The pleiotropic phenotype of FlbA of Aspergillus niger is explained in part by the activity of seven of its downstream-regulated transcription factors.

Inactivation of flbA in Aspergillus niger results in thinner cell walls, increased cell lysis, abolished sporulation, and an increased secretome complexity. A total of 36 transcription factor (TF) genes are differentially expressed in ΔflbA. Here, seven of these genes (abaA, aslA, aslB, azf1, htfA, nosA, and srbA) were inactivated. Inactivation of each of these genes affected sporulation and, with the exception of abaA, cell wall integrity and protein secretion. The impact on secretion was strongest in the case of ΔaslA and ΔaslB that showed increased pepsin, cellulase, and amylase activity. Biomass was reduced of agar cultures of ΔabaA, ΔaslA, ΔnosA, and ΔsrbA, while biomass was higher in liquid shaken cultures of ΔaslA and ΔaslB. The ΔaslA and ΔhtfA strains showed increased resistance to H2O2, while ΔaslB was more sensitive to this reactive oxygen species. Together, inactivation of the seven TF genes impacted biomass formation, sporulation, protein secretion, and stress resistance, and thereby these genes explain at least part of the pleiotropic phenotype of ΔflbA of A. niger.

Compatible solutes determine the heat resistance of conidia.

BACKGROUND: Asexually developed fungal spores (conidia) are key for the massive proliferation and dispersal of filamentous fungi. Germination of conidia and subsequent formation of a mycelium network give rise to many societal problems related to human and animal fungal diseases, post-harvest food spoilage, loss of harvest caused by plant-pathogenic fungi and moulding of buildings. Conidia are highly stress resistant compared to the vegetative mycelium and therefore even more difficult to tackle. RESULTS: In this study, complementary approaches are used to show that accumulation of mannitol and trehalose as the main compatible solutes during spore maturation is a key factor for heat resistance of conidia. Compatible solute concentrations increase during conidia maturation, correlating with increased heat resistance of mature conidia. This maturation only occurs when conidia are attached to the conidiophore. Moreover, conidia of a mutant Aspergillus niger strain, constructed by deleting genes involved in mannitol and trehalose synthesis and consequently containing low concentrations of these compatible solutes, exhibit a sixteen orders of magnitude more sensitive heat shock phenotype compared to wild-type conidia. Cultivation at elevated temperature results in adaptation of conidia with increased heat resistance. Transcriptomic and proteomic analyses revealed two putative heat shock proteins to be upregulated under these conditions. However, conidia of knock-out strains lacking these putative heat shock proteins did not show a reduced heat resistance. CONCLUSIONS: Heat stress resistance of fungal conidia is mainly determined by the compatible solute composition established during conidia maturation. To prevent heat resistant fungal spore contaminants, food processing protocols should consider environmental conditions stimulating compatible solute accumulation and potentially use compatible solute biosynthesis as a novel food preservation target.

Endosymbionts: Bacterial hijacking of fungi?

Bacteria inside fungal hyphae allow the fungus Rhizopus microsporus to form spores and operate via effectors in 'stealth' mode. When the functionality of one effector is taken away, bacteria are captured in septated cells and die.

The C2H2 transcription factor SltA is required for germination and hyphal development in Aspergillus fumigatus.

Germination of inhaled Aspergillus fumigatus conidia is a necessary sequitur for infection. Germination of conidia starts with the breaking of dormancy, which is initiated by an increase of the cellular perimeter in a process termed isotropic growth. This swelling phase is followed by polarized growth, resulting in the formation of a germ tube. The multinucleate tubular cells exhibit tip growth from the hyphae, after which lateral branches emerge to form the mycelial network. The regulatory mechanisms governing conidial germination are not well defined. In this study, we identified a novel role for the transcription factor SltA in the orchestration of germination and hyphal development. Conidia lacking sltA fail to appropriately regulate isotropic growth and begin to swell earlier and subsequently switch to polarized growth faster. Additionally, hyphal development is distorted in a ∆sltA isolate as hyphae are hyper-branching and wider, and show branching at the apical tip. ∆sltA conidia are more tolerant to cell wall stressors on minimal medium compared to the wild-type (WT) strain. A transcriptome analysis of different stages of early growth was carried out to assess the regulatory role of SltA. Null mutants generated for three of the most dysregulated genes showed rapid germ tube emergence. Distinct from the phenotype observed for ∆sltA, conidia from these strains lacked defects in isotropic growth, but switched to polarized growth faster. Here, we characterize and describe several genes in the regulon of SltA, highlighting the complex nature of germination.IMPORTANCEAspergillus fumigatus is the main human fungal pathogen causing aspergillosis. For this fungus, azoles are the most commonly used antifungal drugs for treatment of aspergillosis. However, the prevalence of azole resistance is alarmingly increasing and linked with elevated mortality. Germination of conidia is crucial within its asexual life cycle and plays a critical role during the infection in the human host. Precluding germination could be a promising strategy considering the role of germination in Aspergillus spp. pathogenicity. Here, we identify a novel role for SltA in appropriate maintenance of dormancy, germination, and hyphal development. Three genes in the regulon of SltA were also essential for appropriate germination of conidia. With an expanding knowledge of germination and its different morphotypes, more advances can be made toward potential anti-germination targets for therapy.

Da Vinci's yeast: Blastobotrys davincii f.a., sp. nov.

A new species of the yeast genus Blastobotrys was discovered during a worldwide survey of culturable xerophilic fungi in house dust. Several culture-dependent and independent studies from around the world detected the same species from a wide range of substrates including indoor air, cave wall paintings, bats, mummies, and the iconic self-portrait of Leonardo da Vinci from ca 1512. However, none of these studies identified their strains, clones, or OTUs as Blastobotrys. We introduce the new species as Blastobotrys davincii f.a., sp. nov. (holotype CBS H-24879) and delineate it from other species using morphological, phylogenetic, and physiological characters. The new species of asexually (anamorphic) budding yeast is classified in Trichomonascaceae and forms a clade along with its associated sexual state genus Trichomonascus. Despite the decade-old requirement to use a single generic name for fungi, both names are still used. Selection of the preferred name awaits a formal nomenclatural proposal. We present arguments for adopting Blastobotrys over Trichomonascus and introduce four new combinations as Blastobotrys allociferrii (≡ Candida allociferrii), B. fungorum (≡ Sporothrix fungorum), B. mucifer (≡ Candida mucifera), and Blastobotrys vanleenenianus (≡ Trichomonascus vanleenenianus). We provide a nomenclatural review and an accepted species list for the 37 accepted species in the Blastobotrys/Trichomonascus clade. Finally, we discuss the identity of the DNA clones detected on the da Vinci portrait, and the importance of using appropriate media to isolate xerophilic or halophilic fungi.

Intraspecific variability in heat resistance of fungal conidia.

Microbial species are inherently variable, which is reflected in intraspecies genotypic and phenotypic differences. Strain-to-strain variation gives rise to variability in stress resistance and plays a crucial role in food safety and food quality. Here, strain variability in heat resistance of asexual spores (conidia) of the fungal species Aspergillus niger, Penicillium roqueforti and Paecilomyces variotii was quantified and compared to bacterial variability found in the literature. After heat treatment, a 5.4- to 8.6-fold difference in inactivation rate was found between individual strains within each species, while the strain variability of the three fungal species was not statistically different. We evaluated whether the degree of intraspecies variability is uniform, not only within the fungal kingdom, but also amongst different bacterial species. Comparison with three spore-forming bacteria and two non-spore-forming bacteria revealed that the variability of the different species was indeed in the same order of magnitude, which hints to a microbial signature of variation that exceeds kingdom boundaries.

Multiple Hybridization Events Punctuate the Evolutionary Trajectory of Malassezia furfur.

Malassezia species are important fungal skin commensals and are part of the normal microbiota of humans and other animals. However, under certain circumstances these fungi can also display a pathogenic behavior. For example, Malassezia furfur is a common commensal of human skin and yet is often responsible for skin disorders but also systemic infections. Comparative genomics analysis of M. furfur revealed that some isolates have a hybrid origin, similar to several other recently described hybrid fungal pathogens. Because hybrid species exhibit genomic plasticity that can impact phenotypes, we sought to elucidate the genomic evolution and phenotypic characteristics of M. furfur hybrids in comparison to their parental lineages. To this end, we performed a comparative genomics analysis between hybrid strains and their presumptive parental lineages and assessed phenotypic characteristics. Our results provide evidence that at least two distinct hybridization events occurred between the same parental lineages and that the parental strains may have originally been hybrids themselves. Analysis of the mating-type locus reveals that M. furfur has a pseudobipolar mating system and provides evidence that after sexual liaisons of mating compatible cells, hybridization involved cell-cell fusion leading to a diploid/aneuploid state. This study provides new insights into the evolutionary trajectory of M. furfur and contributes with valuable genomic resources for future pathogenicity studies. IMPORTANCEMalassezia furfur is a common commensal member of human/animal microbiota that is also associated with several pathogenic states. Recent studies report involvement of Malassezia species in Crohn's disease, a type of inflammatory bowel disease, pancreatic cancer progression, and exacerbation of cystic fibrosis. A recent genomics analysis of M. furfur revealed the existence of hybrid isolates and identified their putative parental lineages. In this study, we explored the genomic and phenotypic features of these hybrids in comparison to their putative parental lineages. Our results revealed the existence of a pseudobipolar mating system in this species and showed evidence for the occurrence of multiple hybridization events in the evolutionary trajectory of M. furfur. These findings significantly advance our understanding of the evolution of this commensal microbe and are relevant for future studies exploring the role of hybridization in the adaptation to new niches or environments, including the emergence of pathogenicity.

Abundant Small Protein ICARUS Inside the Cell Wall of Stress-Resistant Ascospores of Talaromyces macrosporus Suggests a Novel Mechanism of Constitutive Dormancy.

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 medium. 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 encoding 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.

Preservation stress resistance of melanin deficient conidia from Paecilomyces variotii and Penicillium roqueforti mutants generated via CRISPR/Cas9 genome editing.

BACKGROUND: The filamentous fungi Paecilomyces variotii and Penicillium roqueforti are prevalent food spoilers and are of interest as potential future cell factories. A functional CRISPR/Cas9 genome editing system would be beneficial for biotechnological advances as well as future (genetic) research in P. variotii and P. roqueforti. RESULTS: Here we describe the successful implementation of an efficient AMA1-based CRISPR/Cas9 genome editing system developed for Aspergillus niger in P. variotii and P. roqueforti in order to create melanin deficient strains. Additionally, kusA- mutant strains with a disrupted non-homologous end-joining repair mechanism were created to further optimize and facilitate efficient genome editing in these species. The effect of melanin on the resistance of conidia against the food preservation stressors heat and UV-C radiation was assessed by comparing wild-type and melanin deficient mutant conidia. CONCLUSIONS: Our findings show the successful use of CRISPR/Cas9 genome editing and its high efficiency in P. variotii and P. roqueforti in both wild-type strains as well as kusA- mutant background strains. Additionally, we observed that melanin deficient conidia of three food spoiling fungi were not altered in their heat resistance. However, melanin deficient conidia had increased sensitivity towards UV-C radiation.

Identifying Conserved Generic Aspergillus spp. Co-Expressed Gene Modules Associated with Germination Using Cross-Platform and Cross-Species Transcriptomics.

Aspergillus spp. is an opportunistic human pathogen that may cause a spectrum of pulmonary diseases. In order to establish infection, inhaled conidia must germinate, whereby they break dormancy, start to swell, and initiate a highly polarized growth process. To identify critical biological processes during germination, we performed a cross-platform, cross-species comparative analysis of germinating A. fumigatus and A. niger conidia using transcriptional data from published RNA-Seq and Affymetrix studies. A consensus co-expression network analysis identified four gene modules associated with stages of germination. These modules showed numerous shared biological processes between A. niger and A. fumigatus during conidial germination. Specifically, the turquoise module was enriched with secondary metabolism, the black module was highly enriched with protein synthesis, the darkgreen module was enriched with protein fate, and the blue module was highly enriched with polarized growth. More specifically, enriched functional categories identified in the blue module were vesicle formation, vesicular transport, tubulin dependent transport, actin-dependent transport, exocytosis, and endocytosis. Genes important for these biological processes showed similar expression patterns in A. fumigatus and A. niger, therefore, they could be potential antifungal targets. Through cross-platform, cross-species comparative analysis, we were able to identify biologically meaningful modules shared by A. fumigatus and A. niger, which underscores the potential of this approach.

Conidial heat resistance of various strains of the food spoilage fungus Paecilomyces variotii correlates with mean spore size, spore shape and size distribution.

Contamination by spores is often the cause of fungal food spoilage. Some distinct strains of the food spoilage fungus Paecilomyces variotii are able to produce airborne conidia that are more heat-resistant than similar species. These ellipsoid asexual spores can vary in size between strains, but also within strains. Here, we compared four measurement techniques to measure conidia size and distribution of five heat-sensitive and five heat-resistant P. variotii strains. Light microscopy (LM), Scanning Electron Microscopy (SEM) and Coulter Counter (CC) were used to measure and compare the spherical equivalent diameter, while CC and flow cytometry were used to study spore size distributions. The flow cytometry data was useful to study spore size distributions, but only relative spore sizes were obtained. There was no statistic difference between the method used of spore size measurement between LM, SEM and CC, but spore size was significantly different between strains with a 2.4-fold volume difference between the extremes. Various size distribution and shape parameters were correlated with conidial heat resistance. We found significant correlations in mean spore size, aspect ratio, roundness and skewness in relation to heat resistance, which suggests that these parameters are indicative for the conidial heat resistance of a P. variotii strain.

Impact of maturation and growth temperature on cell-size distribution, heat-resistance, compatible solute composition and transcription profiles of Penicillium roqueforti conidia.

Penicillium roqueforti is a major cause of fungal food spoilage. Its conidia are the main dispersal structures of this fungus and therefore the main cause of food contamination. These stress resistant asexual spores can be killed by preservation methods such as heat treatment. Here, the effects of cultivation time and temperature on thermal resistance of P. roqueforti conidia were studied. To this end, cultures were grown for 3, 5, 7 and 10 days at 25 °C or for 7 days at 15, 25 and 30 °C. Conidia of 3- and 10-day-old cultures that had been grown at 25 °C had D56-values of 1.99 ± 0.15 min and 5.31 ± 1.04 min, respectively. The effect of cultivation temperature was most pronounced between P. roqueforti conidia cultured for 7 days at 15 °C and 30 °C, where D56-values of 1.12 ± 0.05 min and 4.19 ± 0.11 min were found, respectively. Notably, D56-values were not higher when increasing both cultivation time and temperature by growing for 10 days at 30 °C. A correlation was found between heat resistance of conidia and levels of trehalose and arabitol, while this was not found for glycerol, mannitol and erythritol. RNA-sequencing showed that the expression profiles of conidia of 3- to 10-day-old cultures that had been grown at 25 °C were distinct from conidia that had been formed at 15 °C and 30 °C for 7 days. Only 33 genes were upregulated at both prolonged incubation time and increased growth temperature. Their encoded proteins as well as trehalose and arabitol may form the core of heat resistance of P. roqueforti conidia.

Fungal mycelium classified in different material families based on glycerol treatment.

Fungal mycelium is an emerging bio-based material. Here, mycelium films are produced from liquid shaken cultures that have a Young's modulus of 0.47 GPa, an ultimate tensile strength of 5.0 MPa and a strain at failure of 1.5%. Treating the mycelial films with 0-32% glycerol impacts the material properties. The largest effect is observed after treatment with 32% glycerol decreasing the Young's modulus and the ultimate tensile strength to 0.003 GPa and 1.8 MPa, respectively, whereas strain at failure increases to 29.6%. Moreover, glycerol treatment makes the surface of mycelium films hydrophilic and the hyphal matrix absorbing less water. Results show that mycelium films treated with 8% and 16-32% glycerol classify as polymer- and elastomer-like materials, respectively, while non-treated films and films treated with 1-4% glycerol classify as natural material. Thus, mycelium materials can cover a diversity of material families.

The Third International Symposium on Fungal Stress - ISFUS.

Stress is a normal part of life for fungi, which can survive in environments considered inhospitable or hostile for other organisms. Due to the ability of fungi to respond to, survive in, and transform the environment, even under severe stresses, many researchers are exploring the mechanisms that enable fungi to adapt to stress. The International Symposium on Fungal Stress (ISFUS) brings together leading scientists from around the world who research fungal stress. This article discusses presentations given at the third ISFUS, held in São José dos Campos, São Paulo, Brazil in 2019, thereby summarizing the state-of-the-art knowledge on fungal stress, a field that includes microbiology, agriculture, ecology, biotechnology, medicine, and astrobiology.

From colony to rodlet: "A six meter long portrait of the xerophilic fungus Aspergillus restrictus decorates the hall of the Westerdijk institute."

The extreme xerophilic fungus Aspergillus restrictus is used as a model for a large artwork created out of five microscopic pictures in total measuring 80 cm by 624 cm. The artwork is printed on aluminium and located at the entrance of the Westerdijk Institute, Utrecht, The Netherlands. The first picture is made from a colony of the fungus, which has a dimension of 1 cm and the last picture shows details of ornamentation on conidia and phialides of the fungus. The first two pictures of the artwork are made using a unique method of light microscopy in which many hundreds of pictures are made at different focal depths resulting in high detail and resolution of the pictures. For three other pictures, cryo-electron scanning microscopy was used including both a conventional system for lower magnification and a field emission scanning electron microscope for high resolution micrographs. The range of magnification is, at real size, between 78 and 63,000 times. When the observer passes the artwork it acts like a virtual microscope, just by walking past it you zoom-in to the smallest possible details. This coherent increase of magnification of one fungus, with very high quality light- and electron microscopy micrographs, shows different layers of fungal organization and emergent properties. These include the occurrence of secondary outcrops of hyphae and conidiophores in a colony; the formation of a stipe on a thin aerial hyphae; the presence and formation of characteristic structures on stipes, vesicles and phialides and a continuous zone between the forming conidia and phialides.

Comparing thermal inactivation to a combined process of moderate heat and high pressure: Effect on ascospores in strawberry puree.

High pressure processing is a mild preservation process that inactivates pathogenic and spoilage micro-organisms in food products, but preserves the fresh characteristics of a product. Compared to untreated product, an enhanced shelf life is obtained during refrigerated storage. Knowledge on the use of high pressure pasteurisation aimed for ambient storage is limited. The aim of this research was to investigate if a combination of high pressure and moderate heat could be used to produce a shelf-stable high-acid fruit product. Ascospores of the heat resistant fungi Talaromyces macrosporus and Aspergillus fischeri were added to fresh strawberry puree that served as a model system. The effect of the processing steps and storage at ambient temperature for 2 weeks was studied on viability of the ascospores. A preheating step at 69 °C/2 min resulted in full or partial activation of A. fischeri and T. macrosporus spores, respectively. The pressure build-up by the process without any holding time resulted in additional activation of spores. A combination of moderate heat (maximum 85-90 °C) and high pressure (500-700 MPa) for holding times up to 13 min inactivated these highly resistant spores much faster than a heat treatment alone. At Tmax = 85 °C and 600 MPa the spores of T. macrosporus and A. fischeri were inactivated by 5.0 and 5.5 log10 after 13 and 7 min, respectively. At Tmax = 85 °C the heat treatment alone did not reduce the viability of these spores up to 60 min of treatment. At Tmax = 90 °C the holding time of the combined pressure-heat treatment could be reduced to obtain the same degree of inactivation of the heat resistant fungi. In addition, treated and untreated ascospores in strawberry puree were stored for 14 days at room temperature to evaluate delayed outgrowth of spores. Untreated ascospores of A. fischeri were activated by storage in the puree. However, at conditions combining high pressure ≥ 600 MPa with Tmax ≥ 85 °C for 13 min, heat resistant fungi were successfully inactivated. This research showed that a combination of moderate heat and pressure can drastically improve the effectiveness to inactivate heat-resistant ascospores in a high-acid fruit product compared to a heat treatment, potentially resulting in a better product quality.

Cathelicidin-inspired antimicrobial peptides as novel antifungal compounds.

Fungal infections in humans are increasing worldwide and are currently mostly treated with a relative limited set of antifungals. Resistance to antifungals is increasing, for example, in Aspergillus fumigatus and Candida auris, and expected to increase for many medically relevant fungal species in the near future. We have developed and patented a set of cathelicidin-inspired antimicrobial peptides termed 'PepBiotics'. These peptides were initially selected for their bactericidal activity against clinically relevant Pseudomonas aeruginosa and Staphylococcus aureus isolates derived from patients with cystic fibrosis and are active against a wide range of bacteria (ESKAPE pathogens). We now report results from studies that were designed to investigate the antifungal activity of PepBiotics against a set of medically relevant species encompassing species of Aspergillus, Candida, Cryptococcus, Fusarium, Malassezia, and Talaromyces. We characterized a subset of PepBiotics and show that these peptides strongly affected metabolic activity and/or growth of a set of medically relevant fungal species, including azole-resistant A. fumigatus isolates. PepBiotics showed a strong inhibitory activity against a large variety of filamentous fungi and yeasts species at low concentrations (≤1 µM) and were fungicidal for at least a subset of these fungal species. Interestingly, the concentration of PepBiotics required to interfere with growth or metabolic activity varied between different fungal species or even between isolates of the same fungal species. This study shows that PepBiotics display strong potential for use as novel antifungal compounds to fight a large variety of clinically relevant fungal species.

The most heat-resistant conidia observed to date are formed by distinct strains of Paecilomyces variotii.

Fungi colonize habitats by means of spores. These cells are stress-resistant compared with growing fungal cells. Fungal conidia, asexual spores, formed by cosmopolitan fungal genera like Penicillium, Aspergillus and Peacilomyces are dispersed by air. They are present in places where food products are stored and as a result, they cause food spoilage. Here, we determined the heterogeneity of heat resistance of conidia between and within strains of Paecilomyces variotii, a spoiler of foods such as margarine, fruit juices, canned fruits and non-carbonized sodas. Out of 108 strains, 31 isolates showed a conidial survival >10% after a 10-min-heat treatment at 59°C. Three strains with different conidial heat resistance were selected for further phenotyping. Conidia of DTO 212-C5 and DTO 032-I3 showed 0.3% and 2.6% survival in the screening respectively, while survival of DTO 217-A2 conidia was >10%. The decimal reduction times of these strains at 60°C (D60 value) were 3.7 ± 0.08, 5.5 ± 0.35 and 22.9 ± 2.00 min respectively. Further in-depth analysis revealed that the three strains showed differences in morphology, spore size distributions, compatible solute compositions and growth under salt stress. Conidia of DTO 217-A2 are the most heat-resistant reported so far. The ecological consequences of this heterogeneity of resistance, including food spoilage, are discussed.

The preservative propionic acid differentially affects survival of conidia and germ tubes of feed spoilage fungi.

Propionic acid is widely used as a preservative in (poultry) feed. In this study we have isolated and identified fungal strains from nine samples poultry feed originating from different countries. The majority of the strains were Aspergilli with a eurotium-morph, such as Aspergillus proliferans and A. chevalieri. These and three other species were selected and tested for their sensitivity towards the feed preservative propionic acid, among them Penicillium lanosocoeruleum. The determined MIC values of 6.1-31 mM of these poultry feed specific fungi were well in the range as described in literature. Propionic acid (at 31 mM) damages conidia (spores) in a species dependent fashion after a 24-hour-treatment. The majority of the conidia (over 70%) of P. lanosocoeruleum germinated within 60 h on agar medium, while 50 and 80% of the A. chevalieri and A. proliferans conidia did not, respectively. Dependent on the species, cell damage was visible after incubation with propionic acid. Germ tubes of P. lanosocoeruleum in a biofilm showed extensive (85%) cell death after a 30 min treatment with propionic acid and slightly lower sensitivity was observed with A. proliferans (62% cell death). Microscopic analysis of these fungal biofilms revealed extensive damage to the cell membrane and showed distorted intracellular structures. Fluorescent life-dead staining of the germ tubes showed a clear dose response of propionic acid indicating a fungicidal effect on these growing cells. These results show that conidia can be inactivated by propionic acid, but that germ tubes show a much higher sensitivity. These observations shed new light on the mode of action of this important preservative to prevent fungal contamination of feed.

Fungal spores: Highly variable and stress-resistant vehicles for distribution and spoilage.

This review highlights the variability of fungal spores with respect to cell type, mode of formation and stress resistance. The function of spores is to disperse fungi to new areas and to get them through difficult periods. This also makes them important vehicles for food contamination. Formation of spores is a complex process that is regulated by the cooperation of different transcription factors. The discussion of the biology of spore formation, with the genus Aspergillus as an example, points to possible novel ways to eradicate fungal spore production in food. Fungi can produce different types of spores, sexual and asexually, within the same colony. The absence or presence of sexual spore formation has led to a dual nomenclature for fungi. Molecular techniques have led to a revision of this nomenclature. A number of fungal species form sexual spores, which are exceptionally stress-resistant and survive pasteurization and other treatments. A meta-analysis is provided of numerous D-values of heat-resistant ascospores generated during the years. The relevance of fungal spores for food microbiology has been discussed.