Applied and Industrial Mycology

Filamentous fungi are playing an important role in our daily life as well as in the context of food products in indoor situations. The area of the research group is to study the biodiversity, phylogeny and cell biology of fungi with special relation to food and indoor mycology, with an emphasis on the genera Penicillium and Aspergillus. These genera are significant as spoilage agents of food and beverages, as producers of toxic compounds and as the dominant fungi in human dwellings. They even occasionally occur as human and animal pathogens. On the other hand, many species of these genera are very important industrial microorganisms in food fermentation and biotechnology. The mission of the program is to reach a deeper understanding of applied and fundamental insights of fungi related to food-association and indoor situations. These include a novel polyphasic classification of the subgroups of the genera Penicillium and Aspergillus, and the development of tools for a practical barcoding system of these groups. To understand the biology of the important fungi in these applied fields, the cell biology of the fungal cells, including fungal survival structures (spores) is studied. The research is focussed on the nature of heat resistance of ascospores and the sensitivity of fungal spores for antifungal compounds. The research of the group has always been intertwined with numerous (smaller) projects with external parties (industrial companies and governmental institutions) that request expertise with problems related to food spoilage, indoor environments and industrial applications. This is illustrated by the fact that several large fundamental or applied research projects have been directly initiated as a result of these smaller projects.

Biodiversity of Penicillium and Aspergillus
A major achievement in Penicillium research was the publication of the monograph of subgenus Penicillium, published together with J.C. Frisvad in Studies in Mycology 49 (2004). Studies on other subgenera were started and it is expected that a major part of subgenus Furcatum will be finished in 2008.  Phylogenetical analysis of Penicillium subgenus Biverticillium were collected and expanded by sequencing more genes. The taxonomic placement of this subgenus will lead to a separation from Penicillium and final experiments will be completed in 2008.
The taxonomic research on Aspergillus was greatly stimulated by the appointed of Janos Varga as post-doc. In April 2007 an International workshop on Aspergillus systematics in the genomic era was organised and recommendations and contributions were published in Studies in Mycology 59, and the book Aspergillus in the genomic era which was published in January 2008. Several papers dealing with the various sections of Aspergillus have been completed and it is anticipated that in 2008 and 2009 most of the genus Aspergillus will be completed.

DNA barcoding
DNA barcoding is a taxonomic method which uses a short genetic marker in an organism's (mitochondrial) DNA to quickly and easily identify it as belonging to a particular species. A DNA sequence should meet several criteria to be used successfully for species identification. DNA sequences should be orthologous in the examined organisms, and variable enough to allow species identification, with low levels of intraspecific variation. A DNA barcode should be easily accessible (universally amplified/sequenced by standardized primers from a wide set of organisms), relatively short (≤5-600 bp), simple to sequence, easily alignable, with no recombination. The mitochondrial cytochrome oxidase subunit 1 (cox1, usually referred to as COI in barcoding studies) was proposed to be a good candidate for barcoding animal species including birds, fishes and Lepidopteran insects.
To evaluate the usefulness of the cox1 gene for DNA barcoding, we gathered altogether 47 cox1 sequences and examined their properties for species delimitation. Our data indicate that cox1 is not appropriate to be used as DNA barcode in aspergilli since none of the eight species of the Aspergillus niger species complex could be identified unequivocally. The phylogenetic tree constructed based on the cox1 sequences also shows an overlap between intra- and interspecific variation possibly due to past mitochondrial DNA recombination events as suggested earlier. Although a high degree of heterokaryon incompatibility was observed among isolates of the A. niger species complex, mtDNA transfers occur readily even between incompatible isolates. Comparing the phylogenies based on cox1, ITS, b-tubulin and calmodulin sequences, either b-tubulin and calmodulin could serve as a suitable region for species identification among black aspergilli. Recent attempts to use the cox1 gene for species identification in other fungal groups including Fusarium species and basidiomycetes have also met with limited success (K. Seifert, pers. comm..). Recently, the fungal community decided to use the ITS region as the first choice for DNA barcoding the Fungal Kingdom. If this region does not distinguish all species, a second region could be used to resolve the taxa. In the case of aspergilli, our opinion is to use either b-tubulin or calmodulin sequences for accurate species identification.

Food Mycology
Various aspects such as the fungi producing mycotoxins represented an integral part of the research of the group. Several studies were completed in which a polyphasic taxonomy led to the descriptions of new taxa together with data on their mycotoxin analysis.  In this respect the ecological studies of food-borne fungi are integrated in the taxonomic studies of the biodiversity of Penicillium and Aspergillus as described above.
In August 2006 the group was involved in the organisation of the International Food Mycology Workshop in Cairns (Australia), and in June 2007 they organised together with the International Commission of Food Mycology a three day workshop at Key West (U.S.A.) followed by a two day symposium for the Industry.

Fundamental aspects of spore biology of food-relevant fungi
From 2002 onwards, the biology of the extremely stress-resistant ascospores of the fungus Talaromyces macrosporus was addressed. These spores are comparable with some bacterial spores in their tolerance for high temperatures, drought and high pressure. Moreover, short rigorous environmental triggers (as a pasteurisation treatment or a pressure at 6000 Bar) can activate germination of the dormant cells. Activation is correlated with different changes in the cell wall of the spore as for instance the release of a protein and an increase of permeability. Upon activation, trehalose is degraded and the degradation product, glucose, is released from the cell. Then a sudden ejection of the spore through a very thick outer cell wall occurs which is dubbed “prosilition” (prosilire (Latin), means “to jump out”). The process occurs within seconds and is accompanied by an increase in respiration. This process was identified in more species of the genus Talaromyces. Spin probe studies (ESR) indicated that the cytoplasmic parameters as microviscosity en anisotropy changed most dramatic after prosilition. These data indicate that prosilition is an important process to increase the uptake of nutrients and oxygen (water?) to enable the spore to swell and form a germ tube which was not possible inside the encasement of the isolating outer cell wall.
Further studies have addressed the biology of conidia, air- or water-borne survival structures that are very important for the distribution of food-related fungi. The identification that 1-octen-3-ol, one of the most notable flavour compounds in mushrooms, is also a volatile self-inhibitor of germination of conidia of Penicillium paneum is regarded as a highlight here.
In addition, germination of multicellular conidia has been studied, and it became clear that compartments of these spores initially are alike, but do differentiate and communicate during early germination. When germinating compartments collapse due to an adverse condition, the non-germinating compartments take over and form germ tubes by themselves. In collaboration with the Swedish Agricultural University, the morphology of the sporangiospores of the Rhizopus microsporus groups was studied and novel leads to identification of the members of this important food-related group of fungi were found. Finally, a number of studies were completed on fungal growth and development as a result of collaboration with other groups (including University of Edinburgh (U.K.), University of Wageningen, and the University of Utrecht).
More recently we have studied germination of conidia of P. discolour, and identified an ergosterol- enriched area of the plasma membrane on the site of germ-tube formation. This is part of a STW-funded project on the mode of action of natamycine on fungal spores. The sensitivity of these spores for the polyene-antibiotic during several stages of germination has been studied and several manuscripts are in preparation. We have also done a large study (as part of a SENTER-funded project) on post-harvest diseases, with as a model system the infection of tulip bulbs by the fungus Fusarium oxysporum with the aim to find novel treatments or procedures.

Indoor Mycology
The research on indoor fungi focuses mostly on the taxonomy and biodiversity of the species occurring in dwellings, buildings, archives, etc. Two major studies on aspergilli were completed. In March 2006 an international workshop was organised bringing together the various research disciplines working with fungi in indoor environments. The contributions and recommendation are being prepared for a book “Molds, water and the built environment”. Together with Prof. Olaf Adan, studies on the behaviour of fungi on building materials with emphasis on the role of water were initiated, and this has led to an application for a research grant at STW.  During 2002–2007 Rob Samson gave 2 or 3 courses per year, particularly in Germany, dealing with the identification and detection of indoor fungi. Together with the Landesgesundheitsamt in Stuttgart and some German reference laboratories, a proficiency testing for the identification of common indoor moulds was initiated and performed. This testing is carried out twice a year and between 70 to 80 laboratories working in the field of food and indoor mycology participate.