Sorbaria sorbifolia (L.) A.Braun is a dense, deciduous, perennial shrub that is native to Siberia, the Far East of Russia, northern China, Japan, and Korea (Lee 2007). In China, it is widely planted in parks, gardens, front yards, and promenades as an ornamental, but it also valued for its fragrance and as an air purifier. S. sorbifolia is quite hardy and has few disease reports. The incidence of anthracnose on this shrub in Shandong Province, China is above 60%, and the disease affects all growth stages. From mid-May, spots initially appear on leaves. In the initial stage, the necrotic areas are circular, then expand into large circular or irregular, brown to reddish-brown or black lesions. The center of the lesions is gray-white with brown borders, and some spots are surrounded by a yellow halo ring (Fig. 1a). In the later summer to the middle autumn, most lesions eventually become dry, and the leaves wither and curl (Fig. 1b). While these symptoms are comparable to leaf spots, there is a controversy about the pathogen.

Fig. 1
figure 1

Disease symptoms of Sorbaria sorbifolia anthracnose on leaves in the field. a Initial infection stage. b Later stage of disease

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Diseased leaves of S. sorbifolia were randomly collected from the park in Taian City, Shandong Province, China. Among the fungi isolated from the leaf lesions, 72.5–95.5% of the isolates were Colletotrichum sp., and the rest were Alternaria sp. Twenty-one Colletotrichum isolates were obtained, and they had similar morphological characteristics. One Colletotrichum isolate named CGSS0709 was selected and preserved for further studies.

For pathogenicity tests to fulfill Koch’s postulates, isolate CGSS0709 was cultured for 7 days at 25 °C with 12 h light/dark (3000 lx), then conidia were collected and suspended in sterile distilled water (105 conidia/ml). The suspension was sprayed on healthy leaves of S. sorbifolia grown outdoors in June–July. Leaves were sprayed with sterile distilled water as controls. The inoculated leaves were wrapped with black plastic bags to keep them moist for 2 days. On day 3, all inoculated leaves had typical spots that were circular or subcircular, sunken, water-soaked, and red-brown. At 7 days after inoculation, the center of the lesions had brown borders, but control leaves had no symptoms. The lesions enlarged and often coalesced into large necrotic areas. The fungus was reisolated from the lesions, and characteristics of the colony, acervuli, conidiophores, conidiogenous cells, conidia, appressoria, and daily growth rate were similar to those of isolate CGSS0709. The results confirmed that the isolates caused S. sorbifolia anthracnose disease.

Isolate CGSS0709 was cultured on potato dextrose agar (PDA), potato sucrose agar (PSA), Luria Bertani agar (LA), BPY medium, potato maltose agar (PMA), water agar, and peptone agar at 25 °C. Mycelial growth rates ranged from 0 to 14.2 mm/d, and there were significant differences (n = 5; P < 0.01) according to Tukey HSD test (IBM SPSS Statistics 20 software) (Supplementary Table S1). The growth rates on the media in decreasing order were LA > PDA > PSA, PMA > BPY > Peptone > Agar (Supplementary Table S1). On PDA, the colonies were initially round, radial, white with abundant aerial mycelia, then they turned gray to grayish-black (Fig. 2a). Aerial mycelia were cottony, dense, white to pale gray, and with orange conidial masses near the inoculation site (Fig. 2a); black pigmentation was visible near the center when the reverse side was viewed (Fig. 2b). On LA, colonies were brown, aerial mycelium was villiform, sparse and orange to dark orange, and conidial masses were produced as the colony aged. On agar, mycelium grew hardly. Conidiophores were hyaline, 1-celled, clavate, and spindly. Conidia were hyaline, 1-celled, straight, cylindrical, apex obtuse, truncate at the base (Fig. 2c), 14.8–17.7 × 4.5–6.5 μm (\(\stackrel{-}{x}\) = 16.1 × 5.4, n = 50), length/width (L/W) = 3.0. Conidia were suspended in either 1% (w/v) glucose, 1% (w/v) sucrose, 1% (w/v) maltose, 1% (w/v) urea, or 1% (w/v) ammonium sulfate, all in sterile distilled water, to check germination rates. Sterile distilled water was used as the negative control. In the maltose, glucose, peptone, and sucrose solutions, the germination rate was 73.1, 71.0, 82.6, and 77.3%, respectively, and the rates did not differ significantly among the solutions (n = 5, 100 spores each; P < 0.01) according to Tukey HSD test (IBM SPSS Statistics 20 software) (Supplementary Table S1). In the urea solution, the germination rate was the lowest, 43.2% (Supplementary Table S1). Before germination, the conidium formed a septum that divided the conidium into two equal compartments. From one of the compartments, a germ tube emerged and developed. At the tip of the germ tube, an appressorium formed. The appressoria were brown, ovoid to irregular shaped (Fig. 2d), and the average size was 6.8–7.5 × 5.1–5.8 μm (\(\stackrel{-}{x}\)=7.3 × 5.5, n = 50), L/W = 1.3.

Fig. 2
figure 2

Morphological characteristics of Colletotrichum gloeosporioides sensu stricto isolated from anthracnose lesion of Sorbaria sorbifolia leaves after 7 days on PDA at 25 °C. a Split image of culture, reverse side on left, top side on right; b conidiomata; c conidia; d conidia with germ tube and dark appressorium at tip of germ tube

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The temperature had a remarkable effect on mycelial growth and conidial germination, and the optimum temperature was 25–30 °C. Isolate CGSS0709 grew from 5 to 35 °C (Supplementary Fig. S1). The results were consistent with the type strain C. gloeosporioides sensu stricto (s.s.) IMI 356878 (Cannon et al. 2008), but the daily growth rates at 25 °C of CGSS0709 (13.7 mm/d) and IMI356878 (26.5 mm/d) differed (Cannon et al. 2008).

Isolate CGSS0709 also grew from pH 4.0 to 10.0, showing wide adaptability to hydrogen ion concentration. The growth rates were divided into two groups, pH 5.0 (12.1 mm/d), pH 6.0 (12.0 mm/d), pH 7.0 (12.6 mm/d), pH 8.0 (11.3 mm/d), and pH 9.0 (11.1 mm/d) were in the one group, pH 4.0 and pH 10.0 were in the other group (P < 0.01). Conidia germinated from pH 6.0–10.0, with an optimum at pH 7.0 (79.6%) and pH 8.0 (79.0%) (Supplementary Fig. S2). We found no studies on the effect of hydrogen ion concentration on growth and conidial germination of the type strain IMI356878.

The morphological and cultural characteristics of isolate CGSS0709 fit the descriptions of Colletotrichum gloeosporioides sensu stricto (s.s.), which is indistinguishable in morphology and cultural characteristics from C. gloeosporioides sensu lato (s.l.) (Cannon et al. 2008; Huang et al. 2016) (Table 1).

Table 1 Morphological characteristics of the present pathogens and previously described Colletotrichum gloeosporioides sensu strictoa
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For a multilocus phylogeny to confirm the identity of isolate CGSS0709, genomic DNA was extracted from mycelium, and the primer sequence and PCR conditions to amplify each gene region (ITS, ACT, GAPDH, CHS-1, and TUB2) were done as described previously (Wang et al. 2017). All sequences were deposited in GenBank (Supplementary Table S2). Thirty-eight Colletotrichum isolates sequences were downloaded from GenBank and used for phylogenetic analyses (Supplementary Table S2), and C. boninense MAFF 305972 was used as an outgroup. Each gene sequence was aligned using the Clustal W option in MEGA 7.0 (Kumar et al. 2016). Sequences were manually aligned for maximum sequence alignment and similarity. Gaps were considered to be missing data. Five genes/regions were combined using Fasta alignment joiner software with terms ‘GAPDH-ACT-CHS-1-TUB2-ITS’. Phylogenetic analyses were accomplished with the multiple sequence alignment of five genes using MEGA 7.0 software. Using the maximum parsimony method and maximum likelihood method, two multilocus phylogenetic trees were generated by combining the data sets and drawn to scale, with branch lengths measured as number of substitutions per site. Percentage of bootstrap support for each node was calculated using 1000 replicates.

Five sets of sequence data showed no difference in phylogenetic trees, and the genes were combined. The concatenated sequences of five housekeeping genes (1446 bp) were above 98.5% homologous to C. gloeosporioides s.s. IMI 356878. In the maximum likelihood phylogenetic tree, the tree with the highest log likelihood is − 6727.09. Isolate CGSS0709 was in the same cluster with C. gloeosporioides s.s. with 99% bootstrap support (Fig. 3). The morphological characteristics and phylogenetic analysis thus indicated that the CGSS0709 was C. gloeosporioides s.s.

Fig. 3
figure 3

Phylogenetic tree based on combined sequence data for GPADH, ACT, CHS-1, TUB2, and ITS from fungal strains isolated from Sorbaria sorbifolia anthracnose and allied taxa. The maximum likelihood method was used with 1000 bootstrap replicates (Bootstrap values are at the nodes; the highest log likelihood =  − 6727.09). Colletotrichum boninense MAFF 305972 was used as the outgroup. The scale bar indicates the number of expected changes per site. Ex-type strains or authentic cultures are in bold

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Proper identification of the pathogen is the foundation for developing effective management strategies. C. gloeosporioides is a species complex and contains 38 closely related species (Jayawardena et al. 2016; Weir et al. 2012). In 2019 in a disease note, we first reported that C. gloeosporioides was associated with S. sorbifolia (Li et al. 2019), but failed to determine which species of C. gloeosporioides s.l. caused the disease. Here, we confirmed that C. gloeosporioides s.s. caused S. sorbifolia anthracnose in China by using multilocus phylogeny, morphological features, cultural properties, and biological characteristics. We thus determined that the pathogen was C. gloeosporioides s.s., not the other species in C. gloeosporioides s.l. Because the same host can be affected by different Colletotrichum species and S. sorbifolia is widespread in Northeast, Southwest, and North China, we need to determine whether other Colletotrichum species or new species are associated with S. sorbifolia anthracnose.

In conclusion, our results confirmed the agent causing S. sorbifolia anthracnose and characterized its growth. Future research will focus on understanding the pathogenesis, epidemiology, and other crucial information needed to develop control strategies to manage this disease in China.