DNA Barcoding as a Tool for Surveying Cytospora Species Associated with Branch Dieback and Canker Diseases of Woody Plants in Canada
1
Independent Researcher, Swift Current, SK S9H 4E6, Canada
2
Laboratory of Mycology, Nature Research Centre, LT 08406 Vilnius, Lithuania
*
Author to whom correspondence should be addressed.
DNA 2025, 5(2), 20; https://doi.org/10.3390/dna5020020
Submission received: 30 December 2024
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Revised: 13 April 2025
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Accepted: 17 April 2025
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Published: 21 April 2025
Abstract
:Background/Objectives: Branch dieback and canker diseases caused by Cytospora species adversely impact the health of woody plants worldwide. Results: During this survey, 59 Cytospora isolates were obtained from symptomatic trees and shrubs growing in southwest Ontario and Saskatchewan, Canada. A DNA barcoding approach combined with morphological characterization identified 15 known species of Cytospora associated with these diseases: C. chrysosperma, C. curvata, C. euonymina, C. hoffmannii, C. kantschavelii, C. leucosperma, C. leucostoma, C. nitschkeana, C. piceae, C. populina, C. pruinopsis, C. pruinosa, C. ribis, C. schulzeri, and C. sorbina. The most common species isolated from multiple hosts were C. sorbina (10), C. chrysosperma (8), C. nitschkeana (6), and C. pruinosa (6). A wide range of host associations, including non-conifer species, was observed for C. piceae. Conclusions: The obtained results contribute to the study of diversity, host affiliation, geographical distribution, and pathogenicity of Cytospora species occurring on woody plants in both natural habitats and agricultural systems. The findings support the effectiveness of using DNA barcodes in fungal taxonomy and plant pathology studies.
1. Introduction
DNA barcoding is a standardized approach that can be applied for the correct identification and recognition of fungi while overcoming issues related to the traditional criteria used for the description of fungal species [1]. A DNA barcode is a relatively short gene region of the highly variable parts of a genome, unique for species identification. The main advantage of this approach is that a single specimen can provide information about the species, regardless of its morphology or lifestage characteristics [2]. Currently, the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster is employed as the main fungal barcode. The ITS can be amplified from a small amount of biological material because of multiple repeated copies present in an organism’s genome [3].
The ascomycetous genus Cytospora (Cytosporaceae) includes plant endophytes or latent pathogens with a worldwide distribution. Many Cytospora species are well known as causal agents of branch dieback and canker diseases on different woody plants, especially when their hosts are under stress conditions [4,5,6]. Considered weak pathogens, Cytospora species can reduce the longevity and productivity of related hosts in the long term [7]. Common disease symptoms on branches include wilting, leaf yellowing and defoliation, dark sunken areas, and loose bark. As disease progresses, cankers form in other tree parts, allowing for pathogen overwintering. Cytospora asexual and sexual fruiting structures, when present, mainly appear on infected wood tissues during spring and fall seasons. Under favourable conditions (e.g., high moisture), conidia or spores are released and dispersed on nearby hosts by wind, rain splash, or insects [8].
The traditional approach for Cytospora identification was primarily based on species morphological and ecological characteristics [9,10]. These criteria seem to be insufficient for Cytospora species delimitation because of significant overlap in morphological traits and lack of host specificity within the genus. Cytospora have been recorded on more than 600 plants, and different species of the genus can co-occur on the same host [6,7]. However, the occurrence of some species can be restricted to a single host genus or family [11,12]. Currently, the identification of Cytospora species includes both morphological and molecular (sequence) data analyses. The ITS region was initially employed to resolve the phylogeny of Cytospora [7]. The sequences of gene coding for essential and conserved proteins, such as actin (act1), RNA polymerase II subunit (rpb2), translation elongation factor 1-alpha (tef1-α), and beta-tubulin (tub2), were also used for phylogenetic analyses of the genus to address the issues of species recognition. ITS and protein-coding gene sequence data have been combined in comprehensive analyses to accurately identify and describe Cytospora species, as reported in the most recent studies [13,14,15]. Therefore, a DNA-based approach including multi-locus phylogenetic analysis is critical to uncovering Cytospora species associated with plant diseases.
Trees and shrubs with symptoms of branch dieback and canker diseases were observed during surveys in southwest Ontario and Saskatchewan (Canada) in 2020–2023 (Figure 1). Despite a number of recent taxonomic works on Cytospora, there are few formal reports on this species-rich genus from the country. Therefore, exhaustive knowledge on the identity of Cytospora isolated from symptomatic plants is needed to develop proper control strategies in case of disease outbreaks. Thus, this survey was aimed to identify Cytospora species associated with diseased woody plants by applying the DNA barcoding approach using the ITS and act1 gene regions supported by morphological characterization.
2. Materials and Methods
2.1. Sample Collection and Fungus Isolation
Forest-forming woody plants expressing clear symptoms of branch dieback and canker were examined to detect Cytospora spp. associated with the diseases. Branches and twigs with fruiting structures typical for Cytospora were sampled for fungal isolation and identification in different locations (Table S1). The isolates were mainly obtained using the single spore isolation technique [16]. To isolate pathogen species from necrotic plant tissue, small wood pieces (0.5–1 cm) were surface-sterilized with 70% ethanol for 30 s, following sterilization with 0.5% sodium hypochlorite for 2 m, rinsed three times with sterile water, and plated on malt extract agar (MEA). Cytospora-like colonies were further purified by transferring hyphal tips to new MEA plates. The obtained isolates were grouped into morphotypes, and representative isolates for each morphotype were further selected for sequencing and morphological characterization. Some specimens were deposited in the Canadian National Mycological Herbarium (DAOM), Ottawa, ON, Canada (Table S1).
2.2. Morphological Characterization
Characterization and measurements of asexual reproductive structures (conidiomata (n = 5), conidiophores (conidiogenous cells), and conidia (n = 20) were performed with dissecting (AmScope SE306R-PZ) and compound (AmScope B120C-E5) microscopes (AmScope, Irvine, CA, USA). Radial colony growth and colour (both adverse and reverse sides) were accessed after 7 days of incubation at room temperature in the dark on MEA. Growth rate was defined as either slow-growing (up to 4.5 cm) or fast-growing (up to 9 cm). Pycnidia formation was checked after 21 days of incubation.
2.3. DNA Extraction, PCR Amplification and Sequencing
Total genomic DNA (gDNA) was extracted from 7 to 11 day old pure cultures using DNeasy Plant ProKit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. Polymerase chain reactions (PCRs) were conducted to amplify the internal transcribed spacer rDNA (ITS) and the partial actin (act1) regions (Table 1). The quality of the PCR products was examined using electrophoresis in 1% agarose gel. The procedures (incl., Sanger sequencing) were carried out at the Genome Quebec Innovation Centre (Montreal, QC, Canada).
2.4. Sequence Alignment and Phylogenetic Analysis
The initial identification was performed using the BLASTn tool against the GenBank nucleotide database of the National Center for Biotechnology Information (NCBI) (accessed: October 2024). Sequence data of the related reference strains [6] were downloaded from the GenBank database. The sequences were initially aligned employing CLUSTAL-X2 v.2.1 [19] and manually edited with MEGA-X [20]. Phylogenetic analyses were executed using randomized accelerated maximum likelihood (RAxML) v. 8.0 method [21] for maximum likelihood (ML) analysis. Bayesian posterior (BP) probabilities were defined with MrBayes v.3.2.7 [22] using the TrEase web server [23]. ML analysis was performed using the transition (TIM) substitution model, with the gamma-distributed rate of heterogeneity selected with ModelTest-NG v.0.1.7 [24]. Statistical support values were estimated with bootstrapping of 1000 replicates [25]. The general time reversible (GTR) model was chosen for the BI analysis. The Markov chain Monte Carlo (MCMC) algorithm was used to estimate Bayesian posterior probabilities (BPPs). Six simultaneous Markov chains were run for 1,000,000 generations. A burn-in was implemented with discarding the first 30% of generated trees. The phylograms were visualized using FigTree v. 1.4.4 [26]. The newly generated sequences were deposited in GenBank (Table 2). The final alignment used in the analysis was submitted to TreeBase (www.treebase.org, accessed on 29 December 2024; ID: S31906).
3. Results
3.1. Phylogenetic Analysis
The ML and BP analyses of the combined ITS and act1 sequence data produced phylogenetic trees with similar topologies. The final alignment comprised 679 characters (ITS: 471, act1: 208), including 243 distinct alignment patterns with 22.74% undetermined characters or gaps. The best-scoring ML tree with a log-likelihood value of −3511.991652 is depicted in Figure 2. Estimated base frequencies were as follows: A, C, G, T = 0.250000; substitution rates: AC = 2.518389, AG = 4.873235, AT = 2.518389, CG = 1.000000, CT = 8.755476, GT = 1.000000; gamma distribution shape parameter α = 0.208145.
The obtained strains, clustered into 15 clades with high support values, were assigned to the following known species: C. chrysosperma, C. curvata, C. euonymina, C. hoffmannii, C. kantschavelii, C. leucosperma, C. leucostoma, C. nitschkeana, C. piceae, C. populina, C. pruinopsis, C. pruinosa, C. ribis, C. schulzeri, and C. sorbina.
3.2. Taxonomy
Cytospora chrysosperma (Pers.) Fr., Syst. Mycol. (Lundae) 2(2): 542. 1823. Figure 3A.
Description: Asexual morph (EI-SK-93(A)): Conidiomata pycnidial, immersed in bark, erumpent, discoid, multi-loculate, 1100–1400 μm in diam. Conidiophores mainly reduced to conidiogenous cells, hyaline, smooth-walled, unbranched, enteroblastic, phialidic. Conidia hyaline, allantoid, aseptate, smooth, 3.8–4.4 × 1.3–1.5 μm.
Culture characteristics: Colonies on MEA initially white, relatively fast-growing, with thin, regular texture and sparce aerial mycelium, becoming brownish in adverse and beige in reverse. Sterile mycelium, without fruiting structures after 21 days of incubation.
Cytospora curvata Norph., Bulgakov, T.C. Wen & K.D. Hyde, Mycosphere 8 (1): 57. 2017. Figure 3B.
Description: Asexual morph (EI-203): Conidiomata pycnidial, semi-immersed in bark, erumpent, unilocular, 750–1100 μm in diam. Conidiophores mainly reduced to conidiogenous cells, hyaline, smooth-walled, mainly unbranched, enteroblastic, phialidic. Conidia hyaline, elongated–allantoid, aseptate, slightly curved, smooth, 5.4–5.8 × 1.2–1.5 μm.
Culture characteristics: Colonies on MEA initially white, relatively slow-growing, with thick, irregular texture without aerial mycelium, becoming dark green in adverse and dark grey in reverse. Abundant pycnidia appear after 21 days of incubation.
Cytospora euonymina X.L. Fan & C.M. Tian, Persoonia 45: 21. 2019. Figure 3C.
Description: Asexual morph (EI-250): Conidiomata pycnidial, immersed in bark, erumpent, multi-loculate, 1350–1650 μm in diam. Conidiophores mainly reduced to conidiogenous cells, hyaline, smooth-walled, some branched, enteroblastic, phialidic. Conidia hyaline, elongated–allantoid, aseptate, smooth, 5.6–6.2× 1.4–1.6 μm.
Culture characteristics: Colonies on MEA initially white, relatively fast-growing, with thick, irregular texture without aerial mycelium, becoming brown in adverse and light brown in reverse. Rare pycnidia appear after 21 days of incubation.
Cytospora hoffmannii L. Lin, X.L. Fan & Crous, Stud. Mycol. 109: 354. 2024. Figure 3D.
Description: Asexual morph (EI-SK-231): Conidiomata pycnidial, semi-immersed in bark, erumpent, with a few locules, with ostiolar neck, 950–1300 μm in diam. Conidiophores mainly reduced to conidiogenous cells, hyaline, smooth-walled, unbranched, enteroblastic, phialidic. Conidia hyaline, elongated–allantoid, aseptate, smooth, 6–8 × 1.5–2 μm.
Culture characteristics: Colonies on MEA initially white, relatively slow-growing, with thin, irregular texture and sparce aerial mycelium, becoming olivaceous in adverse and greyish in reverse. Sterile mycelium, without fruiting structures after 21 days of incubation.
Cytospora kantschavelii Gvrit., Mikol. Fitopatol. 7: 547. 1973. Figure 3E.
Description: Asexual morph (EI-SK-44): Conidiomata pycnidial, semi-immersed in bark, erumpent, scattered, with a few locules, 850–1100 μm in diam. Conidiophores mainly reduced to conidiogenous cells, hyaline, smooth-walled, unbranched, enteroblastic, phialidic. Conidia hyaline, elongated–allantoid, aseptate, smooth, 5.2–5.7 × 1.2–1.4 μm.
Culture characteristics: Colonies on MEA initially white, relatively fast-growing, with thin, irregular texture without aerial mycelium, becoming light brown in both adverse and reverse. Sterile mycelium, without fruiting structures after 21 days of incubation.
Cytospora leucosperma (Pers.) Fr., Syst. Mycol. (Lundae) 2(2): 543. 1823. Figure 3F.
Description: Asexual morph (EI-54(A)): Conidiomata pycnidial, semi-immersed in bark, erumpent, discoid, multi-loculate, 1250–1400 μm in diam. Conidiophores mainly reduced to conidiogenous cells, hyaline, smooth-walled, unbranched, enteroblastic, phialidic. Conidia hyaline, allantoid, aseptate, smooth, 3.5–4.2 × 1.2–1.4 μm.
Culture characteristics: Colonies on MEA initially white, relatively slow-growing, with thin, regular texture without aerial mycelium, becoming light brown in both adverse and reverse. Sterile mycelium, without fruiting structures after 21 days of incubation.
Cytospora leucostoma (Pers.) Sacc., Michelia 2(7): 264. 1881. Figure 3G.
Description: Asexual morph (EI-78): Conidiomata pycnidial, semi-immersed in bark, erumpent, multi-loculate, with ostiolar neck, 1250–1400 μm in diam. Conidiophores mainly reduced to conidiogenous cells, hyaline, smooth-walled, mainly unbranched, enteroblastic, phialidic. Conidia hyaline, elongated–allantoid, aseptate, smooth, 6.6–7.1 × 1.6–1.8 μm.
Culture characteristics: Colonies on MEA initially white, relatively slow-growing, with thick, regular texture and sparce aerial mycelium, becoming dark green in adverse and greyish green in reverse. Abundant pycnidia appear after 21 days of incubation.
Cytospora nitschkeana L. Lin, X.L. Fan & Crous, Stud. Mycol. 109: 367. 2024. Figure 3H.
Description: Asexual morph (EI-170): Conidiomata pycnidial, immersed in bark, erumpent, scattered, multi-loculate, 900–1250 μm in diam. Conidiophores mainly reduced to conidiogenous cells, hyaline, smooth-walled, unbranched, enteroblastic, phialidic. Conidia hyaline, elongated–allantoid, aseptate, smooth, 5.5–6.1 × 1.4–1.6 μm.
Culture characteristics: Colonies on MEA initially white, relatively fast-growing, with thick, regular texture and aerial mycelium, becoming brown in both adverse and reverse. Rare pycnidia appear after 21 days of incubation.
Note: The reference strain Cytospora (Valsa) salicina CBS 118.22 is currently designated as C. nitschkeana [6].
Cytospora piceae X.L. Fan, Phytotaxa 383 (2): 188. 2018. Figure 3I.
Description: Asexual morph (EI-SK-154(A)): Conidiomata pycnidial, semi-immersed in bark, erumpent, with a few locules, 700–950 μm in diam. Conidiophores mainly reduced to conidiogenous cells, hyaline, smooth-walled, unbranched, enteroblastic, phialidic. Conidia hyaline, allantoid, aseptate, smooth, 4.7–5.2 × 1.2–1.4 μm.
Culture characteristics: Colonies on MEA initially white, relatively fast-growing, with thick, irregular texture and dense aerial mycelium, becoming grey with brownish tint in both adverse and reverse. Sterile mycelium, without fruiting structures after 21 days of incubation.
Cytospora populina (Pers.) Rabenh., Deutschl. Krypt.-Fl. (Leipzig) 1: 148. 1844. Figure 3J.
Description: Asexual morph (EI-477(B)): Conidiomata pycnidial, semi-immersed in bark, erumpent, multi-loculate, with ostiolar neck, 1100–1300 μm in diam. Conidiophores mainly reduced to conidiogenous cells, hyaline, smooth-walled, mainly unbranched, enteroblastic, phialidic. Conidia hyaline, elongated–allantoid, aseptate, smooth, 5.5–6.2 × 1.3–1.5 μm.
Culture characteristics: Colonies on MEA initially white, relatively fast-growing, with thick, irregular texture without aerial mycelium, becoming dark brown in adverse and light brown in reverse. Rare pycnidia appear after 21 days of incubation.
Cytospora pruinopsis C.M. Tian & X.L. Fan, Mycol. Progr. 14: 74. 2015. Figure 3K.
Description: Asexual morph (EI-SK-38): Conidiomata pycnidial, immersed in bark, erumpent, with a few locules, 850–1050 μm in diam. Conidiophores mainly reduced to conidiogenous cells, hyaline, smooth-walled, mainly unbranched, enteroblastic, phialidic. Conidia hyaline, elongated–allantoid, aseptate, smooth, 5.8–6.4 × 1.5–1.7 μm.
Culture characteristics: Colonies on MEA initially white, relatively fast-growing, with thin, regular texture and dense aerial mycelium, becoming light grey in adverse and grey in reverse. Sterile mycelium, without fruiting structures after 21 days of incubation.
Cytospora pruinosa (Fr.) Sacc., Michelia 1(5): 519. 1879. Figure 3L.
Description: Asexual morph (EI-SK-152): Conidiomata pycnidial, semi-immersed in bark, erumpent, with a few locules, 1200–1500 μm in diam. Conidiophores mainly reduced to conidiogenous cells, hyaline, smooth-walled, unbranched, enteroblastic, phialidic. Conidia hyaline, elongated–allantoid, aseptate, smooth, 6.8–7.4 × 1.6–1.8 μm.
Culture characteristics: Colonies on MEA initially white, relatively fast-growing, with thin, regular texture and sparce aerial mycelium, becoming bright yellow in adverse and brownish in reverse. Sterile mycelium, without fruiting structures after 21 days of incubation.
Cytospora ribis Ehrenb., Sylv. mycol. berol. (Berlin): 28. 1818. Figure 3M.
Description: Asexual morph (EI-396): Conidiomata pycnidial, semi-immersed in bark, erumpent, scattered, multi-loculate, with ostiolar neck, 1050–1300 μm in diam. Conidiophores mainly reduced to conidiogenous cells, hyaline, smooth-walled, mainly unbranched, enteroblastic, phialidic. Conidia hyaline, allantoid, aseptate, smooth, 4.7–5.3 × 1.2–1.4 μm.
Culture characteristics: Colonies on MEA initially white, relatively fast-growing, with thin, regular texture and sparce aerial mycelium, becoming beige in both adverse and reverse. Rare pycnidia appear after 21 days of incubation.
Cytospora schulzeri Sacc. & P. Syd., Syll. fung. (Abellini) 14: 918. 1899. Figure 3N.
Description: Asexual morph (EI-SK-101): Conidiomata pycnidial, immersed in bark, erumpent, scattered, multi-loculate, 1300–1650 μm in diam. Conidiophores mainly reduced to conidiogenous cells, hyaline, smooth-walled, some branched, enteroblastic, phialidic. Conidia hyaline, elongated–allantoid, aseptate, smooth, 6.4–7.0 × 1.5–1.7 μm.
Culture characteristics: Colonies on MEA initially white, relatively fast-growing, with thin, regular texture and dense aerial mycelium, becoming greyish in both adverse and reverse. Rare pycnidia appear after 21 days of incubation.
Cytospora sorbina M. Pan & X.L. Fan, Adverseiers in Plant Science 11 (no. 690): 13. 2020. Figure 3O.
Description: Asexual morph (EI-SK-95(A)): Conidiomata pycnidial, semi-immersed in bark, erumpent, multi-loculate, with ostiolar neck, 950–1200 μm in diam. Conidiophores mainly reduced to conidiogenous cells, hyaline, smooth-walled, mainly unbranched, enteroblastic, phialidic. Conidia hyaline, elongated–allantoid, aseptate, smooth, 5.8–6.3 × 1.4–1.6 μm.
Culture characteristics: Colonies on MEA initially white, relatively fast-growing, with thick, regular texture and sparce aerial mycelium, becoming light orange in adverse and brownish in reverse. Sterile mycelium, without fruiting structures after 21 d of incubation.
4. Discussion
Cytospora species associated with branch dieback and canker diseases of economically important fruit trees have recently been reported in North America [27,28]. In case of disease outbreaks, it can lead to significant yield losses for the growers. Since Cytospora is not host-specific [6,7,29], the fungus may switch from hosts occurring in natural habitats to fruit trees growing in agricultural systems.
This survey was conducted to reveal Cytospora species associated with diseased woody plants in non-agricultural terrains in Canada. A DNA barcoding approach and morphological characterization were applied to properly identify the obtained Cytospora isolates. The analysis, based on combined ITS and act1 sequence data, resolved the phylogenies of the selected Cytospora strains. The morphological characteristics of the obtained samples matched those provided in the original descriptions or recent taxonomy studies on the genus. The results highlighted the relatively rich species diversity of Cytospora isolated from symptomatic plants (15 species amongst 59 isolates). But only four species (C. leucostoma, C. pruinopsis, C. schulzeri and C. sorbina) were isolated from affected Malus spp. in the surveyed areas, while twenty-four species of Cytospora have been found to be related to apple tree diseases worldwide [12,30]. This indicates that more studies employing different techniques (e.g., metabarcoding) should be conducted to fully uncover the pathogenic species in such a diverse genus.
Most of the identified Cytospora have previously been reported as causal agents of tree diseases. The species of C. chrysosperma, C. kantchavelii, C. nivea, C. piceae, C. populina, and C. sorbina were found to be associated with canker disease of common forest-forming tree species such as Juglans nigra, Picea crassifolia, Populus alba, Salix spp., Sorbus tianschanica, and Ulmus pumila [5,6,31,32,33,34,35]. Tree species widely cultivated in agricultural systems or planted for ornamental purposes (e.g., Malus spp., Prunus persica, Olea europaea) can be adversely affected by C. leucostoma, C. parasitica, C. pruinosa, and C. pruinopsis [14,15,36], found in the surveyed areas.
Host specificity can be attributed to a group of Cytospora species (incl., C. piceae) affiliated with conifers [4,31]. Meantime, C. piceae was isolated from symptomatic deciduous trees (ash) and shrubs (staghorn) in the surveyed areas of southwest Saskatchewan. This evidence additionally supports a lack of specific host affiliations among Cytospora spp.
Generally, Cytospora species infect woody plants through bark wounds of different origin, whether accidental or related to plant management practices, such as cold injury or pruning. Perennial cankers originated from pruning wounds can be devastating to stressed plants [8,11]. To minimize the spread of Cytospora infection, dead branch removal and pruning should be performed during dry periods. Moreover, a proper scion selection needs to be applied when performing tree grafting, as it can be an inoculum source [37].
Multiple in vivo pathogenicity assays have recently been conducted to show that Cytospora spp. are able to cause canker symptoms on related hosts [14,38,39]. It was also shown that the species of Cytospora may infect healthy (non-stressed) trees maintained under proper growing conditions [12]. This points out that regular monitoring of trees (incl., asymptomatic) growing in the surroundings of fruit tree orchards should be implemented for the early detection of pathogenic Cytospora species. It is worth noting that different isolates of the same fungal pathogen may have distinct pathogenicity characteristics [40]. Therefore, it is necessary to test the isolates obtained from different hosts and geographical locations to fully access species pathogenicity potential.
5. Conclusions
Overall, the obtained results revealed a strong association of Cytospora species with diseased woody plants (incl., fruit trees), as well as the emergence of new plant hosts infected by pathogenic species of the genus in southwestern Ontario and Saskatchewan, Canada. This survey will contribute to further research on fungal tree pathogens and help to develop effective disease prevention and control strategies in the future.
Supplementary Materials
The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/dna5020020/s1, Table S1. Sample information of Cytospora species collected during the survey.
Author Contributions
Conceptualization, E.I. and S.M.; methodology, E.I. and S.M.; validation, E.I. and S.M.; formal analysis, E.I.; investigation, E.I.; data curation, S.M.; writing—original draft preparation, E.I.; writing—review and editing, S.M.; visualization, E.I. All authors have read and agreed to the published version of the manuscript.
Funding
This research received no external funding.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
The ITS and act1 sequences and alignments generated in this study have been deposited in the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/ accessed on 29 December 2024) and TreeBASE (https://treebase.org/treebase-web/ accessed on 29 December 2024), respectively. The accession numbers are provided in the paper.
Acknowledgments
The authors thank S. Iliukhin for his help with sample collection and data management.
Conflicts of Interest
The authors declare no conflicts of interest.
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Figure 1.
Symptoms of branch dieback and canker diseases observed in (A) Ulmus glabra; (B) Sorbus aucuparia; (C) Syringa vulgaris; (D) Acer ginnala; (E) Salix alba; and (F) Picea glauca.
Figure 1.
Symptoms of branch dieback and canker diseases observed in (A) Ulmus glabra; (B) Sorbus aucuparia; (C) Syringa vulgaris; (D) Acer ginnala; (E) Salix alba; and (F) Picea glauca.
Figure 2.
Phylogram of RAxML tree generated based on the analysis of ITS and act1 sequence data of selected Cytospora strains. Bootstrap support values for ML ≥ 50% and BP ≥ 0.90 are shown as ML/BP above or below the nodes. Reference strains are marked in bold. The tree is rooted to Diaporthe vaccinii (CBS 160.32).
Figure 2.
Phylogram of RAxML tree generated based on the analysis of ITS and act1 sequence data of selected Cytospora strains. Bootstrap support values for ML ≥ 50% and BP ≥ 0.90 are shown as ML/BP above or below the nodes. Reference strains are marked in bold. The tree is rooted to Diaporthe vaccinii (CBS 160.32).
Figure 3.
Seven- to eleven-day-old old pure cultures of (A) C. chrysosperma; (B) C. curvata; (C) C. euonymina; (D) C. hoffmannii; (E) C. kantschavelii; (F) C. leucosperma; (G) C. leucostoma; (H) C. nitschkeana; (I) C. piceae; (J) C. populina; (K) C. pruinopsis; (L) C. pruinosa; (M) C. ribis; (N) C. schulzeri; (O) C. sorbina.
Figure 3.
Seven- to eleven-day-old old pure cultures of (A) C. chrysosperma; (B) C. curvata; (C) C. euonymina; (D) C. hoffmannii; (E) C. kantschavelii; (F) C. leucosperma; (G) C. leucostoma; (H) C. nitschkeana; (I) C. piceae; (J) C. populina; (K) C. pruinopsis; (L) C. pruinosa; (M) C. ribis; (N) C. schulzeri; (O) C. sorbina.
Table 1.
Primers and PCR protocols.
| Gene Region | Primer Pair | PCR Conditions | Reference |
|---|---|---|---|
| ITS | ITS1/ITS4 | 94 °C for 5 min, 35 cycles of 94 °C for 30 s, 55 °C for 50 s, and 72 °C for 1 min, 72 °C for 10 min | [17] |
| act1 | ACT-512F/783R | 95 °C for 5 min, 39 cycles of 95 °C for 30 s, 55 °C for 50 min, and 72 °C for 1 min, 72 °C for 10 min | [18] |
Table 2.
Strains of Cytospora species used in phylogenetic analysis with their GenBank accession numbers. Reference strains are marked in bold. Ex-type strains are marked withT. NA-data not available.
Table 2.
Strains of Cytospora species used in phylogenetic analysis with their GenBank accession numbers. Reference strains are marked in bold. Ex-type strains are marked withT. NA-data not available.
| GenBank Accession Numbers | |||||
|---|---|---|---|---|---|
| Species | Strain | Country | Host | ITS | act1 |
| Cytospora chrysosperma | CFCC 89982 | China | Ulmus pumila | KP281261 | KP310835 |
| EI-101 | Canada | Unknown tree | PQ356607 | PQ728056 | |
| EI-351 | Canada | Rhus sp. | PQ385601 | NA | |
| EI-437 | Canada | Populus tremuloides | PQ678997 | NA | |
| EI-SK-71 | Canada | Populus deltoides | PQ678998 | NA | |
| EI-SK-90 | Canada | Salix bebbiana | PQ679047 | NA | |
| EI-SK-93(A) | Canada | Populus tremula | PQ679032 | NA | |
| EI-SK-127 | Canada | Populus sp. | PQ679040 | NA | |
| EI-SK-196 | Canada | Salix alba | PQ679042 | NA | |
| Cytospora curvata | MFLUCC 15-0865T | Russia | Salix alba | KY417728 | KY417694 |
| EI-132 | Canada | Aronia sp. | PQ677273 | PQ728060 | |
| EI-203 | Canada | Syringa vulgaris | PQ677316 | PQ728061 | |
| Cytospora euonymina | CFCC 89993T | China | Euonymus kiautschovicus | MH933630 | MH933537 |
| EI-250 | Canada | Salix sp. | PQ678925 | NA | |
| EI-316 | Canada | Berberis vulgaris | PQ383411 | NA | |
| Cytospora hoffmannii | CFCC 89641 | China | Elaeagnus angustifolia | KF765683 | KU711006 |
| EI-SK-231 | Canada | Salix bebbiana | PQ677104 | PQ728057 | |
| EI-SK-237 | Canada | Salix sp. | PQ677112 | PQ728058 | |
| Cytospora kantschavelii | MFLUCC 15-0857T | Russia | Populus × sibirica | KY417738 | KY417704 |
| EI-SK-33 | Canada | Ulmus glabra | PQ678995 | PQ728067 | |
| EI-SK-44 | Canada | Acer spicatum | PQ678996 | PQ728068 | |
| Cytospora leucosperma | MFLUCC 18-1199T | Russia | Galega officinalis | MK912128 | MN685810 |
| EI-54(A) | Canada | Berberis vulgaris | PQ281438 | NA | |
| Cytospora leucostoma | CFCC 50022 | China | Prunus padus | MH933627 | MH933534 |
| EI-78 | Canada | Malus sp. | PP751512 | NA | |
| EI-223 | Canada | Vaccinium sp. | PQ368601 | PQ728059 | |
| Cytospora nitschkeana | CBS. 118.22 | Netherlands | Salix alba | MH854712 | KX964746 |
| EI-170 | Canada | Salix babylonica | PQ356735 | NA | |
| EI-193 | Canada | Berberis vulgaris | PQ362651 | NA | |
| EI-429 | Canada | Fraxinus americana | PQ421750 | NA | |
| EI-454 | Canada | Unknown tree | PQ425077 | NA | |
| EI-470 | Canada | Fraxinus nigra | PQ678921 | NA | |
| EI-SK-195 | Canada | Salix alba | PQ678915 | NA | |
| Cytospora piceae | CFCC 52841T | China | Picea crassifolia | MH820398 | MH820406 |
| EI-273 | Canada | Picea pungens | ON352565 | NA | |
| EI-SK-36 | Canada | Rhus sp. | PQ671332 | NA | |
| EI-SK-110 | Canada | Picea sp. | PQ671333 | NA | |
| EI-SK-154(A) | Canada | Fraxinus sp. | PQ666762 | NA | |
| Cytospora populina | CFCC 89644T | China | Salix psammophila | KF765686 | KU711007 |
| EI-434 | Canada | Magnolia sp. | PQ422182 | NA | |
| EI-477(B) | Canada | Sorbus sp. | PQ683229 | PQ728055 | |
| EI-478 | Canada | Acer platanoides | PQ425482 | NA | |
| EI-SK-184 | Canada | Aronia sp. | PQ683280 | NA | |
| Cytospora pruinopsis | CFCC 50034T | China | Ulmus pumila | KP281259 | KP310836 |
| EI-SK-38 | Canada | Populus deltoides | PQ678306 | PQ728066 | |
| EI-SK-75 | Canada | Ulmus americana | PQ678839 | NA | |
| EI-SK-94(A) | Canada | Malus sp. | PQ678815 | NA | |
| EI-SK-223 | Canada | Unknown shrubs | PQ678307 | NA | |
| Cytospora pruinosa | CFCC 50036 | China | Syringa oblata | KP310800 | KP310832 |
| EI-SK-133(A) | Canada | Aronia sp. | PQ677817 | PQ728065 | |
| EI-SK-152 | Canada | Syringa reticulata | PQ677975 | NA | |
| EI-SK-155 | Canada | Unknown shrubs | PQ680075 | NA | |
| EI-SK-211 | Canada | Syringa sp. | PQ677872 | NA | |
| EI-SK-215 | Canada | Fraxinus sp. | PQ677818 | NA | |
| EI-SK-221 | Canada | Unknown shrubs | PQ680079 | NA | |
| Cytospora ribis | CFCC 50026 | China | Ulmus pumila | KP281267 | KP310843 |
| EI-396 | Canada | Fraxinus americana | PQ393077 | PQ728070 | |
| EI-SK-60 | Canada | Syringa vulgaris | PQ683333 | NA | |
| Cytospora schulzeri | CFCC 53173 | China | Berberis sp. | MK673070 | MK673040 |
| EI-378 | Canada | Sorbaria sorbifolia | PQ392014 | NA | |
| EI-414 | Canada | Malus sp. | PQ421083 | NA | |
| EI-480 | Canada | Acer platanoides | PQ432426 | PQ728069 | |
| EI-SK-101 | Canada | Malus sp. | PQ728069 | PQ683213 | |
| Cytospora sorbina | CF 20197660T | China | Sorbus tianschanica | MK673052 | MK673022 |
| EI-SK-25 | Canada | Malus sp. | PQ677620 | PQ728063 | |
| EI-SK-31 | Canada | Aronia sp. | PQ677471 | PQ728062 | |
| EI-SK-67 | Canada | Aronia sp. | PQ677674 | PQ728064 | |
| EI-SK-76 | Canada | Prunus sp. | PQ736325 | NA | |
| EI-SK-82 | Canada | Sorbus aucuparia | PQ677675 | NA | |
| EI-SK-92 | Canada | Prunus padus | PQ677676 | NA | |
| EI-SK-95(A) | Canada | Aronia sp. | PQ680078 | NA | |
| EI-SK-131(A) | Canada | Unknown shrubs | PQ677688 | NA | |
| EI-SK-148 | Canada | Sorbus sp. | PQ680076 | NA | |
| EI-SK-157(A) | Canada | Viburnum cf. trilobum | PQ680077 | NA | |
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Ilyukhin, E.; Markovskaja, S. DNA Barcoding as a Tool for Surveying Cytospora Species Associated with Branch Dieback and Canker Diseases of Woody Plants in Canada. DNA 2025, 5, 20. https://doi.org/10.3390/dna5020020
AMA Style
Ilyukhin E, Markovskaja S. DNA Barcoding as a Tool for Surveying Cytospora Species Associated with Branch Dieback and Canker Diseases of Woody Plants in Canada. DNA. 2025; 5(2):20. https://doi.org/10.3390/dna5020020
Chicago/Turabian StyleIlyukhin, Evgeny, and Svetlana Markovskaja. 2025. "DNA Barcoding as a Tool for Surveying Cytospora Species Associated with Branch Dieback and Canker Diseases of Woody Plants in Canada" DNA 5, no. 2: 20. https://doi.org/10.3390/dna5020020
APA StyleIlyukhin, E., & Markovskaja, S. (2025). DNA Barcoding as a Tool for Surveying Cytospora Species Associated with Branch Dieback and Canker Diseases of Woody Plants in Canada. DNA, 5(2), 20. https://doi.org/10.3390/dna5020020
