Diversity and dynamics of airborne fungi in São Luis , State of Maranhão , Brazil

introduction: This study aimed to identify airborne fungi in São Luis, Maranhão, Brazil, to determine the prevalent genera and to correlate these genera with the area and season. Methods: In total, 1,510 colony-forming units (CFUs) of airborne fungi were isolated from the north, south, east and west sides and from the center of the city from January to December 2007. The samples were collected on Petri dishes that were exposed to the fungi by the gravitational method. Results: Twenty genera of fungi were isolated; the most common were Aspergillus (33.5%), Penicillium (18.8%), Cladosporium (14.2%), Curvularia (10.6%) and Fusarium (7.6%). The CFUs of the fungi were statistically signifi cant (p < 0.0001). Fungal biological diversity was present all year, without any large seasonal variations but with slight increases in May, August and September. Conclusions: The fungal genera identifi ed in this study were correlated with natural systems and could be useful when evaluating the impact of environmental changes on the region.

Fungi, especially fi lamentous fungi, which are common aeroallergens, form a major part of bioaerosols 1 .Fungi are ubiquitous in outdoor air, and their concentrations, aerodynamic diameters and taxonomic compositions have potentially important implications for human health 2 .
The diversity and abundance of anemophilous microorganisms can be infl uenced by and can interfere with environmental conditions.These microorganisms are infl uenced by factors such as season, temperature, the relative humidity of the air and other parameters that exhibit seasonal variation 3,4 .
The relationships among allergic exposure, the fungal presence in indoor and outdoor environments and consequent allergic diseases are not fully understood 5,6 .Therefore, it is important to know both the frequency of certain airborne fungi and their distributions according to the season and the main environment (i.e., indoor or outdoor) in order to evaluate their correlations with respiratory symptoms related to allergic processes [7][8][9] .
These fungi can be used to assess effects on the environment and could contribute to determining the principal changes.The spores of fungi can be present in air particles and can potentially influence the hydrological cycle and climatic changes.In addition, humans are exposed daily to bioaerosols in their personal and professional lives, and these airborne particles represent a potential biological occupational hazard 10 .Biological particles in the air are approximately 40% organic carbon by mass and can be an important source of bioaerosols in the atmosphere above continents 4 .Despite the importance of airborne fungi, very little is known about their diversity 11 , especially of the fungi in São Luis, Maranhão.
For this reason, it was important to perform further observations to determine and characterize the frequencies of the main airborne fungi in outdoor environments and to identify possible correlations with seasonality and the possibility of monitoring allergic respiratory diseases.

MeTHODs
Airborne fungi were collected between January and December 2007 in fi ve outdoor areas of the City of São Luis (northern, southern, eastern, western and central), located in the State of Maranhão, Brazil.São Luis is located at a latitude of 2º31'47" S and a longitude of 44º18'10''W, and it is 24m above sea level.This city covers an area of approximately 830km 2 and has a population of 1,017,772 inhabitants 12 .
Bezerra GFB et al -Diversity and dynamics of airborne fungi resULTs An analysis of the mycobiota was performed using Petri dishes (10cm by 2cm) containing 10mL of Sabouraud agar medium.The dishes were exposed to open air, in selected districts and in a predetermined region, for 15min every month, while placed at a height of 1.5m from the ground to collect fungal spores by the action of gravity 5,10 .Three dishes were exposed each month in each area, resulting in a total of 15 samples per month and 180 samples per year.It is important to emphasize that the areas where the research was conducted were randomly selected.The samples used for microculture and taxonomic analysis were obtained from the colony-forming units (CFUs) 13,14 .

Statistical analysis
The results were analyzed using SPSS software, Chicago, USA, SPSS Inc® version 16.0 for Windows (2007).The study was cross-sectional and observational, and descriptive statistical techniques were used to assess all of the variables, with the aid of graphs and tables of frequencies.Two-tailed analysis of was performed to determine the relationships of the frequencies of the species (from among the fi ve most frequent species) and the months of the year with the number of CFUs.
Twenty genera of fungi were isolated in this study.Depending on the region, it was possible to isolate between 10 and 14 genera, as shown in table 1 (χ 2 = 535.95,p < 0.0001).
Figure 1 shows that the median number of CFUs/dish during the rainy season (January to June) was 20 (maximum = 279 and minimum = 0), and the corresponding number during the dry season was 14 (maximum = 227 and minimum = 0), using the Mann-Whitney test (p = 0.96).
A comparison of the average number of CFUs for the fi ve most frequent fungi (table 2), using Tukey's test, showed that the number for Aspergillus differed signifi cantly (p < 0.05) from the numbers for the genera Cladosporium, Curvularia and Fusarium.Regarding seasonality, we observed the occurrence of fungi throughout the year, with a slight increase in the percentages of fungal genera in the months of May, August and (Figures 2 and 3).
When the percentages of CFUs collected in each of the fi ve (northern, southern, central, eastern and western) areas of São Luis were analyzed, a statistically signifi cant difference was observed (Table 2; χ 2 = 38.99,p < 0.0001).The prevalence in the central and northern areas was lower than in the other areas.

DIscUssIOn
Aspergillus was the most commonly isolated genus in the current study, and Penicillium was the second most commonly isolated genus.In Mexico City, Penicillium is also the second most common genus 15 .However, in studies performed in other countries, such as France, the USA, Chile and Cuba, Cladosporium has stood out as the most prevalent genus [16][17][18] .
Aspergillus, Penicillium, Cladosporium, Curvularia and Fusarium are the most frequent outdoor species, according to previous research 19,20 .The genera of fungi identifi ed in the present study were correlated with natural systems and could be useful in assessing the impact of environmental changes on the region studied.
In Brazil, the occurrence of airborne fungi in indoor and outdoor areas has been investigated in different regions 3,21,22 .In the Northeast, Fortaleza, Natal and Recife are climatically similar cities.In Fortaleza, Ceará reported that the genera Aspergillus and Penicillium prevailed 10 .Additionally, Aspergillus and Penicillium were more frequent genera in Recife and Natal, respectively 23,24 .Curvularia appeared with the highest frequency only in Fortaleza 8 .In Belém, Pará, Aspergillus, Penicillium and Cladosporium were reported to be the most prevalent genera isolated 25 .
Despite differences in climate, in Porto Alegre, Rio Grande do Sul, Aspergillus was the second most frequent genus 26 .In Botucatu, São Paulo, Cladosporium was the most frequent genus 27 .In the metropolitan area of São Paulo, Penicillium spp and Aspergillus spp.were the dominant species both indoors and outdoors 28 .
High relative humidity is essential for the development of fungi, and sunny weather favors the release of spores 29 .High temperature and humidity can result in increased concentrations of fungi 19 .A high concentration of spores in the air is important because this situation can result in increases in allergic diseases of the respiratory system 10 .
In the South and the West, the greatest numbers of airborne fungal genera were isolated (table 1).These regions have greater areas of vegetation covering them.The North is near the sea and presents a low level of air pollution.In Centro (CE), a small number of airborne fungi were isolated, possibly due to higher levels of pollution.
The quantitative analysis of colony counts in the northern, southern, central, eastern and western areas was statistically signifi cant (p = 0.0002) when assessing the fi ve most frequent genera relative to the months of the year.Seasonal fl uctuations were also reported in Santiago, Chile 30 .
The literature reveals that Cladosporium has been repeatedly found indoors, particularly in house dust 13 .However, in the current paper, Cladosporium was found outdoors considerably more often, given that it was the second most isolated genus.
The occurrence of a great number of airborne fungi emphasizes the importance of studying airborne fungi in São Luis, Maranhão.The climate of tropical areas supports the growth of airborne fungi, resulting in high levels of fungal spores in the air, which can increase the incidence of allergic respiratory diseases related to these fungi.

FIGURE 1 -
FIGURE 1 -Frequency distribution of the different genera of fungi isolated in the rainy season (January to June) and the dry season (July to December) in São Luis, State of Maranhão, between January and December 2007.Legend for Figure 1: Box plot.Medians and quartiles.CFUs per period/season.Mann-Whitney test, p = 0.96.CFUs: colony-forming units.

FIGURE 2 -
FIGURE 2 -Fungal growth and the climate variables humidity, temperature and rainfall, recorded monthly in São Luis, State of Maranhão, Brazil, in 2007.CFUs: colony-forming units.

FIGURE 3 -
FIGURE 3 -Seasonal distribution of the fi ve most prevalent genera isolated in São Luis, State of Maranhão, in 2007.CFUs: colony-forming units.

TABLE 1 -
Frequency distribution of colony-forming units of fungi by area in São Luis, State of Maranhão, between January and December 2007.

TABLE 2 -
Comparison of the average number of CFUs for the fi ve most frequent fungi and the frequency distribution of the recovered colony-forming units of fungi by area in São Luis, State of Maranhão, between January and December 2007.