Identifying and characterizing Stagonosporopsis cucurbitacearum causing spot blight on Pinellia ternata in China

Disease sample collection and pathogen isolation

Disease samples with typical spot blight symptoms were collected from three commercial fields in Anguo City (N38°46′32″, E115°27′87″), Hebei Province in August 2020. To isolate the pathogen, disease samples were sterilized with 75% alcohol for 4 min, then washed three times with sterilized distilled water. Samples at the junction of healthy and diseased areas were chopped into pieces (about 0.5 × 0.5 cm²), and then the pieces were plated on potato dextrose agar (PDA) medium containing cefotaxime sodium (100 µg/ml) and incubated at 27 °C in darkness. After the appearance of fungal colonies, hyphae tips were picked from the edges of the colonies with an inoculation needle for purification.

Pathogenicity test

One-month-old healthy P. ternata seedlings were grown in a controlled environment chamber under a 16 h light/8 h dark cycle at 25 °C ± 2 °C, relative humidity 85%. During pathogen inoculation, the healthy leaves and plants were wounded using syringe needles and infected with a 5 × 5 mm mycelial cake of pathogen, and sterile PDA disks were used as the control. The experiments were replicated three times, and a total of 30 seedlings were used. The incidence of spot blight was observed after three days. Fungi were recovered from the diseased leaves to complete Koch’s postulates.

Fungal identification

In this study, the isolated pathogens were identified using conventional morphological and microscopic characteristics. Pathogenic isolates were grown on PDA at 28 °C in darkness for 7–10 days to record colony morphology, color, and growth rate. The hyphae and spores were stained with cotton blue dye. And the size and features of conidia and chlamydospores were observed under a microscope (Olympus, Tokyo, Japan). The DNA of pathogenic fungi was extracted using the CTAB method (Freeman, Katan & Shabi, 1996). The rDNA internal transcribed spacer (ITS) region and 28s large subunit ribosomal RNA (LSU) were then amplified and sequenced using ITS1-ITS4 (Mitchell, Freedman & White, 1994) and LROR-LR5 primers (Moncalvo, Lutzoni & Rehner, 2000). PCR was performed in a 50 μL reaction system that contained 5 μL of 10× buffer, 1 μL of 10 mmol∙L⁻¹ dNTP, 1 μL of 10 μmol∙L⁻¹ forward primer, 1 μL of 10 μmol∙L⁻¹ reverse primer, 1 µL of 5 U∙µL⁻¹ DNA Polymerase, 1 μL of DNA and 40 μL ddH₂O. The thermocycling program was as follows: 95 °C for 3 min, 34 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, and a final extension of 72 °C for 5 min. The PCR products were sequenced and assembled by Tsingke Biological Technology Company (Wuhan, China). All sequences were deposited in GenBank under accession numbers MZ227385 and MZ227377 for ITS and LSU, respectively.

ITS and LSU sequences of other Stagonosporopsis spp. isolates were downloaded from the National Center for Biotechnology Information (NCBI) nucleotide database through BLAST. Alternaria brassicae was used as the outgroup. A phylogenetic tree was constructed using MEGA7 (Kumar, Stecher & Tamura, 2016) and the neighbor-joining (NJ) method (Saitou & Nei, 1987). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches (Felsenstein, 1985). The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura, Nei & Kumar, 2004) and are in units of the number of base substitutions per site.

Statistical analysis

The statistical programme SPSS 18.0 (SPSS Inc., Chicago, IL, USA) was used to analyse the date. ALL results were confirmed by multiple biological repetitions.

Disease incidence and symptoms

In this study, the initial disease symptoms were yellowish-brown spots on leaves that gradually expanded into irregular circular spots with brown centers and greenish-yellow halos surrounding the spots. The spot diameters ranged from 5–10 mm. These small spots connected into larger spots and eventually the entire leaf turned yellow and necrotic. Plants with severe disease also experienced death of all their aboveground parts (Figs. 1A and 1B).

Morphological characteristics of fungal isolates

A total of 15 fungal isolates were obtained from all diseased plant samples, 11 of them showed the same morphology and showed strong pathongenicity on the leaves of P. ternata in vitro. Among the other four fungi, two were Fusarium sp. and two were Penicillium sp. The pathogenicity of two different Fusarium isolates were very weak and the other two Penicillium isolates were also not pathogenic and were generally considered as miscellaneous fungi (Table S1). One strain from the 11 pathogenic isolates were selected and named as AG-3 for further study. The isolate AG-3 colonies grew on PDA for 7 days with a diameter of 60–75 mm at 28 °C. The colonies were regular, white to light gray in color, and had concentric rings seven days after culture. The color further deepened and the surface became gray black and the back became greenish-brown at 15 days (Figs. 2A and 2B). Conidia and chlamydospores formed after two weeks of growth and many small protuberances appeared on the surface of the colony. The conidia were hyaline and oval 4.6 to 8.7 × 1.2 to 2.4 µm (6.62 ± 1.24 × 1.87 ± 0.35, n = 30) in size, and most of them had diaphragms and contained small oil drops (Fig. 2C). Chlamydospores were unicellular, spherical to ellipsoid, 6.3 to 15 × 6 to 11 µm (11.52 ± 3.48 × 8.95 ± 1.64, n = 10) in size, and either single or 4–13 to a chain (Fig. 2D). Based on our morphological observations, the causal fungus was identified as Stagonosporopsis cucurbitacearum (Nuangmek, Aiduang & Suwannarach, 2018; Stewart, Turner & Brewer, 2015).

Molecular identification

The ITS and LSU sequences of isolate AG-3 were uploaded to the GenBank database (accession numbers MZ227385 and MZ227377). BLAST results showed that all of the rDNA-ITS and LSU gene sequences of strain AG-3 showed 99% identity with the existing S. cucurbitacearum sequences in GenBank (JN618358.1, MK519412.1). Moreover, a phylogenetic tree of the ITS gene sequences of AG-3 constructed using the NJ method in MEGA7 software (Zhang, Qian & Zheng, 2019) revealed that AG-3 was closest to S. cucurbitacearum (Fig. 3). Based on morphological and molecular identification, the fungus was determined to be S. cucurbitacearum.

Pathogenicity tests

For the pathogenicity test, three healthy, one-month-old P. ternata plants were infected with a 5 × 5 mm mycelial cake of AG-3. The other three control plants were treated with sterile PDA disks. The treatment group and the control group were placed in a culture room (25 ± 2 °C, relative humidity 85%). One week later, spot blight symptoms had developed on the pathogen-inoculated group, while no disease symptoms were observed in the control group (Fig. 1C). Two weeks later, the leaves of the infected plants had turned yellow and the plants died (Fig. 1D). Koch’s postulates were fulfilled by recovering pathogens from the inoculated plants that were reconfirmed as S. cucurbitacearum through molecular identification.