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Mycological Progress (2023) 22:52
https://doi.org/10.1007/s11557-023-01897-2
ORIGINAL ARTICLE
Fungi associated withstem collar necroses ofFraxinus excelsior
affected byash dieback
SandraPeters1 · SebastianFuchs2 · SteenBien1 · JohannaBußkamp1 · GittaJuttaLanger1 ·
EwaldJohannesLanger3
Received: 16 January 2023 / Revised: 9 May 2023 / Accepted: 11 May 2023
© The Author(s) 2023
Abstract
In recent decades the vitality and physical stability of European ash trees in Germany have been reduced by European ash
dieback, especially when associated with stem collar necroses and rots. This study was carried out to investigate the com-
position of the fungal communities associated with stem collar necroses. Filamentous fungi were isolated from 58 ash trees
out of nine forest stands in northern, eastern, and central Germany. Obtained isolates were identified to a genus or species
level by means of morphological and molecular analyses. In total 162 morphotypes including endophytic, saprotrophic, and
pathogenic fungi were isolated. For 33 species found no prior reports from Fraxinus excelsior were recognised, including
Cryptostroma corticale and Diplodia sapinea. None of the identified species were found at all studied sites, though Diplo-
dia fraxini was the most common fungus with regard to frequency within all isolates, occurring at seven sample sites. This
species is followed by Hymenoscyphus fraxineus, Armillaria spp., Neonectria punicea, Diaporthe cf. eres, Fusarium cf.
lateritium, and Paracucurbitaria sp. in order of frequency within all isolates. The aforementioned species are characterised
and analysed in respect to their occurrence in stem collar necroses and at sample sites. The influence of site conditions on
the fungal composition was described for five intensively sampled sites with a minimum of five studied trees (Schwansee,
Rhüden, Berggießhübel, Satrup, and Schlangen). The sampling site of Schlangen was further subdivided into four subplots
with different positions in the terrain. In the remaining four extensive sample sites, either one or two trees, respectively,
were sampled and analysed (Oranienbaumer Heide, Woltershausen, Wolfenbüttel, and Neuhege). Over all sample sites,
fungal communities of symptomatic stem tissue are similar concerning the most frequent fungi, but vary greatly according
to singularly isolated fungi.
Keywords Fraxinus excelsior· Endophytes· Fungal communities· Ash dieback· Stem collar necroses
Introduction
Since the early 1990s, the European ash (Fraxinus excelsior
L.) is threatened by European ash dieback, caused by Hyme-
noscyphus fraxineus (T. Kowalski) Baral, Queloz, & Hosoya
(Helotiaceae, Ascomycota). First disease reports came from
Poland and Lithuania (Przybyl 2002; Lygis etal. 2005). In
Germany, the severe disease was observed since 2002 and
the causal agent was first proven in the year 2006 (Hey-
deck etal. 2005; Schumacher etal. 2007). Meanwhile, this
invasive fungal pathogen has become widespread in Europe.
Affected European ash (hereafter referred to as ash) trees
of all ages show a broad range of symptoms, such as leaf
necrosis, wilting, shoot blight, inner bark discolorations,
sunken cankers, epicormic shoots, as well as stem collar, and
root necrosis (Gross etal. 2014; Langer 2017). Reduction of
Section Editor: Claus Baessler
* Sandra Peters
Sandra.Peters@nw-fva.de
1 Department ofForest Protection, Northwest German Forest
Research Institute (NW-FVA), D-37079Göttingen, Germany
2 Department ofEnvironmental Control, Northwest German
Forest Research Institute (NW-FVA), D-37079Göttingen,
Germany
3 Fachbereich 10 Naturwissenschaften, Institut für Biologie,
Fachgebiet Ökologie, Universität Kassel, D-34127Kassel,
Germany
Mycological Progress (2023) 22:52
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tree stability and increase of mortality of ash is often con-
nected to stem collar necrosis, which progresses towards
xylem and heartwood. Stem collar necrosis can occur on
trees with or without crown symptoms of ash dieback, but
is often observed on diseased trees (Schumacher etal. 2009;
Husson etal. 2012; Enderle etal. 2017; Langer 2017; Meyn
etal. 2019). Stem collar necroses are defined as basal lesions
with necrotic tissue on the outside and the inside of the stem
mainly caused by fungi (Langer 2017). The actual shape of
stem collar necroses depends on different factors, such as
individual progress or associated fungi. Advanced necroses
are often associated with wood rot caused by fungi colonis-
ing the stem following the initial infection by H. fraxineus
(Langer 2017).
Even though many fungi are reported from F. excelsior
(981 different species according to the USDA website,
Farr and Rossman 2022, retrieved on 17.06.2022) fungi
associated with necrotic stem tissue of ash, specifically in
Germany, have rarely been described (Enderle etal. 2017;
Langer 2017; Meyn etal. 2019). Most frequently isolated
from the necroses at the collar base of diseased ash trees
were Armillaria spp., Diaporthe eres Nitschke, Diplodia
spp., Fusarium avenaceum (Fr.) Sacc., F. lateritium Nees,
Fusarium solani (Mart.) Sacc. (syn. Neocosmospora solani
(Mart.) L. Lombard & Crous), H. fraxineus, and Neonectria
punicea (J. C. Schmidt) Castl. & Rossman (Lygis etal. 2005;
Langer 2017; Meyn etal. 2019; Linaldeddu etal. 2020).
A strong evidence for H. fraxineus being the causal agent
of ash dieback was given by Chandelier etal. (2016), who
proved occurrence of H. fraxineus in the majority of sympto-
matic tissue of ash stem collar. Langer (2017) confirmed fre-
quent isolation from stem collar necroses and the assignment
as primary agent. But not every type of stem collar necrosis
must be primarily caused by H. fraxineus. Langer (2017)
showed that basal necroses can be caused by Phytophthora
under special site conditions, as found in floodplain forests
or by Armillaria spp. on weakened ash trees. The path of
infection by H. fraxineus still remains unknown and little
is understood about the influence of environmental factors
on stem collar necroses. Site characteristics, such as mois-
ture content, are assumed to affect disease severity. Kenigs-
valde etal. (2010) and Marçais etal. (2016) determined
that disease severity correlates positively with soil humidity
conditions. It has been suggested that stem collar necroses
development and extent is also related to moist conditions or
humid topographical positions (Marçais etal. 2016). There-
fore the design of this study covers a wide range of water
supply types at sampling sites. The composition of the forest
stands combined with their nutrient and water availability
could be a factor in assessing differences in fungal diversity
per stand. Most trees moderately and severely damaged due
to ash dieback were observed at forest sites with a high soil
organic matter content and a neutral to slightly alkaline soil
pH (Turczański etal. 2019). Hence, it can be suspected that
fungal composition depends on soil and water availability
just as well (Linaldeddu etal. 2011; Salamon etal. 2020).
This study is conducted as part of the demonstration pro-
ject FraxForFuture and the sub-network FraxPath (Langer
etal. 2022). The aims of this research are to fill knowledge
gaps concerning the α-diversity of cultivable Dikarya Hib-
bett, T. Y. James & Vilgalys associated with stem collar
necroses of trees affected by ash dieback and the composi-
tion of their fungal communities. Therefore, fungi associated
with necrotic stem bases of ash were isolated and identi-
fied from 58 ash trees in order to determine the continuity
and the frequency of H. fraxineus and secondary fungi. The
role of the most frequent fungi in the process of stem collar
necroses formation is discussed.
Materials andmethods
Sampling sites
In total, six federal states of Germany (Lower Saxony, North
Rhine-Westphalia, Saxony, Saxony-Anhalt, Schleswig-
Holstein, and Thuringia) were investigated. Nine mixed
broad-leaved forest stands with a substantial share of F.
excelsior affected by ash dieback were selected in order to
cover different sites with a wide range of soil water supply
types (Table1 and Fig.1). The sample sites are located in
northern, eastern, and central Germany with sub-oceanic to
sub-continental temperate zones. All sites are eutrophic and
cover the most common substrates of ash stands in Germany.
Basic soil and geological data were acquired by using geo-
logical maps with a high resolution (scale 1:25000) of the
respective federal geology departments and the forest inven-
tory and forest site mapping data sets of the federal forestry
authorities. Additional data from soil core sampling, soil
profiles, and soil analyses were available for the sampling
sites of Rhüden, Berggießhübel, Schwansee, and Schlangen
because these sites are part of other studies associated with
soil inventories: The site of Rhüden is part of the national
forest soil inventory. Schwansee and Berggießhübel corre-
spond to the intensive monitoring plots “TH_1 Schwansee”
and “SN_2 Bienhof” of the research cluster FraxForFuture
(Langer etal. 2022). The largest forest stand continuously
including ash trees investigated in this study (Schlangen)
has a pronounced relief and was divided into four subplots
to investigate different positions in the terrain. In this case,
intensive soil exploration was conducted to differentiate
between the four subplots, including pedological assess-
ments and soil sampling from soil profiles.
The shallow sites (Rhüden, Schlangen 2) in exposed
terrain positions on limestone with high coarse soil frac-
tions and low water storage capacities represent the driest
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Table 1 Sampling site information sorted by sampling date; all forest sites are eutrophic
Forest site (coordinates
UTM) Sampling classifica-
tion (sample number;
sample date)
Metres
above sea
level
Exposition and inclina-
tion Climate Soil water supply Soil and bedrock Mixture of tree species
in stand
Oranienbaumer Heide
(33 U 318931
5730457)
Extensive sampling (1;
19.10.2020) 90 Flat Sub-continental Slightly moist, fluctuat-
ing ground water
regime
Diluvial sands, boulder
clay Quercus robur, Fagus
sylvatica, Carpi-
nus betulus, Betula
pendula, Fraxinus
excelsior
Woltershausen (32 U
564973 5757894) Extensive sampling
(2; 20.10.2020 and
04.03.2021)
289 SSE, moderate inclina-
tion (9–18°) Weakly sub-atlantic Slightly moist Clayey loam over creta-
ceous limestone Fagus sylvatica, Acer
pseudoplatanus,
Fraxinus excelsior
Wolfenbüttel (32 U
632092 5778288) Extensive sampling
(1; 27.10.2020) 238 SE, slight inclination
(0–9°) Weakly sub-atlantic Moist, high water hold-
ing capacity Colluvial deposit over
upper Muschelkalk
(triassic limestone)
Fagus sylvatica, Acer
pseudoplatanus,
Fraxinus excelsior
Schwansee (32 U
646432 5660792) Intensive sampling
(8; 10.11.2020) 163 Flat Weakly sub-continental Gleysol, ground water
influenced Carbonatic lake gravel
deposit, lake sediment Fraxinus excelsior,
Quercus robur,
Populus nigra,
European alder
Rhüden (32 U 579914
5757111) Intensive sampling
(10; 22.02.2021) 235 WSW, strong inclina-
tion (18–27°) Weakly sub-atlantic Slightly dry, well
drained, low water
holding capacity
Lower Muschelkalk,
(triassic limestone)
with shallow loess
cover
Fraxinus excelsior,
Fagus sylvatica, Acer
pseudoplatanus,
Prunus avium
Neuhege (32 U 593054
6010661) Extensive sampling
(1; 08.06.2021) 53 ESE, slight inclination
(0–9°) Moderately sub-
atlantic Stagnosol/gleysol,
ground water influ-
enced
Marly till, boulder clay Quercus robur, Alnus
glutinosa, Fraxinus
excelsior, Fagus
sylvatica
Berggießhübel (33 U
426320 5629803) Intensive sampling
(11; 01.07.2021) 475 NNW, slight inclination
(0–9°) Sub-continental, sub-
montane Slightly moist, slope
water influenced,
slightly stagnic
Basalt and gneiss
solifluction soil with
loamy loess cover
Betula pendula, Acer
pseudoplatanus,
Fraxinus excelsior,
Tilia cordata, Quercus
robur, Prunus avium,
Sorbus aucuparia
Satrup (32 U 546373
6072699) Intensive sampling
(5; 06.07.2021) 49 ESE, slight inclination
(0–9°) Moderately sub-
atlantic Slightly moist, high
water holding capac-
ity, slightly stagnic
Marly till, boulder clay Fagus sylvatica,
Fraxinus excelsior
Schlangen 1 (32 U
492453 5740809) Intensive sampling
(3; 01.11.2021) 274 valley bottom, NNW,
slight inclination
(0–9°)
Moderately sub-
atlantic Moist, high water hold-
ing capacity, slightly
stagnic
Colluvial deposit, silty
loam Fagus sylvatica,
Fraxinus excelsior,
Acer pseudoplatanus,
Prunus avium
Schlangen 2 (32 U
492621 5741084) Intensive sampling
(6; 08.11.2021) 299 upper slope, S, strong
inclination (18–27°) Moderately sub-
atlantic Slightly dry, low water
holding capacity Cretaceous limestone,
very shallow loess
cover (clayey loam)
Fagus sylvatica,
Fraxinus excelsior,
Acer pseudoplatanus,
Prunus avium
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end of the ecological niche of ash. Several other sites have
moderate (Schlangen 3, Schlangen 4, Wolterhausen) or
high water storage capacities (Wolfenbüttel, Schlangen 1)
because of medium to deep loamy loess covers or colluvial
deposits. The remaining sites are primarily characterised
by either stagnic soil conditions (Satrup, Berggießhübel),
slope water influence (Berggießhübel), or groundwater
influence (Oranienbaumer Heide, Schwansee, Neuhege).
Soil substrate, water retention capacity, terrain relief, cli-
mate, and presence/absence of groundwater or stagnic soil
properties (Table1) were combined to create a ranking of
site water supplies of the sampling sites (Online Resource
1 and Online Resource 2).
Between one and nineteen trees were excavated and
sampled per site. Hence, the sampling sites were divided
into intensive and extensive sampling sites. The intensive
sampling sites had a minimum of five and a maximum
of 19 sample trees. Intensive sampling was conducted
in Rhüden (10 sampled trees), Satrup (5), Berggießhü-
bel (11), Schwansee (8), and Schlangen (19). Each inten-
sive sampling site or subplot was 0.2–0.5 ha in size. The
extensive sampling sites with only one or two examined
ash trees were taken into account only for fungal occur-
rence: Woltershausen (2 sampled trees), Wolfenbüttel (1),
Neuhege (1), and Oranienbaumer Heide (1). These indi-
vidual trees were included to increase the sample set and
the distribution of investigated stem collar necroses and
their associated fungi.
Sampled trees
In total 58 ash trees were sampled, including six trees ini-
tially selected as control trees (two in Berggießhübel, one
in Satrup, and three in Schlangen; Online Resource 3). The
diameter at breast height of the sampled ash trees ranged
from approximately 7–25 cm. The age of the sample trees
ranged from 15 up to 80 years. The majority was approxi-
mately 40 years old. Classification of stem base and crown
condition of the studied trees was carried out according to
the guidelines of Peters etal. (2021a, b). Additionally, the
neighbouring tree species occurring in the studied stands
were noted (Table1).
Ash trees were felled in the years 2020–2021 and cut at
least 15 cm above the visible necrotic area. Subsequently,
trunk bases and the uppermost parts of the main roots were
dug out with picks and shovels. Depending on soil struc-
ture (rock content) final roots were cut by chainsaw with
a .325-in. Rapid Duro 3 (RD3), 1.6-mm chainsaw chain
(STIHL AG & Co. KG, Dieburg, Germany). The stem col-
lars were transported to laboratory in clean and marked
plastic bags.
Table 1 (continued)
Forest site (coordinates
UTM) Sampling classifica-
tion (sample number;
sample date)
Metres
above sea
level
Exposition and inclina-
tion Climate Soil water supply Soil and bedrock Mixture of tree species
in stand
Schlangen 3
(32 U 492515 5740880) Intensive sampling
(4; 01.11.2021) 305 upper slope, NNW,
slight inclination
(0–9°)
Moderately sub-
atlantic Slightly moist, moder-
ate water holding
capacity
Cretaceous limestone,
moderate loess cover
(clayey loam)
Fraxinus excelsior,
Fagus sylvatica, Acer
pseudoplatanus,
Prunus avium
Schlangen 4 (32 U
492738 5741014) intensive sampling
(6; 01., 02.,
08.11.2021)
316 hill top, W, slight incli-
nation (0–9°) Moderately sub-
atlantic Slightly moist, moder-
ate water holding
capacity
Cretaceous limestone,
moderate loess cover
(clayey loam)
Fagus sylvatica, Acer
pseudoplatanus,
Fraxinus excelsior,
Prunus avium
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Isolation offungi
In preparation for fungal isolation, tree stems were cleaned
with a coarse brush under tap water. After air-drying over-
night, the samples were processed in the laboratory. Each
sample was photographed for documentation. Bark was
removed from symptomatic stem areas at the transition
zones of living and dead woody tissue. Depending on the
thickness of the bark either a sterilised knife or a scalpel
was used for removal of the bark. The exposed tissue was
sprayed with 70% ethanol all over the necrosis. For chipping
of wood tissue samples a chisel and hammer were used. All
tools were sterilised by flame shortly before each use. The
top tissue layer of the sample was discarded and only the
sterile layer directly underneath was incubated. The number
of wood chips taken from each tree varied depending on the
size of the necrosis. Three of the 5–10-mm-long wood chips
were placed in a 90 mm petri dish containing malt yeast
peptone (MYP) agar, modified according to Langer (1994)
containing 0.7% malt extract (Merck, Darmstadt, Germany),
0.05% yeast extract (Fluka, Seelze, Germany), 0.1% peptone
(Merck) and 1.5% agar (Fluka). Once the surface of necroses
was processed, stem collars were cut longitudinally with a
band saw and carefully sanded for better visualisation of
the discolorations. The longitudinal sections were treated
according to the isolation method for the surface of necrosis
described above. Wood chips were taken at the edge of the
necroses and at the transition areas of different discolora-
tions. The process was repeated with the cross section of
the stem (Fig.2).
The petri dishes containing wood chips were incu-
bated at room temperature under ambient daylight for
four weeks. The cultures were checked for isolates once
a week. Emerging mycelia of filamentous fungi were
sub-cultured into pure cultures. The pure cultures were
grouped into morphotypes (MT) based on similarity of
Fig. 1 Sampling sites in Ger-
many divided in intensive (red)
and extensive (yellow) sampling
sites with a detailed view of the
special study site Schlangen and
its feature of splitting in four
subplots with different terrain
positions (resources: QGIS 3.24
© GeoBasis-DE/BKG (2022),
DGM1 © GeoBasis NRW
(2021))
Mycological Progress (2023) 22:52
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colony morphology. At least one representative culture
for each MT was stored in MYP slants at 4 °C at the fun-
gal culture collection of the Northwest German Forest
Research Institute (NW-FVA). Beside the MT assignment,
contaminated or overgrown fungi were summarised under
“Fungus sp.”.
The frequency of each isolated fungal MT within all
isolates (fMT) was specified as the percentage of this par-
ticular MT in all isolates. To measure the ratio of mor-
photype isolates to isolation attempts, the frequency of
isolated fungal MT in relation to the total amount of wood
chips (fWC) is used. Continuity of isolated MT is defined as
the number of sampled trees where the MT was detected
in relation to the total number of sampled trees. Analyses
of the fungal diversity found in this study were conducted
using RStudio (v. 4.1.2, R Core Team 2021). The pack-
ages used were tidyverse (Wickham etal. 2019), ggplot 2
(Wickham 2016), and ggVennDiagram (Gao 2021).
The dependency of fWC of the most commonly isolated
fungal MT on site water supply ranks of the intensive sam-
pling sites was tested by using Dirichlet regression (Maier
2014) and the R add-on package DirichletReg (Maier
2021). A non-parametric test is necessary because site
water supply is an ordinal variable. The ranks are based
on a combination of soil water retention, soil stagnic prop-
erties, relief, topography, and climate characteristics of
the study sites and correspond to the site descriptions in
Table1 and Online Resource 1.
Molecular analysis
For molecular analysis, at least one representative strain
from each MT was chosen. Mycelium was placed in 1.5-ml
Eppendorf tubes with three glass beads (3 mm) and 150 μl
of TE buffer (10 ml 1 mmol Tris HCl (pH 0.8), 2 ml 0.5
mmol EDTA; Carl Roth, Karlsruhe, Germany), and crushed
in a Mixer Mill MM 200 (Retsch, Haan, Germany) with
25 vibrations per second for 90 s. Subsequently, genomic
DNA was extracted following the protocol of Izumitsu etal.
(2012).
The 5.8S nuclear ribosomal gene with the two flanking
internal transcribed spacers ITS-1 and ITS-2 (ITS region)
was amplified for all strains using the primer pair ITS-1F
(Gardes and Bruns 1993) and ITS-4 (White etal. 1990).
Additionally, for a selection of strains belonging to Armil-
laria, a partial sequence of the translation elongation factor
1α (EF-1α) was amplified using the primer pair EF595F +
EF1160R (Kauserud and Schumacher 2001). The PCR mix-
ture consisted of 1 μl of DNA and 19 μl mastermix which
contained 2.5 μl 10× PCR reaction buffer (with 20 mM
MgCl2, Carl Roth, Karlsruhe, Germany), 1 μl of each primer
(10 mmol), 2.5 μl MgCl2 (25 mmol), 0.1 μl Roti®-Pol Taq
HY Taq polymerase (Carl Roth, Karlsruhe, Germany) and
2.5 μl of 2 mmol dNTPs (Biozym Scientific GmbH, Hes-
sisch Oldendorf, Germany). Each reaction was topped up
to a volume of 20 μl by adding sterile water. A StepO-
nePlus™ PCR System (Applied Biosystems, Waltham,
Fig. 2 Fungi associated with the stem collar necrosis of ash tree num-
ber 53 (Online resource 3) from the sampling site Schlangen 2. Iso-
lation loci are numbered. a Sampled stem base from the outside, b
longitudinal-section of stem base with visible wood discoloration, c
basal cross-section of stem base, and d cross-section of the stem base
above ground level every 10–15 cm
Mycological Progress (2023) 22:52
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Massachusetts, US) was used to carry out the DNA ampli-
fications. The PCR conditions for the amplification of the
ITS and EF-1α regions were set according to Bien etal.
(2020) and Guo etal. (2016), respectively. A 1% agarose
gel was used to visualise the PCR products. The products
were sent to Eurofins Scientific Laboratory (Ebersberg,
Germany) for sequencing. Initially, PCR samples of the ITS
region were sequenced using the forward reaction (primer
ITS-1F). In case of imprecise results, additionally, reverse
reactions (primer ITS-4) were sequenced. PCR products of
the EF-1α sequence region were sequenced by the respec-
tive forward and reverse reactions. All resulting sequences
were visually checked and edited as follows using BioEdit
Sequence Alignment Editor (v. 7.2.5; Hall 1999). Consensus
sequences were generated, for all strains with forward and
reverse sequences available. Defective sequence beginnings
and ends were trimmed and erroneous nucleotide allocations
corrected. Sequences were submitted to GenBank (Table2).
Identification offungi
Only cultivatable Dikarya fungi were investigated, however,
yeasts were not taken into account. Only isolates which are
clearly growing from the wood chips were determined.
Obvious contamination with filamentous fungi was not
considered. The genus Trichoderma was not included in the
analyses, because it is difficult to assess whether these very
fast-growing fungi were contaminations or real outgrowth
from the wood.
MT were assigned to fungal taxa based on morphological
observation and molecular analysis of representative strains
following the method of Guo etal. (2000). For fungal taxon
determination blastn searches based on ITS sequences were
conducted on the GenBank database (http:// www. ncbi. nlm.
nih. gov/ genba nk, Altschul etal. 1997) excluding uncultured/
environmental sample sequences from the search. Results
were critically interpreted with emphasis on well-curated
culture collections, such as the Westerdijk Fungal Biodiver-
sity Collection (CBS). In general, blastn results on a species
level below a threshold of 98% identity were not trusted to
be accurate enough for final determination. In case no defi-
nite affiliation to a specific taxonomic level was possible,
for example because of more than one hit with a thresh-
old of over 98% identity, the identification was marked by
cf. (confer) to indicate uncertainties. The results were re-
checked against literature and previously identified cultures
from the institute’s collection for confirmation. In addition
to blastn searches, extended analyses for taxon determination
on a species level were conducted for isolates belonging to
Diplodia and Armillaria due to the considerable number
of isolates from these genera. Phylogenetic analyses were
conducted based on an ITS sequence-dataset and an ITS-
EF-1α concatenated sequence-dataset for Diplodia and
Armillaria isolates, respectively, including appropriate refer-
ence sequences retrieved from GenBank. Both analyses were
performed using RAxML v. 8.2.11 (Stamatakis 2006, 2014)
as implemented in Geneious R11 (Kearse etal. 2012) using
the GTRGAMMA model with the rapid bootstrapping and
search for best scoring ML tree algorithm including 1000
bootstrap replicates (Online Resource 4 and 5).
As a rule, current names were applied according to the
nomenclatorial database MycoBank (Robert etal. 2005).
Two exceptions from this generally applied rule have been
made in the case of the MT designated here as Fusarium
solani s. l. and Armillaria gallica. In the case of F. solani
the authors are aware that there is a currently unsettled dis-
cussion about correct delimitation of this species, or rather
species complex. Here we follow the “classic” nomenclature
substantiated by Geiser etal. (2021) obverse that promoted
by Lombard etal. (2015) and Sandoval-Denis etal. (2019)
who place said species complex in the genus Neocosmos-
pora (N. solani (Mart.) L. Lombard & Crous). The currently
applied name for A. gallica, according to MycoBank is A.
lutea Gillet. However, Marxmüller (1992) stated that A.
lutea is a nomen ambiguum and the later introduced name
A. gallica Marxm. & Romagn. is to be used (Burdsall and
Volk 1993).
Results
Sampled trees
The crown health status regarding ash dieback of the
sampled trees ranged from vital and nearly without die-
back symptoms to almost dead. All sampled trees (Online
Resource 3), except for the six planed control trees, had
obvious stem collar necroses through discoloured, sunken,
and in some cases ruptured bark. In cases of ruptured bark,
subjacent stem collars were neither completely rotting nor
dead. During processing of the sample trees in the labora-
tory two out of the six control trees (trees 32 and 42, Online
Resource 3) showed necrotic woody tissue inside the stem
base. Consequently, these two trees were transferred to and
analysed as sample trees (symptomatic sample trees n = 54,
asymptomatic control trees n = 4).
Isolated fungi
In total, 4401 wood chips of stem collar tissue originating
from 58 trees were incubated. A total of 1511 isolates (from
which 226 were not identifiable due to contaminations;
marked as Fungus sp.) from 1413 wood chips (32%) were
observed. 958 chips (22%) showed no outgrowth at all after
four weeks of incubation, while 960 (22%) chips had been
overgrown by fast-growing fungi from adjacent wood chips
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52 Page 8 of 29
Table 2 List of isolated fungi sorted alphabetically within orders; column “Species” is the name finally determined by the authors; fungi causing wood rot are marked with *; probable first
reports of species isolated from F. excelsior are marked with FR; Phylum: A = Ascomycota. B = Basidiomycota. Datum Blast: 22./23.06.2022
Species Phylum Order NW-FVA ID ITS Accession
no. n isolates Frequency (%) Continuity
(%) Sites
isolated
from
ITS NCBI Blast results
Basis of iden-
tification ID Identity (%) Reference
Acremonium
sp. AHypocreales 8031 OP023272 1 0.07% 2% 1 Acremonium
variecolor KF313108.1 99.63% Min etal. 2014
Akanthomyces
sp.FR AHypocreales 6958 OP023220 1 0.07% 2% 1 Akanthomyces
muscarius MH858126.1 100.00% Vu etal. 2019
Alternaria
infectoria APleosporales 5940 OP023162 3 0.20% 5% 2 Alternaria
infectoria MT561399.1 100.00% Langer and
Bußkamp
(unpublished)
Alternaria sp. A Pleosporales 6119 OP023191 5 0.33% 5% 2 Alternaria
angustio-
voidea
MH861939.1 100.00% Vu etal. 2019
Angustimassa-
rina sp. 1 APleosporales 5951 OP023165 2 0.13% 3% 1 Angustimas-
sarina
lonicerae
KY496759.1 100.00% Tibpromma
etal. 2017
Angustimassa-
rina sp. 2 APleosporales 6207 OP023203 4 0.26% 3% 2 Angustimas-
sarina
lonicerae
KY496759.1 98.61% Tibpromma
etal. 2017
Armillaria
spp.* BAgaricales 5952 OP023166 158 10.46% 50% 5 Armillaria
gallica KX618575.1 99.74% Denman etal.
2017
Armillaria
cepistipes MK966557.1 99.62% Wei (unpub-
lished)
Ascocoryne
sp. 1* AHelotiales 7103 OP023244 3 0.20% 3% 2 Ascocoryne
sp. MH682237.1 100.00% Johnston and
Park (unpub-
lished)
Ascocoryne
sp. 2* AHelotiales 8030 OP023271 1 0.07% 2% 1 Ascocoryne
solitaria HM152545.1 100.00% Griffin etal.
2010
Aureobasidium
pullulans ADothideales 6176 OP023196 5 0.33% 7% 3 Aureoba-
sidium pullu-
lans
KT693733.1 99.82% van Nieuwen-
huijzen et. al
(unpublished)
Beauveria
bassiana AHypocreales 6979 OP023247 2 0.13% 2% 1 Beauveria
bassiana MN122432.1 100.00% Gasmi et. al
(unpublished)
Beauveria
pseudobassi-
ana
AHypocreales 6001 OP023182 1 0.07% 2% 1 Beauveria
pseudobassi-
ana
NR_111598.1 100.00% Schoch etal.
2014
Biscogniauxia
nummularia*AXylariales 5926 OP023156 3 0.20% 5% 1 Biscogniauxia
nummularia NR_153649.1 100.00% Wendt etal.
2018
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Page 9 of 29 52
Table 2 (continued)
Species Phylum Order NW-FVA ID ITS Accession
no. n isolates Frequency (%) Continuity
(%) Sites
isolated
from
ITS NCBI Blast results
Basis of iden-
tification ID Identity (%) Reference
Bispora sp.* A Incertae sedis 8329 OP023300 1 0.07% 2% 1 Bispora anten-
nata KY462800.1 99.03% Baral and Mar-
son (unpub-
lished)
Bjerkandera
adusta*BPolyporales 5943 OP023163 5 0.33% 9% 3 Bjerkandera
adusta MK322310.1 100.00% Chilean Collec-
tion of Micro-
bial Genetic
Resources
(unpublished)
Botrytis cf.
cinerea AHelotiales 6874 OP023212 6 0.40% 9% 3 Botrytis
cinerea MH860108.1 100.00% Vu etal. 2019
Cadophora
dextrinos-
pora FR
AHelotiales 7026 OP023226 11 0.73% 10% 3 Cadophora
dextrinos-
pora
NR_119489.1 100.00% Schoch etal.
2014
Cadophora
melinii AHelotiales 6985 OP023248 1 0.07% 2% 1 Cadophora
melinii MH791342.1 99.02% Alborés 2018
Cadophora
ramosa FR AHelotiales 6194 OP023202 8 0.53% 7% 4 Cadophora
ramosa MN232956.1 100.00% Bien and Damm
2020a
Cadophora
sp. 1 AHelotiales 7100 OP023258 3 0.20% 3% 2 Cadophora
malorum MT561395.1 100.00% Langer and
Bußkamp
(unpublished)
Cadophora
sp. 2 AHelotiales 8478 OP023311 1 0.07% 2% 1 Cadophora
malorum MT561395.1 100.00% Langer and
Bußkamp
(unpublished)
Calycina her-
barum FR AHelotiales 8287 OP023287 1 0.07% 2% 1 Calycina
herbarum MZ159660.1 99.42% Gaya et. al
(unpublished)
Campo-
sporium sp. A Incertae sedis 8336 OP023307 1 0.07% 2% 1 Campo-
sporium
ramosum
MH866030.1 96.17% Vu etal. 2019
Cepha-
lotrichiella
penicillata FR
AMicroascales 7042 OP023232 1 0.07% 2% 1 Cepha-
lotrichiella
penicillata
NR_153893.1 99.82% Crous etal.
2014
Ceratobasidi-
aceae sp. 1 BCantharellales 8003 OP023265 2 0.13% 3% 1 Ceratoba-
sidium sp. KX786242.1 99.84% Lakshman etal.
2017
Ceratobasidi-
aceae sp. 2 BCantharellales 8005 OP023267 1 0.07% 2% 1 Ceratobasidi-
aceae sp. KX610454.1 98.26% Yokoya etal.
2017
Chaetomi-
aceae sp. ASordariales 8332 OP023303 1 0.07% 2% 1 Chaetomium
sp. MK182798.1 99.81% Li and Xu
(unpublished)
Chaetomium
globosum ASordariales 8326 OP023297 1 0.07% 2% 1 Chaetomium
globosum MH858130.1 99.81% Vu etal. 2019
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52 Page 10 of 29
Table 2 (continued)
Species Phylum Order NW-FVA ID ITS Accession
no. n isolates Frequency (%) Continuity
(%) Sites
isolated
from
ITS NCBI Blast results
Basis of iden-
tification ID Identity (%) Reference
Chaetomium
sp. ASordariales 8323 OP023295 1 0.07% 2% 1 Chaetomium
subaffine MH862288.1 100.00% Vu etal. 2019
Chloridium
virescens var.
caudigerum
AChaetospha-
eriales 8290 OP023290 6 0.40% 7% 1 Chloridium
virescens
var. caudi-
gerum
MH857142.1 99.81% Vu etal. 2019
Cladosporium
sp. ACapnodiales 7029 OP023229 2 0.13% 3% 1 Cladosporium
iridis EU167591.1 100.00% Simon etal.
2009
Clonostachys
sp. AHypocreales 5985 OP023174 1 0.07% 2% 1 Clonostachys
rosea f.
catenulata
NR_165993.1 99.25% Vu etal. 2019
Coniochaeta
velutina FR AConi-
ochaetales 8334 OP023305 1 0.07% 2% 1 Coniochaeta
velutina MK656234.1 99.81% van der Merwe
et. al (unpub-
lished)
Coprinellus cf.
domesticus*BAgaricales 6011 OP023184 1 0.07% 2% 1 Coprinellus
domesticus KP132301.1 100.00% Irinyi etal. 2015
Coprinellus cf.
radians 1* BAgaricales 6962 OP023221 1 0.07% 2% 1 Coprinellus
radians JN943117.1 100.00% Nagy (unpub-
lished)
Coprinellus cf.
radians 2* BAgaricales 8590 OP023312 1 0.07% 2% 1 Coprinellus
radians JN943117.1 100.00% Nagy (unpub-
lished)
Coprinellus
dissemi-
nates*
BAgaricales 5991 OP023177 12 0.79% 10% 2 Coprinellus
disseminatus KY977599.1 99.85% Kranjec (unpub-
lished)
Coprinellus
micaceus*BAgaricales 6048 OP023189 22 1.46% 19% 3 Coprinellus
micaceus KJ713992.1 100.00% Jang etal. 2015
Cordycipita-
ceae sp. AHypocreales 6965 OP023223 1 0.07% 2% 1 Samsoniella
hepiali NR_160318.1 100.00% Wang etal.
2015
Cosmospora
sp. 1 AHypocreales 7044 OP023234 1 0.07% 2% 1 Cosmospora
sp. MF495375.1 98.11% Mejia et. al
(unpublished)
Cosmospora
sp. 2 AHypocreales 7095 OP023262 3 0.20% 5% 2 Cosmospora
lavitskiae KU563624.1 100.00% Zeng and
Zhuang 2016
Cryptostroma
corticale* FR AXylariales 5932 OP023158 3 0.20% 5% 2 Cryptostroma
corticale MH857008.1 100.00% Vu etal. 2019
Cyclothyriella
rubronotata FR APleosporales 8333 OP023304 1 0.07% 2% 1 Cyclothyriella
rubronotata NR_147651.1 100.00% Jaklitsch and
Voglmayr
2016
Diaporthe cf.
eres ADiaporthales 5924 OP023155 58 3.84% 43% 7 Diaporthe eres MK024685.1 100.00% Hosseini etal.
2021
Mycological Progress (2023) 22:52
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Page 11 of 29 52
Table 2 (continued)
Species Phylum Order NW-FVA ID ITS Accession
no. n isolates Frequency (%) Continuity
(%) Sites
isolated
from
ITS NCBI Blast results
Basis of iden-
tification ID Identity (%) Reference
Diaporthe cf.
rudis ADiaporthales 6131 OP023194 4 0.26% 7% 1 Diaporthe
rudis KC343234.1 100.00% Gomes etal.
2013
Didymella
sp. 1 APleosporales 5939 OP023161 1 0.07% 2% 1 Didymella
macrostoma MN588154.1 100.00% Patejuk et. al
(unpublished)
Didymella
sp. 2 APleosporales 5982 OP023173 1 0.07% 2% 1 Didymella sp. MW582399.1 99.78% Wang and Pec-
oraro 2021
Didymella
sp. 3 APleosporales 6147 OP023193 1 0.07% 2% 1 Didymella
pinodella MW945409.1 100.00% Zhao etal. 2021
Didymella
sp. 4 APleosporales 6875 OP023213 1 0.07% 2% 1 Didymella
prosopidis NR_137836.1 100.00% Crous etal.
2013
Didymellaceae
sp. APleosporales 8289 OP023289 1 0.07% 2% 1 Didymella sp. MN522472.1 100.00% Rivedal etal.
2020
Diplodia
fraxini ABotryospha-
eriales 5921 OP023153 280 18.49% 71% 7 Diplodia
fraxini MT587349.1 100.00% Zhang etal.
2021
Diplodia
sapinea FR ABotryospha-
eriales 5979 OP023170 1 0.07% 2% 1 Diplodia
sapinea NR_152452.1 100.00% Alves etal.
2006
Geotrichum
candidum FR ASaccharomyc-
etales 6963 OP023222 4 0.26% 3% 1 Geotrichum
candidum MF044044.1 100.00% Rongfeng etal.
2018
Dothiorella sp. A Botryospha-
eriales 5944 OP023164 1 0.07% 2% 1 Dothiorella sp. KF040058.1 100.00% Zlatković etal.
2016
Entoleuca sp. A Xylariales 6018 OP023187 2 0.13% 3% 1 Entoleuca sp. MN538292.1 100.00% Pusz et. al
(unpublished)
Eutypa cf.
petrakii var.
hederae
AXylariales 5994 OP023179 5 0.33% 3% 1 Eutypa
petrakii var.
hederae
MT153641.1 99.82% Bien and Damm
2020b
Eutypa lata* A Xylariales 5937 OP023160 20 1.32% 3% 2 Eutypa lata MK547093.1 99.58% Johnston and
Park (unpub-
lished)
Exophiala sp. A Chaetothyri-
ales 8390 OP023310 4 0.26% 7% 1 Exophiala sp. LC317596.1 92.77% Takahashi
and Yaguchi
(unpublished)
Flammulina
velutipes*BAgaricales 8037 OP023274 4 0.26% 2% 1 Flammulina
velutipes MK934583.1 99.87% Chilean Collec-
tion of Micro-
bial Genetic
Resources
(unpublished)
Fomitopsis
betulina* FR BPolyporales 8324 OP023296 1 0.07% 2% 1 Fomitopsis
betulina MF967582.1 99.68% Yang etal. 2017
Mycological Progress (2023) 22:52
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52 Page 12 of 29
Table 2 (continued)
Species Phylum Order NW-FVA ID ITS Accession
no. n isolates Frequency (%) Continuity
(%) Sites
isolated
from
ITS NCBI Blast results
Basis of iden-
tification ID Identity (%) Reference
Fomitopsis
pinicola*BPolyporales 7048 OP023253 1 0.07% 2% 1 Fomitopsis
pinicola JQ341137.1 100.00% Douanla-Meli
and Langer
2012
Fusariumcf.
lateritium AHypocreales 5919 OP023151 53 3.50% 34% 6 Fusarium
lateritium MN588156.1 100.00% Patejuk et. al
(unpublished)
Fusarium sam-
bucinum AHypocreales 6043 OP023188 1 0.07% 2% 1 Fusarium
sambucinum KM231813.1 100.00% Lombard etal.
2015
Fusarium
solani AHypocreales 6003 OP023183 15 0.99% 12% 2 Fusarium
solani FJ459973.1 100.00% Zhang et. al
(unpublished)
Fusarium sp. 1 A Hypocreales 5920 OP023152 6 0.40% 9% 2 Fusarium
iranicum NR_175069.1 100.00% Sandoval-Denis
(unpublished)
Fusarium sp. 2 A Hypocreales 5933 OP023159 1 0.07% 2% 1 Fusarium
sporotri-
chioides
MT635298.1 100.00% Cudowski
(unpublished)
Fusarium
stercicola AHypocreales 7072 OP023275 1 0.07% 2% 1 Fusarium
stercicola MG250476.1 100.00% Šišić etal. 2018
Geotrichum
sp. ASaccharomyc-
etales 6955 OP023237 2 0.13% 3% 2 Geotrichum
sp. AY787702.2 99.10% Lygis etal. 2005
Gliomastix sp. A Hypocreales 6957 OP023219 2 0.13% 3% 1 Gliomastix
murorum
var. felina
MH864097.1 100.00% Vu etal. 2019
Graphium sp. A Microascales 8007 OP023269 1 0.07% 2% 1 Graphium sp. MF782695.1 98.49% Jankowiak etal.
2019a
Heteroba-
sidion
annosum*
BRussulales 6111 OP023195 2 0.13% 3% 1 Heterobasid-
ion annosum MH859050.1 99.66% Vu etal. 2019
Humicolopsis
cephalo-
sporioides FR
AHelotiales 7045 OP023235 1 0.07% 2% 1 Humicolopsis
cephalo-
sporioides
NR_160150.1 99.24% Vu etal. 2019
Hymenos-
cyphus
fraxineus
AHelotiales 5922 OP023154 179 11.85% 53% 8 Hymenos-
cyphus
fraxineus
MT155386.1 100.00% Panteleev et. al
(unpublished)
Hypholomacf.
acutum*BAgaricales 6345 OP023209 2 0.12% 3% 2 Hypholoma
fasciculare MT573401.1 100.00% Alimadadi 2019
Hypocreales
sp. AHypocreales 6930 OP023217 1 0.07% 2% 1 Albifimbria
verrucaria KF887115.1 99.81% Sun etal. 2014
Hypoxylon
fragiforme*AXylariales 6113 OP023192 4 0.26% 7% 2 Hypoxylon
fragiforme MH855287.1 100.00% Vu etal. 2019
Mycological Progress (2023) 22:52
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Page 13 of 29 52
Table 2 (continued)
Species Phylum Order NW-FVA ID ITS Accession
no. n isolates Frequency (%) Continuity
(%) Sites
isolated
from
ITS NCBI Blast results
Basis of iden-
tification ID Identity (%) Reference
Hypoxylon
howeanum*AXylariales 7046 OP023251 1 0.07% 2% 1 Hypoxylon
howeanum MN588210.1 100.00% Patejuk et. al
(unpublished)
Hypoxylon
rubigino-
sum*
AXylariales 6051 OP023190 6 0.40% 7% 3 Hypoxylon
rubiginosum MZ045853.1 100.00% Langer and
Bußkamp
2021
Ilyonectria
sp. 1 AHypocreales 7051 OP023252 4 0.26% 7% 2 Ilyonectria
lusitanica NR_156240.1 99.80% Cabral etal.
2012
Ilyonectria
sp. 2 AHypocreales 7068 OP023255 1 0.07% 2% 1 Ilyonectria
liliigena JF735297.1 99.80% Cabral etal.
2012
Ilyonectria
sp. 3 AHypocreales 8271 OP023281 1 0.07% 2% 1 Ilyonectria
protearum NR_152890.1 99.12% Lombard et. al
(unpublished)
Jackrogersella
cohaerens*AXylariales 8292 OP023292 3 0.20% 5% 1 Jackrogersella
cohaerens MT561400.1 100.00% Langer and
Bußkamp
(unpublished)
Jackrogersella
sp. AXylariales 6192 OP023200 4 0.26% 7% 3 Jackrogersella
sp. MT153658.1 99.80% Bien and Damm
2020b
Juxtiphoma
eupyrena APleosporales 8182 OP023280 2 0.13% 2% 1 Juxtiphoma
eupyrena MG098275.1 100.00% Bußkamp etal.
2020
Kalmusia sp. A Pleosporales 7032 OP023231 1 0.07% 2% 1 Kalmusia
longispora JX496097.1 99.62% Verkley etal.
2014
Kuehneromy-
ces mutabi-
lis*
BAgaricales 5988 OP023176 1 0.07% 2% 1 Kuehneromy-
ces mutabilis MH855190.1 99.55% Vu etal. 2019
Lasionectria
sp. AHypocreales 6191 OP023199 1 0.07% 2% 1 Lasionectria
vulpina MZ159398.1 99.82% Gaya et. al
(unpublished)
Laxitextum
bicolor BRussulales 8006 OP023268 1 0.07% 2% 1 Laxitextum
bicolor MW742677.1 99.15% Ma and Zhao
(unpublished)
Lepteutypa
fuckelii AAmphisphaeri-
ales 8284 OP023284 1 0.07% 2% 1 Lepteutypa
fuckelii MZ045855.1 100.00% Langer and
Bußkamp
2021
Leptodonti-
dium sp. AHelotiales 8338 OP023309 1 0.07% 2% 1 Leptodonti-
dium sp. AB907597.1 96.72% Fukasawa 2018
Leptosillia
muelleri FR AXylariales 6208 OP023204 2 0.13% 3% 2 Leptosillia
muelleri NR_164065.1 98.12% Voglmayr etal.
2019
Lophiotrema
rubi FR APleosporales 8327 OP023298 1 0.07% 2% 1 Lophiotrema
rubi AF383963.1 99.80% Liew etal. 2002
Mycological Progress (2023) 22:52
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52 Page 14 of 29
Table 2 (continued)
Species Phylum Order NW-FVA ID ITS Accession
no. n isolates Frequency (%) Continuity
(%) Sites
isolated
from
ITS NCBI Blast results
Basis of iden-
tification ID Identity (%) Reference
Lophiotrema
sp. APleosporales 8337 OP023308 1 0.07% 2% 1 Lophiotrema
mucilagi-
nosis
NR_164039.1 100.00% Phookamsak
etal. 2019
Metapochonia
bulbillosa FR AHypocreales 6876 OP023214 2 0.13% 3% 2 Metapochonia
bulbillosa NR_154142.1 100.00% Zare etal. 2000
Metapochonia
suchlasporia
FR
AHypocreales 8272 OP023282 1 0.07% 2% 1 Metapochonia
suchlasporia KU668952.1 100.00% Palmer etal.
2018
Microceracf.
larvarum AHypocreales 6195 OP023210 3 0.20% 5% 3 Microcera
larvarum EU860066.1 100.00% Bills etal. 2009
Microcera
rubra FR AHypocreales 7028 OP023228 1 0.07% 2% 1 Microcera
rubra MH861019.1 100.00% Vu etal. 2019
Micro-
sphaeropsis
olivacea
APleosporales 7039 OP023250 1 0.07% 2% 1 Micro-
sphaeropsis
olivacea
MT561396.1 100.00% Langer and
Bußkamp
(unpublished)
Mycoacia
nothofagi* FR BPolyporales 8043 OP023278 1 0.07% 2% 1 Mycoacia
nothofagi GU480000.1 99.83% Moreno etal.
2011
Nectriaceae
sp. 1 AHypocreales 5987 OP023175 3 0.20% 5% 2 Cylindrocar-
pon sp. EF601608.1 99.14% Belbahri et. al
(unpublished)
Nectriaceae
sp. 2 AHypocreales 8286 OP023286 2 0.13% 3% 1 Dialonectria
ullevolea MH855229.1 100.00% Vu etal. 2019
Nemania
serpens*AXylariales 6193 OP023201 6 0.40% 9% 3 Nemania
serpens MT790321.1 100.00% Blumenstein
etal. 2021
Neoascochyta
sp. APleosporales 6968 OP023225 3 0.20% 5% 2 Neoascochyta
exitialis MT446179.1 100.00% Liu (unpub-
lished)
Neobulgaria
sp.* AHelotiales 8331 OP023302 1 0.07% 2% 1 Neobulgaria
sp. MH191244.1 100.00% Meyn etal.
2019
Neocucurbitaria
acerina FR APleosporales 5981 OP023172 1 0.07% 2% 1 Neocucurbi-
taria acerina MF795768.1 99.82% Jaklitsch etal.
2018
Neocucurbi-
taria sp. APleosporales 7031 OP023230 1 0.07% 2% 1 Neocucurbi-
taria cava KR909135.1 100.00% Travadon etal.
2016
Neofabraea sp. A Helotiales 6125 OP023198 8 0.53% 9% 4 Neofabraea
kienholzii MH864120.1 100.00% Vu etal. 2019
Neonectria
punicea AHypocreales 5980 OP023171 123 8.14% 33% 6 Neonectria
punicea MK955355.1 100.00% Wu (unpub-
lished)
Neopyreno-
chaeta
acicola FR
APleosporales 8328 OP023299 1 0.07% 2% 1 Neopyreno-
chaeta
acicola
KJ395501.1 99.61% Panno etal.
2013
Mycological Progress (2023) 22:52
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Page 15 of 29 52
Table 2 (continued)
Species Phylum Order NW-FVA ID ITS Accession
no. n isolates Frequency (%) Continuity
(%) Sites
isolated
from
ITS NCBI Blast results
Basis of iden-
tification ID Identity (%) Reference
Neopyreno-
chaeta sp. APleosporales 8293 OP023293 2 0.13% 2% 1 Neopyreno-
chaeta
fragariae
MT547823.1 100.00% Bilanski et. al
(unpublished)
Neosetophoma
sp. APleosporales 5928 OP023157 1 0.07% 2% 1 Neosetophoma
rosigena NR_157525.1 99.64% Wanasinghe
etal. 2018
Nigrograna
mycophila APleosporales 8330 OP023301 1 0.07% 2% 1 Nigrograna
mycophila NR_147654.1 99.39% Jaklitsch and
Voglmayr
2016
Obolarina dry-
ophila* FR AXylariales 6324 OP023207 1 0.07% 2% 1 Obolarina
dryophila GQ428313.1 100.00% Pažoutová etal.
2010
Oliveonia sp. B Cantharellales 8004 OP023266 1 0.07% 2% 1 Oliveonia sp. MT235640.1 99.07% Spirin et. al
(unpublished)
Paracucurbi-
taria sp. APleosporales 5999 OP023181 31 2.05% 34% 6 Paracucurbi-
taria corni MT547826.1 99.80% Bilanski et. al
(unpublished)
Paraphae-
osphaeria
neglecta
APleosporales 6014 OP023186 5 0.33% 7% 3 Paraphae-
osphaeria
neglecta
NR_155629.1 99.46% Verkley etal.
2014
Penicillium
daleae FR AEurotiales 6931 OP023218 2 0.13% 3% 1 Penicillium
daleae MH862989.1 100.00% Vu etal. 2019
Peniophora cf.
cinerea 1* BRussulales 6180 OP023208 5 0.33% 7% 3 Peniophora
cinerea MK247860.1 100.00% Zhang (unpub-
lished)
Peniophora cf.
cinerea 2* BRussulales 8039 OP023277 1 0.07% 2% 1 Peniophora
cinerea MZ018635.1 100.00% Volobuev and
Shakhova
2022
Peniophora cf.
incarnata*BRussulales 7034 OP023249 1 0.07% 2% 1 Peniophora
incarnata MW740279.1 99.69% Johnston and
Park (unpub-
lished)
Peniophora
laeta* FR BRussulales 8036 OP023273 1 0.07% 2% 1 Peniophora
laeta MH857617.1 99.33% Vu etal. 2019
Peniophora
lycii*BRussulales 5995 OP023180 19 1.26% 10% 3 Peniophora
lycii MH857624.1 99.83% Vu etal. 2019
Peniophora
quercina* FR BRussulales 8044 OP023279 1 0.07% 2% 1 Peniophora
quercina MT156129.1 99.47% Bien and Damm
2020b
Peniophora
rufomargi-
nata* FR
BRussulales 7027 OP023227 1 0.07% 2% 1 Peniophora
rufomargi-
nata
MH857639.1 100.00% Vu etal. 2019
Peniophora
sp. 1* BRussulales 6927 OP023215 1 0.07% 2% 1 Peniophora
lycii MH857624.1 98.82% Vu etal. 2019
Mycological Progress (2023) 22:52
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52 Page 16 of 29
Table 2 (continued)
Species Phylum Order NW-FVA ID ITS Accession
no. n isolates Frequency (%) Continuity
(%) Sites
isolated
from
ITS NCBI Blast results
Basis of iden-
tification ID Identity (%) Reference
Peniophora
sp. 2* BRussulales 7226 OP023263 1 0.07% 2% 1 Peniophora
lycii JX046435.1 99.83% Fedorova et. al
(unpublished)
Peniophora
sp. 3* BRussulales 8038 OP023276 1 0.07% 2% 1 Peniophora
lycii MH857624.1 97.98% Vu etal. 2019
Peziculacf.
sporulosa AHelotiales 7104 OP023168 5 0.33% 3% 1 Pezicula
sporulosa KR859257.1 100.00% Chen etal. 2016
Pezicula cf.
rostrupii AHelotiales 5976 OP023245 1 0.07% 2% 1 Pezicula ros-
trupii MH665639.1 100.00% Marson and
Hermant
(unpublished)
Pezicula sp. 1 A Helotiales 6112 OP023205 1 0.07% 2% 1 Pezicula
radicicola MH862498.1 95.05% Vu etal. 2019
Pezicula sp. 2 A Helotiales 7154 OP023260 4 0.26% 3% 2 Pezicula sp. KY977580.1 96.74% Kranjec (unpub-
lished)
Pezicula sp. 3 A Helotiales 7060 OP023240 1 0.07% 2% 1 Pezicula
ericae NR_155653.1 97.85% Chen etal. 2016
Phaeosphaeria
sp. APleosporales 7321 OP023264 1 0.07% 2% 1 Phae-
osphaeria
glyceriae-
plicatae
MH862724.1 100.00% Vu etal. 2019
Phanerochaete
sordida s.
lat. Gruppe*
BPolyporales 7052 OP023254 1 0.07% 2% 1 Phanerochaete
sordida KU761238.1 100.00% Dufresne etal.
2017
Phialocephala
fortinii FR AHelotiales 7056 OP023238 1 0.07% 2% 1 Phialocephala
fortinii MT276008.1 100.00% Myrholm and
Ramsfield
(unpublished)
Phialocephala
piceae FR AHelotiales 7038 OP023216 8 0.53% 9% 2 Phialocephala
piceae NR_111319.1 100.00% Schoch etal.
2014
Phialocephala
sp. AHelotiales 6952 OP023236 1 0.07% 2% 1 Phialocephala
oblonga MG553996.1 100.00% Haelewaters
etal. 2018
Phlebia
radiata*BPolyporales 6013 OP023185 3 0.20% 5% 2 Phlebia
radiata MT551932.1 100.00% Lodge et. al
(unpublished)
Pleosporales
sp. APleosporales 8335 OP023306 1 0.07% 2% 1 Phoma herba-
rum MG586981.1 99.81% Kirtsideli
(unpublished)
Pseudeuroti-
aceae sp. 1 AThelebolales 6966 OP023224 1 0.07% 2% 1 Geomyces
asperulatus MH861038.1 100.00% Vu etal. 2019
Pseudeuroti-
aceae sp. 2 AThelebolales 7152 OP023246 1 0.07% 2% 1 Pseudogym-
noascus pan-
norum
MH854615.1 100.00% Vu etal. 2019
Mycological Progress (2023) 22:52
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Page 17 of 29 52
Table 2 (continued)
Species Phylum Order NW-FVA ID ITS Accession
no. n isolates Frequency (%) Continuity
(%) Sites
isolated
from
ITS NCBI Blast results
Basis of iden-
tification ID Identity (%) Reference
Pseudogym-
noascus sp. AThelebolales 6432 OP023211 1 0.07% 2% 1 Pseudogym-
noascus
appendicu-
latus
NR_137875.1 100.00% Malloch etal.
2016
Pseudopitho-
myces char-
tarum FR
APleosporales 8294 OP023294 1 0.07% 2% 1 Pseudop-
ithomyces
chartarum
MH860227.1 99.82% Vu etal. 2019
Ramularia sp. A Mycosphaerel-
lales 7043 OP023233 1 0.07% 2% 1 Ramulariaco-
llo-cygni NR_154944.1 100.00% Videira etal.
2016
Rhexocer-
cosporidium
sp.
AHelotiales 7097 OP023257 1 0.07% 2% 1 Rhexocer-
cosporidium
sp.
MN124205.1 99.41% Padamsee
and Burgess
(unpublished)
Rhizoctonia
fusispora BCantharellales 7057 OP023239 1 0.07% 2% 1 Rhizoctonia
fusispora MH857068.1 99.06% Vu etal. 2019
Sarocladium
cf. dejongiae AHypocreales 7061 OP023241 1 0.07% 2% 1 Sarocladium
dejongiae NR_161153.1 99.62% Lombard
(unpublished)
Schizopora
paradoxa*BHymeno-
chaetales 7155 OP023261 1 0.07% 2% 1 Schizopora
paradoxa MH857218.1 100.00% Vu etal. 2019
Sclerostago-
nospora
cycadis
APleosporales 8285 OP023285 1 0.07% 2% 1 Sclerostago-
nospora
cycadis
KR611890.1 99.22% Crous etal.
2015
Scytalidium
album* FR AHelotiales 6322 OP023206 1 0.07% 2% 1 Scytalidium
album NR_160102.1 99.81% Vu etal. 2019
Scytalidium
lignicola*AHelotiales 7062 OP023242 1 0.07% 2% 1 Scytalidium
lignicola GU934579.1 100.00% Bakys et. al
(unpublished)
Sistotrema
oblongispo-
rum* FR
BCantharellales 8008 OP023270 3 0.20% 3% 1 Sistotrema
oblongispo-
rum
KF218970.1 100.00% Kotiranta and
Larsson 2013
Sordariales sp. A Sordariales 7071 OP023256 1 0.07% 2% 1 Podospora
tetraspora MH859329.1 99.62% Vu etal. 2019
Sporothrix sp. A Ophiostoma-
tales 8291 OP023291 1 0.07% 2% 1 Sporothrix sp. MH740965.1 99.62% Jankowiak etal.
2019b
Steccherinum
sp.* BPolyporales 8283 OP023283 1 0.07% 2% 1 Steccherinum
bourdotii MK795065.1 100.00% Moiseenko etal.
2019
Stereum sp.* B Russulales 5970 OP023167 6 0.40% 5% 2 Stereum arme-
niacum MH862626.1 100.00% Vu etal. 2019
Thelonectria
sp. AHypocreales 5993 OP023178 1 0.07% 2% 1 Thelonectria
sp. OK161009.1 100.00% Lutz etal. 2022
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52 Page 18 of 29
Table 2 (continued)
Species Phylum Order NW-FVA ID ITS Accession
no. n isolates Frequency (%) Continuity
(%) Sites
isolated
from
ITS NCBI Blast results
Basis of iden-
tification ID Identity (%) Reference
Thysanophora
penicillioides
FR
AEurotiales 7063 OP023243 1 0.07% 2% 1 Thysanophora
penicil-
lioides
MH855455.1 100.00% Vu etal. 2019
Trametes versi-
color*BPolyporales 6137 OP023197 5 0.33% 9% 2 Trametes
versicolor LC710148.1 100.00% Mori and Hirai
(unpublished)
Vexillomyces
sp. FR ALeotiales 7102 ON809459 1 0.07% 2% 1 Vexillomyces
verruculosus NR_165533.1 91.83% Bien etal. 2020
Xylaria lon-
gipes*AXylariales 8288 OP023288 1 0.07% 2% 1 Xylaria lon-
gipes MN588219.1 100.00% Patejuk et. al
(unpublished)
Xylaria poly-
morpha*AXylariales 5978 OP023169 7 0.46% 12% 3 Xylaria poly-
morpha MG098262.1 99.82% Bußkamp etal.
2020
Fig. 3 Isolated orders of the Ascomycota, n = 132 of the isolated
morphotypes belonging to the Ascomycota (Microsoft PowerPoint
2013)
before outgrowth could be recognised. The remaining 1070
(24%) wood chips were colonised or contaminated by yeasts,
mould, or fungi which do not belong to Dikarya. The result-
ing pure culture isolates were assigned to 162 MT (excluding
Trichoderma spp.) and all but ten could at least be identified
to a genus level. Eighty-nine isolates could be identified to a
species level (Table2).
The majority of the isolated filamentous fungi from all
samples were Ascomycota (132 MT including Trichoderma
spp., 77.8%), 36 MT (22.2%) belonged to the phylum of
Basidiomycota. Within the Ascomycota, the most frequently
observed orders (Fig.3) were Hypocreales (23.0%) followed
by Pleosporales (22.2%), and Helotiales (19.8%). The
Basidiomycota fungi were mainly represented by Russula-
les (36.1%), Agaricales (25.0%), and Polyporales (22.2%).
Despite the diversity of 162 detected fungal MT, only a
few fungi occurred with high fMT. Sixty-seven MT (41%)
were isolated more than once and from these only 13 fungi
(8%) were obtained ten or more times. The remaining 95
fungi (59%) were only isolated once. Between one and 27
different fungi were found per stem collar. On average, nine
MT were recorded on each tree. None of the identified fungi
were found at all sites (including extensive sampling sites)
and merely 46 MT (28%) were found at more than one site.
In total, 116 fungi (72%) were found solely at one site (the
four subplots of Schlangen are considered one site).
Although morphologically similar to Diplodia mutila
(Fr.) Mont., in the phylogenetic analysis based on ITS
Mycological Progress (2023) 22:52
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Page 19 of 29 52
sequences, the majority of Diplodia isolates from this study
are placed clearly within a clade of strains of Diplodia frax-
ini (Fr.) Fr., including its ex-neotype (Online Resource 4 and
Online Resource 6). One strain from this study (NW-FVA
5979) is placed within a clade formed by strains of Diplodia
sapinea (Fr.) Fuckel including its ex-epitype strain and the
ex-type strain of the synonymised Diplodia intermedia. The
ITS sequences of this clade differ by one nucleotide, how-
ever, the ITS sequence of NW-FVA 5979 is identical to that
of the ex-epitype strain of D. sapinea. Preliminary morpho-
logical observation of strains of Armillaria indicated affili-
ation to A. gallica. However, A. gallica cannot be clearly
distinguished morphologically from Armillaria cepistipes
Velen. (Tsopelas 1999). Of the 35 isolates included in the
phylogenetic analysis of Armillaria, 33 isolates are placed
within a clade containing reference strains of A. gallica.
The two remaining isolates are placed within a clade of A.
cepistipes (Online Resource 5 and Online Resource 7). Since
only a selection of Armillaria isolates was included in the
phylogenetic analysis due to limited lab resources, a clear
distinction could not be made for all isolates of this genus.
Hence, isolates of Armillaria are combined and referred to
as Armillaria spp. in the final assessment of this study.
The most frequent MT isolated were D. fraxini (21.8%),
H. fraxineus (13.9%), Armillaria spp. (12.3%), and N. puni-
cea (9.6%), which account for nearly half of the isolated
fungi. Perithecia of N. punicea were frequently observed
on the ash bark above the necrotic lesions. All other iso-
lated fungi were less frequent with <4% proportion. The
most abundant MT in respect to continuity beside the
aforementioned D. fraxini (71% continuity), H. fraxineus
(53%), Armillaria spp. (50%), and N. punicea (33%), were
Diaporthe cf. eres (43% continuity / 3.8% fMT), Fusarium
cf. lateritium (34% / 3.5%), and Paracucurbitaria sp. (34%
/ 2.1%). These MT were also most abundant in regard to
occurrence at the nine sample sites (H. fraxineus occurs at
eight sites, Diaporthe cf. eres and D. fraxini at seven sites,
Fusarium cf. lateritium, N. punicea, and Paracucurbitaria
sp. at six sites). When taking into account only the intensive
sampling sites with at least five stem collars studied, there
is an overlap of five occurring fungi: Diaporthe cf. eres, D.
fraxini, H. fraxineus, N. punicea, and Paracucurbitaria sp.
The ash dieback pathogen H. fraxineus was isolated from
31 of the 54 stem collar necroses (57%). It was isolated at
all studied sites except at Wolfenbüttel, where only a single
tree with stem collar necrosis was sampled. The fungus was
detected in both, young and advanced necroses, but not in
the four control samples without symptomatic tissue. Hyme-
noscyphus fraxineus was isolated less frequently at the inves-
tigated sites with better water supply (soil water supply and
climate combined; Online Resource 2).
147 MT (91%) were only isolated from necrotic stem
tissue. Almost one-third of all MT according to their
identification are able to decay wood (Table2). A signifi-
cant proportion of the isolated species were found here for
the first time associated with F. excelsior, for example: three
isolates from necrotic stem collar tissue were assigned to the
MT identified as Cryptostroma corticale (Ellis & Everh.)
P.H. Greg. & S. Waller and one isolate from stem collar
tissue was identified as D. sapinea. The isolation of Paracu-
curbitaria sp. from the examined samples is the first proof
of this genus from stem collar necroses of F. excelsior. Fur-
thermore, one isolate from necrotic stem collar tissue was
preliminarily assigned to the genus Vexillomyces S. Bien,
C. Kraus & Damm based on ITS sequence comparison.
Further morphologic as well as multi-locus phylogenetic
investigations based on additional DNA regions (ribosomal
large subunit, EF-1α, and a 200-bp intron of the glyceral-
dehyde-3-phosphate dehydrogenase) revealed this isolate to
represent a novel yet undescribed species of Vexillomyces
(Tan etal. 2022). The MT, which only occurred once in
asymptomatic control samples were assigned to Akantho-
myces sp., Cephalotrichiella penicillata Crous, and Scle-
rostagonospora cycadis Crous & G. Okada. The following
fungi were isolated from stem collar necroses as well as from
symptomless controls: Alternaria infectoria E.G. Simmons,
Alternaria sp., Diaporthe cf. eres, Exophiala sp., Hypoxylon
fragiforme E.G. Simmons, Jackrogersella sp., Paracucur-
bitaria sp., Peniophora sp. 1 and sp. 2, Phaeosphaeria sp.,
Pseudeurotiaceae sp., and Sistotrema oblongisporum M.P.
Christ. & Hauerslev.
Fungal communities instem collar necroses
The number of MT isolated from stem collar necroses at the
different sites ranged from one (Wolfenbüttel, one sample
tree) to ten (Woltershausen, two sample trees) at the exten-
sive sites and from 29 (Schwansee, eight sample trees) to 87
(Schlangen, 19 sample trees) at the intensive sites. Hence,
the fungal communities at the sites studied differed in their
species composition and diversity (Fig.4). In Schlangen 52
MT (32% of all 162 isolated MT of this study) out of 87
MT were found exclusively at this site. Nevertheless, the
most common MT in Schlangen are identical with the most
frequent MT over all sampling sites (Armillaria spp., D.
fraxini, H. fraxineus, and N. punicea). Diplodia fraxini was
isolated from almost 80% and H. fraxineus from almost 70%
of the trees studied at this site. 54 MT were found in the
samples from the site of Berggießhübel and 30 (19%) of
those occurred exclusively at this site. Likewise, the most
common MT at Berggießhübel were D. fraxini, H. fraxineus,
and Armillaria spp. However, N. punicea was isolated only
once there. At the Rhüden study site, 32 MT were found, 13
(8%) of which were found exclusively at this site. The most
frequent MT were the same as the most abundant MT in
respect to continuity from all samples - except N. punicea,
Mycological Progress (2023) 22:52
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52 Page 20 of 29
which was isolated only once in Rhüden. 31 MT were found
in the Satrup samples, 12 of which (8%) exclusively at this
site. The most common MT in Satrup were identical with
the four most common MT in Schlangen. At Schwansee,
29 fungal MT were isolated in total, 12 of which (8%) were
found exclusively at this site. At this location, the most com-
mon MT besides D. fraxini and N. punicea were Coprinellus
species and F. solani s. l.
Discussion
Fungi associated withash 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 stud-
ies as well (Lygis etal. 2005; Langer 2017; Meyn etal. 2019;
Kranjec Orlović etal. 2020). Though 87 taxa (54%) iso-
lated here were not reported by the aforementioned studies.
It has to be taken into account that different sample sizes
and sample site numbers lead to differing numbers of spe-
cies. Considering the mentioned studies, Meyn etal. (2019)
had the smallest sample size with four trees at one sam-
ple 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 etal. (2020) with 68 fungal
species isolated from 90 sample trees examined at three sam-
ple sites. The non-negligible impact of the number of sample
sites on the detectable fungal species diversity is shown in
this study with 162 MT isolated from a smaller sample size
(58 trees), but a higher number of sample sites (9) compared
to the study of Kranjec Orlović etal. (2020). Other factors,
such as number of the incubated wood chips or the isolation
method may also have influence on the number of species
isolated. 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 stem col-
lar 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 etal. 2020; Langer etal. 2021) in this
study the majority of fungi isolated belong to Ascomycota
(77.8%). A reason for the lower frequencies of Basidiomy-
cota compared to Ascomycota might be that fungi belong-
ing to the former often need longer incubation periods in
order to grow out from incubated woody tissues (Oses etal.
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 dis-
crepancy 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 etal. 1990; Bugg etal. 2011). Hence, the
occurrence of white and brown rot fungi in stem collar
necroses is not unusual. Typical white rot fungi like Armil-
laria 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 follow-
ing representatives of this group were found: Biscogniauxia
nummularia (Bull.) Kuntze, Hypoxylon spp., Jackroger-
sella sp., Nemania serpens (Pers.) Gray, and Xylaria spp.
(all Xylariales). Besides 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 MT detected in
this study were listed for F. excelsior in the USDA fungal
database (Farr and Rossman 2022). Only one of the most
Fig. 4 Overlap between the fungi isolated from the main sites (with
at least five samples) with the indication of how many of the isolated
fungi were found at each site and between the different sites (absolute
number as well as percantage), empty white areas indicating no over-
lap between the respective sites. Five fungi at the overlap of all sites
are Diaporthe cf. eres, Diplodia fraxini, Hymenoscyphus fraxineus,
Neonectria punicea, and Paracucurbitaria sp. (RStudio 4.1.2)
Mycological Progress (2023) 22:52
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Page 21 of 29 52
frequent MT 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 fre-
quent MT associated with stem collar necroses in the context
of ash dieback (Langer 2017; Meyn etal. 2019). The other
most abundant MT 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 etal. 2016; Haňáčková
etal. 2017; Langer 2017; Meyn etal. 2019; Linaldeddu
etal. 2020; Kowalski and Bilański 2021; Barta etal. 2022).
Langer (2017) investigated stem collar necroses of 32 ash
trees and determined the aforementioned species as well—
except for Paracucurbitaria sp. Meyn etal. (2019) isolated
D. fraxini (labelled as Botryosphaeria stevensii), H. frax-
ineus, 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 etal.
(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).
Within the genus Armillaria, A. gallica was the most fre-
quently isolated species in the present study. Additionally
to our isolations, mycelial fans and rhizomorphs of Armil-
laria 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 for-
est sites even before ash dieback occurred (Morrison 2004;
Lygis etal. 2005; Bakys etal. 2009b). They are considered
secondary pathogens and wood-decaying fungi infecting
stressed trees, which explains their occurrence in advanced
stem necroses and root rot (Chandelier etal. 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
etal. (2016) in Belgium. Enderle etal. (2017) also detected
A. gallica in stem collar rots in south-western Germany.
These results are in contrast to investigations by Lygis etal.
(2005), who determined A. cepistipes as most frequent in
Lithuania. Nevertheless, regardless of which of the two spe-
cies caused infection, Armillaria spp. most likely acceler-
ate the decline of ash dieback-affected ash trees (Chandelier
etal. 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 etal. (2020) deter-
mined 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, cer-
tainly 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 etal. 2014; Linaldeddu etal. 2020, 2022). Kowal-
ski etal. (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 etal. 2016). These facts might indicate
that infections with H. fraxineus facilitate the colonisation
of affected ash trees by D. fraxini. Another possible expla-
nation for the more frequent occurrence of D. fraxini could
be global warming because this species benefits from warm
temperatures of around 25 °C (Alves etal. 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. Besides 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 etal. (2020), the spe-
cies D. subglobosa could not be isolated in our analysis,
maybe because they investigated branches and not stem col-
lar necroses.
Neonectria punicea has a large host spectrum, includ-
ing F. excelsior (Hirooka etal. 2013). However, this fun-
gus has rarely been documented from this particular host
species before (Langer 2017; Meyn etal. 2019; Karadžić
etal. 2020). N. punicea was found to be associated with
stem collar necroses and cankers of European ash in Ger-
many (Langer 2017; Meyn etal. 2019) and it is able to cause
necroses in juvenile ash trees (Karadžić etal. 2020). Its
perithecia were observed frequently on the bark above the
necrotic ash tissue (ibid. and Karadžić etal. 2020). Neonec-
tria 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 etal. 2014; Linaldeddu etal. 2020).
This species often produces its tiny fruit bodies on dead
woody tissues (Kowalski etal. 2016). In a study by Kowalski
etal. (2017), D. eres showed the least virulence and caused
significantly milder disease symptoms on F. excelsior plants
Mycological Progress (2023) 22:52
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52 Page 22 of 29
than the other tested fungal species. Diaporthe eres could be
considered 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 patho-
genic attack (Kowalski etal. 2016).
In this study Fusarium cf. lateritium Nees has been iso-
lated frequently from symptomatic tissue and once from
healthy wood tissue. The species is already known from F.
excelsior in association with bacterial ash canker (Riggen-
bach 1956) but its virulence seems to be low in comparison
with other fungal species (Bakys etal. 2009b). Kowalski
etal. (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 etal. 2009b; Kowalski etal. 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 shift-
ing 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 etal. (2019) from rootstock. However, Kowalski
and Bilański (2021) detected Paracucurbitaria sp. in previ-
ous year’s ash leaf petioles in Poland, Barta etal. (2022)
isolated it from ash twigs in Slovakia, and Haňáčková etal.
(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 spe-
cies 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 coinci-
dence, because its detection in stem collar necroses of ash
is increasing in ongoing research at the NW-FVA since sam-
pling 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 (Table2). 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 etal. 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 Qui-
jada 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).
Role ofHymenoscyphus fraxineus instem collar
necroses
The ash dieback pathogen H. fraxineus could not be iso-
lated 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 H. frax-
ineus could not be frequently isolated from symptomatic
tissue of ash (Przybyl 2002; Bakys etal. 2009a; Enderle
etal. 2017). A possible explanation for this could be its
slow growth, unfavourable sampling conditions for the
pathogen or too advanced necroses with antagonistic activ-
ity of other colonisers (Kowalski and Holdenrieder 2009;
Hauptman etal. 2013; Gross etal. 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 afore-
mentioned 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 or even DNA
residues if the initial fungus has been suppressed by sec-
ondary invaders (Lindahl etal. 2013). Our results on the
fMT and continuity of the association and localisation of
H. fraxineus in basal stem necroses support the assump-
tion, 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 etal.
2009a; Kowalski and Holdenrieder 2009; Gross etal.
2014). The only lack of evidence for H. fraxineus at the
study site Wolfenbüttel could be explained by the mea-
gre sample size. 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).
Mycological Progress (2023) 22:52
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Page 23 of 29 52
Fungal communities instem collar necroses
The observation of significant differences in the occurrence
of fungal taxa between the investigated forest sites is consist-
ent with the results of Bilański and Kowalski (2022). In the
study of Meyn etal. (2019) only two species were found in
all sample trees and many of the identified fungi were single
isolates. Similarly, Kranjec Orlović etal. (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.
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.
Relation ofthemost common fungi tothesite
characteristics
Independent of age class and site conditions, European ash
trees can be vulnerable to an infection by H. fraxineus (Pau-
tasso etal. 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 colonis-
ing fungal species (Langer etal. 2022), tree vitality, or the
environmental context of the forest site and stand (Havrdová
etal. 2017) are some examples. Ash tree vitality is encour-
aged at fertile and (moderately) wet soils, conditions which
are preferred by ash (Walentowski etal. 2017). It has to be
taken into account that for this study only a selection of for-
est 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 fun-
damental. Furthermore, the selection of sample trees was
subjected to different restrictions. For example the condi-
tion 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
etal. 2011; Salamon etal. 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., Dia-
porthe cf. eres, D. fraxini, Fusarium cf. lateritium, N. puni-
cea, and Paracucurbitaria sp., Dirichlet regression indicated
no correlation between fWC and water supply rank of the site
(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 sugges-
tion 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 etal. 2014;
Erfmeier etal. 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 sig-
nificantly 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 MT Diaporthe
cf. eres, Fusarium cf. lateritium, and Paracucurbitaria sp.
had a consistent fWC over all sites. But Fusarium cf. later-
itium 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 etal. (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 poly-
morpha (Pers.) Grev. (Liers etal. 2011). Furthermore, the
absence of Armillaria spp. isolates in Schwansee, the most
moist 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
eighteenth 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 MT 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
Mycological Progress (2023) 22:52
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52 Page 24 of 29
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 dif-
fer according to tree age, tree species composition, forest
management type, season, and the like (Scholtysik etal.
2013; Tomao etal. 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 etal. 2011; Kowalski etal. 2016; Krah etal. 2018;
Tomao etal. 2020). This is confirmed by the isolation of
sycamore-typical fungi like C. corticale und Cy. rubronotata
from F. excelsior in stands with maple trees. It is further-
more 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. In addition
to its diverse tree species composition, Berggießhübel is the
most eastern sampling site. Satrup is the most northern sam-
pling site and shows high fungal diversity despite its smallest
sample tree amount. This observation suggests that widely
varying sites in Germany lead to differing fungal commu-
nities. 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 etal. 2001; Allen etal. 2003; Unterseher
2011; Muggia etal. 2017).
Conclusion andoutlook
This study provided new insights into the fungal diversity
and communities of endophytes, primary and secondary
pathogens, wood-decaying fungi, and saprotrophic fungi
associated with stem collar necroses of European ash trees.
A rich fungal composition inhabiting symptomatic stem tis-
sue 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 previ-
ous investigations in which 16–75 different species were
detected (Lygis etal. 2005; Enderle etal. 2017; Langer
2017; Meyn etal. 2019). This difference in diversity can be
explained by the larger sampling size (not only tree num-
ber, 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 MT.
Further studies on stem collar necroses can increase the
knowledge of fungal biodiversity on F. excelsior, as clearly
demonstrated by the newly described species, Vexillomyces
fraxinicola (Tan etal. 2022), which was collected in this
study.
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 patho-
gen. Nevertheless, it is possible 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 MT
(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 iso-
lated 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 die-
back, for example H. fraxineus and D. fraxini or N. puni-
cea, can lead to a larger damage in contrast to infection by
only one pathogen (Marçais etal. 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 etal. 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 programmes 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 MT to forest side con-
ditions 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. frax-
ineus 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.
Mycological Progress (2023) 22:52
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Page 25 of 29 52
Supplementary Information The online version contains supplemen-
tary material available at https:// doi. org/ 10. 1007/ s11557- 023- 01897-2.
Acknowledgements Many thanks to Peter Gawehn, Annette Ihlemann,
Martina Hille, Daniel Gaunitz, and Dr. Holger Sennhenn-Reulen for
their technical and statistical support. Additionally, the authors are
grateful to the Forestry departments of Grünenplan, Erfurt-Willrode,
Liebenburg, Neustadt, Satrup, and Nassesand for granting permission
to sample the trees. Special thanks to Maia Ridley for proof-reading the
manuscript. We would also like to thank the reviewers for their valuable
advice on how to improve the manuscript.
Author contribution The study including sampling, lab work, and anal-
ysis was primarily conducted by S. Peters with support from G. Langer,
S. Bien and S. Fuchs. The first draft of the manuscript was written by S.
Peters and revised by G. Langer, S. Bien, J. Bußkamp, and E. Langer.
Funding Open Access funding enabled and organized by Projekt
DEAL. The project receives funding via the Waldklimafonds (WKF)
funded by the German Federal Ministry of Food and Agriculture
(BMEL) and the Federal Ministry for the Environment, Nature Con-
servation, Nuclear Safety and Consumer Protection (BMUV) admin-
istrated by the Agency for Renewable Resources (FNR) under grant
agreement No. 2219WK22A4.
Data availability The DNA sequences generated in this study were
deposited in GenBank (https:// www. ncbi. nlm. nih. gov; Table2). All
sampling data is provided in the online resources (ESM 3). The fungal
strains are stored in the strain collection of the NW-FVA. Text and
images are permanently stored on an internal drive of the NW-FVA.
Declarations
Ethics approval Not applicable
Consent to participate Not applicable
Consent for publication Not applicable
Conflict of interest The authors declare no competing interests.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.
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