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Fungi produce secondary metabolites that are not directly involved in their growth, but often contribute to their adaptation to extreme environmental stimuli and enable their survival. Conidial pigment or melanin is one of the secondary metabolites produced naturally by a polyketide synthesis (PKS) gene cluster in several filamentous fungi and is known to protect these fungi from extreme radiation conditions. Several pigmented or melanized fungi have been shown to grow under extreme radiation conditions at the Chernobyl nuclear accident site. Some of these fungi, including Paecilomyces variotii, were observed to grow towards the source of radiation. Therefore, in this study, we wanted to identify if the pigment produced by P. variotii, contributes to providing protection against radiation condition. We first identified the PKS gene responsible for synthesis of pigment in P. variotii and confirmed its role in providing protection against UV irradiation through CRISPR-Cas9 mediated gene deletion. This is the first report that describes the use of CRISPR methodology to create gene deletions in P. variotii. Further, we showed that the pigment produced by this fungus, was not inhibited by DHN-melanin pathway inhibitors, indicating that the fungus does not produce melanin. We then identified the pigment synthesized by the PKS gene of P. variotii, as a naptho-pyrone Ywa1, by heterologously expressing the gene in Aspergillus nidulans. The results obtained will further aid in understanding the mechanistic basis of radiation resistance.

Byssochlamys is a fungus that causes spoilage of heat processed fruit products by producing heat resistant ascospores. This thesis investigated heat resistant properties of ascospores of Byssochlamys fulva and Byssochlamys nivea. Heat inactivation of the ascospores at temperatures of 82.5 – 90oC showed activation, shoulder, exponential reduction and tailing phases. The D values of the ascospores increased significantlywith culture age over the period of 4 – 24 weeks, and the average increase was 3.5 – 7-fold for B. fulva strains and 2 – 4-fold for B. nivea strains. Ascospores of B. fulva weremore heat resistant than those of B. nivea.Ultrastructure of the ascospores examined by electron microscopy comprised a cellwall, a thick intermediate space (IMS) and a dense cytoplasm. Examining the arrangement of materials in the ascospores with differential scanning calorimetry provided evidence of a glassy state. Aging improved the robustness of the ascosporestructure whereas heating reduced its integrity. Older ascospores occasionally had moresub-layers in the IMS. Ascospores of B. fulva had a more resilient structure and moreelaborated outer coating than those of B. nivea.Flow cytometry revealed heterogeneity in preparations of dormant and heat treatedascospores at different ages. Heating affected the integrity of the ascospore wall, whichwas manifested as increased autofluorescence, permeability to SYTO 9 and clumping ofthe ascospores. Sub-populations of activated and sub-lethally injured ascospores mostlyappeared after heat treatments.Proteomic analysis of ascospores by 2D gel electrophoresis showed that less than 4% oftheir total proteins significantly changed their expression after heating and aging. Mostof the changed proteins were metabolic enzymes, which possibly had been resynthesised and stored in dormant ascospores. For B. nivea, three proteins wereidentified as heat shock proteins 60, 70 and general stress protein 39. Latent proteinsynthesis

Byssochlamys and related Paecilomyces strains are often heat resistant and may produce mycotoxins in contaminated pasteurised foodstuffs. A comparative study of all Byssochlamys species was carried out using a polyphasic approach to find characters that differentiate species and to establish accurate data on potential mycotoxin production by each species. Phylogenetic analysis of the ITS region, parts of the â-tubulin and calmodulin genes, macro- and micromorphological examinations and analysis of extrolite profiles were applied. Phylogenetic analyses revealed that the genus Byssochlamys 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. saturatus. Among these, B. nivea produces the mycotoxins patulin and byssochlamic acid and the immunosuppressant mycophenolic acid. Byssochlamys lagunculariae produces byssochlamic acid and mycophenolic acid and thus chemically resembles B. nivea. Some strains of P. saturatus produce patulin and brefeldin A, while B. spectabilis (anamorph P. variotii s.s.) produces viriditoxin. Some micro- and macromorphological characters are valuable for identification purposes, including the shape and size of conidia and ascospores, presence and ornamentation of chlamydospores, growth rates on MEA and CYA and acid production on CREA. A dichotomous key is provided for species identification based on phenotypical characters.

Byssochlamys and related Paecilomyces strains are often heat resistant and may produce mycotoxins in contaminated pasteurised foodstuffs. A comparative study of all Byssochlamys species was carried out using a polyphasic approach to find characters that differentiate species and to establish accurate data on potential mycotoxin production by each species. Phylogenetic analysis of the ITS region, parts of the â-tubulin and calmodulin genes, macro- and micromorphological examinations and analysis of extrolite profiles were applied. Phylogenetic analyses revealed that the genus Byssochlamys 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. saturatus. Among these, B. nivea produces the mycotoxins patulin and byssochlamic acid and the immunosuppressant mycophenolic acid. Byssochlamys lagunculariae produces byssochlamic acid and mycophenolic acid and thus chemically resembles B. nivea. Some strains of P. saturatus produce patulin and brefeldin A, while B. spectabilis (anamorph P. variotii s.s.) produces viriditoxin. Some micro- and macromorphological characters are valuable for identification purposes, including the shape and size of conidia and ascospores, presence and ornamentation of chlamydospores, growth rates on MEA and CYA and acid production on CREA. A dichotomous key is provided for species identification based on phenotypical characters.