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Sources of variability in the measurement of fungal spore yields.

VARIABILITY IN THE PRODUCTION OF FUNGAL SPORES AND IN THE MEASUREMENT OF SPORE YIELDS WAS INVESTIGATED IN FOUR SPECIES OF FUNGI: Colletotrichum gloeosporioides, Colletotrichum coccodes, Colletotrichum phomoides, and Acremonium strictum. When the fungi were grown on solid medium in microplates and spore yields were measured by counting the subsamples with a hemacytometer, the variability among hemacytometer squares was always the largest source of variation, accounting for 51 to 91% of the total variation. Variability among replicate cultures and results of repeat experiments were generally also significant. The effect of square-to-square variability on the precision of spore yield measurement was minimized by counting a moderate number (ca. 30) of squares per culture. Culture-to-culture variability limited the practical precision of spore production measurements to a 95% confidence interval of approximately the mean +/- 25%. We provide guidelines for determining the number of replicate cultures required to attain this or other degrees of precision. Particle counter-derived spore counts and counts based on spore weights were much less variable than were hemacytometer counts, but they did not improve spore production estimates very much because of culture-to-culture variability. Results obtained by both of these methods differed from those obtained with a hemacytometer; particle counter measurements required a correction for spore pairs, while the relationship between spore weights and spore counts changed as the cultures aged.

Journal Article↗

Glucose-induced pathways for actin tyrosine dephosphorylation during Dictyostelium spore germination.

In the presence of germination signals, dormant spores of Dictyostelium discoideum rapidly germinate to start a new life cycle. Previously we have shown that half of the actin molecules in spores are maintained in a tyrosine-phosphorylated state, and a decline of the actin phosphorylation levels is a prerequisite for spore swelling. In this study, we have established d-glucose as a trigger molecule for the actin dephosphorylation. Present in a nutrient germination medium, d-glucose both may act as a trigger molecule and/or may serve as a substrate within a pathway for actin dephosphorylation depending upon spore age. However, the glucose-induced actin dephosphorylation was insufficient for spores to swell. Other factors in the nutrient medium were required for complete germination of young spores aged 1 to 5 days. In contrast, dispersion in nonnutrient buffer was necessary and sufficient for a decline of actin phosphorylation levels and even the emergence of amoebae in older spores (6 days and beyond). Moreover, the dephosphorylation pathway in the older spores was independent of energy production. We propose that the diversification of the actin dephosphorylation pathway may enable spores to increase their probability of germination upon spore aging.

Actins↗

The effect of volatile and gaseous metabolites of swelling seeds on germination of fungal spores.

Effects of volatile and gaseous metabolites of swelling seeds of pea, bean, wheat, corn cucumber, tomato, lentil, carrot, red papper and lettuce on germination of spores of five genera of fungi were found to depend rather on the fungal than on the plant genus. Germination of spores of Botrytis cinerea, Mucor racemosus and Trichoderma viride was most severely inhibited. Spores of Verticillium dahliae were less sensitive and germination of spores of Fusarium oxysporum was inhibited only in two cases. On the other hand, exudates of pea and bean stimulated germination of spores of Fusarium oxysporum. Also spores of Trichoderma viride germinated better in an atmosphere enriched with exuded metabolites of swelling lettuce seeds. When carbon dioxide produced by the swelling seeds was absorbed in potassium hydroxide, spores of Trichoderma viride and Verticillium dahliae did not germinate at all, the inhibitory effects of volatile and gaseous exudates on germination of spores of Mucor racemosus were accentuated, and also the percentage of germinated spores of Fusarium oxysporum decreased. Germination of spores of Botrytis cinerea was not influenced. Absorption of volatile and gaseous metabolites in a solution of potassium permanganate decreased in most cases their inhibitory effects, particularly in Botrytis cinerea.

Carbon Dioxide↗

Inhibitory effect of 1-methyldodecyldimethylamine oxide and N,N-bis(dodecyldimethyl)-1,2-ethanediammonium dibromide on the spores of Bacillus cereus.

1-Methyldodecyldimethylamine oxide (MDDO) and N,N'-bis(dodecyldimethyl)-1,2-ethanediammonium dibromide (BDED) exhibit a significant affinity for the surface of Bacillus cereus spores and adsorb very rapidly to the cells; they have a pronounced inhibitory effect on spore outgrowth. In order to alter the affinity of the spore surface for these inhibitors, the spores were pretreated with sodium dodecyl sulfate (SDS), and with an electronegative (Tween 80) and electropositive (histone) compound. In SDS-pretreated spores the inhibitory effect of MDDO and BDED was abolished to a considerable extent. Whereas the development of intact spores was inhibited already after germination, in SDS-pretreated spores the postgermination development continued but was not completed. In Tween 80-pretreated spores the addition of BDED led only to a retardation of outgrowth and division; BDED added only during the division stage interrupted further development completely. Histone-pretreated spores stopped their development instantaneously after the addition of BDED at any phase of the postgermination development. The possible mechanisms of the interaction of the compounds used with spore surface or rather with the state of its structures are discussed.

Bacillus cereus↗

Characteristics of developing mitochondrial genetic and respiratory functions in germinating fungal spores.

Spores of the fungus Botryodiplodia theobromae began a cyanide-sensitive oxygen consumption immediately upon exposure to a liquid medium, and spore germination and respiration were not affected by ethidium bromide, D-threochloramphenicol, and acriflavin until later during germ tube emergence. These inhibitors of the mitochondrial genetic system all inhibited total cell protein synthesis to the same intermediate degree from the outset of incubation. When spores were incubated in water under non-germinating conditions, protein synthesis and oxygen uptake proceeded at initial rates almost identical to those seen in spores germinating in the presence of the three mitochondrial system inhibitors. Although the spores respired at rapid rates from the onset of incubation, no cytochrome absorption peaks could be observed in mitochondrial fractions prepared from ungerminated spores; they were readily observed in germinated spores, however. When the spores were germinated in the presence of inhibitors of the mitochondrial system, an excess of cytochrome c was observed in the near absence of cytochromes a and b. The results indicate that the ungerminated spores of this organism contain a preserved, potentially functional aerobic respiratory system which requires cycloheximide-sensitive ribosome activity to become functional when the spores are inoculated into a liquid medium.

Acriflavine↗

Basidiospore allergen release: elution from intact spores.

BACKGROUND: We sought to test the hypothesis that allergens are readily released by intact basidiospores in vitro and that different species release allergens in different patterns that are related to spore wall structure. METHODS: To assess basidiospore allergen releasability, basidiospore allergens were extracted from disrupted spores and eluted from intact spores, and the allergenic potency of the extracts and eluates was compared in Calvatia cyathiformis, Psilocybe cubensis, Lentinus edodes, and Pleurotus ostreatus. RESULTS: There was a significant species difference; on the basis of dry weight, the yield of extract and eluates from P1. ostreatus greatly exceeded the yield from C. cyathiformis extract and eluates. As measured by RAST inhibition, the allergenic potency of P1. ostreatus and L. edodes spore eluates reached the potency of disrupted spore extract in less than 4 hours. Allergen potency of Ps. cubensis and C. cyathiformis eluate approached the potency of disrupted spore extract only after 8 and 24 hours. Allergen staining intensity in sodium dodecyl sulfate polyacrylamide gel electrophoresis IgE immunoblot correlated with RAST activity. C. cyathiformis and Ps. cubensis spores have thick walls, whereas P1. ostreatus and L. edodes spore walls are thin and hyaline (nonpigmented). Thus spore allergen release may relate to spore wall characteristics. CONCLUSIONS: These studies indicate that intact basidiospores of all four species release allergens, but the release patterns differ by species, and these differences may be clinically significant.

Allergens↗

Cellular immunity in salmonids infected with the microsporidial parasite Loma salmonae or exposed to non-viable spores.

Following a per os challenge of naive rainbow trout with live spores of Loma salmonae, head kidney mononuclear cells (MNC) in culture were able to proliferate in response to crude soluble parasite extract or intact dead spores. A significant response was seen by week 2 post-exposure and a maximum response developed by week 6 or 8, respectively. During this initial challenge, spore filled cysts developed on the gills of challenged fish, and the cysts ruptured by week 12 as is typical for microsporidial gill disease of salmonids (MGDS). Two weeks following this, fish were re-challenged with live spores, and in these fish an enhanced in vitro proliferative response of MNC was immediately apparent, and spore filled cysts did not develop. In contrast, when naive trout were given dead spores by intraperitoneal injection, the most pronounced proliferative responses of MNC developed earlier (week 2 PE) and the response was greater when cells were incubated in vitro with dead spores rather than with crude soluble extract. When these fish were re-challenged per os with live spores, a heightened proliferation in MNC was observed 4 weeks after this exposure and the fish likewise resisted development of xenomas. In fish infected orally or injected intraperitoneally with spores, a marked increase in the response to the mitogen concanavalin A was seen for 22 weeks post-exposure when compared to controls not receiving any spores.

Animals↗

A study on the effects of high pressure and heat on Bacillus subtilis spores at low pH.

Bacillus subtilis spore suspensions were subjected to pressure treatments at 100 and 600 MPa at 40 degrees C and over a pH range from 3 to 8. Inactivation of spores under these conditions was maximally 80% and was not increased at low pH. However, higher levels of inactivation were obtained when spores were first pressure treated at neutral pH and then exposed for 1 h to low pH. This large difference in inactivation could be explained by the finding that pressure-induced spore germination, which is known to occur at neutral pH, was inhibited at low pH (< 5). Pressure treatment at low pH made spores more sensitive to heat inactivation, suggesting that demineralized H-spores had been formed. Changes in spore core hydration and pH upon exposure of spores at low pH were studied in a more direct way using green fluorescent protein expressed in recombinant B. subtilis as a reporter protein, and it was confirmed that pressure and heat increase spore permeability for protons. Based on these results, the potential of low temperature, high pressure processes for spore inactivation in acid products is discussed.

Bacillus subtilis↗

Contamination flows of Bacillus cereus and spore-forming aerobic bacteria in a cooked, pasteurized and chilled zucchini purée processing line.

A food processing plant producing pasteurized purées and its zucchini purée processing line were examined for contamination with aerobic and facultative anaerobic bacterial spores during a day's operation. Multiplication of spores was also monitored in the product stored under different conditions. High concentrations of Bacillus cereus spores were found in the soil in which the zucchinis were grown (4.6+/-0.3 log CFU/g), with a background spore population of 6.1+/-0.2 log CFU/g. In the processing plant, no B. cereus or psychrotrophic bacterial spores were detected on equipment. B. cereus and psychrotrophic bacterial spores were detected after enrichment in all samples of raw zucchinis, washed zucchinis, of two ingredients (starch and milk proteins) and in processed purée at each processing step. Steam cooking of raw zucchinis and pasteurization of purée in the final package significantly reduced spore numbers to 0.5+/-0.3 log CFU/g in the processed food. During storage, numbers of spore-forming bacteria increased up to 7.8+/-0.1 log CFU/g in purée after 5 days at 20-25 degrees C, 7.5+/-0.3 log CFU/g after 21 days at 10 degrees C and 3.8+/-1.1 log CFU/g after 21 days at 4 degrees C. B. cereus counts reached 6.4+/-0.5 log CFU/g at 20-25 degrees C, 4.6+/-1.9 log CFU/g at 10 degrees C, and remained below the detection threshold (1.7 log CFU/g) at 4 degrees C. Our findings indicate that raw vegetables and texturing agents such as milk proteins and starch, in spite of their low levels of contamination with bacterial spores and the heat treatments they undergo, may significantly contribute to the final contamination of cooked chilled foods. This contamination resulted in growth of B. cereus and psychrotrophic bacterial spores during storage of vegetable purée. Ways to eliminate such contamination in the processing line are discussed.

Bacillus cereus↗

Interaction of Aspergillus fumigatus spores and pulmonary alveolar macrophages of rabbits.

The in vitro and in vivo interaction of rabbit pulmonary alveolar macrophages (PAM) and Aspergillus fumigatus spores was studied. In vitro experiments showed that PAM from normal rabbits failed to appreciably kill A. fumigatus spores in 4 hours, while A. flavus and A. niger spores were destroyed effectively. Prior opsonization of the spores with normal rabbit serum, rabbit anti-A. fumigatus serum, complement or lung lavage fluid has no profound enhancing effect on the phagocytosis or killing of the spores. Activated macrophages, however, killed slightly more spores than normal macrophages. When A. fumigatus spores were injected intratracheally into rabbits, no dissemination to organs other than the lungs was detected during the first hour, while dissemination to the liver, spleen and kidneys was observed one hour after the inoculation. Free spores in the bronchoalveolar washings and ingested spores in macrophages diminished in 4 hours, while spores in the lung homogenate increased considerably.

Animals↗

Spatiotemporal distribution of airborne mould spores in apartments.

Indoor air contamination with mould spores currently experiences an increasing interest with respect to their relevance to health. To assess adverse health effects, epidemiological studies combine the health outcome of individuals with their concomitant exposure to airborne spores, which is observed, for example, during the current month. While the latter is representative for the studied period, health effects might also be the result of long term-exposure or emerge in consequence of a peak of pollution throughout the year. To consider such questions, additional information about the spatiotemporal distribution of airborne spores is necessary. This paper aims at elucidating the spatial and temporal variation of spore concentrations in Leipzig, Germany. The analysis is based on 1165 matched pairs of indoor and outdoor measurements taken in the period 1998-2002. All data were collected in the frame of previous epidemiological studies and refer to apartments. The analysis comprised spore concentrations (as CFU m(-3) in air) of the most important genera, such as Penicillium, Aspergillus, Alternaria, Mucorales, Cladosporium, and also for yeasts. We found two groups of fungi differing in their spatiotemporal distribution. As this behaviour can be explained by the predominant origin and growing conditions, we call them indoor-relevant and outdoor-relevant genera. Penicillium species are a representative of the former group, while the latter is well represented by Cladosporium. In the studied period we did not observe a clear trend in the spore concentration. Outdoors there is a year-to-year variation of Cladosporium spore concentrations, which follow the prevalent climatic conditions. For the spore concentration of the outdoor-relevant group a significant annual cycle was observed. Highest concentrations occurred during the summer months and were about 100 x the winter burden. That means, for a direct comparison of measurements of spore concentrations taken during different months the season has to be considered. We summarise the findings in a seasonal model, which is fitted to our measurements. Based on the model we developed a procedure for seasonal adjustment, which enabled us to estimate the annual peak spore concentration utilising one monthly observation.

Air Microbiology↗

Protection of bacterial spores in space, a contribution to the discussion on Panspermia.

Spores of Bacillus subtilis were exposed to space in the BIOPAN facility of the European Space Agency onboard of the Russian Earth-orbiting FOTON satellite. The spores were exposed either in dry layers without any protecting agent, or mixed with clay, red sandstone, Martian analogue soil or meteorite powder, in dry layers as well as in so-called 'artificial meteorites', i.e. cubes filled with clay and spores in naturally occurring concentrations. After about 2 weeks in space, their survival was tested from the number of colony formers. Unprotected spores in layers open to space or behind a quartz window were completely or nearly completely inactivated (survival rates in most cases < or = 10(-6)). The same low survival was obtained behind a thin layer of clay acting as an optical filter. The survival rate was increased by 5 orders of magnitude and more, if the spores in the dry layer were directly mixed with powder of clay, rock or meteorites, and up to 100% survival was reached in soil mixtures with spores comparable to the natural soil to spore ratio. These data confirm the deleterious effects of extraterrestrial solar UV radiation. Thin layers of clay, rock or meteorite are only successful in UV-shielding, if they are in direct contact with the spores. The data suggest that in a scenario of interplanetary transfer of life, small rock ejecta of a few cm in diameter could be sufficiently large to protect bacterial spores against the intense insolation; however, micron-sized grains, as originally requested by Panspermia, may not provide sufficient protection for spores to survive. The data are also pertinent to search for life on Mars and planetary protection considerations for future missions to Mars.

Bacillus subtilis↗

Determination of fungal spore release from wet building materials.

The release and transport of fungal spores from water-damaged building materials is a key factor for understanding the exposure to particles of fungal origin as a possible cause of adverse health effects associated to growth of fungi indoors. In this study, the release of spores from nine species of typical indoor fungi has been measured under controlled conditions. The fungi were cultivated for a period of 4-6 weeks on sterilized wet wallpapered gypsum boards at a relative humidity (RH) of approximately 97%. A specially designed small chamber (P-FLEC) was placed on the gypsum board. The release of fungal spores was induced by well-defined jets of air impacting from rotating nozzles. The spores and other particles released from the surface were transported by the air flowing from the chamber through a top outlet to a particle counter and sizer. For two of the fungi (Penicillium chrysogenum and Trichoderma harzianum), the number of spores produced on the gypsum board and subsequently released was quantified. Also the relationship between air velocities from 0.3 to 3 m/s over the surface and spore release has been measured. The method was found to give very reproducible results for each fungal isolate, whereas the spore release is very different for different fungi under identical conditions. Also, the relationship between air velocity and spore release depends on the fungus. For some fungi a significant number of particles smaller than the spore size were released. The method applied in the study may also be useful for field studies and for generation of spores for exposure studies.

Air Microbiology↗

Photosensitization of DNA by dipicolinic acid, a major component of spores of Bacillus species.

The DNA in spores of Bacillus species exhibits a relatively novel photochemistry, as 5-thyminyl-5,6-dihydrothymine (spore photoproduct (SP)) is by far the major UV photoproduct whereas cyclobutane dimers (CPDs) and (6-4) photoproducts (6-4PPs) are the major photoproducts in growing cells. Dehydration and more importantly complexation of DNA by alpha/beta-type small, acid-soluble spore proteins (SASP) have been shown to partly explain the photochemistry of spore DNA. The large amount ( approximately 10% of dry weight) of the spore's dipicolinic acid (DPA) also has been shown to play a role in spore DNA photochemistry. In the present work we showed by exposing spores of various strains of B. subtilis to UVC radiation that DPA photosensitizes spore DNA to damage and favors the formation of SP. The same result was obtained in either the presence or absence of the alpha/beta-type SASP that saturate the spore chromosome. Addition of DPA to dry films of isolated DNA or to frozen solutions of thymidine also led to a higher yield of SP and increased ratio of CPDs to 6-4PPs; DPA also significantly increased the yield of CPDs in thymidine exposed to UVC in liquid solution. These observations strongly support a triplet energy transfer between excited DPA and thymine residues. We further conclude that the combined effects of alpha/beta-type SASP and DPA explain the novel photochemistry of DNA in spores of Bacillus species.

Bacillus↗

Bacillus megaterium spore germination is influenced by inoculum size.

AIMS: The effect of spore density on the germination (time-to-germination, percent germination) of Bacillus megaterium spores on tryptic soy agar was determined using direct microscopic observation. METHODS AND RESULTS: Inoculum size varied from approximately 10(3) to 10(8) cfu ml(-1) in a medium where pH=7 and the sodium chloride concentration was 0.5% w/v. Inoculum size was measured by global inoculum size (the concentration of spores on a microscope slide) and local inoculum size (the number of spores observed in a given microscope field of observation). Both global and local inoculum sizes had a significant effect on time-to-germination (TTG), but only the global inoculum size influenced the percentage germination of the observed spores. CONCLUSIONS: These results show that higher concentrations of Bacillus megaterium spores encourage more rapid germination and more spores to germinate, indicating that low spore populations do not behave similarly to high spore populations. SIGNIFICANCE AND IMPACT OF THE STUDY: A likely explanation for the inoculum size-dependency of germination would be chemical signalling or quorum sensing between Bacillus spores.

Bacillus megaterium↗

Carton sterilization by u.v.-C excimer laser light: recovery of Bacillus subtilis spores on vegetable extracts and food simulation matrices.

AIMS: To determine the recovery of Bacillus subtilis spores loaded onto preformed cartons and irradiated with u.v.-excimer laser (248 nm) light. METHODS AND RESULTS: Bacillus subtilis spores irradiated with u.v.-excimer laser light retained phase brightness, but were blocked at various stages of germination. In the presence of germinant, the majority of spores began to lose phase brightness but only after an extended lag period (ca 90 min). After 6 h ca 9% of the spores had elongated but failed to form new cells, approx. 12% had undergone partial phase darkening (grey spores), 15% remained phase bright whilst the remainder had turned fully phase dark but failed to elongate. No enhanced recovery of u.v.-treated spores (with intact or permeabilized coats) occurred in media containing hen egg white lysozyme or vegetable extracts (celery, carrot, swede or turnip). However, recovery did occur when irradiated spores were incubated for 26 d, semiaerobically, within cartons containing nutrient broth or milk. CONCLUSIONS: The germination ability of B. subtilis spores is altered following u.v.-excimer laser treatment. Recovery of treated spores was found in liquid systems but not on agar plates supplemented with vegetable extracts or lysozyme. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential recovery of u.v.-excimer laser-treated spores in a range of carton-packed food systems requires further investigation.

Animals↗

Tyrosine kinase activation in response to fungal spores is primarily dependent on endogenous reactive oxygen production in macrophages.

Studies from our laboratory (Shahan, T. A., Sorenson, W. G., and Lewis, D. M. (1994) Environ. Res. 67, 98-104) demonstrated that spores from different fungal species differentially activate rat alveolar macrophages as detected by the measurement of superoxide anion and cytokine production (Shahan, T. A., Siegel, P. D., Sorenson, W. G., Kuschner, W. G., and Lewis, D. M. (1998) Am. J. Respir. Cell Mol. Biol. 18, 435-441). Spores from Aspergillus candidus stimulated production of the highest levels of superoxide anion (5.2 nmol/1.0 x 10(6) alveolar macrophages (AMs)/30 min), followed by those from Aspergillus niger (2.4 nmol/1.0 x 10(6) AMs/30 min) and Eurotium amstelodami (0.4 nmol/1.0 x 10(6) AMs/30 min). The mechanism of this differential activation was studied. Our data demonstrate that the tyrosine kinases p56(Hck), p72(Syk), p77(Btk), p62(Yes), p56(Lck), and p59(Fyn) were specifically activated in response to spores from A. candidus, whereas spores from either A. niger or E. amstelodami activated p56(Hck), p72(Syk), and p77(Btk). Kinetic analysis of specific tyrosine kinases demonstrated that p56(Hck), p72(Syk), and p77(Btk) were activated faster and to a greater extent by spores from A. candidus as compared with spores from E. amstelodami. These data suggest a relationship between reactive oxygen species and tyrosine kinase activation. Treatment of AMs with H(2)O(2) (1 mM) caused the activation of p72(Syk) only, whereas treatment with superoxide dismutase and catalase before treatment with the spores had no effect on tyrosine kinase activation. Incubation with NADPH oxidase inhibitors inhibited both superoxide anion production and the activation of p56(Hck), p72(Syk), and p77(Btk) in response to fungal spores. These data indicate that endogenous reactive oxygen species are necessary for the activation of p56(Hck), p72(Syk), and p77(Btk) by spores; they also indicate that some species of spores are capable of activating tyrosine kinases independent of superoxide anion.

Agammaglobulinaemia Tyrosine Kinase↗

mRNA decay is rapidly induced after spore germination of Saccharomyces cerevisiae.

Spores from the yeast Saccharomyces cerevisiae can germinate and resume their vegetative growth when placed in favorable conditions. Biochemical studies on germination have been limited by the difficulty of obtaining a pure population of spores germinating synchronously. Here, we report that spores can be purified and sorted according to their size by centrifugal elutriation and that these spores are able to germinate synchronously. Synchronizing their development has allowed reevaluating certain parameters of germination, and we demonstrate that both transcription and translation are induced very rapidly after germination induction. Spores contain mRNAs that are stable for several months in spores kept at 4 degrees C. Germination induction leads to very rapid degradation of these mRNAs, thus providing a simple model to study induction of mRNA decay in eukaryotes. mRNAs from the spore are polyadenylated, capped, and cosediment on sucrose gradients with ribosomes and polysomes and with components of the mRNA degradation machinery. The presence of polysomes in the spores led us to evaluate the activity of the translation apparatus in these cells. We present evidence that there is ongoing transcription and translation in nongerminating yeast spores incubated in water at 30 degrees C, suggesting that these activities could play a role in spore long term survival.

Base Sequence↗