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

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 identi ﬁ ed 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 Asia d suggesting that the genus Crypho- nectria has developed at two evolutionary centres, one in Europe and Asia Minor, the other in East Asia. Pathogenicity studies con ﬁ rm 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 The on of British Mycological Society. This an


Introduction
In last decades, the generic classification of Cryphonectriaceae (Diaporthales) has been reassessed based on molecular data (Gryzenhout et al., 2006a, b;Jiang et al., 2020). Several wellsupported 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-classified 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 classification 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, was first 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 confirmed 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 sampling 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. radicalis based on the ascospore morphology of herbarium samples preserved in the U.S. National Fungus Collections (BPI) (Gryzenhout 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 conspecific (Rigling and Prospero, 2018). The present study focusses on an additional putative Cryphonectria 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 significance (Sikkema et al., 2016). Until recently, no major pest and disease problems were reported to affect European hornbeam. The powdery 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 first detection of the bacteria 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., 2007Saracchi et al., , 2008, followed later by several central European countries including Germany (Kehr et al., 2016(Kehr et al., , 2017Krauthausen 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 identified as Anthostoma decipiens based on morphological and molecular analyses . The second etiological agent has been reported to produce yellow stromata on the bark, which were assigned to an unknown Endothiella or Cryphonectriaceae species (Ricca et al., 2008;Rocchi et al., 2010;Saracchi et al., 2008Saracchi et al., , 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.
A first 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) on Carpinus species, e.g., on Car. betulus for Abkhazia and Slovakia (labelled as Czechoslovakia), or on Cry. japonica for Japan, Car. laxiflora 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 Fagaceaedincluding 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 identification.
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 first 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 molecular 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.

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 Cryphonectria 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.

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 manufacturer's instructions. Four genomic regions were amplified 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 ml-mix, containing 12.5 ml C. Cornejo, A. Hauser, L. Beenken et al. Fungal Biology 125 (2021) 347e356 JumpStart REDTaq ReadyMix (Sigma Aldrich: Merck KGaA, Darmstadt, Germany), 1 ml of DNA template, 8.5 ml of molecular-grade water (Merck) and 1 ml each primer (10 mM). Thermal cycling parameters 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 final elongation. PCR products were purified by an exonuclease I and alkaline phosphatase treatment following the manufacturer's 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  Cryphonectria specific 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 mL mixtures, using the Big Dye Terminator 3.1 cycle sequencing premix kit (Applied Biosystems, Waltham, Massachusetts, U.S.A.). Cycle sequencing products were purified 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 homopolymer 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 alignments 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).

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 fitted 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 burnin of 2500 generations. Convergence and the consequent proportion of burn-in were assessed using Tracer v1.7 (available from http://beast.community/). To obtain the Bayesian posterior probabilities (PP), a maximum clade credibility tree was generated by analysing the BEAST tree file in TreeAnnotator v.1.8.4 (available in the BEAST package). Bootstrap confidence values (B) were calculated in PhyML for 100 pseudoreplicates (Felsenstein, 1985). Phylograms 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.

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 segments, 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 parafilm 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 magnification with the software ZEN 2.3 (Carl Zeiss Microscopy GMBH, Germany). The mean diameter of the conidia was determined, and the standard deviation was calculated.
Morphology of field collections and cultures were investigated using a Zeiss Discovery.V8 SteREO microscope and hand sections of stromata were studied at 1000 times magnification using Zeiss Axio Scope A1 microscope. The ZEN 2.3 digital equipment was use for photography.

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 woundinoculated in a greenhouse chamber as described by Dennert et al. (2019). For each isolate, five seedlings of each tree species were used. As negative controls, five 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 reisolated 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 significant differences (P 0.05) in mean lesion size between isolates and tree species.

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 five 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 outgroup 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 flat topology that did not resolve most species, except for specimens of the Cryphonectria 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 (genealogical non-discordance) (Dettman et al., 2003(Dettman et al., , 2006Taylor et al., 2000). Since, no well-supported (!70%) conflicting branching was detected among single locus trees, multilocus analyses were performed 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 representation in Fig. 1. This phylogeny confirms the monophyly of the genus Cryphonectria (PP ¼ 1.0; B ¼ 98%), 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 ¼ 1.0; B ¼ 98%). Within lineage B, specimens 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 Cryphonectria 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 homopolymers. 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. decipiens 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.  Isolates from declining hornbeam trees from Italy were clearly positioned together with specimens from Austria, Georgia and Switzerland in both NJ-trees (Fig. 2).

Morphology, growth and mating behaviour
The culture habit of five 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 first week, Cry. japonica developed a transient slightly violet pigmentation, which disappeared and turned into brown pigmentation. The mycelium was, together with Cry. parasitica, fluffy with clearly visible white growth rings with a flat 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 Cryophonectria 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 Â M9290), perithecial necks typically of Cryphonectria spp. were observed (Fig. 4), however, no mature perithecia were present associated with the necks.

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 significant differences between isolates (P ¼ 0.0015) and host species (P ¼ 0.0008). 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 significantly larger lesions than the control on both of these host species. There was no lesion larger   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 significant larger on Car. betulus compared to Car. avellane (P ¼ 0.009) and B. pendula (P ¼ 0.015) but not compared to Cas. sativa (P ¼ 0.84). 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). Reisolations were successful from many lesions, which were not larger than the control. During the entire duration of the experiment, no mortality of the inoculated plants was observed.    MycoBank MB837752 (Fig. 6) Similar to Cryphonectria radicalis, but occurs on species of the family Betulaceae.
Host and distribution: occurring on Carpinus betulus L. in Europe (Austria, Italy, Switzerland) and Carpinus sp. in Georgia.

Notes:
The anamorph of Cryphonectria 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 (Table 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 specificity. While the other Cryphonectria species mainly occur on tree genera of the family Fagaceae, Cry. carpinicola was only found on Carpinus spp. of the family Betulaceae.

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 identified at species level . The present work has studied the second fungus causing hornbeam dieback and confirms the species nov. Cryrphonectria carpinicola as etiological agent. A comprehensive 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 sequences from Italian isolates named in GenBank as Cryphonectriaceae 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 flat 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 confirmed 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 influenced 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).

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 unidentified 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 Fagaceae. In regions, where Cry. carpinicola was found in Europe, extensive sampling of Cas. sativa has been done to study the chestnut blight fungus, but Cry. carpinicola has never been reported on European chestnut in these studies. However, our inoculation tests and a field 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 saprotrophically (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 colonise both Fagaceae and Betulaceae trees seems to be an ancestral character state in the genus Cryphonectria because, e.g., Cry. carpinicola 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 hypothesizes 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 facilitate surviving under dynamic environmental conditions over a long-term timescale. Indeed, host jumps are common for plant pathogenic fungi (Burgess and Wingfield, 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 assume that it can colonise different host families at least as weak parasite or saprotroph, but it was first 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.

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 2007, and 54% of the 300 surveyed hornbeam trees were in 2007 symptomatic (Ricca et al., 2008). Our pathogenicity tests confirmed 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 significant 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 reisolated 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 specificity of the chestnut blight fungus, Cry. parasitica (e.g. Dennert et al., 2019;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 obtained 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 environment often together with A. decipiens . Which combination of environmental factors incites the pathogenic potential of Cry. carpinicola, however, remains to be determined.