Compared to traditional methods of fungal exposure assessment, molecular methods have provided new insight into the richness of fungal communities present in both indoor and outdoor environments. In this study, we describe the diversity of fungi in the homes of asthmatic children located in Kansas City. Fungal diversity was determined by sequencing the internal transcribed spacer (ITS) regions of ribosomal RNA derived from fungi collected in air and dust samples from 31 homes participating in the Kansas City Safe and Healthy Homes Program (KCSHHP). Sequencing results were then compared to data obtained using viable and non-viable fungal exposure assessment methods. ITS clone libraries were predominantly derived from the phylum Ascomycota in both air (68%) and dust (92%) samples and followed by the Basidiomycota and Zygomycota. The majority of Ascomycota clones belonged to four orders including the Pleosporales, Eurotiales, Capnodiales, and Dothideales. ITS sequencing revealed the presence of a number of rarely documented fungal species placed in the Pleosporales. Several species placed in the Basidiomycota were detected in ITS clone libraries but not by viable or non-viable methods. The prevalence of organizational taxonomic units (OTUs) was significantly higher in air than in dust samples (p < 0.0001); however, no differences between OTUs in air samples collected in the subjects’ room and basement were observed. These sequencing results demonstrate a much broader diversity of Ascomycota and Basidiomycota communities in KCSHHP indoor environments than previously estimated using traditional methods of assessment.

Traditional methods of assessing fungal exposure have been confounded by a number of limiting variables. The recent utilization of molecular methods such as internal transcribed spacer (ITS) sequencing of ribosomal RNA genes has provided improved insight into the diversity of fungal bioaerosols in indoor, outdoor and occupational environments. However, ITS analyses may also be confounded by a number of methodological limitations. In this study, we have optimized this technology for use in occupational or environmental studies. Three commonly used DNA extraction methodologies (UltraClean Soil kit, High Pure PCR Template kit, and EluQuik/DNeasy kit) were compared in terms of sensitivity and susceptibility to PCR inhibitors in dust for three common fungal bioaerosols, Aspergillus versicolor, Rhizopus microsporus and Wallemia sebi. Environmental dust samples were then studied using each extraction methodology and results were compared to viable culture data. The extraction methods differed in terms of their ability to efficiently extract DNA from particular species of fungi (e.g. Aspergillus versicolor). In addition, the ability to remove PCR inhibitors from dust samples was most effective using the soil DNA extraction kit. The species composition varied greatly between ITS clone libraries generated with the different DNA extraction kits. However, compared to viable culture data, ITS clone libraries included additional fungal species that are incapable of growth on solid culture medium. Collectively, our data indicated that DNA extraction methodologies used in ITS sequencing studies of occupational or environmental dust samples can greatly influence the fungal species that are detected.

Fusarium graminearum is a significant pathogen of many cereal crops. With its genetic tractability, ease of culture, genome sequence availability and economic significance, F. graminearum has become the subject of intensive molecular research. Although molecular tools have been developed to enhance research into virulence determinants of F. graminearum, simple assays for infection-related development are lacking. As such, the objective of this study was to develop an in vitro protocol for the analysis of infection-related morphogenesis in F. graminearum. We demonstrate that two morphologically distinct hyphal structures are produced by F. graminearum during the invasion of detached wheat glumes: subcuticular hyphae and bulbous infection hyphae. Specialized wheat epidermal cells (papillae) appear to act as sites of invasion by F. graminearum on the adaxial side of detached wheat glumes. In addition, the development of bulbous infection hyphae is dependent on the pathogenicity mitogen-activated protein kinase Gpmk1, further supporting the infection-related nature of these structures. This relatively simple assay will contribute to the tractability of the F. graminearum system and help to uncover molecular requirements for infection-related development.

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