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The occurrence of potentially pathogenic filamentous fungi in recreational surface Publikacja / Publication water as a public health risk, Góralska Katarzyna , Błaszkowska Joanna, Dzikowiec Magdalena DOI wersji wydawcy / Published http://dx.doi.org/10.2166/wh.2020.096 version DOI Adres publikacji w Repozytorium URL / Publication address in https://publicum.umed.lodz.pl/info/article/AMLef8a7608e2854344ad6ba545a71f29a6/ Repository Data opublikowania w Repozytorium 2020-03-31 / Deposited in Repository on Rodzaj licencji / Type of licence Attribution (CC BY) Góralska Katarzyna , Błaszkowska Joanna, Dzikowiec Magdalena: The occurrence of potentially pathogenic filamentous fungi in recreational surface water as a public Cytuj tę wersję / Cite this version health risk, Journal of Water and Health, vol. 18, no. 2, 2020, pp. 127-144, DOI: 10.2166/wh.2020.096 Journal of Water and Health
The occurrence of potentially pathogenic filamentous fungi in recreational surface water as a public health risks --Manuscript Draft--
Manuscript Number: JWH-D-19-00096R2 Full Title: The occurrence of potentially pathogenic filamentous fungi in recreational surface water as a public health risks Article Type: Research Paper Corresponding Author: Katarzyna Góralska, Ph.D. Medical University of Lodz Lodz, POLAND Corresponding Author Secondary Information: Corresponding Author's Institution: Medical University of Lodz Corresponding Author's Secondary Institution: First Author: Katarzyna Góralska, Ph.D. First Author Secondary Information: Order of Authors: Katarzyna Góralska, Ph.D. Joanna Blaszkowska Magdalena Dzikowiec Order of Authors Secondary Information: Abstract: Micro-fungi occurring in surface waters may represent an important health risk. The aim of the study was to assess the diversity of mycobiota in selected artificial bathing reservoirs with regard to its biosafety for the human population. The studies were conducted during the summer of 2016 in three research seasons, June (I), July to August (II) and September (III), taking into account the various periods of recreational activities. Filamentous fungi were isolated from water samples collected at five different ponds utilized for recreation. From 162 water samples, 149 fungal taxa of filamentous fungi were identified: 140 were classified to species level and nine only to genus level. Aspergillus fumigatus was the dominant species. The highest species richness (S) was noted in June, with 93 fungal taxa (Menhinick's index from 2.65 to 4.49). Additionally, in season I the highest diversity of fungal species was revealed (Simpson's diversity index from 0.83 to 0.99). The average number of CFU per 1mL sample ranged between 0.4 and 4.6 depending on the time of sampling and ponds. Of all the isolated species, 128 were clinically relevant (11 from RG-2 and 117 from RG- 1), highlighting the need to introduce seasonal mycological monitoring of such reservoirs.
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1 The occurrence of potentially pathogenic filamentous fungi in recreational surface water as a public 2 3 health risks 4 5 1 2 2 6 Katarzyna Góralska , Joanna Błaszkowska , Magdalena Dzikowiec 7 8 9 1Department of Biomedicine and Genetics, Chair of Biology and Medical Microbiology, Medical University of 10 Lodz 11 12 Pomorska 251, 92-213 Lodz 13 2Department of Biology and Parasitology, Chair of Biology and Medical Microbiology, Medical University of 14 15 Lodz 16 Pomorska 251, 92-213 Lodz 17 18 19 Corresponding author: Katarzyna Góralska 20 21 e- mail: [email protected] 22 Phone: +48 42 272 53 87 23 24 25 Abstract 26 27 Micro-fungi occurring in surface waters may represent an important health risk. Recreational water 28 29 reservoirs are potentially reservoir of pathogenic fungi. The aim of the study was to assess the diversity of 30 mycobiota in selected artificial bathing reservoirs with regard to its biosafety for the human population. The 31 32 studies were conducted during the summer of 2016 in three research seasons, June (I), July to August (II) and 33 September (III), taking into account the various periods of recreational activities. Filamentous fungi were 34 35 isolated from water samples collected at five different ponds utilized for recreation. From 162 water samples, 36 149 fungal taxa of filamentous fungi were identified: 140 were classified to species level and nine only to genus 37 38 level. Aspergillus fumigatus was the dominant species. The highest species richness (S) was noted in June, with 39 93 fungal taxa (Menhinick's index from 2.65 to 4.49). Additionally, in season I the highest diversity of fungal 40 41 species was revealed (Simpson's diversity index from 0.83 to 0.99). The average number of CFU per 1mL 42 sample ranged between 0.4 and 4.6 depending on the time of sampling and ponds. Of all the isolated species, 43 44 128 were clinically relevant (11 from RG-2 and 117 from RG-1), highlighting the need to introduce seasonal 45 mycological monitoring of such reservoirs. 46 47 48 Keywords: aquatic fungi, fungal occurrence, natural bathing areas, human pathogenic fungi, fungal water 49 50 contamination 51 52 53 54 Introduction 55 56 Water reservoir contamination occurs when substances and chemical compounds, and allochthonous 57 organisms, not present under natural conditions are discovered in reservoirs (Libudzisz et al. 2009). 58 59 Microbiological contamination of the aquatic environment can come from natural sources—mainly soil in the 60 61 62 1 63 64 65 Pobrano z https://publicum.umed.lodz.pl / Downloaded from Repository of Medical University of Lodz 2021-10-04 immediate vicinity but also the transmission of microorganisms through air currents—or through anthropogenic 1 sources including wastewater, surface and ground runoff from industrial and agricultural areas and landfills 2 3 (Lucyga et al. 2011). The presence of potentially pathogenic fungi in water reservoirs, as well as the lack of 4 sanitary and epidemiological supervision over such objects, poses a risk of acquiring waterborne infections. 5 6 Throughout the past 30 years in Europe, more than 400 different fungal species have been found in groundwater, 7 surface water, and drinking water; among these species, 46 were classified as Biosafety Level 2 (Novak-Babiˇc 8 9 et al. 2018), meaning they cause various diseases, such as allergies and mycoses, in humans and animals. 10 In natural water environments, autochthonous species are most often represented by microscopic fungi 11 12 from the following classes: Chytridiomycetes, Oomycetes, Trichomycetes and Mucoromycetes (which were once 13 named Zygomycetes). Of the millions of estimated fungal species, only 3000 to 4000 are classified as aquatic 14 15 fungi (Grossart & Rojas-Jimenez 2016) for which the water environment is a natural place of existence. The so- 16 called "water fungi" belong to various taxonomic units and occur abundantly in water reservoirs in the form of 17 18 vegetative mycelium, producing zoospores or other types of spores adapted to spread in water. The optimal 19 temperature for the growth of most temperate aquatic fungi is 25°C, but the fungi can also grow relatively well at 20 21 temperatures as low as 10°C. Genera such as Alternaria, Aureobasidium, Cladosporium and Penicillium detected 22 in aquatic environments are classified as secondary freshwater fungi since they originate from terrestrial habitats 23 24 (Krauss et al. 2011). 25 Although it is very difficult to prove the existence of a causal relationship between the presence of 26 27 microorganisms in recreational water areas and the acquisition of waterborne infection, several studies have 28 29 confirmed the presence of waterborne fungal infections. Some of these studies have found a genetic relationship 30 between waterborne fungal strains and strains of the same species isolated from clinical samples (Anaissie et al. 31 32 2001, 2003). Waterborne outbreaks of fungal infections, with C. albicans, C. parapsilosis, Aspergillus spp., 33 Mucor spp., Trichosporon asahii, Fusarium spp., Scedosporium spp. and Exophiala jeanselmei, associated with 34 35 hospital water containers have been observed among patients, particularly immunosuppressed individuals 36 (Neblett Fanfair et al. 2012; Kanamori et al. 2016). A genetic study of 75 clinical and 156 environmental isolates 37 38 of Fusarium keratoplasticum showed that strains isolated from clinical materials to be identical to those from 39 plumbing biofilm samples (Short et al. 2014). Bandh et al. (2016) investigated the relationship between the 40 41 presence of human pathogenic opportunistic fungi (Aspergillus, Candida, Penicillium, Cryptococcus, Fusarium, 42 Rhizopus and Mucor) in lake water and the incidence of fungal infections in the associated population of 43 44 Kashmir, India: a higher incidence of fungal infection (9.84%) was found in people using lake water than people 45 using only tap water (4.16%). Serious fungal infections (Aspergillus spp., Scedosporium spp., Rhizopus spp.) of 46 47 the lungs and brain resulting from the aspiration of contaminated water have been observed in people who have 48 experienced near-drowning episodes (Signore et al. 2017; Leroy et al. 2006; Jenks & Preziosi 2015; Gerlach et 49 50 al. 2016). Several reports of near-drowning accidents have clearly demonstrated that opportunistic moulds, 51 including Aspergillus species, can cause fungal pneumonia, and only a small amount of water (<150 mL) is 52 53 needed to cause devastating infections (Ter Maaten et al. 1995). A relationship has been confirmed between 54 natural disasters and subsequent fungal infections in people affected by disasters (Benedict & Park 2014). These 55 56 reports show that pulmonary aspergillosis can be waterborne, and that it can be transferred either by air or water 57 aerosol. Cutaneous mucormycosis caused by Apophysomyces trapeziformis occurred among 13 people who were 58 59 severely injured after a tornado (May 22, 2011) in Joplin, Missouri, US. A report from Japan described 60 61 62 2 63 64 65 Pobrano z https://publicum.umed.lodz.pl / Downloaded from Repository of Medical University of Lodz 2021-10-04 disseminated aspergillosis caused by Aspergillus fumigatus to be associated with tsunami lung (Kawakami et al. 1 2012). Other fungal pathogens, such as Rhizopus, Mucor, Fusarium, Scedosporium, Coccidioides immitis and 2 3 Apophysomyces elegans have also been implicated as agents of mycoses affecting the lungs, central nervous 4 system and skin after a tsunami (Benedict & Park 2014). The results of these studies suggest that water should be 5 6 considered as a potential transmission route for pathogenic fungi. 7 The results of research conducted in many countries revealed a high prevalence of potentially 8 9 pathogenic fungi in the waters of natural bathing places, which poses a threat to human health (Dynowska et al. 10 2013; Biedunkiewicz & Góralska 2016). Although the presence of fungi in drinking water, swimming pools and 11 12 natural bathing places and its associated health risks have been documented, there are no international legal 13 regulations for the mycological evaluation of drinking or recreational water (Novak Babiˇc et al. 2017). The 14 15 microbial parameters currently employed in testing (Escherichia coli, intestinal enterococci) have no indicative 16 value regarding fungal contamination (Council Directive 2006/7/EC). The presence of specific species of 17 18 filamentous fungi (Aspergillus spp., Rhizopus spp.) in water reservoir directly indicates a poor sanitary state and 19 hence the epidemiological threat. This fact indicates the need for seasonal microbiological monitoring of the 20 21 aquatic environment, and thus supervision of the quality of bathing water in recreational aquatic areas. The 22 taxonomic and phenological analysis of natural inland bathing sites indicates the highest species diversity of 23 24 fungi during the summer corresponds with the period of time when inhabitants of countries with moderate 25 climates use these types of recreational facilities with the highest frequency. The aim of the study was to assess 26 27 the diversity of the mycobiota in selected natural bathing places, with regard to biosafety implications for the 28 29 human population. The qualitative composition of filamentous fungi before, during and after the bathing season 30 was compared. 31 32 Materials and methods 33 The research area included five artificial water reservoirs used as public bathing places, located in 34 35 recreational areas in Lodz. The city is situated in Central Poland, in a temperate climate zone with the following 36 characteristics: four distinct seasons, a mean annual air temperature of 7.5°C and a mean annual relative air 37 38 humidity of 80%. The area of the city is 293.25 km², making it the fourth largest city in Poland; it is inhabited 39 by 690,422 people (2,354 people / km²). Within the city, there are 19 rivers and streams, partly covered with 40 41 urban infrastructure. 42 The research covered five artificial water reservoirs: Jan's Ponds (SJ), Mlynek (M), Stefanski's Ponds 43 44 (S), Jasien Pond (J) and Arturowek complex (A) (Figure 1). The analyzed reservoirs are used as recreational 45 bathing places by residents during the summer. The Lodz Sanitary Inspectorate recognizes three (SJ, S, A) of the 46 47 analyzed water reservoirs as official bathing areas, while the other two (M and J) are not considered official 48 bathing sites; therefore, they are not subject to sanitary control. 49 50 Jan’s Ponds (SJ) is surrounded by parkland comprising grassy areas and old stands, mainly linden and 51 ash. The area of the pond is 4.3ha and its depth is up to 2.53m. SJ is empty in winter but is filled by the 52 53 Olechowka River in spring. This urban bathing place is accompanied by a rich recreational infrastructure. Its 54 most recent modernization was performed in 1995. 55 56 Mlynek (M) is a water reservoir on the course of the Olechowka River located close to the city limits 57 and woods. It is 3.5ha in surface area and up to 2.75m deep. The shore of the pond is high with a large number of 58 59 reeds but no beaches. There is a varied recreational infrastructure. 60 61 62 3 63 64 65 Pobrano z https://publicum.umed.lodz.pl / Downloaded from Repository of Medical University of Lodz 2021-10-04 Stefanski's Ponds (S) consist of two water bodies located on the course of the Ner River. They cover an 1 area of 11.4ha and their maximum depth is about 4m; their surroundings include rich stands such as black alder, 2 3 white willow, birch, poplar and chestnut. It is used as an urban bathing place, and is accompanied by a rich 4 recreational infrastructure. During the sampling period, the reservoirs were undergoing renovations. 5 6 The Jasien Pond (J) is located in the city center on the course of the Jasien River, within the park 7 surrounding an historical palace. The area of the pond is 3.46ha and the maximum depth is 4m. The shore is 8 9 covered with a gabion system; on one side with beach and large gang-board. There is a varied recreational 10 infrastructure. 11 12 The Arturowek complex (A) is located at the source of the Bzura River and is a part of the Lodz's Hills 13 Landscape Park. The Arturowek complex consists of three interconnected water bodies: upper (area 1.08ha), 14 15 middle (2.58ha) and lower (3.05ha), with maximum depth of 2.1m. Two ponds with a rich recreational 16 infrastructure are used as bathing reservoirs and the third (smallest) is a retention reservoir. The entire Arturowek 17 18 complex was recently modernized during a LIFE+ project entitled "Ecohydrological reclamation of recreation 19 reservoirs". Ponds were desludged and equipped with gabion systems. 20 21 Research was conducted during the summer of 2016 in three research seasons: I in June (before the start 22 of the bathing season), II in July and August (peak bathing season during the holidays) and III in September 23 24 (after the bathing season). The time of sampling was selected to align with the greatest use of these water 25 reservoirs by people. Weather conditions in the first and second seasons of the research period were similar. Due 26 27 to frequent rainfall, water levels were very high. However, before the third season there was a period of drought, 28 29 significantly lowering the water level; this was especially noticeable in the SJ reservoir, where the coastline 30 moved by more than 2m. Samples were always taken at the same time (between 6 AM and 8 AM) and weather 31 32 conditions were similar (windless and rainless). The samples were collected in places most frequently used by 33 people (gang-boards, beaches, designated bathing areas, harbours). 34 35 Water samples were taken in triplicate 1.5-2 m from the shore of the water reservoirs at a layer 15 cm 36 below the surface; the sampling was performed using Whirl-Pak® bags (Nasco, USA) with a volume of 500 mL. 37 38 The number of sampling locations was dependent on the size of the water body: three sampling points on SJ, 39 three on M, four on S, three on J, and five on A. In total, 162 samples were collected. During each collection the 40 41 air and water temperatures and water pH were measured (pHep Tester, Pocket pH Tester, HANNA instruments, 42 Romania). The collected samples were delivered to the laboratory within an hour. The samples (500mL) were 43 44 concentrated by centrifugation to a volume of 10 mL, and then 1 mL of each sample was seeded on Sabouraud 45 dextrose agar (SDA, Biomerieux, France) with chloramphenicol (repeated three times) and Czapek-Dox medium 46 47 (Biomerieux, France) (repeated three times). 48 As the study focused on fungi that could pose a potential threat to human health, Sabouraud's dextrose 49 50 agar was chosen for the growth of clinically relevant species, whereas Czapek-Dox medium was used to obtain 51 sporulation of moulds (mostly of genera Aspergillus and Penicillium) to facilitate identification. Incubation was 52 53 carried out for five to seven days at 24°C. After seven days of incubation, the number of colonies was counted 54 and expressed as the number of colony forming units (CFU)/1 mL for water samples. For slow-growing fungi, 55 56 incubation was prolonged for 14-21 days to achieve sporulation. From the obtained filamentous fungal isolates, 57 microscopic preparations were made according to Gerlach technique by pressing the adhesive tape to the 58 59 60 61 62 4 63 64 65 Pobrano z https://publicum.umed.lodz.pl / Downloaded from Repository of Medical University of Lodz 2021-10-04 mycelium and then transferring it to a microscope slide, using lactofenol and aniline blue staining (Gerlach 1 1972). 2 3 The identification was made on the basis of macroscopic and microscopic characteristics using keys: de 4 de Hoog et al. (2019), Fassatiova (1983) and Watanabe (2002). Current species names were checked according 5 6 to Index Fungorum Website. 7 Clinically relevant species were classified to Risk Groups (RG) and Biosafety Levels (BSL) according 8 9 to the online version of the Atlas of Clinical Fungi (Hoog et al. 2019). These two parameters are commonly used 10 to characterize the occupational health risks and intrinsic virulence associated with the pathogenicity of fungal 11 12 species. 13 Microorganisms used for laboratory work are classified to four Risk Groups-RG (WHO, 2004), but RG- 14 15 4 does not apply to fungi. General criteria for attribution of fungi in different categories were explained by de 16 Hoog (1996), but application to individual species is still under debate. According to Atlas of clinical fungi (de 17 18 Hoog et al. 2019) the categories RG are defined as follows: 19 RG-1: Saprobes or plant pathogens occupying non-vertebrate ecological niches, or commensals. 20 21 Infections are coincidental, superficial, and non-invasive or mild. 22 RG-2: Species principally occupying non-vertebrate ecological niches, but with a relatively pronounced 23 24 ability to survive in vertebrate tissue. In severely immunocompromised patients they may cause deep, 25 opportunistic mycoses. The category also includes pathogens causing superficial infections. 26 27 RG-3: Pathogens potentially able to cause severe, deep mycoses in otherwise healthy patients. 28 29 The levels of laboratory biosafety (BSL) are based on intrinsic virulence (Risk Groups 1-4) and routes 30 of infection (WHO, 2004). Each BS level describes the microbiological practices, safety equipment and facility 31 32 safeguards for the corresponding level of risk associated with handling a particular agent; no special precautions 33 for category BSL-1, while specific procedures are required for BSL-2, BSL-3 and BSL-4 (BSL-4 does not apply 34 35 to fungi). 36 Qualitative and quantitative assessments of biodiversity were applied. The species richness (S) was 37 38 expressed as the number of species found in the water of the examined reservoirs. Additionally, Menhinick’s 39 index (species richness index) and the Simpson’s diversity index were also calculated (Magurran, 2004). 40 41 Menhinick's richness index is calculated as the ratio of the number of species (S) and the square root of the total 42 number of individuals (N). 43 44 푆 퐷푀푒 = 45 √푁 46 Simpson's Diversity Index is a measure of diversity which takes into account the number of species 47 48 present (n), as well as the relative abundance of each species (N). 49 ∑ 푛(푛 − 1) 50 퐷 = 1 − 51 푁(푁 − 1) 52 53 54 The obtained data was analysed using the χ2 test and the Spearman rank correlation. Additionally, for 55 comparison of fungal counts obtained from media, the non-parametric Kruskal-Wallis ANOVA was used. All 56 57 calculations were performed using STATISTICA 13.2 software. For all test, the significance level was assumed 58 to be α≤0.05. 59 60 Results 61 62 5 63 64 65 Pobrano z https://publicum.umed.lodz.pl / Downloaded from Repository of Medical University of Lodz 2021-10-04 In total, 149 different taxa of filamentous fungi were identified: 140 classified to the species and nine to 1 genus level (Table S1). In water reservoirs SJ, M and A, fungi belonging to Oomycota, Zygomycota, Ascomycota 2 3 and Basidiomycota were identified. No representatives of the Zygomycota were found in S, while no fungi from 4 the Basidiomycota were found in J (Table 1, Figure 2). The greatest species richness (S) was found in SJ (79 5 6 species), while the smallest number of species was found in M (47) and A (42) (Figure 3). Significant 7 differences in species richness (S) were observed between research seasons and examined ponds (χ2 =31.20; 8 9 df=8; p=0.0001). Statistically significant differences between species richness and reservoirs were noted for 10 season I (χ2=14.61; df=4; p=0.0056) and for season III (χ2=22.5; df=4; p=0.0002); no significant difference was 11 12 observed for season II (χ2=9.06; df=4; p=0.0595). 13 The highest number of species (93) and number of colonies (308) were noted in water samples in 14 15 research season I; only 68 species and 175 colonies were found in season II, and 70 species and 289 colonies 16 during season III. The Kruskal-Wallis test showed a statistically significant difference in the number of fungal 17 18 colonies obtained on media depending on the research season only for two ponds: S (p=0.013) and A (p=0.021). 19 The average number of CFU/1mL ranged between 0.4 and 4.6. For pond A, the highest differences between 20 21 CFU/1mL were noted in relation to seasons; in season III the value of CFU (2.9/1mL) was over 3.5 and 7 times 22 higher than in season I and II, respectively. More detailed data regarding CFU/1mL is given in Table 2. 23 24 Based on the Menhinick's index, the greatest species richness was observed in SJ (season I and III), S 25 (season I) and A (season I), and the least in S (season III). Simpson's Diversity index was the highest for SJ and 26 27 A for all research seasons, and the lowest in the S pond in season III (Table 2). The lowest species richness 28 29 (0.680) and species diversity indexes (0.144) were obtained for pond S in season III, with the mean number of 30 fungi being 2.1 CFU/1mL of water sample (Table 2). 31 32 In the examined ponds, depending on the sampling date, differences for water pH and water and air 33 temperature were noted. The physicochemical parameters of the analyzed tanks are presented in Table 3. 34 35 A statistically significant negative correlation (Spearman rank correlation) was found between the 36 number of species from the SJ reservoir and the pH of its water (r=-0.737) (Figure 4, 5). There was no 37 38 correlation between the number of species in the M, J and A reservoirs and pH and water temperature, while 39 positive correlations were found between the number of species in the S pond and water pH (r= 0.59) and water 40 41 temperature (r= 0.75) (Figure 4). No significant correlation was observed between number of colonies and air 42 temperature in any pond (Figure 5). 43 44 In the study dominated Aspergillus fumigatus, which was detected in all reservoirs. Alternaria alternata 45 Chrysosporium inops, and Penicillium aurantiogriseum were also frequently isolated (Table S1). In research 46 47 season I, A. fumigatus and P. chrysogenum predominated, but Bjerkandera adusta was also frequent. In research 48 season II, A. fumigatus, followed by A. alternata and C. inops, were most commonly isolated. In the third 49 50 season, Aspergillus fisheri and A. fumigatus were most often identified. In the SJ pond the most frequently 51 recorded species was A. fumigatus, but P. waksmanii, P. citrinum and P. aurantiogriseum were also very often 52 53 found. In the M dominated A. fumigatus; however, Trichoderma harzianum was also frequently identified. In the 54 S reservoir, A. fumigatus and A. alternata were most frequently recorded. In pond J, the most frequently 55 56 identified species was A. niger, but A. fumigatus, P. waksmanii and C. inops were often noted as well. The most 57 common species in reservoir A was A. fumigatus, but A. niger and P. chrysogenum were also very common 58 59 (Table S1). 60 61 62 6 63 64 65 Pobrano z https://publicum.umed.lodz.pl / Downloaded from Repository of Medical University of Lodz 2021-10-04 Almost 86% of all identified fungal species are known to be associated with human infections. Table 2 1 shows the occurrence of clinically-relevant species according to individual research season and ponds. Among 2 3 the isolated species, 11 belong to RG-2, and 117 belong to RG-1; in addition, 24 species were classified as BSL- 4 2 and 104 as BSL-1. More detailed data on the number of species detected in RG-1 and RG-2 in the examined 5 6 ponds in the three research seasons are presented in the supplementary material (Table S2). Statistically 7 significant differences in the numbers of clinically relevant species were observed between ponds and research 8 9 seasons (χ2=24.905, df=8; p=0.0016). The highest prevalence of RG-1 and RG-2 organisms was noted in SJ (81), 10 statistically significant differences were found between the occurrence of clinically relevant species in the 11 12 examined reservoirs (χ2=14.24, df=4, p=0.0065). Species classified as BSL-1 and BSL-2 were most common in 13 SJ (65 and 16, respectively), but similar frequencies were observed in other ponds; however, statistically 14 15 significant differences were found between water reservoirs with regard to the number of species classified as 16 BSL-1 (χ2=11.91, df=4, p=0.0180). A significantly higher total number of BSL-1 and BSL-2 fungi was found in 17 18 season I (119) compared to other seasons (χ2=12.28, df=2, p=0.0022). 19 20 21 Discussion 22 Worldwide studies have indicated that potentially pathogenic fungi are widespread in the waters of 23 24 natural reservoirs, and even in water intended for drinking (tap water and bottled water), which poses a threat to 25 human health (Siqueira et al. 2011; Oliveira et al. 2013; Ashbolt 2015; Fisher et al. 2015; Biedunkiewicz & 26 27 Góralska 2016; Novak-Babiˇc et al. 2016). According to literature data, the filamentous fungi from different 28 29 genera (Acremonium, Alternaria, Aspergillus, Cladosporium, Fusarium, Penicillium, Trichoderma, Mucor, 30 Rhizopus) have often been detected in surface-, ground- and tap water (Siqueira et al. 2011; Novak-Babiˇc et al. 31 32 2016; Bandh et al. 2016). In our study, filamentous fungi from the aforementioned genera predominate, 33 especially Aspergillus spp. and Penicillium spp., which have also been observed by other authors in various 34 35 world regions (Baumgardner 2017, Siqueira et al. 2011, Bandh et al. 2016). It is worth mentioning that in our 36 study Aspergillus fumigatus was the most common species isolated from all analyzed ponds. Similar results from 37 38 samples of water from the Augustow Canal (Poland) were obtained by Cudowski et al. (2015). The species that 39 most frequently appeared in the samples from lakes (Poland) was Aspergillus heteromorphus (Biedunkiewicz & 40 41 Góralska 2016). On the other hand, Aspergillus niger, which is frequently isolated from river water samples in 42 India (Parveen et al. 2011), dominated one of the five examined bathing areas of this study. 43 44 Numerous studies on the presence of typical aquatic fungi, such as zoosporic fungi from the Oomycetes 45 and Chytridiomycetes can be found in the literature (Kiziewicz et al., 2004, Czeczuga et al., 2003, Godlewska et 46 47 al., 2016, Valderrama et al 2016, Hu et al., 2013, Cudowski et al. 2015); much fewer studies concern fungi that 48 secondarily colonize water (Aspergillus, Penicillum, Fusarium, Trichoderma), many of which are potentially 49 50 pathogenic and can cause mycosis in humans and animals (Parveen et al 2011, Di Piazza et al 2017, Arvanitidou 51 et al 2002, Bandh et al. 2016). In our study, only 13 of the isolated 149 taxa, were aquatic. Similarly, species of 52 53 moulds originally associated with terrestrial environments have been found to dominate in bathing lakes in 54 northern Poland (Biedunkiewicz & Góralska 2016), and Parveen et al. (2011) report the presence of 31 species 55 56 of secondary aquatic fungi in water samples from a river in Raipur city (India). In contrast, Czeczuga et al. 57 (2003), report that as many as 108 out of 200 species in the Biebrza River, a natural water body in Poland, 58 59 belonged to aquatic fungi; in addition, of 26 species isolated in the Horodnianka River, Poland, 20 were 60 61 62 7 63 64 65 Pobrano z https://publicum.umed.lodz.pl / Downloaded from Repository of Medical University of Lodz 2021-10-04 originally aquatic fungi (Kiziewicz et al., 2011). This data indicates that primary terrestrial fungi occur more 1 frequently in anthropogenically modified water reservoirs than in natural waters, where autochthonous species 2 3 dominate. 4 Waterborne fungal infections may occur in different ways, such as exposure during sports and 5 6 recreation, drinking contaminated liquids, and from personal and home hygiene activities, including the 7 inhalation of aerosols while showering. Fungal infections resulting from exposure to water of natural and 8 9 anthropogenic water reservoirs (e.g. swimming pools, water parks, interactive fountains, and jacuzzis), primarily 10 affects immunocompromised people or those with trauma, including damaged skin (Baumgardner 2017; 11 12 Hageskal et al. 2009). These infections commonly manifest as allergies and dermatomycoses (mycoses of the 13 skin or nails) but sometimes manifest as keratitis and acute otitis media caused mainly by fungi from the 14 15 following genera: Candida, Fusarium, Aspergillus, Paecilomyces (Kamihama et al. 1997; Baumgardner 2017; 16 Hageskal et al. 2009). Among filamentous fungi, species from the genera Fusarium and Aspergillus are the most 17 18 common causes of fungal keratitis in the US and other parts of the world. A study conducted from 2004 to 2008 19 in Philadelphia revealed an increase in the number of fungal keratitis cases caused by Fusarium, especially 20 21 among contact lens users; four of the 28 patients with keratitis reported specific water exposure: two from 22 lakes and one each from well water exposure and swimming while wearing contact lenses (Baumgardner, 23 24 2017). Fungi from the genus Fusarium were sporadically isolated in the present study, however they are 25 commonly detected in surface water, groundwater and even tap drinking water in Europe (Novak-Babiˇc et al. 26 27 2017). Additionally, surface water can be a possible reservoir of dermatophytes, which are mainly zoophilic 28 29 (Microsporum canis, Trichophyton verrucosum, T. mentagrophytes) and geophilic species (Microsporum 30 gypseum) that are highly transmittable between hosts animal or soil environment and people. On the contrary, 31 32 anthropophilic dermatophyte species (Trichophyton schoenleinii, T. tonsurans, T. violaceum) are most 33 commonly isolated from water used for recreational facilities, such as swimming pools, jacuzzis, baths, and 34 35 saunas (Novak-Babiˇc et al. 2018). It is worth noting that Trichophyton spp. and Epidermophyton floccosum, 36 which cause superficial fungal infections of the hair, fingernails or skin, are the only fungal species considered as 37 38 potential microbial hazards in the WHO guidelines for safe recreational water (WHO, 2006). In these studies 39 three species of dermatophytes (Trichophyton verrucosum, T. schoenleinii, T. violaceum) were detected in 40 41 recreational waters. Contamination of bathing areas with these dermatophytes probably occurred as a result of 42 water coming into contact with bathing people and their animals such as dogs. During sample collection, we 43 44 observed that pet owners often walked there with dogs, which also entered the water. 45 Although the categories RG and BSL were introduced to protect laboratory workers from occupational 46 47 health risks, the classification of species to risk group organisms can also help to estimate the clinical relevance 48 of isolates detected from sources outside the laboratory and determine the risk of environmental exposure to 49 50 fungi. The classification of a species to a specific RG indicates its pathogenicity. It needs to be highlighted that 51 the potentially pathogenic fungi for humans are usually associated with soil, air, water, the human home and 52 53 hospital environments. It is estimated that of all the fungi described in the world, approximately 600 species are 54 opportunistic pathogens (de Hoog et al. 2015), which are etiological agents of mycoses, mainly in 55 56 immunocompromised persons. Over the last two decades, the increase in the number of immunocompromised 57 patients may promote the spread of mycoses caused by environmental RG-1 and RG-2 fungi. In our study, 58 59 almost 86% of the identified taxa of filamentous fungi were clinically relevant species; 11 belonged to RG-2, 60 61 62 8 63 64 65 Pobrano z https://publicum.umed.lodz.pl / Downloaded from Repository of Medical University of Lodz 2021-10-04 and 117 to RG-1, while the same species were respectively classified as BSL-2 (24) and BSL-1 (104). Another 1 study performed in three lakes utilized for recreation in north-eastern Poland found a high percentage (73%) of 2 3 clinically relevant fungal species – eight species out of 45 belonged to BSL-2, and 25 belonged to BSL-1 4 (Biedunkiewicz & Góralska 2016). Additionally, 42 of the 79 identified species isolated from various sites in a 5 6 swimming pool facility by Ekowati et al. (2017) were classified at BSL-1 and RG-1. The presence of such fungi 7 in recreational waters represents a potential threat of fungal infections being spread through water, especially in 8 9 persons with immunodeficiency. 10 The RG and BSL classifications do not always coincide for a particular organism: for example, 11 12 Aspergillus flavus is classified as both RG-1 and BSL-2. In this case in laboratories biosafety level - 2 should be 13 maintained due to the easy spread of conidia and their small size (3.5 μm diameter). Although this species occurs 14 15 in the environment as a saprobe, numerous reports indicate its pathogenicity towards humans: the Atlas of 16 Clinical Fungi (de Hoog et al., 2019) notes about 40 publications on the pathogenicity of A. flavus based on 17 18 cases of mycoses in humans. In our study, A. flavus was detected less frequently than other Aspergillus species, 19 with A. fumigatus being most frequently isolated from water samples. De Hoog et al. (2019) list over 120 20 21 publications on the pathogenicity of A. fumigatus (RG-2, BSL-2). This species is the main agent of pulmonary 22 aspergillosis in patients with impaired immunity (de Hoog et al., 2019). Exposition to fungal propagules via 23 24 water aerosols is believed to represent the most potential route of infection for bathers (Novak Babiˇc et al. 25 2017). 26 27 It has been suggested that human skin might be made more susceptible to fungal infection, especially 28 29 opportunistic black fungi from genera Exophiala and Cladophialophora by softening due to bathing (Lian & de 30 Hoog, 2010; Wang et al. 2018). Two such species (Exophiala jeanselmei, Cladophialophora carrionii) were 31 32 occasionally identified in the water samples in the present study. Exophiala species are often isolated from 33 indoor water sources, such as sinks, swimming pools, and bathing facilities, and also occur in surface water and 34 35 municipal drinking water enabling biofilm formation (Novak Babiˇc et al. 2017, Novak Babiˇc et al. 2018, 36 Oliviera et al., 2013). The clinical manifestations of Exophiala jeanselmei (RG-2/BSL-2) mainly concern 37 38 subcutaneous phaeohyphomycosis and black-grain mycetoma. The propagules are mostly hydrophilic and 39 infections mostly being of traumatic origin (de Hoog et al. 2019). 40 41 Additionally, allergenic fungi were also identified in the present study. Apart from numerous 42 Aspergillus and Penicillium species, Alternaria and Cladosporium species were also detected. Alternaria and 43 44 Cladosporium are cosmopolitan genera of saprobe commonly found on dead plant material and are known to be 45 ubiquitous laboratory contaminants. Alternaria was represented in the present study by seven species, which 46 47 were detected in all examined ponds. Alternaria alternata (RG-1/BSL-1) may cause skin lesions after trauma, 48 often in immunocompromised patients or with chronic underlying metabolic disease, as well as in cases of 49 50 keratitis, cutaneous infections and otitis, onychomycoses (de Hoog et al. 2019). Unlike Alternaria, only one 51 species of Cladosporium (C. sphaerospermum RG-1/BSL-1) was identified in the present study. 52 53 Potentially pathogenic species show the ability to grow at body temperature (37°C) and even within the 54 fever range (38-42°C) of the human host, which is an important requirement for systemic infection. Additionally, 55 56 small spore sizes aid fungal entry and penetration through the host's barriers. Oliviera et al. (2013) reported that 57 66% of the species isolated from different drinking water sources were able to grow at 30°C and they had spore 58 59 sizes below 5µm, while Aspergillus fumigatus, A. viridinutans and Cunninghamella bertholletiae were able to 60 61 62 9 63 64 65 Pobrano z https://publicum.umed.lodz.pl / Downloaded from Repository of Medical University of Lodz 2021-10-04 grow at the higher temperature tested (42°C). It is worth emphasizing that the majority of clinically relevant 1 species isolated from water samples of examined ponds produce spores with a diameter below 5µm (e.g. 2 3 Aspergillus spp., Penicillium spp., Acremonium spp., Trichodetma spp.). Of the species belonging to RG-1, only 4 a few, including Alternaria spp., have spores larger than 5µm. 5 6 Various indicators with different sensitivities are used to assess species richness and biodiversity 7 (Goncalves et al. 2012, Kanieski et al., 2018; Mirzaie et al. 2013) and their application is related to the type of 8 9 population analyzed and the properties of the studied ecosystem. They are used to compare both the richness of 10 the species and the diversity of the studied environments, changes in the structure of the ecosystem over time, 11 12 and to assess the impact of environmental and anthropogenic factors on the studied populations. The indexes 13 commonly used for assessing the structure of the water fungal community are Menhinick’s Species Richness 14 15 Index and Simpson’s Diversity Index. Menhinick's Index is recognized as a species richness indicator for both 16 high- and low-abundance samples (Kanieski et al., 2018; Mirzaie et al. 2013). The Simpson's Diversity Index 17 18 represents the probability that two individuals randomly selected from a sample will belong to different species. 19 These indicators were used in the present study to identify qualitative and quantitative differences in the fungal 20 21 community in the studied recreational ponds at different sampling periods, i.e. June, July-August and September. 22 The Simpson's Diversity Index indicated a high species diversity for most analysed ponds: for 10 out of the 15 23 24 analysed samples (i.e. various ponds and research seasons) the Simpson’s diversity index was above 0.9. It 25 should be emphasized that particularly large discrepancies in the two indices were recorded in pond S (reservoir 26 27 formed on the Ner River), where the indicators fell from 4.02 (Menhinick’s index) and 0.942 (Simpson’s 28 29 diversity index) in June, to 2.109 and 0.603 during period II and then to 0.680 and 0.144 during period III. In the 30 period July-September, no significant changes in pH or water temperature were observed in comparison to other 31 32 reservoirs; however, in September, work began to modernize water pond S to remove sludge from its bottom 33 what could disturb the existing aquatic ecosystem. According to the literature data these indicators display wide 34 35 variation regardless of climate zone: considerable differences in Simpson's Diversity Index values have been 36 found in Antarctic lakes (0.13 to 0.72) (Goncalves et al. 2012), and in urban lakes in China (0.39 to 0.98) (Zhang 37 38 et al, 2018). 39 The abundance and species diversity of fungi depends on the properties of water reservoirs such as 40 41 standing water, flowing water and reservoir volume, as well as the prevailing physicochemical conditions, such 42 as access to light, the amount of oxygen dissolved in water, water pressure and the presence of organic 43 44 compounds (Pejman et al., 2009; Krauss et al. 2011; Pietryczuk et al. 2018). Environmental stress factors such as 45 high concentrations of heavy metals, sulphates and nitrates, as well as low concentrations of oxygen have been 46 47 found to significantly reduce the diversity and biomass of hyphomycetes (Solé et al. 2008). Redundancy analysis 48 (RDA) indicates that variations in water chemistry cause a significant proportion of the change in fungal 49 50 community structure (86.2%), with fungi being negatively correlated with high metal and nutrient 51 concentrations. Nitrates and phosphates stimulate fungal growth but at higher concentrations, this positive 52 53 correlation between nutrients and fungal diversity may become reversed (Krauss et al. 2011). Sridhar et al. 54 (2009) report low fungal diversity at higher concentrations of biogens. The recreational water bodies examined 55 56 in the present study are artificial reservoirs formed on small rivers flowing through the city of Lodz. These rivers 57 are supplied with water from storm canals collecting pollution from the center of the city, recreational plots and 58 59 industrial plants, thus water is rich in organic matter and chemical pollutions. Additionally, they are subject to 60 61 62 10 63 64 65 Pobrano z https://publicum.umed.lodz.pl / Downloaded from Repository of Medical University of Lodz 2021-10-04 periodic increases in pollution due to illegal discharge of industrial wastewater that may affect the fluctuations in 1 the number of micro-fungi in the ponds. 2 3 Most aquatic hyphomycete species tolerate pH within a range between 5 and 7: the number of species in 4 water reservoirs declines below and above these values (Krauss et al. 2011). Anthropogenic acidification of 5 6 water strongly slows the decomposition of leaves and other organic matter by inhibiting the activity of such 7 enzymes as pectin lyase. Low pH values can also raise aluminium concentrations in stream water, which 8 9 severely depresses fungal richness and activity (Baudoin et al., 2008). Additionally, greater increases in 10 temperature resulting from sunlight irradiation, found in slow-flowing surface water, and the DNA-damaging 11 12 effect of UV-radiation may lead to reduce the abundance of fungi and other microorganisms in the surface layer 13 (Dand et al. 2009, Novak Babiˇc et al. 2017). Although the physicochemical conditions in examined ponds 14 15 varied widely depending on the time of sampling (temperature: 10-24 ºC, pH; 6.5-8.5) it seem that these 16 fluctuations did not appear to have significant impact on the abundance of the fungal population. It should be 17 18 emphasized that high air temperatures associated with strong solar radiation were recorded (mean 18°C; 19 maximum 33°C) during the sampling period, both in June and July, that could modulate the abundance fungal 20 21 communities. The abundance of fungi was found to be low in the samples, varying from 0.4 to 4.6 CFU/1mL 22 according to the time of sampling. Comparable CFU values were obtained for three lakes used for recreational 23 24 purposes located in Olsztyn, northern Poland (Biedunkiewicz & Góralska, 2016), where the amounts of fungal 25 propagules ranged between 190 - 550 (CFU)/L before swimming season, 375 - 7000 CFU/L during the season 26 27 and 90 - 1800 CFU/L after the season. Much lower abundance was observed in water samples taken from five 28 29 Antarctic lakes (from 6.5 to 62.0 CFU/1L) (Goncalves et al. 2012), and much higher levels in water samples 30 taken in the Araçá Bay mangrove swamp, São Sebastião, Brazil (Doi et al., 2018) where filamentous fungus 31 4 4 32 colony density ranged from 0.1x10 CFU/ 100 mL to 4.6x10 CFU/100mL. 33 Summary 34 35 Most human mycoses are caused by opportunistic fungi with a widespread occurrence in the 36 environment (genera Aspergillus, Fusarium, Rhizopus). Our study suggests that natural bathing areas can serve 37 38 as reservoirs of potentially pathogenic fungi. Among the 149 taxa of filamentous fungi isolated, 128 (over 85%) 39 were clinically relevant species belonging to risk group 1 and 2 (RG-1 or RG-2). Such a significant percentage of 40 41 species associated with human fungal infections highlights the potential health risk for people bathing in natural 42 bathing areas. Potentially pathogenic species of Aspergillus, Cladosporidium, Alternaria, Fusarium, Penicillium 43 44 and Phialophora, have also been detected in other studies on surface waters used for recreation in other parts of 45 the world. Hence, it is recommended that the degree of fungal contamination of natural and anthropogenic water 46 47 reservoirs should be monitored, especially in bathing season. 48 49 50 References 51 Anaissie E.J., Kuchar R.T., Rex J.H., Francesconi A., Kasai M., Muller F.M., Lozano-Chiu M., Summerbell R. 52 53 C., Dignani M. C., Chanock S. J. & Walsh T., 2001. 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WHO 2004. 26 27 Zhang H., Wang Y., Chen S., Zhao Z., Feng J., Zhang Z., Lu K. & Jia J., 2018. Water bacterial and fungal 28 29 community compositions associated with urban lakes, Xi’an, China. International Journal of Environmenal 30 Research and Public Health 15(469): 1-18. 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 15 63 64 65 Pobrano z https://publicum.umed.lodz.pl / Downloaded from Repository of Medical University of Lodz 2021-10-04 Figure
POLAND
N LODZ W E S
Figure 1. Localization of examined ponds in Lodz city: SJ – Jan’s Pond; M – Mlynek; S – Stefanski’s Pond; J – Pond at Jasien; A – Arturowek complex.
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3.9% 2.7% 2.4%
Oomycota Zygomycota Ascomycota Basidiomycota
91.0%
7.3%
20.2% Alternaria 39.5% Aspergillus Penicillium Talaromyces Trichophyton Fusarium Trichoderma Other Ascomycota
18.2%
6.6% 1.3% 4.3% 2.6% Figure 2. The frequencies of phyla and genera found in water samples of examined ponds
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34
23 16 36 40 21
5 23 20
40 13 No.of species 16 0 22 SJ 16
M 24 8 Season III S 22 Season II J
A Season I
Season I Season II Season III
Figure 3. Species richness (S) in individual water reservoirs in three research seasons.
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26 24 22 20 18 16 14 12 10
C]
o 8 0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20
SJ M S 26 24 22 20
Water temperature [ temperature Water 18 16 14 12 10 8 0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20
J A Number of taxa in ponds
A. The significant positive correlation was found only in pond S. Spearman correlation coefficients were: r=-0.406 (SJ), r=0.067 (M), r=0.745 (S), r=-0.385 (J) and r=-0.475 (A).
9,5 9,0 8,5 8,0 7,5 7,0 6,5 6,0 5,5 5,0 0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20
SJ M S
pH 9,5 9,0 8,5 8,0 7,5 7,0 6,5 6,0 5,5 5,0 0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20
J A Number of taxa in ponds
B. The significant negative correlation was found only in pond SJ and significant positive correlation in pond S. Spearman correlation coefficients were: r=-0.737 (SJ), r=0.472 (M), r=0.587 (S), r=-0.403 (J), r=-0.020 (A).
Figure 4. The number of fungal taxa from water samples depending on water temperature (A) and pH (B).
Pobrano z https://publicum.umed.lodz.pl / Downloaded from Repository of Medical University of Lodz 2021-10-04 Figure
26 24 22 20 18 16 14 12 10 8 6
C]
o 4 0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20 SJ M S 26 24 22
Air temperature[ 20 18 16 14 12 10 8 6 4 0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20 J A Number of taxa in ponds
There was no significant correlation. Spearman correlation coefficients were: r=-0.328 (SJ), r=0.059 (M), r=-0.555 (S), r=-0.418 (J), r=-0.275 (A).
Figure 5. The number of fungal taxa water samples depending on air temperature.
Pobrano z https://publicum.umed.lodz.pl / Downloaded from Repository of Medical University of Lodz 2021-10-04 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Table15 16 17 18 Table 1. Genera of fungi isolated from the examined water reservoirs in three research seasons. 19 Pond [X/A]* Number 20 Total SJ M S J A of 21 number No. Phylum Classis Genus clinically 22 of I II III I II III I II III I II III I II III relevant species 23 species 24 1 Oomycota Oomycetes Globisporangium X/1 1 - 25 2 Phytopythium X/1 X/1 2 - 26 3 Pythium X/1 X/1 X/1 X/2 3 3 27 4 Achlya X/1 X/1 X/1 X/1 1 1 28 5 Aphanomyces X/1 1 - 29 6 Zygomycota Mucoromycetes Lichtheimia X/1 1 1 30 7 Mucor X/1 X/1 X/1 1 1 31 8 Rhizopus X/1 X/2 2 2 32 9 Syncephalastrum X/1 1 1 10 Ascomycota Dothideomycetes Neofusicoccum X/1 X/1 X/1 X/1 X/1 1 - 33 11 Neoscytalidium X/1 1 1 34 12 Cladosporium X/1 X/1 1 1 35 13 Hortaea X/1 1 1 36 14 Tripospermum X/1 1 1 37 15 Sydowia X/1 X/1 X/1 1 1 38 16 Arthrographis X/1 X/1 1 1 39 17 Taeniolella X/1 X/1 1 1 40 18 Alternaria X/4 X/2 X/2 X/1 X/1 X/1 X/2 X/1 X/1 X/3 X/1 X/1 X/2 7 7 41 19 Coniothyrium X/1 1 1 42 20 Didymella X/1 1 - 21 Medicopsis X/1 X/1 1 1 43 22 Phoma X/1 X/1 X/1 2 2 44 23 Stagonosporopsis X/1 X/1 1 1 45 24 Eurotiomycetes Onychocola X/1 X/1 X/1 1 1 46 25 Cladophialophora X/1 1 1 47 26 Cyphellophora X/1 X/1 2 2 48 27 Exophiala X/1 1 1 49 28 Aspergillus X/2 X/3 X/7 X/5 X/6 X/2 X/4 X/3 X/1 X/4 X/6 X/7 X/5 X/2 X/4 23 21 50 29 Neosartorya X/1 X/1 1 1 51 30 Paecilomyces X/1 X/1 1 1 52 31 Penicillium X/7 X/3 X/4 X/3 X/3 X/8 X/2 X/8 X/3 X/8 X/2 X/1 X/3 18 14 32 Talaromyces X/1 X/1 X/1 X/2 X/2 X/1 X/2 X/3 8 8 53 33 Ajellomyces X/1 X/1 X/1 X/1 X/1 X/1 1 1 54 34 Arachniotus X/1 X/1 1 - 55 35 Chrysosporium X/1 X/1 X/1 X/1 X/2 X/1 X/1 2 2 56 36 Trichophyton X/1 X/1 X/1 X/1 X/1 X/1 X/1 X/1 3 3 57 37 Incertae sedis Dissitimurus X/1 X/1 1 1 58 38 Scolecobasidium X/1 X/1 2 2 59 39 Staphylotrichum X/1 1 1 60 40 Leotiomycetes Scytalidium X/1 X/1 X/2 X/1 X/3 X/1 X/1 X/1 3 3 61 41 Orbiliomycetes Arthrobotrys X/1 1 1 42 Sordariomycetes Coniochaeta X/1 X/1 X/1 2 2 62 43 Phaeoacremonium X/1 1 1 63 44 Acremonium X/2 X/1 X/1 X/2 4 4 64 65 Pobrano z https://publicum.umed.lodz.pl / Downloaded from Repository of Medical University of Lodz 2021-10-04 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 45 Bisifusarium X/1 1 1 19 46 Fusarium X/1 X/1 2 1 20 47 Neocosmospora X/1 X/1 X/1 1 3 21 48 Hypomyces X/1 X/1 1 1 22 49 Ilyonectria X/1 1 1 23 50 Cordyceps X/1 X/1 2 2 24 51 Metarhizium X/1 X/1 1 1 25 52 Sarocladium X/2 X/1 X/1 X/1 2 2 53 Trichoderma X/1 X/5 X/1 X/4 X/2 X/2 X/2 X/1 X/1 X/1 5 5 26 54 Papulaspora X/1 X/1 X/1 X/1 X/1 3 3 27 55 Plectosphaerella X/1 X/1 X/1 1 1 28 56 Verticillium X/1 1 - 29 57 Pseudallescheria X/1 1 1 30 58 Scopulariopsis X/1 X/1 2 2 31 59 Cladorrhinum X/1 1 1 32 60 Humicola X/2 X/1 X/2 X/1 X/2 X/1 X/1 4 4 33 61 Thermothelomyces X/1 1 1 34 62 Arthrinium X/1 1 1 35 63 Basidiomycota Agaricomycetes Bjerkandera X/1 X/1 X/1 X/1 X/1 1 1 64 Riopa X/1 X/1 1 1 36 65 Phanerodontia X/1 1 1 37 66 Sporotrichum sp. X/1 1 1 38 Total 149 128 39 *X – occurrence of any species of the genus 40 A – number of species isolated within the genus 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Pobrano z https://publicum.umed.lodz.pl / Downloaded from Repository of Medical University of Lodz 2021-10-04 Table
Table 2. Sampling stations characteristics with species richness (S) and species diversity.
1 SJ M S J A 2 3 I II III I II III I II III I II III I II III 4 Number of species 36 23 34 16 21 16 40 13 5 24 16 23 22 8 22 5 Number of colonies (S) 68 40 69 35 60 21 99 38 54 82 25 57 24 12 88 6 The average number of 7 3.8 2.2 3.8 1.9 3.3 1.2 4.1 1.6 2.2 4.6 3.2 1.4 0.8 0.4 2.9 colonies (CFU/1mL) 8 ±4.965 ±2.901 ±1.618 ±1.862 ±4.935 ±1.200 ±4.267 ±2.570 ±1.180 ±5.227 ±2.333 ±0.916 ±1.215 ±0.674 ±5.413 x̅ ±SD 9 Menhinick’s Species 4.37 3.64 4.09 2.70 2.71 3.49 4.02 2.11 0.68 2.65 3.20 3.05 4.49 2.31 2.35 10 Richness Index^ Simpson's Diversity 11 0.95 0.92 0.96 0.83 0.71 0.98 0.94 0.60 0.14 0.88 0.96 0.95 0.99 0.91 0.92 12 Index^ Number of clinically 13 31 20 30 16 19 14 33 12 5 18 14 18 21 7 21 relevant species* 14 Number of species 15 from: 16 Zygomycota 1 2 1 0 1 0 0 0 0 1 0 1 0 0 1 17 4 2 2 1 1 1 2 1 1 3 1 1 0 0 2 18 Alternaria 19 Aspergillus 2 3 7 5 6 2 3 2 1 3 6 6 4 2 4 20 Cladosporium 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 21 Penicillium 7 2 3 3 2 0 6 2 0 6 3 6 2 1 3 22 1 0 1 1 1 0 1 0 1 0 1 0 0 0 1 23 dermatophytes 24 ^Based on all indentified species 25 *Number of clinically relevant species with defined risk group (RG) and biosafety level (BSL) 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Pobrano z https://publicum.umed.lodz.pl / Downloaded from Repository of Medical University of Lodz 2021-10-04 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Table15 16 17 18 19 20 21 22 Table 3. Physicochemical parameters in analyzed ponds during sampling. 23 Research season* 24 25 June (I) July and August (II) September (III) 26 Average 27 18.8 17.8 15.8 28 temperature [°C] 29 Average humidity 74.8 73.7 70.1 30 31 Pond 32 33 SJ M S J A 34 Season I II III I II III I II III I II III I II III 35 36 Air temperature 21°C 16°C 6.5°C 21°C 17.5°C 7.5°C 16°C 17°C 5.5°C 19°C 24°C 12°C 19°C 15°C 10°C 37 [°C] 38 39 Water temperature 21.3°C 19°C 10°C 21.3°C 19°C 13°C 21°C 19.5°C 14°C 21°C 24°C 14°C 21.4°C 21°C 13°C 40 Water pH 7.67 8.3 6.5 6.5 6.67 6.67 8.5 8.5 7,13 6.5 7.3 6.17 6.8 6.8 6.8 41 42 *Water samples were collected from different ponds during one week in each research season. 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Pobrano z https://publicum.umed.lodz.pl / Downloaded from Repository of Medical University of Lodz 2021-10-04 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Supplementary15 Material 16 17 18 Table S1. Number of colonies of fungi isolated from the examined water reservoirs in three research seasons (acc. Index Fungorum). 19 20 Number of colonies Pond RG/ 21 No. Phylum Classis Species/genus 22 SJ M S J A BSL 23 I II III I II III I II III I II III I II III Oomycota Oomycetes Globisporangium rostratum (E.J. Butler) Uzuhashi, Tojo & 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 - 24 1 Kakish. 25 2 Phytopythium indigoferae (E.J. Butler) P.M. Kirk 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 - 26 Phytopythium oedochilum (Drechsler) Abad, de Cock, Bala, 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 - 3 27 Robideau, Lodhi & Lévesque 28 4 Pythium elongatum V.D. Matthews 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1/1 29 5 Pythium inflatum V.D. Matthews 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1/1 30 6 Pythium sp. 0 0 0 0 0 0 7 0 0 0 1 1 0 0 0 * 31 7 Achlya sp. 0 0 1 0 0 2 0 0 0 0 0 1 0 1 0 * 32 8 Aphanomyces cladogamus Drechsler 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 - 33 9 Zygomycota Mucoromycetes Lichtheimia corymbifera (Cohn) Vuill. 1903 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 2/2 10 Mucor ramosissimus Samouts. 0 0 1 0 1 0 0 0 0 1 0 0 0 0 0 1/1 34 11 Rhizopus arrhizus A. Fisch. 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 2/2 35 12 Rhizopus stolonifer (Ehrenb.) Vuill. 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1/1 36 13 Syncephalastrum racemosum Cohn ex J. Schröt. 0 0 0 0 0 0 0 0 0 0 0 3 0 0 0 ½ 37 Ascomycota Dothideomycetes Neofusicoccum mangiferae (Syd. & P. Syd.) Crous, Slippers & 0 1 1 0 0 0 2 0 0 2 1 0 0 0 0 - 14 38 A.J.L. Phillips 39 15 Neoscytalidium dimidiatum (Penz.) Crous & Slippers 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 2/2 40 16 Cladosporium sphaerospermum Penz. 1 0 0 0 0 0 2 0 0 0 0 0 0 0 0 1/1 41 17 Hortaea werneckii (Horta) Nishim. & Miyaji 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1/1 42 18 Tripospermum myrti 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1/1 19 Sydowia polyspora (Bref. & Tavel) E. Müll. 1 0 1 0 0 0 0 0 0 0 0 1 0 0 0 1/1 43 Arthrographis kalrae (R.P. Tewari & Macph.) Sigler & J.W. 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 1/2 20 44 Carmich. 45 21 Taeniolella stilbospora (Corda) S. Hughes 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1/1 46 22 Alternaria alternata (Fr.) Keissl. 0 1 1 1 1 0 4 1 50 2 1 0 0 0 2 1/1 47 23 Alternaria chartarum Preuss (Ulocladium chartarum) 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1/1 48 24 Alternaria chlamydospora Mouch. 1 0 0 0 0 0 0 0 0 1 0 0 0 0 1 1/1 49 25 Alternaria dianthicola Neerg. 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 1/1 50 26 Alternaria infectoria E.G. Simmons 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1/1 51 27 Alternaria longipes (Ellis & Everh.) E.W. Mason 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1/1 52 28 Alternaria tenuissima (Kunze) Wiltshire 0 1 0 0 0 0 0 0 0 0 0 2 0 0 0 1/1 29 Coniothyrium olivaceum Bonord. (Microsphaeropsis olivacea) 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1/1 53 30 Didymella molleriana (G. Winter) Q. Chen & L. Cai 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 - 54 31 Medicopsis romeroi (Borelli) Gruyter, Verkley & Crous 2 0 2 0 0 0 0 0 0 0 0 0 0 0 0 1/2 55 32 Phoma herbarum Westend. 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1/1 56 33 Phoma minutella Sacc. & Penz. 0 0 0 2 0 0 1 0 0 0 0 0 0 0 0 2/2 57 Stagonosporopsis oculi-hominis (Punith.) Aveskamp, Gruyter & 0 0 0 0 0 0 1 0 0 0 0 0 2 0 0 1/2 34 58 Verkley 59 35 Eurotiomycetes Onychocola canadensis Sigler 0 0 0 0 0 1 0 0 0 0 0 1 0 0 1 1/2 Cladophialophora carrionii (Trejos) de Hoog, Kwon-Chung & 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 2/2 60 36 McGinnis 61 37 Cyphellophora pluriseptata G.A. de Vries, Elders & Luykx 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1/1 62 38 Cyphellophora reptans (de Hoog) Réblová & Unter. 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1/1 63 39 Exophiala jeanselmei (Langeron) McGinnis & A.A. Padhye 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2/2 64 65 Pobrano z https://publicum.umed.lodz.pl / Downloaded from Repository of Medical University of Lodz 2021-10-04 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 40 Aspergillus amstelodami Thom & Church 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1/1 19 41 Aspergillus candidus Link 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1/1 20 42 Aspergillus chevalieri Thom & Church 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1/1 21 43 Aspergillus clavatonanicus Bat., H. Maia & Alecrim 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1/1 22 44 Aspergillus clavatus Desm. 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1/1 23 45 Aspergillus fischeri Wehmer 0 0 5 0 2 1 0 0 0 2 0 4 0 0 1 1/1 24 46 Aspergillus flavipes (Bainier & R. Sartory) Thom & Church 0 0 1 0 0 0 0 0 0 0 1 0 0 0 0 1/1 25 47 Aspergillus flavus Link 0 0 4 0 0 0 0 0 0 0 1 0 0 0 0 1/2 48 Aspergillus fumigatus Fresen. 7 11 9 14 32 2 19 24 0 19 1 1 1 4 54 2/2 26 49 Aspergillus glaucus (L.) Link 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1/1 27 50 Aspergillus hollandicus Samson & W. Gams 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 - 28 51 Aspergillus insolitus (G. Sm.) Houbraken, Visagie & Samson 2 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1/1 29 52 Aspergillus janus Raper & Thom 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 1/1 30 53 Aspergillus neoniveus Samson, S.W. Peterson, Frisvad & Varga 0 0 0 0 1 0 1 0 0 0 1 2 0 0 0 1/1 31 54 Aspergillus nidulans (Eidam) G. Winter 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1/1 32 55 Aspergillus niger Tiegh. 0 0 3 0 0 0 0 0 0 19 4 5 1 0 6 1/1 33 56 Aspergillus repens (Corda) Sacc. 0 0 1 0 0 0 0 0 0 0 1 0 0 0 0 1/1 34 57 Aspergillus reptans Samson & W. Gams 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1/1 35 58 Aspergillus restrictus G. Sm. 0 3 1 0 1 0 0 0 0 0 0 0 0 0 2 1/1 59 Aspergillus sydowii (Bainier & Sartory) Thom & Church 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 1/1 36 60 Aspergillus ustus (Bainier) Thom & Church 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 1/1 37 61 Aspergillus versicolor (Vuill.) Tirab. 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 1/1 38 62 Aspergillus sp. 0 0 0 0 0 0 1 0 0 3 0 0 1 0 0 * 39 63 Neosartorya pseudofischeri S.W. Peterson 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 1/2 40 64 Paecilomyces variotii Bainier 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1/2 41 65 Penicillium albicans Bainier 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 - 42 66 Penicillium albidum Sopp 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 1/1 43 67 Penicillium aurantiogriseum Dierckx 1 1 2 1 0 0 0 0 0 1 1 2 0 0 1 1/1 44 68 Penicillium camemberti Thom 1 0 0 0 0 0 4 0 0 0 0 0 0 0 0 1/1 45 69 Penicillium chrysogenum Thom 5 0 0 4 0 0 10 0 0 1 0 9 1 0 6 1/1 70 Penicillium citrinum Thom 2 2 1 1 0 0 3 0 0 0 0 6 0 0 1 1/1 46 71 Penicillium commune Thom 1 0 0 0 0 0 0 0 0 2 1 0 0 0 0 1/1 47 72 Penicillium decumbens Thom 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1/1 48 73 Penicillium expansum Link 1 0 0 0 0 0 1 1 0 0 0 4 1 0 0 1/1 49 74 Penicillium griseofulvum Dierckx 0 0 0 0 1 0 1 0 0 1 0 1 0 0 0 1/1 50 75 Penicillium italicum Wehmer 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1/1 51 76 Penicillium ochrosalmoneum Udagawa 0 0 0 0 0 0 6 0 0 0 0 0 0 0 0 - 52 77 Penicillium roseopurpureum Dierckx 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1/1 53 78 Penicillium simplicissimum (Oudem.) Thom 0 0 0 0 0 0 0 0 0 11 0 0 0 1 0 1/1 54 79 Penicillium solitum Westling 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1/1 55 80 Penicillium spinulosum Thom 0 0 3 0 0 0 0 0 0 0 0 0 0 0 0 1/1 81 Penicillium waksmanii K.M. Zaleski 0 2 2 0 0 0 2 0 0 4 0 1 0 0 0 - 56 82 Penicillium sp. 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 * 57 Talaromyces duclauxii (Delacr.) Samson, N. Yilmaz, Frisvad & 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1/1 83 58 Seifert 59 84 Talaromyces flavus (Klöcker) Stolk & Samson 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1/1 Talaromyces funiculosus (Thom) Samson, N. Yilmaz, Frisvad & 0 0 0 0 0 0 0 0 0 0 3 0 0 0 0 1/1 60 85 61 Seifert Talaromyces piceae (Raper & Fennell) Samson, N. Yilmaz, 0 0 0 1 0 0 0 0 0 0 0 1 1 0 0 1/1 62 86 63 Houbraken, Spierenburg, Seifert, Peterson, Varga & Frisvad 87 Talaromyces purpureogenus Samson, N. Yilmaz, Houbraken, 0 0 0 0 0 1 0 1 0 0 0 1 0 0 0 1/1 64 65 Pobrano z https://publicum.umed.lodz.pl / Downloaded from Repository of Medical University of Lodz 2021-10-04 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Spierenb., Seifert, Peterson, Varga & Frisvad 19 Talaromyces ruber (Stoll) N. Yilmaz, Houbraken, Frisvad & 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1/1 88 20 Samson Talaromyces rugulosus (Thom) Samson, N. Yilmaz, Frisvad & 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1/1 21 89 22 Seifert Talaromyces variabilis (Sopp) Samson, N. Yilmaz, Frisvad & 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 1/1 23 90 24 Seifert 91 Ajellomyces crescens Sigler 0 1 1 0 1 0 1 0 0 0 0 3 1 0 0 2/2 25 92 Arachniotus dankaliensis (Castell.) J.F.H. Beyma 0 0 1 0 0 0 2 0 0 0 0 0 0 0 0 - 26 93 Chrysosporium anamorph of Nannizziopsis vriesii (CANV) 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1/1 27 94 Chrysosporium inops J.W. Carmich. 0 0 1 0 0 1 0 1 0 2 3 0 1 0 1 1/1 28 Trichophyton schoenleinii (Lebert) Langeron & Miloch. ex 0 0 1 1 0 0 0 0 1 0 0 0 0 0 2 2/2 95 29 Nann. 30 96 Trichophyton verrucosum E. Bodin 1 0 0 0 0 0 2 0 0 0 2 0 0 0 0 1/2 31 97 Trichophyton violaceum Sabour. ex E. Bodin 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1/2 32 98 Incertae sedis Dissitimurus exedrus E.G. Simmons, McGinnis & Rinaldi 2 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1/2 33 99 Scolecobasidium constrictum E.V. Abbott 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1/1 34 100 Scolecobasidium humicola G.L. Barron & L.V. Busch 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1/1 101 Staphylotrichum coccosporum J.A. Mey. & Nicot 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1/1 35 102 Leotiomycetes Scytalidium infestans Iwatsu, Udagawa & Hatai 1 0 0 0 6 0 1 0 0 0 0 0 0 1 0 1/1 36 103 Scytalidium japonicum Udagawa, K. Tominaga & Hamaoka 0 0 1 0 0 0 2 0 0 0 0 0 0 0 0 1/1 37 104 Scytalidium lignicola Pesante 0 1 2 0 0 0 1 0 0 2 0 0 0 0 1 1/1 38 105 Orbiliomycetes Arthrobotrys sp. 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 * 39 Sordariomycetes Coniochaeta hoffmannii (J.F.H. Beyma) Z.U. Khan, Gené & 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1/1 106 40 Guarro Coniochaeta mutabilis (J.F.H. Beyma) Z.U. Khan, Gené & 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 1/1 41 107 42 Guarro 43 108 Phaeoacremonium rubrigenum W. Gams, Crous & M.J. Wingf. 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1/1 44 109 Acremonium alabamense Morgan-Jones 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1/1 110 Acremonium curvulum W. Gams 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1/1 45 111 Acremonium hyalinulum (Sacc.) W. Gams 1 0 0 0 0 1 0 1 0 0 0 0 0 0 0 1/1 46 112 Acremonium spinosum (Negroni) W. Gams 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1/1 47 113 Bisifusarium dimerum (Penz.) L. Lombard & Crous 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 1/1 48 114 Fusarium oxysporum Schltdl. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1/2 49 115 Fusarium sp. 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 * 50 116 Hypomyces chrysospermus Tul. & C. Tul. 0 1 0 0 0 0 2 0 0 0 0 0 0 0 0 1/1 51 117 Ilyonectria destructans (Zinssm.) Rossman, L. Lombard & Crous 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1/1 52 118 Cordyceps farinosa (Holmsk.) Kepler, B. Shrestha & Spatafora 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1/1 53 119 Cordyceps javanica (Bally) Kepler, B. Shrestha & Spatafora 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 1/1 54 120 Metarhizium anisopliae (Metschn.) Sorokīn 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 1/1 121 Neocosmospora falciformis (Carrión) L. Lombard & Crous 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1/2 55 Neocosmospora cyanescens (G.A. de Vries, de Hoog & Bruyn) 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1/2 122 56 Summerb., Schroers & J.A. Scott 57 123 Neocosmospora solani (Mart.) L. Lombard & Crous 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1/2 58 124 Sarocladium kiliense (Grütz) Summerb. 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1/2 59 125 Sarocladium strictum (W. Gams) Summerb. 1 0 1 0 0 0 1 0 0 0 0 0 0 0 0 1/1 60 126 Trichoderma harzianum Rifai 0 2 1 0 2 3 1 0 0 0 0 0 0 0 0 1/1 61 127 Trichoderma koningii Oudem. 0 0 2 0 1 0 0 0 0 0 0 0 0 0 0 1/1 62 128 Trichoderma longibrachiatum Rifai 0 0 6 0 1 0 2 0 0 1 0 0 0 1 0 1/1 63 129 Trichoderma pseudokoningii Rifai 0 0 2 0 1 0 0 0 0 0 0 1 0 0 0 1/1 64 65 Pobrano z https://publicum.umed.lodz.pl / Downloaded from Repository of Medical University of Lodz 2021-10-04 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 130 Trichoderma viride Pers. 0 0 1 1 0 1 0 0 0 1 0 0 0 0 1 1/1 19 131 Papulaspora equi Shadomy & D.M. Dixon 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 1/1 20 132 Papulaspora pallidula Hotson 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1/1 21 133 Papulaspora sp. 0 0 0 0 0 0 2 3 0 0 0 0 0 0 0 * 22 134 Plectosphaerella cucumerina (Lindf.) W. Gams 1 0 0 0 0 0 1 0 0 0 0 0 1 0 0 1/1 23 135 Verticillium sp. 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 * Pseudallescheria boydii (Shear) McGinnis, A.A. Padhye & 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 2/2 24 136 25 Ajello 137 Scopulariopsis asperula (Sacc.) S. Hughes 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1/1 26 138 Scopulariopsis koningii (Oudem.) Vuill. 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1/1 27 139 Cladorrhinum bulbillosum W. Gams & Mouch. 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1/1 28 140 Humicola brevis (J.C. Gilman & E.V. Abbott) J.C. Gilman 0 1 0 0 0 0 0 0 0 1 0 0 1 0 0 1/1 29 141 Humicola fuscoatra Traaen 0 1 0 0 0 1 0 0 0 0 0 0 1 0 1 1/1 30 142 Humicola grisea Traaen 0 0 2 0 0 1 0 0 0 0 0 0 0 0 0 1/1 31 143 Humicola nigrescens Omvik 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1/1 Thermothelomyces thermophilus (Apinis) Y. Marín, Stchigel, 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 1/1 32 144 33 Guarro & Cano 34 145 Arthrinium phaeospermum (Corda) M.B. Ellis 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 1/1 35 146 Basidiomycota Agaricomycetes Bjerkandera adusta (Willd.) P. Karst. 12 0 0 2 0 0 2 0 0 0 0 0 2 1 0 1/1 147 Riopa metamorphosa (Fuckel) Miettinen & Spirin 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 1/1 36 148 Phanerodontia chrysosporium (Burds.) Hjortstam & Ryvarden 0 0 4 0 0 0 0 0 0 0 0 0 0 0 0 1/1 37 149 Sporotrichum sp. 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 * 38 Total 68 40 69 35 60 21 99 38 54 82 25 57 24 12 88 128 39 40 RG – risk group 41 BSL – biosafety level 42 - undefined risk group and biosafety level 43 * risk group and biosafety level defined for some species within the genus 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Pobrano z https://publicum.umed.lodz.pl / Downloaded from Repository of Medical University of Lodz 2021-10-04 Supplementary Material
Table S2. The number of fungal species with a defined risk group (RG) and biosafety level 1 2 (BSL). 3 SJ M S J A 4 5 I II III Total* I II III Total I II III Total I II III Total I II III Total 6 7RG -2 2 3 3 5 3 2 1 4 4 2 1 5 1 1 2 2 4 2 3 6 8RG -1 29 17 27 64 13 17 13 38 29 10 4 42 17 13 16 41 17 5 18 38 9 10BSL -2 7 3 6 11 4 3 3 8 6 2 1 9 2 3 4 8 4 2 6 12 11BSL -1 24 17 24 58 12 16 11 34 27 10 4 38 16 11 14 35 17 5 15 32 12 13 *total number of species isolated in three research seasons; species detected in several seasons was counted only 14 once 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Pobrano z https://publicum.umed.lodz.pl / Downloaded from Repository of Medical University of Lodz 2021-10-04