1 Introduction

Species from genus Alternaria are important and include a wide variety of pathogenic, saprophytic and endophytic fungus widely distributed on worldwide growing on a great variety of substrates (de Hoog et al. 2000). About 300 species have been described and their taxonomic status is being the key to relevant changes based on nucleotide sequence data (Linas et al. 1998; Woudenberg et al. 2013). In general, their conidia are easily identified by the obclavate to obpyriform outline tapering in conical beak at the tip and the transversal and longitudinal septa. Relevance of Alternaria spp. (hereafter Alternaria) and their airborne conidia is related with two main aspects, phytopathology and allergy.

As an allergen, Alternaria is the most important fungus causing allergy (D’Amato et al. 1997; Denning et al. 2006; Kilic et al. 2010; Samson et al. 2009; Tariq et al. 1996), rhinoconjunctivitis and asthma (Andersson et al. 2003; Atkinson et al. 2006), mainly in pediatric population with a significantly high sensitization (Bartra et al. 2009; Peat et al.1993). In addition to that, Alternaria can produce mycotoxins and around other 70 toxic secondary metabolites (López et al. 2016), some of them of a carcinogenic power (Lehmann et al. 2006; Ostry 2008; Schrader et al. 2001; Woody and Chu 1992).

As plant pathogens, they affect a great number of crops; some of them have been traditionally very important in our study area, such as potato (Escuredo et al. 2011; Iglesias et al. 2007), cotton (Bashan et al. 1991), tomato (Somma et al. 2011) and cereal crops (Corden et al. 2003), producing important economic losses worldwide (Rotem 1994). Information about their presence and environmental factors affecting their increase may improve best phytosanitary practices (Aira et al. 2013).

Some general aerobiological studies based on airborne Alternaria conidia have been previously published in Europe (Bergamini et al. 2004; Grinn-Gofroń and Rapiejko 2009; Kasprzyk et al. 2015; Sadyś et al. 2014; Skjøth et al. 2012; Stępalska and Wołek 2005; Thibaudon and Lachasse 2006) and in other parts of the world, like Australia (Mitakakis et al. 1997; Stennett and Beggs 2004), Argentina (Somma et al. 2011) or USA (Timmer et al. 1998; Woudenberg et al. 2015). The Alternaria distribution was also studied in Spain (Aira et al. 2013; Angulo et al. 1999; De Linares et al. 2010; Laín and Bustillo 2003; Rodríguez-Rajo et al. 2005; Sabariego et al. 2012) and with data collected nearby our current study (Aira et al. 2013; Díaz-Pérez et al. 2001; Fernández-Rodríguez et al. 2014a; Fernández-Rodríguez et al. 2015; Maya-Manzano et al. 2012; Paredes et al. 1997).

Nevertheless, this is the first detailed analysis of atmospheric content of Alternaria spores in rural areas of Extremadura region (SW of Iberian Peninsula), which is an extensive territory with a high diversity of landscapes. This is a well known and important factor influencing Alternaria airborne content (Corden et al. 2003; Grinn-Gofron and Rapiejko 2009; Maya-Manzano et al. 2012; Skjøth et al. 2012).

Climate change is a general concern in biology that affects the whole species. In aerobiology, it has been analyzed in relation to airborne pollen and phenological trends showing a clear influence by temperature increase (Besancenot and Thibaudon 2012; García-Mozo et al. 2010). In relation to fungi, three main aspects have been considered, i.e., spore production (a), allergy (b) and mycotoxins (c). With respect to spore production (a), phenology and, consequently, phytopathology, a significant increase in Alternaria propagules has been observed in relation to the rise of CO2 and temperature (Jara et al. 2014; Klironomos et al. 1997; Pakpour et al. 2015; Van de Perre et al. 2015; Wolf et al. 2010). Nevertheless, a delayed and slower response to climate change than plants in an opposite direction to that of pollen has been also found (Damialis et al. 2015; Wu et al. 2016). In relation to allergy (b), several observations have been highlighted taking into account that for Alternaria, the knowledge of hosts phenology is relevant, mainly from crops, which could affect allergic patients due to changes in season of sporulation, and consequently, prevalence of allergies along the year also changes (Ziska and Beggs 2012). Notwithstanding, it is necessary to emphasize the importance of epidemiological studies in various climate conditions (Katotomichelakis et al. 2012; Kilic et al. 2010). Moreover, considering that climate predictions often indicate an increase in drought events in summer, it would be expected that airborne fungal concentrations may be increased in future years, potentially exacerbating human health concerns (Pakpour et al. 2015). Finally, climate change will affect mycotoxins (c) in food for human and animals. Favorable temperature and water activity are crucial for mycotoxins production (Russell et al. 2010). In that way, optimal temperature for mycotoxins production and growth in Alternaria ranges 20-25 °C (Botana and Sainz 2015; Milani 2013; Paterson and Lima 2011; Pose et al. 2010a, b). Climate change can affect infection of crops by toxigenic fungi, the growth of these fungi and the production of mycotoxins (Miraglia et al. 2009; Tirado et al. 2010; Uyttendaele et al. 2015).

The aim of this study is to determinate the seasonal distribution of airborne Alternaria conidia in three rural localities of Extremadura (Plasencia, Don Benito and Zafra), analyzing the main influencing factors, as meteorological parameters, and land uses in order to assess the potential trend of Alternaria spore production in a future scenario of climate change. Knowing the optimal meteorological conditions for airborne Alternaria conidia presence in the air, it will be possible to assess the expected trend within a temperature increase scenario.

2 Materials and methods

2.1 Locations

The study was carried out in three cities located at the SW of the Iberian Peninsula: Plasencia (PL), in the north of Cáceres province; Don Benito (DB) and Zafra (ZA), both of them placed in the province of Badajoz, with maximum distance separation of 182 km (PL–ZA) and minimum of 77 km (DB–ZA) (Fig. 1). The respective heights above sea level are following: 352 m (PL), 253 m (DB) and 508 m (ZA). General vegetation or the most frequent land use around the cities were holm oak and cork oak trees, extensive grazing and some forestal and timber plantation of pines (PL), irrigated crops as fruit trees, corn, tomato, rice (DB), and livestock production, olive trees and holm-oak trees (ZA). The urban surface for the places studied was 415.2 ha (PL), DB 336.5 ha (DB) and 296.1 ha (ZA), and their population in 2014 according with Regional Government of Extremadura was 37011 (PL), 40892 (DB) and 16828 (ZA). Orography shows a quite flat region except for a mountain range with a few hundred meters laid west–east direction between PL and DB (Sierra de San Pedro).

Fig. 1
figure 1

Maps show location of the aerobiological stations in SW Iberian Peninsula (Extremadura region, Spain)

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2.2 Aerobiological monitoring

Spores were collected from March 2011 to December 2014 using Hirst (1952) spore trap samplers. The traps were situated on the terrace of Hospital Virgen del Puerto building in PL (43°10′N, 2°25′W), on the terrace of Donoso Cortés Secondary School in DB (38°58′N, 5°50′W) and on the terrace of the town Hospital building in ZA (38°25′N, 6°25′W). They were located at 17, 6 and 6 m above ground level.

Applied methodology allowed to obtain both hourly and daily mean spore concentrations was used, following those described by the Spanish aerobiology network (REA) (Galán et al. 2007). Slides were examined along two longitudinal transects. Spore concentrations were expressed as the number of spores per cubic meter of air (spore m−3). Records afford hourly concentration; nevertheless, data are usually provided as daily concentration or as average for a period (year or the whole period studied).

2.3 Meteorological data

Meteorological data were provided by the Spanish Meteorological Center (AEMET). Distance between meteorological stations and spore traps was 2.24 km (PL), 0.95 km (DB) and 0.53 km (ZA). Meteorological data that were provided included temperature (°C), rainfall (mm), relative humidity (%) and speed (km h−1) and direction for wind (°). Climatic values from average meteorological data were calculated for the 1961–1990 period.

2.4 Statistical analysis

Nonparametric statistics were used upon preliminary data screening. Spearman’s correlation coefficients were applied comparing daily data for the whole study period between spore concentration and the following meteorological parameters: maximum, minimum and mean temperature (Tmax, Tmin and Tmean), rainfall (Rainfall), relative humidity (RH), direction and speed of the wind (WindDir and WindSpeed) for each station. Level of p < 0.05 was taken into account for statistical significance. Correlation using hourly data was performed for 10 days (n = 240) including the peaks for each year and station. Periods selected were 25/5–3/6 for 2011, 3–12/10 for 2012, 7–16/10 for 2013 and 2–11/10 for 2014. Kruskal–Wallis test was used to compare differences among stations and years using monthly spore concentration data. Friedman test was calculated to compare hourly data among stations.

SPSS package (v.15.0) was used for statistical analysis.

3 Results

3.1 Spore concentration

Annual data of the whole time period are given in Table 1. Annual average concentrations values were 38 spores m−3 in DB, being the place where spores were always more abundant, ZA the second (17 spores m−3) and PL the third (11 spores m−3). The maximum annual average values (Fig. 2) were reached during the first year of the study.

Table 1 Annual average values for Alternaria spore concentrations, air temperature and annual sums of rainfall recorded during the period of 2011–2014 at Plasencia (PL), Don Benito (DB) and Zafra (ZA)
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Fig. 2
figure 2

Average annual concentrations of Alternaria for the three localities and studied years (PL Plasencia, DB Don Benito, ZA Zafra)

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Monthly data showed two periods of maximum concentration, spring (May and June) and autumn (September and October), with some differences between years (Fig. 3). Monthly data, after Kruskal–Wallis test that examined monthly data, confirmed statistically significant differences among stations (χ 2 = 11.92, p = 0.003) and years (χ 2 = 11.94, p = 0.008).

Fig. 3
figure 3

Monthly data including spore concentration, their relationships with mean temperature and rain

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Daily data showed maximum peak concentration in DB (923 spores m−3 obtained on 9/10/2012). Peaks were reached during the first year in spring except for PL (25/8/2011); the rest of studied years peaks were reached in autumn (Fig. 4). Comparison between pairs of stations using daily data showed statistically significant correlation (p < 0.001) for all the compared pairs (PL–DB r = 0.697, PL–ZA r = 0.722, DB–ZA r = 0.725).

Fig. 4
figure 4

Daily data concentration of spores for each year studied (PL Plasencia, DB Don Benito, ZA Zafra)

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Hourly data concentrated around the peaks are shown in Fig. 5. Comparisons among stations are given in Table 2. All cases except for one, PL–ZA 2013, showed statistically significant correlation. Friedman’s test comparing stations showed statistically significant differences (p > 0.001) for the years compared; χ 2 = 95 (2011), 307 (2012), 172 (2013), 170 (2014).

Fig. 5
figure 5

Hourly data concentration around the peak day (PL Plasencia, DB Don Benito, ZA Zafra) for each year studied

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Table 2 Correlation with hourly spore concentrations among stations (PL Plasencia, DB Don Benito, ZA Zafra) (n = 240)
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Hourly pattern showed similar results for all stations in the years studied. Maximum concentration was observed in the evening 17:00–20:00 h and minimum in the morning, between 06:00 and 08:00 h (Fig. 6).

Fig. 6
figure 6

Hourly data distribution (UTC hours) of spores from 2011 to 2014 (PL Plasencia, DB Don Benito, ZA Zafra). Radial scale in spores m−3

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3.2 Relation with meteorology

Average meteorological values for temperature were 17.2 °C (DB), 16.1 °C (PL) and 15.8 °C (ZA); and for rainfall 460.4 mm (DB), 694.0 mm (PL) and 618.9 mm (ZA). Average rainfall is mainly distributed in November–January period. During the first 3-year studied period, annual rainfall was lower than 30 years average values, above all the two firsts (Table 1). March 2013 was a markedly rainy month, with 3–4 times more rainfall (155.2 mm DB, 238.4 mm PL, 168.4 mm ZA) than normal values (41.3 mm DB, 60.5 mm PL, 43.4 mm ZA).

Table 3 shows results for correlation analysis between daily spore concentration and meteorological data. Positive statistically significance was obtained with temperature and negative with relative humidity and rainfall. Fig. 3 shows the spore concentrations for the three cities related with mean temperature and rainfall, and a lightly decrease was observed along the studied years. Graphic analysis of influence of meteorological parameters is shown in Fig. 7. The optimal gradient of temperature for recording more spores in the three stations was ranged between 20 and 30 °C. For relative humidity, the best range is not the same in the three stations; for PL 30–40 % is the best, while for DB and ZA 50–70 %. For wind direction, PL reached maximum values with wind from 90 to 135 grades (ESE), DB 225–270 grades (WSW) and ZA 270–315 grades (WNW). For wind speed, DB reached maximum values with 20–30 km h−1, PL and ZA 30–40 km h−1. For rainfall, this analysis was not performed because on rainy days the presence of airborne conidia was insignificant (Table 4).

Table 3 Results of Spearman’s correlation test between spore concentration and meteorological parameters using daily mean values (PL Plasencia, DB Don Benito, ZA Zafra) (n = 1237–1461)
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Fig. 7
figure 7

Graphic analysis of meteorological parameters (PL Plasencia, DB Don Benito, ZA Zafra) and their relationships with Alternaria spore daily sums

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Table 4 Results of Spearman’s correlation test between for spore concentration and meteorological parameters using hourly mean values (n = 960) (PL Plasencia, DB Don Benito, ZA Zafra)
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4 Discussion

Alternaria showed a continuous presence in the air along all the period studied, with average values ranging from 7 to 18 (PL), from 26 to 44 (DB) and from 10 to 33 spores m−3 (ZA) (Table 1). Higher values were recorded by other authors such as Bergamini et al. (2004) in Módena (Italy), Stennett and Beggs (2004) in Sidney (Australia) or Stępalska and Wołek (2005) in Cracow (Poland). Similar values were obtained by Maya-Manzano et al. (2012) and Paredes et al. (1997) where the analyzed places were nearby the current area of study or Laín and Bustillo (2003) and Sabariego et al. (2012) both toward the center of Spain.

Alternaria peaked in Don Benito with 923 spores m−3 on the October 9, 2012, similar to findings of Aira et al. (2013) in Seville (Spain), or Kasprzyk et al. (2015) in Odessa or Poltava (Ukraine). Nevertheless, this peak is higher than other found by other authors in Spain (Díaz-Pérez et al. 2001; Rodríguez-Rajo et al. 2005; Sabariego et al. 2012; Sánchez-Reyes et al. 2009) and in the center and east of Europe (Kasprzyk et al. 2015), but lower than other findings in Spain (Maya-Manzano et al. 2012; Paredes et al. 1997), UK (Corden et al. 2003), Poland (Kasprzyk et al. 2015) and Ukraine (Kasprzyk et al. 2015).

Maximum hourly peaks were found during the evening, 19:00–20:00 h; the same is found by other authors, such as Paredes et al. (1997), who recorded two peaks in the noon and during evening (Angulo et al. 1999; Oliveira et al. 2009; Skjøth et al. 2012). Rural environment has been barely analyzed aerobiologically; from the literature consulted only in three cases, population is lower than 50,000 inhabitants as in this study: Trives (Rodríguez-Rajo et al. 2005), Vielha (De Linares et al. 2010), Amares (Aira et al. 2013); all of them located in the North of Iberian Peninsula. Population from those environments is more affected by land use in relation to airborne spore sources and the consequences on allergy and impact from climate change (Kinney 2008; Shea et al. 2008). After our results, the riskiest period for the population in relation to the allergy and exposure to Alternaria is May–June and September–November, mainly in the evening hours of the day.

Results from Kruskal–Wallis and Fried tests showed that differences among stations are due to different levels in spore concentrations. Nevertheless, correlations’ significance showed that the pattern of distribution is the same for the three studied localities.

The absence of high mountainous areas between PL, DB and ZA, and the low situated cities, none surpasses about 550 m above sea level, could explain the statistically significant correlation for all the pair cities compared using daily data. Furthermore, this idea is reinforced with the most statistically significant correlations using hourly data in all cities for the entire period. These findings agree with previous studies (Kasprzyk et al. 2015; O’Connor et al. 2014). Hourly data for Alternaria spore concentration were also studied in other city of Extremadura region, Badajoz, to analyze the potential sources of this pathogen (Fernández-Rodríguez et al. 2015).

Several papers showed that regional variations depending on landscape and land use could influence the spore concentration at local scale, for example in Spanish regions such as Galicia (Rodríguez-Rajo et al. 2005), Castilla la Mancha (Sabariego et al. 2012) and Extremadura (Maya-Manzano et al. 2012).

In this study, results of Spearman’s rank correlation analysis showed that the temperature was positively significant in relation to airborne concentrations of Alternaria spores, being agree with other authors (Aira et al. 2013; De Linares et al. 2010; Escuredo et al. 2011; Fernández-Rodríguez et al. 2015; Maya-Manzano et al. 2012; Mitakakis et al. 1997; O’Connor et al. 2014; Rodríguez-Rajo et al. 2005; Sabariego et al. 2012). Negative influence was found for relative humidity and rainfall, which has been reported previously (Aira et al. 2013; Escuredo et al. 2011; Fernández-Rodríguez et al. 2015; Maya-Manzano et al. 2012; O’Connor et al. 2014; Rodríguez-Rajo et al. 2005; Timmer et al. 1998).

Wind direction and wind speed have been studied less frequently in studies of Alternaria, mainly in studies about back trajectories (Fernández-Rodríguez et al. 2015; Sadyś et al. 2014). Wind direction showed negative statistics correlation in Plasencia, however positive in Don Benito and Zafra. On the contrary, wind speed showed the opposite influence in each city. Rodríguez-Rajo et al. (2005) found negative correlations for wind speed, and similar results using weakly data were reported by Grinn-Gofroń and Strzelczak (2013). Wind direction has showed positive and negative influence depending of the site (Rodríguez-Rajo et al. 2005).

Seasonal distribution followed a bimodal distribution during the first year (2011) and afterward (2012–2014) was substituted for another one, unimodal in its spore release season, with peaks recorded in autumn, possibly due to a decrease in temperatures, and an increase in the rainfall during the period studied, as it is shown in Fig. 7. These meteorological conditions were more similar to continental areas. Bimodal distribution is frequent in Mediterranean environments (Maya-Manzano et al. 2012), with two distribution peaks (Fig. 3), the first occurring during spring (mainly in May or June) and the second observed in autumn (September and October), with a decrease during summer and the lowest concentrations in winter. Other authors recorded the same bimodal distribution, such as Angulo et al. (1999) in Córdoba, Díaz-Pérez et al. (2001) in Cáceres, Maya-Manzano et al. (2012) in Badajoz, Cáceres and Mérida or Stennett and Beggs (2004) in Sydney (Australia). Aira et al. (2013) found a stronger presence of this type of distribution in cities in the south of the Iberian Peninsula, probably due to extreme conditions of temperature during summer, while in the north an isolated peak in summer was more frequent. When we compared these results with other studies in central Europe and continental conditions, the same pattern was found. According to this, other studies concluded that seasonal distribution happened from July to October, even with summer peaks (Grinn-Gofron and Rapiejko 2009; Sánchez-Reyes et al. 2009; Stępalska and Wołek 2005), as a consequence of milder temperatures during summer and a progressive decrease in relative humidity, producing a continuous release of Alternaria conidia in this season.

In the current study, for relative humidity the best range acts depending on the station; for PL 30–40 % is the best, for DB and ZA 60–70 %. A 30 years average value for relative humidity in Plasencia is higher than in Don Benito or Zafra.

Maya-Manzano et al. (2012) recorded that decreases in relative humidity below 55 % provoked concentration declines regardless the impact of temperature. However, Timmer et al. (1998) observed that decreases in relative humidity below 50 % stimulated the release of a great number of conidia.

Regional fluctuations in airborne Alternaria conidia depend on land uses and the type of landscape which are surrounding the studied cities (Corden et al. 2003; Fernández-Rodríguez et al. 2015; Maya-Manzano et al. 2012; Sabariego et al. 2012; Skjøth et al. 2012). Wind direction plays a relevant role to explain differences between stations; thus, PL reached maximum values with winds from ESE, where urban area and olive crops are present. In the case of DB, winds from WSW, coming from irrigated crops and pastures, contributed toward high spore concentrations. For ZA, winds from WNW, where oak forests and dehesas are extensive, are associated with higher spore concentrations.

Differences in the height at which spore traps were operating (16 m in PL, 6 m in DB and ZA agl) could also be another reason for explaining the differences among stations, as it was suggested by other authors (Bergamini et al. 2004; Chakraborty et al. 2001; Fernández-Rodríguez et al. 2014b; Khattab and Levetin 2008).

According to Giorgi and Lionello (2008) for a possible scenario in 2071–2100 period, a rain decrease up to 40 % was estimated, mainly in MAM (March, April and May) and SON (September, October and November) monthly periods, and a temperature increase up to 5 °C in JJA (June, July and August) monthly periods and 2–4 °C in MAM and SON periods for the SW of Iberian Peninsula. Those predictions have been also suggested in other papers (Hertig and Jacobeit 2008; Somot et al. 2008). Due to the mean temperatures for the studied localities ranged from 13.8 to 14.7 °C (MAM) and from 16.7 to 17.3 °C (SON), those increases mean that temperature along the two main growing periods of Alternaria is reaching the optimal growing conditions for this fungus, and consequently spore occurrence should increase (Botana and Sainz 2015; Milani 2013; Paterson and Lima 2011; Pose et al. 2010a, b). We will have to take into account the effects of the temperature, which appear to be linked to changes in the seasonal distribution of the spores, as in our case. In the case of rain decrease, the forecast would support that prevision, as rain showed a negative effect on spore production.

5 Conclusions

As differences between stations were recurrent in the same order, environment is the alleged first cause to explain differences among stations; land uses play a relevant role being irrigated crops and pastures important sources of Alternaria conidia, then olive crops and oak forests, more intensively for inhabitants in rural environment. Seasonal meteorology, similar in the three localities in SW of Iberian Peninsula and typical for Mediterranean climate, explains alike seasonal pattern of spore distribution, with May–June and September–October periods with maximum concentration separate for a summer low and winter very low concentrations. Temperature is a main meteorological factor which stimulates spore concentration, within the range of 20–30 °C at the three stations. Relative humidity conditions for the highest spore concentration showed differences among stations. It seems that the weather parameters and land uses were important factors for explaining the Alternaria concentrations. According to climate prevision upon the available literature, an upward trend is expected in Alternaria spore concentration, as the optimal growing temperature will be reached in the two growing periods characteristic for Mediterranean climate.