4.1 Fungi associated with ash stem collars
In total, 162 fungi were isolated from ash stem collars and differentiated within this study. About half of these fungi were isolated from stem collar necroses in comparable studies as well (Lygis et al. 2005; Langer 2017; Meyn et al. 2019; Kranjec Orlović et al. 2020). Though 87 taxa (54%) isolated here were not reported by the aforementioned studies. It has to be taken into account that different samples sizes and sample site numbers lead to differing numbers of species. Considering the mentioned studies, Meyn et al. (2019) had the smallest sample size with four trees at one sample site and reported the smallest diversity with 16 fungal species. Langer (2017) isolated more fungal species (35) from 32 sample trees at seven sample sites. The correlation between sampling size and reported fungal diversity is also confirmed by Kranjec Orlović et al. (2020) with 68 fungal species isolated from 90 sample trees examined at three sample sites. The non-negligible impact of the number of sample sites on the detectable fungal species diversity is shown in this study with 162 MTs isolated from a smaller sample size (58 trees), but a higher number of sample sites (9) compared to the study of Kranjec Orlović et al. (2020). Other factors, such as number of incubated wood chips or the isolation method can also have influence on the number of isolated species. But overall, there seems to be a positive correlation between sample size or number of sampled sites and species richness. Langer (2017) observed that, advanced of stem collar necroses result in a higher number of isolated species. This could be confirmed in the present study when taking into account only the stem collar necroses with isolation of H. fraxineus.
Similar to studies focusing on endophytes of tree woody tissues (Bußkamp et al. 2020; Langer et al. 2021) in this study the majority of fungi isolated belong to Ascomycota (77.8%). A reason for the lower frequencies of Basidiomycota compared to Ascomycota 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. However, the proportion of Basidiomycota (22.2%) in this study is higher than in the aforementioned studies. The reason for this discrepancy might be the focus on different woody tissue types in the mentioned studies, and hence detection of differing fungal communities with divergent ecological functions. Basidiomycota isolated from woody tissues are often related to wood rot, because lignin is primarily decomposed by this fungal group and therefore they are more likely to be found in diseased or necrotic rather than asymptomatic woody tissue (Eriksson et al. 1990; Bugg et al. 2011). Hence, the occurrence of white and brown rot fungi in stem collar necroses is not unusual. Typical white rot fungi like Armillaria spp., Coprinellus spp., Bjerkandera adusta (Willd.) P. Karst., Peniophora spp., Trametes versicolor (L.) Lloyd, and few brown rot fungi like Fomitopsis spp. have been isolated from stem collar necroses in this study. The majority of soft rot fungi isolated here pertain to Ascomycota and the following representatives of this group were found: Biscogniauxia nummularia (Bull.) Kuntze, Hypoxylon spp., Jackrogersella sp., Nemania serpens (Pers.) Gray, and Xylaria spp. (all Xylariales). Beside the occurrence of white and brown rot fungi the frequent association of xylarialean wood decay fungi with stem collar necroses make it plausible that affected ash trees have a massive loss of stability and tend to topple over even without wind as a supporting factor.
Approximately one-third of the isolated MTs detected in this study were listed for F. excelsior in the USDA fungal database (Farr and Rossman 2022). Only one of the most frequent MTs of this study, N. punicea, is not listed there, but other species from the genus Neonectria are mentioned. However, N. punicea was described as one of the most frequent MTs associated with stem collar necroses in the context of ash dieback (Langer 2017; Meyn et al. 2019). The other most abundant MTs isolated in our study Armillaria spp., D. fraxini, H. fraxineus, Diaporthe cf. eres, Fusarium cf. lateritium, and Paracucurbitaria sp. were already described to be associated with ash (Chandelier et al. 2016; Haňáčková et al. 2017; Langer 2017; Meyn et al. 2019; Linaldeddu et al. 2020; Kowalski and Bilański 2021; Barta et al. 2022). Langer (2017) investigated stem collar necroses of 32 ash trees and determined the aforementioned species as well – except for Paracucurbitaria sp. Meyn et al. (2019) isolated D. fraxini (labelled as Botryosphaeria stevensii), H. fraxineus, N. punicea, and Diaporthe cf. eres as well. The most commonly isolated species from stem collar necroses in the present study, except Armillaria sp. and Paracucurbitaria sp., were also isolated in high frequency by Linaldeddu et al. (2020), although they focussed on symptomatic branches of damaged ash trees in Italy. The absence of Armillaria sp. in branches was anticipated, because it is a soil-borne root and stem rot fungus, colonising its host through rhizomorph growth (Morrison 2004).
In this study, isolates belonging to the genus Armillaria were among the most consistently detected MTs, where A. gallica was detected more frequently. Additionally to our isolations from stem collar necroses, mycelial fans and rhizomorphs of Armillaria spp. were observed at all sample sites of this study and the majority of further studied ash stands diseased by ash dieback. Armillaria species are common soil colonisers in Europe and therefore are probably existing in most forest sites even before ash dieback occurs (Morrison 2004; Lygis et al. 2005; Bakys et al. 2009b). They are considered as secondary pathogens and wood decaying fungi infecting stressed trees, which explains their occurrence in advanced stem necroses and root rot (Chandelier et al. 2016). On the one hand, Armillaria spp. can colonise stem collars after the necrosis has already formed by H. fraxineus. On the other hand, they are also able to independently attack a weakened ash tree without a stem collar necroses due to H. fraxineus (Langer 2017). As in our study, the occurrence of A. gallica and A. cepistipes associated with stem collar necroses of trees affected by ash dieback has been shown by Chandelier et al. (2016) in Belgium. Enderle et al. (2017) also detected A. gallica in stem collar rots in south-western Germany. These results are in contrast to investigations by Lygis et al. (2005), who determined A. cepistipes as most frequent in Lithuania. The different distribution of the latter two Armillaria species can be explained with the sampling in different regions of Europe and varying site characteristics and altitudes. The distribution of A. cepistipes appears to correspond with an inverse relationship between latitude and altitude (Guillaumin et al. 1993). For example, A. cepistipes occurred in low altitudes at northern latitudes and in higher altitudes at southern latitudes. However both species are widely distributed and common in Europe (Legrand et al. 1996; Marxmüller and Holdenrieder 2000). In the south of Germany (Bavaria) A. cepistipes tends to occur at higher altitudes (montane) and also colonises conifers, whereas A. gallica was not collected at sites above 600 m.a.s.l. (meters above sea level) (Marxmüller and Holdenrieder 2000). According to (Tsopelas 1999), A. cepistipes occurred more often in higher altitudes, whereas A. gallica is predominant in beech forests. In this study, A. cepistipes was present for forest sites at approx. 300 m.a.s.l. consisting of mixed stand compositions with European beech (Schlangen 1 and Schlangen 4). The occurrence of A. cepistipes at both low and high altitudes (up to 1750 m.a.s.l.) in Germany, is probably related to the fact that there are also regions with continental climate with relatively cold winters over a wide range of altitudes (Guillaumin et al. 1993). According to field observations by Guillaumin et al. (1993) the pathogenicity of A. cepistipes is lower than that of A. gallica, which is a common weak parasite of hardwoods. Nevertheless, regardless of which of the two species caused infection, Armillaria spp. most likely accelerate the decline of ash dieback affected ash trees (Chandelier et al. 2016) and reduce stem stability.
The most frequently isolated species in our study D. fraxini has been recognised as the dominant species in comparable studies as well. Linaldeddu et al. (2020) determined that many reports of D. fraxini on ash have earlier been assigned to D. mutila s. l. Phylogenetic analyses showed, that most of the Diplodia strains isolated in this study, although morphologically similar to D. mutila, certainly match with D. fraxini. It is an aggressive pathogen known to cause bark lesions and wood discoloration or to enlarge necroses, which are primarily caused by H. fraxineus (Alves et al. 2014; Linaldeddu et al. 2020, 2022). Kowalski et al. (2017) classified it as the second most pathogenic fungus after H. fraxineus, though it was not mentioned as a frequent coloniser of F. excelsior before ash dieback disease occurred (Kowalski et al. 2016). These facts might indicate that infections with H. fraxineus facilitate the colonisation of affected ash trees by D. fraxini. Another possible explanation for the more frequent occurrence of D. fraxini could be global warming because this species benefits from warm temperatures of around 25° C (Alves et al. 2014). In our opinion, D. fraxini plays an important role in ash dieback disease and contributes undoubtedly to a greater damage extent, in particular at stem collar necroses. Beside the latter very frequent Diplodia species, to the knowledge of the authors, this is the first report of D. sapinea on ash. In contrast to the study by Linaldeddu et al. (2020), the species D. subglobosa could not be isolated in our analysis, maybe because they investigated branches and not stem collar necroses.
Neonectria punicea has a large host spectrum, including F. excelsior (Hirooka et al. 2013). However, this fungus has rarely been documented from this particular host species before (Langer 2017; Meyn et al. 2019; Karadžić et al. 2020). N. punicea was found to be associated with stem collar necroses and cankers of European ash in Germany (Langer 2017; Meyn et al. 2019) and it is able to cause necroses in juvenile ash trees (Karadžić et al. 2020). Its perithecia were observed frequently on the bark above the necrotic ash tissue (ibid. and Karadžić et al. 2020). Neonectria punicea is mainly known to be a secondary pathogen, but can also express an endophytic lifestyle (Langer 2017). Species of the genus Neonectria invade through natural entrances, like lenticels or artificial wounds, for infection (Flack and Swinburne 1977; Salgado-Salazar and Crouch 2019).
The isolation of strains assigned to Diaporthe cf. eres were made from diseased and also from healthy woody ash tissue in this study. This is in agreement with insights that Diaporthe eres can live as a plant pathogen, endophyte or saprotroph and has a wide host range as well as a widespread distribution (Udayanga et al. 2014; Linaldeddu et al. 2020). This species often produces its tiny fruit bodies on dead woody tissues (Kowalski et al. 2016). In a study by Kowalski et al. (2017), D. eres showed the least virulence and caused significantly milder disease symptoms on F. excelsior plants than the other tested fungal species. Diaporthe eres could be considered as a weak pathogen in comparison to ash dieback on F. excelsior. In case of tree weakening by H. fraxineus the early endophytic presence of D. eres favours a fast pathogenic attack (Kowalski et al. 2016).
In this study Fusarium lateritium Nees has been isolated frequently from symptomatic tissue and once from healthy wood tissue. The species is already known from F. excelsior in association with bacterial ash canker (Riggenbach 1956) but its virulence seems to be low in comparison with other fungal species (Bakys et al. 2009b). Kowalski et al. (2017) showed, that F. lateritium causes none or only small necroses on F. excelsior. In general, Fusarium spp. have a wide host range and are reported as the most common endophytes in ash bark and wood (Kowalski and Kehr 1992; Sieber 2007; Bakys et al. 2009b; Kowalski et al. 2016). The facts described above, indicate that F. lateritium is able to colonise the bark and woody tissue of ash independently of H. fraxineus. In association with ash dieback though it is more likely that the species contributes to the stem collar necroses as secondary pathogen. Thereby, it is non-essential, whether acceleration of ash dieback is established by shifting from endophytic to pathogenic lifestyle or colonising the tree as a secondary pathogen after tree weakening.
As far as it is known, the isolation of Paracucurbitaria sp. from the examined samples is the first proof of this genus in stem collar necroses. It was not isolated by Langer (2017) and Meyn et al. (2019) from rootstock. However, Kowalski and Bilański (2021) detected Paracucurbitaria sp. in previous year’s ash leaf petioles in Poland, Barta et al. (2022) isolated it from ash twigs in Slovakia, and Haňáčková et al. (2017) reported Paracucurbitaria corni (Bat. & A. F. Vital) Valenz.-Lopez, Stchigel, Guarro & Cano as an endophyte of ash leaves and seeds. Therefore the occurrence of species from the genus Paracucurbitaria in plant material of F. excelsior is not striking, but its high frequency in stem collar necroses was unanticipated. It can be assumed that the high frequency of Paracucurbitaria sp. is no coincidence, because its detection in stem collar necroses of ash is increasing in ongoing research at the NW-FVA since sampling for this study.
Besides D. sapinea, there are a few species, which, to the knowledge of the authors, have not been previously reported from F. excelsior (Table 2). One of them is C. corticale, known as the causal agent of the sooty bark disease on maples. Its main host is Acer pseudoplatanus L., but it has been proven that C. corticale can colonise other maple species as well as Aesculus hippocastanum L. (Enderle et al. 2020). This species was found at sampling sites with sycamore. In addition to the first reports of ash as a host, one strain belonging to the genus Vexillomyces was isolated and recognised as undescribed species. The genus Vexillomyces was described in 2020 for two species (V. palatinus, V. verruculosus) isolated from spore traps attached to vine shoots. No host organism is known for these species. Later several species of Claussenomyces and Tympanis were transferred to the genus (Baral and Quijada 2020). The respective species are known from dead or living angiosperm and gymnosperm wood, however, only for V. atrovirens (syn. Claussenomyces atrovirens) an affiliation to the host genus Fraxinus could be recognised (Dennis 1986).
4.2 Role of Hymenoscyphus fraxineus in stem collar necroses
The ash dieback pathogen H. fraxineus could not be isolated from all of the 54 symptomatic stem collars. Only in about half of the trees, the fungus could be determined. It has been already reported by several authors, that the ash dieback pathogen could not be frequently isolated from symptomatic tissue of ash (Przybyl 2002; Bakys et al. 2009a; Enderle et al. 2017). A possible explanation for this could be its slow growth, unfavourable sampling conditions for the pathogen or too advanced necroses with antagonistic activity of other colonisers (Kowalski and Holdenrieder 2009; Hauptman et al. 2013; Gross et al. 2014; Langer 2017). Often, H. fraxineus could be solely isolated from recently discoloured woody tissues of the stem collar necroses (Fig. 2) and is probably supressed in the older parts of the necroses already colonised by secondary fungi. The aforementioned reasons might have contributed to the moderate isolation success of the ash dieback pathogen in this study. Perhaps, fungal community analysis by means of culture-independent methods, such as high throughput sequencing or qPCR could detect H. fraxineus more frequently than by culture based isolation, since these methods have the potential to detect inactively present fungi (spores) or even DNA residues if the initial fungus has been suppressed by secondary invaders (Lindahl et al. 2013). Our results on the fMT and continuity of the association and localisation of H. fraxineus in basal stem necroses confirm the assumption, that this pathogen is very often the main or primary causal agent triggering stem collar necroses. Either way, H. fraxineus is confirmed as the main pathogenic agent of the ash dieback epidemic (Kowalski 2006; Bakys et al. 2009a; Kowalski and Holdenrieder 2009; Gross et al. 2014). The only lack of evidence of H. fraxineus at the study site Wolfenbüttel could be explained by the meagre sample size (single tree, very low number of extracted wood chips). It can be assumed that H. fraxineus may have been isolated if a larger wood chip number or sample tree size was examined. According to information from a co-researcher in FraxPath, H. fraxineus was present in branches of the sample trees at the study site Wolfenbüttel (Maia Ridley, personal communication).
4.3 Fungal communities in stem collar necroses
Kowalski and Kehr (1992) and Sieber (2007) concluded that the fungal communities in F. excelsior are mainly dominated by fungi belonging to Diaporthales (Diaporthe spp.) followed by Pezicula species. In contrast, fungal taxa identified in this study mainly belong to Hypocreales, followed by Pleosporales and Helotiales. Fungi belonging to the Diaporthales (summarised under the category “others” in Fig. 3) account only for a tiny proportion of 1.6% of all isolated ascomycetes from our sampling, including the fifth frequent isolated taxon. Furthermore, Pezicula species are less frequent in this study with 12 isolates representing five MTs. Pezicula cinnamomea (DC.) Sacc. determined by Kowalski and Kehr (1992) as the second most isolated fungus, could not be isolated in this study. This discrepancy in the diversity could be explained by the fact that Kowalski and Kehr (1992) and Sieber (2007) focused on endophytes isolated from asymptomatic plant material. Another possible explanation might be the different geographical regions of the discussed studies, or the use of different plant tissue material, or sampling and isolation methods. The composition of fungal orders inhabiting asymptomatic plant material might not represent the composition in symptomatic stem tissue in specific.
The fact, that the composition of fungi isolated in this study differs with only a little overlap between the sampling sites, leads to the assumption that adding further sampling sites would reveal new sets of fungi not recorded in this study. The observation of significantly different occurrences of fungal taxa between forest sites is confirmed by Bilański and Kowalski (2022). In the study of Meyn et al. (2019) only two species were found in all sample trees and many of the identified fungi were single isolates. Similarly, Kranjec Orlović et al. (2020) revealed just few predominating taxa representing half of all fungal isolates from stem bases of Fraxinus angustifolia Vahl. In addition, species represented only by a single isolate make up one-third of all isolates in the study by the latter authors.
4.3 Relation of the most common fungi to the site characteristics
It is generally accepted that European ash trees independently of age class and site conditions are infected by H. fraxineus (Pautasso et al. 2013). However, the extent of ash dieback in the crown and stem collar necroses and tree mortality, most likely depend on many different factors. Susceptibility of ash trees to the pathogen, the range of subsequent colonising fungal species (Langer et al. 2022), tree vitality, or the environmental context of the forest site and stand (Havrdová et al. 2017) are some examples. Ash tree vitality is encouraged at fertile and (moderately) wet soils, conditions which are preferred by ash (Walentowski et al. 2017). It has to be taken into account that for this study only a selection of forest sites from a rather narrow area out of the wide range of European ash was investigated. An optimal soil and water supply with a sufficient percentage of ash trees was fundamental. Furthermore, the selection of sample trees was subjected to different restrictions. For example the condition of a diameter at breast height less than 25 cm because of logistics and processing abilities in the lab. Besides that, trees with very advanced necroses like completely necrotic or rotten stem base or dead trees were not suitable for investigation.
Our preliminary results indicate that H. fraxineus was isolated less frequent at sites with higher water availability (Online Resource 2). This is in accordance with the guess that the fungal composition of stem collar necroses depends on soil and water availability of the forest stand (Linaldeddu et al. 2011; Salamon et al. 2020). As mentioned before, this assumption refers only to the selection of the investigated forest sites. One explanation could be, that secondary fungi have more favourable conditions at sites with higher water availability and thus are able to overgrow H. fraxineus faster than at drier sites.
For the other most common fungi Armillaria spp., Diaporthe cf. eres, D. fraxini, Fusarium cf. lateritium, N. punicea, and Paracucurbitaria sp. no significant correlation between fWC and water balance could be determined (Online Resource 2). Assuming that H. fraxineus as the sole pathogen influences the extent of damage caused by stem collar necrosis, this this would be in contrast to the suggestion of several authors that stands with wet soil conditions show a higher probability that the individual trees affected by H. fraxineus exhibit greater damage (Gross et al. 2014; Erfmeier et al. 2019). At Schwansee, the wettest sampling site, H. fraxineus was only isolated twice. However, the stem collar necroses were most advanced at these sampling trees, where a lower isolation rate of H. fraxineus was generally expected, as mentioned previously.
It was noticeable, that D. fraxini and N. punicea had a significantly different fWC at the various sampling sites (Online Resource 2), but there was no indication for a correlation with the site characteristics water supply, soil and bedrock, climate, or mixture of trees. However, it was observed that ash trees with a low fWC of D. fraxini had a thinner bark. Compared with D. fraxini and N. punicea, the MTs Diaporthe cf. eres, Fusarium cf. lateritium, and Paracucurbitaria sp. had a consistent fWC over all sites. But Fusarium cf. lateritium was not isolated at Satrup and at the valley bottom in Schlangen. Due to the lower amount of isolations in this study, the authors assume there is also a lower probability of occurrence in stem collar necroses.
Armillaria species were not present at all studied sites and could not be isolated from the trees in Schwansee. This result is contradictory to those of Enderle et al. (2017), who found older necroses to be more often colonised by Armillaria spp. The progress of the necroses formation was clearly visible by their partially ruptured wood surface and presence of fruiting bodies on the necrotic stem areas of wood decay fungi, such as Coprinellus sp. and Xylaria polymorpha (Pers.) Grev. (Liers et al. 2011). Furthermore, the absence of Armillaria spp. isolates in Schwansee, the moistest of all sampling sites which is influenced by its ground water, do not correspond to preference of Armillaria species for continuously moist soil conditions (Whiting and Rizzo 1999). A possible explanation for the lack of this species in Schwansee, could be the specific forest site background as a former lake. The area was earlier used as fishpond until the 18th century. Thus, the soil was subjected to special formation conditions (Welk 2017) and perhaps it was not possible for Armillaria spp. to colonise the soil like in other forest sites.
Many of the other MTs detected in this study were isolated just once, which may indicate no direct correlation with the investigated forest sites, thus site characteristics like soil and water supply relatedness cannot be assumed. However, it cannot be ruled out that the one-time isolated fungi occur in other forest sites, than the investigated ones, too. As well as a higher abundancy is theoretical possible. Additionally, it is to be expected that the composition of fungi might differ according to tree age, tree species composition, forest management type, season and the like (Scholtysik et al. 2013; Tomao et al. 2020). For example, a more diverse tree species composition at a forest site could contribute to the occurrence of a wider spectrum of fungi colonising a tree (Cavard et al. 2011; Kowalski et al. 2016; Krah et al. 2018; Tomao et al. 2020). This is confirmed by the isolation of sycamore typical fungi like C. corticale und C. rubronotata from F. excelsior in stands with maple trees. It is furthermore supported by the fact that the most mixed intensive sampling site of Berggießhübel has one of the highest fungal diversity in relation to its sample tree amount. Besides its diverse tree species composition, in addition Berggießhübel is the most eastern sampling site. Satrup is the most northern sampling site and shows also high fungal diversity despite its smallest sample tree amount. This observation suggests that widely varying sites in Germany lead to differing fungal communities. 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).
4.4 Conclusion and Outlook
This study provided new insights on the fungal diversity and communities of endophytes, primary and secondary pathogens, wood decaying fungi, and saprophytes associated with stem collar necroses of European ash trees. A rich fungal composition inhabiting symptomatic stem tissue has been revealed with four frequent species occurring at most of the studied forest sites, but with little overlap between the sites. The fungal species richness detected in this study (162) is considerably higher compared to previous investigations in which 16–75 different species were detected (Lygis et al. 2005; Enderle et al. 2017; Langer 2017; Meyn et al. 2019). This difference in diversity can be explained by the larger sampling size (not only tree number, but also amount of wood chips taken) and the partially greater number of sites studied. Single trees with only about 20 studied chips of stem collar tissue each (Oranienbaumer Heide, Wolfenbüttel) had the fewest amount of isolated MTs. Further studies on stem collar necroses can increase the knowledge of fungal biodiversity on F. excelsior, as the first proved fungi in this study show.
The ash dieback pathogen was isolated from only about half of the trees sampled. Different reasons like its slow growth can cause a low isolation rate of the primary pathogen. Nevertheless it can be assumed, that stem collar necroses are commonly initiated by this fungus. The occurrence of several pathogenic fungi from necrotic stem tissue of ash beside H. fraxineus is striking, because of their high fMT. It was shown that the different fungal communities of the sample trees are largely dominated by three MTs (D. fraxini, Armillaria spp. and N. punicea) next to H. fraxineus representing almost 50% of all isolates. They are considered to play a major role in the progression of stem collar necroses and rot and therefore also contribute to a loss of tree stability. The remaining fungi which were isolated from the stem collars necroses turned out to be very diverse with much lower fMT, in the majority of cases were represented with only one isolate. Overall, the synergistic interaction of different pathogens in the context of ash dieback, for example H. fraxineus and D. fraxini or N. punicea, can lead to a larger damage in contrast to infection by only one pathogen (Marçais et al. 2010). In this context, N. punicea poses a serious threat to planted ash forests and natural regenerations of F. excelsior, especially if another host tree species, such as European beech (Fagus sylvatica L.) is in mixture. European beech is potentially an inoculum reservoir of N. punicea for future infections of ash stem collars (Karadžić et al. 2020). Therefore, in the future the susceptibility of ash to form stem collar necroses and to be diseased by D. fraxini and N. punicea should be considered in breeding programs to develop more resistant ash trees in relation to ash dieback.
However stem collar necrosis types caused by other fungi than H. fraxineus or Phytophthora spp. (Langer 2017), should not be disregarded. The results of this study show, that at least one fungal pathogen can be found in the necrosis without evidence of H. fraxineus. For example, one of the control samples, which turned out to have necrotic tissue inside the wooden body, was colonised by Armillaria sp. In this case, it is likely that the fungus attacked the weakened tree independently of a pre-colonisation of the stem collar by H. fraxineus.
Since in this study no correlation between the site factors and fungal occurrence could be calculated because most of the isolated fungi were only detected once, further studies should be carried out at additional comparable forest sites. Inventories of stem collar necroses at a higher number of locations may reveal dependence of MTs to forest side conditions and their individual role in the fungal communities in detail. Future studies need to be conducted in order to estimate potentially high risk characteristics of forest sites for pronounced and fast advancing stem collar necroses and rot. Additionally, the investigation of genotypes of H. fraxineus associated with single stem collar necroses could help to better understand the path of infection with H. fraxineus and the secondary colonisation by other fungi.