Currently, the ash dieback causal agent Hymenoscyphus fraxineus is an established invasive pathogen in most European countries. Its potential to spread quickly among invaded forests is based on its propagules: airborne inoculum composed mainly of ascospores originated in apothecia growing on leaf litter infected during the previous vegetation season. The spread of the inoculum by air masses to distant areas is probable and depends on the availability of the ascospores in higher levels of air. Our study aimed to detect the inoculum in an infected area at heights of more than 20 meters. Our study was conducted in a municipal locality (Boršov nad Vltavou) with tens of infected ash trees (Fraxinus excelsior) in South Bohemia (SW Czechia). The infected trees surround an agricultural silo where five rotating arm spore traps (rotorods) were mounted for ten consequent 48h samplings during the peak of the sporulating season (17^th July to 6^th August 2020). The spore traps were mounted 48, 37, 25, 14 and 0,3 meters above ground. Samples were quantified by qPCR. Results clearly proved the ability of the spores to reach a height of 48 meters. Furthermore, H. fraxineus DNA was detected from all five spore traps during all ten samplings. Mostly, the amount of detected spores showed a decreasing trend with height, and varied a lot. During some of the samplings, higher spore concetrations were achieved at the top than at the lower traps, which can be explained by horizontal air transfer of the inoculum from other infected areas. Based on GLM analyses, higher spore concentrations were achieved during days without rain, lower air temperatures, after cloudy, humid and rainy weather without strong winds. A combination of rotorod ROTTRAP 52 with qPCR quantification proved to be an efficient technology for a study focused on the vertical spread of H. fraxineus propagules.

airborne inoculum, ash dieback, riseability, rotorod, ROTTRAP, spore trap

Hymenoscyphus fraxineus (T. Kowalski) Baral, Queloz, and Hosoya is the causal agent of the ash dieback (Kowalski 2006). It is an invasive pathogen introduced to Europe from Eastern Asia (Zhao et al. 2012) probably during the 1990’s (Przybył 2002), first reported in Poland (Kowalski 2006). Its current distribution apart from Eastern Asia covers the whole of Europe apart from Portugal, Greece, Albania, Macedonia, Bulgaria and Moldova (EPPO 2022). H. fraxineus severely attacks native European ash species Fraxinus excelsior and F. angustifolia (Kowalski and Holdenrieder 2009) causing a serious threat to mixed deciduous forest habitats, especially riparian forests where other deciduous tree species are endangered by invasive pathogens (Jankovský and Holdenrieder 2009).

The symptoms of the ash dieback and mechanism of infection of the host trees have been well described in numerous publications (Kowalski 2006; Kowalski and Holdenrieder 2009; Gross et al. 2012, 2014; Husson et al. 2012; Cleary et al. 2013; Kräutler et al. 2015; Chandelier et al. 2016; Fones et al. 2016; Haňáčková et al. 2017)

The predominant way to spread this pathogen is via the airborne ascospores (Gross et al. 2012), although the conidia of the imperfect stage Chalara fraxinea play an important role (Fones et al. 2016). The ascospores are typically released from apothecia growing on shredded infected leaf rachises during the following growing season, mainly from June to September (Timmermann et al. 2011; Gross et al. 2012; Hietala et al. 2013; Chandelier et al. 2014; Dvořák et al. 2016). The ascospores have been shown to be able to span long distances and in France they have been observed to even reach 50 to 100 km (Grosdidier et al. 2018b).

The probability of the long distance spread of the ascospores depends on the height they can reach to be blown with the air masses (Chandelier et al. 2014; Oteros et al. 2015; Aguayo et al. 2021). Existing studies proved that the inoculum of H. fraxineus rises from the ground where it is actively ejected from the apothecia (Timmermann et al. 2011) and it is reliably detectable in aerobiological samples collected by the RNSA (French Network of Aerobiology) at heights of between 10 to 20 meters (Aguayo et al. 2021). According to these authors, ascospores present at this height prove the presence of the pathogen in the landscape scale in the range of tens of kilometres.

The aim of our study was i) to test a methodological approach for describing the vertical spore dispersal pattern of H. fraxineus, and ii) to prove the presence of the inoculum as high as possible, at such a height where the spread of the aerosols is more likely affected by horizontal movement of air masses rather than by convection from the ground surface.

Fresh apothecia were observed on infected rachises at the sampling locality from the beginning of July until the end of the sampling (6^th August, 2020). Consequently, all ten 48-h samplings showed presence of H. fraxineus in the air (Fig. 2).

Figure 2. 

Vertical profiles of spore concentrations recorded during the ten samplings.

Every sampler positively detected inoculum during every sampling. The lowest positively detected spore concentration was detected in samples from the highest sampler R5 from the sampling started on 4^th August (CN = 0.018, Ct = 37.428). This least concentrated sample contained 1.89 ascospores; however, this amount was calculated extrapolating the ascospore suspension standard curve, hence the exact amount cannot be taken in consideration. The highest concentration was recorded at R1 during the following sampling started on 6^th August (CN = 659.063, Ct = 27.181). Taking in account the sampling rate 52 l/min and sampling period of 48 h, we detected average spore concentration in a range from 0.013 to 462.12 spores per m³.

Most of the detected spore concentrations showed a clear decreasing trend following the height of the sampling point. The resulting nonlinear regression model (Fig. 3; residual SE = 99.85, DF = 96), corresponding to the aforementioned parameterization, describing the generally decreasing trend, was estimated as follows:

Figure 3. 

Model of the vertical spread of the inoculum. The nonlinear regression model described the decreasing trend of spore concentrations with increasing height of sampling.

CN = 150.3243⋅exp(-0.2155⋅h)

GLM analyses resulted in 10 significant models to estimate CNs from meteorological variables measured during the sampling and lagged of 2–4, 4–6 and 6–8 days. Their parameters can be found in supplementary file “Parameters of GLM models”. The calculated p and positive or negative meaning of each parameter are displayed in Table 1.

Our results prove for the first time, that propagules of H. fraxineus are reliably detectable at almost 50 meters above ground, where they have their main source in infected rachises (Gross et al. 2012). Repeated sampling revealed H. fraxineus DNA’s presence to be more than twice as higher as in an aerobiological study, where air was sampled by 7-day volumetric spore traps (Hirst 1952) of RNSA (Aguayo et al. 2021).

The results of this study also confirm statements of other authors (Chandelier et al. 2014) who described decreasing trend of ascospore concentrations up to three meters, regardless of the site and time of the sampling. This was a general trend among our samplings. The ground sampling spot R1 always showed higher CN than the top spot R5. In one case even more than 20,000 times.

However, not in every sampling did the CN values descend with increasing height. The sampling spot R4 showed lower values, than R5 for samplings 5, 6 and 7. This was probably due to overloading the sampling rods with dust, which is a critical handicap of rotorods (Lacey and West 2006; Chandelier et al. 2014; Dhingra and Sinclair 2017). The dust apparently came from outlets of ventilators of the silo adjacent to R4 while depositing the harvested grain.

Furthermore, the sampling site R3 gave higher CNs than it could be expected to follow the descendent trend during samplings 8 and 9. Similarly, CNs at R5 from samplings 1, 5 and 6 were also not lower than CN of lower sampling points. An explanation for this anomaly could be the effect of horizontal air currents which could bring a higher amount of inoculum from more distant localities, influencing the results of sampling only in these cases of low local concentrations. Generally, abnormalities in the trend of decreasing CN with the height of sampling can be interpreted as a consequence of the fact that inoculum detected in heights above 10 m might be representative for areas within a perimeter of at least tens of kilometres (Aguayo et al. 2021). Study focused on long distance spread of pollen grains found correlation of pollen data from the sampler (elevated 10–20 m) and land use even 200 km apart (Oteros et al. 2015, 2017). In the case of our experiment, samplers R2, R3, R4 and R5 were mounted higher than 10 meters; R5 even in more than 48 m. Taking into account the previously mentioned studies (Oteros et al. 2015, 2017, Aguayo et al. 2021) there is a theoretical presumption that the detected inoculum can be related to sources at a distance of up to hundreds of kilometres. Also fungal spores’ long distance proofs are known. Based on spatial air sampling and modelling it was revealed that tobacco blue mould outbreak was caused by Peronospora tabacina spores blown from sources several hundred km away (Aylor et al. 1982). Another example of long distance transport of spores was studied by Vasaitis and Enderle (2017) considering a possible way of ash dieback introduction to Great Britain. Agricultural silo used for this study is located very beneficially from this point. Its height is almost three times bigger than the canopy layer of surrounding vegetation, which acts as a natural barrier for the vertical spread of the inoculum (Aylor 1999). Taking into account the geomorphology of surrounding landscape and predominant wind direction (NW), it is highly likely exposed to air masses from an adjacent plane area called “Českobudějovická pánev” (České Budějovice Basin), which is a tectonic ditch oriented NW – SE, elevated 380–410 m a. s. l., almost 70 km long and 10–12 km wide. From personal experiences we know that infected ashes are very frequently present there, especially in alleys along roads, riverside plantations and municipal green spaces. We assume that inoculum detected in R2 – R5 may originate in this area, not only from the sources adjacent to the sampling point. Transfer of spores by air masses up to 100 km has been already proven (Grosdidier et al. 2018b), which is enabling this hypothesis.

Long distance transfers of H. fraxineus by air masses have always been an important issue. It was considered that H. fraxineus had been introduced into Great Britain between 2008 and 2011 via long distance transfer of the air inoculum from mainland Europe. This statement was strongly supported with a model (Vasaitis and Enderle 2017). Recently, H. fraxineus was found for the first time in Spain (Asturias, NW Spain) on matured trees and its surrounding regeneration (Stroheker et al. 2021). The fact that it has not yet been observed in NE Spain, which is adjacent to the closest distribution of the pathogen, implies the possibility of long-distance transport of the inoculum from France via the Bay of Biscay. However, the introduction of the pathogen through plant material import cannot be overlooked.

Results of the GLM modelling of the determination of the detected spore concentrations by meteorological factors discovered numerous relations. Although the low number of repetitions decreases the reliability of the results’ interpretation, we would like to highlight some of them:

Air temperature proved to have significantly positive effect two days preceding the sampling at both R1 and R5. This confirms previous observations of Chandelier et al. (2014), who found a positive influence of higher temperatures on spore concentrations. On the contrary, sooner than two days before and directly during the sampling, the effect of air temperature was significantly negative. This pattern indicates, and confirms, that for the ascospore development and release the high air temperature is not favourable (Grosdidier et al. 2018a). Since the entire sampling period can be characterized like very warm with maximal temperatures exceeding 30 °C, this biological limitation of the pathogen was more apparent within our experiment.

The daily amount of precipitation showed different effects at different sampling heights. R1 was significantly positively affected by precipitations two to four days before sampling. Through the enhanced humidity of the ground surface, rain proved to be essential for successful ascospore maturation and release (Timmermann et al. 2011; Hietala et al. 2013; Dvořák et al. 2016) and the consequent progress of ash dieback (Chumanová et al. 2019). On the other hand, concentrations at R5 and riseability were significantly negatively affected by rain during the days before the sampling, and even highly significantly during the sampling. Two explanations for this phenomenon are probable: i) ascospores currently present in the air are washed out by rain from the aerosol which is a known factor (Aylor 1999), and ii) trapped ascospores are partly splashed from the sticky sampling surface. For the significantly positive effect of precipitation on riseability during sampling we do not have any reasonable explanation; however, the p for this parameter is approaching the limit of significance (p = .048) hence its importance is ambiguous.

Daily duration of sunshine showed highly significantly negative effect on spore concentration at R1 and R5 two days before the sampling and on riseability during the preceding four to six days. However, the sunshine was significantly positive during the sampling at R1 and R5 and during the preceding two to four days it was significantly positively affecting the riseability. This partly confirms and partly neglects results of Burns et al. (2022) who found a clear determination of H. fraxineus spore release after five-day-average net radiation and leaf moisture.

Air humidity was found to be an important factor through our sampling. At R5 the humid air was significantly determining the inoculum load after two to six days; similarly, at R1 there was a significantly positive effect on inoculum concentrations lagged by two to four days. The promoting effect of air humidity on the disease spread and establishment has already been emphasised many times (Timmermann et al. 2011; Hietala et al. 2013; Dvořák et al. 2016; Čermáková et al. 2017; Chumanová et al. 2019; Volke et al. 2019). However, up to two days before the sampling, the air humidity had an opposite effect at R1 and negatively affected also the riseability. This ambiguity of the air humidity effect for sporulation emphasizes the necessity for more detailed study of the ascospore discharge mechanism such as in Ingold (1999).

Wind speed proved to have a significantly negative influence on ascospore concentrations at R1 after two to six days. This effect is probably due to the desiccation of leaf rachises and growing apothecia which are not able to mature and sporulate under such conditions (Timmermann et al. 2011; Hietala et al. 2013). The significantly negative effect of wind speed was also found to affect the sampling directly. Since the wind speed is measured in horizontal direction, it is logical that strong wind cannot be beneficial for vertical spore transfer. Horizontally, moving air masses are supposedly continuously removing the local inoculum, which would otherwise reach higher air levels by convection (Garratt 1994; Lacey and West 2006).

Air pressure did not show much importance in our experiment. It was a significant parameter with negative influence on spore concentrations at R1 after two to four days. At the same period and sampling height two other factors had positive influence on the spore concentrations: precipitations and air humidity. Naturally, rainy and humid weather is characterized by low air pressure, which we assume to be a reason for this result.

From a methodological point of view, rotating arm spore trap ROTTRAP 52 proved to be a reliable tool for the detection of H. fraxineus inoculum. All samplers successfully completed all ten 48h samplings at five sampling spots without any blackout even in hot or rainy weather. This reliability has been improved compared to previous experiments (Dvořák et al. 2016, 2017; Čermáková et al. 2017). Rotorods combined with quantitative real-time PCR detection and quantification of H. fraxineus in the air continuously produce robust and valuable data (Chandelier et al. 2014, Dvořák et al. 2016; Čermáková et al. 2017). Similarly, in our study all samplings yielded positive results. On the other hand, the possibility of overloading by other, more concentrated particles (in this study’s case it was dust) must be always taken into consideration in experiment planning and interpretation of the results.

Our study revealed the permanent presence of the H. fraxineus inoculum up to 48 meters above the ground during the whole sampling period. Its concentration is continuously changing depending on previous and current weather, and decreases with height. It poses a persistent threat to ash trees, either at local or landscape scale. This finding supports a sceptical outlook for the future of ashes in European forests, but also confirms the important role of high height air sampling of the propagules of this invasive alien pathogen to ensure its reliable monitoring.

This work was funded by a HORIZON 2020 project (agreement no. 771271): Holistic Management of Emerging Forest Pests and Diseases (HOMED). We would like to thank to EUROPASTA SE company for cooperation and full access to their silo in Boršov nad Vltavou, namely to the director Ing. Josef Maška, Jaroslav Mrkvička, Ladislav Hakl and Jaroslav Hnilička. Furthermore, we thank Dr. Leticia Botella Sánchez for preparing the plasmid for absolute quantification.