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Received: 29 November 2018    Revised: 6 May 2019    Accepted: 20 May 2019

DOI: 10.1111/efp.12528

ORIGINAL ARTICLE

The first record of Botryodiplodia canker in Poland

Czesław Bartnik1  | Piotr Boroń1  | Jakub Michalcewicz2  | Michał Ciach3

1
Department of Forest Pathology,
Mycology and Tree Physiology, Faculty Abstract
of Forestry, University of Agriculture in This study describes the first observation of Botryodiplodia canker in the Western
Kraków, Kraków, Poland
2 Carpathians in south‐eastern Poland caused by Botryodiplodia hypodermia (Sacc.) Petr.
Department of Forest Protection,
Entomology and Forest Climatology, Faculty (syn. Sphaeropsis hypodermia, S. ulmicola). The canker occurred on an approximately
of Forestry, University of Agriculture in
17‐year‐old Ulmus glabra sapling in a mixed conifer/deciduous stand with elm trees
Kraków, Kraków, Poland
3
Department of Forest Biodiversity, Faculty
severely damaged by Dutch elm disease. This paper describes disease symptoms and
of Forestry, University of Agriculture in provides information on the macro‐ and micromorphology of the fungus isolated from
Kraków, Kraków, Poland
the cankered tissues. The results of BLAST search using DNA sequences obtained for
Correspondence our cultures and subsequent phylogenetic positioning of the fungus among closely
Piotr Boroń, Department of Forest
Pathology, Mycology and Tree Physiology,
related Botryosphaeriaceae indicate that the species is much more closely related to
Faculty of Forestry, University of Agriculture Phaeobotryon than to the other Botryodiplodia or Sphaeropsis species. Moreover, a
in Kraków, al. 29 Listopada 46, 31 – 425
Kraków, Poland.
total of 16 polymorphisms within the ITS region were detected between S. ulmicola
Email: p.boron@ur.krakow.pl associated with Botryodiplodia canker in North America and B. hypodermia associated

Funding information
Ministry of Science and Higher Education of
The Republic of Poland; statutory research
mechanism; Faculty of Forestry; University
of Agriculture in Cracow under the research,
Grant/Award Number: DS 3414
Editor: Thomas Sieber
with the canker observed in Poland. Thus, the “European” variant of “Sphaeropsis”
ulmicola can now be easily identified with our barcode sequences. The Botryodiplodia
canker is much less prevalent in Europe than in North America. Differences in viru‐
lence of “American” and “European” linages and differences in susceptibility of vari‐
ous elm species may be the reason for the higher prevalence of the disease in North
America.

KEYWORDS
Botryodiplodia ulmicola, Botryosphaeriaceae, elm canker, Sphaeropsis ulmicola, Ulmus, wych elm

1 |  I NTRO D U C TI O N
The two most devastating diseases affecting elms worldwide are elm
yellows caused by elm phlöem necrosis mycoplasm and Dutch elm dis‐
ease caused by Ophiostoma ulmi and O. novo‐ulmi (Boudon‐Padieu
et al., 2004; Harwood, Tomlinson, Potter, & Knight, 2010; Thomas,
Stone, & La Porta, 2018; Webber, 2014). Elms may suffer also from
a number of cankers occurring on stems and/or on twigs. These are,
among others, caused by fungi of the Nectria, Neonectria, Diothiorella,
Cytospora, Phomopsis genera and fungal‐like Phytophthora. The list
of elm diseases in Poland is far from complete (Mańka, 2005). This
paper describes a new addition to this list: the Botryodiplodia canker
caused by Botryodiplodia hypodermia (syn. Sphaeropsis hypodermia,
S. ulmicola), identified for the first time on Ulmus glabra in Poland.
According to Riffle (1981), the bark tissue infected with B. hy‐
podermia turns reddish brown to brownish black and becomes
water‐soaked and very soft. The lesion edge is marked by a distinct
demarcation line between living and necrotic bark tissue. The cam‐
bial and sapwood tissues directly beneath infected bark become
reddish brown. Normally, this discoloration extends to the demar‐
cation line. Above the cankers, foliage becomes chlorotic, wilts and
dies. Eventually, the entire section of the stem distal to the girdling
canker dies. Pycnidia develop in patches of dying and dead bark tis‐
sue near canker margins, usually in autumn. Bark wounds are the
entry points for natural infections by other pathogens. Cankers re‐
sulting from infections during the peak of growing season usually
develop rapidly due to the increased demand for water. Early spring,
late autumn and winter infections are usually less severe creating

Forest Pathology. 2019;00:e12528. wileyonlinelibrary.com/journal/efp © 2019 Blackwell Verlag GmbH  |  1 of 6

https://doi.org/10.1111/efp.12528
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2 of 6       BARTNIK et al.
small cankers that are callused over during the following growing
season (Riffle, 1978).
The Botryodiplodia canker was detected in the study site of the
endangered rosalia longicorn (Rosalia alpina) located in the Dutch
elm disease‐affected elm stands in the Beskid Niski Mts (Western
Carpathians, SE Poland). Multiple pathogens were detected in the
studied stands (Bartnik, Michalcewicz, & Ciach, 2015), but a single
tree harboured a characteristic perennial canker reminiscent of a
Botryodiplodia canker. This paper presents a detailed description of
the disease symptoms and a morphological and molecular character‐
ization of cultures isolated from this canker.
2 |  M ATE R I A L A N D M E TH O DS
The study was conducted in 2014 in Beskid Niski Mts (Western
Carpathians) in a ca. 70‐year‐old mixed beech/elm‐dominated stand
(Dentario glandulosae‐Fagetum:30% wych elm, 30% European beech
Fagus sylvatica and 10% for each of sycamore maple Acer pseudo‐
platanus, common ash Fraxinus excelsior, silver fir Abies alba and
Scots pine Pinus sylvestris) (Bartnik et al., 2015). The wych elm trees
were heavily weakened by Dutch elm disease or already dead, and
a number of dead elm trees were removed from the stand what was
evidenced by numerous stumps and confirmed by the forest service.
The history of the stand is not well defined. While European beech
and silver fir occurred mostly due to natural regeneration, other
species were planted according to management plans. The origin of
wych elms seedlings is not known, and they could either come from
seeds collected locally or be traded from regional tree nurseries.
A single wych elm tree with Botryodiplodia‐like canker was ob‐
served on one of the Rosalia longicorn study sites in the vicinity of
Myscowa village (on the Łysa Góra mountain), Dukla Forest District,
southern Poland (49°33′0.00′′N; 21°33′30.00′′E). It occurred on an
approximately 17‐year‐old sapling showing decline symptoms with
the most apparent decreased twig growth resulting in a small num‐
ber of branches. The most probable cause for these symptoms was
the perennial canker girdling approximately half of stem circumfer‐
ence. No readily visible bark beetle galleries were noticed on the
studied tree nor Dutch elm disease characteristic xylem discolor‐
ation of the last growth ring. The trunk section with the canker was
dissected and transported to the laboratory for detailed examination
of the symptoms. Fungal isolations were carried out within 48 hr.
Twelve wood fragments (ca. 5 × 5 × 1 mm) were excised from the
vicinity of the canker under sterile conditions and placed on Petri
dishes containing PDA + T medium (potato dextrose agar, 200 mg/L
tetracycline). All wood fragments were dissected directly from black
or blue‐black smudges visible on the cross‐section of the trunk, ca.
1.5–2.0 cm from the trunk edge and ca. 13 cm below the edge of the
canker. Petri dishes were incubated at room temperature for 7 days
in the dark. Initially, the acquired isolates were identified morpho‐
logically and Botryodiplodia‐like cultures were subjected to detailed
observations for a period of 6 months, followed by sequence‐based
identification. To confirm the ability of cultures to grow on woody
substrate, representative isolates were inoculated on sterilized frag‐
ments of wych elm twigs (⌀ ca. 1 cm, 10 cm long). A portion of my‐
celium‐overgrown PDA + T medium was placed in a notch made on a
twig fragment with a sterile scalpel and incubated in Erlenmeyer flask
covered with cotton wool plug at room temperature. Observations
of both culture development and growth of fungi on elm wood were
carried out for 6 months. All microstructure observations and mea‐
surements were made with either Zeiss Discovery Stereomicroscope
or Zeiss Axiophot light microscope using differential interference
contrast.
Mycelium from individual cultures (⌀ ca. 2 cm) and a culture of
CBS strain 174.63 of B. hypodermia were collected with a sterile scal‐
pel, placed in 2‐ml microcentrifuge tubes with three stainless steel
beads and ca. 0.2 cm3 of bleach‐washed sand and homogenized in a
Retsch MM 200 oscillation mill (Retsch) for 5 min at 25 Hz. Next, a
modified CTAB method (Gawel & Jarret, 1991) was used to extract
the total genomic DNA from the homogenized mycelium samples.
Resulting DNA precipitates were re‐suspended in 50 μl of nuclease‐
free water. 100‐fold diluted (20–100 ng/μl) DNA extracts were used
as templates in PCR amplifications.
In this study, we amplified four barcode DNA regions for a sin‐
gle representative isolate acquired from cankered wood and for CBS
strain 174.63: the ITS region with primers ITS1F (Gardes & Bruns,
1993) and ITS4 (White, Bruns, Lee, & Taylor, 1990), the IGS re‐
gion with primers IGS‐12a and NS1R (Carbone & Kohn, 1999), an
actin gene fragment (ACT) with primers ACT‐512F and ACT_783R
(Carbone & Kohn, 1999) and a calmodulin gene fragment (CAL) with
primers CAL‐228F and CAL‐778R (Carbone & Kohn, 1999). All tar‐
get sequences were amplified in a total volume of 50 μl using the
same reaction mixture and cycling conditions. Reaction mixtures
contained: 1× DreamTaq Green buffer (Thermo Fisher Scientific),
4.5 mM MgCl2, 0.12 mM of each dNTPs, 0.08 μM of each primer
and 1 U of DreamTaq DNA polymerase (Thermo Fisher Scientific).
All amplifications were run in a T100 Thermal Cycler (Bio‐Rad) pro‐
grammed for 5 min of initial denaturation at 94°C, 25 touchdown
cycles of 30 s at 94°C, 30 s at decreasing annealing temperatures
(0.5°/cycle from 67.5°C in the 1st to 55°C in the 25th cycle), 1 min at
72°C and 20 cycles with 30 s at 94°C, 30 s at 55°C and 1 min at 72°C
followed by 10 min at 72°C of final extension. PCR effectiveness
was verified in agarose gel, and positive amplification products were
purified with a Clean‐Up DNA Purification Kit (A&A Biotechnology).
All target fragments were sequenced in both directions using PCR
primers with reagents of BigDye Terminator v3.1 Cycle Sequencing
Kit (Life Technologies) and T100 thermal cycler, and 3,500 Series
Genetic Analyser (Life Technologies) was used to detect sequencing
results.
Sequences were processed with ChromasPro 1.6 software
(Thechnelysium) and queried against the NCBI GenBank database
with the BLAST search tool to retrieve the most similar sequences. As
BLAST search produced inconclusive results, we decided to employ
phylogenetic approach to rectify further the species identification
of our isolates. Apart from our sequences, the phylogenetic analysis
included species of Alanphillipsia, Barriopsis, Diplodia, Lasiodiplodia,
BARTNIK et al.
Phaeobotryon and Sphaeropsis. Sequences of Aplosporella hesperidica
and Septorioides pini‐thumbergii were included as an outgroup. The
complete list of species used with corresponding GenBank accession
numbers is presented in Table S1.
The phylogenetic analysis employed three DNA fragments: the
ITS region, partial sequence of 28S rRNA gene (LSU) and partial se‐
quence of the translation elongation factor 1α (TEF1). Sequences for
all three regions were aligned independently with MAFFT v 7 (Katoh,
Rozewicki, & Yamada, 2017) and refined manually. The resulting ITS
and TEF1 alignments contained highly variable regions and/or com‐
plex overlapping gaps that could not be aligned with high confidence.
These regions were deleted from both, ITS and TEF1 alignments,
prior to the analysis. The pattern of gaps in ITS alignment observed
among our isolate and most similar taxa (B. hypodermia/S. ulmicola
and Phaeobotryon sequences) proved to be much clearer. Gap (indel)
pattern for these taxa was encoded as a present/absent binary ma‐
trix with FastGap 1.2 (Borchsenius, 2009). For the rest of taxa, the
gap regions in ITS alignments were treated as missing data. Thus, our
analysis included three sequence alignments: ITS, LSU and TEF1 and
the gap matrix for ITS sequences that were analysed jointly in a single
run as separate data partitions. All three alignments were tested for
the best‐suited substitution models with jModelTest 2.1.5 (Darriba,
Taboada, Doallo, & Posada, 2012) limiting the number of substitu‐
tion shames to three to allow only evolutionary models implemented
in MrBayes 3.1.2. According to Bayesian information criterion, the
best evolutionary models were as follows: K2P + G (ITS), K2P + I+G
(LSU) and HKY + G (Tef1). MrBayes (Huelsenbeck & Ronquist, 2001),
a Bayesian inference dedicated program, enables to analyse jointly
sequence and binary data using Markov chain Monte Carlo (MCMC)
methods to estimate both the phylogeny itself and posterior proba‐
bility support of clades. The program settings were as follows: two
independent runs with four MCMC chains each (three heated and
one cold), temperature of cold chain: 0.2, burn‐in fraction: 0.25, sam‐
pling frequency: every 100th tree from both runs. The analysis was
run until the standard deviation of split frequencies stop‐value of
0.01 was reached. After the analysis, a 50% majority condensed tree
was generated and visualized in the MEGA 6.06 (Tamura, Stecher,
Peterson, Filipski, & Kumar, 2013) tree‐drawing tool. All alignments
and matrixes used in the analysis, as well as the resulting tree, were
deposited in TreeBASE phylogenetic data repository, available under
the accession URL: http://purl.org/phylo/​treeb​ase/phylo​ws/study/​
TB2:S23569.
3 | R E S U LT S
The observed Botryodiplodia canker was 6.5  cm long and 5.0  cm
wide, and it occurred on the 5.2‐cm‐wide stem that widened to
6.0 cm at the canker opening (Figure 1). A distinct layer of the callus
tissue formed at the rim of the opening surrounding the area of grey‐
coloured dead wood. A gallery of a xylophagous insects (probably
Cossus cossus, Lepidoptera, Cossidae) occurred directly on the edge
of the canker.
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(a) (b)
(c)
F I G U R E 1   Botryodiplodia canker on a stem of wych elm (a),
Botryodiplodia hypodermia culture on PDA + T after 1‐month
incubation at room temperature and ca. 1‐year storage at 4°C (b),
conidium of B. hypodermia (c)
All wood fragments used for fungal isolation and a CBS strain
174.63 of B. hypodermia initiated growth of identical fast growing,
blue‐black, woolly cultures. The representative culture was depos‐
ited in CBS KNAW culture collection. The same type of mycelium
was observed on the surface of inoculated twig fragments. After
3 months, multiple black spherical pycnidia, ⌀ ca. 80 µm, occurred
both, in cultures and on the bark of the inoculated twig sections.
When crushed in a droplet of water on microscope slide, the pycnidia
released unicellular conidia, 25.0‐30.0 μm × 14.0‐18.0 μm, on aver‐
age 28.2 μm × 15.6 μm. The numbers of the released conidia were,
however, very small.
All four barcode sequences, ITS, IGS, ACT and CAL, of the rep‐
resentative isolate from the wood fragment were successfully am‐
plified and sequenced (GenBank accession numbers: MK134682 for
ITS, MK134684 for ACT, MK134686 for CAL and MK134688 for
IGS). The sequences of all four barcode regions were 100% iden‐
tical with those of CBS strain 174.63 of B. hypodermia (GenBank
accession numbers for the CBS strain 174.63 are: MK134681 for
ITS, MK134683 for ACT, MK134685 for CAL and MK134687 for
IGS). Blast search for the ITS region resulted in 99.8% match (sin‐
gle polymorphism per 530‐bp‐long ITS region) with two acces‐
sions: KF800390, an environmental dust sample collected from
indoor air in Kansas City, Missouri, USA (Rittenour et al., 2014), and
KX464232—a sequence deposited as Phaeobotryon sp., culture col‐
lection strain CBS: 123.30 isolated originally in Massachusetts, USA
(Yang et al., 2017). The closest accession submitted with the species
name, 97.4% similarity, was AF243398—S. ulmicola (Zhou & Stanosz,
2001). The three remaining barcode sequences of the isolate pro‐
duced no significant match in the BLAST analysis, that is, the similar‐
ity score was <90%.
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4 of 6       BARTNIK et al.

Phaeobotryon mamane CBS 122980

100
Phaeobotryon mamane CPC 12442
55 Phaeobotryon mamane CPC 12264
Phaeobotryon cupressi CBS 124700
72
100 Phaeobotryon cupressi CBS 124701

Phaeobotryon negundinis MFLUCC 15-0436

95 Phaeobotryon negundinis CAA799
Phaeobotryon
Phaeobotryon sp. NEFU817
85
Phaeobotryon sp. JC-2017a
Phaeobotryon sp. LJR0320
99
Phaeobotryon rhois CFCC 89662
96 Phaeobotryon rhois CFCC 89663
Phaeobotryon sp. 177
ʻPhaeobotryonʼ sp. CBS 123.30
Uncultured air sample CMH299
83
96
ʻSphaeropsisʼ ulmicola 94-13 Botryodiplodia hypodermia
Botryodiplodia hypodermia
Botryodiplodia hypodermia CBS 174.63
100 Bariopsis fusca CBS 174.26
Bariopsis tectonae CBS 137786
99 Bariopsis iraniana MFLUCC 14-1190 Bariopsis
99 Bariopsis iraniana CBS 124698
100 Bariopsis iraniana CBS 124699
Alanphillipsia aloes CPC 21298
Alanphillipsia
100
Alanphillipsia aloeigena CBS 136408
Oblongocollomyces variabilis CBS 121775
100
Oblongocollomyces variabilis CMW 25423
100 Oblongocollomyces variabilis CBS 121774 Oblongocollomyces
Oblongocollomyces variabilis CBS 121776
Sphaeropsis euc ola MFLUCC 11-0579
100
Sphaeropsis euc ola MFLUCC13-0701
70 Sphaeropsis euc ola MFLUCC 11-0654
Sphaeropsis citrigena ICMP 16812
98 Sphaeropsis citrigena ICMP 16818

82 99 Sphaeropsis porosa CBS 110496 Sphaeropsis

Sphaeropsis porosa STE-U 5046
Sphaeropsis visci CBS 186.97
89
Sphaeropsis visci CBS 122527
99 Sphaeropsis visci CBS 100163

Sphaeropsis visci CBS 122526

Diplodia CBS 302.36
Diplodia CJK1
97
Diplodia CBS 112553
Diplodia pyri CBS 121862
82
Diplodia sp. CBS 125.37
Diplodia agrifolia CBS 132777
98 100 Diplodia agrifolia CBS 132841
90 Diplodia agrifolia CBS 124.30

Diplodia africana CBS 120835

Diplodia olivarum CBS 121887
81
Diplodia rosulata CBS 116472
100 Diplodia rosulata CBS 116470

Diplodia sp. CBS 139667

Diplodia
85
68 Diplodia allocellula CBS 130408
94 Diplodia citricarpa CBS 124715
98
Diplodia intermedia CBS 124134
81
Diplodia sapinea CBS 109726
99 Diplodia sapinea CBS 109727
96 58 Diplodia sapinea CBS 109725

Diplodia tsugae CBS418.64

99 Diplodia cupressi CBS 261.85
100 Diplodia cupressi CBS 168.87

Diplodia c ola CBS 112076

95
Diplodia sp. CBS 533.87
100
Diplodia gallae CBS 212.25
99
Diplodia gallae CBS 211.25
Lasiodiplodia pseudotheobromae CBS 374.54
100 Lasiodiplodia pseudotheobromae CBS 116459 Lasiodiplodia
100 Aplosporella hesperidica CBS 732.79

100
Aplosporella hesperidica CBS 208.37 Outgroup
Septorioides pini-thunbergii CBS 473.91
×3

0.05

F I G U R E 2   Phylogenetic positioning of Botryodiplodia hypodermia among related Botryosphaeriaceae genera based on the sequences of
ITS, LSU and TEF1 regions: 50% majority tree generated by combining 31,142 trees sampled in 1,557,000 iterations of Bayesian inference.
Posterior probability support values for each clade are indicated at the nodes, and scale bar indicates 0.05 estimated substitutions per site.
The list of taxa in the tree with GenBank accession numbers for particular sequences is provided in Table S1
BARTNIK et al.
The ITS sequences mentioned above were used in Bayesian
phylogenetic reconstruction, and the resulting tree is presented
in Figure 2. The main feature of this tree is that all included gen‐
era: Alanphillipsia, Barriopsis, Diplodia, Lasiodiplodia, Phaeobotryon
and Sphaeropsis, form well‐defined monophyletic clades. The same
is true for the B. hypodermia clade that included sequences for:
Phaeobotryon sp. CBS 123.30, S. ulmicola isolate 94–13, B. hypo‐
dermia CBS 174.63, an uncultured air sample clone CMH299, and
our isolate. This clade was directly grouped, with high support, with
Phaeobotryon clade as its sister group.
4 | D I S CU S S I O N
The high level of damage to elm trees resulting from B. hypoder‐
mia (syn. S. hypodermia, S. ulmicola) infections has been primarily
reported from North America (Krupinsky & Cunningham, 1993;
Riffle, 1978, 1981). According to the Fungal Database provided by
Farr and Rossman (2018), the range of this pathogen covers North
America. This record was obviously not complete as at least one cul‐
ture from 1963 of B. hypodermia from Helsinki, Finland, is available in
CBS KNAW culture collection. Another culture of the species prob‐
ably from Europe was deposited in the CBS collection, but without
specified location. The literature lacks, however, any report of the
Botryodiplodia canker either in Poland or in the whole range of wych
elm in Europe.
The canker morphology generally followed the descriptions in
the literature (Riffle, 1978, 1981). The significant difference of our
specimen was its close association with the feeding gallery of a
xylophagous insect. It is, however, impossible to determine based
on the specimen whether the damage made by the insect was the
starting point for the fungal infection or the insect used the canker
opening to start a gallery. No symptoms of Dutch elm disease, that
is, brown xylem discoloration in the last growth ring, were present
on the studied tree at the canker level. We observed instead black
or blue‐black irregularly shaped smudges in the sapwood (no heart‐
wood formed yet) visible on the cross‐section of the trunk. A direct
connection between Dutch elm disease and the occurrence of the
Botryodiplodia canker is therefore unlikely. However, the ubiquitous
prevalence of Dutch elm disease in the stand certainly lowered the
vigour of many trees, leading to increased susceptibility to second‐
ary pathogens such as the canker disease described here.
Despite the fact that the canker specimen was relatively old (with
weathered wood surface and well‐developed callus rim), it harboured
a viable fungus that could be easily isolated using standard media.
It took approximately 3 months for cultures to sporulate both on
PDA + T and on sterilized twig. The sizes of both pycnidia and conidia
fall well within the range reported for B. hypodermia (Ellis & Everhart,
1891, 1903; Saccardo, 1892). The sequence‐based identification of
the isolates was, however, somewhat problematic. Two very similar
ITS sequences were identified in BLAST search, both originating from
North America. The first was the ITS region cloned from an airborne
spore collected as a dust sample. This sequence was submitted as
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“uncultured environmental sample,” that is, without the species name
and/or disease symptoms description. Thus, except for the sequence,
no other comparisons with our fungus were possible. Another strain
with a highly similar ITS sequence was strain CBS 123.30, originally
deposited as Botryosphaeria stevensii/Diplodia mutila. However, this
name cannot be correct as the similarity of ITS sequences between
this strain and the D. mutila type culture (NR_144906) is only 93%.
The GenBank accession for the CBS 123.30 strain was made by Yang
et al. (2017) as Phaeobotryon sp., who used this name based on the
results of their phylogenetic analysis: the species was in a sister po‐
sition to all Phaeobotryon species in their phylogenetic tree. No cul‐
tures of B. hypodermia/S. ulmicola were, however, included in their
analysis. The same arrangement, that is, the position of B. hypoder‐
mia clade (instead of a single sequence) at the base of Phaeobotryon
clade, was reproduced in our phylogeny. This obviously puts B. hypo‐
dermia/S. ulmicola lineage outside Sphaeropsis clade and proves that
its classification within the genus Sphaeropsis is incorrect.
The level of difference between the Phaeobotryon and B. hypo‐
dermia clades is substantial, it amounted to 25 polymorphic sites
between the two most similar representatives of both clades in the
ITS region only. The discussion whether or not it constitutes suffi‐
cient divergence to justify describing a new genus to accommodate
B. hypodermia/S. ulmicola linage or transferring it to Phaeobotryon as
Yang et al. (2017) did, far exceeds the scope of this short report and
deserves much more attention in another study involving detailed
morphological and molecular examination of all available cultures.
Another problem to investigate in a more detailed study is the diver‐
gence within the B. hypodermia clade itself. The group is comprised
of at least two lineages separated by minimum 16 polymorphic sites
within the ITS region. The first is “Sphaeropsis” ulmicola represented
by an isolate acquired by Zhou and Stanosz (2001). It is recognized
as the cause of Botryodiplodia canker of elms in North America and
most probably reported and/or studied by (Farr & Rossman, 2018;
Krupinsky & Cunningham, 1993; Riffle, 1978, 1981). Strains CBS
174.63, CBS 123.30, uncultured spore picked by Rittenour et al.
(2014) and our isolates represent the second lineage occurring both
in Europe and in North America.
Despite the controversy regarding the correct name for the
causal fungus, the fact remains that Botryodiplodia canker was de‐
tected for the first time in Poland. Without proper survey, it is im‐
possible to determine the prevalence of this disease but it appears to
be rare, only a single specimen was identified throughout the entire
stand. North American reports (Krupinsky & Cunningham, 1993;
Riffle, 1978) suggest that in proper conditions Botryodiplodia canker
caused by “Sphaeropsis” ulmicola has the potential to severely dam‐
age elm stands. No such situation was observed in our case. One of
the reasons for this difference may be the masking effect of Dutch
elm disease, whose prevalence is much more widespread and dis‐
ease symptoms more pronounced. Another reason may be the dif‐
ference in pathogenicity of the “European” lineage of “Sphaeropsis”
ulmicola and/or lower susceptibility of wych elm. The study aiming to
investigate these factors is currently under way, and the results will
be described in the follow‐up paper.
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Education of The Republic of Poland, statutory research mechanism, bbx108
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Faculty of Forestry, University of Agriculture in Cracow under the
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Mańka, K. (2005). Fitopatologia leśna, 6th ed. Warszawa, Poland:
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