Polygonatum odoratum (Mill.) Druce (Asparagaceae), a flowering ornamental plant native to Asia and Europe, is used in traditional Chinese and Korean medicine. In August 2014, small brown spots were found on the edge of leaves of P. odoratum in a plant nursery at Jilin Agricultural University, Changchun, Jilin, China (125.24° E, 43.48° N). The lesions gradually enlarged to rounded brown spots (Fig. 1b), and leaf margins turned yellow (Fig. 1a).

Fig. 1
figure 1

Symptoms on leaves of Polygonatum odoratum and morphology of causal agent, Colletotrichum spaethianum. a Anthracnose and b brown spots after natural infection. c Water-inoculated leaf with no symptoms 9 days after inoculation. d–f Lesions on detached leaves 5 (d), 7 (e) and 9 (f) days after inoculation (dai) with isolate CCPO34. g Brown spots on inoculated detached leaf 9 dai. h–j Lesions on intact leaves 14 dai with CCPO34. h Mock-inoculated, nonwounded plant. i Mock-inoculated plant, wounded with a fine sterile needle before inoculation with sterile water. j Plant that was wounded before inoculation with CCPO34. k–y Morphology of C. spaethianum. k, l Colonies on PDA (k, l) and on SNA(m, n). o Conidia (o), appressoria (p–t) and conidiophores (u–y) on SNA. Scale bars = 10 μm

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For isolating the causal agent, small leaf pieces were removed from the lesion margins, then surface-sterilized for 1 min in 1.5% (v/v) NaOCl, washed twice with sterile distilled water, then plated onto potato dextrose agar (PDA; Difco, Detroit, MI, USA) slants. Single spores were transferred with a needle using a microscope onto a PDA slant. A representative single-spore isolate was designated as CCPO34 and used in the following tests.

For confirming pathogenicity, a conidial suspension (1 × 106 conidia/ml) of isolate CCPO34 was sprayed onto 21 detached leaves and 10 leaves on young plants of P. odoratum following the method of Liu et al. (2013, 2017). Distilled water served as a negative control. These plants were covered with plastic bags for 6 days and kept in a greenhouse at 25–28 °C with a 12-h photoperiod using fluorescent light and 90% of relative humidity. This experiment was repeated twice. After 5, 7 and 9 days, all detached leaves (Fig. 1d–g) and intact leaves on inoculated plants (Fig. 1j) had symptoms but control plants had no symptoms (Fig. 1c, h, i). Isolate CCPO34 was consistently re-isolated from the lesions (Fig. 1g), but not from the control leaves (Fig. 1c, h, i). Brown spots on naturally diseased leaves of P. odoratum (Fig. 1b) and on the inoculated leaves were similar (Fig. 1d, f, g). By 14 days after inoculation with CCPO34, leaves on the plants had turned yellow and died (Fig. 1j).

Morphological characteristics of isolate CCPO34 were examined with a microscope after incubation on synthetic nutrient-poor agar (SNA) (Damm et al. 2009) at 25 °C with 12 h light/12 h dark period for 7 days. Conidia were 1-celled, hyaline, smooth-walled, aseptate, curved to slightly curved, with a more rounded or somewhat acute apex and a truncate base, (15.5 −)16 − 23.2 (− 28) × (3.0 −)3.5 − 4.1(− 5.3) μm, mean ± SD = 20.5 ± 2.8 × 3.9 ± 0.5 μm, L/W ratio = 5.3 (Fig. 1o). Spore curvature ratios were calculated using the method of Sato et al. (2015): the average outer curvature of the conidia was 32.2, average inner curvature was 7.5, and average curvature deviation was 1.19. The colonies on PDA developed abundant white, aerial mycelium with olivaceous white mycelium below with a white margin (Fig. 1k, l). Appressoria were single or in loose groups, dark brown, irregular shapes, sometimes more or less lobed, smooth-walled, (3.2 −)8.1 − 9.4(− 15.4) × 3.2 − 6.2(− 12.6) μm, mean ± SD = 9.3 ± 2.5 × 7.5 ± 1.6 μm, L/W ratio = 1.2 (Fig. 1p–t). Colonies on SNA were flat with an entire margin, having short grayish white, aerial mycelium (Fig. 1m, n). The conidiophores formed directly on hyphae (Fig. 1u–y). Morphological and cultural characterizations were almost consistent with the description of Colletotrichum spaethianum (Allesch.) Damm et al. (2009). Setae were not observed.

For molecular support of these observations, genomic DNA of isolate CCPO34 was extracted using the method of Liu et al. (2013). The 5.8S nuclear ribosomal gene with the two flanking internal transcribed spacers (ITS), a 200-bp intron of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a partial sequence of the actin (ACT), chitin synthase 1(CHS-1) and beta-tubulin (TUB2) genes were amplified and sequenced using primer pairs ITS-1 + ITS-4 (Gardes and Bruns 1993; White et al. 1990), GDF1 + GDR1 (Guerber et al. 2003), ACT-512F + ACT-783R (Carbone and Kohn 1999), CHS-345R + CHS-79F (Carbone and Kohn 1999), and T1 (O’Donnell and Cigelnik 1997) + Bt-2b (Glass and Donaldson 1995) (the PriMicro database (https://primicro.jpn.org/)), respectively.

The ITS, GAPDH, ACT, CHS-1 and TUB2 sequences of isolate CCPO34 were deposited in GenBank (accession MH020771 for ITS, MH020772 for GAPDH, MH045677 for ACT, MH020773 for CHS-1, and MH045678 for TUB2). A BLAST search revealed that all the sequences had more than 99% identity to C. spaethianum (isolate CBS 167.49; GU227807 for ITS, GU228199 for GAPDH, GU227905 for ACT and GU228297 for CHS-1, and GU228101 for TUB2). Phylogenetic trees were constructed using MEGA7 (Kumar et al. 2016) for the combined data set of the ITS, GAPDH, ACT, CHS-1 and TUB2 genes for 22 sequences from Colletotrichum (Table 1). Percentage bootstrap support > 60% (1000 replications) by the maximum parsimony (MP) methods are shown on the respective branch. Isolate CCPO34 of P. odoratum clustered in the C. spaethianum clade (Fig. 2).

Table 1 Strains and isolates of Colletotrichum spp. and the accessions used in this study
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Fig. 2
figure 2

Phylogenetic tree constructed using combined data set for ITS, GAPDH, ACT, CHS-1 and TUB2 sequences from 22 accessions of Colletotrichum. Percentage bootstrap support > 60% (1000 replications) using maximum parsimony (MP) methods are shown on the respective branches. Isolate CCPO34 from leaf spots on Polygonatum odoratum clustered with Colletotrichum spaethianum

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Isolate CCPO34 was placed in the Herbarium of Mycology of Jilin Agricultural University. This pathogen has been previously reported on Peucedanum praeruptorum Dunn, Lilium lancifolium Thunb and Atractylodes japonica Koidzumi in China (Guan et al. 2018; Guo et al. 2013; Zhao et al. 2016); Hemerocallis flava Linn and Allium fistulosum L. in Brazil (Santana et al. 2016; Vieira et al. 2014); A. fistulosum, Crinum latifolium L., Iris germanica L. and Raphanus sativus L. in Japan (Sato et al. 2005, 2015); and on Hosta plantaginea Aschers in Korea (Cheon and Jeon 2016). In May 2001, C. spaethianum was also found on potted plants of Polygonatum falcatum A.Gray in Japan (Sato et al. 2015; Tomioka et al. 2008). This study on the etiology of leaf spot on P. odoratum based on pathogenicity experiments, morphological and molecular characterization is the first report of C. spaethianum causing leaf spot on P. odoratum in China.