Indirect Immunodetection of Fungal Fragments by Field Emission Scanning Electron Microscopy.

Submicronic fungal fragments have been observed in in vitro aerosolization experiments. The occurrence of these particles has therefore been suggested to contribute to respiratory health problems observed in mold-contaminated indoor environments. However, the role of submicronic fragments in exacerbating adverse health effects has remained unclear due to limitations associated with detection methods. In the present study, we report the development of an indirect immunodetection assay that utilizes chicken polyclonal antibodies developed against spores from Aspergillus versicolor and high-resolution field emission scanning electron microscopy (FESEM). Immunolabeling was performed with A. versicolor fragments immobilized and fixed onto poly-l-lysine-coated polycarbonate filters. Ninety percent of submicronic fragments and 1- to 2-µm fragments, compared to 100% of >2-µm fragments generated from pure freeze-dried mycelial fragments of A. versicolor, were positively labeled. In proof-of-concept experiments, air samples collected from moldy indoor environments were evaluated using the immunolabeling technique. Our results indicated that 13% of the total collected particles were derived from fungi. This fraction comprises 79% of the fragments that were detected by immunolabeling and 21% of the spore particles that were morphologically identified. The methods reported in this study enable the enumeration of fungal particles, including submicronic fragments, in a complex heterogeneous environmental sample.

Profile and Morphology of Fungal Aerosols Characterized by Field Emission Scanning Electron Microscopy (FESEM).

Fungal aerosols consist of spores and fragments with diverse array of morphologies; however, the size, shape, and origin of the constituents require further characterization. In this study, we characterize the profile of aerosols generated from Aspergillus fumigatus, A. versicolor, and Penicillium chrysogenum grown for 8 weeks on gypsum boards. Fungal particles were aerosolized at 12 and 20 L min-1 using the Fungal Spore Source Strength Tester (FSSST) and the Stami particle generator (SPG). Collected particles were analyzed with field emission scanning electron microscopy (FESEM). We observed spore particle fraction consisting of single spores and spore aggregates in four size categories, and a fragment fraction that contained submicronic fragments and three size categories of larger fragments. Single spores dominated the aerosols from A. fumigatus (median: 53%), while the submicronic fragment fraction was the highest in the aerosols collected from A. versicolor (median: 34%) and P. chrysogenum (median: 31%). Morphological characteristics showed near spherical particles that were only single spores, oblong particles that comprise some spore aggregates and fragments (<3.5 µm), and fiber-like particles that regroup chained spore aggregates and fragments (>3.5 µm). Further, the near spherical particles dominated the aerosols from A. fumigatus (median: 53%), while oblong particles were dominant in the aerosols from A. versicolor (68%) and P. chrysogenum (55%). Fiber-like particles represented 21% and 24% of the aerosols from A. versicolor and P. chrysogenum, respectively. This study shows that fungal particles of various size, shape, and origin are aerosolized, and supports the need to include a broader range of particle types in fungal exposure assessment.

Submicronic fungal bioaerosols: high-resolution microscopic characterization and quantification.

Submicronic particles released from fungal cultures have been suggested to be additional sources of personal exposure in mold-contaminated buildings. In vitro generation of these particles has been studied with particle counters, eventually supplemented by autofluorescence, that recognize fragments by size and discriminate biotic from abiotic particles. However, the fungal origin of submicronic particles remains unclear. In this study, submicronic fungal particles derived from Aspergillus fumigatus, A. versicolor, and Penicillium chrysogenum cultures grown on agar and gypsum board were aerosolized and enumerated using field emission scanning electron microscopy (FESEM). A novel bioaerosol generator and a fungal spores source strength tester were compared at 12 and 20 liters min(-1) airflow. The overall median numbers of aerosolized submicronic particles were 2 × 10(5) cm(-2), 2.6 × 10(3) cm(-2), and 0.9 × 10(3) cm(-2) for A. fumigatus, A. versicolor, and P. chrysogenum, respectively. A. fumigatus released significantly (P < 0.001) more particles than A. versicolor and P. chrysogenum. The ratios of submicronic fragments to larger particles, regardless of media type, were 1:3, 5:1, and 1:2 for A. fumigatus, A. versicolor, and P. chrysogenum, respectively. Spore fragments identified by the presence of rodlets amounted to 13%, 2%, and 0% of the submicronic particles released from A. fumigatus, A. versicolor, and P. chrysogenum, respectively. Submicronic particles with and without rodlets were also aerosolized from cultures grown on cellophane-covered media, indirectly confirming their fungal origin. Both hyphae and conidia could fragment into submicronic particles and aerosolize in vitro. These findings further highlight the potential contribution of fungal fragments to personal fungal exposure.

Inhibition of fungal plant pathogens by synergistic action of chito-oligosaccharides and commercially available fungicides.

Chitosan is a linear heteropolymer consisting of ß 1,4-linked N-acetyl-D-glucosamine (GlcNAc) and D-glucosamine (GlcN). We have compared the antifungal activity of chitosan with DPn (average degree of polymerization) 206 and FA (fraction of acetylation) 0.15 and of enzymatically produced chito-oligosaccharides (CHOS) of different DPn alone and in combination with commercially available synthetic fungicides, against Botrytis cinerea, the causative agent of gray mold in numerous fruit and vegetable crops. CHOS with DPn in the range of 15-40 had the greatest anti-fungal activity. The combination of CHOS and low dosages of synthetic fungicides showed synergistic effects on antifungal activity in both in vitro and in vivo assays. Our study shows that CHOS enhance the activity of commercially available fungicides. Thus, addition of CHOS, available as a nontoxic byproduct of the shellfish industry, may reduce the amounts of fungicides that are needed to control plant diseases.

Characterization of field isolates of Trichoderma antagonistic against Rhizoctonia solani.

The aim of the present study was to characterize sixteen isolates of Trichoderma originating from a field of sugar beet where disease patches caused by Rhizoctonia solani were observed. Use of both molecular and morphological characteristics gave consistent identification of the isolates. Production of water-soluble and volatile inhibitors, mycoparasitism and induced systemic resistance in plant host were investigated using in vitro and in vivo tests in both sterilized and natural soils. This functional approach revealed the intra-specific diversity as well as biocontrol potential of the different isolates. Different antagonistic mechanisms were evident for different strains. The most antagonistic strain, T30 was identified as Trichoderma gamsii. This is the first report of an efficient antagonistic strain of T. gamsii being able to reduce the disease in different conditions. The ability to produce water-soluble inhibitors or coil around the hyphae of the pathogen in vitro was not related to the disease reduction in vivo. Additionally, the strains collected from the high disease areas in the field were better antagonists. The antagonistic activity was not characteristic of a species but that of a population.

Effect of Germination Initiation on Competitive Capacity of Trichoderma atroviride P1 Conidia.

ABSTRACT Trichoderma biocontrol isolates are most effective as highly concentrated inocula. Their antagonism to other fungi may be a result of pregermination respiration. In a nutrient-rich medium, almost all Trichoderma atroviride P1 (P1) conidia initiated germination processes and increased respiration, even in dense suspensions. When 1 x 10(7) P1 conidia/ml were coinoculated with 1 x 10(5) Botrytis cinerea conidia/ml, dissolved oxygen fell to <1% within 2 h and the pathogen failed to germinate. More dilute P1 suspensions consumed oxygen slowly enough to allow coinoculated B. cinerea to germinate. On nutrient-poor media, fewer P1 conidia initiated germination. Oxygen consumption by the inoculum and inhibition of B. cinerea were enhanced when P1 conidia were nutrient activated before inoculation. Pregermination respiration also affected competitive capacity of the antagonist on solid substrates, where respiratory CO(2) stimulated germination rate and initial colony growth. These parameters were directly correlated with inoculum concentration (R(2) >/= 0.97, P < 0.01). After initiating germination, Trichoderma conidia became more sensitive to desiccation and were killed by drying after only 2 h of incubation on a nutrient-rich substrate at 23 degrees C. These results indicate that nutrient-induced changes preceding germination in Trichoderma conidia can either enhance or decrease their biological control potential, depending on environmental conditions in the microhabitat.