Unmanaged grasslands are a reservoir of Alternaria and other important fungal species with differing emission patterns.

Alternaria is a ubiquitous fungal genus with many allergenic and pathogenic species inhabiting grasslands. We hypothesise that grasslands (natural/man-made) host a diversity of fungal species whose spores have varying emission patterns. Therefore, the purpose of this study was to examine the potential of grasslands for emission, diversity and composition of Alternaria and other fungal species. To test the hypothesis, Hirst-type and multi-vial Cyclone samplers collected air samples from two grassland sites (unmanaged and managed) and a non-grassland site at Lakeside campus of the University of Worcester, United Kingdom for the period May to September 2019. The unmanaged grassland was originally planted with grasses and left uncut for three years. The managed grassland was a roadside verge that was cut once every year, typically after most grasses have flowered. We used optical microscopy and Illumina MiSeq sequencing to investigate the emission, abundance, diversity and composition of the fungal spores from each site alongside meteorological variables. Kruskal-Wallis and Wilcoxon tests examined differences in the bi-hourly Alternaria concentrations between the sites. Shannon's and Simpson's Index determined the diversity of the fungal spores between the unmanaged and non-grassland sites. The results showed that grasslands are a strong source of Alternaria spores with considerably higher numbers of clinically important days compared with the non-grassland site. The managed grassland varied in Alternaria spore emission pattern from the unmanaged, probably due to differences in environmental variables and cutting frequency. The unmanaged grassland and non-grassland sites showed a high diversity of fungi including Alternaria, Cladosporium, Ascochyta, Botrytis and Aureobasidium. Overall, the study shows that grasslands are a strong source of fungal spores with allergenic and pathogenic potential and have varying emission patterns, compared with nearby urban areas where monitoring stations are located. This information is useful for atmospheric modelling of airborne fungal spore sources and has implications for allergy sufferers in particular.

Outdoor airborne allergens: Characterization, behavior and monitoring in Europe.

Aeroallergens or inhalant allergens, are proteins dispersed through the air and have the potential to induce allergic conditions such as rhinitis, conjunctivitis, and asthma. Outdoor aeroallergens are found predominantly in pollen grains and fungal spores, which are allergen carriers. Aeroallergens from pollen and fungi have seasonal emission patterns that correlate with plant pollination and fungal sporulation and are strongly associated with atmospheric weather conditions. They are released when allergen carriers come in contact with the respiratory system, e.g. the nasal mucosa. In addition, due to the rupture of allergen carriers, airborne allergen molecules may be released directly into the air in the form of micronic and submicronic particles (cytoplasmic debris, cell wall fragments, droplets etc.) or adhered onto other airborne particulate matter. Therefore, aeroallergen detection strategies must consider, in addition to the allergen carriers, the allergen molecules themselves. This review article aims to present the current knowledge on inhalant allergens in the outdoor environment, their structure, localization, and factors affecting their production, transformation, release or degradation. In addition, methods for collecting and quantifying aeroallergens are listed and thoroughly discussed. Finally, the knowledge gaps, challenges and implications associated with aeroallergen analysis are described.

Using qPCR and microscopy to assess the impact of harvesting and weather conditions on the relationship between Alternaria alternata and Alternaria spp. spores in rural and urban atmospheres.

Alternaria is a plant pathogen and human allergen. Alternaria alternata is one of the most abundant fungal spores in the air. The purpose of this study was to examine whether Alternaria spp. spore concentrations can be used to predict the abundance and spatio-temporal pattern of A. alternata spores in the air. This was investigated by testing the hypothesis that A. alternata dominates airborne Alternaria spp. spores and varies spatio-temporally. Secondarily, we aimed at investigating the relationship between airborne Alternaria spp. spores and the DNA profile of A. alternata spores between two proximate (~ 7 km apart) sites. These were examined by sampling Alternaria spp. spores using Burkard 7-day and cyclone samplers for the period 2016-2018 at Worcester and Lakeside campuses of the University of Worcester, UK. Daily Alternaria spp. spores from the Burkard traps were identified using optical microscopy whilst A. alternata from the cyclone samples was detected and quantified using quantitative polymerase chain reaction (qPCR). The results showed that either A. alternata or other Alternaria species spores dominate the airborne Alternaria spore concentrations, generally depending on weather conditions. Furthermore, although Alternaria spp. spore concentrations were similar for the two proximate sites, A. alternata spore concentrations significantly varied for those sites and it is highly likely that the airborne samples contained large amounts of small fragments of A. alternata. Overall, the study shows that there is a higher abundance of airborne Alternaria allergen than reported by aerobiological networks and the majority is likely to be from spore and hyphal fragments.

Environmental DNA reveals diversity and abundance of Alternaria species in neighbouring heterogeneous landscapes in Worcester, UK.

Alternaria is a pathogenic and allergenic fungus affecting 400 plant species and 334 million people globally. This study aimed at assessing the diversity of Alternaria species in airborne samples collected from closely located (7 km apart) and heterogeneous sites (rural, urban and unmanaged grassland) in Worcester and Lakeside, the UK. A secondary objective was to examine how the ITS1 subregion varies from ITS2 in Alternaria species diversity and composition. Airborne spores were collected using Burkard 7-day and multi-vial Cyclone samplers for the period 5 July 2016-9 October 2019. Air samples from the Cyclone were amplified using the ITS1and ITS2 subregions and sequenced using Illumina MiSeq platform whereas those from the Burkard sampler were identified and quantified using optical microscopy. Optical microscopy and eDNA revealed a high abundance of Alternaria in the rural, urban and unmanaged sites. ITS1 and ITS2 detected five and seven different Alternaria species at the three sampling sites, respectively. A. dactylidicola, A. metachromatica and A. infectoria were the most abundant. The rural, urban and unmanaged grassland sites had similar diversity (PERMANOVA) of the species due to similarity in land use and proximity of the sites. Overall, the study showed that heterogeneous and neighbouring sites with similar land uses can have similar Alternaria species. It also demonstrated that an eDNA approach can complement the classical optical microscopy method in providing more precise information on fungal species diversity in an environment for targeted management. Similar studies can be replicated for other allergenic and pathogenic fungi. Supplementary Information: The online version contains supplementary material available at 10.1007/s10453-022-09760-9.