbs.knaw.nl
Fungi are found in a wide variety of environments and colonization of new substrata is by means of survival vehicles as air- or waterborne spores. These conidia are characterized by a dormant state and moderate stress-resistance. Upon contact with a moist substrate, germination of conidia occurs including a change from a dormant stabilized state towards a growing vegetative cell.
Dormancy and germination were studied by an analysis of the transcriptome of conidia of Aspergillus niger in the absence or presence of the antifungal compound natamycin. Most changes in the transcriptome were observed during early germination. During the first two hours, the complexity of the transcriptome decreased by 23%, but the abundance of transcripts increased with 35%. This was accompanied by a reduction in cellular microviscosity and initiation of carbon metabolism. The RNA complexity increased gradually with 36% between 2 and 8 h. During this time period spores grew initially isotropically, followed by polarized growth, 1 cycle of mitosis and formation of germ tubes. The transcriptome of natamycin-treated conidia was nearly identical to untreated conidia at early germination, but showed severely affected gene expression after 8 h. The spores did not exhibit mitosis and germ tube formation.
Heat resistance of Byssochlamys ascospores: a study of ascospore age and ultrastructure
Anh Linh Nguyen1, Nai Tran-Dinh2, Ailsa Hocking2*, and Graham Fleet1
1 School of Chemical Engineering, The University of New South Wales, Sydney, NSW 2052, Australia
2 CSIRO Food and Nutritional Sciences, North Ryde, NSW 2113, Australia
* Presenter: Ailsa.Hocking@csiro.au
Byssochlamys spp. are major spoilage and public health concerns for the fruit industry mostly because of their heat resistant ascospores. However, research on mechanisms of the heat resistance is very limited.
To gain a better understanding of thermotolerance, effects of ascospore age on heat resistance were studied on four food-related strains of two common species, B. fulva and B. nivea. Ascospores aged between 4 and 24 weeks were heat inactivated at 85 – 90OC for up to 90 minutes (B. fulva) or 82.5 – 87.5oC for up to 60 minutes (B. nivea). Inactivation of the four strains was not linear but displayed a four-phase pattern comprising activation, shoulder, linear reduction and tailing. Despite the evident non-linearity of the inactivation curves, D values were derived from the linear portion of the curves and used to compare heat resistance. The results showed that age significantly increased the heat resistance of Byssochlamys ascospores. For 4 and 24 week old samples, D values at 85OC and 87.5OC of B. fulva ascospores changed by up to seven-fold while D82.5 of B. nivea ascospores also increased by up to three-fold. The heat resistance of Byssochlamys ascospores often peaked at 12 weeks with little change at 24 weeks. The age-induced thermotolerance was not significant at higher inactivation temperatures where D values were not statistically different.
One strain of each species was further studied by electron microscopy for changes in ascospore structure with age (4 and 24 weeks), activation (75OC, 30 minutes) and inactivation (95OC, 30 minutes). All samples had a common three-region ultrastructure consisting of cell wall, relatively thick intermediate space (IMS) and highly dense cytoplasm. Dormant and activated ascospores were very similar at both ages with respects to cell wall thickness and area ratios. B. fulva but not B. nivea ascospores developed a multilayered IMS and retained a resistant coating which was removed by heating. Inactivation treatment caused some damage to the cell wall and severe distortion to ascospore shape.
Findings of this study are beneficial for food processors and manufacturers, indicating that delay in processing material should be avoided. They may also suggest an explanation for the thermotolerance and a possible mechanism of heat inactivation in Byssochlamys ascospores.
Compatible solutes in the fungal cell
Timon Wyatt1*, Richard van Leeuwen1, Jan Dijksterhuis1
1CBS Fungal Biodiversity Centre, Royal Dutch Academy of Sciences (KNAW), Uppsalalaan 8, 3584 CT, Utrecht, the Netherlands.
*Presenter: t.wyatt@cbs.knaw.nl
Fungi survive and even grow during adverse conditions including extreme temperatures, or oxidative- and osmotic stresses. These stresses often evoke an accumulation of so-called “compatible solutes”, which are compatible with metabolic processes and other cell functions even when present at high concentrations. The solutes are small molecules generally belonging to three groups of chemicals, namely the saccharides (mono- and di-), polyols (sugar alcohols) and amino acids. Trehalose is the most well known compatible solute occurring all over the fungal kingdom, but also occurs among insects, nematodes, protists and crustaceans. Trehalose is an excellent stabilizer of proteins and membranes and protects them against heat and drought. Trehalose can be present in both vegative and reproductive stages, but generally has higher concentrations in survival structures, like spores and sclerotia. Trehalose accumulates intracellular, when fungi are exposed to stresses like: heat stress, starvation, high osmolarity or desiccation. Polyols, as glycerol and mannitol, are also accumulated in fungi during or after stress. The precise mechanism of action of these molecules is not clear but may include glass formation, viscosity, water replacement and cosmotrophic effects. The solutes may function differently, while glycerol, the smallest polyol, is more important in osmotolerance, and trehalose has a correlation with longevity and survival of spores. In addition proteins (like heatshock proteins), ions (like organic acids) and other unknown factors may build the stress resistance of fungal cells, some of them nearly as resistant as bacterial spores.
Applied situations and the fungal collection: panels of test strains
Jan Dijksterhuis1*, Tineke van Doorn1, Jaap Postma2, Ad Fluit3, Wietske Botterhuis3, Tridia van der Laan4, Dick van Soolingen4, Emilia Rico5 and Shawn Johnson5.
1Applied and Industrial Mycology, CBS Fungal Biodiversity Centre, Royal Academy of Sciences, Utrecht. 2 Ecofide BV, Weesp. 3UMC, Utrecht. 4RIVM, Bilthoven Affiliations1-4 in The Netherlands. 5BCN Research Laboratories, Knoxville, USA
*Presenter: j.dijksterhuis@cbs.knaw.nl
Large collections of fungal species offer many possibilities for applied research. In this contribution several examples are given were panels of relevant selected fungal species that are well characterized (morphological and molecular) at the species level are used in the evaluation of among others medical and ecotoxicological situations. These examples include the identification of a possible novel antibiotic; the evaluation of ecotoxicology of agrochemicals in surface water ecosystems; evaluating the effect of different sanitizers on fungal survival structures. As will be explained, a sound identification of the fungal species is very important in these situations. Surprisingly and counter intuitively, from this approach leads into fundamental research may be found.
ICARUS, a protein related to the extreme stress-resistant ascospores of the fungus Talaromyces macrosporus
Daniela Buchner1*, Timon Wyatt1, Micha Hanssen1, Folkert Hoekstra2, Elena Golovina2,3, Rob Samson1, Han Wösten3, Luis Lugones3 and Jan Dijksterhuis1
1CBS Fungal Biodiversity Centre, Royal Dutch Academy of Sciences (KNAW), Uppsalalaan 8, 3584 CT, Utrecht, The Netherlands,
2Laboratory of Plant Physiology, Wageningen University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands,
3Laboratory of Biophysics, Wageningen University, and Wageningen NMR Center, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands,
4Department of Molecular Microbiology, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
*Presenter: d.buchner@cbs.knaw.nl
The ascospores of Talaromyces macrosporus survive ultrahigh pressure (6000 Bar), temperature (85 °C) and drought. They exhibit constitutive dormancy and are only activated to germination after a rigorous physical trigger including high temperature- or pressure pulses. Dormant ascospores contain a very high concentration of mainly trehalose and exhibit a high density and internal microviscosity. These parameters drop strongly during early germination, which is also characterised by a sudden ejection of the inner cell through the ruptured outer cell wall.
Heat activation is correlated with a change of the permeability and structure of the very thick outer cell-wall. It also triggers trehalose degradation into glucose, which is secreted into the environment. Activation is also accompanied with a release from the cell wall of large amounts of a small (7 kD) abundant protein (≈ 5% of the total cell protein). The protein was dubbed ICARUS. The ICARUS gene was successfully disrupted and mutants are currently characterised. The growth rates are similar compared to the wild type , however upon heating the mutants do not show release of the protein and have an impaired ascospore production. Furthermore, the mutants release a deep red pigment into the agar medium. The spores are identical as judged by cryoSEM, with a slightly different colour than the wild type. Mutant spores do form very loose pellets after centrifugation and also differ in cell-wall permeability for fluorescent dyes and dormancy.
Taxonomic review of food relevant Paecilomyces variotii and related
species
Jos Houbraken1*, Jens Frisvad2, and Rob Samson1
1CBS-KNAW Fungal Biodiversity Centre, dept. Applied and Environmental Mycology, Utrecht, The Netherlands.
2Centre for Microbial Biotechnology, Technical University of Denmark, Søltofds Plads, Building 221, DK 2800 kgs. Lyngby, Denmark
*Presenter: j.houbraken@cbs.knaw.nl
Byssochlamys and related Paecilomyces strains are commonly occurring and have previously been isolated from various foodstuffs, such as pasteurised products and sorbate containing foods. The taxonomy of P. variotii and related species is investigated using a polyphasic approach. Combining phenotypic and physiological characters, extrolite profiles and sequences, a robust taxonomy was obtained. These results show that Paecilomyces sensu stricto includes nine species, five of which form a teleomorph, i.e. B. fulva, B. lagunculariae, B. nivea, B. spectabilis and B. zollerniae, while four are asexual, namely P. brunneolus, P. divaricatus, P. formosus and P. dactylethromorphus. Each species has specific extrolite profiles, and accurate data on potential mycotoxin production by each species is established. Byssochlamys nivea produces the mycotoxins patulin and byssochlamic acid, and the immunosuppressant mycophenolic acid. Some strains of P. dactyelethromorphus produce patulin and brefeldin A, while B. spectabilis (anamorph P. variotii s.s.) produces viriditoxin. An overview on the occurrence of members of this phylogenetically closely related group is presented.
