Fungal Richness of Cytospora Species Associated with Willow Canker Disease in China

Species of Cytospora are considered important plant pathogens of a wide range of plant hosts, especially Salicaceae plants. Salix (Salicaceae, Malpighiales) has been widely cultivated in China because of its strong ecological adaptability, fast growth, and easy reproduction. In this study, a total of eight species of Cytospora were discovered on Salix in China, including C. ailanthicola, C. alba, C. chrysosperma, C. gigaspora, C. nivea, C. paracinnamomea, C. rostrata, and C. sophoriopsis. Among them, C. alba and C. paracinnamomea were identified as novel species based on morphology and phylogenetic analyses of ITS, act, rpb2, tef1-α, and tub2 gene sequences and were confirmed as pathogens of willow canker disease by pathogenicity tests. The mycelial growth rates of strains from these two novel species (C. alba and C. paracinnamomea) had optimum temperatures of 21 to 22 °C and an optimum pH value of 5 to 6. The effectiveness of six carbon sources on the mycelial growth showed that fructose and maltose had the highest influence. Cytospora species richness was significantly positively correlated with dry and wet areas. This study represents a significant evaluation of Cytospora associated with willow canker disease in China and provides a theoretical basis for predicting the potential risk of willow canker disease.


Introduction
Willow (Salix) trees have been widely cultivated in China because of their strong ecological adaptability, fast growth, easy reproduction, and short rotation period [1]. In terms of horizontal distribution, they are distributed all over the country, with the most concentrated distribution in the northeast, northwest, and southwest regions. These regions have higher elevations or latitudes, and the horizontal distribution of species is extremely rich [2]. However, a large number of willow trees suffer from diseases caused by several fungal pathogens, such as over 20 species of Cytospora causing canker disease [3,4] (https: //nt.arsgrin.gov/fungaldatabases, accessed on 6 April 2022); over 50 species of Melampsora causing rust disease [5][6][7][8][9] (https://nt.ars-grin.gov/fungaldatabases, accessed on 6 April 2022); Colletotrichum salicis causing anthracnose disease [10]; and Rhytisma filamentosum causing tar-spot disease [11,12]. Among various diseases that affect willow trees, canker and dieback disease caused by Cytospora are the main branch and stem diseases in China, especially in the north ( Figure 1) [4,[13][14][15][16]. The pathogens in Cytospora are host-dominant, and usually invade weak trees. There are many factors that cause tree weakness, such as meteorological factors including temperature, humidity, rainfall, sunshine, airflow, etc. Meanwhile, tree age, tree species, slope aspect, soil, nursery management, planting density, and stand tending management technology are also factors that cause Cytospora canker disease. These factors influence each other and work together [17]. Symptoms of Cytospora canker disease associated with willow trees vary according to the stage of disease development. The disease infects the inner bark and causes the sapwood to sink slightly and discolor [18]. Several prominent fruiting bodies form and are buried or semi-buried under the bark, solitary, or in clusters [4]. Under moist conditions, the conidia emerge from the conidiomata in the form of colored and coiled tendrils [4]. Usually, these conidiomata develop in the cankers on bark but may also exist in healthy plant tissue and can be isolated from sound bark, xylem, and leaves of many tree species [19,20]. The genus Cytospora (Cytosporaceae, Diaporthales) was established by Ehrenberg [21], which comprises important phytopathogenic, saprobic, and endophytic fungi [4,18]. Species of Cytospora inhabit a wide variety of hosts that include economically and ecologically important trees (e.g., Elaeagnaceae, Juglandaceae, Rosaceae, Salicaceae, Ulmaceae) [22][23][24][25][26]. About 150 species of Cytospora in total have been discovered on dieback and stem canker in over 130 species of woody hosts [18,[27][28][29][30][31][32][33].
Over 20 Cytospora species infect willow trees all over the world (https://nt.arsgrin.gov/fungaldatabases, accessed on 6 April 2022). An increase in the number of Cytospora species isolated from willow cankers has also been reported in China recently. Fan et al. [22] concluded that six species of Cytospora from Salix were observed in northern China: C. chrysosperma, C. fugax, C. leucosperma, C. nivea, C. populina, and C. rostrata. Wang et al. [3] identified six Cytospora species occurring on poplar and willow: C. atrocirrhata, C. chrysosperma, C. davidiana, C. fugax, C. kantschavelii, and C. translucens. The diversity of Cytospora on willows deserves further exploration for better disease management. There is an urgent need for systematic studies and re-evaluation to facilitate the identification of Cytospora from Salix in China. The objectives of this study were (1) to determine species of Cytospora from willow trees in China; (2) to confirm the pathogenicity of two new Cytospora species; (3) to determine the effects of temperature, pH, and carbon source on the mycelial growth of the new Cytospora species; and (4) to evaluate the geographical distribution of Cytospora species associated with willow canker disease in China. Symptoms of Cytospora canker disease associated with willow trees vary according to the stage of disease development. The disease infects the inner bark and causes the sapwood to sink slightly and discolor [18]. Several prominent fruiting bodies form and are buried or semi-buried under the bark, solitary, or in clusters [4]. Under moist conditions, the conidia emerge from the conidiomata in the form of colored and coiled tendrils [4]. Usually, these conidiomata develop in the cankers on bark but may also exist in healthy plant tissue and can be isolated from sound bark, xylem, and leaves of many tree species [19,20]. The genus Cytospora (Cytosporaceae, Diaporthales) was established by Ehrenberg [21], which comprises important phytopathogenic, saprobic, and endophytic fungi [4,18]. Species of Cytospora inhabit a wide variety of hosts that include economically and ecologically important trees (e.g., Elaeagnaceae, Juglandaceae, Rosaceae, Salicaceae, Ulmaceae) [22][23][24][25][26]. About 150 species of Cytospora in total have been discovered on dieback and stem canker in over 130 species of woody hosts [18,[27][28][29][30][31][32][33].
Over 20 Cytospora species infect willow trees all over the world (https://nt.arsgrin.gov/fungaldatabases, accessed on 6 April 2022). An increase in the number of Cytospora species isolated from willow cankers has also been reported in China recently. Fan et al. [22] concluded that six species of Cytospora from Salix were observed in northern China: C. chrysosperma, C. fugax, C. leucosperma, C. nivea, C. populina, and C. rostrata. Wang et al. [3] identified six Cytospora species occurring on poplar and willow: C. atrocirrhata, C. chrysosperma, C. davidiana, C. fugax, C. kantschavelii, and C. translucens. The diversity of Cytospora on willows deserves further exploration for better disease management. There is an urgent need for systematic studies and re-evaluation to facilitate the identification of Cytospora from Salix in China. The objectives of this study were (1) to determine species of Cytospora from willow trees in China; (2) to confirm the pathogenicity of two new Cytospora species; (3) to determine the effects of temperature, pH, and carbon source on the mycelial growth of the new Cytospora species; and (4) to evaluate the geographical distribution of Cytospora species associated with willow canker disease in China.

Specimens and Strains
Sixty-two specimens were collected from diseased branches or twigs of Salix matsudana, Salix psammophila, and Salix cupularis distributed in China. Sixty-six Cytospora strains were isolated by removing a mucoid spore mass from conidiomata and/or ascomata. The suspension was spread over the surface of potato dextrose agar (PDA) (potato, 200 g; glucose, 20 g; agar, 20 g; distilled water, to complete 1000 mL) media, which were incubated at 25 • C in darkness until spores germinated. Hyphal tips were removed to a new PDA plate twice to obtain a pure culture. Specimens were preserved in the Museum of Beijing Forestry University (BJFC). Strains were deposited in the China Forestry Culture Collection Centre (CFCC).

Microscopic Observations
The ascomata and conidiomata formed on the branches and/or twigs of Salix were photographed using the Leica stereomicroscope (M205) (Leica Microsystems, Wetzlar, Germany). Over 30 conidiomata (and/or ascomata) and 50 conidia and/or ascospores were selected randomly to measure their lengths and widths using a Nikon Eclipse 80i microscope (Nikon Corporation, Tokyo, Japan) equipped with a Nikon digital sight DS-Ri2 high-definition color camera with differential interference contrast (DIC). Cultural characteristics of strains plated onto PDA and incubated at 25 • C in darkness were recorded after seven and thirty days according to the color charts of Rayner [34].

DNA Extraction, Amplification, and Phylogeny
Total genomic DNA was harvested from axenic cultures on PDA using CTAB method [35]. PCR amplifications and sequencing of ITS, act, rpb2, tef1-α, and tub2 genes were performed. The PCR conditions and primers are listed in Table S1. PCR products were electrophoresed in 1% agarose gel and the DNA was sequenced by the Tsingke Biotechnology Company Limited. A preliminary identification of strains was based on BLAST using the ITS sequences to confirm the strains obtained in the current study grouped in the genus Cytospora. Twenty-two representative strains were selected for the following analyses excluding the strains having parallel ITS sequence, morphological features, host, and location for each species. The sequences generated from this study were supplemented with other sequences obtained from GenBank (Table S2) based on recent publications of Cytospora [4,26,32]. For the five individual datasets (ITS, act, rpb2, tef1-α, and tub2), sequences alignments were made using MAFFT v. 6.0 [36] and adjusted using MEGA v. 6.0 [37]. Ambiguous regions were excluded from alignments.
Phylogenetic analyses were carried out with maximum parsimony (MP), maximum likelihood (ML), and Bayesian Inference (BI) analyses. MP analysis was computed with PAUP v. 4.0b10 [38] using a heuristic search option of 1000 random-addition sequences with a tree bisection and reconnection (TBR) branch swapping algorithm. ML analysis was computed with GTR+G+I model of site substitution using PhyML v. 3.0 [39] following the instruction of Fan et al. [4]. BI analysis was computed using the best-fit evolutionary models for each partitioned locus estimated in MrModeltest v. 2.3 [40], with a Markov Chain Monte Carlo (MCMC) algorithm in MrBayes v. 3.1.2 [41]. Phylograms were viewed in Figtree v. 1.3.1 [42]. Sequence data were deposited in GenBank. The multilocus sequence alignment and the trees obtained were deposited in TreeBASE (study ID S29245).

Pathogenicity Test
After species identification, healthy cutting plants of Salix matsudana inoculated with two novel species (C. alba and C. paracinnamomea) to determine the relative pathogenicity. For mycelial inoculation, the ex-holotype strains (C. alba: CFCC 55462; C. paracinnamomea: CFCC 55453) were cultured for a duration of five days at room temperature (25 • C) in darkness. Cuttings of plants were scalded using a 5-mm-diameter sterile inoculation loop, putting a PDA mycelial plug on each burn wound. Moistened cotton was placed directly above each disc, and all were covered with parafilm. Ten inoculation dots were set for each isolate. Ten replicates were inoculated using uncolonized PDA plugs to serve as controls. After a week, the parafilm and cotton wool were removed. The inoculated plants were maintained in the field to observe the symptoms of willow canker and expansion of lesion length. Re-isolation and morphological observation of fungal pathogens were performed to verify Koch's postulates [43].

Temperature, pH, and Carbon Source Tests
Ex-holotype strains (C. alba: CFCC 55462; C. paracinnamomea: CFCC 55453) were selected to evaluate the effects of temperature, pH, and carbon source on mycelial growth of Cytospora. For the temperature variation test, a 5-mm-diameter mycelial plug was inoculated onto PDA media in triplicate and incubated in the dark at 0-40 • C with a 5 • C gradient. For the pH variation test, the PDA was adjusted with 1 mol/L NaOH and 1 mol/L HCl to obtain pH values ranging from 3.0 to 11.0 at an interval of 1.0. A 5-mm-diameter mycelial plug was inoculated in the medium in triplicate and the cultures were incubated in the dark at 25 • C [44,45]. For the investigation of the utilization of carbon sources, the strains were incubated in triplicate in the dark at 25 • C and the PDA was used as the base medium replacing glucose by the tested carbon sources. The 20 g of glucose was replaced by 20 g of fructose, galactose, maltose, sucrose, and xylose [44,45]. Measurements of colony diameter were made every 24 h for four days. Data were analyzed using IBM SPSS Statistics v. 19.0 by one-way analysis of variance (ANOVA). Linear Regression was used to estimate optimum growth temperature and pH value. Figures were output using Microsoft Excel.

Phylogenetic Analyses
The combined matrix of five genes of Cytospora included 3189 characters with gaps (ITS, act, rpb2, tef1-α, and tub2, included 604, 351, 726, 843, and 665, respectively). The concatenated alignment comprised sequences from 255 strains and Diaporthe vaccinii CBS 160.32 was selected as the outgroup. The alignment contained 189 parsimony-uninformative characters, 1445 characters were variable and parsimony-informative. MP analysis generated 200 equally parsimonious trees with similar clade topologies, one of which was presented in Figure   The 21 strains obtained in this study clustered in eight clades based on the combined ITS/act/rpb2/tef1-α/tub2 tree as well as each gene tree except for ITS tree (Figures S1-S5 and 2). The three strains CFCC 55461-55463 grouped as one clade were phylogenetically distinct from all Cytospora species with a high statistical support (MP/ML/BI = 100/100/1). Nine strains (CFCC 55452-CFCC 55460) were grouped together (MP/ML/BI = 99/100/1) and distinguished from other Cytospora species. These two separate clades are herein described as novel species.

Taxonomy
Cytospora species on Salix herein include two novel species (C. alba and C. paracinnamomea) and six known species collected from Salix (C. ailanthicola, C. chrysosperma, C. gigaspora, C. nivea, C. rostrata, and C. sophoriopsis). The asexual morph of C. alba as well as the sexual and asexual morph of C. paracinnamomea were described in this study.

Taxonomy
Cytospora species on Salix herein include two novel species (C. alba and C. paracinnamomea) and six known species collected from Salix (C. ailanthicola, C. chrysosperma, C. gigaspora, C. nivea, C. rostrata, and C. sophoriopsis). The asexual morph of C. alba as well as the sexual and asexual morph of C. paracinnamomea were described in this study.
Culture characteristics: Cultures on PDA initially white, growing to 6.5 cm after 2 days, entirely covering the 9-cm Petri dish after 3 days and becoming greyish yellow green after 7 days. Colonies pale mouse grey and flat with a uniform texture after one month. Notes: Cytospora alba is associated with canker disease of Salix matsudana in China. It is close to C. mali-spectabilis in the phylogenetic diagram ( Figure 2). It can be distinguished from C. mali-spectabilis by smaller conidia (6.0-9.0 × 1.5-2.0 vs. 9.0-10.0 × 1.5-2 µm in C. mali-spectabilis) and the lack of a central column in the conidiomata [26]. Cytospora alba has black conceptacle surrounding the asexual stroma, whereas C. mali-spectabilis lacks a conceptacle [26]. Furthermore, C. alba has multiloculate conidiomata sharing a larger single ostiole (93-216 vs. 60-84 µm) than C. mali-spectabilis [26].
Cytospora ailanthicola X.L. Notes: Cytospora ailanthicola was introduced by Fan et al. [4] causing canker disease on Ailanthus altissima in China. In the current study, we collected specimens of this species associated with twigs and branches of Salix matsudana. Although C. ailanthicola was closely related to C. salicacearum and C. melnikii in the phylogram, they can be distinguished by morphological characters. Morphologically, C. ailanthicola has multiple locules subdivided frequently by invaginations with common walls, whereas C. salicacearum and C. melnikii have conidiomata with 1-2 locule(s) and a single locule, respectively [4,22]. In addition, this is the first time that C. ailanthicola has been reported from willow tree. Notes: Cytospora chrysosperma, the type species of Cytospora [53], is considered a significant plant pathogen causing dieback and canker disease [4,18]. Previous studies have concluded that many species of Cytospora have a similar morphology (such as conidiomata cytosporoid and conceptacle absent) as C. chrysosperma, which indicated that these species should be regarded as a species complex [4,18]. Recently, Fan et al. [4] revisited ten related Cytospora species as belonging to the C. chrysosperma complex based on multigene analyses.  Notes: Cytospora gigaspora was originally observed on twigs and branches of Salix psammophila in Shaanxi Province [24]. After that, Fan et al. [4] added specimens from Shanxi. Cytospora gigaspora differs from Cytospora nivea by having flat locules and larger conidia [4].
Culture characteristics: Cultures on PDA initially white, growing up to 9 cm after 2 days, and becoming umber after 14 days in center. Colonies thick, concentric circles with a uniform texture, lacking aerial mycelium.
Specimens  Culture characteristics: Cultures on PDA initially white, growing up to 9 cm after 2 days, and becoming umber after 14 days in center. Colonies thick, concentric circles with a uniform texture, lacking aerial mycelium.

Cytospora rostrata
Cytospora sophoriopsis X.L. Fan  Notes: Cytospora sophoriopsis has scattered conidiomata without conceptacles, numerous locules subdivided by invaginations with common wall, and conidia measuring 4.0-4.5 × 1.0-1.5 µm [4]. Phylogenetically, the isolate in the current study (CFCC 55469) formed a fully supported clade with sequences from the ex-type isolate of C. sophoriopsis. In addition, the current study extends the host range of C. sophoriopsis to Salix matsudana in China.

Pathogenicity Test
Through pathogenicity tests, both C. alba (CFCC 55462) and C. paracinnamomea (CFCC 55453) were confirmed as pathogens on the willow stems ( Figure 5). Sunken and resinous cankers were obvious on the stems when the parafilm was removed after 7 days. The enlarged cankers lead further to wilting and consequent death of the plants. No symptoms were observed in the non-inoculated controls. Cytospora alba (CFCC 55462) was found to be more virulent than C. paracinnamomea (CFCC 55453) with the lesion presence frequency reaching 100% and canker length averaging 53 mm after 7 days. The average lesion caused by C. paracinnamomea (CFCC 55453) was 36 mm in length after 7 days. All Cytospora species were reisolated from the lesions or reproductive structures on inoculated plants.

Temperature, pH, and Carbon Source Tests
The effects of temperature, pH, and carbon source tests were performed with the exholotype strains (C. alba: CFCC 55461 and C. paracinnamomea: CFCC 55453). The regression equations and the estimated optimum growth temperature and pH value are presented in Table S3.
Colonies of both C. alba (CFCC 55461) and C. paracinnamomea (CFCC 55453) grew in the temperature range of 0-30 °C, but not at 35 °C or 40 °C. The fastest mycelial growth of C. alba (CFCC 55461) occurred at 20 °C, and C. paracinnamomea (CFCC 55453) occurred at 20 °C or 25 °C, with colonies of both reaching a diameter of 90 mm after four days. The least mycelial growth of C. alba (CFCC 55461) and C. paracinnamomea (CFCC 55453) all occurred at 0 °C, reaching only 15 mm and 14 mm, respectively, after four days. Based on the regression analysis, the optimal growth after incubation was estimated to occur at 21.1 °C for C. alba (CFCC 55461) and at 21.9 °C for C. paracinnamomea (CFCC 55453) ( Figure 6).

Temperature, pH, and Carbon Source Tests
The effects of temperature, pH, and carbon source tests were performed with the exholotype strains (C. alba: CFCC 55461 and C. paracinnamomea: CFCC 55453). The regression equations and the estimated optimum growth temperature and pH value are presented in Table S3.
Colonies of both C. alba (CFCC 55461) and C. paracinnamomea (CFCC 55453) grew in the temperature range of 0-30 • C, but not at 35 • C or 40 • C. The fastest mycelial growth of C. alba (CFCC 55461) occurred at 20 • C, and C. paracinnamomea (CFCC 55453) occurred at 20 • C or 25 • C, with colonies of both reaching a diameter of 90 mm after four days. The least mycelial growth of C. alba (CFCC 55461) and C. paracinnamomea (CFCC 55453) all occurred at 0 • C, reaching only 15 mm and 14 mm, respectively, after four days. Based on the regression analysis, the optimal growth after incubation was estimated to occur at 21.1 • C for C. alba (CFCC 55461) and at 21.9 • C for C. paracinnamomea (CFCC 55453) ( Figure 6). Colonies of both C. alba (CFCC 55461) and C. paracinnamomea (CFCC 55453) grew on PDA in the pH range of 3.0-10.0, but not at a pH of 11.0. For C. alba (CFCC 55461), mycelium grew most rapidly at pH 5.0, reaching 90 mm after four days, followed by pH 4.0 and 6.0. Mycelium grew slowly at pH values of 10.0 and 11.0, attaining colony diameters of no more than 30 mm after four days. For C. paracinnamomea (CFCC 55453), mycelium grew most rapidly at pH 6.0, reaching 90 mm after three days, followed by pH 5.0 and 7.0. Mycelium grew slowly at pH values of 10.0, attaining colony diameters of no more than 14 mm after four days. Based on the regression analysis, the optimal growth after incubation was estimated to occur at pH 5.6 for C. alba (CFCC 55461) and at pH 5.4 for C. paracinnamomea (CFCC 55453) (Figure 7). Both C. alba (CFCC 55461) and C. paracinnamomea (CFCC 55453) have the ability to grow on all six carbon sources tested. After two days, the growth of mycelia of C. alba (CFCC 55461) on the fructose and maltose media was apparently faster than that on other media (p-value = 0.0001), and the growth of mycelia of C. paracinnamomea (CFCC 55453) on the fructose, glucose, and maltose media was apparently faster than that on other media after two days (p-value = 0.0001). The colonies grown on other carbon sources were Colonies of both C. alba (CFCC 55461) and C. paracinnamomea (CFCC 55453) grew on PDA in the pH range of 3.0-10.0, but not at a pH of 11.0. For C. alba (CFCC 55461), mycelium grew most rapidly at pH 5.0, reaching 90 mm after four days, followed by pH 4.0 and 6.0. Mycelium grew slowly at pH values of 10.0 and 11.0, attaining colony diameters of no more than 30 mm after four days. For C. paracinnamomea (CFCC 55453), mycelium grew most rapidly at pH 6.0, reaching 90 mm after three days, followed by pH 5.0 and 7.0. Mycelium grew slowly at pH values of 10.0, attaining colony diameters of no more than 14 mm after four days. Based on the regression analysis, the optimal growth after incubation was estimated to occur at pH 5.6 for C. alba (CFCC 55461) and at pH 5.4 for C. paracinnamomea (CFCC 55453) (Figure 7). Colonies of both C. alba (CFCC 55461) and C. paracinnamomea (CFCC 55453) grew on PDA in the pH range of 3.0-10.0, but not at a pH of 11.0. For C. alba (CFCC 55461), mycelium grew most rapidly at pH 5.0, reaching 90 mm after four days, followed by pH 4.0 and 6.0. Mycelium grew slowly at pH values of 10.0 and 11.0, attaining colony diameters of no more than 30 mm after four days. For C. paracinnamomea (CFCC 55453), mycelium grew most rapidly at pH 6.0, reaching 90 mm after three days, followed by pH 5.0 and 7.0. Mycelium grew slowly at pH values of 10.0, attaining colony diameters of no more than 14 mm after four days. Based on the regression analysis, the optimal growth after incubation was estimated to occur at pH 5.6 for C. alba (CFCC 55461) and at pH 5.4 for C. paracinnamomea (CFCC 55453) (Figure 7). Both C. alba (CFCC 55461) and C. paracinnamomea (CFCC 55453) have the ability to grow on all six carbon sources tested. After two days, the growth of mycelia of C. alba (CFCC 55461) on the fructose and maltose media was apparently faster than that on other media (p-value = 0.0001), and the growth of mycelia of C. paracinnamomea (CFCC 55453) on the fructose, glucose, and maltose media was apparently faster than that on other media after two days (p-value = 0.0001). The colonies grown on other carbon sources were Both C. alba (CFCC 55461) and C. paracinnamomea (CFCC 55453) have the ability to grow on all six carbon sources tested. After two days, the growth of mycelia of C. alba (CFCC 55461) on the fructose and maltose media was apparently faster than that on other media (p-value = 0.0001), and the growth of mycelia of C. paracinnamomea (CFCC 55453) on the fructose, glucose, and maltose media was apparently faster than that on other media after two days (p-value = 0.0001). The colonies grown on other carbon sources were not significantly different from one another. After three days, mycelium growing on fructose and maltose were the first cultures to reach 90 mm in diameter. For C. alba (CFCC 55461), the utilization of sucrose was apparently lower than that of the others, reaching no more than 87 mm growth in diameter after four days. For C. paracinnamomea (CFCC 55453), the utilization of xylose was apparently lower than that of the others, reaching no more than 65 mm growth in diameter after four days (Figure 8). not significantly different from one another. After three days, mycelium growing on fructose and maltose were the first cultures to reach 90 mm in diameter. For C. alba (CFCC 55461), the utilization of sucrose was apparently lower than that of the others, reaching no more than 87 mm growth in diameter after four days. For C. paracinnamomea (CFCC 55453), the utilization of xylose was apparently lower than that of the others, reaching no more than 65 mm growth in diameter after four days (Figure 8).

Geographic Distribution
The geographical distribution of Cytospora species from Salix shows that C. chrysosperma and C. leucosperma are the main species observed on Salix, followed by C. nivea (Figure 9). Cytospora chrysosperma and C. leucosperma can be found almost in the entire distribution area of Salix (12 provinces); C. nivea and C. populina are mainly distributed in the Yellow River Basin (four and two provinces, respectively). Gansu province has the highest species diversity of Cytospora (nine species). The results show that the number of Cytospora species from Salix in China is more abundant in the northwest and less in the southeast (Figure 9). Poisson regression analysis showed that Cytospora species richness was significantly positively correlated with dry and wet areas (p-value = 0.024 < 0.05, Estimate = 0.33296). The more drought conditions there were, the more Cytospora species richness was recorded. There was no correlation with host Salix species richness (p-value = 0.303).

Geographic Distribution
The geographical distribution of Cytospora species from Salix shows that C. chrysosperma and C. leucosperma are the main species observed on Salix, followed by C. nivea (Figure 9). Cytospora chrysosperma and C. leucosperma can be found almost in the entire distribution area of Salix (12 provinces); C. nivea and C. populina are mainly distributed in the Yellow River Basin (four and two provinces, respectively). Gansu province has the highest species diversity of Cytospora (nine species). The results show that the number of Cytospora species from Salix in China is more abundant in the northwest and less in the southeast (Figure 9). Poisson regression analysis showed that Cytospora species richness was significantly positively correlated with dry and wet areas (p-value = 0.024 < 0.05, Estimate = 0.33296). The more drought conditions there were, the more Cytospora species richness was recorded. There was no correlation with host Salix species richness (p-value = 0.303).
In previous studies, five species (C. atrocirrhata, C. fugax, C. leucosperma, C. populina, and C. translucens) associated with willow canker disease have also been reported in China [3,4,13,24,51,57]. Cytospora atrocirrhata was first reported on branches of willow in Georgia [58], which was described as erumpent conidiomata with distinct conceptacles and single locules and ostiole [24]. Subsequently, this species was reported on poplar and walnut in Inner Mongolia and Qinghai [3,24]. Spielman [53] re-described the sexual and asexual morph of C. fugax, which provided important information for the accurate identification of this species. In China, Cytospora fugax was previously recorded in Heilongjiang, Inner Mongolia, and Jilin [3]. Cytospora leucosperma was recorded in Beijing, Gansu, Hebei, Heilongjiang, Jiangsu, Jilin, Liaoning, Ningxia, Qinghai, Shanxi, Xinjiang, and Zhejiang Provinces in China [4,13,51,57]. Cytospora populina was redescribed by Fan et al. [24] on branches of Salix psammophila in China. It can be distinguished from other Cytospora species by its asci with four ascospores [24]. Cytospora translucens was previously discovered on willow and poplar in Beijing, Heilongjiang, Inner Mongolia, and Jilin [3]. Vu et al. [59] added their DNA data; however, they still require a modern illustration and description.
Cytospora atrocirrhata, C. chrysosperma, C. fugax, C. leucosperma, C. nivea, and C. translucens were also reported to infect Salix spp. in other countries, e.g., Canada, England, Iran, New Zealand, Poland (https://nt.ars-grin.gov/fungaldatabases/index.cfm, accessed on 6 April 2022). Among these species, C. chrysosperma can infect over 110 host species, which is a prominent species of Cytospora in the world [4,18]. Cytospora ailanthicola, C. gigaspora, C. rostrata, C. sophoriopsis have only been found in China, of which C. rostrata has been only found in Salix cupularis [3,4,22,23]. Although C. alba and C. paracinnamomea have been confirmed as pathogens of willow canker disease, they have only been found in Gansu province. Whether they are distributed elsewhere and infect local willow trees are unknown.
The results of pathogenicity tests showed that C. alba and C. paracinnamomea were pathogens to Salix matsudana, which had a high degree of damage. The optimal growth was estimated to occur at 21.1 • C for C. alba (CFCC 55461) and at 21.9 • C for C. paracinnamomea (CFCC 55453); and at pH 5.6 for C. alba (CFCC 55461) and at pH 5.4 for C. paracinnamomea (CFCC 55453). Fructose and maltose were utilized better than other tested carbon sources. Based on the biological characterization of these two novel Cytospora species, more attention is needed to prevent the occurrence of Cytospora canker disease in the region with an average monthly temperature of approximately 20 • C, such as April, May, September, and October in Southwest, Northwest, and North China. This indicates that both spring and autumn may be a time of high disease risk. Therefore, the understanding of biological characteristics of these Cytospora species can be used for distribution prediction. Preventive measures can be taken in advance.
The geographical distribution analysis showed that Cytospora on Salix was less commonly known in the humid regions than in the arid region, semiarid region, and semihumid region. Previous studies showed that drought-stressed plants are more prone to Cytospora cankers [60,61]. However, Kristen et al. [62] demonstrated that reduced water availability and a 2-3 • C increase in temperature did not significantly increase the incidence or severity of cankers in inoculated willow plants. Therefore, willows that occur in dry areas are more likely to be infected by Cytospora because the trees are weakened by drought stress and poor soil conditions. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jof8040377/s1, Refs. [63][64][65][66][67], Figure S1: Phylogenetic tree of Cytospora species based on ML analyses of the ITS gene sequences; Figure S2: Phylogenetic tree of Cytospora species based on ML analyses of the act gene sequences; Figure S3: Phylogenetic tree of Cytospora species based on ML analyses of the rpb2 gene sequences; Figure S4: Phylogenetic tree of Cytospora species based on ML analyses of the tef1-α gene sequences; Figure S5: Phylogenetic tree of Cytospora species based on ML analyses of the tub2 gene sequences; Table S1: Genes used in this study with PCR primers, primer DNA sequence, optimal annealing temperature; Table S2: Strains of Cytospora used in the molecular analyses in this study; Table S3: The regression equations and the estimated optimum growth temperature, pH value, and carbon source.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
Alignments generated during the current study are available in Tree-BASE (accession http://purl.org/phylo/treebase/phylows/study/TB2:S29245, accessed on 6 April 2022). All sequence data are available in NCBI GenBank following the accession numbers in the manuscript.