Fungi in the air at the mountain trails of the Czarna Góra Massif. Preliminary study

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1 Prace oryginalne / Original articles Mikologia Lekarska 2012, 19 (4): Copyright 2012 Cornetis ISSN X Fungi in the air at the mountain trails of the Czarna Góra Massif. Preliminary study Grzyby występujące w powietrzu na szlakach górskich masywu Czarnej Góry. Doniesienie wstępne Rafał Ogórek Division of Plant Pathology and Mycology, Department of Plant Protection, Wrocław University of Environmental and Life Sciences ABSTRACT Introduction: Fungi present in the air are in the form of bioaerosols and retain in it their infectious potential. They can cause infections, secrete mycotoxins and can cause the such symptoms of the respiratory system as allergies. Aim of the study: The research aim was the evaluation of the mycological air pollutions through determination of species number and species assemblage founded in the air of mountain trail, depending on the height above sea level in different time. Material and methods: Air samples were taken from 6 locations at the mountain trails of the Czarna Góra Massif. Air Ideal 3 P sampler was used to examine the air. The studies were conducted twice: on 10 th September, 2011 and 3 rd May,2012. Results: Mycological purity of the air varied and depended on the measurement location and research date. In late summer more fungi were isolated from the air than in late spring. The most frequently isolated fungus in both research dates was Cladosporium. Conclusions: The number and composition of fungi species isolated from the air depend on the height above sea level, research date and culture medium. In late summer the air in the Czarna Góra Massif was characterized by higher number of fungi than in late spring. Fungi, which could be the cause of allergies, dominated in both research dates. The number of fungi which occur in the air, were not a threat for healthy tourists. Key words: fungi, air pollutions, mountain trails STRESZCZENIE Wprowadzenie: Grzyby w powietrzu występują w postaci bioaerozoli i zachowują w nim swój potencjał infekcyjny. Mogą one powodować infekcje, wydzielać mikotoksyny oraz powodować dolegliwości górnych dróg oddechowych w postaci alergii. Cel pracy: Określenie stopnia zanieczyszczenia zarodnikami grzybów powietrza na szlaku górskim, w zależności od wysokości nad poziomem morza w różnych terminach badań. Materiał i metody: Materiał do badań stanowiło powietrze pobrane z 6 miejsc na szlakach Czarnej Góry. Analizę powietrza przeprowadzono metodą zderzeniową (urządzenie Air Ideal 3 P). Badania były realizowane w dwóch terminach: i r. Wyniki: Czystość mikologiczna powietrza była zróżnicowana i zależała od miejsca pomiaru i terminu badań. Pod koniec okresu letniego wyizolowano więcej grzybów z powietrza niż pod koniec okresu wiosennego. Najczęściej izolowanym rodzajem grzyba w obu terminach badań był Cladosporium. Wnioski: Liczba i skład gatunkowy grzybów izolowanych z powietrza zależał od wysokości nad poziomem morza, terminu badań i podłoża hodowlanego. Pod koniec lata powietrze masywu Czarnej Góry charakteryzowało się większą liczbą grzybów niż pod koniec wiosny. W obu terminach badań dominowały grzyby, które mogą powodować alergie. Liczba grzybów uzyskanych z powietrza nie była zagrożeniem dla zdrowych turystów. Słowa kluczowe: grzyby, zanieczyszczenia powietrza, szlaki górskie 147

2 Fungi in the air at the mountain trails of the Czarna Góra Massif Mikologia Lekarska 2012, 19 (4) 148 Introduction Microorganisms in the air occur in the form of bioaerosols, what are systems contain the dispersing phase in the form of air and the dispersed phase consisting of biological materials (viruses, protozoa, bacterial cells, cell fragments of plants and animals, fragments of fungal mycelium, spores) [1]. Bioaerosols represent about 5-3 4% of indoor air pollution, and the dispersed phase is usually μm in diameter [2]. For example, a single bacterial cells and spores of bacteria are size from 0.5 to 2.0 μm (Bacillus spp., Pseudomonas spp.), fungal spores are larger ( μm Trichoderma harizanum, μm Aspergillus fumigatus, μm Cladosporium macrocarpum and μm Epiccocum nigrum) [3 ]. Harmful biological agents can be divided into: infectious and invasive agents, allergens, biological toxins, carcinogens and biological vectors [4]. The qualitative and quantitative composition of pollution in the air is variable in time and space, constantly changing as a result of air movements and impacts with their molecules [5]. The content of microorganisms in the air depends on many factors including geographical ones inter alia area, season [6]. However, the most important factors affecting the survival of the fungi include humidity and temperature, because the development of all of these microorganisms is faster in increased humidity [7]. Literature data reports, that in Poland from the mountain air (the Śnieżnik Massif ) isolated following species of fungi: Rhodotorula, Rhizopus, Penicillium, Epicoccum, Cladosporium, Botrytis and Alternaria [8]. Among listed above genus of fungi, there are fungi, which cause serious illnesses, and those, which cause allergies. For example Rhizopus spp. may cause mucormycosis of lungs, sinuses and generalized, Candida spp. may cause candidiasis of the mucosal membranes of the mouth and throat and genital and systemic candidiasis [9, 10] and Alternaria spp. or Cladosporium spp. may cause allergies [11]. Fungi can cause in humans and other mammals fungal infections (mycoses) and allergic reactions [12]. The mycoses are the diseases caused by fungi colonizing the surface of the skin or developing inside the human organism. The fungal infections (mycoses) usually are caused by yeasts and fungi of the genus Aspergillus. A special type of fungus infections are infections caused by dermatophytes. These are the only fungi which attack skin, nails and hair. Fungi, which cause dermatophyte infections belong to genus Microsporum (attack hair and skin), Epidermophyton (attack nails and cuticle) and Trichophyton (cause fungal nails, skin and hair) [13 ]. The symptoms of allergic reactions can include: skin lesions, mucous membrane, chronic rhinitis or cough. The most common allergens are fungi of genus Cladosporium, Alternaria, Fusarium, Penicillium and Aspergillus. However, the concentrations of fungi considered to be harmless (such as Acremonium spp., Trichoderma spp.) for health is very large, but they can be much more harmful than pathogenic fungi found in lower concentrations [12]. Aim of the study The research objective was to evaluate the extent of the mycological air pollutions through determination of species number and species assemblage founded in the air of the mountain trail, depending on the height above sea on different research dates. Materials and methods Air samples were taken on 10 th of September 2011 (the first research date) and 3 rd May 2012 (the second research date) from 6 locations at the mountain trails of the Czarna Góra Massif. To examine the air, two culture media were used: PDA (Potato Dextrose Agar), Biocorp and acidified PDA, Biocorp with 0.5% acetic acid. The sampler (Air Ideal 3 P) was programmed for sample volumes of 50 L, 100 L and 150 L of the air. Measurement in places was performed in six-plicate for each volume. The sampler was positioned 1.5 m above the ground. The incubation of cultures on Petri dishes (90mm diameter) was carried out at room temperature (±22 C) for 2-7 days. After the incubation the number of CFU/1 m 3 (Colony Forming Unit/1 m 3 of air) was calculated. The temperature and relative humidity was measured using thermohygrometer AB-171 Data Logger (Abtronic). The longitude and latitude was determined using navigation Go Live 825 (TomTom). The specific identification of the isolates of the sampled fungi was performed using macro- and microscopic observations of the colonies which have grown on culture media, according to the commonly accepted methods used in mycological laboratories. The fungi were identified using diagnostic keys [14-16]. Results The prevalent temperature in the investigated locations on both research dates ranged between 19.3 and 25.4 C and was so beneficial for the development of fungi. The relative humidity of air was low and it was not very beneficial for development of fungi especially during the second research date (tab. I, fig. 1). The mycological analysis of the air samples has shown, that fungal air pollution varied between studied locations. Furthermore, the number of fungal colonies isolated from the sampled air depended on research date and medium tab. II, III, fig. 2. The highest numbers of Colony Forming Unit in 1m 3 of air (CFU/m 3 ) on both mediums were isolated from the forth location (height 910 meters above sea level, lower subalpine of vegetation layers) for the first research date and from the sixth location (height 1203 meters above sea level, upper subalpine of vegetation layers) for the second research date. The least numbers of CFU/m 3 were isolated from the air for the first research date from the sixth location (height 1203 meters above sea level, upper subalpine of vegetation layers) on both mediums and for the second research date from the second location on PDA and the third location on acidified PDA medium (height 653 and 825 meters above sea level, lower subalpine of vegetation layers) tab. II, III, fig. 2. From all of studied locations on both research dates and both mediums 19 species of fungi were isolated from the air. In the first research on both mediums 10 species of fungi (7 on PDA medium and 9 on acidified PDA) were isolated and in the second research 18 species of fungi (14 on PDA medium and 17 on acidified PDA). Cladosporium spp. occurred in the largest number on both research dates fig. 3. The fungi such as Fusarium avenaceum, Fusarium culmorum, Mucor mucedo, Penicillium chrysogenum, Rhizopus stolonifer, Sclerotinia sclerotiorum and the yeasts such as Candida albicans, Rhodotorula glutinis and Rhodotorula rubra were isolated only from the air during the second research. Whereas Trichoderma spp. was only isolated during the first research tab. II, III, fig. 3. The highest percentage of Cladosporium spp. to other fungi isolated from the air was noted for PDA medium in the first rese-

3 Grzyby występujące w powietrzu na szlakach górskich masywu Czarnej Góry Table I: Tabela I: Temperature and humidity of air during measurements Temperatura i wilgotność powietrza podczas pomiarów Location of measurement Miejsce pomiaru I Layers of vegetation Piętra roślinności Lower subalpine Piętro regla dolnego Time of measurement Godzina pomiaru Height ASL Wysokość n.p.m. Longitude and latitude Długość i szerokość geograficzna During the study Podczas badań Air temperature [ o C] Temperatura powietrza [ o C] Relative humidity of air [%] Wilgotność względna powietrza [%] o 45` E 50 o 13 ` N II o 45` E 50 o 14` 0.60 N III o 45` E 50 o 14` N IV o 46` E 50 o 14` N V VI (Mountain peak / Szczyt góry) Upper subalpine Piętro regla górnego o 47` E 50 o 14` N o 48` E 50 o 15` N Height above sea level / Wysokość n.p.m Air temperature [ o C] research date / Temperatura powietrza [ o C] data badań Air temperature [ o C] research date / Temperatura powietrza [ o C] data badań Relative humidity of air research date / Wilgotność względna powietrza [%] data badań Relative humidity of air research date / Wilgotność względna powietrza [%] data badań Fig. 1. Temperature and humidity of air during measurements Ryc. 1. Temperatura i wilgotność powietrza podczas pomiarów arch and the least for acidified PDA medium during the second research fig. 4. Discussion The content of microorganisms in the air is conditioned by many factors e.g. the strength, direction of winds, the amount and structure of plant species, which are responsible indirectly for the development, number and species composition of microorganism in the air [8]. Environmental conditions in the Czarna Góra Massif are varied and influenced the number of fungi isolated from the air. Flora in Czarna Góra is similar to flora of Śnieżnik and is characterized by intermediate character of flora, between Western Sudety and Western Karpaty [17]. During the first research (end of summer 10 th September, 2011) from the air were isolated more fungi than in the second research (end of spring 3 rd May, 2012). This was probably caused by low air relative humidity and start of growing season of vegetation during measurements. Most of fungal spores in the first research were isolated from the air in lower subalpine vegetation layers (the forth location) and in the second research from the sixth location (upper subal- 149

4 Fungi in the air at the mountain trails of the Czarna Góra Massif Mikologia Lekarska 2012, 19 (4) Table II: Valuation of mycological pollutions of the air at the Czarna Góra Massif research date Tabela II: Ocena zanieczyszczeń mikologicznych powietrza w masywie Czarnej Góry data badań r. Location of measurement Miejsce pomiaru Fungus Grzyb Medium PDA / Podłoże PDA Medium acidified PDA / Podłoże PDA zakwaszone I Alternaria alternata Keissl Acremonium strictum W.Gams Aspergillus niger Tiegh Botrytis cinerea Pers Cladosporium herbarum Pers Epicoccum nigrum Link Penicillium expansum Link II Alternaria alternata Keissl Cladosporium herbarum Pers Epicoccum nigrum Link Penicillium expansum Link Trichoderma harzianum Rifai III Alternaria alternata Keissl Cladosporium cladosporioides Vries Cladosporium herbarum Pers Epicoccum nigrum Link Penicillium citrinum Thom Penicillium expansum Link IV Alternaria alternata Keissl Botrytis cinerea Pers Cladosporium herbarum Pers Epicoccum nigrum Link Penicillium expansum Link V Alternaria alternata Keissl Botrytis cinerea Pers Cladosporium cladosporioides Vries Cladosporium herbarum Pers Epicoccum nigrum Link VI Acremonium strictum W.Gams Botrytis cinerea Pers Cladosporium herbarum Pers Epicoccum nigrum Link Penicillium expansum Link pine vegetation layers). However, last concentration of spores for the first research was in upper subalpine vegetation layers (the sixth location) and for the second research in lower subalpine vegetation layers (PDA the second location, acidified PDA the third location). This was probably due to humidity of the air, which during the first period of research was more than 63 % for all measuring points. However, during the second period of the study was low and it reached the highest values in the upper subalpine vegetation levels. In summer, average daily temperatures are higher than in spring and most of the plants is already in the final period of growth, acting in good conditions for development of fungi. In the second research most of fungal spores was isolated from the sixth location. This was probably caused by the influx of air masses of spores, which can easily reach the place of research located on a top of a mountain. In the air of the Czarna Góra Massif on both research dates, fungi of the genus Cladosporium were dominated. This is confirmed by reports of Ogórek et al. [8], who examined fungi in the mountain trails of the Śnieżnik Massif. Domsch et al. [18] also reported that, Cladosporium spp. are common in many parts of the world, they are cosmopolitan organisms and their spores can be found in air, soil and water. The level of concentrations of Cladosporium spores in the air (in 1 m 3 ) during a day has a very large variation over the year: from zero to several thousand spores. In

5 Grzyby występujące w powietrzu na szlakach górskich masywu Czarnej Góry Table III: Valuation of mycological pollutions of the air at the Czarna Góra Massif research date Tabela III: Ocena zanieczyszczeń mikologicznych powietrza w masywie Czarnej Góry data badań r. Location of measurement Miejsce pomiaru Fungus Grzyb Medium PDA / Podłoże PDA Medium acidified PDA / Podłoże PDA zakwaszone I Alternaria alternata Keissl Acremonium strictum W.Gams Aspergillus niger Tiegh Cladosporium cladosporioides Vries Cladosporium herbarum Pers Fusarium avenaceum (Fr.) Sacc Mucor mucedo de Bary & Woron Penicillium chrysogenum Thom Penicillium citrinum Thom Rhizopus stolonifer Vuill Sclerotinia sclerotiorum (Lib.) de Bary II Alternaria alternata Keissl Aspergillus niger Tiegh Cladosporium herbarum Pers Penicillium expansum Link Rhizopus stolonifer Vuill Sclerotinia sclerotiorum (Lib.) de Bary III Alternaria alternata Keissl Acremonium strictum W.Gams Aspergillus niger Tiegh Candida albicans (C.P. Robin) Berkhout Cladosporium herbarum Pers Fusarium culmorum (W.G. Sm.) Sacc Penicillium expansum Link Rhizopus stolonifer Vuill Rhodotorula glutinis (Fresen.) F.C. Harrison Rhodotorula rubra (Schimon) F.C. Harrison Sclerotinia sclerotiorum (Lib.) de Bary IV Aspergillus niger Tiegh Alternaria alternata Keissl Candida albicans (C.P. Robin) Berkhout Cladosporium cladosporioides Vries Cladosporium herbarum Pers Fusarium avenaceum (Fr.) Sacc Fusarium culmorum (W.G. Sm.) Sacc Penicillium chrysogenum Thom Penicillium citrinum Thom Rhizopus stolonifer Vuill Rhodotorula rubra (Schimon) F.C. Harrison Sclerotinia sclerotiorum (Lib.) de Bary V Alternaria alternata Keissl Aspergillus niger Tiegh Botrytis cinerea Pers Cladosporium cladosporioides Vries Cladosporium herbarum Pers Penicillium expansum Link Rhizopus stolonifer Vuill VI Alternaria alternata Keissl Aspergillus niger Tiegh Cladosporium cladosporioides Vries Cladosporium herbarum Pers Epicoccum nigrum Link Fusarium culmorum (W.G. Sm.) Sacc Penicillium citrinum Thom Penicillium expansum Link Rhizopus stolonifer Vuill Sclerotinia sclerotiorum (Lib.) de Bary

6 Fungi in the air at the mountain trails of the Czarna Góra Massif Mikologia Lekarska 2012, 19 (4) Air total CFU/m 3 / Powietrze ogólna liczba jtk/m I II III IV V VI Location of measurement / Miejsce pomiaru Medium PDA research date / Podłoże PDA data badań Medium acidified PDA research date / Podłoże PDA zakwaszone data badań Medium PDA research date / Podłoże PDA data badań Medium acidified PDA research date / Podłoże PDA zakwaszone data badań Fig. 2. Total number of fungi isolated from the air in the studied locations of the Czarna Góra Massif Ryc. 2. Ogólna liczba grzybów wyizolowanych z powietrza badanych miejsc masywu Czarnej Góry Trichoderma harzianum Sclerotinia sclerotiorum Rhodotorula rubra Rhodotorula glutinis Rhizopus stolonifer Penicillium expansum Penicillium citrinum Penicillium chrysogenum Mucor mucedo Fusarium culmorum Fusarium avenaceum Epicoccum nigrum Cladosporium herbarum Cladosporium cladosporioides Candida albicans Botrytis cinerea Aspergillus niger Acremonium strictum Alternaria alternata 0.0% 10.0% 20.0% 3 0.0% 40.0% 50.0% 60.0% 70.0% 80.0% 90.0% Medium acidified PDA research date / Podłoże PDA zakwaszone data badań Medium PDA research date / Podłoże PDA data badań Medium acidified PDA research date / Podłoże PDA zakwaszone data badań Medium PDA research date / Podłoże PDA data badań Fig. 3. The percentage participation of fungi genus isolated from the air in all studied locations of the Czarna Góra Massif Ryc. 3. Procentowy udział rodzajów grzybów wyizolowanych z powietrza wszystkich badanych miejsc masywu Czarnej Góry

7 Grzyby występujące w powietrzu na szlakach górskich masywu Czarnej Góry Medium acidified PDA research date Podłoże PDA zakwaszone data badań Medium PDA research date Podłoże PDA data badań Medium acidified PDA research date Podłoże PDA zakwaszone data badań Medium PDA research date Podłoże PDA data badań % 20.0% 40.0% 60.0% 80.0% Fig. 4. The percentage of Cladosporium spp. to all other fungi isolated from the air in all studied locations of the Czarna Góra Massif Ryc. 4. Procentowy udział Cladosporium spp. do wszystkich pozostałych grzybów wyizolowanych z powietrza badanych miejsc z masywu Czarnej Góry the European countries, the peak of season of Cladosporium is in the months from June to September [19, 20]. Cladosporium spp. can cause mainly allergies and rarely opportunistic infections. This problem is relate mainly to people with compromised immune systems such as patients with hematological diseases or AIDS. There are also reports about infections in healthy people by Cladosporium carrionii, which may cause changes chromoblastomycosis [21]. These fungi can cause diseases of plants such as black point of cereals, scab of cucurbits and brown spots on tomato leaves [22, 23 ]. People who experience allergic symptoms should avoid contact with a fungus. During the rainless weather, the best time to go for a walk, is morning. It is caused by lowest concentration of fungal spores in the air. It is also worth to order tests for the presence of fungi in the apartment. Conclusions 1. The number and composition of fungi species isolated from the air depend on the height above sea level, research date and culture medium. 2. In late summer the air in the Czarna Góra Massif was characterized by higher number of fungi than in late spring. 3. Fungi, which could be the cause of allergies, dominated on both research dates. 4. The number of fungi which occur in the air, were not a threat for healthy tourists. References 1. An HA, Mainelis G, Yao M. Evaluation of a high-volume portable bioaerosol sampler in laboratory and field environments. Indoor Air. 2004;14: Gołofit-Szymczak M, Skowron J. Zagrożenia mikrobiologiczne w pomieszczeniach biurowych. Bezpieczeństwo pracy. 2005;3 : Srikanth P, Sudharsanam S, Steinberg R. Bio-aerosols in Indoor Environment: composition, health effects and analysis. Indian J Med Microbiol. 2008;26: Menetrez MY, Foarde KK, Dean TR. An evaluation of the protein mass of particulate matter. Atmospheric Environ. 2007;4: Krajewska-Kułak E, Gniadek A, Łukaszuk CR, et al. Wstępne porównanie wyników badań zanieczyszczenia powietrza grzybami z wykorzystaniem aparatu SAS SUPER 100 i MAS 100. Doniesienie wstępne. Mikol Lek. 2009;16: Kaiser K. Zalecenia normatywne dla systemów wentylacji i klimatyzacji w szpitalach, cz.1. Technika Chłodnicza i Klimatyzacyjna. 2005;3 : Przondo-Mordarska A. Zakażenia szpitalne. Etiologia i przebieg. Wrocław: Continuo; p Ogórek R, Lejman A, Pląskowska E, Bartnicki M. Fungi in the mountain trails of the Śnieżnik Massif. Mikol Lek. 2012;2: Adamski Z, Henke K, Zawirska A, et al. Grzybice narządowe. W: Baran E, red. Mikologia co nowego? Wrocław: Cornetis; p Adamski Z, Henke K, Zawirska A, et al. Grzybice błon śluzowych. W: Baran E, red. Mikologia co nowego? Wrocław: Cornetis; p Adamski Z, Henke K, Zawirska A, et al. Rola grzybów w etiopatogenezie chorób alergicznych. W: Baran E, red. Mikologia co nowego? Wrocław: Cornetis; p Zykubek K. Bioareozole grzybowe w magazynach archiwalnych zagrożenie dla ludzi i zasobu. Problemy jakości powietrza wewnętrznego w Polsce Warszawa: Inst. Ogrzewnictwa i Wentylacji; p Macura AB. Diagnostyka grzybów część III. Diagnostyka dermatofitów. Diagnosta Laboratoryjny. 2009;1: Ellis MB. Dematiaceous Hyphomycetes. Kew, Surrey: Commonwealth Mycol. Inst.; Raper KB, Fennell DI. The genus Aspergillus. Baltimore: Willims and Wilkins Co.; Raper KB, Thom C. A manual of the Penicillia. New York: Hafner Publishing Co.; Fabiszewski J, Brej T. Dynamika przemian flory i roślinności. W: Jahn A, Kozłowski S, Pulina M, red. Masyw Śnieżnika. Zmiany w środowisku przyrodniczym. Warszawa: PAE; p Domsch KH, Games W, Anderson TH. Compendium of soil fungi. London: Academic Press; p D Amato G, Spieksma FThM. Aerobiologic and clinical aspects of mould allergy in Europe. Allergy. 1995;50: Lipiec A. Mould hypersensitivity in allergic rhinitis patients. In Rev Allergol Clin Immunol. 2000;6: Plomer-Niezgoda E, et al. Pathogenicity of fungi from the genus of Alternaria, Cladosporium and Chrysosporium. Mikol Lek. 1998;5: Kochman J. Zarys mikologii dla fitopatologów. Warszawa: SGGW; p , 23. Kryczyński S, Weber Z. Fitopatologia. Choroby roślin uprawnych (tom 2). Poznań: PWRiL; p Publication is co-financed by the European Union as part of the European Social Fund. Received: Approved: Conflict of interests: none declared Address for correspondence: M.Sc. Eng. Rafał Ogórek Pl. Grunwaldzki 24a Wrocław, Poland tel.: rafal.ogorek@up.wroc.pl 153