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Cryphonectria carpinicola sp. nov. Associated with hornbeam decline in Europe

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Since the early 2000s, reports on declining hornbeam trees (Carpinus betulus) are spreading in Europe. Two fungi are involved in the decline phenomenon: One is Anthostoma decipiens, but the other etiological agent has not been identified yet. We examined the morphology, phylogenetic position, and pathogenicity of yellow fungal isolates obtained from hornbeam trees from Austria, Georgia and Switzerland, and compared data with disease reports from northern Italy documented since the early 2000s. Results demonstrate distinctive morphology and monophyletic status of Cryphonectria carpinicola sp. nov. as etiological agent of the European hornbeam decline. Interestingly, the genus Cryphonectria splits into two major clades. One includes Cry. carpinicola together with Cry. radicalis, Cry. decipiens and Cry. naterciae from Europe, while the other comprises species known from Asia—suggesting that the genus Cryphonectria has developed at two evolutionary centres, one in Europe and Asia Minor, the other in East Asia. Pathogenicity studies confirm that Car. betulus is a major host species of Cry. carpinicola. This clearly distinguished Cry. carpinicola from other Cryphonectria species, which mainly occur on Castanea and Quercus.
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Cryphonectria carpinicola sp. nov. Associated with hornbeam decline in
Europe
Carolina Cornejo
a
,
*
, Andrea Hauser
a
, Ludwig Beenken
a
, Thomas Cech
b
, Daniel Rigling
a
a
Swiss Federal Research Institute WSL, Zuercherstrasse 111, 8903, Birmensdorf, Switzerland
b
Bundesforschungszentrum für Wald, Institut für Waldschutz, Seckendorff-Gudent-Weg 8, 1131, Wien, Austria
article info
Article history:
Received 3 August 2020
Received in revised form
30 October 2020
Accepted 30 November 2020
Available online xxx
Keywords:
Pathogen
Cryphonectriaceae
Carpinus
Castanea
Phylogeny
A
bstract
Since the early 2000s, reports on declining hornbeam trees (Carpinus betulus) are spreading in Europe.
Two fungi are involved in the decline phenomenon: One is Anthostoma decipiens, but the other etiological
agent has not been identied yet. We examined the morphology, phylogenetic position, and pathoge-
nicity of yellow fungal isolates obtained from hornbeam trees from Austria, Georgia and Switzerland, and
compared data with disease reports from northern Italy documented since the early 2000s. Results
demonstrate distinctive morphology and monophyletic status of Cryphonectria carpinicola sp. nov. as
etiological agent of the European hornbeam decline. Interestingly, the genus Cryphonectria splits into two
major clades. One includes Cry. carpinicola together with Cry. radicalis,Cry. decipiens and Cry. naterciae
from Europe, while the other comprises species known from Asiadsuggesting that the genus Crypho-
nectria has developed at two evolutionary centres, one in Europe and Asia Minor, the other in East Asia.
Pathogenicity studies conrm that Car. betulus is a major host species of Cry. carpinicola. This clearly
distinguished Cry. carpinicola from other Cryphonectria species, which mainly occur on Castanea and
Quercus.
©2020 British Mycological Society. Published by Elsevier Ltd. All rights reserved.
1. Introduction
In last decades, the generic classication of Cryphonectriaceae
(Diaporthales) has been reassessed based on molecular data
(Gryzenhout et al., 2006a,b;Jiang et al., 2020). Several well-
supported clades were recognized within Cryphonectriaceae,
which correlate with morphological and eco-geographical features
and were proposed to represent distinct generic lineages within the
family. Some of these lineages relocated species formerly named as
Cryphonectria in new erected genera, e.g., Crysoporthe,Rostraureum,
Elaeocarpus,Amphilogia (Gryzenhout et al., 2006a), or Microthia,
Holocryphia and Ursicollum (Gryzenhout et al., 2006b). In other
cases, species known from other genera have been re-classied as
Cryphonectria (e.g., Cry. citrina;Jiang et al., 2020), and new species
are being discovered continuously, expanding the species list
within this genus (e.g., Cry. quercus and Cry. quercicola,Jiang et al.,
2018;Cry. neoparasitica,Jiang et al., 2019). The present study
follows recent, revised classication and takes a critical look at
some species within the genus Cryhonectria that occur in Europe.
The genus Cryphonectria is best known for its famous member,
Cry. parasitica, the causal agent of chestnut blight (Rigling and
Prospero, 2018). In Europe, three additional Cryphonectria species
have been reported to occur together with the invasive chestnut
blight fungus. One species, Cry. radicalis,wasrst reported for
North America but also well-documented for Europe and Japan at
the beginning of the 20th century. It has, however, apparently
disappeared in North America and seems to be rare in Europe since
the introduction of the chestnut blight fungus (Hoegger et al.,
2002). The other, Cry. naterciae, was recently described based on
morphology as well as molecular data and has been conrmed for
Portugal on Castanea sativa and Quercus suber (Bragança et al.,
2011), and for Algeria and Italy on declining Q. suber (Pinna et al.,
2019;Smahi et al., 2018). Both Cry. radicalis and Cry. naterciae
have often been accidently isolated from Cas. sativa during sam-
pling campaigns for the chestnut blight fungus (Bragança et al.,
2011;Hoegger et al., 2002;Sotirovski et al., 2004). A putative
third species is Cry. decipiens, which was separated from Cry. radi-
calis based on the ascospore morphology of herbarium samples
preserved in the U.S. National Fungus Collections (BPI) (Gryzenhout
Abbreviations: Cryphonectria, Cry.; Castanea, Cas.; Carpinus, Car.; Corylus, Cor..
*Corresponding author. Forest Health and Biotic Interactions, Swiss Federal
Research Institute WSL, Zuercherstrasse 111, 8903, Birmensdorf, Switzerland.
E-mail address: carolina.cornejo@wsl.ch (C. Cornejo).
Contents lists available at ScienceDirect
Fungal Biology
journal homepage: www.elsevier.com/locate/funbio
https://doi.org/10.1016/j.funbio.2020.11.012
1878-6146/©2020 British Mycological Society. Published by Elsevier Ltd. All rights reserved.
Fungal Biology xxx (xxxx) xxx
Please cite this article as: C. Cornejo, A. Hauser, L. Beenken et al.,Cryphonectria carpinicola sp. nov. Associated with hornbeam decline in Europe,
Fungal Biology, https://doi.org/10.1016/j.funbio.2020.11.012
et al., 2009). While there is no isolate deposition of Cry. decipiens
linked to the holotype BPI 1112743, it has been assumed that Cry.
decipiens and Cry. naterciae are conspecic(Rigling and Prospero,
2018). The present study focusses on an additional putative Cry-
phonectria species, which has been claimed to be involved in the
decline of hornbeam trees in Europe.
The European hornbeam, C. betulus L. (Betulaceae), is a widely
distributed deciduous tree with a natural range extending from the
Pyrenees to southern Sweden and eastwards over the Caucasus to
western Iran (Sikkema et al., 2016). It is one of few shade tolerant
tree species, playing an important role as a secondary species in
mixed stands dominated by oak (Postolache et al., 2017), or as
ornamental tree in urban parks, gardens and along roadsides
(Imperato et al., 2019;Saracchi et al., 2007). Although the wood of
the hornbeam is very hard and strong, trees tend to have an
irregular form and are therefore of minor commercial signicance
(Sikkema et al., 2016). Until recently, no major pest and disease
problems were reported to affect European hornbeam. The pow-
dery mildew Erysiphe arcuata is known parasitizing the European
hornbeam (Braun et al., 2006;Vajna, 2006;Wołcza
nska, 2007), and
E. kenjiana found on hornbeam was recently reported as new alien
species for Ukraine (Heluta et al., 2009). In addition, Moradi-
Amirabad et al. (2018) presented the rst detection of the bacte-
ria Brenneria spp. and Rahnella victoriana, which are associated with
hornbeam trees in the western forests of Iran and causes symptoms
similar to acute oak decline.
Since the early 2000s, however, declining hornbeam trees have
been repeatedly reported in Europedstarting from northern Italy
(Dallavalle and Zambonelli, 1999;Ricca et al., 2008;Rocchi et al.,
2010;Saracchi et al., 2007,2008), followed later by several cen-
tral European countries including Germany (Kehr et al., 2016,2017;
Krauthausen and Fischer, 2018), Austria (Cech, 2019) and
Switzerland (Queloz and Dubach, 2019)das well as from the most
eastern distribution limit of Car. betulus in Iran (Mirabolfathy et al.,
2018). Trees are described to be infected by two fungi, either
individually or both at the same time, and die within a few years if
heavily attacked. One fungus produces large bark necrosis with red
resin-like clumps on trunks and main branches, and could be
clearly identied as Anthostoma decipiens based on morphological
and molecular analyses (Rocchi et al., 2010). The second etiological
agent has been reported to produce yellow stromata on the bark,
which were assigned to an unknown Endothiella or Cryphonec-
triaceae species (Ricca et al., 2008;Rocchi et al., 2010;Saracchi
et al., 2008,2015). The term Endothiella refers to a historical
generic name for the asexual form of Cryphonectria species and is
considered here obsolete according to the International Code of
Nomenclature for Algae, Fungi, and Plants (Melbourne Code; McNeill
et al., 2012). For this reason, hereafter, we refer to the fungus with
yellow stromata on the European hornbeam as Cryphonectria taxon.
Arst species hypothesis for the Cryphonectria taxon tested the
relationship to Cry. parasitica.Dallavalle and Zambonelli (1999)
isolated a Cryphonectria-like strain from hornbeam trees in the
city of Parma and based on mating and vegetative compatibility
experiments ruled out that it belongs to Cry. parasitica. Another
hypothesis related the Cryphonectria taxon with Cry. radicalis based
on the ascospore morphology (Dallavalle et al., 2003). In fact, the
fungal collection BPI registers several herbarium samples of Cry.
radicalis (syn., Endothia radicalis)onCarpinus species, e.g., on Car.
betulus for Abkhazia and Slovakia (labelled as Czechoslovakia), or
on Cry. japonica for Japan, Car. laxiora for Korea, and Carpinus sp.
for the U.S.A. In contrast to these records, most Cryphonectria
species are known to occur on members of the family Fagaceae-
dincluding mainly Castanea and Quercus (Gryzenhout et al.,
2006b). In fact, only a few Cryphonectria species are reported on a
wider host range than Fagaceae. Examples include the said Cry.
radicalis and Cry. japonica (syn., Cry. nitschkei; Gryzenhout et al.,
2009) with six different tree families listed as hosts (Myburgh
et al., 2004a). However, many of historical reports should be
taken with caution, as the identity of the Cryphonectria species
remains uncertain due to the lack of molecular identication.
During phytosanitary surveys in Switzerland, several fungal
cultures were isolated from the bark of hornbeam trees that
showed sporulation as reported for the Cryphonectria taxon
(Queloz et al., 2019). These isolates shared morphological features
with the isolates M9290 from Austria and M5717 from Georgia
preserved in our isolate collection at the Swiss Federal Research
Institute WSL. First attempts to taxonomically assign the Austrian
and Georgian isolates based on the fungal barcode ITS reached high
identity scores with undetermined Cryphonectriaceae sp.
(KC894698eKC894672) and Endothiella sp. (AM400898) (last
search on 2020/07/17 on www.ncbi.nlm.nih.gov). Starting from
these preliminary data, the present study rst investigates the
taxonomic position of the Cryphonectria taxon on hornbeam under
the hypotheses that it belongs (i) to one of the four Cryphonectria
species present in Europe, or that it represents (ii) a distinctive
Cryphonectria, yet undescribed species. For this purpose, a molec-
ular phylogeny was generated based on four genetic markers: the
large subunit (LSU) and the internal transcribed spacer (ITS) of the
ribosomal RNA gene, two different sections of the
b
-Tubulin gene
(TUB) and a partial sequence of the RNA polymerase II gene (RPB2).
In addition, we analysed phenotypic traits such as culture
morphology and conidia size, which were compared with features
of Cry. naterciae,Cry. radicalis and Cry. parasitica, which are present
in Europe. Since sexual reproduction is common in Cryphonectria
species (Milgroom et al., 1993;Wilson et al., 2015), isolates of the
Cryphonectria taxon were crossed on hornbeam twigs to test their
mating behaviour. Finally, the pathogenic characteristics of the
isolates from Austria, Georgia and Switzerland was assessed in an
inoculation experiment on Carpinus,Corylus, and Betula species as
well as C. sativa.
2. Material and methods
2.1. Isolates used in this study
Voucher information for all isolates included in this study is
listed in Table 1. From the Cryphonectria taxon, four isolates were
molecularly characterized, one isolate from Carpinus sp. in Georgia,
and three isolates from Car. betulus in Austria and Switzerland.
Isolates of Cry. parasitica and Cry. radicalis from Switzerland, Cry.
japonica from Japan, and Cry. naterciae from Portugal were used to
compare morphological features between the different Crypho-
nectria species. The species Cry. decipiens was only assessed
molecularly at three loci (LSU, ITS and TUB) based on GenBank
entries as no isolate of the holotype BPI 1112743 is available.
2.2. DNA extraction, PCR and sequencing
Strains were grown on Potato Dextrose Agar (PDA; 39 g/l; Difco
Laboratories, Detroit, U.S.A.) for a period of 7 d at 25
C in the dark.
Thereafter, mycelia were harvested, transferred to 2 ml Eppendorf
tubes, and lyophilized overnight. Genomic DNA was extracted from
10 to 20 mg of lyophilized and milled fungal mycelium using the
DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the
manufacturers instructions. Four genomic regions were amplied
by polymerase chain reaction (PCR): (1) LSU (primer used: LR0R/
LR5 and LR3/LR7; Vilgalys and Hester, 1990); (2) ITS (ITS1/ITS4;
White et al., 1990); (3) TUB (Bt1a/Bt1b and Bt2a/Bt2b; Glass and
Donaldson, 1995); and (4) RPB2 (fRPB2-5F/fRPB2-7cR; Liu et al.,
1999). All reactions used a 25
m
l-mix, containing 12.5
m
l
C. Cornejo, A. Hauser, L. Beenken et al. Fungal Biology xxx (xxxx) xxx
2
JumpStart REDTaq ReadyMix (Sigma Aldrich: Merck KGaA, Darm-
stadt, Germany), 1
m
l of DNA template, 8.5
m
l of molecular-grade
water (Merck) and 1
m
l each primer (10
m
M). Thermal cycling pa-
rameters for all reactions were: 2 min 94
C initial denaturation, 35
cycles of 30 sec 94
C, 30 sec 55
C, 1 min 72
C, and 10 min at 72
C
nal elongation. PCR products were puried by an exonuclease I
and alkaline phosphatase treatment following the manufacturers
instructions (GE Healthcare, Chicago, Illinois, U.S.A.).
The forward and reverse DNA strands were Sanger sequenced
using the same primer as for the PCR reactions, except for locus
RBP2 that exceeded the available sequencing length. Therefore, an
internal forward primer Cryph_RPB2seq_F (5
0
- TCTACGGCTGGTGT
CTCTCA-3
0
) was newly designed and combined with the
Table 1
Isolates used in the present study.
Species Specimen ID
a
Host Origin GenBank accession numbers
b
LSU ITS TUB RPB2
Bt1 Bt2
Cryphonectria carpinicola M5717 Carpinus sp. Georgia MT311229 MT330389 MW086463 MW086449
M9615
c
Carpinus betulus Switzerland MT311233 MT330391 MW086465 MW086451
M9290 Carpinus sp. Austria MT311230 MT330390 MW086464 MW086450
M10525 Carpinus betulus Switzerland MT311232 MT330393 MW086466 MW086452
GIALLO-A
d
Carpinus betulus Italy NA AM400898 AM920692 NA NA
AR2_5
d
Carpinus betulus Italy NA KC894698 NA NA NA
AR9-3_2
d
Carpinus betulus Italy NA KC894699 NA NA NA
AR6-2
d
Carpinus betulus Italy NA KC894700 NA NA NA
AR1_7
d
Carpinus betulus Italy NA KC894701 NA NA NA
AR3_8
d
Carpinus betulus Italy NA KC894702 NA NA NA
C. citrina CBS 109758 Quercus mongolica Russia EU255074 MN172407 NA EU219342
C. decipiens CMW 10484 Castanea sativa Italy NA AF368327 AH011606 NA
CMW 10436 Quercus suber Portugal JQ862750 AF452117 AF525703 AF525710 NA
C. naterciae M3659 Quercus suber Portugal MT311226 EU442649 MW086470 MW086455
M3660 Quercus suber Portugal MT311227 EU442650 MW086471 MW086456
M3664 Quercus suber Portugal MT311228 EU442657 MW086472 MW086457
C 0084 Quercus suber Portugal NA NA EU442658 NA
C 0608 Quercus suber Portugal NA NA EU442659 NA
C 0614 Quercus suber Portugal NA NA EU442660 NA
C 0679/CBS 129352 Castanea sativa Portugal NA NA EU442661 NA
C 0685 Castanea sativa Portugal NA NA EU442662 NA
C 0691 Castanea sativa Portugal NA NA EU442663 NA
C. japonica M9605 Castanea crenata Japan MT311220 MT330397 MW086473 MW086458
M9606 Castanea crenata Japan MT311221 MT330396 MW086474 MW086459
M9607 Castanea crenata Japan MT311222 MT330398 MW086475 MW086460
CMW 10527 Quercus mongolica Russia AF408341 DQ120761 AH015162 NA
CFCC 52148 Quercus spinosa China MH514023 MH514033 MH539686 MH539696 NA
CMW 13742 Quercus grosseserrata Japan NA AY697936 AH014588 NA NA
C. neoparasitica CFCC 52146 Castanea mollissima China MH514019 MH514029 MH539682 MH539692 NA
CFCC 52147 Castanea mollissima China MH514020 MH514030 MH539683 MH539693 NA
C. macrospora CMW 10463 Castanopsis cuspidata Japan NA AF368331 AH011608 NA
CMW 10914 Castanea cuspidata Japan JQ862749 AY697942 AH014594 NA
CBS 109764 Quercus mongolica Russia AF408340 EU199182 NA EU220029
C. parasitica M2671 Castanea sativa Switzerland MT311218 MT330394 MW086476 MW086461
M4023 Castanea sativa Switzerland MT311219 MT330395 MW086477 MW086462
ATCC 38755 Castanea dentata EU199123 AY141856 NA DQ862017
C. radicalis M2268 Castanea sativa Switzerland MT311223 NA MW086467 NA NA
M2269 Castanea sativa Switzerland MT311224 AF548744 MW086468 MW086453
M2270 Castanea sativa Switzerland MT311225 AF548745 MW086469 MW086454
CMW 10455 Quercus suber Italy NA AF452113 NA NA
CMW 7051 Castanea sativa Italy NA AF368328 NA NA
CMW 13754 Fagus japonica Japan NA NA AH014584 NA
CMW 10477 Quercus suber Italy NA NA AH011607 NA
C. quercicola CFCC 52140 Quercus wutaishansea China NA MG866026 MG896113 MG896117 NA
CFCC 52141 Quercus wutaishansea China NA MG866027 MG896114 MG896118 NA
C. quercus CFCC 52138 Quercus aliena China NA MG866024 MG896111 MG896115 NA
CFCC 52139 Quercus aliena China NA MG866025 MG896112 MG896116 NA
Amphilogia gyrosa
e
CMW 10469 Elaeocarpus New Zealand AY194107 AF452111 AF525707 AF525714 NA
Endothia gyrosa
e
CMW 2091 Quercus palustris USA AY194114 AF368325 AH011601 AH011601 NA
Chrysoporthe cubensis
e
CBS 101281 Eucalyptus urophylla Cameroon NA NA NA EU219341
a
ATCC ¼American Type Culture Collection, Manassas, Virginia, U.S.A.; CBS ¼Centraalbureau voor Schimmelcultures, Westerdijk Fungal Biodiversity Institute, Utrecht, The
Netherlands; CFCC ¼China Forestry Culture Collection Center, Research Institute of Forest Ecology, Environment and Protection, Beijing, China; CMW ¼Culture Collection of
the Forestry and Agricultural Biotechnology Institute, University of Pretoria, Pretoria, South Africa; C¼Collection of the Instituto Nacional de Recursos Biologicos (INRB), I.P.,
Oeiras, Portugal; M¼culture collection of the Swiss Federal Institute for Forest, Snow and Landscape Research (WSL), Birmensdorf, Switzerland.
b
Sequences obtained in the present study are highlighted in bold type. All other sequences were acquired from GenBank (https://www.ncbi.nlm.nih.gov/). LSU ¼28S large
subunit of the nrDNA gene; ITS ¼internal transcribed spacer of the nrDNA gene, including the 5.8S gene; TUB ¼
b
-tubulin gene, Bt1 and Bt2 according to Glass and Donaldson
(1995);RPB2 ¼RNA polymerase II gene.
c
Holotype specimen MB837752.
d
Strain IDs of specimens used in Rocchi et al. (2010).
e
Sequences of Amphilogia gyrosa,Endothia gyrosa, and Chrysoporthe cubensis were used as outgroup in phylogenetic analyses.
C. Cornejo, A. Hauser, L. Beenken et al. Fungal Biology xxx (xxxx) xxx
3
Cryphonectria specic variant of the degenerated primer fRPB27cR
(Cryph_RPB2seq_R, 5
0
-TCCTCGTCATCTTTCTTTCT-3
0
), for nested
sequencing reactions. Sequencing reactions were then conducted
in 10
m
L mixtures, using the Big Dye Terminator 3.1 cycle
sequencing premix kit (Applied Biosystems, Waltham, Massachu-
setts, U.S.A.). Cycle sequencing products were puried using the
BigDye XTerminator Solution (Applied Biosystems). Sequences
were detected on an ABI PRISM 3130 Genetic Analyzer (Applied
Biosystems).
Forward and reverse sequences were assembled using the
program CLC Main Workbench v.7 (Qiagen). If sequences contained
long runs of a single nucleotide repeats, the Sanger read quality
declined rapidly after the so-called homopolymer. This was the case
within some ITS and TUB sequences, which contained long poly
(dA) and poly (dT) stretches. In such cases, reference sequences
were used for assembling the forward and reverse reads. The ho-
mopolymer itself was regarded as missing data and denoted with
poly (dN) for each of four nucleotides according to guidelines of the
National Center for Biotechnology Information (NCBI) (www.ncbi.
nlm.nih.gov). Homopolymer regions and other ambiguous align-
ments were excluded from analyses by processing all datasets with
Gblocks 0.91b (Castresana, 2000;Talavera and Castresana, 2007)on
the Phylogeny.fr platform (Dereeper et al., 2008).
2.3. Phylogenetic reconstructions
Phylogenetic trees were reconstructed by Bayesian and
maximum likelihood (ML) analyses, using BEAST 1.8.4 (Drummond
and Rambaut, 2007) on desk computer and PhyML 3.0 (Guindon
and Gascuel, 2003;Guindon et al., 2010) on the ATGC platform
(www.atgc-montpellier.fr). To select the model that best tted our
data, the Smart Model Selection SMS (Lefort et al., 2017) and the
Akaike Information Criterion (AIC) (Akaike, 1973) were used on the
ATGC platform. BEAST analysis was run with 10 million generations
and sampled every 1000th generation, following a discarded burn-
in of 2500 generations. Convergence and the consequent propor-
tion of burn-in were assessed using Tracer v1.7 (available from
http://beast.community/). To obtain the Bayesian posterior proba-
bilities (PP), a maximum clade credibility tree was generated by
analysing the BEAST tree le in TreeAnnotator v.1.8.4 (available in
the BEAST package). Bootstrap condence values (B) were calcu-
lated in PhyML for 100 pseudoreplicates (Felsenstein, 1985). Phy-
lograms were displayed in TreeGraph 2 (Stoever and Mueller, 2010).
The neighbor joining (NJ) algorithm (Saitou and Nei, 1987)was
applied exclusively to assess the genetic diversity of all available
sequences linked to the hornbeam decline in Europe. Early Italian
studies on hornbeam decline submitted several ITS sequences
(KC894698eKC894672) and one TUB sequence (AM920698) to
GenBank named as Cryphonectriaceae sp. or Endothiella sp. (cf.
Table 1). The analyses were performed with SplitsTree v.4.11.3
(Huson and Bryant, 2006) on each data matrix separately. Support
values for branch lengths were computed from 1000 bootstrap
replicates.
2.4. Morphology, growth and mating behaviour
Mycelial plugs (0.5 cm diameter) of the Cryphonectria taxon
M9290, M5717, and M9615 were excised with a sterile cork borer
from the edge of actively growing PDA cultures and placed in the
centre of a 9 cm PDA plate. One set of subcultures was incubated in
a climate chamber at 25
C with a cycle of 10 h at dark and 14 h at
light and the morphology was recorded for a period of three weeks.
The second set was incubated at 10, 15, 20, 25, and 30
C in the dark
to assess the effect of temperature on fungal growth. Ten replicated
plates were prepared for each fungal strain and temperature. The
radial growth (mm) of the colonies was assessed after 2 and 4 days
and the mean and standard deviation calculated. Isolates of Cry.
japonica, Cry. naterciae, Cry. parasitica and Cry. radicalis were
included in this experiment for comparison of culture morphology,
but growthetemperature correlation of these species was not
assessed, since this has been already studied elsewhere (cf.
Bragança et al., 2011;Gryzenhout et al., 2009;Hoegger et al., 2002).
To test the development of sexual fruiting bodies, three isolates
(M9290, M5717, and M9615) were crossed on C. betulus stem seg-
ments, either with themselves or with each other.Small stems of
Car. betulus with a diameter of approx. 2 cm were cut into 5 cm long
segments, split lengthwise and then autoclaved for 15 min at
121
C. The autoclaved segments were individually placed in 9 cm
diameter petri dishes and PDA medium was poured around them.
The isolates to be crossed were inoculated onto the agar medium at
both ends of the segments. The plates were incubated at 25
C
under a 16 h photoperiod for 3 weeks. Conidia produced by the
isolates were then suspended in sterile water and distributed over
the stem segments to induce mating. The mating plates were sealed
with paralm and incubated at 20
C under a 12 h photoperiod. The
plates were periodically examined for the presence of perithecia
under a dissecting microscope for one year. To prevent desiccation,
sterile water was added to the plates if necessary.
The mating plates were also used to harvest conidia of the
Cryphonectria taxon for size measurements. After incubation for
one year, conidia were taken under sterile conditions from the
blister-like conidiomata produced on the hornbeam stems and
dissolved in a water drop on a glass-slide. A Zeiss Axio Scope A1
microscope was used to measure 50 conidia of each isolate at 1000
times magnication with the software ZEN 2.3 (Carl Zeiss Micro-
scopy GMBH, Germany). The mean diameter of the conidia was
determined, and the standard deviation was calculated.
Morphology of eld collections and cultures were investigated
using a Zeiss Discovery.V8 SteREO microscope and hand sections of
stromata were studied at 1000 times magnication using Zeiss Axio
Scope A1 microscope. The ZEN 2.3 digital equipment was use for
photography.
2.5. Pathogenicity studies
To assess the pathogenicity of the Cryphonectria taxon, three
isolates (M9290, M5717, and M9615) were inoculated into C. betulus
and two additional tree species belonging to the family Betulacea,
Corylus avellane and Betula pendula. Because Castanea spp. are
major hosts for many Cryphonectria species, we also included
C. sativa in this inoculation experiment. Two-year-old seedlings of
Swiss provenances were used, except for Cas. sativa, which was of a
German provenance. The stem of each seedling was wound-
inoculated in a greenhouse chamber as described by Dennert
et al. (2019). For each isolate, ve seedlings of each tree species
were used. As negative controls, ve seedlings of each species
(three for B. pendula) were inoculated with an agar plug. Two
months after inoculations, the length and width of the lesions were
measured and the lesion size calculated using the formula of an
ellipse area. Sporulation of the isolates was assessed by recording
presence or absence of fungal stromata on each lesion. In the end of
the experiment, all lesions were sampled to recover the inoculated
fungus as described by Dennert et al. (2020). The identity of the re-
isolated cultures was assessed visually by their typical orange
culture morphology when growing on PDA plates. Linear model
with Scheffe post hoc test (calculated using DataDesk 6.3, Data-
Description Inc, Ithaca, NY) were used to test for signicant dif-
ferences (P0.05) in mean lesion size between isolates and tree
species.
C. Cornejo, A. Hauser, L. Beenken et al. Fungal Biology xxx (xxxx) xxx
4
3. Results
3.1. Phylogenetic analyses
In total, ten ITS, 15 RPB2, 16 LSU, and 16 TUB sequences were
obtained and submitted to GenBank (Table 1). The ITS and TUB
sequences contained highly variable and repetitive regions that
poses analytical problems related to the substitution model, which
does not account for fast-evolving, repetitive mutations. Therefore,
homopolymers and ambiguously aligned regions were processed
with the software Gblocks. Information on data matrices, such as
the number of excluded and polymorphic sites, is listed in Table 2.
The RPB2 dataset was mainly composed of one exon coding for a
354 amino acid sequence and containing around 18% polymorphic
single nucleotides (SNP) as well as one indel (insertion/deletion)da
codon that was present in the outgroup species Chrysoporthe
cubensis but not in Cryphonectria spp. TUB sequences included four
introns and ve exons, which resulted in protein sequences of
160e163 amino acids. After the exclusion of homopolymers and
ambiguously aligned regions, the TUB dataset was composed of c.
24% and the ITS matrix of c. 11% informative SNPs. Although the LSU
sequences were highly conserved, the c. 2.5% informative SNPs
were mainly concerned to the studied lineages Cryphonectria taxon,
Cry. decipiens,Cry. naterciae and Cry. radicalis as well as the out-
group species. In single-locus analyses (Supplemental Fig. S1), RPB2
topology resulted in well-supported monophyletic clades for the
six analysed species. On contrary, the reduced ITS and TUB datasets
failed to discriminate between already described speciesdsuch as
Cry. naterciae and Cry. decipiens (ITS and TUB) or Cry. quercus and
Cry. quercicola (TUB). The LSU tree resulted in a at topology that
did not resolve most species, except for specimens of the Crypho-
nectria taxon, Cry. decipiens and Cry. radicalis, but not among Cry.
decipiens and Cry. naterciae. In the present study, a species was
considered strongly supported if a lineage exhibited monophyly in
a majority of sampled loci (genealogical concordance), which was
not contradicted by phylogenetic patterns in other loci (genealog-
ical non-discordance)(Dettman et al., 2003,2006;Taylor et al.,
2000). Since, no well-supported (70%) conicting branching was
detected among single locus trees, multilocus analyses were per-
formed based on a concatenated dataset.
The concatenated dataset included only specimens that were
represented by three or four sequences in order to improve the
detection of monophyly. For this reason, species like Cry. quercus
and Cry. quercicola, which were described on two loci (ITS and TUB)
only, were not included in this dataset. The resulting data matrix
comprised 2804 sites and was composed of 23 sequences of nine
Cryphonectria species and two outgroup species, Endothia gyrosa
and Amphilogia gyrosa. Of the 2804 sites, 329 were polymorphic.
Both the Bayesian and PhyML analyses resulted in almost identical
topologies. Therefore, the Bayesian tree was selected for repre-
sentation in Fig. 1. This phylogeny conrms the monophyly of the
genus Cryphonectria (PP ¼1.0; B ¼98%), which splits into two
highly supported lineages (Fig. 1, A, and B). Within lineage A, the
isolates from declining hornbeam trees are separated from Cry.
decipiens,Cry. naterciae and Cry. radicalis in a strongly supported
monophyletic clade (PP ¼1.0; B ¼98%). Within lineage B, speci-
mens of Cry. japonica,Cry. macrospora,Cry. neoparasitica, and Cry.
parasitica each also represented a well-supported monophyletic
clade.
The ITS dataset for NJ analysis contained 19 sequences of Cry-
phonectria taxon, Cry. decipiens, Cry. naterciae and Cry. radicalis.
Thirty-four ambiguous positions were excluded from the dataset
and, of the 503 analysed characters, 34 were polymorphic. On
contrary, 78% of all positions were excluded from the TUB dataset
due to ambiguously aligned sites and highly repetitive homopoly-
mers. Finally, the dataset contained 20 sequences and reached a
length of 339 positions including nine polymorphic sites. Similar to
the single-locus topology, the ITS-tree failed to separate Cry. deci-
piens from Cry. naterciae at species level, but TUB data contained
some genetic variability within the Cryphonectria taxon and
abundant polymorphism between Cry. decipiens and Cry. naterciae.
Table 2
Summary of molecular data matrices used in phylogenetic analyses including the substitution model applied.
Alignment per locus LSU ITS TUB RPB2 Combined
Number of sequences 27 35 32 19 25
Number of characters 581 549 833 1098 e
Excluded ambiguous sites
a
e127 142 ee
Final alignment 569 423 691 1098 2804
b
Polymorphic sites 22 46 154 153 329
Substitution model GTR þI GTR þI HKY þG GTR þI GTR þGþI
a
Ambiguous aligned sites were automatically excluded using Gblocks.
b
Data matrices were combined after Gblocks analyses.
Fig. 1. Phylogeny and culture morphology of specimens isolated from declining
hornbeam trees, named as Cryphonectria carpinicola. Left: Phylogram resulting from
Bayesian analysis of combined ITS, LSU, RPB2 and TUB sequences. Bifurcations with
posterior probabilities <0.9 were collapsed. Numbers above branches represent pos-
terior probabilities and bootstrap values of maximum likelihood analysis (bootstraps
values >70%). Species names written in bold highlight cultures shown at right. Right:
Photographs of some representative species of the genus Cryphonectria grown on PDA
plates under lab conditions. At top the habit of Cry. carpinicola.
C. Cornejo, A. Hauser, L. Beenken et al. Fungal Biology xxx (xxxx) xxx
5
Isolates from declining hornbeam trees from Italy were clearly
positioned together with specimens from Austria, Georgia and
Switzerland in both NJ-trees (Fig. 2).
3.2. Morphology, growth and mating behaviour
The culture habit of ve Cryphonectria species examined in the
present study are shown in Fig. 1 (right side). To assess growth
characteristics and culture morphology, all isolates were grown on
PDA plates incubated at 25
C under a 14 h photoperiod. Under
these conditions, the Cry. parasitica and Cry. radicalis isolates grew
the fastest and reached the margin of the 90 mm agar plates after
one-week incubation. While all other isolates reached the margin
after two weeks, the Georgian isolate M5717 grew very slowly and
reached the margin only after three weeks. The pigmentation of all
species started after two to three days from the centre of the plates
and extended to the edge of the cultures after two weeks. The
Georgian isolate M5717 developed pigmentation in the third week.
The Cryphonectria taxon showed orange pigmentation on a beige
background, whereas the saturation of the orange colour was
higher around the central area and faded out towards the margins
of the culture. The Georgian isolate M5717 was beige to brown
pigmented and had only a small central orange area. The mycelium
of Cry. parasitica was orange-brown, similar to Cry. naterciae,
whereas Cry. radicalis showed luteous to orange pigmentation with
a dark brown central area. After the rst week, Cry. japonica
developed a transient slightly violet pigmentation, which dis-
appeared and turned into brown pigmentation. The mycelium was,
together with Cry. parasitica,uffy with clearly visible white
growth rings with a at central area. In contrast, Cry. radicalis and
Cry. naterciae, as well as the Cryphonectria taxon had rather less
visible growth rings. The margins of the cultures were smooth
except for the crenate margins of Cry. parasitica and the Georgian
isolate M5717. The bright orange-beige coloured conidiomata of
Cry. japonica were grouped as round droplets along the growth
rings, whereas in the other cultures conidiomata were less visible.
The effect of temperature on mean colony diameter of the Cry-
ophonectria taxon after two and four days is shown in Fig. 3.
Initially, colonies expanded fastest at 25e30
C, but this early
behaviour decreased rapidly and, after four days, all isolates grew
optimally at 20e25
C. The Georgian isolate M5717 exhibited a
slower growth than the other two isolates and did not grow above
temperatures of 25
C. In contrast, the Swiss isolate M9615 grew up
to 30
C and the Austrian isolate M9290 even at 35
C.
Conidia dimensions are listed in Table 3. The mean conidia
width of the Cryphonectria taxon and Cry. naterciae was similar but
the mean length was shorter compared with the conidia length of
Cry. naterciae and Cry. parasitica, but longer than Cry. radicalis.
No sexual fruiting bodies (perithecia) were produced on the
mating plates, even after an extended incubation time of more than
one year. In one cross (M9290 M9290), perithecial necks typically
of Cryphonectria spp. were observed (Fig. 4), however, no mature
perithecia were present associated with the necks.
3.3. Pathogenicity studies
The lesions produced by the Cryphonectria taxon after wound
inoculations varied depending on the isolate and the host species
(Table 4,Fig. 5). The general linear model revealed signicant dif-
ferences between isolates (P¼0.0015) and host species
(P¼0.0008). The largest lesions were produced by the isolates
M9290 on Car. betulus and to smaller extend on Cas. sativa. The
other two isolates did not produce signicantly larger lesions than
the control on both of these host species. There was no lesion larger
Fig. 2. Unrooted phylograms resulting from NJ analyses of (A) ITS, and (B) TUB se-
quences. Numbers beside branches represent bootstrap values. The species nova Cry-
phonectria carpinicola is highlighted in orange rectangles. Specimen vouchers are listed
beside nodes. Italian specimens shared identical ITS (A) or highly similar TUB (B) se-
quences with isolates from Austria, Georgia and Switzerland analysed in the present
study. (For interpretation of the references to colour in this gure legend, the reader is
referred to the Web version of this article.)
Fig. 3. Relationship between culture growth (cm) and temperature (C) after 2 days
(dashed line) and 4 days (dragged line) on PDA medium. Each data point represents
the mean value of ten repetitions and vertical bars the corresponding standard
deviation.
Table 3
Conidia size measure of Cryphonectria carpinicola and comparison with related
species.
Taxon IsolateeID n Mean þSD (
m
m) Range (
m
m)
a
C. carpinicola 150 3.5 ±0.3 1.3 ±0.1 3.0e4.7 0.9e1.8
M5717 50 3.5 ±0.2 1.4 ±0.1 3.1e3.6 1.2e1.5
M9615 50 3.4 ±0.3 1.2 ±0.1 3.0e4.6 0.9e1.5
M9290 50 3.7 ±0.4 1.4 ±0.1 3.1e4.7 1.2e1.8
C. decipiens
b
3.0e5.0 1.5e2.0
C. naterciae
c
150 3.7 ±0.4 1.3 ±0.1 2.9e4.9 1.0e1.6
C. radicalis
c
150 3.4 ±0.3 1.4 ±0.1 2.7e4.1 1.2e1.8
C. parasitica
c
150 3.6 ±0.4 1.4 ±0.1 2.9e4.5 1.1e1.7
a
Range is given as minimum and maximum dimension measured.
b
Gryzenhout et al. (2009).
c
Bragança et al. (2011).
C. Cornejo, A. Hauser, L. Beenken et al. Fungal Biology xxx (xxxx) xxx
6
than the control on Cor. avellana and B. pendula for all isolates. In
many cases, the inoculated wounds became completely overgrown
by the callusing reaction of trees (Supplemental Fig. S2). Overall,
lesions were signicant larger on Car. betulus compared to Car.
avellane (P¼0.009) and B. pendula (P¼0.015) but not compared to
Cas. sativa (P ¼0.84). Sporulation was observed only for isolate
M9290 on two lesions on Car. betulus and two lesions on Cas. sativa
(Table 4). This isolate also showed the highest re-isolation rate (15
out of 20 lesions), followed by M9615 (12 out of 20 lesions). The
isolate M5717 was only recovered from three lesions on B. pendula
and one lesion on Cas. sativa (totally 4 out of 20 lesions). Re-
isolations were successful from many lesions, which were not
larger than the control. During the entire duration of the experi-
ment, no mortality of the inoculated plants was observed.
Fig. 4. Cryphonectria carpinicola in culture. (A) Halved stem of Carpinus betulus over-
grown with mycelium. (BeC) Conidiomata with conidial tendrils. (D) Long neck on a
fake perithecia. (E) Conidia.
Table 4
Pathogenic characteristics of Cryphonectria carpinicola isolates on different tree
species, assessed two months after inoculation.
Host, Isolate N Lesion size (cm
2
) Sporulation
a
Re-Isolations
b
Carpinus betulus
M5717 5 0.57 ±0.21 a
c
00
M9290 5 3.85 ±3.05 b 2 5
M9615 5 1.00 ±0.30 ab 0 4
Control 5 0.60 ±0.12 a 0 0
Corylus avellana
M5717 5 0.48 ±0.16 a 0 0
M9290 5 0.40 ±0.05 a 0 2
M9615 5 0.43 ±0.07 a 0 1
Control 5 0.43 ±0.07 a 0 0
Betula pendula
M5717 5 0.51 ±0.15 a 0 3
M9290 5 0.50 ±0.07 a 0 3
M9615 5 0.52 ±0.11 a 0 4
Control 3 0.46 ±0.10 a 0 0
Castanea sativa
M5717 5 1.05 ±0.23 a 0 1
M9290 5 1.94 ±0.63 b 2 5
M9615 5 1.00 ±0.05 a 0 3
Control 5 0.93 ±0.27 a 0 0
a
Number of lesions with fungal stromata development.
b
Number of lesions from which C. carpincola was successfully re-isolated.
c
Means followed by different letters were signicant different (P<0.05).
Fig. 5. Pathogenicity test on Carpinus betulus using three isolates of Cryphonectria
carpinicola (A) M5717, (B) M9290, and (C) M9615. For each isolate, ve two-year-old
seedlings were inoculated. (D) As negative control, ve seedlings of Car. betulus were
inoculated with an agar plug.
Fig. 6. Cryphonectria carpinicola from eld collections. (AeC) Conidiomata breaking
through the bark of Carpinus betulus, conidial mass emerging in orange tendrils. (D)
Section through a multilocular stroma. (EeG) Microscopic view of section of stroma.
(E) Prosenchymatose outermost layer. (F) Conidiogenous cells. (G) Part of pseudopar-
enchymatous layer surrounding conidial locules. Scale bars: AeD¼1 mm;
EeG¼10
m
m.
C. Cornejo, A. Hauser, L. Beenken et al. Fungal Biology xxx (xxxx) xxx
7
3.4. Taxonomy
Cryphonectria carpinicola D. Rigling, T. Cech, Cornejo &L.
Beenken, sp. nov.
MycoBank MB837752 (Fig. 6)
Similar to Cryphonectria radicalis, but occurs on species of the
family Betulaceae.
Etym.: carpinicola means growing on Carpinus, the host genus.
Sexual state: ascomata not observed. Asexual state: uni- to
multilocular, stromatic conidiomata immersed in the bark,
pustular, erupting through the bark, surface shiny smooth,
bright orange, discolouring wine-red to purple with 2.5% KOH,
1e5(10)0.5e1.5 mm (sometimes several conuent to larger
patches), up to 1 mm high; outermost layer pseudoparenchy-
matous up to 60
m
m thick, with globose to angulate cells 2e6
m
m
in diameter, cell walls up to 1.5(2)
m
m thick, orange coloured;
inner part pulvinate, eustromatic throughout, only basal, close
to the substrate pseudostromatic, prosenchymatose, hyphae (1)
1.5 e2.5(3.5)
m
m wide, with colourless or yellow content, orange
incrustations on the hyphal walls dissolving and discolouring
pink to purple in 2.5% KOH; many refracting, colourless crystal
grains (roundish, up to 40
m
m in diameter) between the stromal
hyphae. Up to 8 locules per conidioma, locules ovoid bottle to
irregularly shaped and convoluted, 250e850
m
m wide, 150e900
m
m high, non-ostiolate, locules surrounded by a colourless
pseudoparenchymatous layer of angulate cells 2e54e10
m
m,
cell walls up to 1
m
m thick. Conidiophores straight, cylindrical,
10e50
m
m long, 1.5e1.8
m
m wide, septate, rarely branched.
Conidiogenous cells slightly tapered towards apex, phialidic.
Conidia minute, bacilloid, 3.0e4.7 0.9e1.8
m
m, hyaline, cylin-
drical, aseptate, conidial mass emerging in orange tendrils of
70e250
m
m diameter.
Host and distribution: occurring on Carpinus betulus L. in Europe
(Austria, Italy, Switzerland) and Carpinus sp. in Georgia.
Type:SWITZERLAND, Basel, Cemetery Wolfgottesacker,
47.54020 N, 7.60829 E, 278 m altitude, on dead trunk of Carpinus
betulus, 16 Jan. 2018, leg. S. Ramin (M9615). Holotype ZT Myc
61307, culture ex-holotype CBS 147194. GenBank accession
numbers: MT311233 (LSU), MT330391 (ITS), MW086451 (RPB2)
and MW086465 (TUB).
Additional specimens examined:SWITZERLAND, canton of Basel
Landschaft, Birsfelden, forest «Oberi Hard», 47.53900 N, 7.65550
E, 275 m altitude, on dead trunk of Carpinus betulus, 13 Jun. 2019,
V. Queloz (M10525), ZT Myc 61308, culture CBS 147195.
dcanton of Jura, Del
emont, Le B
eridier, forest la Vigne, 47.37836
N, 7.35399 E, 550 m altitude, on dead trunk of Carpinus betulus,
03 May 2020. V. Queloz (WSS 13609)
AUSTRIA, Biedermannsdorf, 48.090261 N, 16.348137 E, 190 m
altitude, on dead trunk of Carpinus betulus in a hedge, 24 Nov.
2009, Thomas Cech (M9290), CBS 147196. GEORGIA, Tkibuli, N
42.372981, E 43.016217, 1050 m altitude, on a dead stem of
Carpinus sp. 31 March 2010. D. Rigling (M5717), CBS 147197.
Notes:TheanamorphofCryphonectria carpinicola shows only
slight differences in morphology and anatomy to the closely
related species Cry. radicalis and Cry. naterciae (Fig. 1)
(Bragança et al., 2011). Small differences can be found in the
dimensions of the conidia that show a large overlap (Ta bl e 3).
The teleomorph of this new species, which could show more
differentiating features, has not yet been found and mating
experiments resulted in fake perithecia that did not produce
any asci and ascospores. Therefore, the differentiation of the
new species is mainly based on molecular sequence data and to
some extend to its host specicity. While the other Crypho-
nectria species mainly occur on tree genera of the family
Fagaceae, Cry. carpinicola was only found on Carpinus spp. of
the family Betulaceae.
4. Discussion
4.1. Phylogenetic analyses
During last decades, many cases of dieback of C. betulus trees
were reported in northern Italy and central Europe. Even though,
there was major effort to characterize both etiological agents
associated with dieback, only A. decipiens could be identied at
species level (Rocchi et al., 2010). The present work has studied the
second fungus causing hornbeam dieback and conrms the species
nov. Cryrphonectria carpinicola as etiological agent.A comprehen-
sive phylogenetic analysis, including all Cryphonectria species
known to date (Jiang et al., 2020), show that this fungus belongs to
the genus Cryphonectria as it is clearly integrated within the
ingroup and forms monophyletic clades in three of four sampled
loci (Supplemental Fig. S1). Additionally, NJ analysis of the ITS se-
quences from Italian isolates named in GenBank as Cryphonec-
triaceae sp. and Endothiella sp. were identical to all our isolates of
Cry. carpinicola as well as the TUB sequence AM920692 from the
Lombardy (Italy). The phylogeny of Cryphonectria splits into two
major clades (Fig.1). One includes Cry. carpinicola together with Cry.
radicalis,Cry. decipiens and Cry. naterciae from Europe, while the
other comprises species spread in eastern Asia, such as Cry. citrina,
Cry. japonica,Cry. macrospora or Cry. parasitica.
Morphologically, Cry. carpinicola shared many characteristics
with Cry. radicalis, demonstrating the close relationship between
both species. On PDA, the mycelium was at in both species, but
Cry. radicalis developed purple colour when grown in the dark
(Hoegger et al., 2002). However, we also observed some variation in
culture morphology among the Cry. carpinicola isolates. For
example, the Georgian isolate grew very slowly and only up to 25
C
(Fig. 3). However, molecular data clearly conrmed its taxonomic
position together with all other isolates of Cry. carpinicola (Figs. 1
and 2). Additionally, the conidia of all three isolates had similar
shape and size. Culture morphology can be inuenced by many
factors and it is well-known that it can change during sub-culturing
in the laboratory. Virus infection is also known to affect culture
morphology in Cryphonectria spp. (Hillman and Suzuki, 2004).
4.2. Host range and distribution of Cry. carpinicola
So far, Cry. carpinicola was found in Europe only on Car. betulus
and in the Caucasus region on an unidentied Carpinus species,
probably either Car. betulus or Car. orientalis.Carpinus spp. (family
Betulaceae) noticeably is the main host of Cry. carpinicola. This
clearly distinguishes this species from other Cryphonectria species,
which mainly occur on Castanea and Quercus in the family Faga-
ceae. In regions, where Cry. carpinicola was found in Europe,
extensive sampling of Cas. sativa has been done to study the
C. Cornejo, A. Hauser, L. Beenken et al. Fungal Biology xxx (xxxx) xxx
8
chestnut blight fungus, but Cry. carpinicola has never been reported
on European chestnut in these studies. However, our inoculation
tests and a eld study by Saracchi et al. (2010) demonstrate that Cry.
carpinicola possesses the potential to invade bark tissue of Cas.
sativa, although it acts rather as weak pathogen because no girdling
cankers were observed. Likewise, Cry. naterciae and Cry. japonica
were primarily reported to colonise chestnut wood saprotrophi-
cally (Dennert et al., 2020).
Cryphonectria japonica has also been reported as weak parasite
on Carpinus tschonoskii in Japan (as the syn. Cry. nitschkei;Myburgh
et al., 2004a). Although, Car. tschonoskii is only a minor host
amongst the main host plants of the Fagaceae, the ability to colo-
nise both Fagaceae and Betulaceae trees seems to be an ancestral
character state in the genus Cryphonectria because, e.g., Cry. carpi-
nicola and Cry. japonica belong to different lineages within this
genus and the most recent common ancestor is placed at the basal
genus node (Fig. 1). For this reason, we assume that, depending on
the prevailing environmental conditions, Cryphonectria species
have the potential to behave as pathogen, as weak parasite or as
saprophyte on both Fagaceae and Betulaceae in the sense of the
endophytic continuum (Schulz and Boyle, 2005). This concept hy-
pothesizes that there are no neutral interactions, but rather that
endophyteehost interactions involve a balance of antagonisms
with at least a degree of virulence on the part of the fungus
enabling infection. The ability to maintain a wide host range facil-
itate surviving under dynamic environmental conditions over a
long-term timescale. Indeed, host jumps are common for plant
pathogenic fungi (Burgess and Wingeld, 2016;Sieber, 2007;
Slippers et al., 2005) and previous studies have shown that
different species in the Cryphonectriaceae undergo regularly host
jumps (Chen et al., 2016;Gryzenhout et al., 2009;Heath et al.,
2006;Vermeulen et al., 2011). Hence, for Cry. carpinicola, we as-
sume that it can colonise different host families at least as weak
parasite or saprotroph, but it was rst discovered as conspicuous
pathogen on hornbeam trees.
Additionally, Cry. radicalis has been documented on Carpinus
trees in old herbarium specimens. An explanation for these records
is that in the past several closely related species were jointly
interpreted as Cry. radicalis due to scarce morphological features
useable for species discrimination. In fact, Myburg et al. (2004a,b)
reported phylogenetically distinctive lineages of specimens
labelled as Cry. radicalis that resulted in the separation of the new
species Cry. decipiens from Cry. radicalis sensu stricto (Gryzenhout
et al., 2009). It is thus possible that Cry. carpinicola was reported
on Car. betulus under the name of the morphologically very similar
Cry. radicalis and not recognized as a distinctive species.
4.3. Pathogenic potential of Cry. carpinicola
Due to heavy dieback in the Lombardy and Piedmont at early
2000s, the disease affecting Car. betulus trees was called hornbeam
decline in Italy (Ricca et al., 2008;Rocchi et al., 2010;Saracchi et al.,
2007). In Torino, e.g., mortality increased by 11% from 2004 to 20 07,
and 54% of the 300 surveyed hornbeam trees were in 2007
symptomatic (Ricca et al., 2008). Our pathogenicity tests conrmed
that Car. betulus is a main host species of Cry. carpinicola. Two
isolates (one each from Austria and Switzerland) produced clearly
visible lesions, when inoculated into the stems of Car. betulus
(Table 4,Fig. 5) and both could be re-isolated at high frequency two
months after inoculation. This result is consistent with a previous
inoculation study using a Cry. carpinicola (named Endothiella sp.)
isolate from Italy, which produced signicant larger lesions on Car.
betulus than on other potential host trees (Saracchi et al., 2015). The
isolate from Austria proved to be particularly virulent by producing
sporulating lesions on both Car. betulus and Cas. sativa. None of the
isolates caused lesions on hazelnut and birch, but still could be re-
isolated to some extend in the end of the experiment, most notably
from inoculated birch seedlings.
To assess the pathogenicity potential of Cry. carpinicola, we used
a wound inoculation method, which has been widely applied to
determine virulence and host specicity of the chestnut blight
fungus, Cry. parasitica (e.g. Dennert et al., 2019;2020;Peever et al.,
2000). Upon inoculations of susceptible chestnut seedlings, Cry.
parasitica isolates typically produced large lesions within a few
weeks, which lead to high seedling mortality (Dennert et al., 2019).
In comparison, lesions produced by Cry. carpinicola on hornbeam
were much smaller and did not cause host mortality, suggesting
that Cry. carpinicola is rather a secondary than a primary pathogen
on its main host tree. The isolates used in this study were all ob-
tained from dead hornbeams trees, which in the cases of the Swiss
isolates suffered from drought periods. In Italy, Cry. carpinicola has
been mainly reported on stressed hornbeam trees in urban envi-
ronment often together with A. decipiens (Saracchi et al., 2010).
Which combination of environmental factors incites the patho-
genic potential of Cry. carpinicola, however, remains to be
determined.
Acknowledgement
We thank Valentin Queloz for providing Swiss samples of Cry.
carpinicola for this study. This work was supported by the Swiss
Federal Ofce for the Environment, FOEN.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.funbio.2020.11.012.
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... Some fungi of the ascomycetous genus Cryphonectria can cause important disease in tree species native to temperate regions of the Northern Hemisphere, particularly in chestnut (Castanea), oak (Quercus) [18,19], and hornbeam (Carpinus) [20]. Cryphonectria species have a pronounced regional distribution, e.g., C. naterciae is until today only known from the Mediterranean Basin (Portugal [21] and Algeria [22]), and C. carpinicola has only been found in Central Europe and the Caucasus mountains. ...
... Two additional Cryphonectria species, i.e., C. radicalis and C. carpinicola, are known to occur in Europe and potentially undergo cross-species virus transmission. Phylogenetic study has shown that these two species are closely related to C. naterciae, while C. parasitica belongs to another clade together with other Asian species [20,38]. ...
... The virus designated as Cryphonectria naterciae fusagravirus 1 (CnFGV1) is composed of a non-segmented dsRNA genome with two open reading frames (ORFs), able to cross the incompatibility barrier and to persistently infect C. naterciae and other Cryphonectria species. In addition, CnFGV1 was found to severely reduce the fitness in the experimentally infected C. carpinicola-which is the etiological agent of the European hornbeam disease [20]. The data obtained indicated that CnFGV1 is a potential biological control agent against C. carpinicola and possibly against other Cryphonectria species. ...
Article
Full-text available
Cryphonectria is a fungal genus associated with economically significant disease of trees. Herein we characterized a novel double-stranded RNA virus from the fungal species Cryphonectria naterciae, a species unexplored as a virus host. De novo assembly of RNA-seq data and Sanger sequencing of RACE (rapid amplification of cDNA ends) clones gave the complete, non-segmented genome (10,164 bp) of the virus termed Cryphonectria naterciae fusagravirus (CnFGV1) that was phylogenetically placed within the previously proposed viral family Fusagraviridae. Of 31 field- collected strains of C. naterciae, 40% tested CnFGV1-positive. Cocultivation resulted in within-species transmission of CnFGV1 to virus-free strains of C. naterciae. Comparison of the mycelium phenotype and the growth rate of CnFGV1-infected and virus-free isogenic strains revealed frequent sectoring and growth reduction in C. naterciae upon virus infection. Co-culturing also led to cross-species transmission of CnFGV1 to Cryphonectria carpinicola and Cryphonectria radicalis, but not to Cryphonectria parasitica. The virus-infected C. naterciae and the experimentally infected Cryphonectria spp. readily transmitted CnFGV1 through asexual spores to the next generation. CnFGV1 strongly reduced conidiation and in some cases vegetative growth of C. carpinicola, which is involved in the European hornbeam disease. This study is the first report of a fusagravirus in the family Cryphonectriaceae and lays the groundwork for assessing a hypovirulence effect of CnFGV1 against the hornbeam decline in Europe.
... The virus designated as Cryphonectria naterciae fusagravirus 1 (CnFGV1) is composed of a non-segmented dsRNA genome with two open reading frames (ORFs) and able to cross the incompatibility barrier and to infect persistently C. naterciae and other Cryphonectria species. In addition, CnFGV1 was found to severely reduce the fitness in the experimentally infected C. carpinicola-which is the etiological agent of the European hornbeam disease [20]. The data obtained indicated that CnFGV1 is a potential biological control agent against C. carpinicola and possibly against other Cryphonectria species. ...
... Another possible explanation might be a strong transmissibility barrier. Previous phylogenetic study [20] of the genus Cryphonectria demonstrated that C. naterciae and C. parasitica belong to distinct European and Asian evolutionary lineages, respectively (Supplementary Figure S1), what we hypothesise here to result in a strong interspecies transmission barrier between the representatives of both lineages. The fact that transmission of CHV1 from C. parasitica to C. radicalis (from the European lineage) was also not successful by co-cultivation, although CHV1 can replicate in C. radicalis following protoplast fusion [65], additionally supports the idea of a strong vegetative rejection during hyphal contact. ...
... For other mycoviruses, the disruption of conidia formation is strongly associated with hypovirulence, e.g., CHV1 infection of C. parasitica [68], FgV-DK21 [69] or FgV-ch9 [70] infections of Fusarium graminearum, and ChNRV1 infection of Colletotrichum higginsianum [71,72]. It is worth noting here that C. carpinicola spread mainly through conidia [20]. In this context, viral disruption of spore production could significantly slow down the disease epidemic by affecting spread of the fungus. ...
Preprint
Full-text available
Cryphonectria is a fungal genus associated with economically significant disease of trees. Herein we characterized a novel double-stranded RNA virus from the fungal species Cryphonectria naterciae, a species unexplored as a virus host. De novo assembly of RNA-seq data and Sanger sequencing of RACE (rapid amplification of cDNA ends) clones gave the complete, non-segmented genome (10,164 bp) of the virus termed Cryphonectria naterciae fusagravirus (CnFGV1) that was phylogenetically placed within the previously proposed viral family Fusagraviridae. Of 31 field-collected strains of C. naterciae, 40% tested CnFGV1-positive. Co-cultivation resulted in within-species transmission of CnFGV1 to virus-free strains of C. naterciae. Comparison of the mycelium phenotype and the growth rate of CnFGV1-infected and virus-free isogenic strains revealed frequent sectoring and growth reduction in C. naterciae. Co-culturing also led to cross-species transmission of CnFGV1 to Cryphonectria carpinicola and Cryphonectria radicalis, but not to Cryphonectria parasitica. The virus-infected C. naterciae and the experimentally infected Cryphonectria spp. readily transmitted CnFGV1 through asexual spores to the next generation. CnFGV1 strongly reduced conidiation and in some cases vegetative growth of C. carpinicola, which is involved in the European hornbeam disease. This study is the first report of a fusagravirus in the family Cryphonectriaceae and lays the groundwork for assessing a hypovirulence effect of CnFGV1 against the hornbeam decline in Europe.
... The genus Cryphonectria contains an important plant pathogen, Cryphonectria parasitica, causing one of the three most destructive tree diseases in addition to other plant pathogenic and non-pathogenic fungi [23][24][25]. It is noteworthy that C. parasitica serves as a model filamentous fungal host suitable for studying host antiviral RNA silencing/viral counterdefense responses [26,27]. ...
... Among them, a Japanese strain, JS13, was originally identified as C. nitschkei, and the authors claimed it to be the strain infected with an RNA virus unrelated to hypoviruses most prevailing in C. parasitica (family Hypoviridae, well-known Cryphonectria viruses with a positive-sense, single-stranded (+)ssRNA genome) [33]. This fungal strain was re-classified as Cryphonectria carpinicola (see Figure 1), which is a recently established species within the genus Cryphonectria [25]. ...
Article
A novel dsRNA virus (Cryphonectria carpinicola fusagravirus 1, CcFGV1), isolated from a Japanese strain (JS13) of Cryphonectria carpinicola, was thoroughly characterized. The biological comparison of a set of isogenic CcFGV1-infected and -free (JS13VF) strains indicated asymptomatic infection by CcFGV1. The sequence analysis showed that the virus has a two open reading frame (ORF) genome of 9.6 kbp with the RNA-directed RNA polymerase domain encoded by ORF2. The N-terminal sequencing and peptide mass fingerprinting showed an N-terminally processed or degraded product (150 kDa) of the 5′-proximal ORF1-encoded protein (1462 amino acids) to make up the CcFGV1 spherical particles of ~40 nm in diameter. Interestingly, a portion of CcFGV1 dsRNA co-fractionated with a host protein of 70 kDa. The purified CcFGV1 particles were used to transfect protoplasts of JS13VF as well as the standard strain of an experimental model filamentous fungal host Cryphonectria parasitica. CcFGV1 was confirmed to be associated with asymptomatic infection of both fungi. RNA silencing was shown to target the virus in C. parasitica, resulting in reduced CcFGV1 accumulation by comparing the CcFGV1 content between RNA silencing-competent and -deficient strains. These results indicate the transfectability of spherical particles of a fusagravirus associated with asymptomatic infection.
... An RNA silencing deficient mutant Δdcl2 of C. parasitica was a generous gift from Dr. Donald L. Nuss at the University of Maryland (Segers et al., 2007). A European strain, DR1 (WSL collection code, M4733), of Cryphonectria radicalis (Hoegger et al., 2002;Shahi et al., 2021), a Japanese strain JS13 of Cryphonectria carpinicola (Cornejo et al., 2021a;Liu et al., 2007), a Japanese strain E16 (MAFF code, 410155) of Cryphonectria nitchkei and a Japanese strain AVC53 of Valsa ceratosperma (order Diaporthales) (Sasaki et al., 2002) were described earlier (Shahi et al., 2021). These fungal strains were grown on Difco potato dextrose agar (PDA) or potato dextrose broth (PDB) medium (Becton, Dickinson and Co., New Jersey, USA) on the benchtop at 23-26 • C. ...
... These observations suggest the intimate interactions between CnSpV1 and host factors specifically present in C. naterciae and C. carpinicola. Of note is that C. naterciae is phylogenetically closer to C. carpinicola than to C. parasitica or C. nitschkei (Cornejo et al., 2021a). ...
Article
Positive-sense (+), single-stranded (ss) RNA viruses with divided RNA-dependent RNA polymerase (RdRP) domains have been reported from diverse filamentous ascomycetes since 2020. These viruses are termed splipalmiviruses or polynarnaviruses and have been characterized largely at the sequence level, but ill-defined biologically. Cryphonectria naterciae, from which only one virus has been reported, is an ascomycetous fungus potentially plant-pathogenic to chestnut and oak trees. We molecularly characterized multiple viruses in a single Portuguese isolate (C0614) of C. naterciae, taking a metatranscriptomic and conventional double-stranded RNA approach. Among them are a novel splipalmivirus (Cryphonectria naterciae splipalmivirus 1, CnSpV1) and a novel fusagravirus (Cryphonectria naterciae fusagravirus 1, CnFGV1). This study focused on the former virus. CnSpV1 has a tetra-segmented, (+)ssRNA genome (RNA1 to RNA4). As observed for other splipalmiviruses reported in 2020 and 2021, the RNA-dependent RNA polymerase domain is separately encoded by RNA1 (motifs F, A and B) and RNA2 (motifs C and D). A hypothetical protein encoded by the 5′-proximal open reading frame of RNA3 shows similarity to a counterpart conserved in some splipalmiviruses. The other RNA3-encoded protein and RNA4-encoded protein show no similarity with known proteins in a blastp search. The tetra-segment nature was confirmed by the conserved terminal sequences of the four CnSpV1 segments (RNA1 to RNA4) and their 100% coexistence in over 100 single conidial isolates tested. The experimental introduction of CnSpV1 along with CnFGV1 into a virus free strain C0754 of C. naterciae vegetatively incompatible with C0614 resulted in no phenotypic alteration, suggesting asymptomatic infection. The protoplast fusion assay indicates a considerably narrow host range of CnSpV1, restricted to the species C. naterciae and C. carpinicola. This study contributes to better understanding of the molecular and biological properties of this unique group of viruses.
... Die Gruppe Phytopathologie der WSL hat vor Kurzem durch eine genaue phylogenetische Analyse sowie Kultur-und Infektionsexperimente gezeigt, dass es sich bei dem Pilz um eine neue, eigenständige, auf Carpinus spezialisierte Cryphonectria-Art handelt. Sie wurde daher als Cryphonectria carpinicola neu beschrieben (Cornejo et al., 2020). ...
Article
Full-text available
Invasions by non-native pathogens represent a major threat to managed and natural ecosystems worldwide. Although necessary for adopting preventive strategies, the identification of invasive species before they are introduced is particularly difficult. Indeed, most pathogenic species that have become established in the last decades were first described only after they became invasive. To prevent further biological invasions, not only the early identification of potential new invasive plant pathogens is crucial, but also the assessment of their potential host range. In this study, we determined the pathogenicity and the saprotrophic ability of three Cryphonectria species toward three potential hosts in the family Fagaceae. For this, seedlings and dormant stems of European chestnut (Castanea sativa), pedunculate oak (Quercus robur) and European beech (Fagus sylvatica) were inoculated with different genotypes of C. parasitica (Asian species, invasive in Europe), C. naterciae (European species), and C. japonica (Asian species, not present in Europe). Lesion growth was measured and mortality assessed for 4 months. The highest damage was caused by C. parasitica on European chestnut, while C. japonica and C. naterciae induced significantly smaller lesions on this host species. All three Cryphonectria species did not grow saprophytically on F. sylvatica and Q. robur, but successfully colonized dormant stems of C. sativa. In the context of biological invasions, our study shows that the Asian C. japonica most likely represents a much less severe threat than C. parasitica for the tested European host species. Nonetheless, the ability of C. naterciae and C. japonica to saprotrophically colonize fresh chestnut wood may suggest that they could become established in chestnut forests and eventually infect weakened chestnut trees or other hosts not tested in this study.
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Cryphonectria species associated with canker disease are presently difficult to identify, because of lack of ex-type cultures with molecular data and few distinguishable characters. During our survey about chestnut blight in western China, Cryph-onectria parasitica was confirmed as the most important canker pathogen based on ten morphologically atypical isolates. Two novel species with orange stromata are introduced based on multigene sequence (ITS, TUB1, TUB2) analyses. This is the first study that has established the Cryphonectria species associated with oak canker in China using a multiphasic approach .