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Fungal diversity and seasonal succession in ash leaves infected
by the invasive ascomycete Hymenoscyphus fraxineus
Hugh Cross
1
, Jørn Henrik Sønstebø
1
, Nina E. Nagy
1
, Volkmar Timmermann
1
, Halvor Solheim
1
, Isabella Børja
1
,
Havard Kauserud
2
, Tor Carlsen
2
, Barbara Rzepka
3
, Katarzyna Wasak
4
, Adam Vivian-Smith
1
and Ari M. Hietala
1
1
Norwegian Institute of Bioeconomy Research, Pb. 115,
As NO-1431, Norway;
2
Department of Biosciences, Section for Genetics and Evolutionary Biology, University of Oslo, Pb. 1066
Blindern, Oslo NO-0316, Norway;
3
Faculty of Chemistry UJ, Jagiellonian University, Ingardena 3, Krakow 30-060, Poland;
4
Department of Pedology and Soil Geography, Institute of
Geography and Spatial Management, Jagiellonian University, Gronostajowa 7, Krakow 30-387, Poland
Author for correspondence:
Hugh Cross
Tel: +47 98005527
Email: hugh.cross@nibio.no
Received: 29 January 2016
Accepted: 15 August 2016
New Phytologist (2016)
doi: 10.1111/nph.14204
Key words: ash dieback, Hymenoscyphus
fraxineus, indigenous fungi, internal tran-
scribed spacer (ITS), invasive pathogens,
metabarcoding.
Summary
High biodiversity is regarded as a barrier against biological invasions. We hypothesized that
the invasion success of the pathogenic ascomycete Hymenoscyphus fraxineus threatening
common ash in Europe relates to differences in dispersal and colonization success between
the invader and the diverse native competitors.
Ash leaf mycobiome was monitored by high-throughput sequencing of the fungal internal
transcribed spacer region (ITS) and quantitative PCR profiling of H. fraxineus DNA.
Initiation of ascospore production by H. fraxineus after overwintering was followed by
pathogen accumulation in asymptomatic leaves. The induction of necrotic leaf lesions
coincided with escalation of H. fraxineus DNA levels and changes in proportion of biotrophs,
followed by an increase of ubiquitous endophytes with pathogenic potential.
H. fraxineus uses high propagule pressure to establish in leaves as quiescent thalli that
switch to pathogenic mode once these thalli reach a certain threshold –the massive feedback
from the saprophytic phase enables this fungus to challenge host defenses and the resident
competitors in mid-season when their density in host tissues is still low. Despite the general
correspondence between the ITS-1 and ITS-2 datasets, marker biases were observed, which
suggests that multiple barcodes provide better overall representation of mycobiomes.
Introduction
A continental scale dieback threatens the future existence of com-
mon ash (Fraxinus excelsior) and poses a set of cascading impacts
upon the biodiversity associated with this keystone tree species in
Europe (Pautasso et al., 2013; Mitchell et al., 2014). Dieback of
common ash is caused by the invasive ascomycete Hymenoscyphus
fraxineus (syn. H. pseudoalbidus, anamorph Chalara fraxinea)
(Kowalski, 2006; Queloz et al., 2011; Baral et al., 2014). In Asia,
the presumed native range, this fungus has been regarded as a leaf
saprophyte of Manchurian ash (F. mandshurica) (Zhao et al.,
2013; Han et al., 2014; Zheng & Zhuang, 2014), which is a close
relative of common ash (Wallander, 2008). Recent studies indi-
cate that the fungus is a leaf endophyte of Manchurian ash
(Cleary et al., 2016) and can also show some pathogenic potential
in its native range (Drenkhan et al., 2016).
Dieback of common ash was first recorded in Poland in the
early 1990s (Przybył, 2002). Since 2001, an intensive spread of
the disease has been observed in central, northern, eastern and
western Europe (Juodvalkis & Vasiliauskas, 2002; Przybył, 2002;
Kowalski & Łukomska, 2005; Lygis et al., 2005; Kowalski, 2006;
Timmermann et al., 2011), and only populations at the southern
and eastern range margins of common ash currently remain
disease-free (McKinney et al., 2014).
The pathogen has the ability to colonize the compound leaf,
shoots, main stem and even the roots of common ash (Kirisits &
Cech, 2009; Kowalski & Holdenrieder, 2009; Schumacher et al.,
2010). Young trees often die within a few years of infection,
while older trees become chronically diseased and susceptible to
secondary diseases such as root rot caused by the white-rot fungi
Armillaria (Skovsgaard et al., 2010). Ash trees are affected by the
disease not only in the forest, but also in nurseries, on roadsides,
in associated plantations, and in parks and gardens.
Hymenoscyphus fraxineus is an outcrossing heterothallic fungus,
and the airborne ascospores have a significant role in primary
infection and long-distance dispersal (Bengtsson et al., 2012;
Gross et al., 2012a,b, 2014b;Kraj et al., 2012; Hamelin et al.,
2016). During the epidemic stage, H. fraxineus shows the ability
to simultaneously liberate ascospores on massive scales early in
the morning (Timmermann et al., 2011; Hietala et al., 2013),
indicating that propagule pressure may be an important strategy
for colonization success. According to the current model,
H. fraxineus ascospores germinate on the leaf surface, giving rise
to mycelia that spread to the leaf petiole and further into
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connecting stem tissues to cause shoot dieback (Gross et al.,
2014a). The invasive behavior of H. fraxineus is obviously inti-
mately linked with efficient pathogen capture of the leaf vein sys-
tem, its primary sporulation substrate and main niche also in its
native range in Asia. By contrast, ash shoot infection by
H. fraxineus can be considered a dead-end in the life cycle of this
fungus, because its ascomata are rarely formed on twigs and stems
of common ash (Gross et al., 2014b). The nutritional modes of
H. fraxineus in common ash leaves remain to be clarified. Several
studies have suggested that local biodiversity represents an impor-
tant line of defense against the spread of invaders (e.g. Kennedy
et al., 2002; Bairey et al., 2016).
Fungal community studies have been strengthened in recent
years through a combination of high-throughput sequencing
(HTS) and a well-curated database of fungal internal transcribed
spacer (ITS) sequences (K~oljalg et al., 2013). DNA metabarcod-
ing studies have shown that highly diverse fungal communities
are associated with healthy leaves of other angiosperm trees
(Jumpponen & Jones, 2009; Cordier et al., 2012; Balint et al.,
2013; Vorıskova & Baldrian, 2013) , and we predicted that
already during early summer, before the sporulation period of
H. fraxineus, leaves of common ash would be exposed to high col-
onization pressure by a wide range of functionally divergent
fungi. There is increasing evidence that propagule pressure is an
important ecological trait that influences the success of introduc-
tion as well as the transition of invasive species to subsequent
stages of local establishment, spreading outside the area of intro-
duction and eventual widespread dominance (Lockwood et al.,
2005; Colautti et al., 2006). We hypothesized that the ability of
H. fraxineus to capture the leaf vein system is a result of advan-
tages in propagule pressure and colonization strategies. To test
these hypotheses, compound ash leaf samples were collected
throughout the growing season in two consecutive years from a
stand exhibiting an epidemic level of ash dieback. Leaflet and
petiole tissues were subjected to quantitative PCR (qPCR) profil-
ing of H. fraxineus DNA content and to HTS of fungal sequences
separately amplified from the ITS-1 and -2 of the rDNA gene
cluster. As reference, airborne fungal spores captured from the
experimental stand during the peak sporulation period of
H. fraxineus were also subjected to HTS. Our data reinforce the
fact that, during the ascospore production period, H. fraxineus
accumulates in ash leaves as quiescent thalli that switch to the
pathogenic growth phase once the initial tissue colonization
reaches a specific threshold. The massive mid-season sporulation
provides a crucial signal from the saprophytic phase and enables
this fungus to challenge host defense and the resident competitors
when their density in host tissues is still low.
Materials and Methods
Plant and spore material
Randomly chosen compound leaves from the understory of com-
mon ash trees were sampled throughout the growing season in
2011 and 2012 in a stand located 30 km south of Oslo (
As
municipality, 59°40044″N, 10°46031″E,100 m above sea level
(asl)), exhibiting epidemic levels of ash dieback.
In 2011, leaves were sampled from three selected trees on a
weekly basis from the beginning of July until the leaves shed at
the end of August; altogether 27 compound leaves were collected.
In that year the monthly precipitation in June and July in the
experimental region was 20–60 mm above the long-term averages
for the area, while the mean temperatures were similar to (June)
or slightly above (July) the long-term averages for the area (Sup-
porting Information Fig. S1).
In 2012, 12 trees were sampled from the end of June
until the period of leaf shed in mid-September. These con-
sisted of six healthy appearing trees and six that had shoot
symptoms of ash dieback in the beginning of the season
(Fig. S2). As an addition, the twig region directly below the
axillary bud of the sampled compound leaves was also col-
lected (Fig. 1). Altogether 50 compound leaf/twig samples
were collected. In 2012 the monthly precipitation for June
and July in the experimental region was 10–30 mm above
the long-term average for the area, whereas the mean tem-
peratures were 1–2.5°C below the average (Fig. S1).
Fig. 1 Tissue samples (shown as blue frames)
taken from the compound ash leaf. 1, leaflets
(three leaflets, apical, central and basal,
pooled together); 2, rachis, upper; 3, rachis,
middle; 4, petiole, upper; 5, petiole, middle;
6, petiole, base; 7, twig.
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DNA from airborne spores, collected by a volumetric Burkard
spore sampler at the experimental stand at four time-points in
June and August 2010 in our previous study (Hietala et al.,
2013), were used as reference material in sequencing.
DNA isolation and qPCR
For each tree and sampling time, the apical leaflet, one leaflet from
themiddle,andonefromthebaseofthecompoundleafwere
excised and pooled together to form a balanced sample. In addition,
separate, c. 5-mm-long samples were taken from the upper and mid-
dle rachis and likewise from the upper, middle and basal parts of the
petiole. In 2012 the subsampling was modified, omitting the middle
part of petiole, and taking a sample of the twig region directly below
the axillary bud of the sampled compound leaves (Fig. 1). Samples
were processed separately and the FW of each sample was recorded
for normalization of qPCR data. Dissected and subsampled ash tis-
sues were stored at 20°C until DNA extraction.
Leaflet samples were frozen with liquid nitrogen and pulver-
ized with a mortar and pestle, while the other tissues were pulver-
ized in liquid N
2
-chilled Eppendorf tubes with a Retsch 300 mill
(Retsch Gmbh, Haan, Germany). Up to 50 mg tissue was pro-
cessed with DNeasy Plant Mini Kit (Qiagen) according to the
manufacturer’s instructions, to a final elution volume of 50 ll.
The real-time PCR quantification of Hymenoscyphus fraxineus
(T. Kowalski) Baral, Queloz, Hosoya was performed as described
by Ioos et al. (2009) (see Methods S1). The primer and probe
concentrations per assay were 300 and 100 nM, respectively (Ioos
et al., 2009). A standard curve with known concentrations of
H. fraxineus DNA was prepared from pure cultures as previously
described (Hietala et al., 2013). To ensure that the cycle thresh-
old values from the experimental samples were within the stan-
dard curves, and that PCR inhibitory compounds potentially
present in undiluted samples remained low in the assay, a 3-log
dilution series was prepared so that for each sample the undiluted
DNA and the 10- and 100-fold dilutions were used as templates
for real-time PCR. Standard curves were constructed by plotting
the Ct values against log-transformed DNA amounts. The calcu-
lated linear regression equation was used for interpolation of
pathogen DNA amount in unknown samples.
Many of the undiluted leaf and twig tissue DNA samples
showed a higher Ct value than the 10-fold diluted template,
whereas the differences in Ct values between 10- and 100-fold
dilutions were in the range obtained for the log dilutions of the
corresponding standard curve samples. Thus, many undiluted
templates appeared to be compromised by PCR inhibitory com-
pounds. Therefore, Ct values from the 10-fold diluted templates
were used in subsequent calculations. Sampling time-specific dif-
ferences in H. fraxineus DNA amount were tested by ANOVA
and Fisher’s least significant difference (LSD) post hoc test, and
considered statistically significant at P<0.05.
Pyrosequencing of ITS1 region
DNA extracted from the leaflet, petiole upper and petiole
base tissues collected in 2011 and the reference spore material
was subjected to 454 pyrosequencing following the protocol
of Lindner et al. (2013). DNA from the leaf tissue samples
collected from three selected trees was pooled at equimolar
concentrations so that one DNA sample per tissue type and
sampling time was processed: altogether, 27 pooled DNA
samples (one leaflet, one petiole upper and one petiole base
sample for each of the nine sampling dates) were analyzed.
This entailed a nested PCR approach, using the fungal-
specific primers ITS1F and ITS4 (White et al., 1990; Gardes
& Bruns, 1993) in the first step and fusion primers ITS5
and ITS2 (White et al., 1990) in the nested step, using 50 9
diluted product from the first PCR. Fusion primers contained
16 different 10 bp unique tags and 454 pyrosequencing adap-
tors A and B to both ITS5 and ITS2, respectively (see Methods
S1 for details). PCR products were normalized to a single
DNA concentration using the SequelPrep Normalization
Plate (Invitrogen) and then cleaned with Wizard_SV PCR
Clean-Up System (Promega). GS FLX sequencing of the tagged
amplicons was performed at the Norwegian High-Throughput
Sequencing Centre (http://www.sequencing.uio.no) using one
454 plate divided into eight compartments. We included two
negative controls through all analyses from the DNA extraction
step.
Ion Torrent PGM sequencing of ITS2 region
The same 27 pooled DNA samples from leaf tissues processed for
ITS-1 sequencing were also used for ITS-2 sequencing, along
with the reference spore material. The fungal ITS-2 region was
amplified using the degenerate gITS7 primer (Ihrmark et al.,
2012) together with the ITS4 primer (White et al., 1990; Meth-
ods S1). Each reaction was cleaned with 1.1 volumes of Ampure
XP (Beckman Coulter Inc., Pasadena, CA, USA), and the prod-
ucts were ligated to barcoded adaptors as outlined in the Ion
Amplicon Library Preparation user guide and Ion Xpress frag-
ment kit (PN 4468326 rev B and P/N 4471252, respectively),
and where the A adaptor contained a sample specific Ion
Xpress_barcode identification sequence (Thermo Fisher Scien-
tific, Waltham, MA, USA; catalogue no. 4471250). The resulting
products were pooled in equal amounts and purified using
another round of Ampure XP cleaning, and analyzed on a Bioan-
alyzer 2100 High Sensitivity DNA Chip (Agilent Biosciences,
Santa Clara, CA, USA). Subsequently library pools were diluted
and sequenced according to Ion Torrent manufacturer specifica-
tions (Thermo Fisher Scientific) on the Ion PGM using 314 v2
chips with 400 bp chemistry. Sequences were inspected using
FastQC (Andrews, 2010).
Extrapolation of total fungal biomass in ash leaf tissues
The total DNA amount of all fungi present in ash leaf tissues was
extrapolated using the following formula: total fungal DNA
amount (ng) =(Hfrax
DNA
9100%)/Hfrax
seq,
where Hfrax
DNA
is
the H. fraxineus DNA amount (ng) determined by qPCR, and
Hfrax
seq
is the corresponding ITS-2 sequence proportion (%) of
H. fraxineus in the sample at a given time.
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Bioinformatics and statistical analyses
Processing of raw sequences All sequence reads from 454 and
Ion Torrent were processed using the programs CUTADAPT
(Martin, 2011) and FASTX-Toolkit (http://hannonlab.cshl.
edu/fastx_toolkit/index.html) in a pipeline with custom bash and
python scripts (Methods S1). Briefly, all adapter and primer
sequences were trimmed at both ends of each sequence, and then
filtered for minimum length (100 bp) and quality (at least 90%
of reads with a phred score of 20). All files were renamed and
converted to the FASTA format for downstream analyses, and
lodged with the NCBI Sequence Read Archive (ID
PRJNA305543).
Operational taxonomic unit (OTU) clustering and taxonomy
assignment The overall method for clustering reads and assign-
ing taxonomy followed a modified and customized version of
open-reference OTU clustering, as described by Rideout et al.
(2014), as this method has been found to be a good compromise
between OTU stability and the inclusion of novel taxa (He et al.,
2015). The analyses proceeded in three major steps. Initially, all
sequence reads were dereplicated and then clustered into OTUs
using the program SWARM (Maheet al., 2015); these OTUs were
used as queries for searching against the UNITE-INSD fungal
ITS and NCBI nt databases, and the hits from these searches
were used to construct a reference sequence database. In the sec-
ond phase, all reads were clustered with these reference sequences
to assign taxonomy (‘closed reference clustering’) using USEARCH
v.8 (Edgar, 2010) at minimum 97% similarity. In the last step,
any reads that did not cluster closely with the reference sequences
(33.5% of ITS-1 and 39.5% of ITS-2 reads) were reclustered into
OTUs using USEARCH (‘open reference clustering’); taxonomy for
these OTUs was assigned using the USEARCH utax algorithm and
RDP method in QIIME (Caporaso et al., 2010). Taxonomy for
open reference clustering was limited to the genus level to avoid
inflation of rare species/OTUs as a result of sequence error or
gaps in the sequence database. For details of this approach, see
Methods S1.
Statistical and taxonomic analyses The program QIIME (Capo-
raso et al., 2010) was used to summarize taxonomic tables and
plot relative abundance of taxa across all samples, and by date
and tissue type. Abundance tables were imported into the pro-
gram MEGAN, v.5.10.3 (Huson et al., 2011), to map and chart
the taxonomy, and compare the ITS-1 and ITS-2 results. Alpha
rarefaction curves were calculated in MEGAN by repeatedly sub-
sampling the dataset (1000 replicates) and computing the num-
ber of taxa in the subsample. All OTU sequence read totals for
which taxonomy could be assigned at least to genus level were
combined by genus for ordination analyses. The sample totals for
the 50 most abundant genera were normalized using the CSS
method (Paulson et al., 2013) in QIIME. In order to determine
gradients of taxonomic composition, we conducted principal
component analyses (PCA) on normalized OTU abundance
tables using the R package VEGAN (Oksanen et al., 2016). To con-
sider sampling time and tissue type-specific differences in
numbers of genera, as well as differences in genus read propor-
tions, and positions along PC1 and PC2, we applied one-way
ANOVA with the LSD post hoc test, with P≤0.05 using the
SPSS 22.0 (IBM Inc., Armonk, NY, USA). Pearson product
moment correlation coefficients were calculated between the
qPCR and the ITS-2 datasets for selected taxa.
All Hymenoscyphus reads were remapped to a single representa-
tive sequence (FJ597975, type specimen of H. fraxineus) using
the BWA mem algorithm (Li & Durbin, 2009). Additionally,
reads assigned to other taxa of relatively high abundance (>1or
2%) were extracted and compared with reference sequences and
OTUs to determine their relative species composition and diver-
sity. These reads and references were aligned using GENEIOUS v.6
(Kearse et al., 2012) for comparison.
Results
Accumulation of pathogen DNA in ash tissues
In 2011 necrotic lesions on leaf veins were induced during the
first week of August. The presence of H. fraxineus DNA in all ash
leaf tissues was first detected by qPCR on 11 July (day 192), after
which the pathogen DNA level showed a generally continuous
increase (Fig. 2a), which coincided with the vigorous increase of
airborne ascospores of H. fraxineus at the stand in July (Fig. 2c).
The first significantly higher pathogen DNA levels compared
with those observed on 11 July (day 192) occurred on leaflets on
18 July (day 199), on 2 August (day 214) for the upper part of
the rachis and on 12 August (day 224) for the remaining leaf tis-
sues. The largest fold-changes between two consecutive sampling
times occurred between 2 and 12 August (days 212–224) when
the pathogen DNA showed between five- and 161-fold increases
(up to 18-fold increase in leaflet, petiole base, and rachis tissues,
and 90–161-fold increase in the upper and middle petioles);
excluding the upper part of the rachis and petiole base, these
increments were statistically significant.
In 2012, the amount of necrotic leaf lesions increased rapidly
after the second week of August in all trees. H. fraxineus DNA
was first detected in all sampled tissues on the 3 August (day 216;
Fig. 2b); the first significantly higher pathogen DNA levels com-
pared with those observed on 3 August occurred on leaflets on 10
August (day 223) and, for the remaining leaf tissues, during the
period between 23 August and 7 September (days 236 and 251).
Twig tissues showed a unique pattern in pathogen DNA accumu-
lation in which the peak on 17 August (day 230), differing signif-
icantly from that on 3 August, was followed by a steep decline.
There was no clear relationship between the pathogen DNA titer
in leaf tissues and the general health of the tree over the course of
the season (Figs S2, S3).
Fungal community profiling by ITS-1
After processing of raw reads and filtering for quality and length,
there were a total of 98 113 ITS-1 sequences from 454 sequenc-
ing (average of 2803 per sample over 31 samples). About 66.5%
of the total reads clustered with 342 reference sequences, and the
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remaining proportion of reads were de novo-clustered into 969
OTUs.
The vast majority of sequences was associated with fungi, with
a small proportion (5.7%) left unassigned. A very small fraction
of reads (<0.3%) were identified as plants (mostly the host
species Fraxinus excelsior), algae, and protists. All subsequent
analyses were confined to fungal taxa. Although there were
smaller numbers of reads in some individual leaf samples (includ-
ing leaflet, and petiole upper and lower parts), rarefaction analy-
ses across most samples suggest that the number of reads
sequenced had sampled most of the species diversity (Fig. S4a).
The taxonomic assignments of ITS-1 sequences revealed a fungal
community comprising a wide range of basidiomycete and
ascomycete taxa (Figs 3, S5).
Overall, most differences were observed between spore and
plant tissues (Tables S1–S3). The PCA analysis of ITS-1 data
from leaf tissues indicated significant differences in fungal species
composition, both between all time categories (P<0.0001), and
between leaflet and petiole tissues (P<0.005) (Fig. 4a). The
arrows on the PCA biplot (Fig. 4a) indicate a range of species
highly correlated with each other (Oksanen et al., 2016).
Despite the high taxonomic diversity across all samples, a
striking omission was that almost no reads were assigned to
Hymenoscyphus species, in stark contrast with both the qPCR
data and the visual observation of disease symptoms. Exami-
nation of reference sequences showed a very large insert in
the ITS-1 region of this genus. Therefore we suspected that
the PCR product size resulting from the ITS5/ITS2 primer
pair (581 bp) for H. fraxineus was too long for efficient
sequencing and that this was the primary cause of low
Hymenoscyphus read numbers.
Fungal community profiling by ITS-2
As Hymenoscyphus sequences were rarely detected with the ITS5/
ITS2 primer pair, ITS-2 amplicons were sequenced using the
gITS7/ITS4 primers with the Ion Torrent PGM, as the product
size is 280 bp for H. fraxineus, well within the range for Ion Tor-
rent and about the average size for fungal species. Ion Torrent
sequencing produced 177 681 ITS-2 reads after filtering (average
of 5923 per sample over 30 samples). The overall results were
similar to ITS-1: 60.5% of reads clustered with 481 reference
sequences, and the remaining sequences were clustered into 1539
de novo OTUs. The majority of taxonomic matches were to
fungi, with a small fraction matching plants or algae (<1%), and
3.7% were left unassigned.
The measures of alpha and beta diversity were similar to
ITS-1, exhibiting a highly diverse fungal community with a wide
range of ascomycetes and basidiomycetes in each sample, and suf-
ficient read coverage (Figs 3, 4, S4b). Between samples, the pri-
mary differences were found between leaf tissues and spores
(Tables S1, S2, S4). The PCA analysis of ITS-2 data from leaf tis-
sues indicated significant differences in fungal species composi-
tion between all time categories (P<0.006), but not between
leaflet and petiole tissues (Fig. 4b). The PCA biplot shows corre-
lation between taxa (Fig. 4b). Taxa that were abundant late in the
season, such as the genera Phyllactinia and Phoma, were positively
correlated with Hymenoscyphus, while taxa that were more abun-
dant early in the season, such as Taphrina,Tilletiopsis,
Cladophialophora, were negatively correlated with
Hymenoscyphus.
(a)
(b)
(c)
Fig. 2 Hymenoscyphus fraxineus DNA amount in ash tissues (ng DNA mg
–1
tissue) sampled throughout the summers of 2011 (a) and 2012 (b), and
the amount of airborne pathogen ascospores at the experimental stand
during 2009–2011 (c), either analyzed by a real-time PCR assay specific
to the DNA of the fungus or by microscopy (spore data). For leaflet
tissues from 2011 and spores, the data are obtained from Hietala et al.
(2013). Calendar days with missing values in (a) and (b) indicate that the
fungus was not detected. Note that, for spores, the sampling covered
only part of the sporulation season in 2009 and 2011. In panel (c) the
continuous line indicates a model fitted to the data, while the dashed
lines show 95% predictive intervals calculated as described in Supporting
Information Methods S1.
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All the c. 13 000 reads identified as Hymenoscyphus were
aligned and evaluated in the GENEIOUS software program. While
the most common genotype showed 100% sequence similarity to
the H. fraxineus type specimen in ITS-2 (Fig. S6), two base pair
changes in one variant were in sufficient quantity to identify a
distinct H. fraxineus assignment. Across most samples, the puta-
tive additional genotype is near the frequency of that of the refer-
ence genotype (Fig. S7).
Seasonal and tissue-specific patterns in sequence
abundance of indigenous fungal species
There was a strong correlation between the H. fraxineus ITS-2
sequence percentages and qPCR-based DNA amounts
(Fig. 5a–c): the Pearson product moment correlation coeffi-
cients between sample datasets for the leaflet, the upper part
of the petiole and the petiole base were 0.83, 0.93 and 0.88,
respectively. Extrapolation of total fungal DNA amount in
the leaf material from 2011 (Fig. 5d) suggested that no major
change took place in July, whereas the total fungal biomass
increased rapidly in all leaf tissues after 2 August, and that by
the end of August the petiole tissues hosted more fungal
biomass than the leaflets.
There was a good overall correspondence between the ITS-1
and -2 datasets as both generally showed a similar seasonal pat-
tern for a given genus (Table S1) –the disparities between the
ITS-1 and ITS-2 datasets were primarily related to the relative
abundance of basidiomycetes in the order Tremellales, (Fig. S5a),
and ascomycetes in the orders Taphrinales, (Fig. S5b),
Chaetothyriales and Helotiales (Fig. S5c).
The vast majority of fungi associated with ash leaves showed
essentially stable read percentages throughout July (Fig. 6; Tables
S1, S2). As exceptions, towards the end of July H. fraxineus
showed significant increases in ITS-2 read percentages in leaflets
and petiole base, the biotrophic genus Exobasidium showed sig-
nificant increase in ITS-1 and ITS-2 read proportions in leaflets,
while the biotrophic genus Phyllactinia showed a nonsignificant
trend of increase in ITS-1 and -2 read percentages in leaflets. By
contrast, the biotrophic genus Taphrina showed a significant
decline in ITS-1 read percentages in leaflets towards the end of
July, while the epiphyte genus Tilletiopsis showed a general
decline in ITS-1 and ITS-2 read percentages in petiole tissues.
Most of the significant changes in ITS read percentages of
fungi associated with ash leaves occurred in August. Besides
H. fraxineus, both epiphytic yeasts (Bullera,Rhodotorula),
biotrophs (Exobasidium,Phyllactinia) and endophytes with
pathogenic potential (Boeremia,Diaporthe,Epicoccum,Fusarium,
Knufia,Phoma,Pleospora) showed significant increases in ITS-1
or -2 read percentages in one or several leaf tissues (Fig. 6; Tables
S1, S2). Genera that showed a significant decline in ITS-1 or -2
read proportions towards the end of summer in one or several leaf
tissues included Aureobasidium,Tilletiopsis,Sporobolomyces and
Taphrina (Table S1).
Fungi that, across the season, showed significant positive corre-
lation with ITS-2 sequence proportions of H. fraxineus in one or
several leaf tissues included Exobasidium,Phyllactinia,Devriesia,
Fig. 3 Overview of fungal taxonomic
diversity of internal transcribed spacer-1
(ITS-1) and ITS-2 results, at the class
taxonomic rank as visualized with the MEGAN
software, and lined up by taxon. The size of
the circle at the tips indicates the relative
number of reads assigned to each sample.
Classes in blue indicate those groups that
were found in ITS-1 but not in ITS-2, and
taxa in red indicate those found in ITS-2 but
not in ITS-1.
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Knufia and Phoma, whereas the genera Aureobasidium,
Tilletiopsis, Sporobolomyces and Taphrina showed significant nega-
tive correlations with ITS-2 sequence proportions of H. fraxineus
(Table S5).
Regarding tissue specificity, the majority of fungi showed rela-
tively similar read percentages between the leaflet and upper and
basal parts of the petiole at any given time (Tables S1, S2).
However, fungi in the ascomycetous genera Naevala,Gyoerffyella
and Phyllactinia showed, across the season, generally higher read
percentages in leaflet than in the petiole tissues. Fungi that
showed generally higher read percentages in one or both of the
petiole tissues than in the leaflet included basidiomycetes in the
genera Exobasidium,Rhodotorula and Phallus and ascomycetes in
the genus Leptosphaeria.
−4 −2 0 2 4
−4 −2 0 2 4
PC1 (27% of Variation)
(a)
(b)
PC2 (15% of variation)
Fusarium
Mrakiella
Rhodotorula
Curvibasidium
Claviceps
Cladophialophora
Colletotrichum
Zymoseptoria
Itersonilia
Gyoerffyella
Dioszegia
Tilletiopsis
Cryptosporiopsis
Davidiella
Exobasidium
Cryptococcus
Naevala
Monographella
Kabatiella
Entyloma
Lalaria
Epicoccum
Phoma
Cystofilobasidium
Boeremia
Steccherinum
Mrakia
Peniophora
Phallus
Udeniomyces
Bensingtonia
Bullera
Erythrobasidium
Sarcinomyces
Microdiplodia
Botrytis
Diaporthe
Ceramothyrium
Aureobasidium
Peyronellaea
Taphrina
Phomopsis
Ascochyta
Endoconidioma
Phyllactinia
Articulospora
Devriesia
Lophiostoma
Leptosphaeria
Sporobolomyces
Dates
29 June−4 July
11−18 July
25 July−12 August
17−25 August
Tissues
Leaf
Upper petiole
Lower petiole
−4 −2 0 2 4
−4 −2 0 2 4
PC1 (27% of Variation)
PC2 (15% of variation)
Fusarium
Mrakia
Rhodotorula
Curvibasidium
Neosetophoma
Knufia
Neofabraea
Ceramothyrium
Zymoseptoria
Malassezia
Acicuseptoria
Dioszegia
Exobasidium
Exophiala
Cryptococcus
Naevala
Occultifur
Pleospora
Kabatiella
Lalaria
Epicoccum
Phoma
Cystofilobasidium
Boeremia
Mrakiella
Capronia
Udeniomyces
Bullera
Tilletiopsis
Erythrobasidium
Cadophora
Hymenoscyphus
Entyloma
Cladophialophora
Phaeoramularia
Aureobasidium
Stagonosporopsis
Peyronellaea
Rhinocladiella
Taphrina
Penicillium
Phomopsis
Cladosporium
Phyllactinia
Articulospora
Devriesia
Phlyctis
Leptosphaeria
Paraleptosphaeria
Sporobolomyces
29 June−4 July
11−18 July
25 July−12 August
17−25 August
Tissues
Leaf
Upper petiole
Lower petiole
Fig. 4 Principal component analysis (PCA)
biplots of 50 most abundant genera of
internal transcribed spacer-1 (ITS-1) (a) and
ITS-2 (b) datasets. The samples are indicated
by symbols and colors, with the symbols
corresponding to the time periods (circles,
sampling dates 29 June and 4 July; squares,
11 and 18 July; diamonds, 25 July and 2 and
12 August; triangles, 17 and 25 August).
Colors indicate the tissue type of each sample
(purple for leaf, green for upper petiole, and
blue for lower petiole). The placement of
generic names indicates the samples with
which they are correlated (e.g.
Hymenoscyphus is correlated with late
season samples in ITS-2 (b)). The arrows
pointing to each genus represent
eigenvectors showing the correlation of one
taxon to another; genera with a small angle
between their vectors are strongly positively
correlated, genera with angles at 180°are
expected to be strongly negatively
correlated, and genera perpendicular to each
other (angles of 90 or 270°) are not
correlated to each other.
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Less than 40% of the fungal genera detected by ITS-1 or
ITS-2 sequencing in leaf tissues in the period 11 July–12 August
2011 were also detected in the sequenced spore samples that had
been captured at the experimental stand by a volumetric air sam-
pler in the previous year (Tables S3, S4). Tilletiopsis was by far
the most common genus in both datasets and showed a decline
in sequence proportion in the spore material towards the end of
the sampling period, similar to that observed in leaf material.
Regarding ITS-2, the stably high read proportions of H. fraxineus
in the spore material at the end of July and beginning of August
coincided with a drastic increase in its read proportions in leaf tis-
sues. The maximum ITS-2 read proportions of genera
Cladosporium and Cladophialophora in the spore material at the
end of the sampling period were in contrast to the decline
observed in their read proportions in leaf tissues. Biotrophs
(Exobasidium,Phyllactinia) generally showed a trend of increase
in read proportions in the spore material towards the end of the
sampling period, which was in line with their increased read pro-
portions in leaf tissues towards autumn. At class level, the average
ITS-1 and ITS-2 sequence proportions for sporulating endo-
phytic genera in Dothideomycetes (Cladosporium excluded),
Eurotiomycetes (Cladophialophora excluded), Helotiales (H. frax-
ineus excluded) and Sordariomycetes were below 0.5%.
Discussion
Dieback of common ash is caused by the invasive ascomycete
Hymenoscyphus fraxineus, which is considered to originate from
Asia. The disease was first observed in Europe in the 1990s and is
currently threatening the existence of common ash across Europe.
An increase in population density is a prerequisite for introduced
species to become widespread and dominant in a new environ-
ment, and the current profiling of fungal community structure in
ash leaves increases our understanding of life cycle traits that con-
tribute to the invasiveness of H. fraxineus in Europe. Through a
combination of approaches, we tracked the colonization pressure
of the pathogen and indigenous fungi in ash leaf tissues, docu-
menting the establishment of first contact between the tree,
indigenous fungi and the pathogen, the following quiescent
phase, and finally the intraspecific fungal competition upon colo-
nization of weakened host tissues.
Methodological considerations
The utility of metabarcoding for accurately estimating species
abundances is unclear, as studies have found both a strong corre-
lation (Amend et al., 2010a) and little correlation (Deagle &
Tollit, 2007; Pompanon et al., 2012) between number of reads
and species abundance. In our study, the absence of H. fraxineus
sequences from the ITS-1 data as a result of its large insertion
serves as an extreme example of barcode length bias affecting the
results, and controlling for this variable produced accurate pre-
dictions for Hymenoscyphus at least. Both ITS-1 and ITS-2 are
associated with many potential biases (Aird et al., 2011; Ihrmark
et al., 2012; Blaalid et al., 2013; Lindahl et al., 2013; Tedersoo
et al., 2015; Wang et al., 2015), and these seem to have a much
greater effect than differences in HTS platform (Yergeau et al.,
2012). In our study, the correlation between ITS-1 and ITS-2
was generally high for abundant species, although it varied for
specific fungal groups. Our results demonstrate that multiple
barcode markers provide a more complete representation of the
fungal community.
Fig. 5 Comparison of Hymenoscyphus fraxineus DNA amount estimates
as determined by real-time PCR (qPCR) and sequencing of internal
transcribed spacer-2 (ITS-2) region (sequence) for ash leaflet (a), petiole
upper (b) and petiole base (c) samples collected in 2011, and estimates of
total fungal DNA amount in the three leaf tissue types (d).
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Changes in fungal community structure during the
asymptomatic phase
Sporulation of H. fraxineus usually starts at the end of June at the
ash stand under study (Hietala et al., 2013). Our data indicated a
continuous accumulation of pathogen biomass in ash leaves from
the initiation of sporulation throughout the season. As symptoms
of necrotic leaf lesions were first observed at the beginning of
August, the interaction period of H. fraxineus with leaves of com-
mon ash clearly involves a long latent phase.
Already in the symptomless period, leaves of common ash
hosted a phylogenetically and functionally highly diverse myco-
biota: epiphytic fungi comprised mostly yeasts, obligatory para-
sites that depend on living plant tissue to complete their life
cycle, and endophytes comprising many facultatively parasitic fil-
amentous ascomycetes (Fig. S5). The same functional groups
were documented in HTS studies of leaf-associated fungal com-
munities in bur oak (Quercus macrocarpa; Jumpponen & Jones,
2009), European beech (Fagus sylvatica; Cordier et al., 2012), ses-
sile oak (Quercus petraea; Vorıskova & Baldrian, 2013) and bal-
sam poplar (Populus balsamifera;Balint et al., 2013).
The strong correlation between the increments of ITS-2
sequence read proportions of H. fraxineus and the qPCR-
based pathogen DNA amount estimates across the season
implies that changes in the biomass of H. fraxineus in ash
leaf tissues were more pronounced than those concerning
coinhabiting fungi. Among co-associated fungi, only the
biotrophic genus Exobasidium showed a significant increase
in read proportion during the asymptomatic phase, whereas
the biotrophic genus Taphrina showed a significant decline.
The peaking of the genus Taphrina early in the growing sea-
son was also observed in leaves of bur oak (Jumpponen &
Jones, 2010).
The general invariability of read percentages of fungi in July
would suggest that during the asymptomatic phase H. fraxineus
accumulates in leaf tissues as quiescent epiphytic and endophytic
thalli that do not induce major physiological changes in host tis-
sue or interact with other fungi to an extent that would influence
the general stability of the fungal community.
Changes in fungal community during the symptomatic
phase
In 2011 the amount of airborne H. fraxineus ascospores at the
experimental stand reached a maximum by mid-July. Necrotic
lesions in ash leaves occurred during the first week of August,
(a)
(b)
Fig. 6 Seasonal changes in internal
transcribed spacer-1 (ITS-1) (a) and ITS-2 (b)
read proportions of Hymenoscyphus
fraxineus, and dominant biotrophic
(Phyllactinia,Exobasidium,Taphrina),
epiphytic (Tilletiopsis) and endophytic
(Phoma anamorph/related teleomorph
genera) fungi in ash leaf tissues.
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coincident with a strong increase in H. fraxineus DNA level and
ITS-2 read proportions in leaflets. The high concentrations of
H. fraxineus DNA detected on necrotic leaf tissues in comparison
to green ash leaf tissues suggest a cause–effect relationship
(Steinb€ock, 2013). The genus Hymenoscyphus belongs to the
order Helotiales, which includes many species that shift between
endophytic and pathogenic growth phases, and Sieber (2007)
postulated that once the density of endophytes exceeds a certain
tissue-specific threshold, the endophytic thalli resume growth
and kill the host tissues.
The total fungal biomass in leaf tissues was extrapolated from
H. fraxineus qPCR quantities and ITS-2 sequence proportions,
because H. fraxineus has a generally average genome size for fungi
(http://www.zbi.ee/fungal-genomesize). However, the copy num-
ber of ITS rDNA gene cluster can vary between species, and
therefore these results need to be considered as relative and rough
estimates. Similar variation in species-specific conversion factors
also applies to the more conventional fungal biomass assays based
on chitin and ergosterol assays (e.g. Eikenes et al., 2005). The
extrapolated vigorous increase in total fungal biomass in ash leaf
tissues after formation of necrotic leaf lesions during the first
week of August in 2011 implies that a range of fungi resumed
growth in weakened tissues. The fungi that showed significant
increases in read percentages in one or several leaf tissues after the
first week of August included saprophytic epiphytes (Bullera) and
biotrophs (Phyllactinia) (Fig 4b), coincident also with their read
percentage increases in the spore samples towards autumn. The
powdery mildew fungus Phyllactinia fraxini is a widespread asso-
ciate of common ash and other ash species (Braun, 1995). The
other fungi that increased significantly in read percentages in one
or several leaf tissues after the first week of August included endo-
phytes having pathogenic potential (Fusarium,Pleospora, the
anamorph genus Phoma and related teleomorph genera Boeremia
and Epicoccum), but these were hardly detected in the spore mate-
rial. The amount of fungal biomass typically increases in leaves of
deciduous trees towards autumn (e.g. Vorıskova & Baldrian,
2013). Before ash dieback appeared in Germany, fungal isola-
tions throughout the season from healthy leaves of common ash
showed a trend of increase in isolation frequency of these endo-
phytes towards autumn (Reiher, 2011). In the current epidemic
stage of ash dieback, the foliage of all ash trees at a stand become
obviously infected by H. fraxineus, and it is difficult to assess to
what extent a late-summer increase in biomass of an endophyte is
triggered by natural host senescence. The increase of read propor-
tions of resident endophytes directly after the escalation of
H. fraxineus biomass in leaves and the formation of leaf necrotic
lesions is presumably at least partly triggered by host tissue weak-
ening by H. fraxineus.
Dimorphic ascomycetes in the genus Aureobasidium are com-
mon epiphytes in the phyllosphere of trees, common ash
included (Slavikovaet al., 2007), and tend to increase in fre-
quency across the season (e.g. Jumpponen & Jones, 2010). In the
present study, this genus showed a significant decline in sequence
read percentages in the leaflet and petiole base after the first week
of August. The most detailed studies on the association of
Aureobasidium species with trees are in relation to apple, where
these fungi show increased colonization on leaf veins across the
season (McGrath & Andrews, 2006). While their spatial localiza-
tion on ash leaves remains to be established, a primary localiza-
tion on leaf veins could mean competition with the vein specialist
H. fraxineus, and could account for the observed decline in
sequence proportion of this genus.
The significance of primary inoculum in the invasiveness of
H. fraxineus
The observed high propagule pressure of H. fraxineus is typical of
invasive species (Simberloff, 2009; Hamelin et al., 2016). The
low presence of functionally related native endophytes in the
spore material during the peak sporulation period of H. fraxineus,
and the general increase of sequence proportions of these fungi in
leaf tissues right after the development of necrotic lesions are con-
sistent with a life cycle that involves early-season establishment
by a small primary inoculum followed by a quiescence phase,
eventual resumption of growth, and production of secondary
inoculum in weakened host tissues. Species of Aureobasidium,
Phoma and Fusarium have been detected in buds of common ash
during winter (Chen, 2011), suggesting that their primary inocu-
lum to leaf infection may originate from propagules that over-
winter in meristematic tissues. Based on our DNA level profiling
of H. fraxineus in planta, the capacity of this fungus to produce
symptoms on leaves depends on a large inoculum. In this respect,
the aggressiveness of this fungus may well be comparable to many
common ash endophytes, but it is presumably compensated by
the huge primary inoculum of this invader. This would be in line
with the propagule pressure theory (Lockwood et al., 2005) that
is used to explain the success of invasive species. Sustained invest-
ment in the mid-season production of ascopores may enable
H. fraxineus to overcome defense responses of common ash and
to challenge the resident endophyte competitors, whose density
in leaf tissues is still low at this time in summer. We propose that
seasonal and quantitative differences in sporulation between
H. fraxineus and indigenous competitors facilitate the invasive-
ness of this pathogen. There are very few other studies available
that have compared the propagule pressure of invasive fungal
plant parasites and their native competitors. When monitoring
the spread of Heterobasidion irregulare, a North American conifer
pathogen introduced to central Italy, Garbelotto et al. (2010)
concluded that a seasonal difference in spore production between
this invader and a native competitor facilitates the establishment
and spread of this alien species. While mate limitation impacts
the spreading rate of outcrossing fungal plant pathogens
(Hamelin et al., 2016), the equal frequency of occurrence of the
two mating types in European populations of H. fraxineus (Gross
et al., 2012b) has obviously facilitated the rapid spread of
H. fraxineus.
The role of diseased and asymptomatic ash individuals in
the pathogen life cycle
To examine the relationship between tree health condition and
pathogen growth, in 2012 we sampled leaves from ash trees with
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no crown symptoms and trees that showed shoot dieback. Both
phenotypes showed comparable rates of pathogen DNA accumu-
lation in leaf tissues, suggesting that the difference in the degree
of shoot symptoms cannot be a result of tree-specific variation in
pathogen leaf colonization. As H. fraxineus ascomata form on
overwintered leaf tissues, this implies that symptom-free trees also
support the build-up of infection pressure within a forest stand,
an observation that may have ramifications for ash management
strategies in Europe. Considering the apparently low aggressive-
ness of H. fraxineus, it seems likely that, following a local intro-
duction, several years are required to build up a propagule
pressure that is sufficient for this fungus to cause disease. Such a
scenario would explain the wave-like spread of ash dieback at the
invasion frontier (e.g. Hamelin et al., 2016).
Future prospects
According to the current model, infection of ash leaf tissues by
H.fraxineus ascospores is followed by mycelial spread through the
petiole into twigs and shoots to cause shoot dieback (Gross et al.,
2014a, and references therein). In the unusually cold summer of
2012, accumulation of H. fraxineus DNA in leaf tissues appeared
delayed by 2–3 wk compared with 2011, presumably because of
delayed onset of sporulation. It remains to be clarified how varia-
tion between years and different climatic regions affect propagule
pressure and success of shoot infection by H. fraxineus.
The huge propagule pressure exerted by H. fraxineus can be
envisaged to result in leaf colonization by a large number of genets.
This scenario is supported by the fairly similar frequency of two
ITS-2 sequence variants of H. fraxineus in all ash leaf tissues by the
end of the season. The territorial behavior of H. fraxineus is mani-
fested as vegetative incompatibility in nonself confrontations
(Brasier & Webber, 2013). Besides interactions with indigenous
fungi, the interactions between the different genets of H. fraxineus
need to be explored in order to increase further our understanding
of factors that contribute to invasion success of this pathogen.
Acknowledgements
We thank Inger Heldal for excellent technical assistance, Jørn-
Frode Nordbakken for help with statistical analyses, the Norwe-
gian University of Life Sciences for placing their ash forest at our
disposal, and the Research Council of Norway (grants 203822/
E40 and 235947/E40), Norwegian Institute of Bioeconomy
Research and Erasmus Student Mobility for Placements Program
(grants to B.R. and K.W.) for financial support.
Author contributions
V.T., N.E.N., I.B., H.S. and A.M.H. designed the research and
collected the material, T.C. and H.K. performed ITS-1 sequenc-
ing and J.H.S. and A.V-S. performed ITS-2 sequencing. B.R.
and K.W. performed DNA extraction and qPCR. H.C. carried
out all bioinformatics analyses, and, together with N.E.N. and
A.M.H., performed the statistical analyses. H.C. and A.M.H.
wrote the first version of the manuscript and revised it based on
comments from all other co-authors.
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Supporting Information
Additional Supporting Information may be found online in the
Supporting Information tab for this article:
Fig. S1 Monthly precipitation and mean temperature in 2011
and 2012.
Fig. S2 Damage assessment of sampled ash trees in early (22
June) and late (23 August) summer 2012.
Fig. S3 Hymenoscyphus fraxineus DNA amount in tissues col-
lected from healthy and diseased ash trees throughout the sum-
mer 2012 and analyzed by a qPCR assay specific to the DNA of
the fungus.
Fig. S4 Alpha rarefaction curves of individual samples for next
generation sequencing ITS-1 and ITS-2 datasets.
Fig. S5 Seasonal changes in ITS-1 and ITS-2 read proportions of
fungal taxa at the experimental stand.
Fig. S6 Portion of nucleotide alignment of Hymenoscyphus ITS-2
sequences including a putative new undescribed genotype of H.
fraxineus.
Fig. S7 Seasonal changes in H. fraxineus DNA amount, deter-
mined by qPCR and by ITS-2 read percentages of the two main
sequence variants assigned to this species.
Table S1 The most common fungal genera present in both ITS-
1 and -2 datasets, and their sequence proportions (%) in leaf tis-
sues in early summer (29 June–11 July), mid-summer and late
summer (12–25 August) in 2011
Table S2 The most common fungal genera detected in one ITS
dataset only and their sequence proportions in leaf tissues in early
summer (29 June–11 July), mid-summer (18 July–02 August)
and late summer (12–25 August) in 2011
Table S3 ITS-1 sequence percentages of fungi detected in air
samples captured by a volumetric spore sampler at the experi-
mental ash stand in 2010
Table S4 ITS-2 sequence percentages of fungi detected in air
samples captured by a volumetric spore sampler at the experi-
mental ash stand in 2010
Table S5 Pearson product moment correlation between ITS-2
sequence proportions of Hymenoscyphus fraxineus and the most
common fungal general in different ash leaf tissues across the sea-
son 2011
Methods S1 Detailed laboratory, bioinformatics and statistical
analyses.
Please note: Wiley Blackwell are not responsible for the content
or functionality of any Supporting Information supplied by the
authors. Any queries (other than missing material) should be
directed to the New Phytologist Central Office.
Ó2016 The Authors
New Phytologist Ó2016 New Phytologist Trust
New Phytologist (2016)
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New
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