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

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Prace oryginalne / Original articles Mikologia Lekarska 2012, 19 (4): 147-153 Copyright 2012 Cornetis www.cornetis.pl ISSN 123 2-986X 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: 10.09.2011 i 3.05.2012 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

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 0.3-100 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 (2.8-3.2 μm Trichoderma harizanum, 3.5-5.0 μm Aspergillus fumigatus, 5.0-8.0 μm Cladosporium macrocarpum and 15.0-25.0 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-

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 [%] 10.09.2011 03.05.2012 10.09.2011 03.05.2012 10.09.2011 03.05.2012 10 50 12 3 5 590 16 o 45` 40.10 E 50 o 13 ` 17.40 N II 11 40 13 3 0 653 16 o 45` 3 3.50 E 50 o 14` 0.60 N III 12 3 5 14 20 825 16 o 45` 3 0.3 0 E 50 o 14` 3 0.3 0 N IV 13 3 5 15 10 910 16 o 46` 48.00 E 50 o 14` 3 7.50 N V VI (Mountain peak / Szczyt góry) Upper subalpine Piętro regla górnego 14 3 0 16 20 1057 16 o 47` 54.80 E 50 o 14` 51.60 N 15 20 17 00 1203 16 o 48` 17.80 E 50 o 15` 6.3 9 N 20.7 23.1 71.3 51.4 21.2 25.4 69.7 3 8.6 21.0 23.0 66.6 3 8.8 21.2 23.1 65.1 40.6 21.3 22.9 65.6 57.6 21.9 19.3 63.5 57.5 80 70 60 50 40 3 0 20 10 0 590 653 825 910 1057 1203 Height above sea level / Wysokość n.p.m Air temperature [ o C] research date 10.09.2011 / Temperatura powietrza [ o C] data badań 10.09.2011 Air temperature [ o C] research date 10.09.2011 / Temperatura powietrza [ o C] data badań 10.09.2011 Relative humidity of air research date 03.05.2012 / Wilgotność względna powietrza [%] data badań 03.05.2012 Relative humidity of air research date 03.05.2012 / Wilgotność względna powietrza [%] data badań 03.05.2012 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

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 10.09.2011 Tabela II: Ocena zanieczyszczeń mikologicznych powietrza w masywie Czarnej Góry data badań 10.09.2011 r. Location of measurement Miejsce pomiaru Fungus Grzyb Medium PDA / Podłoże PDA Medium acidified PDA / Podłoże PDA zakwaszone I Alternaria alternata Keissl. 770 15 620 2.4 Acremonium strictum W.Gams 10 1.3 5 0.8 Aspergillus niger Tiegh. 5 0.8 Botrytis cinerea Pers. 3 0 4.8 Cladosporium herbarum Pers. 73 0 94.8 550 88.7 Epicoccum nigrum Link 25 3.2 5 0.8 Penicillium expansum Link 5 0.6 10 1.6 II Alternaria alternata Keissl. 3 5 815 4.3 50 705 7.1 Cladosporium herbarum Pers. 73 0 89.6 63 0 89.4 Epicoccum nigrum Link 45 5.5 15 2.1 Penicillium expansum Link 5 0.6 Trichoderma harzianum Rifai 10 1.4 III Alternaria alternata Keissl. 20 775 2.6 45 605 7.4 Cladosporium cladosporioides Vries 70 9.0 Cladosporium herbarum Pers. 450 58.1 270 44.6 Epicoccum nigrum Link 45 5.8 3 0 5.0 Penicillium citrinum Thom 0 0.0 13 0 21.5 Penicillium expansum Link 190 24.5 13 0 21.5 IV Alternaria alternata Keissl. 15 880 1.7 80 790 10.1 Botrytis cinerea Pers. 10 1.3 Cladosporium herbarum Pers. 840 95.5 650 82.3 Epicoccum nigrum Link 25 2.8 40 5.1 Penicillium expansum Link 10 1.3 V Alternaria alternata Keissl. 575 20 500 4.0 Botrytis cinerea Pers. 5 0.9 20 4.0 Cladosporium cladosporioides Vries 60 10.4 Cladosporium herbarum Pers 470 81.7 460 92.0 Epicoccum nigrum Link 40 7.0 VI Acremonium strictum W.Gams 10 3 25 3.1 40 270 14.8 Botrytis cinerea Pers. 3 0 9.2 Cladosporium herbarum Pers. 270 83.1 200 74.1 Epicoccum nigrum Link 5 1.5 Penicillium expansum Link 10 3.1 3 0 11.1 150 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

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 03.05.2012 Tabela III: Ocena zanieczyszczeń mikologicznych powietrza w masywie Czarnej Góry data badań 03.05.2012 r. Location of measurement Miejsce pomiaru Fungus Grzyb Medium PDA / Podłoże PDA Medium acidified PDA / Podłoże PDA zakwaszone I Alternaria alternata Keissl. 11 287 3.8 3 3 67 0.8 Acremonium strictum W.Gams 13 3.5 Aspergillus niger Tiegh. 3 2 11.1 Cladosporium cladosporioides Vries 15 5.2 Cladosporium herbarum Pers. 146 50.9 13 7 3 7.3 Fusarium avenaceum (Fr.) Sacc. 2 0.7 Mucor mucedo de Bary & Woron 67 18.3 Penicillium chrysogenum Thom 2 0.7 20 5.4 Penicillium citrinum Thom 2 0.7 20 5.4 Rhizopus stolonifer Vuill 3 0 10.5 7 1.9 Sclerotinia sclerotiorum (Lib.) de Bary 47 16.4 100 27.2 II Alternaria alternata Keissl. 8 163 4.9 7 145 4.8 Aspergillus niger Tiegh. 13 8.0 23 15.9 Cladosporium herbarum Pers. 72 44.2 48 3 3.1 Penicillium expansum Link 6 3.7 Rhizopus stolonifer Vuill 16 9.8 3 4 23.4 Sclerotinia sclerotiorum (Lib.) de Bary 48 29.4 3 3 22.8 III Alternaria alternata Keissl. 58 176 3 3.0 13 129 10.1 Acremonium strictum W.Gams 4 2.3 Aspergillus niger Tiegh. 22 12.5 8 6.2 Candida albicans (C.P. Robin) Berkhout 2 1.6 Cladosporium herbarum Pers. 65 3 6.9 43 3 3.3 Fusarium culmorum (W.G. Sm.) Sacc. 4 2.3 Penicillium expansum Link 3 1.7 18 14.0 Rhizopus stolonifer Vuill 15 8.5 3 5 27.1 Rhodotorula glutinis (Fresen.) F.C. Harrison 2 1.6 Rhodotorula rubra (Schimon) F.C. Harrison 2 1.6 Sclerotinia sclerotiorum (Lib.) de Bary 5 2.8 6 4.7 IV Aspergillus niger Tiegh. 23 220 10.5 255 Alternaria alternata Keissl. 45 17.6 Candida albicans (C.P. Robin) Berkhout 4 1.8 Cladosporium cladosporioides Vries 13 5.9 Cladosporium herbarum Pers. 85 3 8.6 140 54.9 Fusarium avenaceum (Fr.) Sacc. 4 1.8 2 0.8 Fusarium culmorum (W.G. Sm.) Sacc. 2 0.9 1 0.4 Penicillium chrysogenum Thom 12 4.7 Penicillium citrinum Thom 45 20.5 14 5.5 Rhizopus stolonifer Vuill 3 0 13.6 5 2.0 Rhodotorula rubra (Schimon) F.C. Harrison 1 0.4 Sclerotinia sclerotiorum (Lib.) de Bary 14 6.4 3 5 13.7 V Alternaria alternata Keissl. 282 7 251 2.8 Aspergillus niger Tiegh. 23 8.2 17 6.8 Botrytis cinerea Pers. 6 2.4 Cladosporium cladosporioides Vries 116 41.1 Cladosporium herbarum Pers 93 3 3.0 180 71.7 Penicillium expansum Link 10 3.5 14 5.6 Rhizopus stolonifer Vuill 40 14.2 27 10.8 VI Alternaria alternata Keissl. 25 287 8.7 17 3 57 4.8 Aspergillus niger Tiegh. 3 1.0 3 0.8 Cladosporium cladosporioides Vries 6 2.1 Cladosporium herbarum Pers. 217 75.6 264 73.9 Epicoccum nigrum Link 3 1.0 2 0.6 Fusarium culmorum (W.G. Sm.) Sacc. 3 1.0 1 0.3 Penicillium citrinum Thom 7 2.4 3 0.8 Penicillium expansum Link 15 5.2 2 0.6 Rhizopus stolonifer Vuill 7 2.4 15 4.2 Sclerotinia sclerotiorum (Lib.) de Bary 1 0.3 50 14.0 151

Fungi in the air at the mountain trails of the Czarna Góra Massif Mikologia Lekarska 2012, 19 (4) 1000 900 Air total CFU/m 3 / Powietrze ogólna liczba jtk/m 3 800 700 600 500 400 3 00 200 100 0 I II III IV V VI Location of measurement / Miejsce pomiaru Medium PDA research date 10.09.2011 / Podłoże PDA data badań 10.09.2011 Medium acidified PDA research date 10.09.2011 / Podłoże PDA zakwaszone data badań 10.09.2011 Medium PDA research date 03.05.2012 / Podłoże PDA data badań 03.05.2012 Medium acidified PDA research date 03.05.2012 / Podłoże PDA zakwaszone data badań 03.05.2012 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 03.05.2012 / Podłoże PDA zakwaszone data badań 03.05.2012 Medium PDA research date 03.05.2012 / Podłoże PDA data badań 03.05.2012 Medium acidified PDA research date 10.09.2011 / Podłoże PDA zakwaszone data badań 10.09.2011 Medium PDA research date 10.09.2011 / Podłoże PDA data badań 10.09.2011 152 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

Grzyby występujące w powietrzu na szlakach górskich masywu Czarnej Góry Medium acidified PDA research date 03.05.2012 Podłoże PDA zakwaszone data badań 03.05.2012 Medium PDA research date 03.05.2012 Podłoże PDA data badań 03.05.2012 Medium acidified PDA research date 10.09.2011 Podłoże PDA zakwaszone data badań 10.09.2011 Medium PDA research date 10.09.2011 Podłoże PDA data badań 10.09.2011 0.0% 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. 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