For diseased sample collections, we sampled three strawberry varieties including “Hongjia,” “Zhangji,” and “Tianxianzui.” For each variety, ten strawberry plants were randomly sampled from two greenhouses. The strawberry varieties “Hongjia” and “Zhangji” were collected from four greenhouses in Zhoushan, varieties “Hongjia,” “Zhangji,” and “Tianxianzui” were collected from six greenhouses in Jiande. A total of 100 strawberry plants were collected from 10 greenhouses in the main growing areas of Zhejiang Province (27°02′ to 31°11′ N, 118°01′ to 123°10′ E), China.

For Colletotrichum spp. isolation, the diseased tissues were washed with running tap water, cut into 5 × 5 mm pieces using sterilized scissors, soaked in 75% alcohol for 30 s, and then soaked in 3% sodium hypochlorite solution for 2 min, washed with sterile distilled water three times, and dried on sterile filter paper. Each tissue piece was placed on a plate containing potato dextrose agar (PDA) medium supplemented with kanamycin sulfate and streptomycin sulfate (100 mg/L) and incubated at 25°C. After 3 to 5 days incubation, the corresponding mycelia were then transferred to a new PDA plate (Phoulivong et al., 2010; Noman et al., 2018). The single-conidium isolates were stored on PDA slants at 4°C.

Morphological and cultural characterizations were performed according to the methods described by Cai et al. (2009). Briefly, the mycelia plugs (5 mm in diameter) taken from the margin of 5-day-old colony of tested isolates were transferred to the new PDA plates and incubated at 25°C. The experiment was designed as a randomized complete block, with three replicates for each isolate. After 7 days, the colony diameter was measured, and the growth rate was calculated; the colony color of the aerial hyphae and morphology were recorded as previously described (Cai et al., 2009; Weir et al., 2012).

Conidial suspensions of each isolate were prepared in lactic acid from conidial masses on PDA. The shape and color of the conidia were observed, and the sizes of 50 conidia from each isolate were measured at 400× magnification under a light microscope (Carl Zeiss Microscopy GmbH, Gottingen, Germany). The sporulation and germination rates of each isolate were calculated as previously described (Johnny et al., 2011).

The total DNA of each tested isolate was extracted using the fungi genomic DNA rapid extraction kit (B518229-0100; Sangon Biotech). The target genes selected in this study were actin (ACT), calmodulin (CAL), chitin synthase (CHS), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and internal transcribed spacer (ITS). The cycling parameters and primers to amplify ACT, CAL, CHS, GAPDH, and ITS used in this study were the same as those described by Damm et al. (2012) and Weir et al. (2012). The primers selected for corresponding PCR amplification are shown in Table 1. The PCR products from a 50 μL amplification system were verified on 1.0% agarose gel at 254 nm (UV) and further sequenced by Shanghai Sangon Biological.

All sequences were grouped and aligned using DNAMAN v. 7.0. Sequences with single nucleotide polymorphisms were amplified and sequenced three times to ensure that the observed polymorphisms were not due to sequencing errors. The 100 tested isolates and referenced standard isolates (Weir et al., 2012; Diao et al., 2017) used in this study are listed in Supplementary Table 1. C. boninense (CBS: 123755) was used as the outgroup (Weir et al., 2012). All sequences were aligned using Clustal X 2.0.10 (Larkin et al., 2007). Gene sequences were compared and corrected using the “W” function in MEGA 5.0 (Tamura et al., 2011). The gene sequences were concatenated. Modeltest3.7.win, Win-paup4b10-console, and Mrmodeltest2, as implemented in MrMTgui, were used to estimate the best model of nucleotide substitution (Vaidya et al., 2011). Bayesian inference (BI) phylogenies were constructed using Mr. Bayes v. 3.1.2 (Ronquist and Huelsenbeck, 2003). Six simultaneous Markov chains were run for 1,000,000 generations each, and trees were sampled every 100th generation. The first 2,000 trees, representing the burn-in phase of the analyses, were discarded, and the remaining 8,000 trees were used to calculate the posterior probability (PP) in the majority rule consensus tree. Phylogenetic trees were drawn using treeView (Page, 1996). The alignments and trees were deposited in treeBase.

Each of the 100 tested isolate was cultured on a PDA medium for 5 days at 25°C. The spore suspensions were prepared following Gale et al. (2005). Five hyphae blocks (diameter of 5 mm) taken from the colony margin were transferred to 100 mL PD liquid medium and shaken at 160 r/min and 25°C to induce conidia production. After 5 days, spores were collected and adjusted to 1.0 × 10⁶ conidia/mL using a hemocytometer.

Strawberry seedlings (“Hongjia,” “Zhangji,” and “Tianxianzui” varieties) were planted in pots filled with sterilized nutrient soil and placed at 25 ± 5 °C under 12 h light and 12 h darkness. Five-week-old healthy strawberry plants had not been treated with any chemicals, were adopted for inoculation. Each leaf was inoculated with 10 μL spore suspension drop, and each crown was sprayed with 30 mL of the spore suspension. The negative control was inoculated with sterile water, three plants were inoculated for each isolate and all 100 isolates were tested. The plants were then placed back in a greenhouse at 25 ± 5°C with 90% humidity for 7 days under a 12 h light/dark cycle. The pathogens were re-isolated from the diseased leaves or crowns to complete Koch’s postulates (Chen et al., 2020).

We randomly selected representative isolates to determine the pathogenicity on leaves (“Hongjia,” “Zhangji,” “Tianxinazui,” “Hongyu”) and the crown (“Hongjia,” “Zhangji,”, “Tianxinazui”). These included, JD-J-ZJ-3-2, JD-J-ZJ-5, and JD-J-ZJ-9 from C. fructicola obtained from crown rot (CFCR), ZS-J-ZJ-54, ZS-J-ZJ-111, and JD-J-HX-A-6 from C. siamense obtained from crown rot (CSCR), JD-HX-Y-H-22, JD-ZJ-Y-H-69, and ZS-ZJ-Y-2-2 from C. fructicola obtained from leaf anthracnose (CFLA), JD-HX-Y-B-7, JD-ZJ-Y-H-60, and ZS-HX-Y-8 from C. siamense obtained from leaf anthracnose (CSLA) were adopted.

The spore suspension was prepared, and the leaves and crown were inoculated according to the methods described above. Both wounded and non-wounded plants were inoculated, the wounded condition using pin-pricking method (Karimi et al., 2019). Depending on the size of the leaf, using a sterile sharp needle pricking 4–12 wounds on the adaxial surface of each leaf. Before inoculation, leaves were surface sterilized by dipping in 1% sodium hypochlorite for 30 s and ethanol 70% followed by washed three times with distilled water, and then left to dry on sterile paper. For crown, using sterilizing blades caused several wounds at the crown. For each isolate, the inoculation was repeated three times with three leaves and crowns for each repeat. The diameter of the lesions on the leaves was measured 7 days after inoculation (Han et al., 2016). The severity standard of crown rot was recorded following (Haack et al., 2018). One-way ANOVA was performed to analyze the significance difference using IBM SPSS Statistics V22 software, and the least significant difference (LSD) test was applied to separated mean values for different species in the pathogenicity test at P = 0.05 level.

Three Colletotrichum spp. were characterized to determine the phenotype using the Biolog standard procedure (Bochner et al., 2001; Bochner, 2003; von Eiff et al., 2006). The concentration of the spore suspension was adjusted to 1 × 10⁶ conidia/mL. Metabolic plates 1–10 were used to assess the metabolic pathways of carbon (PM1–2), nitrogen (PM3), phosphorus and sulfur (PM4), bio-synthetic pathways (PM5), peptide nitrogen (PM 6-8), osmotic and ionic conditions (PM9), and pH conditions (PM10). To each well of the PM plates, 100 μL of a cell suspension was added with 62% transmittance. The metabolic phenotype test plate was placed in the Biolog system incubator at 28°C for 7 days. Phenotypic data were recorded every 15 min by the OmniLog 2.4 system. Heatmaps of phenotype analysis were generated with HemI software (Heatmap Illustrator, version 1.0; Zhao et al., 2017).

A total of 309 Colletotrichum spp. were isolated, with 150 from the crown and 159 from leaves. Pathogenicity was determined according to the Koch’s postulate, and all isolates were pathogenic. We selected 50 strawberry plants at random, from which Colletotrichum spp. were successfully isolated from both the crown and leaves. In Jiande, 10, 10, and 9 plants were selected for “Hongjia,” “Zhangji,” and “Tianxianzui,” respectively. In Zhoushan, 11 and 10 plants were selected for “Hongjia” and “Zhangji,” respectively. A total of 100 isolates were collected for further characterization, including 50 for LA and 50 for CR.

According to morphological and cultural characterizations, all the 100 isolates were grouped into three Colletotrichum species. The colony morphology of the above three Colletotrichum species on PDA media did not differ substantially (Figure 1). C. fructicola produced a gray-white colony with irregular edges and colony was black on the back on PDA plates; the conidium heap was orange, and conidia were cylindrical, with both ends obtuse or pointed. Appressoria were single or scattered, and most were light brown to dark brown, spherical, cylindrical, or fusiform, rare for irregular type; a few produced two suborbicular appressoria. The C. siamense colony was fluffy, gray in the middle and white at the edge, neat, yellow in the middle on the back, and white at the edge. The conidia were dark yellow, straight, cylindrical, blunt and round at both ends, colorless, and smooth. Appressoria were brown, and/or oval or spindle-shaped, with complete edges, and a few irregularities. The aerial hyphae of C. aenigma were sparse, light gray to white. The colony was pale white on the back and gray–green in the center. Conidia heaps were rare, and the conidia were stick-like, blunt, and round at both ends, single, or loosely distributed; appressoria were dark brown and ovate or irregularly shaped.

The significant morphological differences among three Colletotrichum spp. were listed in Table 2. C. fructicola had the largest spores, and C. aenigma had the largest appressoria. C. siamense had a higher growth rate, sporulation ability, and spore germination rate than that of C. fructicola and C. aenigma.

Phylogenetic trees were constructed using combined ACT, GADPH, CHS, CAL, and ITS datasets consisting of 100 Colletotrichum isolates with Colletotrichum boninense (CBC 123755) as the outgroup taxa. The concatenated alignment included 1256 characters. The boundaries of the loci used in the alignment were as follows: ACT: 1–152; CAL: 153– 448; CHS: 449–658; GADPH: 659–866; and ITS: 867–1256. All isolates were identified as C. gloeosporioides species complex and fell into three clades, with 91 isolates clustered in C. siamense, 8 isolates clustered in C. fructicola, and one isolate clustered in C. aenigma (Figure 2).

We conducted the pathogenicity analysis of each isolate on leaves using four varieties: “Hongjia,” “Zhangji,” “Hongyu,” and “Tianxianzui.” Under wounded conditions, C. siamense and C. fructicola had significant differences (P < 0.01) in the pathogenicity on “Zhangji” and “Hongyu” (Figures 3A,B), and the pathogenicity of C. siamense was significantly higher than that of C. fructicola. C. siamense showed stronger pathogenicity to the “Hongyu” and “Tianxianzui” varieties but weaker pathogenicity to “Hongjia” and “Zhangji” (Figure 4A).

The disease spot diameters on wounded leaves caused by C. siamense on “Hongyu” and “Tianxianzui” were 19.65 and 18.44 mm, respectively, and on “Hongjia” and “Zhangji,” they were 13.33 and 12.65 mm, respectively. C. fructicola showed stronger pathogenicity to “Tianxianzui,” with a disease spot diameter of 15.5 mm. Under non-wounded conditions, C. siamense and C. fructicola had significantly different pathogenicity indicated by mean disease spot diameters on “Hongjia,” “Hongyu,” and “Tianxianzui” (P < 0.01; Figures 3C,D). The pathogenicity of C. siamense was significantly higher than that of C. fructicola on “Hongyu” and “Tianxianzui” but weaker than that of C. fructicola on “Hongjia.”

On the crown, we tested the pathogenicity of three strawberry varieties: “Hongjia,” “Zhangji,” and “Tianxianzui.” Under wounded conditions (Figures 4A,B), the pathogenicity of C. siamense and C. fructicola was significantly different on “Hongjia” and “Tianxianzui” but not on “Zhangji.” The pathogenicity of C. siamense was significantly higher than that of C. fructicola on “Tianxianzui”; the disease index achieved by C. siamense and C. fructicola were 68.83 and 50.00, respectively. However, the pathogenicity of C. siamense was significantly weaker than that of C. fructicola on “Hongjia”; the disease index were 113.12 and 126.9, respectively. In general, C. siamense and C. fructicola showed the strongest pathogenic ability toward “Hongjia,” followed by “Tianxianzui” and “Zhangji” (Figures 4A,C). There were no significant differences under the non-wounded conditions (Figure 4D).

Using PM plates 1–10, three Colletotrichum spp. were characterized. Overall, 950 different growth nutrient conditions were tested, including 190 carbon substrates (PM1-2), 95 nitrogen substrates (PM3), 59 phosphorus substrates (PM4), 35 sulfur substrates (PM4), 94 bio-synthetic pathways (PM5), 285 peptide nitrogen substrates (PM6-8), 96 osmotic and ionic conditions (PM9), and 96 pH environments (PM10) (Figure 5).

For carbon source, all three Colletotrichum spp. could metabolize 190 carbon sources, such as sugars, nucleotides, and carboxylic acids, and produce color reactions (Figures 5A,B). C. fructicola had a relatively high availability, especially in L-arabinose (PM1, A02), glycerin (PM1, B03), and dulcitol (PM1, A12), whereas C. siamense and C. aenigma had a relatively low availability. The three species showed higher utilization rates with arbutin (PM2, B08) (Figure 5C). For amino acid nitrogen substrate metabolization, C. siamense, C. fructicola, and C. aenigma had high utilization rates. For phosphorus and sulfur substrate metabolization (Figure 5D), C. fructicola had a relatively high availability, and C. siamense and C. fructicola had a relatively low availability. For the bio-synthetic pathways (Figure 5E), C. aenigma had the highest overall utilization of nutritional supplement elements, followed by C. siamense and C. fructicola. For peptide nitrogen substrate metabolization (Figures 5F–H), C. fructicola had the best utilization of various peptide nitrogen elements, especially in Ala-Pro (PM6, A12), Arg-Ala (PM6, B05), Arg-Arg (PM6, B06), and Gly-Arg (PM6), E03), and had the highest utilization capacity, followed by C. aenigma and C. siamense. For osmotic and ionic adaptability (Figure 5I), C. aenigma had the highest utilization rate. The three Colletotrichum spp. had basically the same metabolic profiles under the condition of 80 mM sodium nitrite (PM9, H11), but the utilization rate was not high. In the pH environmental adaptability tests, C. aenigma had the highest utilization of pH under different conditions (Figure 5J). The three Colletotrichum spp. had basically the same metabolic profile under 5% ethylene glycol (PM10, D09) and showed extremely high utilization rates.

The LA and ACR in Zhejiang province were caused by different Colletotrichum species. Total 50 isolates from LA samples were grouped into two clusters with 8% (4 isolates) as C. fructicola and 92% (46 isolates) as C. siamense. For ACR, 4 isolates (8%) were identified as C. fructicola, 45 isolates (90%) were identified as C. siamense, and one isolate (2%) was identified as C. aenigma. Moreover, C. siamense was the most frequently pathogenic species, causing both LA and ACR in sampled greenhouses.

In Jiande, C. siamense, C. fructicola, and C. aenigma accounted for 87.99, 10.31, and 1.7% of all collected isolates, respectively. They caused 82.76, 13.79, and 3.45% ACR, and 93.10, 6.89, and 0.0% LA, respectively. In Zhoushan, C. siamense and C. fructicola accounted for 95.23 and 4.76% for ACR, 90.48% and 9.52% for LA, respectively (Figure 6).

The size of appressoria and conidia, and the growth rate of hyphae of the five types of Colletotrichum spp. are shown in Table 3. The sporulation and germination abilities were significantly different between the same Colletotrichum species obtained from plants showing LA or CR symptoms. The sporulation of C. fructicola causing crown rot (CFCR) was less than C. fructicola causing leaf anthracnose (CFLA), the C. siamense causing crown rot (CSCR) was higher than C. siamense causing leaf anthracnose (CSLA). The spore germination rate of the CSCR and CFCR was lower than the CSLA and CFLA. However, there was no significant difference (P > 0.05) in the size of spores, appressorium size, hyphal growth rate, and sporulation between the same Colletotrichum species obtained from plants showing LA and CR symptoms.

All the Colletotrichum spp. obtained from plants showing both LA and CR symptoms can cause leaf and crown disease when re-inoculated on strawberry, but the pathogenicity was significantly different (Supplementary Figure 1). Under wounded conditions, there were no significant differences in the pathogenicity of CSCR and CSLA to the leaves of “Hongjia” and “Hongyu.” The pathogenicity of CSLA was higher than that of CSCR to the leaves of “Zhangji”; the disease spot diameters caused by CSLA and CSCR were 13.39 and 11.85 mm, respectively. While in “Tianxianzui,” the pathogenicity of CSLA was weaker than that of CSCR, the disease spot diameters caused by CSLA and CSCR were 16.88 and 20.13 mm, respectively. Under non-wounded conditions, the pathogenicity of CSLA was weaker than that of CSCR in the “Zhangji” and “Hongyu” varieties (Table 4). For the crown, under wounded conditions, the pathogenicity of CSCR in different varieties was higher than that of CSLA (P < 0.05). Under non-wounded conditions, there was no significant difference in the pathogenicity of CSCR and CSLA (Table 5).

CFLA and CFCR had significant differences in pathogenicity toward the leaves of the four varieties (Table 4). Under wounded and non-wounded conditions, the pathogenicity of CFLA to leaves was higher than that of CFCR (P < 0.01). For the crown, under wounded conditions, CFLA and CFCR showed significant differences in pathogenicity to the three varieties; the pathogenicity of CFCR on different varieties was higher than that of CFLA (Table 5). Under non-wounded conditions, there was no significant difference in the pathogenicity of CFCR and CFLA. CFCR and CFLA were the most pathogenic to “Hongjia,” followed by “Hongyu” and “Zhangji.”