The preparation,germination properties and stability of superdormant spores of Bacillus cereus

Aims: To determine yields, germination and stability of superdormant Bacillus cereus spores. Methods and Results: Superdormant B. cereus spores were isolated by germination with high concentrations of inosine or l ‐alanine in 2–5% yield and did not germinate with high concentrations of either of these germinants, but germinated like starting spores with Ca‐DPA, dodecylamine, l ‐alanine plus inosine or concentrated complete medium. Yields of superdormant spores from germinations with low inosine concentrations were higher, and these spores germinated poorly with low inosine, but relatively normally with high inosine. Yields of superdormant spores were also higher when nonheat‐activated spores were germinated. Superdormant spores stored at 4°C slowly recovered some germination capacity, but recovery was slowed significantly at ?20°C and ?80°C. Conclusions: Factors that influence levels of superdormant B. cereus spores and the properties of such spores are similar to those in B. megaterium and B. subtilis, suggesting there are common mechanisms involved in superdormancy of Bacillus spores. Significance: Superdormant spores are a major concern in the food industry, because the presence of such spores precludes decontamination strategies based on triggering spore germination followed by mild killing treatments. Studies of the properties of superdormant spores may suggest ways to eliminate them.     

PHOTOSYNTHETIC PHYSIOLOGY AND CHEMICAL COMPOSITION OF SPORES OF THE KELPS MACROCYSTIS PYRIFERA,NEREOCYSTIS LUETKEANA,LAMINARIA FARLOWII,AND PTERYGOPHORA CALIFORNICA (PHAEOPHYCEAE)1

Recently released spores of the kelps Macrocystis pyrifera (L.) C. Ag., Nereocystis luetkeana (Mert.) Post. and Rupr., Laminaria farlowii Setch., and Pterygophora californica Rupr. had different levels of net photosynthesis. Spore-specific photosynthesis–irradiance relationships were similar in many respects for M. pyrifera, N. luetkeana, and L. farlowii spores. All three species had low rates of net light-saturated photosynthesis. In contrast, spores of P. californica had higher photosynthetic potential and overall net photosynthesis than the other three species. On a cell carbon basis, however, photosynthetic rates in N. luetkeana spores were similar to those of P. californica spores and higher than those of M. pyrifera spores. Chlorophyll a content of spores varied 10-fold among species. The rank order of significant differences in chlorophyll a content was P. californica > L. farlowii > N. luetkeana > M. pyrifera. As a result, chlorophyll-specific measurements suggest M. pyrifera and N. luetkeana spores had much higher quantum efficiency and photosynthetic potential than either P. californica or L. farlowii spores. Maternal carbon and nitrogen investment significantly differed in spores of M. pyrifera, N. luetkeana, and P. californica with P. californica > M. pyrifera > N. luetkeana. Carbon content in spores of each of these three species increased by about 30% during 12 h of saturating irradiance. We suggest that the photosynthetic capabilities of and maternal investment in spores may be related to the spore as a unit of dispersal, to the reproductive ecology of the parental sporophytic stages, and to the growth and physiology of the germling gametophyte stages.     

Intradiurnal periodicity of fungal spore concentrations (Alternaria, Botrytis, Cladosporium, Didymella, Ganoderma) in Cracow, Poland

Aerobiological monitoring enables the definition of seasonal fungal spore concentrations and also intradiurnal time when the highest concentrations of spores could cause or increase allergy symptoms. These data are useful to estimate symptoms of disease, duration of infection and how advanced the illness is in people suffering from fungal allergens. The aim of the study was to compare the concentrations of fungal spores (Alternaria, Botrytis, Cladosporium, Didymella, Ganoderma) during dry and rainy periods and to analyse their intradiurnal changes. Average daily spore concentrations in dry and rainy periods were compared, using z test, separately for each taxon, season and for a combined 3-year period. Intradiurnal periodicity of fungal spore concentrations was analysed on the basis of three complementary diagrams. These spore concentrations were presented using three curves for all, dry and rainy days in 1997–1999 (April–November). The spore percentage in particular hours was normalized in relation to the daily spore sum accepted as 100%. Two further diagrams enabled the more precise analysis of the highest concentrations in dry days. Daily Botrytis and Cladosporium spore concentrations did not show significant differences between dry and rainy periods. In the case of Didymella and Ganoderma spore concentrations, there were no significant differences between both weather types in the single years, although there was a significant difference when a 3-year period was considered. The differences between daily concentrations of Alternaria spores in dry and rainy periods occurred in 1997 and in a 3-year period. Intradiurnal periodicity of spore concentrations was different for ‘dry’ and ‘wet’ fungal spores. Dry spores are released from the spore-producing parts of the fungus under conditions of decreasing humidity and increasing airflow. Examples of dry spores are those from Alternaria, Cladosporium and Botrytis. Wet spores, such as those from many Ascomycetes (Didymella) and Basidiomycetes (Ganoderma), are released into the atmosphere by processes related to humidity conditions or rain. The highest concentrations of ‘dry’ spores were observed early in the afternoon, while highest values of ‘wet’ spore concentrations occurred in the predawn hours. Statistically non-significant differences between daily spore concentrations in dry and rainy periods of single seasons were found except for Alternaria. Statistically significant differences could occur when the studied period was longer than one season (Alternaria, Didymella, Ganoderma). The highest concentrations of Alternaria, Botrytis and Cladosporium spores were recorded at noon and early in the afternoon. Concentrations of Didymella and Ganoderma spores were highest in the predawn hours.     

Effect of Pyroquilon, an Inhibitor of Melanin Synthesis, on Sporulation and Secondary Infection of Magnaporthe grisea

The effect of pyroquilon. an inhibitor of meianin synthesis. on the sporulation and secondary infection of Magnaporthe grisea spores was investigated. Spore formation of M. grisea was significantly inhibited on the pyroquilon-containing medium. but mycelial growth was not impaired. Moreover, although the colour of the spore suspension obtained from control medium without pyroquilon was black, the suspension prepared from spores which had grown on the pyroquilon-containing medium was red-brown. The cell walls of the spores consisted of two layers. the outer of which was highly electron-dense and saw-like in cross section, regardless of treatment. Both the outer and the inner layers of the cell walls of spores which had grown on pyroquilon-containing medium were thin compared with those of control spores. When M. grisea spores which had formed on the pyroquilon-containing medium were inoculated onto rice leaf sheaths, red brown appressoria were formed. Compared with the control, appressorial penetration and hyphal growth in the host cells were inhibited. The inhibitory effect pyroquilon exerted upon the infection behavior of M. grisea spores was dependent on the dose of the chemical.     

Population dynamics of an ice-associated diatom, Thalassiosira australis Peragallo, under fast ice near Syowa Station, East Antarctica, during austral summer

A clear shift from vegetative cells to auxospores and resting spores in Thalassiosira australis was observed in the water column and sinking fluxes under the fast ice near Syowa Station in the austral summer of 2005/2006. This is the first report of the auxosporulation by T. australis in situ. Resting spores were also observed in the sediment even before new spore formation, suggesting that T. australis can overwinter in the sediment. Heterotrophic dinoflagellates ingested and digested vegetative cells and auxospores but did not digest resting spores, suggesting a high tolerance of resting spores to grazing by heterotrophic dinoflagellates. We discuss the possible life history and overwintering strategy that T. australis uses in an Antarctic coastal area to cope with the unpredictable timing of sea ice growth and decay.     

FREE AMINO ACID CHANGES DURING GERMINATION OF TELIOSPORES OF THE WHEAT BUNT FUNGUS,TILLETIA CARIES

Teliospores were aerated and agitated in a mineral salts medium and their free amino acid contents were analyzed at eight different times, from shortly after imbibition of water until just before germ tube emergence. In addition to the common amino acids, eight unidentified ninhydrin-positive components were detected. About 50 % or more of nearly each of the amino acids diffused out of the spores during the initial phase of germination. These released amino acids were actively taken up by the spores during the latter stages of germination. The free amino acids in largest amounts in the dormant spores of T. caries were arginine 15.0, glutamic acid 6.3, and alanine 3.7 μmoles per g dry spores. Together these three amino acids accounted for about 71 % of the total free amino acids in dormant spores of T. caries and T. controversa. The total amounts of free amino acids in spores of common bunt were much higher than in spores of dwarf bunt.     

Observations on the pathogenicity of Pasteuria penetrans, a parasite of root-knot nematodes

A simple test was used to determine whether or not Pasteuria penetrans spores would attach to 17 species of nematodes. All susceptible individuals had spores attached to their cuticles after 24 h of gentle agitation in suspensions containing 10⁵spores/ml. Spores of P. penetrans from six populations of Meloidogyne only adhered to species of Meloidogyne and they adhered in greatest numbers to the species from which they had been originally isolated. Sonication of spores from infected females increased attachment but the effect was dependent on pH and whether the test was conducted in tap or distilled water. Invasion of tomato roots was reduced by up to 86% when, rather than using healthy juveniles, second-stage juveniles bearing 15 or more spores were added to soil at high densities (1000 or 3000/plant); at low densities (500/plant) invasion was not significantly affected. The rate of development of M. incognita juveniles infected with P. penetrans was slower than that of healthy juveniles. The numbers of second-generation of M. incognita were reduced by 82–93% when juveniles encumbered with 1–15 spores were added to soil instead of those bearing no spores. Pasteuria penetrans populations differed in their aggressiveness and when juveniles encumbered with the same number of spores from two populations were added to soil there were differences in the numbers of females that became infected. The implications of these results for the development of P. penetrans as a biological control agent are discussed.     

Abundance of arbuscular mycorrhizal fungi spores in different environments in a tropical rain forest, Veracruz, Mexico

 The aim of this study was to compare mycorrhizal abundance and diversity in sites with different regimes of disturbance in a tropical rain forest at Los Tuxtlas, Veracruz, Mexico. Arbuscular mycorrhizal spores were quantified at two sites: closed canopy and gaps in the forest. Data were recorded during dry, rainy, and windy ("nortes") seasons. Spores of eight Glomus species, sporocarps of three Sclerocystis species, three species of Acaulospora and two of Gigaspora were found. Significant differences in the number of species and spores were found among seasons. The highest numbers of species and spores were observed during the dry season, with a marked decrease during the rainy season. Our results show that disturbance does not but seasonality does affect abundance and richness of mycorrhizal spores in this tropical wet forest. Accepted: 11 October 1998     

Study of airborne fungal spores in Madrid, Spain

The concentration of fungal spores in the atmosphere of Madrid was recorded and analyzed for the year 2003. Airborne spores were sampled continuously with a Hirst-type spore trap located on the roof of a building of the School of Pharmacy, at about 8 m above ground level. Correlation between the mean daily spore concentrations and meteorological variables were explored by means of Spearman’s correlation analyses. Seventy spore types were identified, of which the most numerous were Cladosporium, Aspergillaceae (conidia), Coprinus, Agaricales (basidiospores), Ustilago (teliospores) and Pleospora (ascospores). These six types of spores represented more than 70% of the total. Cladosporium represented 41% of the total fungal spores, while Ustilago spores, the concentrations of which in May and June exceeded 47% of the monthly total spore count, constituted the second most important group. Spores reached their highest concentrations in the spring months, and in the autumn, mainly in October. A␣positive significant correlation was found between airborne spore counts and temperature and relative humidity. The results provide a picture of the spectrum of airborne fungal spores present in the atmosphere of Madrid and of the `peak' periods of their presence. Future studies will provide more detailed information on the seasonal dynamics of the spores most frequently found in the air as well as on the extent to which atmospheric conditions influence their release, dispersion and sedimentation processes.     

Glycoconjugates enhanced the intracellular killing of Bacillus spores, increasing macrophage viability and activation

Infections caused by Bacillus spores can be attenuated if the intracellular killing of the organism by macrophages can be enhanced. Glycoconjugate-bearing polymers, which selectively bind to Bacillus spores, were tested for modulation of intracellular killing when added prior to, during, and following macrophage exposure to B. cereus spores. In the absence of glycoconjugates, murine macrophages were ineffective at killing Bacillus spores. In presence of glycoconjugates, however, macrophages efficiently killed spores, whether the glycoconjugates were added to the cells prior to, during, and following spore addition. Glycoconjugates were shown to exert a protective influence on macrophages and increase their activation, as evidenced by viability and lactate dehydrogenase release assays. Increased levels of nitric oxide production by macrophages pretreated with glycoconjugates suggest that, under these conditions, glycoconjugates provide an activation signal to macrophages. These results indicate that glycoconjugates promote killing of Bacillus spores, while increasing macrophage activation level and viability. The selection of glycoconjugate ligands bearing immunomodulating properties could be exploited for vaccine and/or immunomodulator development and/or for the improvement of existing vaccines against B. cereus and B. anthracis.     

Effects of Mn levels on resistance of Bacillus megaterium spores to heat, radiation and hydrogen peroxide

Aims: To determine the effects of Mn levels in Bacillus megaterium sporulation and spores on spore resistance. Methods and Results: Bacillus megaterium was sporulated with no added MnCl₂ and up to 1 mmol l^?1 MnCl₂. The resultant spores were purified and loosely bound Mn removed, and spore Mn levels were found to vary c. 100‐fold. The Mn level had no effect on spore γ‐radiation resistance, but B. megaterium spores with elevated Mn levels had higher resistance to UVC radiation (as did Bacillus subtilis spores), wet and dry heat and H₂O₂. However, levels of dipicolinic acid and the DNA‐protective α/β‐type small, acid‐soluble spore proteins were the same in spores with high and low Mn levels. Conclusions: Mn levels either in sporulation or in spores are important factors in determining levels of B. megaterium spore resistance to many agents, with the exception of γ‐radiation. Significance and Impact of the Study: The Mn level in sporulation is an important factor to consider when resistance properties of B. megaterium spores are examined, and will influence the UV resistance of B. subtilis spores, some of which are used as biological dosimeters.     

The occurrence and effect of a protozoan parasite, Ophryocystis elektroscirrha (Neogregarinida: Ophryocystidae) on overwintering monarch butterflies, Danaus plexippus (Lepidoptera: Danaidae) from two California winter sites

Abstract.

• 1 Monarch butterflies, Danaus plexippus, from two overwintering populations, were found to have Ophryocystis elektroscirrha spores on their scales at rates between 53% and 68%. The frequency of butterflies with O.elektroscirrha spores remained about the same between sites and throughout the winter.
• 2 The spores, recovered from all parts of the body of the butterfly, were most numerous on the abdomen, particularly near the posterior third.
• 3 Butterflies with spores survived as long as those without detectable spores at 10.1°C ±0.4 SE and 78.3% r.h. ±0.6SE. Insects with spores held at 19.4°C ±0.4SE and 44.9% r.h. ±1.5SE showed a significantly higher rate of moisture loss and survived a shorter period than monarch butterflies without detectable spores.

     

The aerobiological significance of smut spores in Tulsa,Oklahoma

Few aerobiological studies have focused on smut spores, teliospores of fungi within the order Ustilaginales, but the scientific literature provides evidence of the potential aerobiological significance of these plant pathogens. The atmosphere in Tulsa, Oklahoma was monitored for the presence of smut teliospores using a Burkard Volumetric Spore Trap. Smut spores were identified in the atmospheric samples every day from May to October during 1991 and 1992 at concentrations that were normally below 1000 spores/m³. The peak concentration observed during this study was almost 6000 spores/m³. Daily concentrations fluctuate due to a variety of factors such as precipitation, relative humidity, percent sunshine, and the phenology of fungi in relation to their hosts. In northeastern Oklahoma, the most prevalent species of smuts in the atmosphere during the spring includeSphacelotheca occidentalis, Ustilago tritici, andU. kolleri. In the fall, spores ofU. brumivora, U. bullata, andU. maydis are more common.     

Daily and seasonal variations ofAlternaria andCladosporium airborne spores in León (North-West, Spain)

The spores ofAlternaria andCladosporium are present throughout the year in the atmosphere of León (NW Spain), although they show an important seasonal variation. To understand the relationship between the number of spores and climatic factors,Alternaria andCladosporium spores counts for January 1994 to December 1995 were examined by means of correlation analyses. The results of weekly samples of both years showed that the spores concentration of two taxa are significantly and positively correlated with maximum and minimum temperature and sunshine hours and negatively with relative humidity. The statistical analysis of daily samples generally showed the same results. In the hourly distribution of spore concentrations we can see a similar behaviour ofAlternaria andCladosporium, with most spores collected in the 12–14 h period.     

The aerobiological significance of smut spores in Tulsa,Oklahoma

Few aerobiological studies have focused on smut spores, teliospores of fungi within the order Ustilaginales, but the scientific luterature provides evidence of the potential aerobiological significance of these plant pathogens. The atmosphere in Tulsa, Oklahoma was monitored for the presence of smut teliospores using a Burkard Volumetric Spore Trap. Smut spores were identified in the atmospheric samples every day from May to October during 1991 and 1992 at concentrations that were normally below 1000 spores/m³. The peak concentration observed during this study was almost 6000 spores/m³. Daily concentrations fluctuate due to a variety of factors such as precipitation, relative humidity, percent sunshine, and the phenology of fungi in relation to their hosts. In northeastern Oklahoma, the most prevalent species of smuts in the atmosphere during the spring includeSphacelotheca occidentalis, Ustilago tritici, andU. kolleri. In the fall, spores ofU. brumivora, U. bullata, andU. maydis are more common.     

长白山哈泥泥炭地丘间生境泥炭藓孢子的寿命

长寿有性繁殖体对于植物种群的长存具有重要意义,迄今,泥炭地苔藓植物孢子长寿性研究还很少。在长白山哈泥泥炭地钻取丘间表层泥炭样品,测定泥炭腐殖化度和烧失量,逐层提取和培养泥炭藓孢子,研究埋藏时间对孢子萌发的影响。结果表明,丘间泥炭藓孢子埋藏环境中,随着埋深的增加即埋藏年限的增加,泥炭腐殖化度和烧失量总体上分别呈现增加和递减的趋势,而地层泥炭藓孢子萌发率呈现直线递减的规律,但在埋藏近150余年后孢子萌发率仍可达40%。研究进一步证明泥炭藓具有长期持久孢子库,根据推算,泥炭地丘间埋藏环境中,泥炭藓孢子最大寿命可超过400a。     

Effects of media composition on submerged culture spores of the entomopathogenic fungus,Metarhizium anisopliae var. acridum Part 2: Effects of media osmolality on cell wall characteristics,carbohydrate concentrations,drying stability,and pathogenicity

This study evaluates osmolality of a submerged conidia-producing medium in relation to the following spore characteristics: yield, morphology (dimensions and cell wall structure), chemical properties of cell wall surfaces (charge, hydrophobicity, and lectin binding), cytoplasmic polyols and trehalose, and performance (drying stability and pathogenicity). Spore production was increased by the addition of up to 150 g l^?1 polyethylene glycol 200 (PEG). Spores from high osmolality medium (HOM spores) containing 100 g l^?1 PEG had thin cell walls and dimensions more similar to blastospores than submerged conidia or aerial conidia. However, a faint electron-dense layer separating primary and secondary HOM spores’ cell walls was discernable by transmission electron microscopy as found in aerial and submerged conidia but not found in blastospores. HOM spores also appeared to have an outer rodlet layer, unlike blastospores, although it was thinner than those observed in submerged conidia. HOM spores’ surfaces possessed hydrophobic microsites, which was further evidence of the presence of a rodlet layer. In addition, HOM spores had concentrations of exposed N-acetyl-β-d-glucosaminyl residues intermediate between blastospores and submerged conidia potentially indicating a masking of underlying cell wall by a rodlet layer. All spore types had exposed α-d-mannosyl and/or α-d-glucosyl residues, but lacked oligosaccharides. Similar to blastospores, HOM spores were less anionic than submerged conida. Although HOM spores had thin cell walls, they were more stable to drying than blastospores and submerged conidia. Relative drying stability did not appear to be the result of differences in polyol or trehalose concentrations, since trehalose concentrations were lower in HOM spores than submerged conidia and polyol concentrations were similar between the two spore types. HOM spores had faster germination rates than submerged conidia, similar to blastospores, and they were more pathogenic to Schistocerca americana than submerged conidia and aerial conidia.     

Airborne fungal spores in a sawmill environment in Palakkad District, Kerala, India

Concentration of airborne fungal spores inindoor and outdoor environments of a sawmill in Palakkad district of Kerala, India was studied with Burkard Personal Slide Sampler from January to December 1997. Total spore concentration in the indoor and outdoor showed a 3:2 ratio. Higher spore count was observed in indoor in January and in outdoor in October. Thirty three fungal spore types were identified from the indoor and twenty six from the outdoor. Aspergillus/Penicillium, Cladosporium, Nigrospora, Ganoderma, `other basidiospores' and ascospores were the dominant components of the airspora. Aspergillus/Penicillium, the most dominant spore type in the indoor contributed 51.19% and Cladosporium, the most dominant spore type in the outdoor contributed 44.75% of the total spores. The study revealed high prevalence of predominantly allergenic fungal spores in the sawmill environment.     

Insect feeding on spores of a bracket fungus, Elfvingia applanata (Pers.) Karst. (Ganodermataceae, Aphyllophorales)

Insects visiting sporocarps of Elfvingia applanata, a wood-rotting bracket fungus, were examined in Kyoto, central Japan. Mycodrosophila flies (Drosophilidae: Diptera) were predominant and visited the spore-producing sporocarps exclusively. They were observed feeding on the spores, and a number of spores seemed to be alive even after having passed through insects digestive tracts. In addition, the insects attached a number of spores on their body surfaces. In a rearing experiment with insects caught from E. applanata sporocarps, Mycodrosophila flies excreted 7700–469 000 and dropped 10–000–329 000 of viable spores during 48 h after collection. They were supposed to migrate among the sporocarps of other bracket fungi growing on different logs or stumps, suggesting that Mycodrosophila flies may act as spore-dispersal agents for E. applanata.     

Inhibitory Effect of 8-(N,N-Diethylamino)octyl-3, 4, 5-Trimethoxybenzoate Hydrochloride (TMB-8) on Germination of Bacillus cereus T Spores

Effect of 8-(N,N-diethylamino)octyl-3, 4, 5 - trimethoxybenzoate hydrochloride (TMB-8), a calcium antagonist, on germination of Bacillus cereus T spores induced by L -alanine and inosine was investigated. TMB-8 had no effect on the germination of heat-activated spores, whereas it inhibited that of nonactivated spores. The TMB-8 inhibitory effect was antagonized competitively by inosine, but not by L -alanine. Addition of Ca²⁺ reversed the inhibitory effect of TMB-8 in a dose-related fashion. Based on the results, a role of inosine and a site(s) for inhibitory action of TMB-8 in the process leading to the germination of nonactivated spores were discussed.