Elsevier

Fungal Biology

Volume 116, Issue 2, February 2012, Pages 214-224
Fungal Biology

DNA analysis of outdoor air reveals a high degree of fungal diversity, temporal variability, and genera not seen by spore morphology

https://doi.org/10.1016/j.funbio.2011.11.004Get rights and content

Abstract

Fungi are ubiquitous with many capable of causing disease by direct infection, toxicoses, or allergy. Fungal spores are present in outdoor air throughout the year, yet airborne diversity is poorly characterised. Airborne fungal spores are routinely counted by microscopy, enabling identification to genera at best. We generated traditional microscopic counts over a year, then used environmental sequencing techniques to assess and compare 3 d selected from the main fungal spore season. The days selected corresponded to one with a high quantity of spores unidentifiable by microscopy, and two representing dry and wet summer periods. Over 86 % of genera detected by sequencing were not routinely identifiable by microscopy. A high degree of temporal variability was detected, with the percentage of clones attributed to Basidiomycota or Ascomycota, and composition of genera within each phylum varying greatly between days. Throughout the year Basidiomycota spores were found at higher levels than Ascomycota, but levels fluctuated daily with Ascomycota comprising 11–84 % of total spores and Basidiomycota 7–81 %. No significant difference was found between the proportion of clones attributed to each morphological group detected by sequencing to that counted by microscopy (P = 0.477, 0.985, and 0.561). The majority of abundant genera detected by DNA analysis are not routinely identified by microscopy (>75 %). Of those, several are known human and plant pathogens, and may represent unrecognised aeroallergens.

Highlights

► Fungal bioaerosols are ubiquitous, yet airborne diversity is poorly characterised. ► Outdoor microscopic fungal spore counts were generated over the course of a year. ► Environmental clone sequencing was used to determine fungal diversity on 3 d. ► >86 % of genera detected by sequencing were not routinely identifiable by microscopy. ► Levels of spores and DNA correlated & high degree of temporal variability detected.

Introduction

Aerobiology as a term was first used in the 1930s, and is defined as the study of biological particles present in the air, both outdoors (extramural) and indoors (intramural); and includes the study of airborne pollen grains and fungal spores. The pollen component is well characterised, with pollen monitoring networks established in many countries. In contrast, the airborne fungal load is poorly defined with few sites actively monitoring daily levels, due in part to limitations of traditional identification methodologies. Fungi are ubiquitous and fungal spores are present in outdoor air throughout the year, with many fungi exhibiting seasonal periodicity. The number of fungal spores per cubic metre of air can often exceed pollen concentrations by 100–1000 fold (Horner et al. 1995). Many airborne fungi are capable of causing disease by direct infection, toxicoses, or allergy. Incidences of allergy are rising, with fungal respiratory allergy affecting up to 30 % of atopic individuals. There is a clear association between life-threatening asthma and sensitisation to fungal allergens, and studies have correlated outdoor spore concentrations with asthma symptoms (Black et al. 2000). The prevalence of fungal allergy in severe asthma ranges from 35 % to 70 % (Denning et al. 2006). With regard to agriculture, many important fungal plant pathogens are dispersed by wind or rain-splash, and are capable of causing severe losses in susceptible crops with significant economic consequences. Long-distance dispersal is an important survival strategy for many fungi, and has caused the spread of important diseases on a continental or global scale. The ability to detect these fungal pathogens from air samples can be used in an effort to devise or improve disease control methods (West et al. 2008).

Traditionally, airborne fungal biodiversity studies were based on culture-dependent methods which inevitably underestimate diversity, and grossly bias studies towards fungi that can be cultured on generic fungal growth media. The current standard device for most aerobiological studies is an automatic volumetric spore trap, with morphological identification of spores; limiting studies by the time and skill required to count them. Of the 40 or so fungal categories that can be recognised, some can be classified to genus, a few to species, but many have to be recorded in groups with similar characteristics (Lacey 1996).

PCR-based methods can detect and quantify biological material in air samples; and a number of total fungal and species-specific assays have been developed (Williams et al., 2001, Haugland et al., 2004). These assays, however, have primarily been designed to quantify fungi whose presence had previously been demonstrated from culture-based studies, and are therefore limited to the same biased view of fungal diversity. Before an assay to measure airborne spores can be developed, a comprehensive understanding of common airborne fungi is required without the bias of culture or limitations of morphological studies.

In contrast to indoor air, which has been analysed from both residential and occupational environments, very few studies have used a molecular approach to study outdoor airborne fungal diversity. Those that have targeted various regions of the fungal nuclear ribosomal operon (rDNA) which is present in multiple copies, universally applicable, and has far greater representation in publicly available databases than any other region (Ward et al. 2004). Three studies have targeted 18s rDNA, analysing air samples from Phoenix Arizona, USA (Boreson et al. 2004), Boulder Colorado, USA (Fierer et al. 2008), and San Diego California, USA (Urbano et al. 2011). Three others have targeted internal transcribed spacer region (ITS) rDNA, two analysing air samples from Germany (Despres et al., 2007, Frohlich-Nowoisky et al., 2009), the third analysing air collected from Seoul, Korea (Lee et al. 2010). The time period during which individual air samples were collected varied from a few hours (Fierer et al., 2008, Urbano et al., 2011), to 24 h (Boreson et al., 2004, Lee et al., 2010) to several days (Despres et al., 2007, Frohlich-Nowoisky et al., 2009). Surprisingly, given the risk of bias being introduced through primer selection (Anderson et al. 2003), none of the previous airborne diversity studies have compared data generated using a molecular approach to data from more traditional microscopic analysis sampled simultaneously; although one compared a culture-dependant method to a DNA-based method (Urbano et al. 2011), unsurprisingly finding samples collected by culture were not similar to clones in the 18S rRNA gene clone library. This present study used environmental cloning and sequencing techniques to assess and compare airborne fungal diversity from a central UK location on 3 d during the main fungal spore season, and to compare the level of diversity detectable by the molecular approach to data generated using traditional microscopic analysis.

Section snippets

Sample collection and microscopic analysis

Each sample was collected over a 24 h period from midnight, representing an individual day. Outdoor air samples were collected using two traps located 2 m apart on the roof of a building on the University of Leicester campus, 12 m above ground level in an urban area 60 m above sea level and approximately 1 km south of the city centre, recently shown to be sufficient for aeroallergen analysis for a 41 km area (Pashley et al. 2009). These traps sample air that has been thoroughly mixed by the

Results

An environmental clone sequencing approach was developed to analyse airborne fungal diversity in greater detail than currently available using microscopic identification of airborne fungal spores. An overview of the methodology is shown in Fig 1. In brief, airborne particles were collected directly into a microcentrifuge tube sampling 16.5 L min−1 for 24 h, from which total genomic DNA was extracted. The fungal component was targeted by amplifying LSU rDNA with universal fungal primers. The

Discussion

This is the first study that we are aware of that has used a molecular approach to determine airborne fungal biodiversity and compared the data generated to results from direct microscopic identification of airborne fungal spores. This is also the first study to apply a molecular approach to identify air sampled within the UK. Traditional aerobiological studies identify some spores to genera, but many are recorded under broader morphological classifications. In this study nearly half the fungal

Acknowledgements

We would like to thank Richard Edwards for assistance with outdoor fungal spore counts, Adam Berg for assistance with mothur, and the European Regional Development fund and the Midlands Asthma and Allergy Research Association (MAARA) for funding.

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