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The debates over the requirement of the International Code of Nomenclature for algae, fungi, and plants (ICNafp) for a viable specimen to represent the name-bearing type material for a species or infraspecific taxon have a long history. Taxonomy of fungi commonly studied as living cultures exemplified by yeasts and moulds, strongly depend on viable reference material. The availability of viable cultures is also particularly useful for several groups of filamentous and dimorphic fungi. While the preservation of metabolically inactive cultures is permitted and recommended by the ICNafp, there is room for improvement. Below, we review the history and current status of cultures as the name-bearing type material under the Code. We also present a roadmap with tasks to be achieved in order to establish a stable nomenclatural system that properly manages taxa typified by viable specimens. Furthermore, we propose setting up rules and defining the nomenclatural status of ex-type cultures under Chapter F, the section of the ICNafp that includes provisions specific to names of fungi.

Pineapple Fruitlet Core Rot (FCR) is a fungal disease characterized by a multi-pathogen pathosystem. Recently, Fusarium proliferatum, Fusarium oxysporum, and Talaromyces stollii joined the set of FCR pathogens until then exclusively attributed to Fusarium ananatum. The particularity of FCR relies on the presence of healthy and diseased fruitlets within the same infructescence. The mycobiomes associated with these two types of tissues suggested that disease occurrence might be triggered by or linked to an ecological chemical communication-promoting pathogen(s) development within the fungal community. Interactions between the four recently identified pathogens were deciphered by in vitro pairwise co-culture bioassays. Both fungal growth and mycotoxin production patterns were monitored for 10 days. Results evidenced that Talaromyces stollii was the main fungal antagonist of Fusarium species, reducing by 22% the growth of Fusarium proliferatum. A collapse of beauvericin content was observed when FCR pathogens were cross-challenged while fumonisin concentrations were increased by up to 7-fold. Antagonism between Fusarium species and Talaromyces stollii was supported by the diffusion of a red pigmentation and droplets of red exudate at the mycelium surface. This study revealed that secondary metabolites could shape the fungal pathogenic community of a pineapple fruitlet and contribute to virulence promoting FCR establishment.

The study focused on the examination of the different fungal species isolated from commercial rice samples, applying conventional culture techniques, as well as different molecular and phylogenic analyses to confirm phenotypic identification. Additionally, the mycotoxin production and contamination were analyzed using validated liquid chromatography-tandem mass spectrometry (LC-MS/MS). In total, 40 rice samples were obtained covering rice berry, red jasmine rice, brown rice, germinated brown rice, and white rice. The blotting paper technique applied on the 5 different types of rice samples detected 4285 seed-borne fungal infections (26.8%) for 16,000 rice grains. Gross morphological data revealed that 19 fungal isolates belonged to the genera Penicillium/Talaromyces (18 of 90 isolates; 20%) and Aspergillus (72 of 90 isolates; 80%). To check their morphologies, molecular data (fungal sequence-based BLAST results and a phylogenetic tree of the combined ITS, BenA, CaM, and RPB2 datasets) confirmed the initial classification. The phylogenic analysis revealed that eight isolates belonged to P. citrinum and, additionally, one isolate each belonged to P. chermesinum, A. niger, A. fumigatus, and A. tubingensis. Furthermore, four isolates of T. pinophilus and one isolate of each taxon were identified as Talaromyces ( T. radicus, T. purpureogenum, and T. islandicus). The results showed that A. niger and T. pinophilus were two commonly occurring fungal species in rice samples. After subculturing, ochratoxin A (OTA), generated by T. pinophilus code W3-04, was discovered using LC-MS/MS. In addition, the Fusarium toxin beauvericin was detected in one of the samples. Aflatoxin B1 or other mycotoxins, such as citrinin, trichothecenes, and fumonisins, were detected. These preliminary findings should provide valuable guidance for hazard analysis critical control point concepts used by commercial food suppliers, including the analysis of multiple mycotoxins. Based on the current fi

The respiratory tree maintains sterilizing immunity against human fungal pathogens. Humans inhale ubiquitous filamentous molds and geographically restricted dimorphic fungal pathogens that form small airborne conidia. In addition, pathogenic yeasts, exemplified by encapsulated Cryptococcus species, and Pneumocystis pose significant fungal threats to the lung. Classically, fungal pneumonia occurs in immune compromised individuals, specifically in patients with HIV/AIDS, in patients with hematologic malignancies, in organ transplant recipients, and in patients treated with corticosteroids and targeted biologics that impair fungal immune surveillance in the lung. The emergence of fungal co-infections during severe influenza and COVID-19 underscores the impairment of fungus-specific host defense pathways in the lung by respiratory viruses and by medical therapies to treat viral infections. Beyond life-threatening invasive syndromes, fungal antigen exposure can exacerbate allergenic disease in the lung. In this review, we discuss emerging principles of lung-specific antifungal immunity, integrate the contributions and cooperation of lung epithelial, innate immune, and adaptive immune cells to mucosal barrier immunity, and highlight the pathogenesis of fungal-associated allergenic disease. Improved understanding of fungus-specific immunity in the respiratory tree has paved the way to develop improved diagnostic, pre-emptive, therapeutic, and vaccine approaches for fungal diseases of the lung.

Diplomarbeit Universität Innsbruck 2022

The long-term effects of agricultural management such as different fertilization strategies on soil microbiota and soil suppressiveness against plant pathogens are crucial. Therefore, the suppressiveness of soils differing in fertilization history was assessed using two Rhizoctonia solani isolates and their respective host plants (lettuce, sugar beet) in pot experiments. Further, the effects of fertilization history and the pathogen R. solani AG1-IB on the bulk soil, root-associated soil and rhizosphere microbiota of lettuce were analyzed based on amplicon sequencing of the 16S rRNA gene and ITS2 region. Organic fertilization history supported the spread of the soil-borne pathogens compared to long-term mineral fertilization. The fertilization strategy affected bacterial and fungal community composition in the root-associated soil and rhizosphere, respectively, but only the fungal community shifted in response to the inoculated pathogen. The potential plant-beneficial genus Talaromyces was enriched in the rhizosphere by organic fertilization and presence of the pathogen. Moreover, increased expression levels of defense-related genes in shoots of lettuce were observed in the soil with organic fertilization history, both in the absence and presence of the pathogen. This may reflect the enrichment of potential plant-beneficial microorganisms in the rhizosphere, but also pathogen infestation. However, enhanced defense responses resulted in retarded plant growth in the presence of R. solani (plant growth/defense tradeoff).

Diplomarbeit Universität Innsbruck 2022

Fungal quinones can be used for a variety of applications, such as pharmaceuticals, food colorants, textile dyes, and battery electrolytes. However, when producing quinones by fungal cultivation, many considerations arise regarding the feasibility of a production system, such as the quinone yield, purity, ease of extraction, and the co-production of mycotoxins. In this work, we display the initial screening of filamentous fungi for quinone production and evaluate their potential for future optimization. We investigated toluquinone (TQ) potentially produced by Penicillium cf. griseofulvum, terreic acid (TA) produced by Aspergillus parvulus and A. christenseniae, and anthraquinone (AQ) monomers and dimers produced by Talaromyces islandicus. The strains grew on various agar and/or liquid media and were analyzed by ultra-high-performance liquid chromatography–diode array detection–quadrupole time-of-flight mass spectrometry (UHPLC-DAD-QTOF MS). In the case of AQs, feature-based molecular networking (FBMN) was used for the identification of AQ analogs. TQ was not observed in the production strains. TA constituted one of the major chromatogram peaks and was secreted into the growth medium by A. parvulus. The AQs constituted many major chromatogram peaks in the mycelium extracts and endocrocin and citreorosein were observed extracellularly in small amounts.

The long-term effects of agricultural management such as different fertilization strategies on soil microbiota and soil suppressiveness against plant pathogens are crucial. Therefore, the suppressiveness of soils differing in fertilization history was assessed using two Rhizoctonia solani isolates and their respective host plants (lettuce, sugar beet) in pot experiments. Further, the effects of fertilization history and the pathogen R. solani AG1-IB on the bulk soil, root-associated soil and rhizosphere microbiota of lettuce were analyzed based on amplicon sequencing of the 16S rRNA gene and ITS2 region. Organic fertilization history supported the spread of the soil-borne pathogens compared to long-term mineral fertilization. The fertilization strategy affected bacterial and fungal community composition in the root-associated soil and rhizosphere, respectively, but only the fungal community shifted in response to the inoculated pathogen. The potential plant-beneficial genus Talaromyces was enriched in the rhizosphere by organic fertilization and presence of the pathogen. Moreover, increased expression levels of defense-related genes in shoots of lettuce were observed in the soil with organic fertilization history, both in the absence and presence of the pathogen. This may reflect the enrichment of potential plant-beneficial microorganisms in the rhizosphere, but also pathogen infestation. However, enhanced defense responses resulted in retarded plant growth in the presence of R. solani (plant growth/defense tradeoff).

Aspergillus section Candidi encompasses white- or yellow-sporulating species mostly isolated from indoor and cave environments, food, feed, clinical material, soil and dung. Their identification is non-trivial due to largely uniform morphology. This study aims to re-evaluate the species boundaries in the section Candidi and present an overview of all existing species along with information on their ecology. For the analyses, we assembled a set of 113 strains with diverse origin. For the molecular analyses, we used DNA sequences of three house-keeping genes (benA, CaM and RPB2) and employed species delimitation methods based on a multispecies coalescent model. Classical phylogenetic methods and genealogical concordance phylogenetic species recognition (GCPSR) approaches were used for comparison. Phenotypic studies involved comparisons of macromorphology on four cultivation media, seven micromorphological characters and growth at temperatures ranging from 10 to 45 degrees C. Based on the integrative approach comprising four criteria (phylogenetic and phenotypic), all currently accepted species gained support, while two new species are proposed (A. magnus and A. tenebricus). In addition, we proposed the new name A. neotritici to replace an invalidly described A. tritici. The revised section Candidi now encompasses nine species, some of which manifest a high level of intraspecific genetic and/or phenotypic variability (e.g., A. subalbidus and A. campestris) while others are more uniform (e.g., A. candidus or A. pragensis). The growth rates on different media and at different temperatures, colony colours, production of soluble pigments, stipe dimensions and vesicle diameters contributed the most to the phenotypic species differentiation.

Penicillium species are common fungi found worldwide from diverse substrates, including soil, plant debris, food products and air. Their diversity in aquatic environments is still underexplored. With the aim to explore the fungal diversity in Spanish freshwater sediments, numerous Penicillium strains were isolated using various culture-dependent techniques. A preliminary sequence analysis of the beta-tubulin (tub2) gene marker allowed us to identify several interesting species of Penicillium, which were later characterized phylogenetically with the barcodes recommended for species delimitation in the genus. Based on the multi-locus phylogeny of the internal transcribed spacer region (ITS) of the ribosomal DNA, and partial fragments of tub2, calmodulin (cmdA), and the RNA polymerase II largest subunit (rpb2) genes, in combination with phenotypic analyses, five novel species are described. These are P. ausonanum in section Lanata-Divaricata, P. guarroi in sect. Gracilenta, P. irregulare in sect. Canescentia, P. sicoris in sect. Paradoxa and P. submersum in sect. Robsamsonia. The study of several isolates from samples collected in different locations resulted in the reinstatement of P. vaccaeorum into section Citrina. Finally, P. heteromorphum (sect. Exilicaulis) and P. tardochrysogenum (sect. Chrysogena) are reported, previously only known from Antarctica and China, respectively.

Aspergilus series Versicolores members occur in a wide range of environments and substrates such as indoor environments, food, clinical materials, soil, caves, marine or hypersaline ecosystems. The taxonomy of the series has undergone numerous re-arrangements including a drastic reduction in the number of species and subsequent recovery to 17 species in the last decade. The identification to species level is however problematic or impossible in some isolates even using DNA sequencing or MALDI-TOF mass spectrometry indicating a problem in the definition of species boundaries. To revise the species limits, we assembled a large dataset of 518 strains. From these, a total of 213 strains were selected for the final analysis according to their calmodulin (CaM) genotype, substrate and geography. This set was used for phylogenetic analysis based on five loci (benA, CaM, RPB2, Mcm7, Tsr1). Apart from the classical phylogenetic methods, we used multispecies coalescence (MSC) model-based methods, including one multilocus method (STACEY) and five single-locus methods (GMYC, bGMYC, PTP, bPTP, ABGD). Almost all species delimitation methods suggested a broad species concept with only four species consistently supported. We also demonstrated that the currently applied concept of species is not sustainable as there are incongruences between single-gene phylogenies resulting in different species identifications when using different gene regions. Morphological and physiological data showed overall lack of good, taxonomically informative characters, which could be used for identification of such a large number of existing species. The characters expressed either low variability across species or significant intraspecific variability exceeding interspecific variability. Based on the above-m

Quinones represent an important group of highly structurally diverse, mainly polyketide-derived secondary metabolites widely distributed among filamentous fungi. Many quinones have been reported to have important biological functions such as inhibition of bacteria or repression of the immune response in insects. Other quinones, such as ubiquinones are known to be essential molecules in cellular respiration, and many quinones are known to protect their producing organisms from exposure to sunlight. Most recently, quinones have also attracted a lot of industrial interest since their electron-donating and -accepting properties make them good candidates as electrolytes in redox flow batteries, like their often highly conjugated double bond systems make them attractive as pigments. On an industrial level, quinones are mainly synthesized from raw components in coal tar. However, the possibility of producing quinones by fungal cultivation has great prospects since fungi can often be grown in industrially scaled bioreactors, producing valuable metabolites on cheap substrates. In order to give a better overview of the secondary metabolite quinones produced by and shared between various fungi, mainly belonging to the genera Aspergillus, Penicillium, Talaromyces, Fusarium, and Arthrinium, this review categorizes quinones into families such as emodins, fumigatins, sorbicillinoids, yanuthones, and xanthomegnins, depending on structural similarities and information about the biosynthetic pathway from which they are derived, whenever applicable. The production of these quinone families is compared between the different genera, based on recently revised taxonomy.

Ascospores of Talaromyces.macrosporus belong to the most stress resistant eukaryotic cells and show a constitutive dormancy, i.e., no germination occurs in the presence of rich growth me-dium. Only an extreme trigger as very high temperature or pressure is able to evoke synchronized germination. In this study, several changes within the thick cell wall of these cells are observed after a heat treatment: (i.) a change in its structure as shown with EPR and X-ray diffraction; (ii.) a release of an abundant protein into the supernatant, which is proportional to the extent of heat activation; (iii.) a change in the permeability of the cell wall as judged by fluorescence studies in which staining of the interior of the cell wall correlates with germination of individual ascospores. The gene encod-ing the protein, dubbed ICARUS, was studied in detail and was expressed under growth conditions that showed intense ascomata (fruit body) and ascospore formation. It encodes a small 7–14 kD protein. Blast search exhibits that different Talaromyces species show a similar sequence, indicating that the protein also occurs in other species of the genus. Deletion strains show delayed ascomata formation, release of pigments into the growth medium, higher permeability of the cell wall and a markedly shorter heat activation needed for activation. Further, wild type ascospores are more heat-resistant. All these observations suggest that the protein plays a role in dormancy and is related to the structure and permeability of the ascospore cell wall. However, more research on this topic is needed to study constitutive dormancy in other fungal species that form stress-resistant ascospores.

Since the last revision in 2015, the taxonomy of section Flavipedes evolved rapidly along with the availability of new species delimitation techniques. This study aims to re-evaluate the species boundaries of section Flavipedes members using modern delimitation methods applied to an extended set of strains (n = 90) collected from various environments. The analysis used DNA sequences of three house-keeping genes (benA, CaM, RPB2) and consisted of two steps: application of several single-locus (GMYC, bGMYC, PTP, bPTP) and multi-locus (STACEY) species delimitation methods to sort the isolates into putative species, which were subsequently validated using DELINEATE software that was applied for the first time in fungal taxonomy. As a result, four new species are introduced, i.e. A. alboluteus, A. alboviridis, A. inusitatus and A. lanuginosus, and A. capensis is synonymized with A. iizukae. Phenotypic analyses were performed for the new species and their relatives, and the results showed that the growth parameters at different temperatures and colonies characteristics were useful for differentiation of these taxa. The revised section harbors 18 species, most of them are known from soil. However, the most common species from the section are ecologically diverse, occurring in the indoor environment (six species), clinical samples (five species), food and feed (four species), droppings (four species) and other less common substrates/environments. Due to the occurrence of section Flavipedes species in the clinical material/hospital environment, we also evaluated the susceptibility of 67 strains to six antifungals (amphotericin B, itraconazole, posaconazole, voriconazole, isavuconazole, terbinafine) using the reference EUCAST method. These results showed some potentially clinical

The identity of the fungi responsible for fruitlet core rot (FCR) disease in pineapple has been the subject of investigation for some time. This study describes the diversity and toxigenic potential of fungal species causing FCR in La Reunion, an island in the Indian Ocean. One-hundred-and-fifty fungal isolates were obtained from infected and healthy fruitlets on Reunion Island and exclusively correspond to two genera of fungi: Fusarium and Talaromyces. The genus Fusarium made up 79% of the isolates, including 108 F. ananatum, 10 F. oxysporum, and one F. proliferatum. The genus Talaromyces accounted for 21% of the isolated fungi, which were all Talaromyces stollii. As the isolated fungal strains are potentially mycotoxigenic, identification and quantification of mycotoxins were carried out on naturally or artificially infected diseased fruits and under in vitro cultures of potential toxigenic isolates. Fumonisins B1 and B2 (FB1-FB2) and beauvericin (BEA) were found in infected fruitlets of pineapple and in the culture media of Fusarium species. Regarding the induction of mycotoxin in vitro, F. proliferatum produced 182 mg kg⁻1 of FB1 and F. oxysporum produced 192 mg kg⁻1 of BEA. These results provide a better understanding of the causal agents of FCR and their potential risk to pineapple consumers.

The identity of the fungi responsible for fruitlet core rot (FCR) disease in pineapple has been the subject of investigation for some time. This study describes the diversity and toxigenic potential of fungal species causing FCR in La Reunion, an island in the Indian Ocean. One-hundred-and-fifty fungal isolates were obtained from infected and healthy fruitlets on Reunion Island and exclusively correspond to two genera of fungi: Fusarium and Talaromyces. The genus Fusarium made up 79% of the isolates, including 108 F. ananatum, 10 F. oxysporum, and one F. proliferatum. The genus Talaromyces accounted for 21% of the isolated fungi, which were all Talaromyces stollii. As the isolated fungal strains are potentially mycotoxigenic, identification and quantification of mycotoxins were carried out on naturally or artificially infected diseased fruits and under in vitro cultures of potential toxigenic isolates. Fumonisins B1 and B2 (FB1-FB2) and beauvericin (BEA) were found in infected fruitlets of pineapple and in the culture media of Fusarium species. Regarding the induction of mycotoxin in vitro, F. proliferatum produced 182 mg kg⁻1 of FB1 and F. oxysporum produced 192 mg kg⁻1 of BEA. These results provide a better understanding of the causal agents of FCR and their potential risk to pineapple consumers.

A novel fungal species able to synthesize enzymes with potential synergistic actions in lignocellulose conversion was isolated from the biomass of Arundo donax during biodegradation under natural conditions in the Gussone Park of the Royal Palace of Portici (Naples, Italy). In this work, this species was subjected to morphological and phylogenetic analyses. Sequencing of its genome was performed, resulting in 28 scaffolds that were assembled into 27.05 Mb containing 9744 predicted genes, among which 396 belong to carbohydrate-active enzyme (CAZyme)-encoding genes. Here we describe and illustrate this previously unknown species, which was named Talaromyces borbonicus, by a polyphasic approach combining phenotypic, physiological, and sequence data.

A novel fungal species able to synthesize enzymes with potential synergistic actions in lignocellulose conversion was isolated from the biomass of Arundo donax during biodegradation under natural conditions in the Gussone Park of the Royal Palace of Portici (Naples, Italy). In this work, this species was subjected to morphological and phylogenetic analyses. Sequencing of its genome was performed, resulting in 28 scaffolds that were assembled into 27.05 Mb containing 9744 predicted genes, among which 396 belong to carbohydrate-active enzyme (CAZyme)-encoding genes. Here we describe and illustrate this previously unknown species, which was named Talaromyces borbonicus, by a polyphasic approach combining phenotypic, physiological, and sequence data.

Duclauxins are dimeric and heptacyclic fungal polyketides with notable bioactivities. We characterized the cascade of redox transformations in the biosynthetic pathway of duclauxin from Talaromyces stipitatus. The redox reaction sequence is initiated by a cupin family dioxygenase DuxM that performs an oxidative cleavage of the peri-fused tricyclic phenalenone and affords a transient hemiketal-oxaphenalenone intermediate. Additional redox enzymes then morph the oxaphenoalenone into either an anhydride or a dihydrocoumarin-containing monomeric building block that is found in dimeric duxlauxins. Oxidative coupling between the monomers to form the initial C-C bond was shown to be catalyzed by a P450 monooxygenase, although the enzyme responsible for the second C-C bond formation was not found in the pathway. Collectively, the number and variety of redox enzymes used in the duclauxin pathway showcase Nature's strategy to generate structural complexity during natural product biosynthesis.

Infections with geographically constrained dimorphic fungi cause the endemic mycoses, which include blastomycosis, coccidioidomycosis, emmonsiosis, histoplasmosis, paracoccidioidomycosis, sporotrichosis, and penicilliosis. In the last 5 years, our understanding of the epidemiology, diagnostics, and to a lesser extent management of these diseases has advanced. Specifically, the application of molecular techniques for genotyping fungal pathogens has resulted in the recognition of cryptic species within several genera, including Blastomyces, and Paracoccidioides; the reclassification of Penicillium marneffei, the agent of penicilliosis, to the genus Talaromyces; and the global emergence of dimorphic fungi of the genus Emmonsia, cause disease in immunocompromised persons. New and refined diagnostic tests are available based on the detection of circulating antigens and antibodies, mass spectrometry, and targeted gene amplification. In contrast, the development of new therapeutic options remains stalled, although isavuconazole may hold promise. Finally, advances have been made in the prospect of viable vaccines for preventing animal and human disease.

Ascospores of Neosartorya, Byssochlamys and Talaromyces can be regarded as the most stress-resistant eukaryotic cells. They can survive exposure at temperatures as high as 85°C for 100 min or more. Neosartorya fischeri ascospores are more viscous and more resistant to the combined stress of heat and desiccation than the ascospores of Talaromyces macrosporus which contain predominantly trehalose. These ascospores contain trehalose-based oligosaccharides (TOS) that are novel compatible solutes, which are accumulated to high levels. These compounds are also found in other members of the genus Neosartorya and in some other genera within the order Eurotiales that also include Byssochlamys and Talaromyces. The presence of oligosaccharides was observed in species that had a relatively high growth temperature. TOS glasses have a higher glass transition temperature (Tg ) than trehalose, and they form a stable glass with crystallizing molecules, such as mannitol. Our data indicate that TOS are important for prolonged stabilization of cells against stress. The possible unique role of these solutes in protection against dry heat conditions is discussed.

UHPLC-DAD-HRMS based dereplication guided the detection of new halogenated alkaloids co-produced by Talaromyces wortmannii. From the fungal growth in large scale, the epimers 2,8-dichlororugulovasines A and B were purified and further identified by means of a HPLC-SPE/NMR hyphenated system. Brominated rugulovasines were also detected when the microbial incubation medium was supplemented with bromine sources. Studies from 1D/2D NMR and HRMS spectroscopy data allowed the structural elucidation of the dichlorinated compounds, while tandem MS/HRMS data analysis supported the rationalization of brominated congeners. Preliminary genetic studies revealed evidence that FADH2 dependent halogenase can be involved in the biosynthesis of the produced halocompounds.

Aspergillus, Penicillium, and Talaromyces are among the most chemically inventive of all fungi, producing a wide array of secondary metabolites (exometabolites). The three genera are holophyletic in a cladistic sense and polythetic classes in an anagenetic or functional sense, and contain 344, 354, and 88 species, respectively. New developments in classification, cladification, and nomenclature have meant that the species, series, and sections suggested are natural groups that share many extrolites, including exometabolites, exoproteins, exocarbohydrates, and exolipids in addition to morphological features. The number of exometabolites reported from these species is very large, and genome sequencing projects have shown that a large number of additional exometabolites may be expressed, given the right conditions ("cryptic" gene clusters for exometabolites). The exometabolites are biosynthesized via shikimic acid, tricarboxylic acid cycle members, nucleotides, carbohydrates or as polyketides, non-ribosomal peptides, terpenes, or mixtures of those. The gene clusters coding for these compounds contain genes for the biosynthetic building blocks, the linking of these building blocks, tailoring enzymes, resistance for own products, and exporters. Species within a series or section in Aspergillus, Penicillium, and Talaromyces have many exometabolites in common, seemingly acquired by cladogenesis, but some the gene clusters for autapomorphic exometabolites may have been acquired by horizontal gene transfer. Despite genome sequencing efforts, and the many breakthroughs these will give, it is obvious that epigenetic factors play a large role in evolution and function of chemodiversity, and better methods for characterizing the epigenome are needed. Most of the individual species of the three genera produce a consistent and characteristic profile of exometabolites, but growth medium variations, stimulation by exometabolites from other species, and variations in abiotic intrinsi

Ascospores of the fungus Talaromyces macrosporus are dormant and extremely stress resistant, whereas fungal conidia--the main airborne vehicles of distribution--are not. Here, physical parameters of the cytoplasm of these types of spores were compared. Cytoplasmic viscosity and level of anisotropy as judged by spin probe studies (electron spin resonance) were extremely high in dormant ascospores and during early germination and decreased only partly after trehalose degradation and glucose efflux. Upon prosilition (ejection of the spore), these parameters fell sharply to values characteristic of vegetative cells. These changes occurred without major volume changes that suggest dramatic changes in cytoplasmic organization. Azide reversibly inhibited prosilition as well as the decline in cytoplasmic parameters. No organelle structures were observed in etched, cryoplaned specimens of ascospores by low-temperature scanning electron microscopy (LTSEM), confirming the high cytoplasmic viscosity. However, cell structures became visible upon prosilition, indicating reduced viscosity. The viscosity of fresh conidia of different Penicillium species was lower, namely, 3.5 to 4.8 cP, than that of ascospores, near 15 cP. In addition the level of anisotropic motion was markedly lower in these cells (h(0)/h(+1) = 1.16 versus 1.4). This was confirmed by LTSEM images showing cell structures. The decline of cytoplasmic viscosity in conidia during germination was linked with a gradual increase in cell volume. These data show that mechanisms of cytoplasm conservation during germination differ markedly between ascospores and conidia.

Ascospores of the fungus Talaromyces macrosporus are dormant and extremely stress resistant, whereas fungal conidia--the main airborne vehicles of distribution--are not. Here, physical parameters of the cytoplasm of these types of spores were compared. Cytoplasmic viscosity and level of anisotropy as judged by spin probe studies (electron spin resonance) were extremely high in dormant ascospores and during early germination and decreased only partly after trehalose degradation and glucose efflux. Upon prosilition (ejection of the spore), these parameters fell sharply to values characteristic of vegetative cells. These changes occurred without major volume changes that suggest dramatic changes in cytoplasmic organization. Azide reversibly inhibited prosilition as well as the decline in cytoplasmic parameters. No organelle structures were observed in etched, cryoplaned specimens of ascospores by low-temperature scanning electron microscopy (LTSEM), confirming the high cytoplasmic viscosity. However, cell structures became visible upon prosilition, indicating reduced viscosity. The viscosity of fresh conidia of different Penicillium species was lower, namely, 3.5 to 4.8 cP, than that of ascospores, near 15 cP. In addition the level of anisotropic motion was markedly lower in these cells (h(0)/h(+1) = 1.16 versus 1.4). This was confirmed by LTSEM images showing cell structures. The decline of cytoplasmic viscosity in conidia during germination was linked with a gradual increase in cell volume. These data show that mechanisms of cytoplasm conservation during germination differ markedly between ascospores and conidia.

AIMS: Ascospores of Talaromyces macrosporus are constitutively dormant and germinate after a strong external shock, classically a heat treatment. This fungus is used as a model system to study heat resistance leading to food spoilage after pasteurization. This study evaluates the effect of high pressure on the germination behaviour of these spores.METHODS AND RESULTS: Ascospore containing bags were subjected to ultra high pressure and spores were plated out on agar surfaces. Untreated suspensions showed invariably very low germination. Increased germination of ascospores occurred after short treatments at very high pressure (between 400 and 800 MPa). Activation is partial compared with heat activation and did not exceed 6.9% (65 times that of untreated suspensions) of the spore population. Maximum activation was attained shortly (10 s-3 min) after the pressure was applied and accompanied by cell wall deformations as judged by scanning electron microscopy. The spores observed in this study were harvested from cultures that were 39-58 days old. The maturity of spores at similar developmental stages was measured by assessing the heat resistance of ascospores. Between 20 and 40 days heat resistance increased 2.4-fold, but only an additional increase of 1.3-fold was observed at later stages (40-67 days).CONCLUSIONS: Our investigations show that high pressure constitutes a second type of shock that can activate heat-resistant ascospores to germinate. Activation is maximal after very short treatments and accompanied with changes in the cell wall structure. High-pressure activation is not the result of immaturity of the ascospores.SIGNIFICANCE AND IMPACT OF THE STUDY: These observations are relevant for the application of high pressure as a novel pasteurization method.

AIMS: Ascospores of Talaromyces macrosporus are constitutively dormant and germinate after a strong external shock, classically a heat treatment. This fungus is used as a model system to study heat resistance leading to food spoilage after pasteurization. This study evaluates the effect of high pressure on the germination behaviour of these spores.METHODS AND RESULTS: Ascospore containing bags were subjected to ultra high pressure and spores were plated out on agar surfaces. Untreated suspensions showed invariably very low germination. Increased germination of ascospores occurred after short treatments at very high pressure (between 400 and 800 MPa). Activation is partial compared with heat activation and did not exceed 6.9% (65 times that of untreated suspensions) of the spore population. Maximum activation was attained shortly (10 s-3 min) after the pressure was applied and accompanied by cell wall deformations as judged by scanning electron microscopy. The spores observed in this study were harvested from cultures that were 39-58 days old. The maturity of spores at similar developmental stages was measured by assessing the heat resistance of ascospores. Between 20 and 40 days heat resistance increased 2.4-fold, but only an additional increase of 1.3-fold was observed at later stages (40-67 days).CONCLUSIONS: Our investigations show that high pressure constitutes a second type of shock that can activate heat-resistant ascospores to germinate. Activation is maximal after very short treatments and accompanied with changes in the cell wall structure. High-pressure activation is not the result of immaturity of the ascospores.SIGNIFICANCE AND IMPACT OF THE STUDY: These observations are relevant for the application of high pressure as a novel pasteurization method.

Talaromyces macrosporus forms ascospores that survive pasteurization treatments. Ascospores were dense (1.3 g ml(-1)), relatively dry [0.6 g H(2)O (g dry weight)(-1)] and packed with trehalose (9-17% fresh weight). Trehalose was degraded to glucose monomers between 30 and 100 min after heat activation of the spores. The maximal activity of trehalase was calculated as 400-520 nmol glucose formed min(-1) (mg protein)(-1) as judged by measurements of the trehalose content of spores during germination. During early germination, glucose was released from the cell (10% of the cell weight or more). The intracellular concentration of glucose only peaked briefly. After 160-200 min, the protoplast encompassed by the inner cell wall was ejected through the outer cell wall in a very quick process. Subsequently, respiration of spores increased strongly. The data suggested that trehalose is primarily present for the protection of cell components as glucose is released from the cell. Then, an impenetrable outer cell wall is shed before metabolic activity increases.

Talaromyces macrosporus forms ascospores that survive pasteurization treatments. Ascospores were dense (1.3 g ml(-1)), relatively dry [0.6 g H(2)O (g dry weight)(-1)] and packed with trehalose (9-17% fresh weight). Trehalose was degraded to glucose monomers between 30 and 100 min after heat activation of the spores. The maximal activity of trehalase was calculated as 400-520 nmol glucose formed min(-1) (mg protein)(-1) as judged by measurements of the trehalose content of spores during germination. During early germination, glucose was released from the cell (10% of the cell weight or more). The intracellular concentration of glucose only peaked briefly. After 160-200 min, the protoplast encompassed by the inner cell wall was ejected through the outer cell wall in a very quick process. Subsequently, respiration of spores increased strongly. The data suggested that trehalose is primarily present for the protection of cell components as glucose is released from the cell. Then, an impenetrable outer cell wall is shed before metabolic activity increases.

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