Species of the Colletotrichum spp., the Causal Agents of Leaf Spot on European Hornbeam (Carpinus betulus)

European hornbeam (Carpinus betulus L.) is widely planted in landscaping. In October 2021 and August 2022, leaf spot was observed on C. betulus in Xuzhou, Jiangsu Province, China. To identify the causal agent of anthracnose disease on C. betulus, 23 isolates were obtained from the symptomatic leaves. Based on ITS sequences and colony morphology, these isolates were divided into four Colletotrichum groups. Koch’s postulates of four Colletotrichum species showed similar symptoms observed in the field. Combining the morphological characteristics and multi-gene phylogenetic analysis of the concatenated sequences of the internal transcribed spacer (ITS) gene, Apn2-Mat1-2 intergenic spacer (ApMat) gene, the calmodulin (CAL) gene, glyceraldehyde3-phosphate dehydrogenase (GAPDH) gene, Glutamine synthetase (GS) gene, and beta-tubulin 2 (TUB2) genes, the four Colletotrichum groups were identified as C. gloeosporioides, C. fructicola, C. aenigma, and C. siamense. This study is the first report of four Colletotrichum species causing leaf spot on European hornbeam in China, and it provides clear pathogen information for the further evaluation of the disease control strategies.


Introduction
European hornbeam (Carpinus betulus Linnaeus) belongs to the family Betulaceae and is mainly distributed in temperate and subtropical regions. It is native to peripheral forests around Europe, Asia Minor, and the Caspian Sea, and is often mixed with oak and beech. It can grow well above 1000 m above sea level [1]. In Iran, European hornbeam is the main tree species in the wood industry, with excellent technical performance and great application potential. It is mainly used for manufacturing tool handles, furniture, and paper, and it is also an excellent wood for railway sleeper production and dam reinforcement after preservative treatment [2]. European hornbeam is also very popular in urban green spaces and parks, and it has excellent characteristics of cold resistance, drought resistance, and pruning resistance. European hornbeam has been selected as an important tree species in garden construction since the Italian Renaissance [3]. In addition, it has been reported that many anticancer substances can be extracted from the young stems and leaves of European hornbeam, such as pheophorbide A (PHA) and some triterpenoids [4,5]. Therefore, European hornbeam has great research value and practical potential.
The genus Colletotrichum Corda is the only genus of Glomerellaceae [6] and one of the ten most important plant pathogenic fungi in the world [7]. The fungi of the genus Colletotrichum are distributed worldwide, with diverse host plants, including more than 3000 species of monocot and dicot plants [8,9]. Some Colletotrichum species can also cause human infection and inflammation, such as Colletotrichum gloeosporioides [10,11].
Before the 1990s, the classification of Colletotrichum was mainly based on morphological characteristics. The morphological classification of the genus Colletotrichum is mainly

Sampling and Fungal Isolation
The field survey was investigated in Xuzhou Urban Garden Company (34.28 • N, 118.03 • E) in October 2021 and August 2022 in Xuzhou, Jiangsu Province. Xuzhou has a temperate monsoon climate with four distinct seasons, the annual sunshine hours are 2284-2495 h, the sunshine rate is 52-57%, the average annual precipitation is 800-930 mm, and the rainy season precipitation accounts for 56% of the whole year.
Diseased leaves were collected from a 1-2 m part of European hornbeam. Approximately 30 diseased samples were collected from 10 European hornbeam trees which were scattered in the field. Fungi isolation was conducted on the second day after field survey. The diseased leaves were disinfected in 1% sodium hypochlorite for 90 s, rinsed in sterile water twice for 30 s, and dried with sterile paper. Then, the tissues from the margin of the lesions (0.2 cm × 0.2 cm) were excised, incubated on 2% potato dextrose agar (PDA) supplemented with 100 mg/L ampicillin sodium, and incubated in the dark at 25 • C for 4 days. Fungal hyphae grown from the leaf tissues were picked up and transferred to fresh PDA within 2-4 days [38].

Pathogenicity Tests
Healthy European hornbeam saplings with a height of approximately 1 m were obtained from Xuancheng Garden Greening Co., Ltd. in Xuancheng, Anhui Province.
Before the pathogenicity experiment, the surfaces of the leaves were sprayed with 75% alcohol 2-3 times, and then the above operation was repeated with sterile water to remove the residual alcohol; then, they were dried with absorbent paper, or we waited for the surfaces to dry. The spore suspension (10 6 conidia·mL −1 ) was sprayed 2-3 mL onto the leaves using a 10 mL plastic sprinkling can, and hornbeam leaves were treated with sterile water as the control.
Each of the treatment and control groups contained five leaves, and each treatment consisted of one seedling. All of the seedlings under different treatments were kept in a 25 • C greenhouse with high humidity under natural light conditions, and the development of symptoms was observed daily. The experiments were conducted twice.
To complete Koch's postulates, as previously mentioned, the fungus was reisolated from the margin tissue of the diseased lesions that developed from the inoculated tissue and were identified via molecular and phylogenetic analysis.

Morphological Characteristics
Fresh mycelium blocks were cut from the edge of three-day-old colonies and transferred to fresh PDA medium. After 4 days of incubation in the dark at 25 • C, the colony morphology was observed and recorded.
To observe the morphology of conidia, fresh mycelium pieces were cut off and transferred to fresh potato dextrose broth (PDB) supplemented with 100 mg/L ampicillin sodium. Then, the PDB bottles containing the mycelium pieces were placed on a shaking table and shaken at a rotating speed of 200 rpm in the dark at a temperature of 25 • C. After 2 days, the culture solution was collected and filtered with sterile filter cloths to collect the conidia. Appressoria were induced via cultivation on the surface of a hydrophobic coverslip [39]. Asci or ascospores were obtained from the ascomata that grew for 2-3 weeks on PDA or SNA in darkness at 25 • C. Then, each structure was observed to generate 30 measurements using a ZEISS Axio Imager A2m microscope (ZEISS), and the size of each structure was measured using the cross-assay method [40].

Phylogenetic Analysis
Fungal hyphae were collected from fresh colonies using sterilized scalpels. Genomic DNA was extracted using a CTAB Extraction Solution Kit (Leagene Biotechnology, Beijing, China). Then, all of the DNA extracts were stored at −20 • C for subsequent use.
Six nuclear gene regions were amplified and sequenced, including the ITS, CAL, GAPDH, TUB2, ACT, and CHS-1 regions. The primers and PCR conditions are shown in Table 1. Amplification was performed in an Eppendorf Nexus Thermal Cycler (Eppendorf) in a volume of 50 µL, which consisted of 4 µL of genomic DNA, 2 µL of forward/reverse primer (0.01 nmol/µL), 25 µL of 2× Green Taq Mix (Vazyme, Nanjing), and 17 µL of doubledistilled H 2 O. PCR products were sequenced by Sangon Biotech Co., Ltd. (Nanjing, China). The sequences were analyzed using MAFFT [49] in PhyloSuite v. 1.2.2 [50] and manually trimmed to ensure maximum sequence similarity.
Maximum likelihood (ML) analysis and Bayesian inference (BI) analysis were used to mutually corroborate the phylogenetic reconstructions. IQ-TREE v. 1.6.8 [51] was used for inferring the ML phylogenies under the edge-linked partition model for 100,000 ultrafast bootstraps. MrBayes v. 3.2.6 [52] was used for inferring BI phylogenies, and the initial quarter of the sampled data was discarded as burn-in. ModelFinder [53] was used to select the best-fit model on the basis of the Akaike information criterion (AIC). According to the AIC, the best-fitting model for ML analysis was GTR + F + I + G4, with 1,000,000 ultrafast [54] bootstrap replicates determining the branch stability, while the model for BI analysis was GTR + F + I + G4 under 2 parallel runs of 1,000,000 generations. The phylogenetic tree was viewed by FigTree v. 1.4.4.

Field Survey and Symptoms in the Field
The field survey was investigated in Xuzhou Urban Garden Company (34.28  Most of the spots were distributed along the edge of the European hornbeam leaves, and the spot wounds tended to expand inward. In addition, some serious disease spots caused leaf shape loss or leaf curling. The spots were brown to dark brown, some areas of the lesion appeared to be grayish-white, and the margin of a part of the lesions appeared as a pale green halo ( Figure 1).

Fungal Isolation
A total of 23 fungal strains were isolated from the diseased leaf samples of European hornbeam. Based on ITS sequences, 23 strains belonged to the genus Colletotrichum. According to the density of hyphae and the distribution of pigment on the reverse side of colonies, 23 Colletotrichum strains were divided into 4 groups, with quantities of 5 (group 1), 12 (group 2), 4 (group 3), and 2 (group 4).

Fungal Isolation
A total of 23 fungal strains were isolated from the diseased leaf samples of European hornbeam. Based on ITS sequences, 23 strains belonged to the genus Colletotrichum. According to the density of hyphae and the distribution of pigment on the reverse side of colonies, 23 Colletotrichum strains were divided into 4 groups, with quantities of 5 (group 1), 12 (group 2), 4 (group 3), and 2 (group 4).

Morphological Characteristics
One representative isolate was selected from each Colletotrichum group for further study (XZEC11 from group 1, XZEC21 from group 2, XZEC31 from group 3, and XZEC41 from group 4).
The colonies of XZEC11 isolates produced white aerial hyphae with loose marginal hyphae, and the back of the colonies was light orange-red (Figure 2A,B). The colonies of XZEC21 had fluffy aerial hyphae with loose marginal hyphae, and both sides were all white. The center of the reverse side appeared to be irregular and slightly grayish-green ( Figure 3A,B). The aerial hyphae of XZEC31 were compact and raised in the center, and the reverse side was pale orange ( Figure 4A,B). The colonies of XZEC41 exhibited fluffy aerial hyphae with loose marginal hyphae, and both sides were all white. The center of the front side was gray, and the reverse side showed blackish-green annular concentric rings ( Figure 5A,B).

Morphological Characteristics
One representative isolate was selected from each Colletotrichum group for further study (XZEC11 from group 1, XZEC21 from group 2, XZEC31 from group 3, and XZEC41 from group 4).
The colonies of XZEC11 isolates produced white aerial hyphae with loose marginal hyphae, and the back of the colonies was light orange-red (Figure 2A,B). The colonies of XZEC21 had fluffy aerial hyphae with loose marginal hyphae, and both sides were all white. The center of the reverse side appeared to be irregular and slightly grayish-green ( Figure 3A,B). The aerial hyphae of XZEC31 were compact and raised in the center, and the reverse side was pale orange ( Figure 4A,B). The colonies of XZEC41 exhibited fluffy aerial hyphae with loose marginal hyphae, and both sides were all white. The center of the front side was gray, and the reverse side showed blackish-green annular concentric rings ( Figure 5A,B).         The colonies of XZEC11, XZEC21, and XZEC41 on SNA produced white, sparse aerial hyphae, and the center area of the colonies of XZEC31 was slightly dense compared with the marginal hyphae; the colonies of XZEC31 produced sparser aerial hyphae than those of the other three Colletotrichum groups ( Figure 2C,D, Figure 3C,D, Figure 4C,D and Figure  5C,D). The colonies of XZEC11, XZEC21, and XZEC41 on SNA produced white, sparse aerial hyphae, and the center area of the colonies of XZEC31 was slightly dense compared with the marginal hyphae; the colonies of XZEC31 produced sparser aerial hyphae than those of the other three Colletotrichum groups ( Figure 2C,D, Figure 3C,D, Figure 4C,D and Figure 5C,D).
The conidia of the four Colletotrichum groups were obtained after shaking cultivation with a rotating speed of 200 rpm in the dark at a temperature of 25 • C. Generally, the structures of the four groups appeared to be cylindrical, straight, and hyaline, and they were all aseptate. Additionally, the conidia of XZEC11 were blunt and rounded at both ends, and the longitudinal middle was slightly concave ( Figure 2E-H). The conidia of XZEC21 were thinner than those of the other groups, and one end of the conidia was slightly convex ( Figure 3E-H). The conidia of XZEC31 showed a slightly standard semicircle at both ends, and one end was convex ( Figure 4E-H). The conidia of XZEC41 had a slightly sharp end and were slightly concave in the middle ( Figure 5E-H). The size of each group is shown in Table 2. The description of the size is length (µm) × width (µm). The number of each structure observed is 30.
The appressoria of the four Colletotrichum groups were induced via cultivation on the surface of the hydrophobic coverslip in darkness at 25 • C for 12 h. The appressoria were all olive green. The shape ranged from nearly round to nearly oval, and irregular shapes were observed. Most of the conidia of the four Colletotrichum groups extended from one end to form appressoria, and a few conidia could extend from both ends to form an appressorium ( Figures 2I-L, 3I-L, 4I-L and 5I-L). The sizes of the appressoria of the four Colletotrichum groups were similar ( Table 2).
The ascomata developed on the surface of the colony or under the mycelium, and sterile blades were used to pick the ascomata out of the colony and cut them into pieces to obtain the asci and ascospores. The ascomata of XZEC11 were irregular, and the ascomata produced in the medium were black, while those produced on the surface of the medium were brown (Figure 2M-P). The ascospores of XZEC11 were aseptate, spindle-shaped, slightly curved, and with round ends (Figure 2Q-T); the ascospores of XZEC21 were hyaline, one-celled, and aseptate ( Figure 3N). The asci of XZEC21 were clavate, thin-walled, and eight-spored ( Figure 3M).

Pathogenicity Tests
For each Colletotrichum group, one representative isolate was selected for the pathogenicity test (XZEC11 from group 1, XZEC21 from group 2, XZEC31 from group 3, and XZEC41 from group 4). Four isolates of Colletotrichum were pathogenic, and the inoculated European hornbeam leaves showed lesions similar to the previous symptoms that were observed naturally; nevertheless, the controls remained healthy 10 days after inoculation. Most of the lesions occurred at the edge of the leaves, and a few occurred in some central areas of the leaves ( Figure 6). According to the appearance of the lesions, lesions caused by XZEC1 and XZEC4 were scattered, and their area was small. Lesions caused by XZEC31 were mainly distributed along the edge of leaves with a long and narrow shape, and some infected areas of leaves were missing. Lesions caused by XZEC21 were mainly distributed along the edge of leaves and were wider than those of XZEC31.

Phylogenetic Analysis
Eleven representative Colletotrichum strains (three strains of group 1, three strains of group 2, three strains of group 3, and two strains of group 4) were selected for phylogenetic analysis on the basis of the sequences of the six nuclear gene regions. The sequences of the 11 Colletotrichum isolates were deposited in GenBank ( Table 3) Table 3.

Phylogenetic Analysis
Eleven representative Colletotrichum strains (three strains of group 1, three strains of group 2, three strains of group 3, and two strains of group 4) were selected for phylogenetic analysis on the basis of the sequences of the six nuclear gene regions. The sequences of the 11 Colletotrichum isolates were deposited in GenBank ( Table 3) Table 3.   Center, National Institute of Technology and Evaluation, Japan. C. hippeastri (CBS 241.78) was added as an outgroup. b : ITS, internal transcribed spacer gene; CAL, partial calmodulin gene; CHS-1, partial chitin synthase; GAPDH, partial glyceraldehyde 3-phosphate dehydrogenase gene; ACT, partial actin gene; TUB2, partial beta-tubulin 2 gene; c : isolates used for morphological and biological analysis and pathogenicity tests.

Figure 7.
Phylogenetic tree generated with the concatenated sequences of the ITS, ACT, ApMat, CAL, CHS−1, GAPDH, GS, and TUB2 genes using maximum likelihood analysis. The tree generated by Bayesian inference had a similar topology. Bootstrap support values above 60% (before the slash marks) and Bayesian posterior probabilities above 0.60 (after the slash marks) are given at each node (BP/BPP). Colletotrichum hippeastri (CBS 241.78) was used as an outgroup. Ex-type strains are marked with (*).

Discussion
A graceful appearance with strong phenotypic plasticity and excellent technical properties of timber gives European hornbeams an important role in urban landscaping and economy. However, leaf spot deteriorates the leaf appearance and affects apical dominance, reducing the quality of wood [55]. In this study, C. gloeosporioides, C. fructicola, C. aenigma, and C. siamense were identified as the causal agents of leaf spot on European hornbeam.
Generally speaking, the morphological structure will be identified initially to determine the genus of Colletotrichum. In early studies, most of the identification of the Colletotrichum species was based on the shape of the conidia [56,57]. The size of the conidia of C. gloeosporioides in this study was similar to that reported by Huang et al. [58], Kim et al. [59], and Chen et al. [60], but larger than that reported by Chen et al. [61]. The conidia of C. siamense were similar to those reported by Kim et al. [59] and Cao et al. [62], but smaller than those reported by Zhang et al. [63]. The conidia of C. aenigma were similar to those reported by Zheng et al. [64] but larger than those reported by Wang et al. [65]. The conidia of C. fructicola were similar to those reported by Cai et al. [66] and Huang et al. [58] but shorter than those reported by Costa et al. [67] and Zheng et al. [64]. Other structures, such as appressoria, also exhibit various degrees of difference. This phenomenon in which the sizes of the same structure are not similar could be because of different growth conditions or a loss or change under repeated subculturing [20], similar to the results reported for the asci and ascospores of C. siamense and C. aenigma, which we failed to induce in this study. Significantly, we isolated C. aenigma from Acer rubrum in 2020 in the same nursery in Xuzhou [68], and the method of inducing asci and ascospores was developed during the cultivation of C. aenigma (2020). Except for asci and ascospores, the colonies of the two C. aenigma species were not quite the same (Figure 8). The colonies of C. aenigma (2020) were relatively flat, with a relatively fluffy texture. The middle area of C. aenigma colonies (2022) was raised, the height dropped gradually from the middle to the edge, and the texture was relatively tight.

Discussion
A graceful appearance with strong phenotypic plasticity and excellent technical properties of timber gives European hornbeams an important role in urban landscaping and economy. However, leaf spot deteriorates the leaf appearance and affects apical dominance, reducing the quality of wood [55]. In this study, C. gloeosporioides, C. fructicola, C. aenigma, and C. siamense were identified as the causal agents of leaf spot on European hornbeam.
Generally speaking, the morphological structure will be identified initially to determine the genus of Colletotrichum. In early studies, most of the identification of the Colletotrichum species was based on the shape of the conidia [56,57]. The size of the conidia of C. gloeosporioides in this study was similar to that reported by Huang et al. [58], Kim et al. [59], and Chen et al. [60], but larger than that reported by Chen et al. [61]. The conidia of C. siamense were similar to those reported by Kim et al. [59] and Cao et al. [62], but smaller than those reported by Zhang et al. [63]. The conidia of C. aenigma were similar to those reported by Zheng et al. [64] but larger than those reported by Wang et al. [65]. The conidia of C. fructicola were similar to those reported by Cai et al. [66] and Huang et al. [58] but shorter than those reported by Costa et al. [67] and Zheng et al. [64]. Other structures, such as appressoria, also exhibit various degrees of difference. This phenomenon in which the sizes of the same structure are not similar could be because of different growth conditions or a loss or change under repeated subculturing [20], similar to the results reported for the asci and ascospores of C. siamense and C. aenigma, which we failed to induce in this study. Significantly, we isolated C. aenigma from Acer rubrum in 2020 in the same nursery in Xuzhou [68], and the method of inducing asci and ascospores was developed during the cultivation of C. aenigma (2020). Except for asci and ascospores, the colonies of the two C. aenigma species were not quite the same (Figure 8). The colonies of C. aenigma (2020) were relatively flat, with a relatively fluffy texture. The middle area of C. aenigma colonies (2022) was raised, the height dropped gradually from the middle to the edge, and the texture was relatively tight. During the cultivating of these two Colletotrichum aenigma, we used the same PDA medium with the same formula, cultivated them in the same incubator in the darkness at 25 °C, and the positions in the incubator were also very close. However, despite this, the colony morphology and the ability to produce asci and ascospores changed. It is confusing that the morphology of the same Colletotrichum species changed just because of the different hosts. Therefore, it is not accurate to identify the Colletotrichum species only from morphology, even if it is a 100% identical species. So, more accurate identification methods are needed to distinguish the Colletotrichum species, such as multi-genecombined phylogenetic analysis.
According to the previous literature, ACT, CHS-1, GAPDH, HIS3, ITS, and TUB2 could be used to classify the majority of the Colletotrichum species [6], and three additional During the cultivating of these two Colletotrichum aenigma, we used the same PDA medium with the same formula, cultivated them in the same incubator in the darkness at 25 • C, and the positions in the incubator were also very close. However, despite this, the colony morphology and the ability to produce asci and ascospores changed. It is confusing that the morphology of the same Colletotrichum species changed just because of the different hosts. Therefore, it is not accurate to identify the Colletotrichum species only from morphology, even if it is a 100% identical species. So, more accurate identification methods are needed to distinguish the Colletotrichum species, such as multi-gene-combined phylogenetic analysis.
According to the previous literature, ACT, CHS-1, GAPDH, HIS3, ITS, and TUB2 could be used to classify the majority of the Colletotrichum species [6], and three additional loci (ApMat, CAL, and GS) have been used for the C. gloeosporioides species complex [20,69].
Five conventional genes (ACT, CHS-1, GAPDH, ITS, and TUB2), four specific genes (ApMat, CAL, and GS), and one additional specific gene (APN2) were used in this study, and four species in this study were separated from the C. gloeosporioides species complex. The combined phylogenetic tree was consistent with trees presented in other studies [61,[70][71][72].
Due to its strong environmental adaptability, the Colletotrichum species can cause leaf spot and fruit diseases with huge losses in agricultural and forestry production worldwide [21,65,[73][74][75][76][77][78][79][80][81][82]. Furthermore, changes in the climate, human activities, and other factors may cause fungi host jumping within the plants in the nursery [83]. It is very likely that C. aenigma, which we isolated in 2020 and 2022, has experienced this, and maybe C. aenigma and the other three species also jumped to the other hosts (except for Acer rubrum and European hornbeams), though we have not found this yet. Therefore, more reports about local leaf spot diseases caused by Colletotrichum species may be produced in the future.

Conclusions
In conclusion, this study identified C. gloeosporioides, C. fructicola, C. aenigma, and C. siamense as the pathogens causing leaf blight on European hornbeam, posing a new and emerging threat to European hornbeam. This research represents the first detailed study of the pathogenicity, morphology, and phylogeny of four Colletotrichum species on European hornbeam in China. Further research exploring the infection cycle of this emerging disease in European hornbeam remains to be conducted, and strategies for the control of this new pathological system should be identified.  Institutional Review Board Statement: Not applicable for studies not involving humans or animals.

Informed Consent Statement: Not applicable.
Data Availability Statement: All data generated or analyzed during this study are included in this article.

Conflicts of Interest:
The authors declare no conflict of interest.