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DNA Barcoding Reveals Diversity of Cytospora Species Associated with Branch Dieback and Canker Diseases of Woody Plants in Canada

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06 January 2025

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07 January 2025

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Abstract
Branch dieback and canker diseases caused by Cytospora species negatively affect health of woody plants worldwide. In this study, 59 Cytospora isolates were obtained from symptomatic trees and shrubs growing in southwest Ontario and Saskatchewan, Canada. DNA barcoding approach combined with morphological (culture) characterization identified 15 known species of Cytospora associated with the 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 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 also support the effectiveness of using sequence barcodes (ITS, act1) in fungal taxonomy and plant pathology studies.
Keywords: 
branch dieback
;  
canker disease
;  
Cytospora
;  
DNA barcoding
;  
pathogen
Subject: 
Biology and Life Sciences  -   Life Sciences

1. Introduction

DNA barcoding is a standardized approach that can be applied for correct identification and recognition of fungi while overcoming the issues with traditional criteria used for description of fungal species [1]. DNA barcode is a relatively short gene region of the highly variable parts of a genome, unique for the 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 [3].
Cytospora species are well known as causal agents of branch dieback and canker diseases on different woody plants [4,5,6]. Considered as weak pathogens, the species of Cytospora can reduce longevity and productivity of related hosts in the long term [7]. Disease symptoms can initially be observed on twigs and branches in a form of sunken areas with dark discoloration. As disease progresses, cankers form in other tree parts allowing for pathogen overwintering. Cytospora fruiting structures (pycnidia) appear on infected wood tissues during spring and fall seasons mainly. Under favorable conditions (e.g. high moisture), conidia or spores eject from pycnidia and disseminate on nearby hosts with rain splash or wind infecting plant tissue through bark wounds [8].
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. Besides, different species of Cytospora can co-occur on the same host [7]. However, occurrence of some species can be restricted to a single host genus or family [11,12]. Currently, identification of Cytospora species includes both morphological and molecular (sequence) data analyses. ITS region was initially employed to resolve the phylogeny of Cytospora [7]. The partial protein-coding genes (PCG), such as actin (act1), RNA polymerase II subunit (rpb2), translation elongation factor 1-alpha (tef1-α), and beta-tubulin (tub2) were further applied to phylogenetic analyses of the genus to address the issues with species recognition. Combined multi-gene (ITS+PCGs) sequence data have been used to correctly identify or describe Cytospora species in the most recent studies [13,14,15]. Therefore, a DNA-based approach including multi-locus phylogenetic analysis is critical to uncovering Cytospora species diversity associated with plant diseases.
Trees and shrubs with symptoms of branch dieback and canker diseases (Figure 1) have been observed during the surveys in southwest Ontario and Saskatchewan (Canada) in 2020-23. Since Cytospora are among the main causal agents of these diseases, exhaustive knowledge on the identity of the fungi isolated from symptomatic plants is needed to develop proper control strategies in case of disease outbreaks. Thus, this study was aimed to identify Cytospora species associated with diseased woody plants by applying DNA barcoding approach and morphological (culture) characterization.

2. Materials and Methods

2.1. Sample Collection and Fungus Isolation

Branches and twigs with symptoms of dieback and canker were sampled for isolation and identification of Cytospora spp. associated with the diseases. The isolates were mainly obtained using the single spore isolation technique [16]. To isolate pathogen species from necrotic plant tissue, small wood pieces (0.5-1cm) were surface sterilized with 70% ethanol for 30s, following sterilization with 0.5% sodium hypochlorite for 2m, 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 in morphotypes and representative isolates for each morphotype were further selected for morphological (culture) characterization and sequencing.

2.2. Morphological Characterization

Fungus fruiting structures (conidio- or ascomata) were observed and sectioned under a dissecting microscope (AmScope SE306R-PZ). To confirm sample affiliation with Cytospora species, microstructures (conidia, spores) were examined using a compound microscope (AmScope B120C-E5). Radial colony growth and color (both adverse and reverse sides) were accessed after 7d of incubation at room temperature in the dark on MEA. Growth rate was defined as either slow growing (up to 4.5cm) or fast growing (up to 9cm). Pycnidia formation was checked after 21d of incubation.

2.3. DNA Extraction, PCR Amplification and Sequencing

Total genomic DNA (gDNA) was extracted from 7-11d old pure cultures using DNeasy Plant ProKit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The primers ITS1/ITS4 [17] were used to amplify internal transcribed spacer (ITS) region. Additionally, the partial actin (act1) gene region was amplified with the primer pair ACT-512F and ACT-783R [18] for the selected strains to improve species resolution. The quality of PCR products was examined using electrophoresis in 1% agarose gel. 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). 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]. The ML analysis was performed using the transition (TIM) substitution model with gamma-distributed rate of heterogeneity selected with ModelTest-NG v.0.1.7 [24]. The statistical support values were estimated with bootstrapping of 1,000 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 (BPP). 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 1). The final alignment used in the analysis was submitted to TreeBase (www.treebase.org; 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 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.
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: See [7].
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, withouth fruiting structures after 21d of incubation.
Material examined: Canada, Southwest Ontario: 43°13'15.8"N 79°13'32.2"W, from fallen branches of unknown tree, May 2020, E. Ilyukhin (EI-101); 43°03'51.1"N 79°17'10.1"W, from branches of Rhus sp., September 2020, E. Ilyukhin (EI-351); 43°08'21.7"N 79°10'09.3"W, from fallen branches of Populus tremuloides, July 2021, E. Ilyukhin (EI-437). Southwest Saskatchewan: 50°16'59.6"N 107°45'49.4"W, from fallen branches of Populus deltoides, July 2023, E. Ilyukhin (EI-SK-71); 50°38'50.8"N 108°00'18.2"W, from twigs of Salix bebbiana, August 2023, E. Ilyukhin (EI-SK-90); 50°39'35.2"N 108°02'55.5"W, from branches of Populus tremula, August 2023, E. Ilyukhin (EI-SK-93(A)); 50°40'24.6"N 107°57'16.2"W, from fallen branches of Populus sp., August 2023, E. Ilyukhin (EI-SK-127); 50°16'35.1"N 108°24'47.8"W, from twigs of Salix alba, September 2023, E. Ilyukhin (EI-SK-196).
Cytospora curvata Norph., Bulgakov, T.C. Wen & K.D. Hyde, Mycosphere 8 (1): 57. 2017. Figure 3B.
Description: See [27].
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 21d of incubation.
Material examined: Canada, Southwest Ontario: 43°06'36.3"N 79°15'04.7"W, from branches of Aronia sp., May 2020, E. Ilyukhin (EI-132); 43°11'59.5"N 79°13'37.1"W, from twigs of Syringa reticulata, May 2020, E. Ilyukhin (EI-203).
Cytospora euonymina X.L. Fan & C.M. Tian, Persoonia 45: 21. 2019. Figure 3C.
Description: See [28].
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 21d of incubation.
Material examined: Canada, Southwest Ontario: 43°11'27.3"N 79°15'53.2"W, from twigs of Salix sp., June 2020, E. Ilyukhin (EI-250); 43°06'23.2"N 79°13'39.0"W, from twigs of Berberis vulgaris, August 2020, E. Ilyukhin (EI-316).
Cytospora hoffmannii L. Lin, X.L. Fan & Crous, Stud. Mycol. 109: 354. 2024. Figure 3D.
Description: See [27] (as C. nivea).
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, withouth fruiting structures after 21d of incubation.
Material examined: Canada, Southwest Saskatchewan: 50°59'43.0"N 106°25'45.1"W, from twigs of Salix bebbiana, October 2023, E. Ilyukhin (EI-SK-231); 50°59'34.9"N 106°25'33.5"W, from branches of Salix sp., October 2023, E. Ilyukhin (EI-SK-237).
Cytospora kantschavelii Gvrit., Mikol. Fitopatol. 7: 547. 1973. Figure 3E.
Description: See [27] (as C. parakantschavelii).
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, withouth fruiting structures after 21d of incubation.
Material examined: Canada, Southwest Saskatchewan: 50°17'05.5"N 107°47'06.0"W, from twigs of Ulmus glabra (plant tissues plated), September 2022, E. Ilyukhin (EI-SK-33); 50°16'00.8"N 107°46'53.5"W, from twigs of Acer spicatum, October 2022, E. Ilyukhin (EI-SK-44).
Cytospora leucosperma (Pers.) Fr., Syst. Mycol. (Lundae) 2(2): 543. 1823. Figure 3F.
Description: See [29] (as C. galegicola).
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, withouth fruiting structures after 21d of incubation.
Material examined: Canada, Southwest Ontario: 43°12'40.4"N 79°14'31.0"W, branches of Bereberis vulgaris, April 2020, E. Ilyukhin (EI-54(A)).
Cytospora leucostoma (Pers.) Sacc., Michelia 2(7): 264. 1881. Figure 3G.
Description: See [27] (as C. erumpens).
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 21d of incubation.
Material examined: Canada, Southwest Ontario: 43°13'07.3"N 79°13'42.3"W, from twigs of Malus sp., May 2020, E. Ilyukhin (EI-78); 43°11'05.2"N 79°13'23.8"W, from branches of Vaccinium sp., May 2020, E. Ilyukhin (EI-223).
Cytospora nitschkeana L. Lin, X.L. Fan & Crous, Stud. Mycol. 109: 367. 2024. Figure 3H.
Description: See [6].
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 21d of incubation.
Material examined: Canada, Southwest Ontario: 43°12'03.0"N 79°14'25.6"W, from twigs of Salix babylonica, May 2020, E. Ilyukhin (EI-170); 43°12'59.7"N 79°12'51.7"W, from branches of Berberis vulgaris, May 2020, E. Ilyukhin (EI-193); 43°08'29.3"N 79°10'09.0"W, from twigs of Fraxinus americana, July 2021, E. Ilyukhin (EI-429); 43°05'33.3"N 79°18'13.1"W, from branches of Syringa sp., October 2021, E. Ilyukhin (EI-454); 43°05'29.4"N 79°18'19.6"W, from branches of Fraxinus nigra, October 2021, E. Ilyukhin (EI-470). Southwest Saskatchewan: 50°16'35.1"N 108°24'47.8"W, from twigs of Salix alba, September 2023, E. Ilyukhin (EI-SK-195).
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: See [30].
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, withouth fruiting structures after 21d of incubation.
Material examined: Canada, Southwest Ontario: 43°11'36.8"N 79°16'02.4"W, from branches of Picea pungens (plant tissue plated), August 2020, E. Ilyukhin (EI-273). Southwest Saskatchewan: 50°16'50.6"N 107°47'09.1"W, from branches of Rhus sp., September 2022, E. Ilyukhin (EI-SK-36); 50°40'43.3"N 107°56'38.9"W, from branches of Picea sp. (plant tissue plated), August 2023, E. Ilyukhin (EI-SK-110); 50°16'30.5"N 108°25'02.6"W, from twigs of Fraxinus sp., September 2023, E. Ilyukhin (EI-154(A)).
Cytospora populina (Pers.) Rabenh., Deutschl. Krypt.-Fl. (Leipzig) 1: 148. 1844. Figure 3J.
Description: See [31].
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 21d of incubation.
Material examined: Canada, Southwest Ontario: 43°08'29.0"N 79°10'23.0"W, from branches of Magnolia sp., July 2021, E. Ilyukhin (EI-434); 43°05'23.6"N 79°18'25.1"W, from branches of Sorbus sp., October 2021, E. Ilyukhin (EI-477(B)); 43°05'26.5"N 79°18'21.7"W, from twigs of Acer platanoides, October 2021, E. Ilyukhin (EI-478). Southwest Saskatchewan: 50°16'36.6"N 108°24'52.3"W, from branches of Aronia sp., September 2023, E. Ilyukhin (EI-SK-184).
Cytospora pruinopsis C.M. Tian & X.L. Fan, Mycol. Progr. 14: 74. 2015. Figure 3K.
Description: See [32].
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, withouth fruiting structures after 21d of incubation.
Material examined: Southwest Saskatchewan: 50°16'44.5"N 107°47'10.0"W, from branches of Populus deltoides, October 2022, E. Ilyukhin (EI-SK-38); 50°16'51.8"N 107°45'37.2"W, from twigs of Ulmus americana, June 2023, E. Ilyukhin (EI-SK-75); 50°40'00.5"N 108°02'59.7"W, from twigs of Malus sp., August 2023, E. Ilyukhin (EI-SK-94(A)); 51°00'01.3"N 106°25'59.3"W, from branches of unknown shrubs, October 2023, E. Ilyukhin (EI-SK-223).
Cytospora pruinosa (Fr.) Sacc., Michelia 1(5): 519. 1879. Figure 3L.
Description: See [6].
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, withouth fruiting structures after 21d of incubation.
Material examined: Southwest Saskatchewan: 50°38'58.5"N 107°56'52.1"W, from twigs of Aronia sp., August 2023, E. Ilyukhin (EI-SK-133(A)); 50°16'34.8"N 108°24'45.6"W, from twigs of Syringa reticulata, September 2023, E. Ilyukhin (EI-SK-152); 50°16'38.4"N 108°25'01.1"W, from twigs of unknown shrubs, September 2023, E. Ilyukhin (EI-SK-155); 50°59'42.2"N 106°25'22.6"W, from branches of Syringa sp., October 2023, E. Ilyukhin (EI-SK-211); 50°59'37.6"N 106°25'35.4"W, from twigs of Fraxinus sp., October 2023, E. Ilyukhin (EI-SK-215); 50°59'43.8"N 106°25'53.2"W, from branches of unknown shrubs, October 2023, E. Ilyukhin (EI-SK-221).
Cytospora ribis Ehrenb., Sylv. mycol. berol. (Berlin): 28. 1818. Figure 3M.
Description: See [6].
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 21d of incubation.
Material examined: Southwest Ontario: 43°08'01.3"N 79°22'50.9"W, from twigs of Fraxinus americana, May 2021, E. Ilyukhin (EI-396). Southwest Saskatchewan: 50°16'51.8"N 107°45'40.6"W, from twigs of Syringa vulgaris, June 2023, E. Ilyukhin (EI-SK-60).
Cytospora schulzeri Sacc. & P. Syd., Syll. fung. (Abellini) 14: 918. 1899. Figure 3N.
Description: See [33] (as C. parasitica).
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 21d of incubation.
Material examined: Southwest Ontario: 43°04'00.5"N 79°17'06.3"W, from twigs of Sorbaria sorbifolia, September 2020, E. Ilyukhin (EI-378); 43°08'21.5"N 79°22'26.2"W, from branches of Malus sp., May 2021, E. Ilyukhin (EI-414); 43°05'26.5"N 79°18'21.0"W, from twigs of Acer platanoides, October 2021, E. Ilyukhin (EI-480). Southwest Saskatchewan: 50°40'16.2"N 108°00'11.3"W, from twigs of Malus sp., August 2023, E. Ilyukhin (EI-SK-101).
Cytospora sorbina M. Pan & X.L. Fan, Adverseiers in Plant Science 11 (no. 690): 13. 2020. Figure 3N.
Description: See [34].
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, withouth fruiting structures after 21d of incubation.
Material examined: Southwest Saskatchewan: 50°17'26.2"N 107°47'17.3"W, from branches of Malus sp., September 2022, E. Ilyukhin (EI-SK-25); 50°17'11.5"N 107°47'24.6"W, from branches of Aronia sp., September 2022, E. Ilyukhin (EI-SK-31); 50°16'55.4"N 107°45'43.9"W, from twigs of Aronia sp., June 2023, E. Ilyukhin (EI-SK-67); 50°16'51.8"N 107°45'37.2"W, from twigs of Prunus sp., June 2023, E. Ilyukhin (EI-SK-76); 50°38'45.8"N 107°58'42.0"W, from branches of Sorbus aucuparia, August 2023, E. Ilyukhin (EI-SK-82); 50°38'50.8"N 108°00'18.2"W, from branches of Prunus padus, August 2023, E. Ilyukhin (EI-SK-92); 50°39'52.4"N 108°02'38.0"W, from branches of Aronia sp., August 2023, E. Ilyukhin (EI-SK-95(A)); 50°38'59.6"N 107°56'52.0"W, from twigs of unknown shrubs, August 2023, E. Ilyukhin (EI-SK-131(A)); 50°16'42.4"N 108°24'36.0"W, from twigs of Sorbus sp., September 2023, E. Ilyukhin (EI-SK-148); 50°16'38.2"N 108°24'52.3"W, from twigs of Viburnum cf. trilobum, September 2023, E. Ilyukhin (EI-SK-157(A)).

4. Discussion

Cytospora species associated with branch dieback and canker diseases of economically important fruit trees have been recently reported in North America [35,36]. In case of disease outbreaks, it can lead to significant yield losses for the growers. Since Cytospora is not host-specific [7,28], the fungus may switch from hosts occurring in natural habitats to fruit trees growing in agricultural systems.
This study was conducted to reveal Cytospora species associated with diseased woody plants in non-agricultural terrains in Canada. DNA barcoding approach and morphological (culture) characterization were applied to properly identify the obtained Cytospora isolates. The analysis based on combined ITS and act1 sequence data resolved phylogenies of the selected Cytospora strains. 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 24 species of Cytospora were found to be related to apple tree diseases worldwide [12,34]. It indicates that more studies employing different techniques (e.g., metabarcoding) should be conducted to fully uncover 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 [5,30,32,34,37,38]. Host specificity can be attributed to a group of Cytospora species (incl., C. piceae) affiliated with conifers [4,30]. Meantime, C. piceae was isolated from symptomatic deciduous trees (ash) and shrubs (staghorn) in this study. This evidence additionally supports a lack of specific host affiliations among Cytospora spp. Other tree species widely cultivated in agricultural systems (e.g., Malus spp., Prunus persica, Olea europaea) can also be significantly affected by C. leucostoma, C. parasitica, C. pruinosa, and C. pruinopsis [14,15,39] found in the surveyed areas.
Multiple in-vivo pathogenicity assays have been recently conducted to show that Cytospora spp. are able to cause canker symptoms on related hosts [14,40,41]. It was also shown that the species of Cytospora may infect healthy (not stressed) trees maintained under proper growing conditions [12]. It points out that regular monitoring of trees (incl., asymptomatic) growing in the surroundings of fruit tree orchards should be implemented for early detection of pathogenic Cytospora species.
Overall, the obtained results revealed a strong association of Cytospora species with diseased woody plants, as well as the emergence of new hosts in southwestern Ontario and Saskatchewan, Canada. This study will contribute to the further research of fungal tree pathogens and help to develop effective disease prevention and control strategies.

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

Not applicable.

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/) and TreeBASE (https://treebase.org/treebase-web/), respectively. The accession numbers were provided in the paper.

Acknowledgments

The authors thank Simon 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.
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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).
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Figure 3. 7-11d 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. 7-11d 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.
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Table 1. 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 1. 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 Berberissp. 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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