Synergistic Effects in Sonochemical Sterilization

The synergistic effects observed during the sterilization of Bacillus subtilis var. niger ATCC 9372 by the combined action of ethylene or propylene oxide with high-intensity airborne sound waves (34.8 kc/sec) were investigated. It has been shown that the number of surviving spores deposited on paper strips decreases exponentially with the sound intensity at sample level. Reductions of the order of one-third of the time required for standard propylene oxide sterilization have been observed by using the combined action of sound waves with gaseous sterilization. At the present time, maximal synergistic effects seem to be achieved for the following experimental conditions: propylene oxide concentration, 500 to 1,000 mg/liter; acoustic intensity, 161 to 162 db; contact time, 80 min; temperature, 60 C; and relative humidity, 40%. The basic mechanism involved in sonochemical sterilization seems to be more of a physical (accelerated gas diffusion) than a chemical nature.     

Characteristics of electrical responses by the medial geniculate bodies in Vespertilionidae and Rhinolophidae to ultrasonic stimuli with different frequencies

The total electrical responses and action potentials of the neurons in the medial geniculate bodies in Vespertilionidae and Rhinolophidae were investigated. Maximum sensitivity to ultrasonic stimuli was recorded inMyotis oxygnathus (Vespertilionidae) in the range 10–40 kc/sec and 65–80 kc/sec, and in Rhinolophidae in the ranges 10–70 kc/sec and 81–86 kc/sec. Low thresholds were observed inMyotis oxygnathus for the frequencies covered by their echo-location cries, whereas the thresholds recorded in Rhinolophidae in the 80 kc/sec band (the principal frequency of their echo location cries) were 15–30 dB higher than those for adjacent frequencies. Minimum thresholds of off-responses were observed inMyotis oxygnathus in the range 50–60 kc/sec, and in Rhinolophidae in the range 78–80 kc/sec. The regions of neuronal response in both species of bat were generally similar in form to those of responses recorded in the medial geniculate bodies of other mammals. Some of the neurons in Rhinolophidae with a characteristic frequency of about 80 kc/sec were also sensitive to stimuli with one-half and one-third of the principal frequency. In Rhinolophidae the greatest selectivity for frequencies was possessed by neurons that responded within the range from 80 to 90 kc/sec.A. A. Zhadanov Leningrad State University. Translated from Neirofiziologiya, Vol. 3, No. 2, pp. 138–144, March–April, 1971.     

Characteristics of electrical responses by cochlear nuclei in Vespertilionidae and Rhinolophidae to ultrasonic stimuli with various fill frequencies

The total electrical response and action potentials of separate neurons in the cochlear nuclei in Vespertilionidae and Rhinolophidae were investigated. Maximum sensitivity to ultrasound was recorded in Vespertilionidae in the frequency ranges 10–30 and 70–80 kc/sec, and in Rhinolophidae in the frequency ranges 10–30 and 84–86 kc/sec. Mininum off-response thresholds were observed in Vespertilionidae in the range 50–60 kc/sec, and in Rhinolophidae in the range 78–80 kc/sec. The areas of responses by neurons in the cochlear nuclei in both species of bats were similar in shape to those recorded in the same structure in other animals. An exception was provided by Rhinolophidae, in which three peculiar types of neurons were observed: 1) neurons whose response area lay in the frequency ranges up to 78 kc/sec or from 80 to 90 kc/sec; 2) neurons responding in the range 40–90 kc/sec, but not sensitive to stimuli with a fill frequency of 78–80 kc/sec; and 3) neurons whose response area lay in the range 78–80 kc/sec, but in which the character of the response changed from tonic to phasic when there was a change in the fill frequency of the stimulus. Maximum selectivity with regard to fill frequency of stimulus was observed in the neurons of Rhinolophidae in the frequency range 70–90 kc/sec.The term "fill frequency" can be rendered as frequency — Consultants Bureau.A. A. Zhdanov Leningrad State University. Translated from Neirofiziologiya, Vol. 3, No. 4, pp. 379–385, July–August, 1971.     

Communication ecology of webbing clothes moth: attractiveness and characterization of male-produced sonic aggregation signals

Abstract:  We tested the hypothesis that the webbing clothes moth, Tineola bisselliella (Hum.) (Lep., Tineidae), uses sonic signals in addition to pheromonal signals for communication. To record sound from individual or groups of moths of either or both sexes, we used a digital recording system, microphones sensitive to sonic and/or ultrasonic frequencies, and speakers capable of emitting sonic and ultrasonic sound. In a soundproof environment, male T. bisselliella produced sounds of 27 decibels (dB, sound pressure level; 0 dB corresponds to 20  μ Pa), with a base frequency of 40–50 Hz and a harmonic frequency of 80–100 Hz. Sound intensity and frequency increased to 55 dB and 65–75 Hz (with ≥3 harmonic frequencies), respectively, when calling males were near (<2 cm) conspecifics of either sex. There was no evidence that females produce sound and no evidence for ultrasonic sound production by either sex. In Y-tube bioassay experiments, virgin male and female T. bisselliella preferred played-back sonic signals from males to silent control stimuli, whereas mated females showed no preference for either stimulus. In arena bioassay experiments, males as well as virgin and mated females preferred played-back sonic signals from males over a white noise control. Use of pheromonal and sonic signals by T. bisselliella would be adaptive, because the capacity for sonic communication persists even if sensory adaptation or habituation to pheromonal signals occurred. This hypothesis is consistent with the fact that other inhabitants of enclosed microhabitats, such as the greater wax moth, Galleria mellonella L., and Indian meal moth, Plodia interpunctella (Hb.), have also evolved analogous multimodal communication systems.     

Viscoelastic relaxations in solutions of poly(glutamic acid) and gelatin at ultrasonic frequencies

Complex shear viscosity η* = η′ – iη″ of poly (L -glutarmic acid) solution was measured by the torsional crystal method at 50 kc./sec. as a function of pH. A sharp peak was found at the midpoint of the helix-coil transition region in both η′ and η″. The relaxation time is calculated from η′ and η″ assuming a single relaxation process and the peak value at the midpoint of transition is estimated at 10^?6 sec. Such behavior agrees well with the prediction from the theory of Schwarz. The attenuation of longitudinal sound waves at,3 Me./sec. was measured as a function of pH for solutions of poly(glutamic acid), glutamic acid, and gelatin. A small attenuation peak was observed for the three solutions, the peak height being almost, the same for them. The peak is interpreted in terms of the dissociation reaction of side chains.     

Induced Sporicidal Activity of Chlorhexidine against Clostridium difficile Spores under Altered Physical and Chemical Conditions

Background

Chlorhexidine is a broad-spectrum antimicrobial commonly used to disinfect the skin of patients to reduce the risk of healthcare-associated infections. Because chlorhexidine is not sporicidal, it is not anticipated that it would have an impact on skin contamination with Clostridium difficile, the most important cause of healthcare-associated diarrhea. However, although chlorhexidine is not sporicidal as it is used in healthcare settings, it has been reported to kill spores of Bacillus species under altered physical and chemical conditions that disrupt the spore’s protective barriers (e.g., heat, ultrasonication, alcohol, or elevated pH). Here, we tested the hypothesis that similarly altered physical and chemical conditions result in enhanced sporicidal activity of chlorhexidine against C. difficile spores.

Principal Findings

C. difficile spores became susceptible to heat killing at 80°C within 15 minutes in the presence of chlorhexidine, as opposed to spores suspended in water which remained viable. The extent to which the spores were reduced was directly proportional to the concentration of chlorhexidine in solution, with no viable spores recovered after 15 minutes of incubation in 0.04%–0.0004% w/v chlorhexidine solutions at 80°C. Reduction of spores exposed to 4% w/v chlorhexidine solutions at moderate temperatures (37°C and 55°C) was enhanced by the presence of 70% ethanol. However, complete elimination of spores was not achieved until 3 hours of incubation at 55°C. Elevating the pH to ≥9.5 significantly enhanced the killing of spores in either aqueous or alcoholic chlorhexidine solutions.

Conclusions

Physical and chemical conditions that alter the protective barriers of C. difficile spores convey sporicidal activity to chlorhexidine. Further studies are necessary to identify additional agents that may allow chlorhexidine to reach its target within the spore.     

Airborne basidiospores of Coprinus and Ganoderma in a Caribbean region

The present study sought to examine Coprinus and Ganoderma airborne spore counts, analysing seasonal variations, the influence of meteorological variables and intraday behaviour with a view to charting the aerobiological dynamics of both genera in Havana (Cuba) during a 2-year period (November 2010–October 2012). A Hirst-type volumetric air sampler was used as sampling methodology. The spores registered in the air were identified and counted following the model proposed by the Spanish Aerobiological Network based on two longitudinal transects along the slides. Coprinus spores outnumbered those of the Ganoderma (28,468 annual total spores vs. 1,921 spores). Moreover, both genera were recorded in the atmosphere on a large number of days over the two-year study period, with daily maximum concentrations in the rainy months. The daily maximum value for Coprinus was 880 spores/m³ (30 September 2011), whereas for Ganoderma 44 spores/m³ (4 September 2011). The analysis of the Spearman correlation coefficient showed a significant positive correlation of the airborne Coprinus and Ganoderma spores with the temperature, relative humidity and rainfall, whereas the degree of association is negative with the wind speed. Regarding the intraday behaviour, both basidiospores were more abundant in the atmosphere during the night, with a maximum peak at 5–6 a.m.     

Comparative bioacoustical study of diurnal and nocturanl rodents from closed ranges

Studies have been made on the evoked potentials at the level of the colliculus inferior in the nocturnal rodent Glis glis and diuranl one--Eutamias sibiricus. In G. glis, the evoked potentials were recorded in a wide range of sonic and ultrasonic frequencies up to 90 kc, while in E. sibiricus these potentials were recorded in a range of frequencies up to 60 kc. The auditory threshold curves for G. glis exhibited two areas of minimum thresholds-- a low frequency area and an area at a boundary between sonic and ultrasonic frequencies. Threshold curves for E. sibiricus have no clear sensitivity maximum. In G. glis, correlation between the spectrum of their communication signals and acoustic noise in the habitat was observed. The species investigated differ in the rate of the recovery of the evoked potentials to paired stimuli and in the values of critical summation time.     

Dispersal of Septoria nodorum Pycnidiospores by Simulated Rain and Wind

The influence of wind on the splash dispersal of Septoria nodorum pycnidiospores was studied in a raintower/wind tunnel complex with single drops or simulated rain falling on spore suspensions or infected stubble with windspeeds of 1.5 to 4 m/sec. When single drops fell on spore suspensions (depth 0.5 mm, concentration 7.8 × 10⁵ spores/ml) most of the spore-carrying droplets collected on fixed photographic film between 0–4 m downwind (windspeed 3 m/sec) were >200 μm in diameter. However, most spores were carried in droplets with diameter > 1000 μm, 70 % of which carried more than 100 spores. When simulated rain fell on infected stubble most of the spore-carrying droplets collected beyond 1 m downwind (windspeeds 1.4 and 4 m/sec) were <200 μm in diameter and none were >600 μm; most of these droplets carried only one spore. The distribution of splash droplets (with diameter >100 μm) deposited on chromatography paper showed a maximum at 40–50 cm upwind of the target but many more droplets were deposited 20–30 cm downwind, when single drops fell on a spore suspension (concentration 1.2 × 10⁵ spores/ ml) containing fluorescein dye with a windspeed of 2 m/sec; droplets were collected up to 3 m downwind but not more than 70 cm upwind. With a windspeed of 3 m/sec, numbers of sporecarrying droplets and spores collected on film decreased with increasing distance downwind; most were collected within 2 m of the target but some were found up to 4 m. When simulated rain fell on infected stubble, increasing the windspeed from 1.5 to 4 m/sec greatly increased the number of spores deposited more than 1 m downwind. At 1.5 m/sec none were collected beyond 2 m downwind, whereas at 4 m/sec some were collected at 4 m. A few air-borne S. nodorum spores were collected by suction samplers at a height of 40 cm at distances up to 10 m downwind of a target spore suspension on which simulated rain fell.     

Airborne allergenic microorganisms associated with mushroom cultivation

Exposure to microorganisms, including thermophilic actinomycetes and fungal spores, and to airborne dust produced during compost production and mushroom picking may cause work-related respiratory symptoms. Previous studies have implicated Thermoactinomyces vulgaris and Faenia rectivirgula, the aetiological agents in farmer's lung disease, as causes of these symptoms but these species have been rare in aerobiological studies of mushroom farms. As part of a study of the respiratory health of the exposed workers, we carried out an aerobiological survey of all the stages of commercial mushroom production. Samples of viable airborne microorganisms were collected at a farm from eight locations on two occasions using Andersen cascade impactors. Large numbers of airborne thermophilic actinomycetes, yielding > 10⁶ colony-forming units (cfu) m^?3 air sampled, were associated with compost handling. These were predominantly Thermomonospora spp., while Thermoactinomyces spp. and Faenia rectivirgula were few. Because the compost was largely undisturbed, few airborne actinomycete spores were found in mushroom growing houses, but concentrations of fungal spores exceeded 10⁵ cfu m^?3 when mushrooms were being harvested. Most were Penicillium spp. and Aspergillus fumigatus but Peziza ostracoderma and Trichoderma spp. were also isolated. Workers are thus exposed to a wide range of airborne microorganisms, but the role of many of these in mushroom workers' respiratory symptoms is not yet fully understood.     

Nosema ceranae Can Infect Honey Bee Larvae and Reduces Subsequent Adult Longevity

Nosema ceranae causes a widespread disease that reduces honey bee health but is only thought to infect adult honey bees, not larvae, a critical life stage. We reared honey bee (Apis mellifera) larvae in vitro and provide the first demonstration that N. ceranae can infect larvae and decrease subsequent adult longevity. We exposed three-day-old larvae to a single dose of 40,000 (40K), 10,000 (10K), zero (control), or 40K autoclaved (control) N. ceranae spores in larval food. Spores developed intracellularly in midgut cells at the pre-pupal stage (8 days after egg hatching) of 41% of bees exposed as larvae. We counted the number of N. ceranae spores in dissected bee midguts of pre-pupae and, in a separate group, upon adult death. Pre-pupae exposed to the 10K or 40K spore treatments as larvae had significantly elevated spore counts as compared to controls. Adults exposed as larvae had significantly elevated spore counts as compared to controls. Larval spore exposure decreased longevity: a 40K treatment decreased the age by which 75% of adult bees died by 28%. Unexpectedly, the low dose (10K) led to significantly greater infection (1.3 fold more spores and 1.5 fold more infected bees) than the high dose (40K) upon adult death. Differential immune activation may be involved if the higher dose triggered a stronger larval immune response that resulted in fewer adult spores but imposed a cost, reducing lifespan. The impact of N. ceranae on honey bee larval development and the larvae of naturally infected colonies therefore deserve further study.     

Seasonal variation of fungal propagules in a fruit market environment, Nagpur (India)

The concentration of airborne fungal spores in a marketenvironment was examined to provide basic information needed to evaluatethe importance of varying levels and heterogeneity. Sampling has beencarried out by rotorod sampler and exposed Petri plate method to obtainthe quantitative and qualitative estimations respectively.Aspergillus was the most frequent and predominant genusdetected. Cladosporium, Penicillium and Alternariaspores were also fairly abundant which are well known as allergenic andpathogenic. The high concentration of airborne spores was recordedduring December–January. While the maximum concentration ofAspergillus was found during summer months in the marketenvironment.     

Production of Aseptic Spores of Vesicular-Arbuscular Endophytes and Their Viability After Chemical and Physical Stress

The survival of germinating spores of vesicular-arbuscular endophytes after treatments with oxidizing agents, antibiotics, moist heat, ultrasonic radiation, and ultraviolet radiation was compared with that of their contaminating microbes. Spores of three species were rapidly decontaminated by treatment with 0.42% (wt/vol) chlorine available from 5.0% (wt/vol) chloramine-T at 30°C for 20 to 40 min depending on the species and the soil from which they were extracted. This treatment did not change spore viability. The survival of spores was reduced by exposure for 20 min to 1.11% chlorine at 30°C for Glomus caledonius or at 35°C for Acaulospora laevis. Growth of any bacteria surviving treatment with oxidizing agents was inhibited by 100 μg of chloramphenicol per ml in agar; however, spore germination and germ tube growth were reduced only by concentrations greater than 200 μg/ml in agar. Spore germination was decreased by concentration of pimaracin, which controlled fungal growth. The spores survived moist heat at 40°C for 80 min, 55°C for 10 min, and 60°C for less than 1 min. The viability of spores was unaffected by ultrasonic irradiation for up to 4 min. Spores of G. caledonius and A. laevis were extremely resistant to ultraviolet radiation. Their viability was unaffected by exposure to 5 × 10⁸ ergs cm⁻² from an ultraviolet source of 253.7nm. The spores had very thick, pigmented walls, and the possibility that these provided some protection against the physical and chemical treatments is discussed. The degree of physiological damage to the spores caused by the treatments demonstrated some adverse effects of basic laboratory procedures. This information, together with that on the comparative sensitivity of contaminating microbes to the treatments, was used in the development of protocol for producing large numbers of uncontaminated spores.     

A Quantitative Dynamic Simulation of Bremia lactucae Airborne Conidia Concentration above a Lettuce Canopy

Lettuce downy mildew, caused by the oomycete Bremia lactucae Regel, is a major threat to lettuce production worldwide. Lettuce downy mildew is a polycyclic disease driven by airborne spores. A weather-based dynamic simulation model for B. lactucae airborne spores was developed to simulate the aerobiological characteristics of the pathogen. The model was built using the STELLA platform by following the system dynamics methodology. The model was developed using published equations describing disease subprocesses (e.g., sporulation) and assembled knowledge of the interactions among pathogen, host, and weather. The model was evaluated with four years of independent data by comparing model simulations with observations of hourly and daily airborne spore concentrations. The results show an accurate simulation of the trend and shape of B. lactucae temporal dynamics of airborne spore concentration. The model simulated hourly and daily peaks in airborne spore concentrations. More than 95% of the simulation runs, the daily-simulated airborne conidia concentration was 0 when airborne conidia were not observed. Also, the relationship between the simulated and the observed airborne spores was linear. In more than 94% of the simulation runs, the proportion of the linear variation in the hourly-observed values explained by the variation in the hourly-simulated values was greater than 0.7 in all years except one. Most of the errors came from the deviation from the 1:1 line, and the proportion of errors due to the model bias was low. This model is the only dynamic model developed to mimic the dynamics of airborne inoculum and represents an initial step towards improved lettuce downy mildew understanding, forecasting and management.     

Responses of the less sensitive acoustic sense cells in the tympanic organs of some noctuid and geometrid moths

The separate impulses contributed by the A1 and A2 acoustic sense cells in the tympanic organs of the noctuids, Autographa pseudogamma and Noctua c.-nigrum, and by the A1, A2, and A3 sense cells in the tympanic organ of the geometrid, Ennomos magnarius, were identified and counted from oscillograms grams made as the moths were exposed to ultrasonic pulses of different intensities. These data were used to construct curves relating the response/intensity characteristics of the less sensitive acoustic sense cells to that of the most sensitive unit, A1. The A2 sense cells of the noctuids were found to be from 20 to 30 dB less sensitive than A1 at sound frequencies to which these ears are most sensitive. In the geometrid it was found that the A2 sense cell was 15 dB less sensitive than A1 and 12 dB more sensitive than A3. Only traces of the response of the fourth geometrid acoustic sense cell (A4) could be identified at high sound intensities. In both noctuids and geometrids the acoustic sensitivity of A2 relative to A1 remained unchanged when tested at selected ultrasonic frequencies between 28 and 50 kHz. This confirms the conclusion that the ears of these moths are incapable of pitch discrimination over this frequency range. Each of the systems had a dynamic range of 40 to 45 dB, that of the geometrid showing greater range overlap of the four A cells and hence greater capacity for sound intensity discrimination.     

Methods for Integrated Air Sampling and DNA Analysis for Detection of Airborne Fungal Spores

Integrated air sampling and PCR-based methods for detecting airborne fungal spores, using Penicillium roqueforti as a model fungus, are described. P. roqueforti spores were collected directly into Eppendorf tubes using a miniature cyclone-type air sampler. They were then suspended in 0.1% Nonidet P-40, and counted using microscopy. Serial dilutions of the spores were made. Three methods were used to produce DNA for PCR tests: adding untreated spores to PCRs, disrupting spores (fracturing of spore walls to release the contents) using Ballotini beads, and disrupting spores followed by DNA purification. Three P. roqueforti-specific assays were tested: single-step PCR, nested PCR, and PCR followed by Southern blotting and probing. Disrupting the spores was found to be essential for achieving maximum sensitivity of the assay. Adding untreated spores to the PCR did allow the detection of P. roqueforti, but this was never achieved when fewer than 1,000 spores were added to the PCR. By disrupting the spores, with or without subsequent DNA purification, it was possible to detect DNA from a single spore. When known quantities of P. roqueforti spores were added to air samples consisting of high concentrations of unidentified fungal spores, pollen, and dust, detection sensitivity was reduced. P. roqueforti DNA could not be detected using untreated or disrupted spore suspensions added to the PCRs. However, using purified DNA, it was possible to detect 10 P. roqueforti spores in a background of 4,500 other spores. For all DNA extraction methods, nested PCR was more sensitive than single-step PCR or PCR followed by Southern blotting.     

Characteristics of Airborne Actinomycete Spores

Airborne actinomycete spores, important contaminants in occupational and residential environments, were studied with respect to their (i) release into the air, (ii) aerodynamic and physical size while airborne, and (iii) survival after collection onto agar with an impactor. Three actinomycete species were selected for the tests to exemplify the three main spore types: Streptomyces albus for arthrospores, Micromonospora halophytica for aleuriospores, and Thermoactinomyces vulgaris for endospores. The results show that the incubation conditions (temperature, time, and nutrients) needed for the development of spores for their release into air are different from the conditions that are needed for colony growth only. Additional drying of M. halophytica and T. vulgaris cultures was needed before spores could be released from the culture. The aerodynamic sizes of the spores, measured with an aerodynamic particle sizer, ranged from 0.57 (T. vulgaris) to 1.28 μm (M. halophytica). The physical sizes of the spores, when measured with a microscope and an image analysis system, were found to be smaller than previously reported in the literature. The relative recovery of the spores on agar media ranged from 0.5 (T. vulgaris) to 35% (S. albus). The results indicate that the culturability of the collected airborne actinomycete spores varies widely and is affected by several variables, such as the species and the sampling flow rate. Therefore, alternatives to commonly used cultivation methods need to be developed for the enumeration of actinomycete spores.     

Soluble CD40 Ligand in Sera of Subjects Exposed to Leishmania infantum Infection Reduces the Parasite Load in Macrophages

Background

While CD40L is typically a membrane glycoprotein expressed on activated T cells and platelets that binds and activates CD40 on the surface on antigen presenting cells, a soluble derivative (sCD40L) that appears to retain its biological activity after cleavage from cell membrane also exists. We recently reported that sCD40L is associated with clinical resolution of visceral leishmaniasis and protection against the disease. In the present study we investigated if this sCD40L is functional and exerts anti-parasitic effect in L. infantum-infected macrophages.

Methodology/Principal Findings

Macrophages from normal human donors were infected with L. infantum promastigotes and incubated with either sera from subjects exposed to L. infantum infection, monoclonal antibodies against human CD40L, or an isotype control antibody. We then evaluated infection by counting the number of infected cells and the number of parasites in each cell. We also measured a variety of immune modulatory cytokines in these macrophage culture supernatants by Luminex assay. The addition of sCD40L, either recombinant or from infected individuals’ serum, decreased both the number of infected macrophages and number of intracellular parasites. Moreover, this treatment increased the production of IL-12, IL-23, IL-27, IL-15, and IL1β such that negative correlations between the levels of these cytokines with both the infection ratio and number of intracellular parasites were observed.

Conclusions/Significance

sCD40L from sera of subjects exposed to L. infantum is functional and improves both the control of parasite and production of inflamatory cytokines of infected macrophages. Although the mechanisms involved in parasite killing are still unclear and require further exploration, these findings indicate a protective role of sCD40L in visceral leishmaniasis.     

Airborne fungi associated with ornamental plant propagation in greenhouses

The objective was to determine potential exposure to airborne fungi in greenhouses and to characterize the temporal patterns of airborne fungi in relation to environmental conditions. We analyzed air samples collected in two greenhouses. Results showed that the top 5 fungi in greenhouse 1 were Trichoderma, hyphal fragments, Aspergillus/Penicillium-like, Cladosporium, and Botrytis in a descending order. Those in greenhouse 2 were Aspergillus/Penicillium-like, Cladosporium, Botrytis, yeast-like, and hyphal fragments. Maximum concentrations of Trichoderma and total spores in greenhouse 1 were 36,426 and 49,729 spores/m³, respectively. Maximum concentrations of Aspergillus/Penicillium and total spores in greenhouse 2 were 46,961 and 71,037 spores/m³, respectively. Airborne fungal populations fluctuated dramatically within 2 h during work hours, tenfold for Aspergillus/Penicillium, 66-fold for Trichoderma, and sevenfold for total spores. QPCR detected Trichoderma harzianum ranging from 7 to 3,500 conidia E/m³. Aspergillus/Penicillium and Botrytis showed diurnal patterns, but not Trichoderma. Aspergillus/Penicillium and Cladosporium were positively correlated with temperature, relative humidity, dew point, heat index, and light and negatively with air movement and air pressure. Botrytis and Trichoderma were not correlated with the environmental factors. Greenhouse workers were potentially exposed up to 71,037 spores/m³ of airborne fungi.