Fraction Of Spores Research Articles

Catching some air: a method to spatially quantify aerial triazole resistance in Aspergillus fumigatus.

Airborne triazole-resistant spores of the human fungal pathogen Aspergillus fumigatus are a significant human health problem as the agricultural use of triazoles has been selecting for cross-resistance to life-saving clinical triazoles. However, how to quantify exposure to airborne triazole-resistant spores remains unclear. Here, we describe a method for cost-effective wide-scale outdoor air sampling to measure both spore abundance as well as antifungal resistance fractions. We show that prolonged outdoor exposure of sticky seals placed in delta traps, when combined with a two-layered cultivation approach, can regionally yield sufficient colony-forming units (CFUs) for the quantitative assessment of aerial resistance levels at a spatial scale that was up to now unfeasible. When testing our method in a European pilot sampling 12 regions, we demonstrate that there are significant regional differences in airborne CFU numbers, and the triazole-resistant fraction of airborne spores is widespread and varies between 0 and 0.1 for itraconazole (∼4 mg/L) and voriconazole (∼2 mg/L). Our efficient and accessible air sampling protocol opens up extensive options for fine-scale spatial sampling and surveillance studies of airborne A. fumigatus.IMPORTANCEAspergillus fumigatus is an opportunistic fungal pathogen that humans and other animals are primarily exposed to through inhalation. Due to the limited availability of antifungals, resistance to the first choice class of antifungals, the triazoles, in A. fumigatus can make infections by this fungus untreatable and uncurable. Here, we describe and validate a method that allows for the quantification of airborne resistance fractions and quick genotyping of A. fumigatus TR-types. Our pilot study provides proof of concept of the suitability of the method for use by citizen-scientists for large-scale spatial air sampling. Spatial air sampling can open up extensive options for surveillance, health-risk assessment, and the study of landscape-level ecology of A. fumigatus, as well as investigating the environmental drivers of triazole resistance.

An extension of mathematical model for severity of rice blast disease

<abstract><p>This paper aims to extend the spore dispersal model to the Healthy-Latent-Infectious-Removed (HLIR) epidemic model for assessing the severity of rice blast disease. The model was solved by the Finite Difference Method (FDM). The results of the model were compared to data from the Prachinburi Rice Research Center (PRRC) on the severity of rice blast disease. Because of a small error, the comparison results showed good agreement between the PRRC data and the simulation by looking at the value of Willmott's index of agreement ($ d $). The first bed $ d $ was 0.7166, while the second bed $ d $ was 0.6421, indicating the model's performance. Furthermore, the optimal parameter, the fraction of spores deposited on the crop, was determined to be 0.173 and 0.016 for beds 1 and 2, respectively. The model can simulate and analyze rice blast outbreaks for educational purposes in future preparedness planning.</p></abstract>

Character expression, reproductive barriers, and origin of the rare fern hybrid Asplenium ×aran-tohanum (Aspleniaceae)

Hybridization is a ubiquitous force in plant evolution. In ferns, hybrids are often easily recognized by their intermediate morphology and abortive spores and thus provide a good model for studying reproductive isolating barriers between species. Asplenium ×aran-tohanum is a rare fern hybrid, despite wide coexistence of its parental species (A. billotii and A. trichomanes subsp. quadrivalens) in Western Europe. We made a complete characterization of its three known individuals, including macro- and micromorphology, sporogenesis, gametophyte reproduction, and chloroplast DNA inheritance and evolution. The hybrid expressed morphological characters that were mostly intermediate between those of the parents, but some characters were more similar to one parent or the other. Sizes of both guard cells and spores indicate that the hybrid is tetraploid, as are both parents, and one parent (A. billotii) consistently acted as the female. A very small fraction of spores (~ 7%) were viable and the resulting gametophytes could not form sporophytes, either sexually or apogamously, suggesting that effective postzygotic barriers exist between the parents. The lineages of these taxa diverged about 35 million years ago, which may explain the strong reproductive isolation and rarity of the hybrid. Asplenium ×aran-tohanum appears to be an evolutionary dead end, probably formed recurrently at the places where it grows, but incapable of completing its life cycle or producing viable offspring.

New protocol for successful isolation and amplification of DNA from exiguous fractions of specimens: a tool to overcome the basic obstacle in molecular analyses of myxomycetes.

Herbarium collections provide an essential basis for a wide array of biological research and, with development of DNA-based methods, they have become an invaluable material for genetic analyses. Yet, the use of such material is hindered by technical limitations related to DNA degradation and to quantity of biological material. The latter is inherent for some biological groups, as best exemplified by myxomycetes which form minute sporophores. It is estimated that ca. two-thirds of myxomycete taxa are represented by extremely scanty material. As DNA isolation methods applied so far in myxomycete studies require destructive sampling of many sporophores, a large part of described diversity of the group remains unavailable for phylogenetic studies or barcoding. Here, we tested several procedures of DNA isolation and amplification to seek for an efficient and possibly non-destructive method of sampling. Tests were based on herbarium specimens of 19 species representing different taxonomic orders. We assayed several variants of isolation based on silica gel membrane columns, and a newly designed procedure using highly reduced amount of biological material (small portion of spores), based on fine disruption of spores and direct PCR. While the most frequently used column-based method led to PCR success in 89.5% of samples when a large amount of material was used, its performance dropped to 52% when based on single sporophores. Single sporophores provided amplicons in 89.5% of samples when using a kit dedicated to low-amount DNA samples. Our new procedure appeared the most effective (94.7%) while it used only a small fraction of spores, being nearly non-destructive; it was also the most cost-effective. We thus demonstrate that combination of adequate handling of spore micro-disruption coupled with application of direct PCR can be an efficient way to circumvent technical limitations for genetic studies in myxomycetes and thus can substantially improve taxon sampling for phylogeny and barcoding. Additionally, this approach gives a unique possibility to apply both molecular and morphological assays to the same structure (sporophore), which then can be further stored as documentation.

Inactivation of aerosolized Bacillus anthracis surrogate spores in close proximity to the flame: Simulation study

Survival of bio-warfare aerosol agents exposed to different environmental conditions has gained substantial attention in the biodefense and biosecurity research communities. The present simulation study aimed at investigating the survival of aerosolized bacterial spores in close proximity to a flame under test conditions relevant to a fire or explosion in a bio-weapon facility. The tests were conducted with aerosolized spores of Bacillus thuringiensis var. kurstaki (Btk), a well-recognized surrogate of Bacillus anthracis, which is a common bio-warfare agent. The loss in spore viability resulting from their exposure to a peripheral area of an air-acetylene flame at two particle-to-flame proximity levels was experimentally determined for different exposure time intervals ranging approximately from 0.1 to 6.0 s. The spore inactivation was quantified through comparison of the viability levels obtained for exposed and non-exposed (control) samples. At relatively short time intervals, the inactivation rate was close to exponential. However, as the exposure time increased, the spore viability decrease slowed down for both proximity levels. The “breaking point” was found to be dependent on the particle-to-flame proximity. The findings point to a small fraction of spores that exhibits particularly high resistance to the stress from the flame-originated heat. To verify the existence of such a sub-population, a separate experiment was performed, in which the aerosolized spores that survived the stress caused by exposure to flame were subsequently subjected to an additional (post-exposure) stress by being placed in an oven for 5, 10 and 20 s. The study results demonstrated the presence of “super-resistant” Btk spores. This justifies the need of developing special materials with pronounced biocidal capabilities (well in excess of those associated with an air-acetylene flame) in order to effectively inactivate “super-resistant” aerosolized bio-agents.

Probing the germination kinetics of ethanol-treated Bacillus thuringiensis spores.

Bacillus thuringiensis (Bt) is the most widely used microbial insecticide. To clarify the mechanism of bacterial resistance to ethanol toxicity, the present study investigated the effects of 70% (v/v) ethanol at a moderate temperature (65°C) on Bt spore germination by single-cell Raman spectroscopy and differential interference contrast microscopy. We found that over 80% of Bt spores were inviable after 30 min of treatment. Moreover, ethanol treatment affected spore germination; the time for initiation of rapid calcium dipicolinate (CaDPA) release (i.e., lag time, Tlag), time taken for rapid CaDPA release (i.e., ΔTrelease), and time required for complete hydrolysis of the peptidoglycan cortex of spores (i.e., ΔTlys) were increased with longer treatment times. Alanine-initiated germination upon ethanol treatment for 30-90 min showed a 2- to 4-fold longer Tlag, 2- to 3.5-fold longer ΔTrelease, and ∼2-fold longer ΔTlys relative to the control. Dodecylamine-initiated germination treated for 15-30 min had 3- to 5-fold longer Tlag and 1.4- to 1.7-fold longer ΔTrelease than the control. Germination induced by exogenous CaDPA was observed only in a small fraction of spores treated with ethanol for 5 min. Single-cell Raman spectroscopy revealed that more than 52% of spores lost CaDPA after 30 min of ethanol treatment; these showed reductions in the intensity of 1280 and 1652 cm-1 bands (corresponding to protein α-helical structure) and increases in that of 1245 and 1665 cm-1 bands (attributed to irregularities in protein structure). These results indicate that CaDPA in the core of Bt spores confers resistance to ethanol, and that damage to the spore inner membrane by ethanol treatment results in CaDPA leakage. Additionally, moderate-temperature ethanol treatment and consequent denaturation of germination-related proteins affected spore germination, specifically by inactivating the cortex-lytic enzyme CwlJ. Our findings provide a theoretical basis for the development of more effective methods for killing spore-forming bacteria; microscopy imaging and Raman spectroscopy can provide novel insight into the effects of chemical agents on microbial cells.

Ice nucleation by fungal spores from the classes <i>Agaricomycetes</i>, <i>Ustilaginomycetes</i>, and <i>Eurotiomycetes</i>, and the effect on the atmospheric transport of these spores

Abstract. We studied the ice nucleation properties of 12 different species of fungal spores chosen from three classes: Agaricomycetes, Ustilaginomycetes, and Eurotiomycetes. Agaricomycetes include many types of mushroom species and are widely distributed over the globe. Ustilaginomycetes are agricultural pathogens and have caused widespread damage to crops. Eurotiomycetes are found on all types of decaying material and include important human allergens. We focused on these classes because they are thought to be abundant in the atmosphere and because there is very little information on the ice nucleation ability of these classes of spores in the literature. All of the fungal spores investigated contained some fraction of spores that serve as ice nuclei at temperatures warmer than homogeneous freezing. The cumulative number of ice nuclei per spore was 0.001 at temperatures between −19 °C and −29 °C, 0.01 between −25.5 °C and −31 °C, and 0.1 between −26 °C and −31.5 °C. On average, the order of ice nucleating ability for these spores is Ustilaginomycetes > Agaricomycetes ≃ Eurotiomycetes. The freezing data also suggests that, at temperatures ranging from −20 °C to −25 °C, all of the fungal spores studied here are less efficient ice nuclei compared to Asian mineral dust on a per surface area basis. We used our new freezing results together with data in the literature to compare the freezing temperatures of spores from the phyla Basidiomycota and Ascomycota, which together make up 98% of known fungal species found on Earth. The data show that within both phyla (Ascomycota and Basidiomycota), there is a wide range of freezing properties, and also that the variation within a phylum is greater than the variation between the average freezing properties of the phyla. Using a global chemistry–climate transport model, we investigated whether ice nucleation on the studied spores, followed by precipitation, can influence the transport and global distributions of these spores in the atmosphere. Simulations suggest that inclusion of ice nucleation scavenging of these fungal spores in mixed-phase clouds can decrease the annual mean concentrations of fungal spores in near-surface air over the oceans and polar regions, and decrease annual mean concentrations in the upper troposphere.

Large-eddy simulation of turbulence and particle dispersion inside the canopy roughness sublayer

AbstractModelling the dispersion of small particles such as fungal spores, pollens and small seeds inside and above plant canopies is important for many applications. Transport of these particles is driven by strongly inhomogeneous and non-Gaussian turbulent flows inside the canopy roughness sublayer, the region that extends from the ground to approximately three canopy heights. A large-eddy simulation (LES) approach is refined to study particle dispersion within and above the canopy region. Effects of plant reconfiguration are parameterized through a velocity-dependent drag coefficient, which is shown to be critical for accurate reproduction of velocity statistics and mean spore concentrations. The model yields predictions of turbulence statistics that are in good agreement with measurements. This is particularly true of the stress fractions carried by strong events, as revealed by standard quadrant analysis of the resolved velocity fluctuations, which is a known weakness of earlier LES studies of canopy flow using a constant drag coefficient. Experimental data on spore dispersal inside and above a maize canopy are reproduced successfully as well. Characteristics of the particle plume are analysed using LES results, and a pre-existing theoretical framework is adapted to model particle dispersal above the canopy. The results suggest that the plume above the canopy can be approximated using a simple analytical solution if the fraction of spores that escape the canopy region is known. Source height and gravitational settling have strong effects on the plume inside the canopy region and consequently determine the escape fraction. These effects are parameterized in the theoretical model by using the escape fraction to rescale the source strength.

Interpreting three-dimensional spore concentration measurements and escape fraction in a crop canopy using a coupled Eulerian–Lagrangian stochastic model

Plant disease epidemics caused by pathogenic spores are common threat to agricultural crops. Pathogenic spores are often produced and released inside plant canopies but are transported out of the canopy region by turbulent motions and advected longitudinally over long distances so as to infect other fields. Hence, the fraction of spores that “escape” the canopy sets the effective source strength that determines how fast and far plant diseases can spread. To explore the governing variables of this escape and spatial spread of spores, extensive spore release and recapture experiments were conducted in a maize field and interpreted using a coupled Eulerian–Lagrangian stochastic model (LSM). Spores were released from point sources located at three prototypical depths inside the canopy. Concentration measurements were obtained inside and above the canopy with a 3-dimensional grid of spore collectors. The experimental measurements of mean spore concentration were then used to evaluate an LSM for spore dispersion. The drift and dispersion terms of the LSM were predicted employing a conventional second-order closure model of turbulence within plant canopies thereby allowing this combined Eulerian–Lagrangian formulation to be applied to a broad set of agricultural crops. The dispersion model includes spore deposition on and rebound from canopy elements. The combination of experimental and numerical simulations was then used to quantify the fraction of spores that escape the canopy. Effects of release height and friction velocity on the escape fraction of spores were explored with the LSM.

Quantification of the impact of single and multiple mild stresses on outgrowth heterogeneity of Bacillus cereus spores

Outgrowth heterogeneity of bacterial spore populations complicates both prediction and efficient control of spore outgrowth. In this study, the impact of mild preservation stresses on outgrowth of Bacillus cereus ATCC 14579 spores was quantified during the first stages of outgrowth. Heterogeneity in outgrowth of heat-treated (90°C for 10min) and non-heat-treated germinated single spores to the maximum micro-colony stage of 256 cells was assessed by direct imaging on Anopore strips, placed on BHI plates at pH7 and pH5.5, without and with added NaCl or sorbic acid (HSA). At pH7 non-heated and heat-treated germinated spores required 6h to reach the maximum microcolony stage with limited heterogeneity, and these parameters were only slightly affected with both types of spores when incubated at pH7 with added NaCl. Notably, the most pronounced effects were observed during outgrowth of spores at pH5.5 without and with added NaCl or HSA. Non-heat-treated germinated spores showed again efficient outgrowth with limited heterogeneity reaching the maximum microcolony stage after 6h at pH5.5, which increased to 12h and 16h with added NaCl and HSA, respectively. In contrast, heat-treated spores displayed a strong delay between initial germination and swelling and further outgrowth at pH5.5, resulting in large heterogeneity and low numbers of fastest growers reaching the maximum microcolony stage after 10, 12 and 24h, without and with added NaCl or HSA, respectively. This work shows that Anopore technology provides quantitative information on the impact of combined preservation stresses on outgrowth of single spores, showing that outgrowth of germinated heat-treated spores is significantly affected at pH5.5 with a large fraction of spores arrested in the early outgrowth stage, and with outgrowing cells showing large heterogeneity with only a small fraction committed to relatively fast outgrowth.

The use of biodosimetry to measure the UV-C dose delivered to a sphere, and implications for the commercial treatment of fruit

Commercialization of UV-C treatment of horticultural produce in order to induce beneficial responses in the produce following treatment requires both accurate dose delivery and a method of treating large quantities of produce efficiently. Furthermore, it has long been assumed that such effects require the entire surface of the horticultural commodities – typically fruit – to be exposed to UV-C. This has invariably been achieved by manually rotating the fruit in a UV-C field whilst reducing the dose delivered at each rotation in direct proportion to the number of rotations. However, the resulting UV-C dose distributions achieved under these circumstances are generally not reported in the literature. In the work described here a polystyrene sphere (dia. 70mm) was used to simulate fruits such as tomatoes, apples, peaches, etc., that have an approximately spherical form in order to provide a means of measuring the total doses of UV-C accumulated during treatment and comparing such estimates to theoretically-derived ones. This was achieved using dosimetry based on spores of Bacillus subtilis in which spore-impregnated membranes were attached to the surface of the sphere. The fraction of spores surviving exposure was used to estimate dose from a dose–response curve for the spores. Under irradiation conditions leading to a theoretically calculated dose of 10.6J, spore dosimetry yielded estimates of 9.1, 10.7 and 6.1J for UV-C delivered in, respectively, one, two or four exposures. In the case of exposure of the sphere during continuous mechanical rotation for the same length of time (80s) a value of only 3.5J was obtained. Irradiation conditions resulting in the spores being subject to intermittent exposure to UV-C led to dose estimates below the theoretically derived ones. The circumstances under which spore dosimetry can be used to obtain surface dose distributions are discussed.

Destruction of Aspergillus versicolor, Penicillium chrysogenum, Stachybotrys chartarum, and Cladosporium cladosporioides spores using chemical oxidation treatment process

AbstractThe survival of aqueous suspensions of Penicillium chrysogenum, Stachybotrys chartarum, Aspergillus versicolor, and Cladosporium cladosporioides spores was evaluated using various combinations of hydrogen peroxide and Fe2+ as catalyst. Spore concentrations of 106–107 colony forming units per milliliter (CFU/mL) were suspended in water and treated with initial hydrogen peroxide and iron concentrations ranging from 0.05 to 10 percent and 100 to 200 ppm, respectively. After four hours of reaction time, samples were plated on agar plates, and the viable fraction of spores was determined by the number of colonies formed. Hydrogen peroxide concentrations above 50,000 ppm resulted in greater than 6‐log10 reduction of viable spores for both catalyzed and noncatalyzed reactions. Iron had a strong catalytic effect when added to solutions with hydrogen peroxide concentration above 5,000 ppm and resulted in two to three orders of magnitude greater reduction compared to hydrogen peroxide alone. Additional samples taken after 24 hours of reaction time showed that the effect of the addition of 100 and 200 ppm of Fe2+ catalyst was mostly kinetic, and noncatalyzed hydrogen peroxide had sporicidal effects similar to catalyzed hydrogen peroxide. This study identified initial reagent concentrations of hydrogen peroxide and Fe2+ that accomplish a 6‐log10 reduction of viable mold spores within reaction times of 4 and 24 hours. © 2007 Wiley Periodicals, Inc.

DNA segregation by the bacterial actin AlfA during Bacillus subtilis growth and development

We here identify a protein (AlfA; actin like filament) that defines a new family of actins that are only distantly related to MreB and ParM. AlfA is required for segregation of Bacillus subtilis plasmid pBET131 (a mini pLS32-derivative) during growth and sporulation. A 3-kb DNA fragment encoding alfA and a downstream gene (alfB) is necessary and sufficient for plasmid stability. AlfA-GFP assembles dynamic cytoskeletal filaments that rapidly turn over (t(1/2)< approximately 45 s) in fluorescence recovery after photobleaching experiments. A point mutation (alfA D168A) that completely inhibits AlfA subunit exchange in vivo is strongly defective for plasmid segregation, demonstrating that dynamic polymerization of AlfA is necessary for function. During sporulation, plasmid segregation occurs before septation and independently of the DNA translocase SpoIIIE and the chromosomal Par proteins Soj and Spo0J. The absence of the RacA chromosome anchoring protein reduces the efficiency of plasmid segregation (by about two-fold), suggesting that it might contribute to anchoring the plasmid at the pole during sporulation. Our results suggest that the dynamic polymerization of AlfA mediates plasmid separation during both growth and sporulation.

Assessment of the aerosolization potential for fungal spores in moldy homes

The airborne fungal concentration measured with air samplers during specific time intervals may not adequately represent the indoor air quality because of the sporadic nature of spore release from sources. The conventional source evaluation (e.g. swab and tape sampling) characterizes the mold source but does not relate to the fraction of spores that can be aerosolized from a contaminated material. As an alternative to these methods, we have recently developed and laboratory-tested a novel Fungal Spore Source Strength Tester (FSSST). It allows assessing the potential of aerosolization of fungal spores from contaminated surfaces under the most favorable release conditions. In this study, the FSSST was used to characterize the release of spores from four building materials in mold-problem homes. The spores of different species were efficiently aerosolized by the FSSST, exhibiting a total spore release rate ranging approximately from 10(2) to 10(3) cm2/min. For all tested materials, <2% of the spores on the contaminated surface were released during the tests. The airborne spore concentration estimated from the release rate data was found in most cases to be significantly greater than the concentration actually measured in these environments with simultaneous air sampling. The results suggest that the FSSST can be used for the assessment of maximum potential exposure to airborne spores released from identified sources in homes. A recently developed FSSST was found to be suitable to measure the aerosolization potential of indoor fungal sources at the most favorable release conditions. The FSSST generates the data that allows assessing the strength of mold sources in homes with respect to their maximum ability to contaminate indoor air with fungi. The novel approach bridges two conventional methods, the air sampling and the direct source evaluation (e.g. swab sampling), thus providing a better representation of the airborne fungal exposure than these methods individually. The device prototype can be used for evaluating the effectiveness of environmental interventions by taking samples before and after the intervention. As a broader application, the FSSST can be utilized for assessing the release of various hazardous biological and non-biological particles from contaminated surfaces.

Inactivation of Bacillus cereus spores in milk by mild pressure and heat treatments

The objective of this work was to study the germination and subsequent inactivation of Bacillus cereus spores in milk by mild hydrostatic pressure treatment. In an introductory experiment with strain LMG6910 treated at 40 °C for 30 min at 0, 100, 300 and 600 MPa, germination levels were 1.5 to 3 logs higher in milk than in 100 mM potassium phosphate buffer (pH 6.7). The effects of pressure and germination-inducing components present in the milk on spore germination were synergistic. More detailed experiments were conducted in milk at a range of pressures between 100 and 600 MPa at temperatures between 30 and 60 °C to identify treatments that allow a 6 log inactivation of B. cereus spores. The mildest treatment resulting in a 6 log germination was 30 min at 200 MPa/40 °C. Lower treatment pressures or temperatures resulted in considerably less germination, and higher pressures and temperatures further increased germination, but a small fraction of spores always remained ungerminated. Further, not all germinated spores were inactivated by the pressure treatment, even under the most severe conditions (600 MPa/60 °C). Two possible approaches to achieve a 6 log spore inactivation were identified, and validated in three additional B. cereus strains. The first is a single step treatment at 500 MPa/60 °C for 30 min, the second is a two-step treatment consisting of pressure treatment for 30 min at 200 MPa/45 °C to induce spore germination, followed by mild heat treatment at 60 °C for 10 min to kill the germinated spores. Reduction of the pressurization time to 15 min still allows a 5 log inactivation. These results illustrate the potential of high-pressure treatment to inactivate bacterial spores in minimally processed foods.

Fluorescence based rRNA sensor systems for detection of whole cells of Saccharomonospora spp. and Thermoactinomyces spp.

Airborne thermophilic actinomycetes (TPAs) are a growing hygienic challenge in different occupational situations e.g. large scale composting. This study describes first results of a new approach for highly specific and rapid detection of organisms of this group using fluorescently labelled oligonucleotide probes as sensors for whole cells. Three genus-specific 16S rRNA-targeted probes, two for Saccharomonospora spp. and one for Thermoactinomyces spp. were developed and evaluated in a fluorescence in situ hybridisation (FISH) format with agar-grown whole cells. For optimal sensitivity and specificity of FISH, conditions for cell wall permeabilisation and hybridisation stringency were evaluated independently for both genera. Performing specified pretreatment protocols, all three probes yielded strong fluorescence signals. However, the relative fraction of detectable cells or spores clearly depended on the single bacterial species. The probes can serve as cell sensors for direct detection of TPAs in natural samples.

Sterilization of plastic containers using electron beam irradiation directed through the opening.

Studies were performed to demonstrate the efficacy of a novel electron beam irradiation system for the on-line sterilization of polymeric containers using energetic electrons directed through the container opening. The distribution of dosage delivered during conveyance beneath the electron beam treatment system was determined for two sizes (8 or 16 ounces) of high-density polyethylene (HDPE) blow-moulded bottles. The biological effects of treatment were then determined using Bacillus pumilus ATCC 27142 spores inoculated as 10 microl droplets dried (i) onto the surface of flat coupons of the high-density polyethylene bottle material, and (ii) onto the region of lowest delivered dose (the bottom side wall) within each bottle. The inactivation obtained was determined by examining the level of survival after swab recovery of the inoculated spores, with reference to the level recovered from untreated control samples. The inactivation of B. pumilus spores during treatment was reproducible and proportional to the applied dose. In each instance, the logarithm of the surviving fraction of spores was well fitted by linear regression models and in good general agreement with values reported for inactivation of the same organism using gamma irradiation. The results of this work demonstrate the potential of a new physical method capable of high throughput for in-line sterilization of polymeric containers. The ability of the process to eliminate the washing step involved with traditional chemical sterilants, such as hydrogen peroxide, greatly reduces the size and environmental impact of these systems.

A PHYSICALLY BASED MODEL OF MACROALGAL SPORE DISPERSAL IN THE WAVE AND CURRENT-DOMINATED NEARSHORE

Propagule dispersal in seaweeds is a process influenced by a variety of biological and physical factors, the complexity of which has hindered efforts to understand colonization, persistence, post-disturbance recovery, and dynamics of algal populations in general. In view of this limitation, we employ here modifications to an existing turbulent-transport model to explore the mechanics of nearshore macroalgal spore dispersal and its relationship to coastal hydrodynamic conditions. Our modeling efforts focus on four example species of seaweed whose reproductive propagules span a wide range in sinking speed and height of release above the sea floor: the giant kelp Macrocystis pyrifera, the erect fucoid Sargassum muticum, the small filamentous brown alga Ectocarpus siliculosus, and the flaccid red alga Sarcodiotheca gaudichaudii. Results indicate that both propagule sinking speed and release height can affect dispersal distance substantially, but that the influence of these biological parameters is modulated strongly by the intensity of turbulence as dictated by waves and currents. In rapid flows with larger waves, it is primarily fluid dynamic processes, in particular current velocities, that determine dispersal distance. Additional simulations suggest that patterns of spore dispersal are highly skewed, with most propagules encountering the sea floor within a few meters to hundreds of meters of their parents, but with a sizeable fraction of spores also dispersing as far as kilometers. Such model predictions imply a much greater potential for longer range dispersal than has typically been assumed, a finding with important implications for understanding the demographics of algal populations and for predicting levels of connectivity among them.

Ability of the Gaussian plume model to predict and describe spore dispersal over a potato crop

The Gaussian plume model (GPM) is considered as a valuable tool in predictions of the atmospheric transport of fungal spores and plant pollen in risk assessments. The validity of the model in this important area of application has not been extensively evaluated. A field experiment was set up to test and—if necessary—adapt the GPM, as applied to the dispersal of spores. Spores of the fern Lycopodium clavatum were released artificially over a period of 10 min from a source placed 70 cm above the surface in a potato crop. Spore catches were made with a network of Rotorod and Burkhard samplers, placed up to 100 m downwind from the source and at several heights and crosswind distances from the anticipated plume axis. The width and height of Gaussian plumes depend on atmospheric mixing, as affected by weather. Mixing parameters in risk assessments are commonly predicted on the basis of weather conditions. We found a low correlation ( R=0.4) between measured spore concentrations and predicted spore concentrations, using a widely used prediction method (GPM Pasquill Atmospheric Diffusion, 2nd ed., Wiley, New York, 1974), based on cloud cover, wind speed, season and time of day. More precise methods for predicting the width and height of Gaussian plumes require detailed site-specific information (measurements of wind speed and temperature at two heights above the vegetation), and are therefore not readily applicable in risk assessments. An alternative that is often adequate is to use a worst case approach, in which the dispersal parameters are used that give the highest spore concentration at the location of interest. Predictability could be improved by measuring atmospheric stability during and just after weather conditions conducive to release of the pollen or spores of interest. We calibrated the model with a weighted least-squares method. Calibrating the model led to a more than 100-fold decrease in the sum of weighted squares. A comparison of estimated concentrations with the measurements confirmed that spore clouds originating from a point source take the form of a Gaussian plume: the coefficient of correlation between measured spore concentrations and fitted concentrations was 0.8. The fraction of spores that escaped the canopy and was available for long distance dispersal amounted to 64±17%. We found an 83% correlation between this so-called escape fraction and wind speed.

Localization of GerAA and GerAC germination proteins in the Bacillus subtilis spore.

The GerAA, -AB, and -AC proteins of the Bacillus subtilis spore are required for the germination response to L-alanine as the sole germinant. They are likely to encode the components of the germination apparatus that respond directly to this germinant, mediating the spore's response; multiple homologues of the gerA genes are found in every spore former so far examined. The gerA operon is expressed in the forespore, and the level of expression of the operon appears to be low. The GerA proteins are predicted to be membrane associated. In an attempt to localize GerA proteins, spores of B. subtilis were broken and fractionated to give integument, membrane, and soluble fractions. Using antibodies that detect Ger proteins specifically, as confirmed by the analysis of strains lacking GerA and the related GerB proteins, the GerAA protein and the GerAC+GerBC protein homologues were localized to the membrane fraction of fragmented spores. The spore-specific penicillin-binding protein PBP5*, a marker for the outer forespore membrane, was absent from this fraction. Extraction of spores to remove coat layers did not release the GerAC or AA protein from the spores. Both experimental approaches suggest that GerAA and GerAC proteins are located in the inner spore membrane, which forms a boundary around the cellular compartment of the spore. The results provide support for a model of germination in which, in order to initiate germination, germinant has to permeate the coat and cortex of the spore and bind to a germination receptor located in the inner membrane.