The observed widespread occurrence of SBD in Germany coincides with information from neighbouring central European countries (Kelnarová et al. 2017; Cech 2019; Queloz et al. 2020) As shown by the SBD reports documented in this study, there are no cases of the disease in the forests of the most northern federal state of Germany, Schleswig-Holstein. This might be due to a more maritime, and thus more humid, climate. When comparing the site conditions of the forest stand where the endophytes were isolated, it can also be observed that the site in Nehmten, Schleswig-Holstein has a different bedrock than the other sites (Table 1). The Forest plot in Nehmten was the only site with a high water storing capacity (WSC). The WSC is highly dependent on the amount of skeleton in the ground since it takes up valuable space that cannot be used for storing water. The sandy stand in Nehmten has a larger soil depth, as well as less skeleton (less than 5%), in the soil compared to the sites in Hesse (Basalt, 10% – 15% skeleton on average, which increases with soil depth). Thus, the site in Nehmten has a higher WSC (Paar et al. 2016). Additionally, the site in Nehmten is influenced by ground water due to being located in a lake region. This indicates that even in drought periods, the plant water availability is more stable in Nehmten, resulting in less plant stress. When comparing climate data for 2018 and 2019 between Hesse and Schleswig-Holstein, the data for 2018 shows that the average summer temperature (AST) in Hesse was 19.6°C, 3.4°C above the average of the reference period (ARP) and the AST for Schleswig-Holstein was at 18.4°C, only 2.6°C above the ARP (DWD 2018b). The difference between the two states was similar for 2019, for Hesse the AST was 19.1°C, 2.9°C above the ARP and for Schleswig-Holstein 17.9°C, which was 2.1°C above the ARP (DWD 2019b). For 2018 and 2019 the precipitation deficit both for the summer period and the entire year was lower for Schleswig-Holstein than for Hesse (DWD 2018b, 2019b). This shows that temperatures in Hesse were in general higher, as well as in relation to the ARP, with less precipitation in comparison to Schleswig-Holstein, indicating potential higher drought stress and thus more favourable circumstances for the outbreak of SBD in Hessen.
The result of Kelnarová et al. (2017) that in a quarter of all studied urban sycamore trees in Prague C. corticale could be detected as endophyte was confirmed by our observations of the latent non-symptomatic stage of SBD in forest trees. Our result that 26% of all studied, apparently healthy sycamores, harboured C. corticale non-symptomatically, regardless of the occurrence of external symptoms in the studied forest stands or area, lead to the assumption that latent infection with C. corticale is widespread in Germany. Therefore the risk of mortality due to C. corticale for apparently healthy sycamore trees rises with the increase of years with drought and extraordinary high temperatures, since SBD is triggered by drought and hot temperatures (Dickenson 1980; Ogris et al. 2021). Even though only vital trees with no signs of distress or injury were chosen for our sampling, 35% of the increments exhibited wood discoloration and/or wood rot. In Kelnarová et al. (2017), discoloration was observed in 59% of all sampled trees and was correlated strongly with the presence of C. corticale. In this study C. corticale was only isolated once from discoloured areas. This might have been the result of analysing vital trees, not focusing on discoloured tissue. The amount of discoloured tissue observed in this study was also rather low and thus not a basis for adequate characterization. Furthermore, direct correlation of discolouration with C. corticale is speculative, since the discolouration could have as well been caused by other agents.
The total amount of endophytic species isolated from woody sycamore tissue in this study (91) is significantly higher than in previous investigations were 10–52 different species were detected (Butin and Kowalski 1986; Kowalski and Kehr 1992; Unterseher et al. 2005; Brglez et al. 2020a) This difference in diversity can be explained by the larger sampling size and the greater number of sites studied here in contrast to the other studies.
Many of the fungal species detected in our study were isolated just once, which may indicate a sporadic occurrence or sampling bias since only ten trees per site were studied at one specific height. However, it cannot be ruled out that these fungi occur in other stands as well as in higher abundancy and simply were not isolated from the sampled material. Due to the rather small sample size of two or three increments compared to the entire wood body of the tree, the listed fungi are likely just a small fraction of the fungal community. Additionally, it is to be expected that the composition of fungi might differ within the tree, depending on the host tissue type (Gennaro et al. 2003) and tree age (Halley et al. 1994; Maherali and Klironomos 2007). Furthermore, a possible underestimation of fungal diversity in the studied trees may occur since not all fungi are detectable through standardised culture based methods or in general (Guo et al. 2001; Allen et al. 2003; Unterseher 2007; Muggia et al. 2017). The composition of the forest stands combined with the nutrient and water availability could also be a factor in assessing the differences in fungal diversity per stand. The stand in Nehmten, stocked only with sycamore, had the lowest fungal diversity of all studied sites and at the same time the lowest nutrient availability.
Similar to other studies on endophytes of tree woody tissues (Langer et al. 2021; Singh et al. 2017; Bußkamp 2018; Ghobad-Nejhad et al. 2018), in this study mainly Ascomycota (85.17%) and significantly less Basidiomycota (9.89%) were isolated. This also corresponds to studies focusing on endophytes colonising tree tissues (Petrini and Fisher 1988; Kowalski and Kehr 1992; Peršoh et al. 2010; Martínez-Álvarez et al. 2012; Sanz-Ros et al. 2015). A reason for the lower frequencies of Basidiomycota compared to Ascomycota detected in this study might be that fungi belonging to the former often need longer incubation periods in order to grow out from incubated woody tissues (Oses et al. 2008) but since the incubated increment segments were kept for four weeks on nutrient media, it has to be assumed that enough time was given for fungi to grow out.
Sieber (2007) concluded that the fungal communities in the Aceraceae are mainly dominated by species belonging to Diaporthales. Furthermore, Pleosporales and Xylariales can be dominant endophytes in angiosperms. The species identified in this study mainly belong to Pleosporales, followed by Xylariales and Hypocreales. Species belonging to the Diaporthales constitute only the fourth most common group. In comparable studies focusing solely on woody sycamore tissue (e.g. Butin and Kowalski 1986, Kowalski and Kehr 1992, Unterseher et al. 2005 and Brglez et al. 2020) the most commonly isolated orders include Diaporthales, Helotiales, Hypocreales, and Pleosporales. Diaporthales did not unequivocally dominate in any of these studies. This discrepancy in the diversity could be explained by the fact that Sieber (2007) focused on different forest trees and endophytes isolated from leaves and woody tissue except roots. The composition of fungal orders inhabiting leaves and woody tissue, as subsumed by Sieber (2007), might not represent the composition solely in woody tissue of Acer trees in specific. The fungal community of this and the aforementioned studies were not dominated by a few host-specific species as stated by Sieber (2007). In our study only five species (C. corticale, L. muelleri, L. turgidum, Penicillium sp., and X. longipes) were found in 10% or more of the examined trees. Only one of these is genus-specific, namely the most abundant, invasive species C. corticale. This might be due to the small geographical region that the analysed sites were located in, in comparison to the geographical area covered in the study of Sieber (2007), as well as the diversity of the studied plants or even the differing sampling and isolation methods used.
The ten most abundant fungi were all isolated from healthy wood tissue. Therefore, it must be assumed that all of them exhibit an endophytic life stage, even though three of them were isolated from discoloured tissue once as well (B. nummularia, C. corticale and L. muelleri). Cryptostroma corticale appears to exhibit an endophytic life stage, as it was isolated from healthy, as well as from discoloured, wood. This observation is supported by its affiliation to the Xylariales, which is an order hosting many fungi with endophytic life stages (Hendry et al. 2002; Bußkamp et al. 2020). Xylaria longipes was the second most abundant species in the study and is a known endophyte in a number of different tree species. Xylaria species often grow and sporulate on lying deadwood and stumps (Scholtysik et al. 2013). The third most abundant species, L. turgidum, belongs to the Xylariales as well.. Leptosillia muelleri, being the fourth most abundant species, belongs to Xylariales like C. corticale and Xylaria sp. and thus can be assumed to have at least an endophytic life stage. Cadophora prunicola was only described in 2020 by Bien and Damm (2020) and to the authors’ knowledge has not been reported elsewhere since. This is the first study to report C. prunicola from A. pseudoplatanus, and furthermore it was isolated from healthy tissue. With C. prunicola belonging to the Helotiales, an endophytic life stage is probable, since several other fungi belonging to the Helotiales are known endophytes (Petrini and Fisher 1988; Langer et al. 2021). Trichoderma species can occur as endophytes as described by Evans et al. (2003) and belong to the Hypocreales, which is a group hosting several other endophytic species as well. Diaporthe pustulata was originally isolated from A. pseudoplatanus (Gomes et al. 2013). As described in Suryanarayanan (2011), Bußkamp et al. (2020) and Langer et al. (2021), species belonging to the Diaporthales can occur as endophytes, making it likely for D. pustulata to also have an endophytic stage in its life cycle. The pleosporalean fungus D. macrostroma is presumed to have an endophytic life stage as well. Many other fungi in the Pleosporales also have endophytic life stages (Langer et al. 2021). Biscogniauxia nummularia is a known and widespread multi-host endophyte isolated from several different tree species (Petrini-Klieber 1985; Nugent et al. 2005, (Petrini-Klieber 1985, p.; Chapela and Boddy 1988; Chapela 1989; Bußkamp et al. 20) belonging to the Xylariales as well, and very common on beech trees (Chapela and Boddy 1988; Chapela 1989).
The fungus isolated with the highest frequency, but very low abundance, was one belonging to the family of Hymenochaetaceae. Even though this species has the highest frequency, it is not the most important or common species since all but one isolate originate from one tree. The respective cores showed signs of white rot and the species was isolated from many pieces of one of the cores.
Detection of some fungal groups, like Penicillium sp. or Trichoderma sp. can be explained, since these groups have in general a wide host range as well as a wide geographical distribution and are frequently reported in comparable studies (Cotter and Blanchard 1982; Chapela 1989; Ragazzi et al. 1999; Hendry et al. 2002; Baum et al. 2003). Several wood decay fungi were isolated and three species them were isolated from discoloured woody tissue (B. nummularia, Hymenochaetaceae sp., and J. cohaerens).
The composition of fungi isolated in this study differed significantly between the studied forest sites, with very little overlap between the sites. It can be assumed that adding another differing site would reveal an entirely new set of endophytes not recorded in this study. While some of the isolated fungi (29.1%) were already described as associated with maple (see Ellis and Ellis 1985; Butin and Kowalski 1986; Chlebicki 1988; Kowalski and Kehr 1992; Unterseher et al. 2005; Brglez et al. 2020), most were not recorded in the aforementioned articles (70.9%). Only ten of the isolated species (C. corticale, D. pustulata, D. rudis, D. macrostroma, Eutypa maura (Fr.) Fuckel, N. cinnabarina, N. acerina, Petrakia irregularis Aa, T. vestitum and X. longipes) were listed for A. pseudoplatanus in the USDA fungal database (Farr and Rossman 2022). Three of them were also reported from sycamore in Germany. This further illustrates the current lack of knowledge about endophytes, specifically in woody tissue of A. pseudoplatanus in Germany.
Cryptostroma corticale was isolated from four out of five sampled stands. It was found in Melsungen, a site with trees without obvious symptoms of SBD and located more than 30 km away from the next known area of infection. Furthermore, it was detected in Nehmten, however, symptoms of SBD have not yet been reported for the entire federal state of Schleswig-Holstein. Isolation of C. corticale in Beerfelden was unexpected, due to the high wood quality and tree vitality of the sampled stand. The detection of C. corticale in Nidda was expected, due to obvious symptoms of SBD at the site. In contrast, the fungus was not detected at the stand of Fulda despite of obvious symptoms recognised. Similar to the results of Kelnarová et al. (2017), who also used both a culture and a non-culture based isolation method sampling 112 trees on seven different sites, the number of trees from which C. corticale was isolated was lower than expected. The continuity of C. corticale in the presented study is 26%, similar to the results of Kelnarová et al. (2017) where the continuity, based on both isolation methods combined was 25%.
The spread of C. corticale in its endophytic stage should be further investigated in Germany as well as in Europe in order to estimate the potential risk for Acer cultures in this region. The presented data functions as a basis in this endeavour. An increase of negative effects due to climate change, such as rising temperature and insufficient precipitation, need to be expected for the future, and constitute major risks for the forest sector. In the specific case of SBD, the location of Acer plantations should be carefully considered in order to increase the potential resilience of the tree. Additionally, the discovery of potential biological control agents against C. corticale among the determined endophytes could help improve resilience of sycamore trees against SBD.