Colletotrichum Species Associated with Anthracnose in Salix babylonica in China

Salix babylonica L. is a popular ornamental tree species in China and widely cultivated in Asia, Europe, and North America. Anthracnose in S. babylonica poses a serious threat to its growth and reduces its medicinal properties. In 2021, a total of 55 Colletotrichum isolates were isolated from symptomatic leaves in three provinces in China. Phylogenetic analyses using six loci (ITS, ACT, CHS-1, TUB2, CAL, and GAPDH) and a morphological characterization of the 55 isolates showed that they belonged to four species of Colletotrichum, including C. aenigma, C. fructicola, C. gloeosporioides s.s., and C. siamense. Among them, C. siamense was the dominant species, and C. gloeosporioides s.s. was occasionally discovered from the host tissues. Pathogenicity tests revealed that all the isolates of the aforementioned species were pathogenic to the host, and there were significant differences in pathogenicity or virulence among these isolates. The information on the diversity of Colletotrichum spp. that causes S. babylonica anthracnose in China is new.

Salix babylonica L. (Salicaceae) is distributed mostly in the northern hemisphere [30]. Since S. babylonica has a high ornamental value with its slender and graceful branches, it is widely planted by rivers and roadsides [31][32][33]. Salix babylonica also possesses a wide range

Multi-Locus Phylogenetic Analyses
Seventeen representative isolates of Colletotrichum from different areas were selected for sequencing and analyses. The BLAST result of the ITS sequences showed that the 17 isolates belonged to the C. gloeosporioides species complex. They were analyzed using multi-locus sequences (ITS, ACT, CHS-1, TUB2, CAL, and GAPDH) and compared with 42 In this study, a total of six diseased sample batches were collected from six areas in the three provinces of China (Table 1). Thirty leaves were collected for each sample batch. A total of 55 Colletotrichum isolates were isolated according to their colony morphology on PDA and the ITS sequence data. Among these isolates, 12 isolates were from Suzhou, 10 isolates from Zibo, 10 isolates from Wuhan, and 23 isolates from Nanjing. Based on their ITS sequence data and colony characteristics on PDA, the isolates were divided into four types. Of these, 17 representative isolates were selected for further study and were sent to the China Forestry Culture Collection Center (CFCC).

Pathogenicity Tests
At 7 dpi, 17 representative isolates of the four Colletotrichum species developed dark brown lesion symptoms of anthracnose on the leaves of S. babylonica inoculated by a spore suspension. The infection incidence was 100%. No lesions were observed on the leaves of the control plants (Figure 7). However, different isolates had different levels of virulence, resulting in different lesions sizes. Among them, four out of the nine isolates of C. siamense had the most virulence, and C. aenigma had the least virulence ( Table 2). The virulence

Pathogenicity Tests
At 7 dpi, 17 representative isolates of the four Colletotrichum species developed dark brown lesion symptoms of anthracnose on the leaves of S. babylonica inoculated by a spore suspension. The infection incidence was 100%. No lesions were observed on the leaves of the control plants (Figure 7). However, different isolates had different levels of virulence, resulting in different lesions sizes. Among them, four out of the nine isolates of C. siamense had the most virulence, and C. aenigma had the least virulence ( Table 2). The virulence within the same species of C. siamense and C. gloeosporioides s. s. varied significantly. The fungus was re-isolated from the infected tissues, and the morphology of the colony and the ITS sequence data matched the inocula. No fungi were isolated from the control leaves. The re-isolation rate was 100%. Thus, all 17 isolates were pathogens of anthracnose in S. babylonica.

Discussion
Salix babylonica is endemic in China and has a high ornamental value. Rec thracnose in S. babylonica has been discovered, seriously affecting the ecologica S. babylonica. The identification of fungal pathogens is the most important firs disease management [47]. In this study, we collected 55 isolates from six region provinces where S. babylonica is grown and identified four known species o trichum.  C. siamense YH2-2 5.6 ± 0.2 c Data were analyzed with SPSS Statistics 19.0 by one-way ANOVA, and means were compared using Duncan's test at a significance level of p = 0.05. Letters indicate the significant difference at the p = 0.05 level.

Discussion
Salix babylonica is endemic in China and has a high ornamental value. Recently, anthracnose in S. babylonica has been discovered, seriously affecting the ecological value of S. babylonica. The identification of fungal pathogens is the most important first step for disease management [47]. In this study, we collected 55 isolates from six regions in three provinces where S. babylonica is grown and identified four known species of Colletotrichum.
Current identification systems for Colletotrichum species have included traditional morphological features, molecular phylogeny, and other traits [48]. However, these morphological features show plasticity under different conditions of growth (host, media, temperature, light regime, etc.), and some can be lost or change with repeated subculturing [22]. The conidia and ascospores developed on S. babylonica in this study are larger than those of the ex-type of C. fructicola (ICMP 18581) from Coffea arabica. Our morphological analyses also showed that the Colletotrichum species had the same sexual state characteristics under the same conditions. For example, C. fructicola and C. aenigma tend to develop asci and ascospores on PDA, resulting in the coexistence of sexual and asexual states. Thus, the identification of fungal pathogens in plants includes not only morphology but also multi-locus phylogenetic analyses [49,50]. For instance, Wang et al. [51] used three DNA sequences of ITS, TUB2, and TEF1-α to confirm a Pestalotiopsis-like species causing gray blight disease in tea plants in China. Poudel et al. [52] used ITS sequences to identify Erysiphe fallax causing powdery mildew on phasey beans in the United States. In this study, concatenated sequences of ITS, ACT, CHS-1, TUB2, CAL, and GAPDH were used to construct phylogenetic trees, and we identified the 17 isolates to be C. aenigma, C. fructicola, C. gloeosporioides s.s., and C. siamense.
The pathogenicity tests indicated pathogenic differences among the four species. Colletotrichum siamense had the highest virulence. In this study, C. siamense had the fastest colony growth rate on PDA, and correspondingly, it showed the highest virulence in the pathogenicity test. Secondly, the appressoria of C. siamense germinated easily. Colletotrichum aenigma had the slowest colony growth rate and showed the least virulence. The results indicated that the pathogenicity of the isolates was closely related to the colony growth rate and the appressorial germination rate. Colletotrichum siamense is an important pathogen that can infect many trees and fruits. For instance, C. siamense has been shown to cause anthracnose in pears, a number of host species in Proteaceae, and Cunninghamia lanceolata [25,53,54]. Colletotrichum fructicola was first reported in coffee berries from Thailand [44] and was later reported in Pyrus pyrifolia in Japan [22]. Subsequently, this species was widely recognized as the pathogen that caused pear anthracnose [55]. However, it can also infect other fruits, for instance, Averrhoa carambola, Prunus sibirica, and Amygdalus persica [56][57][58].
Based on pathogenicity test, C. aenigma, C. fructicola, C. gloeosporioides s.s., and C. siamense were identified as the pathogens of anthracnose in S. babylonica. Of them, C. siamense was the dominant species, and C. gloeosporioides s.s. was occasionally discovered from the host tissues. All of the isolates belong to the C. gloeosporioides species complex. The difference in the dominant species in the six regions may be due to different geographical locations, climates, host varieties, host health conditions, planting methods, and collection times [29]. Actually, many reports have shown that a host plant can be infected by several different Colletotrichum species. For example, chili is reported to be infected by C. fioriniae, C. fructicola, C. gloeosporioides s.s., C. scovillei, etc. [3]. Anthracnose in mango is caused by C. asianum, C. fructicola, C. siamense, C. tropicale, etc. [59]. Therefore, further studies are required to identify the host range and distribution of different Colletotrichum species.
It has been reported that C. siamense, C. gloeosporioides s.s., and C. acutatum can infect S. babylonica [33,60], but this study proved that C. fructicola and C. aenigma can also infect the leaves of S. babylonica. It is uncertain whether other Colletotrichum species can cause anthracnose in S. babylonica; extensive sampling in all distribution areas is required. In addition, the sensitivity of different Colletotrichum species to fungicides needs to be further studied. This is the first report on the diversity of Colletotrichum species associated with S. babylonica anthracnose worldwide. For controlling S. babylonica anthracnose effectively, these data will help us to select appropriate strategies for managing this disease.

Sample Collection and Fungi Isolation
From June to October 2021, the symptoms and pathogenesis of anthracnose in S. babylonica in different areas were assessed. Leaves with typical symptoms of anthracnose were randomly collected from six areas in three provinces (Jiangsu, Shandong, Hubei), China, and the samples (10 leaves/tree) were collected from three trees in each region. The samples were rinsed with running water for 10 min and dried in sterilized Petri dishes [61]. Small pieces of infected tissue (3-4 mm 2 ) were surface-sterilized in 75% ethanol for 30 s followed by 1% NaClO for 90 s, rinsed three times in sterile water, dried on sterilized filter paper, plated on potato dextrose agar (PDA), and incubated at 25 • C in the dark [62,63]. Fungal growth was checked daily. Pure cultures were obtained by cutting hyphal tips and the monosporic isolation method [64]. All isolates were transferred to fresh PDA plates.
The representative isolates were selected for further analyses and were sent to the China Forestry Culture Collection Center (CFCC).

Phylogenetic Analyses
The ITS, ACT, CHS-1, TUB2, CAL, and GAPDH sequences with high similarities to the genes/region sequences of Colletotrichum species in GenBank using BLAST were selected, and in total the sequences of 42 Colletotrichum isolates (23 species) were obtained from GenBank for phylogenetic analyses ( Table 4). The sequences of Colletotrichum boninense (CBS 123755) were used as an outgroup. Nucleotide sequences of each gene/region of the selected isolates were aligned by the MAFFT ver. 7.313 [71]. The aligned sequences were edited using BioEdit version 7.0.9.0 [72]. Six locus sequences (ITS, ACT, CHS-1, TUB2, CAL, and GAPDH) were concatenated by PhyloSuite software [73]. After selecting the best model with ModelFinder [74], phylogenetic relationships were inferred using maximum likelihood (ML) estimation and Bayesian inference (BI). The ML analysis employed IQtree ver. 1.6.8 using the GTR+F+I+G4 model, with the bootstrapping method of 1000 replicates [75,76]. A bootstrap posed statistical support at ≥50%. BI analysis used the GTR+I+G+F model by MrBayes ver. 3.2.6, including 2 parallel runs and 2,000,000 generations [76]. Branches that received Bayesian posterior probabilities of 0.90 (BPP) were set as significantly supported. Phylogenetic trees were constructed with FigTree ver. 1

Morphological Study
Morphological examinations focused on the colony characteristics, acervuli, conidiophores, conidiogenous cells, conidia, setae, appressoria, ascomata, asci, and ascospores of representative isolates that were randomly selected from each Colletotrichum species. Mycelial plugs (5 mm diam) from the margin of cultures were transferred to PDA and incubated at 25 • C in the dark. Colony characteristics were photographed with a Canon EOS M50 Mark II camera after 4 d, and colony diameters were measured daily to calculate the mycelial growth rates (mm/d). In order to induce appressorium formation, 10 µL of conidial suspension (10 6 conidia/mL) was placed on a slide, placed inside plates containing a piece of moistened filter paper with sterile water, and then incubated at 25 • C in dark [77]. Measurements and morphological descriptions of acervuli, conidiophores, conidiogenous cells, conidia, setae, appressoria, ascomata, asci, and ascospores of the representative isolates were observed using a Zeiss Axio Imager A2m microscope (Carl Zeiss Microscopy, Oberkochen, Germany). Fifty individuals of per structure were measured for each isolate.

Pathogenicity Tests
Seventeen representative isolates of four Colletotrichum species were used for pathogenicity tests. Healthy 2-yr-old seedlings with 10 leaves per seedling were wound with a sterile needle and inoculated with conidial suspensions (10 6 conidia/mL) in each leaf. The conidial suspensions were sprayed onto the wound. Control plants were treated with sterile water in the same way. Seedlings were covered with plastic bags after inoculation and maintained in a greenhouse at 25 ± 2 • C and 80% RH for seven days. The experiments were conducted three times, and each treatment had three replicates. Eventually 54 seedlings were used. Seven days after inoculation, the diameter of the lesion on the leaves was measured and the inoculated leaves were used for re-isolation.  Institutional Review Board Statement: Not applicable for studies not involving humans or animals.

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