1. Introduction
The ash dieback pathogen
Hymenoscyphus fraxineus [
1], which is native to Asia [
2,
3,
4] and previously known as
Hymenoscyphus pseudoalbidus, [
5] is meanwhile widespread throughout Europe [
6]. Symptoms of the disease range from brown lesions on leaves and branches, wilting of leaves and crown-dieback to discolorations in the wood. The ascomycete threatens ash trees (
Fraxinus excelsior) of all age classes in all their growth areas, in lowlands, floodplain or mountain forests. Above all, young trees are severely affected [
7] but more and more trees in older ash stands also die within a few years of infection. In the extent of this study, severity and progression of the disease in south-eastern Germany have been analyzed for a period of four years in 22 study sites. As there is currently no method to cure the trees or slow down the disease expansion, special study attention was drawn to potentially resistant trees. According to the data from studies in different countries, where the disease was described earlier, it can be expected that the resistance in ash stands against
H. fraxineus is low [
8,
9]. Recommendations for the management of affected ash stands provide for not removing healthy or less-affected
F. excelsior trees depending on their crown defoliation as the best strategy to retain possibly resistant trees [
10]. Furthermore, it was suggested that trees under infection stress by
H. fraxineus are more susceptible to secondary pathogens [
11]. Some bark beetles are specialized on Genus
Fraxinus or
Oleacea. The most abundant species are
Hylesinus fraxini, but also
Hylesinus oleiperda and
Hylesinus crenatus [
12]. During nearly all of their life cycle they live inside the tissue and feed on the phloem [
13]. Before the impact of ash dieback these species were not of any economic importance in the forest industry. There is some concern that these secondary biotic pest agents may increase in abundance on weakened trees and become primary pest agents [
11,
14]. Therefore, the role of ash bark beetles was closely investigated in the different study sites to determine if such beetles are still secondary pests even in
H. fraxineus infected ash stands.
An additional problem results from the species of
Armillaria. These basidiomycetes are distributed worldwide and play variable ecological roles, including causing root disease of diverse forest trees [
15]. In Europe, seven
Armillaria species can be distinguished. Some of them are considered as weak parasites, whereas other species, like
A. mellea,
A. ostoyae and
A. cepistipes, belong to a group of serious pathogens [
16].
A. cepistipes and
A. ostoyae can frequently be found in the same forest stand, whereby their virulence depends on the production of rhizomorphs [
17]. For Lithuania, where the first reports of
H. fraxineus infected trees came from, the presence of
A. cepistipes in declining ash stands was already reported [
18]. In Denmark and south-west Germany
A. gallica was associated with ash dieback, although the fungus was not considered as the main causal agent [
7,
19]. Not only on ash trees, but also on oaks, some
Armillaria species were found jointly responsible for a large-scale mortality in Arkansas, USA [
20]. Some species are able to infect the non-wounded root bark directly [
21].
Armillaria species cause central butt rots, leading to a severe loss in the production of wood, and are therefore economically important. This rot normally does not spread more than a couple of meters [
22].
In ash dieback affected stands in south-eastern Germany, resistance against
Armillaria species has been lowered and the danger of a fungal infection has increased drastically. The interaction between
Armillaria species and
H. fraxineus in declining ash stands is examined. It becomes evident that
Armillaria reduces not only the timber quality but also the stability of the infected trees, which are at high risk for falling down even under windless conditions [
19]. Increasing mortality by combined infections of
H. fraxineus and
Armillaria spp. causes large tree gaps within the stands. Also potentially resistant trees are endangered within dissolving ash stands, as there is a high inoculum of rhizomorphs of
Armillaria spp. within the soil and because of changing groundwater levels caused by the high mortality rate of ash trees. Therefore, forest conversion must be carried out for future prospects, and seeds of potentially resistant trees must be urgently harvested for further breeding, for the possible conservation of this endangered tree species. The aims of this study are therefore (i) to study the development and severity of the ash dieback disease in different geographic areas in south-eastern Germany in young growth, pole and matured forests; (ii) to screen for potentially resistant trees; (iii) and to clarify the role of the secondary pathogens
Hylesinus fraxini and
Armillaria spp. in the decline process to derive further recommendations from these findings and to prove the urgency to act resolutely in these affected regions.
4. Discussion
Since its first identification in Germany in 2007 [
29], the fungus
H. fraxineus has spread nationwide. In south-eastern Germany, it was described for Bavaria in 2009 [
30]. Since then, vitality in all ash stands has been observed to be continuously decreasing (
Figure 2), and is still expected in the coming years, as the infection pressure has not lowered. At the beginning of the monitoring of the ash stands, no disease free areas were found, suggesting that since its arrival a fast spread of the disease has taken place accompanied by an intense development of the symptoms in the affected stands. Also, the degree of fungal infestation differed massively between the different study sites and it is impossible to determine retrospectively at which time the fungus arrived in each stand. We also could not find an apparent correlation between the extent of the disease and the geographical position of the stands, although first symptoms could be observed in the south. Nevertheless, we assume that in study sites located close together, a nearly simultaneous infestation of the fungus has occurred. By the analysis of the data regarding the mean annual changes in vitality, we could find notable differences between study sites that had stands of different age classes. By comparison with pole or matured stands, symptoms occur faster in young growth stands and the mortality rate is higher there (
Figure 3). This can be explained by their smaller crowns and thinner stem diameters, where the fungus more quickly colonizes the whole tissue. The high mortality rates of up to 95% (
Figure 2) reflect similar findings already known from Lithuania, where starting in 2005 progeny trials only showed 10% survival after eight years of constant infections with
H. fraxineus [
31]. Also in pole forests, a massive increase of mortality was monitored in three out of six study sites. Matured ash stands show a rapid decrease in healthy or less affected trees. The majority of the trees are moderate affected, belonging to the vitality classes 2–3 (
Figure 2). The mortality within these stands is comparably low. Surprisingly, the mean annual changes of vitality were slightly higher in matured stands than in pole stands (
Figure 3, top row). Therefore, we assume that the shift from the vitality classes 0–1 to the vitality classes 2–3 in matured stands proceeds faster than the shift from the vitality classes 2–3 to the vitality classes 4–5 in pole stands. In summary, these data clearly show for the first time the longer process of dying in older stands in comparison to young growth stands. It is important to follow up the disease, but, up to now, there has been a lack of clear trends in the damage of the trees that makes it difficult to predict the future development of the disease. At present, no successful treatments for the affected ash stands exist to cure or slow down the disease. For the conservation of the species, it is therefore important to screen for potentially resistant trees, which still could be found in all areas, except for two study sites (
Table 4). Apparently healthy individuals stood alongside severely affected trees. As each study site has been infested by the fungus for different amounts of time, a comparison between the resistance levels is impossible. In time delayed infected ash stands, we expect also a high number of falsely identified resistant trees. The most reliable data can be obtined from the stands with the fastest disease progression. That means in young growth stands (
Figure 3) and from the stands that were already massively impaired by the disease in 2010; for example, the pole stands No. 4, No. 7 and No. 12 (
Figure 2). In young growth stands with only 4% and in the three mentioned pole stands with only 1%–2%, few potentially resistant candidate trees were left (
Table 4). This relatively low number is comparable to the findings in other countries, where the disease has arrived earlier [
8,
32]. It thus seems that the total ash population has only a small fraction of genotypes that will enable it to withstand the disease [
8,
31,
32,
33]. Therefore, the advice of not removing healthy looking trees is necessary not only to be able to provide further recommendations for disease management to practitioners but also to find and protect potentially resistant ash trees for further natural or artificial selection following breeding in this area which is planned for the future.
In different countries, clones were already tested for susceptibility and it could be shown that there are no totally resistant clones, but that there are some individuals that remain in relatively good health. In southern Sweden, some clones exhibited reduced susceptibility and retained this resistance after six years under heavy infection pressure [
33], thus giving hope to the likelihood of being able to conserve
Fraxinus excelsior in Europe. Not only the screening for candidates is important but also the characterization of the underlying resistance mechanisms, to evaluate which genetic material can be utilized for further breeding [
9] and to prove the robustness of the observed partial resistance when new virulent strains evolve. Also in south-eastern Germany [
25] and other countries, a high level of fungal genetic variation was already found [
34] and the virulence of the different strains has to be proven in the future. The survival of potentially resistant trees is endangered not only by the infections with new evolving virulent strains of
H. fraxineus itself, but also by secondary pathogens, like the ash bark beetles or other species of
Armillaria that can be increasingly found in weakened ash stands as it was proven in this study. There was some concern that the abundance of ash bark beetles increases drastically and that they could become primary pest agents [
11,
14]. Therefore, the role of ash bark beetles was examined in four study sites to prove if they act as primary or secondary pathogens in weakened ash stands. It could be shown that ash bark beetles are not playing a major role in the decline process at this juncture. Breeding galleries from
H. fraxini can only be found in recently died back trees (
Table 6). These findings are in line with observations made in Denmark, where ash trees that had died during spring were heavily colonized by bark beetles in the same growth season [
7]. The amount of breeding galleries (
Figure 6) as well as ash roses that appear as a consequence of maturation feeding (
Figure S3) was mostly higher on the lower parts of the stem than near the top. Nevertheless, these insects should be carefully observed in the future and, if required, sanitation measures could be considered. Our findings will facilitate the detection of these beetles, because it was shown that they are not only found on the crown but also at lower heights.
A more serious problem occurs through
Armillaria infections. These infections depend on the conditions of the ecosystem [
35] and can cause major disease symptoms on woody plants and thus affect the ecosystem itself in its structure and function. The fungus is often associated with forest decline events and is generally considered as a contributing factor ultimately responsible for tree death following some other predisposing stresses [
20]. Symptoms of the disease can be found on collar rots and were firstly described in Lithuania and Denmark [
18,
21,
36], where ash dieback was found earlier. Recently, also reports from south-western Germany describe the disease symptoms [
19]. Although it was shown that
Armillaria acts as a secondary pathogen in affected ash stands [
7,
18], it is still difficult to distinguish if
Armillaria is the primary or secondary agent responsible for the lesions at the stem base. In France, where still disease-free areas exist,
Armillaria alone was never observed, which suggests a primary role for
H. fraxineus in initial lesion formation [
37]. However, ash trees were also found to belong to the vitality classes 0–1, which means less affected trees show stem necroses (
Figure 4). These findings are in accordance with similar and earlier findings in Lithuania, where also colonization of the fungus on 80% of sound-looking trees was reported in [
18]. This speaks against
H. fraxineus as a primary inducer. Also in study sites in south-west Germany, a spatial dependence of collar rots was shown. This points to
Armillaria as a primary inducer because of the infection path via roots and because it is not spatially independent through airborne ascospores [
19]. In south-eastern Germany, no
H. fraxineus free plots are available and can be examined regarding
Armillaria infection alone. Investigated necrotic lesions of trees in Gaden were positive for both pathogens. Therefore, a higher amount of infected trees have to be tested in the future. Nevertheless, it can be suggested that high amounts of
Armillaria inoculum in the soil, abundantly present through dying trees, facilitate the infection even of healthier trees. We could show that trees with the stem necroses also possess a significantly higher ash dieback intensity, which is in accordance with former results from different countries [
7,
19,
36]. When starting the monitoring in 2009, as the infections became visible, the vast majority of affected ash trees showed a gradual decline from one vitality class to the next class (exemplarily shown for Freising). Three years later, in 2012, necrotic lesions and signs of
Armillaria infection at the stem base became apparent in many study sites. Since then, vitality drops from several trees to more vitality classes have been observed (
Table 5). Therefore, we conclude that
Armillaria spp. contributes severely to the acceleration of ash mortality.
The high mortality in ash stands affected by H. fraxineus and Armillaria leads to economic losses and also compromises the survival of potentially resistant trees, as trees of all vitality classes are affected. For this reason, seeds of potentially resistant trees in severely affected ash stands should be harvested for further breeding and, taking our results into consideration, the planning of reforestation in the ash stands by using the resting shield of ash trees should be assigned more importance. The use of specifically selected alternative tree species must be considered to avoid tree gaps on a large scale. Also for forest practitioners, there must be an increased awareness of the significant risk when trees are falling spontaneously, because of their impaired stability.