Potential for preventing spread of fungi in air-conditioning systems constructed using copper instead of aluminium

Aims:  As copper has been previously suggested as an antimicrobial surface, we tested the effectiveness of copper as an antifungal surface which could be used in air-conditioning systems as an alternative to aluminium.
Methods and Results:  Coupons of copper (C11000) and aluminium were inoculated with fungal isolates ( Aspergillus spp., Fusarium spp., Penicillium chrysogenum and Candida albicans ) for various time periods. Culture on potato dextrose agar and an in situ viability assay using the fluorochrome FUN-1 were used to determine whether spores had survived. The results showed increased die off of fungal isolates tested compared to aluminium. In addition, copper also prevented the germination of spores present, thereby reducing the risk of the release of spores.
Conclusions:  Copper offered an antifungal surface and prevented subsequent germination of spores present. FUN-1 demonstrated that fungal spores entered into a viable but not culturable (VBNC) state on copper indicating the importance of using such methods when assessing the effect of an antifungal as culture alone may give false results.
Significance and impact of study:  Copper offers a valuable alternative to aluminium which could be used in air-conditioning systems in buildings, particularly in hospital environments where patients are more susceptible to fungal infections.     

Fungal spore transport by omnivorous mycophagous slug in temperate forest

Slugs are important consumers of fungal fruiting bodies and expected to carry their spores. In this study, we examined whether slugs (Meghimatium fruhstorferi) can act as effective dispersers of spores of basidiomycetes. The microscopic observation confirmed the presence of basidiospores in feces of field‐collected slugs, and the DNA metabarcoding study revealed that Ascomycota and Basidiomycota were major fungal taxa found in the feces. In Basidiomycota, the dominant order was Agaricales followed by Trichosporonales and Hymenochaetales. The laboratory experiments using Tylopilus vinosobrunneus showed that slugs carried a large number of spores in their digestive tracts. It was also observed that Pleurotus, Armillaria, and Gymnopilus spores excreted by slugs had a higher germination capacity than control spores collected from spore prints. The field experiments showed that slugs traveled 10.3 m in 5 h at most by wandering on the ground, litter layers, wood debris, and tree trunks. These results suggest that slugs could carry spores of ectomycorrhizal, saprophytic, and wood‐decaying fungi to appropriate sites for these fungi to establish colonies.     

Natural folding of airborne fungal spores: a mechanism for dispersal and long-term survival?

Analysis of numerous air samples has indicated that dormant, viable fungal spores are highly present, which suggests that aerial dispersion is important for fungi. Whereas the majority of the spores may travel only very short distances, there is indication that a notable number of them cover much longer distances. Harmomegathy is a terminology coined by Wodehouse (1935) describing the natural folding of pollen to accommodate controlled and reversible water loss. Here, we discuss evidence that this concept may also apply to airborne fungal spores that face similar challenges and have to survive periods of drought and low temperatures while retaining viability to germinate after deposition upon a suitable moist substrate. In fact, (air)dried conidia, appear collapsed, survive for much longer times compared to spores in liquid, that deteriorate in time. This indicates that for some types of fungal spores, true dormancy is reached in the desiccated state. For these airborne spores this might be regarded as a pre-adaptation that supports long-distance transport of viable cells through air. We state that spores are naturally folded during transport in air if the humidity is low enough. We hypothesize that this is a pre-adaptation supporting release, dispersal and survival of airborne spores. Moreover, the smaller size of dry naturally-folded spores may also be relevant, e.g. for the opportunistic pathogenic fungus Aspergillus fumigatus reduced spore size supports deposition within the alveoli in the lung.     

Fungal Fragments as Indoor Air Biocontaminants

The aerosolization process of fungal propagules of three species (Aspergillus versicolor, Penicillium melinii, and Cladosporium cladosporioides) was studied by using a newly designed and constructed aerosolization chamber. We discovered that fungal fragments are aerosolized simultaneously with spores from contaminated agar and ceiling tile surfaces. Concentration measurements with an optical particle counter showed that the fragments are released in higher numbers (up to 320 times) than the spores. The release of fungal propagules varied depending on the fungal species, the air velocity above the contaminated surface, and the texture and vibration of the contaminated material. In contrast to spores, the release of fragments from smooth surfaces was not affected by air velocity, indicating a different release mechanism. Correlation analysis showed that the number of released fragments cannot be predicted on the basis of the number of spores. Enzyme-linked immunosorbent assays with monoclonal antibodies produced against Aspergillus and Penicillium fungal species showed that fragments and spores share common antigens, which not only confirmed the fungal origin of the fragments but also established their potential biological relevance. The considerable immunological reactivity, the high number, and the small particle size of the fungal fragments may contribute to human health effects that have been detected in buildings with mold problems but had no scientific explanation until now. This study suggests that future fungal spore investigations in buildings with mold problems should include the quantitation of fungal fragments.     

Spore dissemination by mycophagous adult drosophilids

Although most fungal spores are transported by wind, some remain on lamellae even after fungal fruiting bodies start to decay. This raises the question: are these remaining spores useless or instead transported by other means? In this study, we accordingly investigated whether adult dipteran insects transport fungal spores. Our examination revealed that fungal spores were present in intestines of most drosophilid adults but almost completely absent from those of other dipteran adults. At least some spores excreted by Drosophila angularis and D. brachynephros retained the capacity to germinate. Structural damage to spores that passed through the digestive tract of these two drosophilid species varied among fungal species, with a greater number of colorless spores generally damaged than colored ones. These results suggest that adult drosophilid flies, but not other dipterans, can transport fungal spores.     

Synthesis of ribonucleic acids during the germination of Rhizopus stolonifer sporangiospores

A number of ribonucleic acids initiated synthesis during the first 15 min of germination of Rhizopus stolonifer sporangiospores. They included ribosomal, 5S, and transfer RNA, and a heterogeneously-sedimenting fraction that composed about 5% of the total cellular RNA; this fraction was unmethylated, did not compete with ribosomal RNA for hybridization to DNA, and was bound to poly dT-cellulose in a manner characteristic of RNAs containing polyadenylate segments. The nearly simultaneous onset of synthesis of the various RNAs with germination contrasts with the sequential onset of RNA synthesis reported for other fungal spores.     

Lipid and elemental composition as indicators of the physiological state of sporangiospores in Mucor hiemalis cultures of different ages

The number of sporangiospores used as inocula may significantly affect the development of the morphogenetic programs in mucoraceous fungi, including manifestations of dimorphism. In order to assess the physiological state of sporangiospores differing in their viability and germination patterns, lipid composition of sporangiospores from 6-, 14-, and 20-day cultures of Mucor hiemalis F-1431 was determined and the element ratios in these spores (Ca/K and P/S) were measured using X-ray microanalysis. While the lipid composition of the “old” (20 days) and “young” (6 days) spores was generally similar, older spores contained no cerebrosides, had half the level of polar lipids and γ-linolenic acid, and also contained 6 times more monoacylglycerols and 2.5 times more phosphatidic acid. The ratio of esterified to free sterols (ES/FS) characterizing the sterol storage pool was lowest in the spores from 20-day cultures. X-ray microanalysis revealed the highest P/S ratio in 6-day spores and lowest ratio in 14-day ones. Mature 14-day spores had the highest Ca/K ratio, ten times exceeding that for the 20-day spores. Higher values of P/S and Ca/K ratios in young (ripening) spores than in old 20-day spores indicate their higher metabolic activity and correlate with their higher viability and mycelial type of germination. Together with the lipid characteristics, the Ca/K and P/S ratios are the parameters which may be used to develop the criteria for assessment of the physiological state of the cells, including viable fungal sporangiospores. This complex may be also used to assess the capacity of mucoraceous fungi for dimorphism, including the species (M. hiemalis) for which this capacity has not been demonstrated previously.     

Variation in Tolerance and Virulence in the Chestnut Blight Fungus-Hypovirus Interaction

Chestnut blight, caused by the fungus Cryphonectria parasitica, has been effectively controlled with double-stranded RNA hypoviruses in Europe for over 40 years. The marked reduction in the virulence of C. parasitica by hypoviruses is a phenomenon known as hypovirulence. This virus-fungus pathosystem has become a model system for the study of biological control of fungi with viruses. We studied variation in tolerance to hypoviruses in fungal hosts and variation in virulence among virus isolates from a local population in Italy. Tolerance is defined as the relative fitness of a fungal individual when infected with hypoviruses (compared to being uninfected); virulence is defined for each hypovirus as the reduction in fitness of fungal hosts relative to virus-free hosts. Six hypovirus-infected isolates of C. parasitica were sampled from the population, and each hypovirus was transferred into six hypovirus-free recipient isolates. The resulting 36 hypovirus-fungus combinations were used to estimate genetic variation in tolerance to hypoviruses, in hypovirus virulence, and in virus-fungus interactions. Four phenotypes were evaluated for each virus-fungus combination to estimate relative fitness: (i) sporulation, i.e., the number of asexual spores (conidia) produced; (ii) canker area on field-inoculated chestnut trees, (iii) vertical transmission of hypoviruses into conidia, and (iv) conidial germination. Two-way analysis of variance (ANOVA) revealed significant interactions (P < 0.001) between viruses and fungal isolates for sporulation and canker area but not for conidial germination or transmission. One-way ANOVA among hypoviruses (within each fungal isolate) and among fungal isolates (within each hypovirus) revealed significant genetic variation (P < 0.01) in hypovirus virulence and fungal tolerance within several fungal isolates, and hypoviruses, respectively. These interactions and the significant genetic variation in several fitness characters indicate the potential for future evolution of these characters. However, biological control is unlikely to break down due to evolution of tolerance to hypoviruses in the fungus because the magnitudes of tolerance and interactions were relatively small.     

Volatile and gaseous metabolites released by germinating seeds of lentil and maize cultivars with different susceptibilities to fusariosis and smut

The effect of volatile and gaseous metabolites released by germinating seeds of lentil cultivars more and less susceptible to fusariosis on the germination of spores ofMucor racemosus, Trichoderma viride, Verticillium dahliae andBotrytis cinerea was found to depend rather on the fungal genus than on the lentil cultivar. However, spores ofFusarium oxysporum reacted more sensitively during germination to the presence of exudates of both cultivars, when the more susceptible lentil displayed a stimulation, the less susceptible one an inhibition of spore germination. The greatest difference in the effect of exudates was observed in the more and less susceptible maize cultivars with respect to the germination of chlamydospores ofUstilago maydis, especially during the first hours of seed germination. Analysis of the exudates of germinating seeds showed the release of a greater amount of ethanol and methanol with acetaldehyde by the more susceptible cultivars of lentil and particularly maize.     

Direct investigation of viscosity of an atypical inner membrane of Bacillus spores: A molecular rotor/FLIM study

We utilize the fluorescent molecular rotor Bodipy-C₁₂ to investigate the viscoelastic properties of hydrophobic layers of bacterial spores Bacillus subtilis. The molecular rotor shows a marked increase in fluorescence lifetime, from 0.3 to 4 ns, upon viscosity increase from 1 to 1500 cP and can be incorporated into the hydrophobic layers within the spores from dormant state through to germination. We use fluorescence lifetime imaging microscopy to visualize the viscosity inside different compartments of the bacterial spore in order to investigate the inner membrane and relate its compaction to the extreme resistance observed during exposure of spores to toxic chemicals. We demonstrate that the bacterial spores possess an inner membrane that is characterized by a very high viscosity, exceeding 1000 cP, where the lipid bilayer is likely in a gel state. We also show that this membrane evolves during germination to reach a viscosity value close to that of a vegetative cell membrane, ca. 600 cP. The present study demonstrates quantitative imaging of the microscopic viscosity in hydrophobic layers of bacterial spores Bacillus subtilis and shows the potential for further investigation of spore membranes under environmental stress.     

Effects of nutrient medium composition and temperature on the germination of conidia and the entomopathogenic activity of the fungi Beauveria bassiana and Metarhizium anisopliae

The effects of nutrient medium composition and temperature on the germination of conidia of the fungi Beauveria bassiana (strain AlG) and Metarhizium anisopliae (strain M-99) and their entomopathogenic activity have been studied. It was demonstrated that the presence of carbohydrates alone was sufficient for the spores of M. anisopliae M-99 to germinate, whereas the germination of B. bassiana AlG spores was inhibited by carbohydrates. Addition of KJ, ZnSO₄, or KBr into the Czapek medium increased the entomopathogenic activity of B. bassiana. The optimum temperature for spore germination was 20–35°C in both fungal species.     

Soil burrowing Muscina angustifrons (Diptera: Muscidae) larvae excrete spores capable of forming mycorrhizae underground

Muscina angustifrons (Diptera: Muscidae) is a mycophagous species that exploits a variety of fungi, including ectomycorrhizal fungi. Larvae of this species have been shown to feed on sporocarps (including spores), and full-grown larvae leave sporocarps and pupate 0–6?cm below the soil surface. In this study, we examined whether M. angustifrons larvae are capable of transporting ectomycorrhizal fungal spores and enhancing ectomycorrhiza growth on host-plant roots. Full-grown larvae were found to move horizontally 10–20?cm from their feeding sites and burrow underground. These wandering larvae retained ectomycorrhizal fungal spores in their intestines, which were excreted following relocation to underground pupation sites. Excreted spores retained germination and infection capacities to form ectomycorrhiza on host-plant roots. In the infection experiments, ectomycorrhizal fungal spores applied in the vicinity of underground host-plant roots were more effective in forming ectomycorrhiza than those applied to the ground surface, suggesting that belowground transportation of spores by M. angustifrons larvae could enhance ectomycorrhizal formation. These results suggested that M. angustifrons larvae act as a short-distance spore transporter of ectomycorrhizal fungi.     

An antifungal compound produced by Bacillus subtilis YM 10-20 inhibits germination of Penicillium roqueforti conidiospores

AIMS: To identify and characterize an antifungal compound produced by Bacillus subtilis YM 10-20 which prevents spore germination of Penicillium roqueforti. METHODS AND RESULTS: The antifungal compound was isolated by acid precipitation with HCl. This compound inhibited fungal germination and growth. Identification by HPLC and mass spectrometry analysis showed high similarity to iturin A. Permeabilization and morphological changes in P. roqueforti conidia in the presence of the inhibitor were revealed by fluorescence staining and SEM, respectively. CONCLUSOINS: The iturin-like compound produced by B. subtilis YM 10-20 permeabilizes fungal spores and blocks germination. SIGNIFICANCE AND IMPACT OF THE STUDY: Fluorescence staining in combination with flow cytometry and scanning electron microscopy are efficient tools for assessing the action of antifungal compounds against spores. Iturin-like compounds may permeabilize fungal spores and inhibit their germination.     

The impact of urban and forest fires on the airborne fungal spore aerobiology

The occurrence of airborne fungal spores is driven by several biogeographic and climatic factors. In addition, the occurrence of fires near fungal ecosystems seems to play an important role on the aerobiology of fungal spores. Wildfires are prevalent throughout the world and particularly so in several South European countries with Mediterranean climate. The present survey aimed at analysing the impact of urban and forest fires on the airborne fungal spore content of Madeira Island. Data suggest that after a fire occurrence, the concentration of fungal spores tends to increase in the air, peaking on the 10th day after a fire event. It is likely that fire-induced convections promote release of fungal spores from their natural habitat and that the soil heating conditions could trigger the germination of fungi colonizing post-fire plant residues and soil. It is also possible that the registered low precipitation and wind intensity could help increase the concentration of fungal spores in the atmosphere during 1–2 weeks. These findings need further research, not only at a regional but also at a larger scale, in order to clarify the specific impact of fires on such biological particles and the possible consequences on public health.     

Viability testing and characterization of germination of fungal spores by automatic image analysis

Fungal spores are used in the laboratory for culture maintenance and at laboratory and other scales as inocula for fermentations. The spore swelling and germination processes constitute a major part of the lag phase, and the subsequent culture morphology and productivity can be greatly influenced by the initial concentration and condition of the spores. An image analysis method has been developed for assessing the viability and the germination characteristics of fungal spores in submerged cultures. Structural variations during germination, i.e., swelling, germ tube formation, and germ tube elongation, are measured in terms of distributions of spore volumes and of germ tube lengths and volumes. These measurements are fully automatic and give a very rapid assessment of spore viability. This image analysis method might be used as a tool in culture maintenance and for determining the quality of inocula for fungal fermentations. (c) 1993 John Wiley & Sons, Inc.     

Germination of Bacillus thuringiensis var. israelensis spores in the gut of Aedes larvae (Diptera: Culicidae)

In laboratory experiments, germination and growth of Bacillus thuringiensis israelensis in the gut of Aedes aegypti and A. vexans larvae (Culicidae: Diptera) was observed. The number of spores and vegetative cells in the gut of living larvae and in cadavers was estimated by plaing homogenized larvae on selective agar plates. The number of spores per gut increased in the first 40–140 min of exposure to a maximum, and decreased in the subsequent time, demonstrating spore germination in living larvae, moribunds, and in cadavers. Twenty-four hours after the death of the larvae, a minimal amount of spores, but an increased number of vegetative cells, was found in cadavers. In A. aegypti larvae, germination and growth of B. thuringiensis israelensis in the larval gut was photographically documented.     

Plate assay for determining the time of production of protease, cellulase, and pectinases by germinating fungal spores

A new method for detecting enzymes produced by fungal spores during germination is described here. With this method, the production of enzymes such as protease, cellulase, or pectinase can be correlated with the extent of spore germination. Germination is studied in vitro on agar-based media containing protein, cellulose, or pectin. The spores are immobilized on a permeable membrane mounted on the substrate-containing medium. At various times after inoculation the membrane-bound spores are removed and the medium is stained. The extent of germination is assessed by microscopic examination of the spores and the presence of active hydrolytic enzymes is revealed by the staining. The staining methods are sensitive; detection limits are 1 X 10(-3) unit of cellulase; 2 X 10(-4) unit of protease; 3 X 10(-3) unit of pectin lyase; 3.5 units of polygalacturonase; 2 X 10(-3) unit of pectin methyl esterase. The method has been demonstrated by studying the production of enzymes by germinating conidia of Botrytis cinerea. Cellulase and protease were present before any spores germinated. Pectin lyase was first observed when at least 80% of the spores had germinated. Pectin methyl esterase and polygalacturonase were not produced by the spores.     

Germination of spores of mycelial fungi in relation to exogenous dormancy

Comparative analysis of germination of asexual sporulation spores (conidia and sporangiospores) and of specific features of dormancy release was carried out for ascomycete mycelial fungi Aspergillus tamarii VKM F-64 and A. sydowii VKM F-441, as well as for zygomycete fungi Cunninghamella echinulata VKM F-663 and Umbelopsis ramanniana VKM F-582. The spores of these strains were shown to be in a state of exogenous dormancy and differed in lag phase duration and germination rate, which depended on the presence of nutrients in the medium. Only the strain C. echinulata VKM F-663 exhibited 100% spore germination, with the germination rate and lag phase duration not depending on the composition of the medium. While in A. tamarii strain VKM F-64, the total number of spores germinating on rich and poor media was also almost the same, in the absence of nutrients lag phase duration increased and the germination rate decreased. For strains U. ramanniana VKM F-582 and A. sydowii VKM F-441, the degree of spore germination in the absence of nutrients in the medium was considerably lower than on the rich medium, while the lag phase was longer. These data indicate that the spores of C. echinulata VKM F-663 are in the state of exogenous dormancy, which does not require for release any compounds except water. The spores of U. ramanniana strain VKM F-582 and of the Aspergillus strains exhibited another variant of exogenous dormancy, which required for release, apart from water, also the sources of carbon and nitrogen. Thus, the character of dormancy release may differ even within a single genus (Aspergillus).     

Protein Composition of Infectious Spores Reveals Novel Sexual Development and Germination Factors in Cryptococcus

Spores are an essential cell type required for long-term survival across diverse organisms in the tree of life and are a hallmark of fungal reproduction, persistence, and dispersal. Among human fungal pathogens, spores are presumed infectious particles, but relatively little is known about this robust cell type. Here we used the meningitis-causing fungus Cryptococcus neoformans to determine the roles of spore-resident proteins in spore biology. Using highly sensitive nanoscale liquid chromatography/mass spectrometry, we compared the proteomes of spores and vegetative cells (yeast) and identified eighteen proteins specifically enriched in spores. The genes encoding these proteins were deleted, and the resulting strains were evaluated for discernable phenotypes. We hypothesized that spore-enriched proteins would be preferentially involved in spore-specific processes such as dormancy, stress resistance, and germination. Surprisingly, however, the majority of the mutants harbored defects in sexual development, the process by which spores are formed. One mutant in the cohort was defective in the spore-specific process of germination, showing a delay specifically in the initiation of vegetative growth. Thus, by using this in-depth proteomics approach as a screening tool for cell type-specific proteins and combining it with molecular genetics, we successfully identified the first germination factor in C. neoformans. We also identified numerous proteins with previously unknown functions in both sexual development and spore composition. Our findings provide the first insights into the basic protein components of infectious spores and reveal unexpected molecular connections between infectious particle production and spore composition in a pathogenic eukaryote.     

Electrical Sensing Zone Particle Analyzer for Measuring Germination of Fungal Spores in the Presence of Other Particles

Microscopic, respirometric, and electronic sizing methods for measuring germination of fungal spores were compared. With the electronic sizing method, early stages of germination (i.e., spore swelling) were detected long before germ tube emergence or significant changes in respiratory rates were observed. This method, which is rapid, easy, sensitive, and reproducible, also permits measuring the germination of spores when similar-size particles are present in concentrations considerably in excess of the number of spores.