Fungi in the mountain trails of the Śnieżnik Massif

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1 PRACE ORYGINALNE / ORIGINAL ARTICLES Mikologia Lekarska 2012, 19 (2): Copyright 2012 Cornetis ISSN X Grzyby występujące w powietrzu na szlakach górskich masywu Śnieżnika Rafał Ogórek 1, Agnieszka Lejman 2, Elżbieta Pląskowska 1, Michał Bartnicki 1 1 Division of Plant Pathology and Mycology, Department of Plant Protection, Wrocław University of Environmental and Life Sciences, Poland 2 Department of Agroecosystems and Green Areas Management, Wrocław University of Environmental and Life Sciences, Poland ABSTRACT Introduction: Fungi present in the air are in the form of bioaerosols and retain in it their infectious potential. They can secrete mycotoxins and can cause symptoms of the respiratory system as allergies. Aim of the study: The research was focused on evaluation of mycological air pollution through determination of the number of species and species assemblage found in the air of mountain trails, depending on the height above the sea level. Material and methods: Air samples were taken from 6 locations on the mountain trails of the Śnieżnik Massif. To examine the air, Air Ideal 3P sampler and two culture media: PDA and acidified PDA were used. Results: Mycological air pollution analysis showed that CFU/m 3 air (Colony Forming Unit of 1 m 3 air) values for different culture media varied and depended on the height above the sea level. The highest numbers of CFU/m 3 air on both media were isolated from the 2 nd location (height 825 ASL) and the 5 th location (height 1306 ASL). The least numbers of CFU/m 3 air on both media were isolated from the 4 th location (height 1167 ASL). The most frequently isolated genus of fungus on both culture media was Cladosporium. Conclusions: At the end of summer time, in the air of the Śnieżnik Massif, fungi which could be the cause of allergies (espescially Cladosporium spp.) were dominating. The number of isolated fungi from the air, was not a threat for healthy tourists, but it could be the cause of upper respiratory symptoms in allergic patients. The number and species composition of fungi isolated from the air depend on the environment (temperature, humidity, vegetation, air movement), which depends on the height above the sea level. 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 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. Materiał i metody: Materiał do badań stanowiło powietrze pobrane z 6 miejsc na szlakach Śnieżnika. Analizę powietrza przeprowadzono metodą zderzeniową (urządzenie Air Ideal 3P) z użyciem podłoża hodowlanego PDA i PDA zakwaszonego. Wyniki badań: Analiza mikologiczna pobranych próbek powietrza wykazała, że wartości CFU (colony forming unit jtk) w 1 m 3 powietrza były zróżnicowane i zależały od rodzaju użytego w doświadczeniu podłoża oraz wysokości n.p.m. Największą wartość CFU/m 3 powietrza na obu podłożach hodowlanych została uzyskana z drugiego miejsca pomiarów (wysokość 825 m n.p.m.) oraz z piątego miejsca (wysokość 1306 m n.p.m.). Najmniejszą wartość CFU/m 3 powietrza na obu podłożach uzyskano z czwartej lokalizacji (wysokość 1167 m n.p.m.). Najczęściej izolowanym rodzajem grzyba na obu podłożach był Cladosporium. Wnioski: Pod koniec lata w powietrzu masywu Śnieżnika dominowały grzyby, które mogą być przyczyną alergii (w szczególności Cladosporium spp.). Liczba wyizolowanych grzybów z powietrza nie była zagrożeniem dla zdrowych turystów, ale mogła być przyczyną m.in. dolegliwości górnych dróg oddechowych u alergików. Liczba i skład gatunkowy grzybów izolowanych z powietrza zależał od środowiska (temperatura i wilgotność powietrza, szata roślinna, ruchy mas powietrza), na które ma wpływ wysokości n.p.m. SŁOWA KLUCZOWE: grzyby, zanieczyszczenia powietrza, szlaki górskie 57

2 Introduction Atmospheric air contains fixed amounts of oxygen, nitrogen and carbon dioxide, as well as various contaminants including the biological ones [1]. These include viruses, protozoa, bacteria, cells and tissue fragments of plants and animals, fragments of fungal mycelia and fungal spores [2]. Microbial air pollutions may emit secondary metabolites such as mycotoxins, endotoxins, enterotoxins and enzymes, that may adversely affect human organism and its health [3]. Harmful biological agents appearing in the air, due to the pathogenicity of the human body can be divided into: infectious and invasive agents (e.g. viruses, bacteria, fungi), allergens, biological agents (e.g. plant and animal particles, fungi), biological toxins (e.g. bacterial endotoxin, mycotoxins), carcinogens (e.g. aflatoxin) and biological vectors such as ticks and mosquitoes that carry the germs of diseases [2]. Most of mycotoxins are not susceptible to heat, and as a result, they are stable during normal food preparation processes and feeding. They can cause many animal diseases and health problems in humans. They penetrate into the body not only through the gastrointestinal systems, but also by inhalation and through the skin. High concentrations of mycotoxins can cause strong damage to internal organs, which can lead to a severe clinical disease, whereas low concentrations of mycotoxins may cause their accumulation, which with time can lead to chronic diseases in humans and animals such as kidney and liver cancers [4]. Therefore, it is important to establish the type of toxin [5] for example the fungi of genus Cladosporium secrete emodin and cladosporin [6, 7] and Alternaria spp. secrete alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), altertoxins I, II, III (ATX-I, II, III), tenuazonic acid (TeA) [8]. Literature data report that in Poland from the air of the buildings, the following genus of fungi were isolated: Alternaria, Acremonium, Aspergillus, Botrytis, Cladosporium, Candida, Epicoccum, Fusarium, Penicillium, Rhizopus, Mucor and Verticilium [9-14], but there is no literature data about fungi in the air on mountain trails. Among the types of fungi listed above, there are fungi that cause serious illnesses, and those which cause allergens. The first group includes fungi such as Aspergillus spp. (aspergillosis of the Mikologia Lekarska 2012, 19 (2) lungs, sinuses, cornea, orbit, skin, nails, and of ear canal), Rhizopus spp. (mucormycosis of lungs, sinuses and generalized), Fusarium spp. (generalized fusariosis) and Candida spp. (candidiasis of the mucosal membranes of the mouth and throat, genital and systemic candidiasis) [15, 16]. The second group are the microbial agents causing allergies. The fungi classified above, in the first group, belong here as well, but apart from them, there are also such taxa as Alternaria spp., Acremonium spp., Cladosporium spp. and Penicillium spp. [17]. One of the most universal techniques which are used to study microbial air pollution is the collisions method. It uses air samplers and Petri dishes containing solidified culture medium and involves colliding the sampled air with a layer of medium. The impact force causes the microorganisms and their spores to stick to the medium [18]. The type of culture medium is a very important factor in determining microbial air pollution by means of the collisions method. The type of medium directly affects the number and species composition of fungi and bacteria isolated from the air. This effect is related to the variable availability of nutrients, such as sources of carbon, nitrogen and microelements, to the sampled and cultured microorganisms, in different types of media. It has been demonstrated by the study of Ogórek et al. [9, 10], who had used different culture media (PDA medium Potato Dextrose Agar, Biocorp, MEA medium Malt Extract Agar, Biocorp, Sabouraud medium glucose 4%, agar 2%, peptone 1% and Czapek-Dox Agar medium, Biocorp) to determine the mycological quality of air. The results have shown that, depending on the culture medium, different numbers and species composition of fungi were obtained from the air samples taken in the same room. The best media for the isolation of dermatophytes were Czapek-Dox Agar medium and the PDA and Saburo medium, similar results are achieved in terms of overall value of CFU in 1 m 3 of air and compositions of fungi species. Aim of the study The research objective was to evaluate the extent of mycological air pollutions through determination of number of species and species assemblage found in the air of mountain trails, depending on the height above sea level. Table I: Tabela I: Temperature and humidity of air during measurements Temperatura i wilgotność powietrza podczas pomiarów 58 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 o 47` 29,10 E 50 o 13` 17,43 N II o 47` 32,15 E 50 o 13` 40,25 N III Upper subalpine Piętro regla górnego o 48` 50,49 E 50 o 12` 45,21 N IV o 49` 47,81 E 50 o 12` 29,42 N V VI (mountain peak / szczyt góry) Floor of subalpine Piętro subalpejskie o 50` 20,50 E 50 o 12` 14,40 N o 50` 50,35 E 50 o 12` 27,00 N Air temperature [ o C] Temperatura powietrza [ o C] 24,2 64,0 25,0 60,0 25,3 58,0 26,7 48,1 32,0 41,3 29,0 46,8 Relative humidity of air [%] Wilgotność względna powietrza [%]

3 Grzyby występujące w powietrzu na szlakach górskich masywu Śnieżnika Material and methods Air samples were taken on 11 th of September 2011 from 6 locations on the mountain trails of the Śnieżnik Massif (tab. I). To examine the air, two culture media were used: PDA (Potato dextrose agar), Biocorp and acidifed PDA, Biocorp with 0.5% acetic acid. The sampler (Air Ideal 3P) was programmed for sample volumes of 50 L and 100 L. Measurement in places was performed in sixplicate for every volume. The sampler was positioned 1,5 m above the ground. The incubation of cultures on Petri dishes (90 mm diameter) was carried out at room temperature (±22 C) for 2-7 days. After the incubation the number of CFU/1 m 3 of air (Colony Forming Unit / 1 m 3 of air) was calculated. The temperature and relative humidity was measured using thermo-hygrometer AB-171 Data Logger (Abtronic). The longitude and latitude were determined using navigation Go Live 825 (Tom -Tom). The specific identification of the isolates of the sampled fungi was performed using macro- and microscopic observations of the colonies that had grown on culture media, according to the commonly accepted methods used in mycological laboratories. The fungi were identified using diagnostic keys [19-21]. Table II: Tabela II: Mycological pollution of the air of the Śnieżnik Massif Zanieczyszczenie mikologiczne powietrza masywu Śnieżnika Location of measurement Miejsce pomiaru Fungi species Gatunek grzyba Medium PDA / Podłoże PDA Air CFU/m 3 Species [%] Powietrze jtk/m 3 Gatunek [%] Species Gatunki In total Ogólnie Medium acidifed PDA / Podłoże PDA zakwaszone Air CFU/m 3 Species [%] Powietrze jtk/m 3 Gatunek [%] Species Gatunki In total Ogólnie I Botrytis cinerea Pers , ,8 Cladosporium herbarum Pers , ,4 Epicoccum nigrum Link , ,1 Penicillium expansum Link 5 0,6 20 3,7 Rhodotorula mucilaginosa Harrison 5 0,9 II Alternaria alternata Keissl , ,8 Botrytis cinerea Pers. 15 1,4 60 8,4 Cladosporium herbarum Pers , ,7 Epicoccum nigrum Link 50 4,6 15 2,1 Penicillium expansum Link 5 0,5 III Alternaria alternata Keissl ,1 495 Cladosporium cladosporioides Vries 20 3,0 15 3,0 Cladosporium herbarum Pers , ,8 Epicoccum nigrum Link , ,1 Penicillium expansum Link 15 3,0 Rhizopus stolonifer Vuill 15 3,0 IV Alternaria alternata Keissl , ,0 Botrytis cinerea Pers. 15 6,0 Cladosporium cladosporioides Vries 60 16,4 Cladosporium herbarum Pers , ,0 Epicoccum nigrum Link 30 8,2 10 4,0 Rhizopus stolonifer Vuill 5 1,4 V Alternaria alternata Keissl , ,8 Botrytis cinerea Pers. 0,0 3 0,4 Cladosporium herbarum Pers , ,2 Epicoccum nigrum Link 40 3,8 43 5,9 Penicillium expansum Link 5 0,7 VI Alternaria alternata Keissl , ,2 Botrytis cinerea Pers. 4 0,9 Cladosporium cladosporioides Vries 85 11,2 Cladosporium herbarum Pers , ,7 Epicoccum nigrum Link 65 8,6 1 0,2 Penicillium expansum Link 5 1,1 59

4 Mikologia Lekarska 2012, 19 (2) 1200 medium PDA / podłoże PDA medium acidified PDA / podłoże PDA zakwaszone 1000 Fig. 1. Total number of fungi isolated from the air in the studied locations of the Śnieżnik Massif on PDA and acidified PDA Ryc. 1. Ogólna liczba grzybów wyizolowanych z powietrza badanych miejsc masywu Śnieżnika na podłożu PDA i PDA zakwaszonym Air total CFU/m 3 Powietrze ogólna jtk/m I II III IV V VI Location of measurement / Miejsce pomiaru Rhodotorula spp. Rhizopus spp. Penicillium spp. Epicoccum spp. Cladosporium spp. Fig. 2. The percentage proportion of fungi genera isolated from the air in all the studied locations of the Śnieżnik Massif on PDA and acidified PDA Ryc. 2. Procentowy udział rodzajów grzybów wyizolowanych z powietrza wszystkich badanych miejsc masywu Śnieżnika na podłożu PDA i PDA zakwaszonym Botrytis spp. Alternaria spp. 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% medium acidified PDA / podłoże PDA zakwaszone medium PDA / podłoże PDA 60 Results The temperature prevalent in the investigated locations ranged between 24,2 and 32,0 C, being therefore advantageous for the development of fungi. On the contrary, the relative humidity of the air was low and as such was not very beneficial for them (tab. I). Mycological analysis of air samples has shown that microbial air pollution varied between the studied locations. Furthermore, the number of fungal colonies isolated from the sampled air depended on the type of media used in the experiment (tab. II, fig. 1). The highest numbers of Colony Forming Unit/1 m 3 of air (CFU/m 3 ) on both media were isolated from the air in the second location (height 825 m above sea level, lower subalpine of vegetation layers) and from the fifth location (height 1306 m above the sea level, floor of subalpine). The least numbers of CFU/m 3 of air on both media were isolated from the air in/of the fourth location (height 1167 m above sea level, upper subalpine of vegetation layers) tab. II, fig. 1. From the air of all of the studied locations, 7 genus of fungi were isolated. 6 were isolated on the PDA medium and 7 on acidified PDA. Fungi of the genus Cladosporium occurred in the largest number and in the least number, the yeasts of the genus Rhodotorula (fig. 2). Fungi of the genus Cladosporium and Epicoccum were isolated on both media from the air of/in all the locations of the Śnieżnik Massif, whereas Rhodotorula spp. were isolated from the air in only the first place of research on acidified PDA (tab. II). The highest percentage of the Cladosporium spp. to other fungi isolated from the air was noted for locations in the floor of subalpine, the fifth localisation (94.3%) for the medium PDA and the sixth (95.7%) for the acidified PDA medium, whereas, the lowest

5 Grzyby występujące w powietrzu na szlakach górskich masywu Śnieżnika Location of measurement / Miejsce pomiaru VI V IV III II I 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% medium acidified PDA / podłoże PDA zakwaszone medium PDA / podłoże PDA Fig. 3. The percentage proportion of Cladosporium spp. in comparison to all other fungi isolated from air in the studied locations of the Śnieżnik Massif on PDA and acidified PDA Ryc. 3. Procentowy udział Cladosporium spp. w stosunku do wszystkich pozostałych grzybów wyizolowanych z powietrza badanych miejsc masywu Śnieżnika na podłożu PDA i PDA zakwaszonym for both media in the lower subalpine of vegetation layers (first location), which amounted to 70.8% of the PDA and acidified PDA medium 69.5% (fig. 3). Discussion The content of microorganisms in the air is conditioned by many factors e.g. geographical area and time of the year [22]. Other factors include: the strength, direction of winds, the amount and structure of plant species which are responsible indirectly for the development, number and species composition of microorganisms in the air. Environmental conditions in the Śnieżnik Massif are varied and influence the number of fungi isolated from the air. Flora in the Śnieżnik Massif is characterized by intermediate character of the flora between the western Sudetes and western Carpathians [23]. Most of fungal spores were isolated on both media, from the second and the fifth location of the measurements. They are in the lower subalpine and the floor of subalpine. Vegetation layers of the lower subalpine are mainly covered by spruce monocultures [24] and were characterized by the highest levels of air relative humidity during the studies. On the peak of the Śnieżnik Massif the floor of subalpine is located, which was characterized by the lowest levels of air relative humidity. In this area phytocenosis of high mountain alpine grassland (Carem bigelowii-festuca airoides, Deschampsia flexuosa-nardus stricta) dominates in addition to crowberries (Empetrum hermaphroditum-vaccinium uliginosum). There are also shrubs, introduced in the XIX century, the dwarf pine, and a single dwarf spruce [25]. The high concentration of spores in the second measurement (lower subalpine) was probably influenced by: high density of trees and high humidity of the air, which resulted in favorable conditions for development of fungi and their sporulation. The fifth localisation of measurements was in the floor of subalpine, but it was located at 119 m below the peak of the Śnieżnik Massif. There was a rich vegetation of grasses and perennials, in comparison to the peak, and numerous exaples of dwarf pine and dwarf spruce could be found. The rich vegetation of undergrowth and under peak (shrubs and trees to a height of 1-6 m), contributed probably to the creation of favorable conditions for development of fungi and their protection against the wind, which causes the movement of spores. Whereas the third and fourth place of measurement were characterized by a very small amount of isolated fungi. They are in the floor of subalpine, which is characterized by floristic poverty the total number of vascular plants is only species. In the high mountain meadows mainly spruce forest Plagiothecio-Piceetum hercynicum develops [25]. Poor vegetation probably influenced the growth of fungi and the number of spores in the air. In the air of the Śnieżka Massif, fungi of the genus Cladosporium (C. herbarium and C. cladosporioides) were dominating, which accounted for over 80% of all the isolated types of fungi. This is confirmed by the report of Domsch et al. [26] that fungi of the genus Cladosporium Link ex Fr. are common in many parts of the world, they are cosmopolitan organisms and their spores can be found in air, soil and water. Moreover, the studies of atmospheric air of various regions of Europe, similarly show that the spore of Cladosporium spp. dominates in 80% of all the caught spores and the spore of Alternaria spp. in about 10% [27]. However, the level of concentrations of Cladosporium spores in the air (in 1m 3 ) in a day has a very large variation over the year: from zero to several thousand spores. In European countries, the peak of the season of Cladosporium spp. and Alternaria spp. sporulation is in the months from June to September, when the concentration of Cladosporium spores reaches several thousand per cubic meter of air, and Alternaria spores a few hundred [27, 28]. Despite the greater occurrence of the spores of Cladosporium spp., the spores of Alternaria spp. are more allergenic. The number of spores necessary to induce symptoms of allergic respiratory system disease in most patients with hypersensitivity to these allergens for the Polish population was estimated at 2800 spores in 1 m 3 of air for Cladosporium spp. and only 100 for the spores of Alternaria spp. [29]. Therefore, the number of fungi isolated from the air of the Śnieżnik Massif, could not have influence on the health of tourists who don t have problems with the immune system. 61

6 Cladosporium spp. can cause also opportunistic infections, but very rarely. This problem is related mainly to people with compromised immune systems such as patients with hematological diseases or AIDS. There are also reports about infections by Cladosporium carrionii in healthy people, which may cause changes, for example chromoblastomycosis [30]. These fungi can cause diseases of plants such as black point of cereals, scab of cucurbits and brown spots on tomato leaves [31, 32]. Conclusions 1. At the end of summer time, in the air of the Śnieżnik Massif, the fungi which may be the cause of allergies (espsecially Cladosporium spp.) were dominating. 2. The number of the isolated fungi from the air, was not a threat for healthy tourists, but it could be the cause of upper respiratory symptoms in allergic patients. 3. 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Treshold pollen concentration necessary to evoke allergic symptoms. Internat Rev Allergol Clin Immunol. 2004;10: Plomer-Niezgoda E, Baran E, Maj J, et al. Pathogenicity of fungi from the genus of Alternaria, Cladosporium and Chrysosporium. Mikol Lek. 1998;5: Kochman J. Zarys mikologii dla fitopatologów. Warszawa: Wyd SGGW; p Kryczyński S, Weber Z. Fitopatologia. Choroby roślin uprawnych (tom 2). Poznań: Wyd PWRiL; p Received: Approved: Conflict of interests: none declared ADDRESS FOR CORRESPONDENCE: Rafał Ogórek, M.Sc. Eng. pl. Grunwaldzki 24a Wrocław, Poland tel.: rafal.ogorek@up.wroc.pl 62