A fungal spore calendar for the atmosphere of Szczecin , Poland

A calendar of fungal spore seasons for Szczecin during 2013 was established using a 7-day volumetric Lanzoni trap. Thirty-five spore taxa were identified. The dominant spore types detected were Cladosporium (66%), Didymella (29%), Alternaria (1.67%), and Leptosphaeria type (1.21%). The spores were present throughout the study year. However, there was a wide daily fluctuation in the concentration values with a tendency towards an increase during the summer months. Seasonally, the spore levels of Cladosporium, Alternaria, and Leptosphaeria type peaked in summer (June–September), while those of Didymella mainly in July. Most of the other spore types had the highest concentrations in summer but occurred in the air from spring to late fall.


Introduction
Air-borne particles can have many effects, including plant, animal and human diseases, allergies, plant pollination, and colonization of new habitats.Fungi, as one of the main component of air, tend to be especially difficult to study since their production, maturation, release, and concentration in the air depend on multiple biotic and abiotic factors.In Szczecin, there have been few studies on atmospheric mycobionta.Aeromycological research in outdoor environments has been carried out in Szczecin only for selected allergenic taxa (Alternaria, Cladosporium, Ganoderma, Didymella) [1][2][3][4].The main goal of this study was to create a spore calendar for 2013 and show the percentage of all identified fungal spores.In the previous work [5], the annual and seasonal variations between 2004 and 2006 were examined, separating 10 spore types from the total spore numbers.This was not a quantitative investigation of the range of airborne spores in the atmosphere of Szczecin.Thus, a daily spore calendar was established for Szczecin, Poland, studying the year 2013 in detail and using a Lanzoni spore trap.mounted on microscope slides in glycerol jelly containing fuchsine (0.1%) and protected with a cover glass.The spores were observed and identified using a Zeiss Primo Star microscope at a magnification of 400× along one longitudinal traverse.The spore concentrations were estimated as the number of spores per cubic meter of air sampled according to the British Aerobiology Federation [6].

Location of the Lanzoni spore trap
The air sampler was placed on the roof of the Faculty of Biology building at the University of Szczecin at 21 m above ground level, near the city center.The area is completely urbanized with green spaces (Kasprowicza Park) and abundant trees.
Szczecin is situated in the Odra River valley in the northwest part of Poland (53°26' N, 14°32' E; Fig. 1).The altitude varies between 0.1 m under sea level and 148 m above sea level.The present ''Baltic'' climate in Szczecin is influenced by the air masses from over the Northern Atlantic and is characterized by mild winters and cool summers.The average relative humidity is 84%, the average air temperature ranges from 8 to 8.4°C.The average annual rainfall total is 550 mm and within a year there are approximately 167 days with precipitation.
Fungal spores that did not fit into the above categories were designated as ''other spores'' , which include partial or unidentifiable fungal spores.In order to verify the accuracy of the calculations performed under the microscope, most of the samples were reviewed using a microscopic camera connected to a computer screen.For the analysis of microscopic fungal spore images, the software Carl Zeiss AxioVision SE64 was used.

Results
Thirty-five spore taxa were identified (Tab. 1) and on the basis of this the spore calendar was created (Fig. 2).The cumulative annual total of daily spore counts for 2013 was 595 199.Spores of the genus Cladosporium dominated, providing 66% of the total count, about two times more than the next most abundant genus.The six most frequently detected spore groups, accounting for 98.75% of the total count, were Cladosporium, Didymella, Alternaria, Leptosphaeria type, Ganoderma, and Epicoccum (Fig. 3) The remaining 29 spore types were present in low numbers, seven less than 0.15% and 22 less than 0.1% of the annual total.Tab. 1 List and percentage contribution of airborne fungal spores in the atmosphere of Szczecin, 2013.

Fusarium
The average daily total fungal spore count for 2013 was 1630 spores m 3 of air.Most spore types were present almost throughout the year.Cladosporium spores were abundant the entire year with a peak spanning late spring, summer and early fall.The spore concentration of Alternaria, Didymella, Epicoccum, Stemphylium, Pithomyces, and Torula peaked mainly in summer, Ganoderma, Leptosphaeria type, Drechslera type, Pleospora, Parapheosphaeria, Polythrincium in late summer and fall.The other taxa appeared sporadically from spring to fall.

Discussion
Monitoring of fungal spore concentrations in the air is important from the aerobiological, agricultural, and medical point of view.Aerobiologists can compare the qualitative and quantitative analyses of aero-plankton in different locations.Moreover, the effects of biotic and abiotic factors on the changes in the concentrations of individual taxa can be indicated.In agriculture, effects of air monitoring are very important in order to study of the life cycle of parasites and to develop plant protection plans.From the human health point of view, the indication of exceeded threshold concentrations for allergenic taxa (among others, Alternaria and Cladosporium) helps in the diagnosis and treatment of inhaled allergens.
Currently, aerobiological monitoring is carried out on all continents and covers from a few to a dozen types of spores.In Australia (Melbourne) 29 genera and five spore groups were identified.The dominant spore types detected were Cladosporium, Leptosphaeria, and Coprinus [7].Cladosporium spore levels peaked in spring and summer, Leptosphaeria toward summer and fall, whilst Coprinus fluctuated all year round.Troutt and Levetin [8] described six spore types and three spore groups in the air of Tulsa (United States of America).The three most abundant spore types, which occurred in the air, were as follows: Cladosporium, Alternaria, and Epicoccum, and the most important meteorological factors influencing the presence of airborne spores were air temperature and dew point.Airborne spore monitoring in South America is represented by four locations: Argentina (La Plata) [9], Uruguay (Montevideo City) [10], Chile (Santiago) [11], and Cuba (Havana) [12].The most taxa were described from Argentina (79) and Uruguay (56), followed by Cuba (34), and only 14 in Chile.In every monitoring station, the most numerous taxon was Cladosporium, followed by the following taxa: in three stations Leptosphaeria, in two Alternaria and Coprinus, while in single points Agaricus, Didymella, and Aspergillus/Penicillium.
In West Africa, three taxa were isolated from desert samples: Cladosporium, Alternaria, and Aspergillus [16], while on the Arabian Peninsula up to 30 taxa with the most numerous spore types belonging to Cladosporium and Alternaria genera [17].
In Europe, the largest number of monitoring stations is found compared to other continents.The biggest aerobiological monitoring networks are in Spain, Portugal, the United Kingdom, and Poland.For example, in Catalonia (Spain) 20 fungal taxa were identified with the most numerous Agrocybe and Cladosporium type [18].In Portugal (Porto) 22 spore types were noted in 2003, and Cladosporium and Ganoderma reached the highest concentrations.The remaining spore types did not exceed 3% of the total annual sum [19] The first investigations into airborne fungal spores in Poland were conducted in two cities: Cracow and Rabka [20], where 15 types of fungal spores were identified.Spore monitoring is conducted in Cracow to this day, and the last analysis of the content of spores in the air was published by Stępalska and Wołek in 2005 [21].They described 13 spore taxa during one fungal season in which Cladosporium occurred as the most numerous fungal spore.Ten taxa were identified in Rzeszów during 2-year monitoring, and Cladosporium, Alternaria, Epicoccum, and Torula reached the highest concentrations [22].In northwest Poland (Szczecin), detailed fungal monitoring has been carried on since 2004 [23].The highest concentrations were found for Cladosprium, Alternaria, Ganoderma, and Didymella.
In recent years, the content of airborne fungal spores has been reported from Slovakia and Romania [24,25].Ščevková et al. [24] described annual trends for three spore taxa: Alternaria, Epicoccum, and Stemphylium.Ianovici [25] analyzed concentrations of five spore types: Cladosporium, Alternaria, Epicoccum, Pithomyces, and Torula.In both publications, the authors put special emphasis on climate change and concluded that temperature increase affects the concentration levels of airborne spores.
Comparing the results of this study, it can be concluded that in every continent Cladosporium is the dominant spore type in most monitoring stations.In our investigation, Cladosporium was present almost year round, except for the time with snow cover.The period in which the highest concentrations are noted, regardless of the continent, always occurs in the summer.This is associated with the respective air temperature and relative humidity, which can increase mycelium growth and spore release.Due to its allergenic properties, Alternaria is also a frequently monitored spore type.The higher prevalence of Alternaria sensitization is possibly explained by Alternaria allergens cross reacting with a range of genera, including Cladosporium, Stemphylium, Epicoccum, Fusarium, Drechslera, and Culvularia [26].
Didymella and Leptosphaeria spores, identified and recorded in Szczecin, represent proportionately "high" counts, compared to studies from other countries; this observation might be attributed to the humid and mild climate in Szczecin.Both types are classified as "wet spores", so the meteorological parameters in northwest Poland are suitable for their development.
The basidiospores of Ganoderma, commonly known as a bracket fungus or wood decay fungus, are an important and prevalent group of airborne fungal spores worldwide.Studies from various parts of the world have clearly implicated Ganoderma spores as aeroallergens [27], and they have been implicated in the elicitation of respiratory allergic diseases.The high concentration Ganoderma spores in the air of Szczecin is the result of the proximity of two major forest complexes: Beech Forest and Wkrzańska Forest.
Other types of spores occurred seasonally in low concentrations.Most of them are also recorded in most aerobiological stations across the world, but only a few achieve relatively high concentrations.The highest spore concentration peaks were coincident with the peaks of some allergenic pollen types.In Poland, an increase in allergy symptoms related to pollen during summer is observed.At the same time, high concentrations of fungal spores occur, mainly Cladosporium and Alternaria, which can interact with airborne pollen aggravating allergic problems.
In further study, we intend to consolidate these results in order to develop a useful database that could be used to assist clinicians to identify and control fungal allergy symptoms as well as by farmers to prevent plant diseases.

Fig. 2 A
Fig. 2 A fungal spore calendar for the atmosphere of Szczecin, Poland, for the year 2013.

Fig. 3
Fig.3The percentage of frequently found fungal spores in the area.