Colletotrichum Species Associated with Peaches in China

Colletotrichum is regarded as one of the 10 most important genera of plant pathogens in the world. It causes diseases in a wide range of economically important plants, including peaches. China is the largest producer of peaches in the world but little is known about the Colletotrichum spp. affecting the crop. In 2017 and 2018, a total of 286 Colletotrichum isolates were isolated from symptomatic fruit and leaves in 11 peach production provinces of China. Based on multilocus phylogenetic analyses (ITS, ACT, CAL, CHS-1, GAPDH, TUB2, and HIS3) and morphological characterization, the isolates were identified to be C. nymphaeae, C. fioriniae, and C. godetiae of the C. acutatum species complex, C. fructicola and C. siamense of the C. gloeosporioides species complex, C. karsti of the C. boninense species complex, and one newly identified species, C. folicola sp. nov. This study is the first report of C. karsti and C. godetiae in peaches, and the first report of C. nymphaeae, C. fioriniae, C. fructicola, and C. siamense in peaches in China. C. nymphaeae is the most prevalent species of Colletotrichum in peaches in China, which may be the result of fungicide selection. Pathogenicity tests revealed that all species found in this study were pathogenic on both the leaves and fruit of peaches, except for C. folicola, which only infected the leaves. The present study substantially improves our understanding of the causal agents of anthracnose on peaches in China.


Introduction
The peach (Prunus persica (L.) Batsch) originated in China [1] and has been grown in many temperate climates around the world. China is the largest peach producer in the world, accounting for 55.28% of the total peach acreage in the world and 61.12% of global peach production [2]. The country produced 15,016,103 metric tons on 779,893 ha in 2020 [2].
When the temperature and humidity are favorable, Colletotrichum spp. can infect peaches and other fruits and cause massive economic losses [3]. Colletotrichum spp. pathogenic on peaches mainly infect the fruit but may also cause leaf or twig lesions. Fruit lesions appear as firm, brown, sunken (Figure 1a,c,d) areas often displaying concentric rings (Figure 1e) of small orange acervuli (Figure 1b,c,f). The acervuli produce conidia that are primarily spread by rainfall and splashing [4]. If a conidium lands on susceptible host plant tissue, it can cause secondary infection. Gumming can be observed when Colletotrichum spp. infect fruitlets (Figure 1a). Infected fruitlets do not reach maturity (Figure 1i), display atrophy, and eventually shrink from water loss (Figure 1i,j). Several lesions on green or mature fruit may coalesce (Figure 1a,f). Colletotrichum can also infect leaves with brown lesions (Figure 1g,h) and orange acervuli (Figure 1h). Severe twig infections can lead to twig dieback (Figure 1j). Colletotrichum species overwinter in fruit mummies and affected twigs, and form conidia in early spring [5]. In addition to asexual reproduction, they may also produce ascospores in perithecia, which were observed on apples in dead wood and on pears in leaves [6][7][8]. fruit mummies and affected twigs, and form conidia in early spring [5]. In addition to asexual reproduction, they may also produce ascospores in perithecia, which were observed on apples in dead wood and on pears in leaves [6][7][8]. In the past, the taxonomy of the genus Colletotrichum mainly relied on host range and morphological characteristics [9]. However, these characteristics are not suitable for species-level identification since they are dependent on environmental conditions, many Colletotrichum species are polyphagous, and multiple species can infect the same host plant [10][11][12][13]. Molecular identification based on multilocus phylogenetic analyses or specific gene sequencing has been used for the classification and description of species concepts [3]. To date, 15 Colletotrichum species complexes and 22 individual species have been identified [14][15][16].
The causal agents of peach anthracnose were first reported as Colletotrichum acutatum and Colletotrichum gloeosporioides [17][18][19][20]. However, the use of molecular tools for the classification of anthracnose pathogens revealed that peach anthracnose in the USA was mostly caused by Colletotrichum nymphaeae and Colletotrichum fioriniae of the C. acutatum species complex [21], and Colletotrichum siamense and Colletotrichum fructicola of the C. gloeosporioides species complex [22]. C. nymphaeae was also reported in Brazil on peaches [23], and C. fioriniae, C. fructicola, and C. siamense were identified in South Korea on peaches [24]. Peach infections by Colletotrichum truncatum and Colletotrichum acutatum are rare [25,26].
The objective of this study was to systematically identify Colletotrichum spp. associated with peach fruit and leaf anthracnose in China using morphological characterization and multilocus phylogenetic analyses.

Isolation of Colletotrichum spp. from Peach Samples
During 2017 and 2018, the fruit and leaves of peaches with anthracnose symptoms were collected from 14 commercial peach orchards and two nurseries (Wuhan, Hubei and Fuzhou, Fujian) in 11 provinces of China, which were dry-farmed and sprayed with fungicides for anthracnose control. Conidia on diseased tissues were dipped in a cotton swab and spread on a potato dextrose agar (PDA, 20% potato infusion, 2% glucose, and 1.5% agar, and distilled water) medium and picked up with a glass needle under a professional single spore separation microscope (Wuhan Heipu Science and Technology Ltd., Wuhan, China). If no conidia were present, leaf and fruit pieces (5 × 5 mm) at the intersection of healthy and diseased tissues were surface sterilized with a sodium hypochlorite solution (1%) for 30 s and washed three times in sterilized water, followed by 75% ethanol for 30 s, then washed three times in sterilized water again. After the tissue pieces were dried, they were placed on PDA and incubated at 25 • C with a 12 h/12 h fluorescent light/dark cycle for about seven days to produce spores. Cultures were transferred to 15% diluted oatmeal agar (0.9% oatmeal, 1.5% agar, and distilled water) plates if there was no sporulation on PDA [27]. The ex-type living culture of novel species in this study was deposited in the China Center for Type Culture Collection (CCTCC), Wuhan, China.

Morphological Characterization
Mycelial plugs (5 mm) were transferred from the edge of actively growing cultures to fresh PDA plates and incubated at 25 • C in the dark. Colony diameters were measured after three days to calculate the mycelial growth rates (mm/d). The shape and color of colonies were investigated on the sixth day. Sexual morphs of some species were produced after four weeks. The characteristics of conidiomata were observed using fluorescence stereo microscope (Leica M205 FA, Leica Microsystem Ltd., Wetzlar, Germany). Moreover, the shape and color of conidia, conidiophores, appressoria, ascomata, asci, ascospores, and setae were recorded using a light microscope (Nikon Eclipse E400, Nikon Instruments Inc., San Francisco, CA, USA), and the length and width of 30 randomly selected conidia and 30 appressoria were measured for each representative isolate. Appressoria were induced by dropping 50 µL conidial suspension (10 5 conidia/mL) on a microscope slide, which was placed inside a plate containing moistened filter papers with distilled water, and incubated at 25 • C in the dark for 24 to 48 h [28].

DNA Extraction, PCR Amplification, and Sequencing
From the 286 obtained isolates, 51 were selected for further multilocus phylogenetic analyses. They represented each geographical population, colony type, conidia morphology, and host tissue.
Fungal DNA was extracted as described previously [29]. The 5.8S nuclear ribosomal gene with the two flanking internal transcribed spacers (ITS), partial sequences of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), chitin synthase 1 gene (CHS-1), actin gene (ACT), beta-tubulin gene (TUB2), histone3 gene (HIS3), and calmodulin gene (CAL) were amplified and sequenced using the primer pairs described in Table S1. The PCR conditions were 4 min at 95 • C, followed by 35 cycles of 95 • C for 30 s, annealing for 30 s at different temperatures for different genes/loci (Table S1), and 72 • C for 45 s, with a final extension at 72 • C for 7 min. DNA sequencing was performed at Tianyi Huiyuan Biotechnology Co., Ltd. (Wuhan, China) with an ABI 3730XL sequencer from Thermo Fisher Scientific (China) Co., Ltd. (Shanghai, China). The consensus sequences were assembled from forward and reverse sequences with MEGA v. 7.0 [30]. All sequences of 51 representative Colletotrichum isolates in this study were submitted to GenBank and the accession numbers are listed in Table S2.

Phylogenetic Analyses
Isolates were divided into four groups based on multilocus phylogenetic analyses, and type isolates of each species were selected and included in the analyses (Table 1). Multilocus phylogenetic analyses with concatenated ITS, GAPDH, CHS-1, HIS3, ACT, and TUB2 sequences were conducted for the C. acutatum species complex [31]; ACT, CAL, CHS-1, GAPDH, ITS, and TUB2 sequences were concatenated for the analysis of the C. gloeosporioides species complex [32]; the combined ITS, GAPDH, CHS-1, HIS3, ACT, TUB2, and CAL sequences were used to analyze the C. boninense species complex [33]; and the ITS, GAPDH, CHS-1, ACT, and TUB2 sequences were applied for remaining species [34]. Multiple sequences were aligned and combined using MAFFT v.7 [35] and MEGA v.7.0 [30].
Bayesian inference (BI) was used to construct phylogenetic trees in MrBayes v.3.2.2 [36]. Best-fit models of nucleotide substitution were selected using MrModeltest v.2.3 [37] based on the corrected Akaike information criterion (AIC) ( Tables 2-5). BI analyses were launched with two MCMC chains that were run for 1 × 10 6 generations (C. acutatum species complex and C. boninense species complex) [31,33], and trees sampled every 100 generations; or run 1 × 10 7 generations (C. gloeosporioides species complex, and remaining species) [8,34], and trees sampled every 1000 generations. The calculation of BI analyses was stopped when the average standard deviation of split frequencies fell below 0.01. On this basis, the first 25% of generations were discarded as burn-in. Maximum parsimony (MP) analyses were implemented by using Phylogenetic Analysis Using Parsimony (PAUP*) v.4.0b10 [38]. Goodness of fit values including tree length (TL), consistency index (CI), retention index (RI), rescaled consistency index (RC), and homoplasy index (HI) were calculated for the bootstrap analyses (Tables 2-5). Phylogenetic trees were generated using the heuristic search option with Tree Bisection Reconnection (TBR) branch swapping and 1000 random sequence additions, with all characters equally weighted and alignment gaps treated as missing data. Maximum likelihood (ML) analyses were carried out by using the CIPRES Science Gateway v.3.3 (www.phylo.org, accessed on 29 December 2021), while RAxML-HPC BlackBox was selected with default parameters. Phylogenetic trees were visualized in FigTree v.1.4.2 [39]. TreeBASE was used to store the concatenated multilocus alignments (submission number: 29227).

Culture a Host Location
GenBank Accession Number

Culture a Host Location
GenBank Accession Number   New species and their most closely related neighbors were analyzed using the Genealogical Concordance Phylogenetic Species Recognition (GCPSR) model by performing a pairwise homoplasy index (PHI) test [40]. The PHI test was carried out on SplitsTree v.4.14.6 [41,42] using concatenated sequences (ITS, GAPDH, CHS-1, ACT, and HIS3). The result of pairwise homoplasy index below a 0.05 threshold (Φw < 0.05) indicated the presence of significant recombination in the dataset. The relationship between closely related species was visualized by constructing a splits graph. In addition, the results of relationships between closely related species were visualized by constructing EqualAngle splits graphs, using both LogDet character transformation and split decomposition distances options.

Pathogenicity Test
Two to five isolates of each Colletotrichum sp. were used in pathogenicity tests on detached fruit and leaves. The experimental varieties for fruit and leaf inoculations were "Xiaohong" and "Xiahui No. 5", respectively. Commercially mature fruit (still firm but with no green background color) and asymptomatic, fully developed leaves with short twigs (1-2 cm) were washed with soap and water, and surface sterilized in 1% sodium hypochlorite for 2 min and 30 s, respectively, then rinsed with sterile water and air-dried on sterile paper. Fruit was stabbed with sterilized toothpicks to produce wounds of about 5 mm deep, while leaves were punctured with sterile, medical needles. For inoculation, a 10-µL droplet of conidia suspension (1.0−2.0 × 10 5 conidia/mL) was dropped on each wounded site, and control fruit or leaves received sterile water without conidia. Each fruit and leaf had two inoculation sites. Three fruits and three leaves were used for each isolate. Inoculated fruit and leaves were placed in a plastic tray onto 30 mm diameter plastic rings for stability. The bottom of the tray (65 cm × 40 cm × 15 cm, 24 peaches or leaves per tray) contained wet paper towels and the top was sealed with plastic film to maintain humidity. Peaches and leaves were incubated at 25 • C for six days. Pathogenicity was evaluated by the infection rates and lesion diameters. The infection rates were calculated by the formula (%) = (infected inoculation sites/all inoculation sites) × 100%. The lesion size was determined as the mean of two perpendicular diameters. The experiment was performed twice.
The fungus was re-isolated from the resulting lesions and identified as described above, thus fulfilling Koch's postulates.

Results
From 2017 to 2018, a total of 286 Colletotrichum isolates were obtained from 11 provinces in China (Table 6; Figure 2a); 33 isolates were from leaves and 253 isolates were from fruit ( Table 6). Although we tried to collect samples in Gansu and Shanxi provinces in northern China, no symptomatic leaves or fruit were found. C. nymphaeae was the most widespread and most prevalent species (Figure 2b,c), with presence in Hubei, Guizhou, Guangxi, Fujian, and Sichuan provinces. C. fioriniae was found in three centrally located provinces (Zhejiang, Guizhou, and Jiangxi). C. siamense was only found in the northernmost orchards of the collection area in Shandong and Hebei provinces, while C. fructicola was only found in the southernmost provinces of the collection area of Guangdong and Guizhou provinces. C. folicola, C. godetiae, and C. karsti were only found in Yunnan province in the westernmost border of the collection area (Table 6; Figure 2a).

Phylogenetic Analyses
Phylogenetic trees were constructed based on the concatenated gene/locus sequences. MP and ML trees are not shown because the topologies were similar to the displayed BI tree (Figure 3, Figure 4, Figure 5 and Figure 6). The number of taxa, aligned

Phylogenetic Analyses
Phylogenetic trees were constructed based on the concatenated gene/locus sequences. MP and ML trees are not shown because the topologies were similar to the displayed BI tree (Figures 3-6). The number of taxa, aligned length (with gaps), invariable characters, uninformative variable characters, and phylogenetically informative characters of each gene/locus and combined sequences are listed in Tables 2-5. (gene/locus boundaries in the alignment: ITS: 1-546, GAPDH: 551-815, CHS-1: 820-11 HIS3: 1106-1492, ACT: 1497-1744, TUB2: 1749-2240) of 27 isolates from peaches in study, 44 reference strains of C. acutatum species complex and one Colletotrich species (C. orchidophilum strains CBS 632.80) as the outgroup, 2240 characters includ the alignment gaps were processed. For the Bayesian analysis, a HKY + I model w selected for ITS, a HKY + G model for GAPDH, a K80 + I model for CHS-1, a GTR + I model for HIS3, and a GTR + G model for ACT and TUB2, and all were incorporated the analysis ( Table 2). As the phylogenetic tree shows in Figure 3, the 27 isolates of th acutatum species complex were clustered in three groups: 11 with C. nymphaeae, ei with C. fioriniae, and eight with C. godetiae. Although in the same general cluster nymphaeae from China were genetically distinct from C. nymphaeae isolates from USA and Brazil.  the Bayesian analysis, a HKY + G model was selected for ACT, a GTR + G model for CAL, a K80 + G model for CHS-1, a HKY + I model for GAPDH and TUB2, and a SYM + I + G model for ITS, and they were all incorporated in the analysis (Table 3). In the phylogenetic tree of the C. gloeosporioides species complex, 10 isolates clustered with C. fructicola and nine isolates clustered with C. siamense (Figure 4). They clustered together with isolates from South Korea and the USA. strain C. gloeosporioides CBS 112999, 2763 characters including the alignment gaps we processed. For the Bayesian analysis, a SYM + I + G model was selected for ITS, HKY for GAPDH and TUB2, K80 + G for CHS-1, GTR + I + G for HIS3, GTR + G for ACT, an HKY + G for CAL, and they were all incorporated in the analysis (Table 4). In Figure three Chinese isolates clustered with C. karsti in the C. boninense species complex. For the remaining phylogenetic analyses, the alignment of combined DN sequences was obtained from 50 taxa, including two isolates from peaches in this stud For the C. acutatum species complex, in the multilocus sequence analyses (gene/locus boundaries in the alignment: ITS: 1-546, GAPDH: 551-815, CHS-1: 820-1101, HIS3: 1106-1492, ACT: 1497-1744, TUB2: 1749-2240) of 27 isolates from peaches in this study, 44 reference strains of C. acutatum species complex and one Colletotrichum species (C. orchidophilum strains CBS 632.80) as the outgroup, 2240 characters including the alignment gaps were processed. For the Bayesian analysis, a HKY + I model was selected for ITS, a HKY + G model for GAPDH, a K80 + I model for CHS-1, a GTR + I + G model for HIS3, and a GTR + G model for ACT and TUB2, and all were incorporated in the analysis ( Table 2). As the phylogenetic tree shows in Figure 3, the 27 isolates of the C. acutatum species complex were clustered in three groups: 11 with C. nymphaeae, eight with C. fioriniae, and eight with C. godetiae. Although in the same general cluster, C. nymphaeae from China were genetically distinct from C. nymphaeae isolates from the USA and Brazil. analysis (Table 5). In the phylogenetic tree, two isolates (YNHH2-2 and YNHH10-1 (CCTCC M 2020345)) clustered distantly from all known Colletotrichum species and are described herein as a new species, C. folicola ( Figure 6). The PHI test result (Φw = 1) of C. folicola and its related species C. citrus-medicae ruled out the possibility of gene recombination interfering with the species delimitation (Figure 7). This is further evidence that C. folicola is a new species.  For the Bayesian analysis, a HKY + G model was selected for ACT, a GTR + G model for CAL, a K80 + G model for CHS-1, a HKY + I model for GAPDH and TUB2, and a SYM + I + G model for ITS, and they were all incorporated in the analysis (Table 3). In the phylogenetic tree of the C. gloeosporioides species complex, 10 isolates clustered with C. fructicola and nine isolates clustered with C. siamense (Figure 4). They clustered together with isolates from South Korea and the USA.
For the remaining phylogenetic analyses, the alignment of combined DNA sequences was obtained from 50 taxa, including two isolates from peaches in this study, 47 reference isolates of Colletotrichum species, and one outgroup strain Monilochaetes infuscans CBS 869.96. The gene/locus boundaries of the aligned 1981 characters (with gaps) were: ITS: 1-571, GAPDH: 576-896, CHS-1: 901-1165, ACT: 1170-1448, TUB2: 1453-1981. For the Bayesian analysis, a GTR + I + G model was selected for ITS and CHS-1, and HKY + I + G for GAPDH, ACT, and TUB2, and they were incorporated in the analysis (Table 5). In the phylogenetic tree, two isolates (YNHH2-2 and YNHH10-1 (CCTCC M 2020345)) clustered distantly from all known Colletotrichum species and are described herein as a new species, C. folicola ( Figure 6). The PHI test result (Φw = 1) of C. folicola and its related species C. citrus-medicae ruled out the possibility of gene recombination interfering with the species delimitation ( Figure 7). This is further evidence that C. folicola is a new species. Figure 7. PHI test of C. folicola and phylogenetically related species using both transformation and splits decomposition. PHI test value (Φw) < 0.05 indicate sig recombination within the datasets.
Notes: Colletotrichum acutatum var. fioriniae was first isolated from Fiorinia externa [44] and host plants of the scale insect as an endophyte [45] in New York, USA. In 2009, Shivas and Tan identified it from Acacia acuminate, Persea americana, and Mangifera indica in Australia as a separate species and named it Colletotrichum fioriniae [46]. C. fioriniae was mainly isolated from wide host plants and fruits in the temperate zones [3,31]. In this study, the C. fioriniae isolates clustered in two subclades, which is consistent with the results of Damm's study [31].
Notes: Colletotrichum godetiae was first reported on the seeds of Godetia hybrid in Denmark by Neergaard in 1943 [47], and given detailed identification seven years later [48]. C. godetiae was also recovered from fruits of Fragaria × ananassa, Prunus cerasus, Solanum betaceum, Citrus aurantium, and Olea europaea [49]; leaves of Laurus nobilis and Mahonia aquifolium; twigs of Ugni molinae; and canes of Rubus idaeus [31]. In this study, the isolates were obtained from peach leaves and could infect both the peach fruit and leaf.
in Australia as a separate species and named it Colletotrichum fioriniae [46]. C. fioriniae wa mainly isolated from wide host plants and fruits in the temperate zones [3,31]. In thi study, the C. fioriniae isolates clustered in two subclades, which is consistent with the results of Damm's study [31].
Notes: Colletotrichum godetiae was first reported on the seeds of Godetia hybrid in Denmark by Neergaard in 1943 [47], and given detailed identification seven years late [48]. C. godetiae was also recovered from fruits of Fragaria × ananassa, Prunus cerasus, So lanum betaceum, Citrus aurantium, and Olea europaea [49]; leaves of Laurus nobilis and Ma  honia aquifolium; twigs of Ugni molinae; and canes of Rubus idaeus [31]. In this study, the isolates were obtained from peach leaves and could infect both the peach fruit and leaf.
Culture characteristics: Colonies on PDA attained 16-21 mm diameter in three days at 25 • C and 7-10 mm diameter in three days at 30 • C; greenish-black, white at the margin, and aerial mycelium scarce.

Discussion
This study is the first large-scale investigation of Colletotrichum species ca anthracnose fruit and leaf diseases in peaches in China. The most co Colletotrichum species were C. nymphaeae and C. fioriniae of the C. acutatum s complex and C. fructicola and C. siamense of the C. gloeosporioides species com The same species were also identified in the southeastern USA [17,21,22], where over time appeared to favor C. gloeosporioides species complex in South Carolin authors speculated that inherent resistance of C. acutatum to benzimidazole fung (MBCs) may have given this species complex a competitive advantage when MBC frequently used [22]. As MBCs were replaced by other fungicides (including qu outside inhibitors and demethylation inhibitors), that competitive advantage may disappeared and C. gloeosporioides species may have increased in prevalence [22, support of this hypothesis is previous research showing a higher virulence gloeosporioides on peaches, pears, and apples compared to C. acutatum [8,56,57] this study and others show that the C. gloeosporioides species complex may be adapted to the hot South Carolina climate compared to the C. acutatum species co [3]. MBCs are still popular fungicides in Chinese peach production regions. Theref is possible that the dominance of C. acutatum species complex, specifically C. nymp is, at least in part, a result of fungicide selection.

Discussion
This study is the first large-scale investigation of Colletotrichum species causing anthracnose fruit and leaf diseases in peaches in China. The most common Colletotrichum species were C. nymphaeae and C. fioriniae of the C. acutatum species complex and C. fructicola and C. siamense of the C. gloeosporioides species complex. The same species were also identified in the southeastern USA [17,21,22], where a shift over time appeared to favor C. gloeosporioides species complex in South Carolina. The authors speculated that inherent resistance of C. acutatum to benzimidazole fungicides (MBCs) may have given this species complex a competitive advantage when MBCs were frequently used [22]. As MBCs were replaced by other fungicides (including quinone outside inhibitors and demethylation inhibitors), that competitive advantage may have disappeared and C. gloeosporioides species may have increased in prevalence [22,55]. In support of this hypothesis is previous research showing a higher virulence of C. gloeosporioides on peaches, pears, and apples compared to C. acutatum [8,56,57]. Also, this study and others show that the C. gloeosporioides species complex may be better adapted to the hot South Carolina climate compared to the C. acutatum species complex [3]. MBCs are still popular fungicides in Chinese peach production regions. Therefore, it is possible that the dominance of C. acutatum species complex, specifically C. nymphaeae is, at least in part, a result of fungicide selection.
The high prevalence of C. nymphaeae in Chinese peach orchards is consistent with other local studies reporting the same species affecting a wide variety of other fruit crops in China. For example, C. nymphaeae was reported in Sichuan province on blueberries and loquats [58,59], in Hubei province on strawberries and grapevines [60,61], and in Zhejiang province on pecans [62]. Internationally, it is one of the most common species affecting pome fruits, stone fruits, and small fruits [23,63,64].
C. godetiae, C. karsti, and C. folicola were reported on peaches for the first time. The three species were geographically isolated and only present in Yunnan province. Rare occurrences of Colletotrichum species have also been formerly observed on peaches, i.e., C. truncatum was only found in one of many orchards examined in South Carolina, USA [25]. C. godetiae and C. karsti are well-known pathogens of fruit crops. C. godetiae was reported to cause disease on apples, strawberries, and grapes [65][66][67][68], while C. karsti was reported to affect apples and blueberries [69,70]. It is, therefore, possible that these pathogens migrated from other hosts into Yunnan province peach orchards. The observed occurrence, however, does point to either a rather rare host transfer event or to environmental conditions that favor these species. Yunnan province is located in southwestern China and peach production is popular in the Yunnan-Guizhou high plateau, a region with low latitude and high altitude [71]. The complicated local topography and diverse climate lead to highly abundant biodiversity [72], which may explain the emergence of the new species C. folicola.
As mentioned above, regional differences in Colletotrichum species composition in commercial orchards may be influenced by fungicide selection pressure. For example, C. acutatum is less sensitive to benomyl, thiophanate-methyl, and other MBC fungicides compared with C. gloeosporioides [56,73,74]. Meanwhile, all C. nymphaeae strains in this study have been confirmed to be resistant to carbendazim (MBC) [75]. C. nymphaeae was reported to be less sensitive to demethylation inhibitor (DMIs) fungicides (flutriafol and fenbuconazole) compared with C. fioriniae, C. fructicola, and C. siamense [21] and C. gloeosporioides was reported to be inherently tolerant to fludioxonil [76,77]. Most of the peach farms in China are small and there is vast diversity in the approaches to managing diseases. However, MBC (i.e., carbendazim and thiophanate-methyl) fungicides are commonly used to control peach diseases, followed by DMIs (i.e., difenoconazole). Whether fungicide selection had an impact on the Colletotrichum species distribution is unknown, but the high prevalence of C. acutatum species complex and their resilience to MBCs (and, in the case of C. nymphaeae, to DMIs) would allow for such a hypothesis.
In conclusion, this study provides the morphological, molecular, and pathological characterization of seven Colletotrichum spp. occurring on peaches in China. This is of great significance for the prevention and control of anthracnose disease in different areas in China.

Data Availability Statement:
Alignments generated during the current study are available from TreeBASE (http://treebase.org/treebase-web/home.html; study 29227). All sequence data are available in the NCBI GenBank, following the accession numbers in the manuscript.