Identification of Corynespora cassiicola causing leaf spot on Syringa species
Abstract: Lilacs (Syringa spp.), a genus of trees and shrubs in the Oleaceae, are known for their beautiful, fragrant flowers and are widely cultivated as ornamentals. In September 2017, a new leaf spot disease was observed on S. oblata in the Sui and Tang Ruins Dynasties Botanical Garden in Luoyang, Henan Province, China. Leaf symptoms appeared as small brown lesions at earlier stages, which with age turned into suborbicular brown, faintly zonate and depressed. Fully mature lesions were mostly irregular to circular, co… Show more
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First Report of Corynespora cassiicola Causing Leaf Spot on Nanhaia speciosa in China
Nanhaia speciosa, commonly known as Niudali, is a medicinal woody vine belonging to the Leguminosae family. Valued for its culinary and medicinal properties, it is extensively cultivated, covering approximately 5,973 hm2 in the Guangxi Zhuang Autonomous Region of China. The edible tubers of this plant are reported to possess antibacterial and antioxidant effects (Luo et al., 2023; Shu et al., 2020). In July 2021, a Niudali plantation in Yulin, Guangxi, China (22°64′N; 110°29′E) exhibited leaf spot symptoms, with an incidence rate exceeding 40% across a 46,690 m2 area. Initially, small circular, pale yellow spots appeared on the leaves, which subsequently evolved into dark brown lesions surrounded by yellow halos, ultimately leading to foliage wilting. Leaves exhibiting typical symptoms were collected for pathogen investigation. The leaves were thoroughly washed with sterile water and small tissue fragments (5×5 mm) were excised from the lesion periphery. These fragments were surface-sterilized with 75% ethanol and 1% NaClO, rinsed three times with sterile water, and subsequently cultured on potato dextrose agar (PDA) at 28 °C in darkness for 7 days. Through single-spore isolation, seven isolates with similar morphological traits were obtained. After 7 days of incubation on PDA at 28 °C in dark, the colonies exhibited a white to grey coloration on the upper surface with abundant aerial hyphae, while the underside appeared dark black. The conidia, cylindrical or obclavate in shape, were straight, pale brown, and measured 30.1-128.9 μm × 4.8-15.0 μm (n=50). The morphological characteristics matched those of Corynespora sp.(Wang et al. 2021). For molecular identification, the isolate N5-2 underwent DNA sequence analysis using genomic DNA and primers ITS1/ITS4 and EF1-688F/EF1-1251R. The sequences (ITS: OP550425; TEF1-α: OQ117118) were deposited in GenBank, exhibiting 98% identity to C. cassiicola (OP981637) for TEF1-α and 99% homology to C. cassiicola (OP957070) for ITS. Based on the concatenated ITS and TEF1-α, a maximum likelihood phylogenetic analyses using MEGA7.0 clustered the isolate with C. cassiicola. Consequently, the fungus was identified as C. cassiicola based on its morphological and molecular features. In the pathogenicity test on 1-year-old Nanhaia speciosa seedlings, leaves were gently scratched and inoculated with mycelial plugs (5 mm). Control seedlings received PDA plugs. Five leaves per plant and five plants per treatment were selected for assessment. All seedling were maintained in a greenhouse (12/12h light/dark cycle, 25 ± 2°C, 90% humidity). After a 7-day incubation period, all leaves subjected to fungal inoculation exhibited symptoms consistent with those observed in the field, while control plants remained symptom-free. The fungus was successfully reisolated from the infected leaves in three successive trials, fulfilling Koch's postulates. While C. cassiicola is well-documented for inducing leaf spots on various plant species, including Jasminum nudiflorum, Strobilanthes cusia, Acanthus ilicifolius, Syringa species (Hu et al., 2023; Liu et al., 2023; Xie et al., 2021; Wang et al., 2021), this study represents the first report of C. cassiicola causing leaf spots on Nanhaia speciosa in China. The identification of this pathogen in Nanhaia speciosa has significant implications for future epidemiological investigations and serves as a valuable reference for controlling leaf spot disease in Nanhaia speciosa.
First Report of Corynespora cassiicola Causing Leaf Spot on Nanhaia speciosa in China
Nanhaia speciosa, commonly known as Niudali, is a medicinal woody vine belonging to the Leguminosae family. Valued for its culinary and medicinal properties, it is extensively cultivated, covering approximately 5,973 hm2 in the Guangxi Zhuang Autonomous Region of China. The edible tubers of this plant are reported to possess antibacterial and antioxidant effects (Luo et al., 2023; Shu et al., 2020). In July 2021, a Niudali plantation in Yulin, Guangxi, China (22°64′N; 110°29′E) exhibited leaf spot symptoms, with an incidence rate exceeding 40% across a 46,690 m2 area. Initially, small circular, pale yellow spots appeared on the leaves, which subsequently evolved into dark brown lesions surrounded by yellow halos, ultimately leading to foliage wilting. Leaves exhibiting typical symptoms were collected for pathogen investigation. The leaves were thoroughly washed with sterile water and small tissue fragments (5×5 mm) were excised from the lesion periphery. These fragments were surface-sterilized with 75% ethanol and 1% NaClO, rinsed three times with sterile water, and subsequently cultured on potato dextrose agar (PDA) at 28 °C in darkness for 7 days. Through single-spore isolation, seven isolates with similar morphological traits were obtained. After 7 days of incubation on PDA at 28 °C in dark, the colonies exhibited a white to grey coloration on the upper surface with abundant aerial hyphae, while the underside appeared dark black. The conidia, cylindrical or obclavate in shape, were straight, pale brown, and measured 30.1-128.9 μm × 4.8-15.0 μm (n=50). The morphological characteristics matched those of Corynespora sp.(Wang et al. 2021). For molecular identification, the isolate N5-2 underwent DNA sequence analysis using genomic DNA and primers ITS1/ITS4 and EF1-688F/EF1-1251R. The sequences (ITS: OP550425; TEF1-α: OQ117118) were deposited in GenBank, exhibiting 98% identity to C. cassiicola (OP981637) for TEF1-α and 99% homology to C. cassiicola (OP957070) for ITS. Based on the concatenated ITS and TEF1-α, a maximum likelihood phylogenetic analyses using MEGA7.0 clustered the isolate with C. cassiicola. Consequently, the fungus was identified as C. cassiicola based on its morphological and molecular features. In the pathogenicity test on 1-year-old Nanhaia speciosa seedlings, leaves were gently scratched and inoculated with mycelial plugs (5 mm). Control seedlings received PDA plugs. Five leaves per plant and five plants per treatment were selected for assessment. All seedling were maintained in a greenhouse (12/12h light/dark cycle, 25 ± 2°C, 90% humidity). After a 7-day incubation period, all leaves subjected to fungal inoculation exhibited symptoms consistent with those observed in the field, while control plants remained symptom-free. The fungus was successfully reisolated from the infected leaves in three successive trials, fulfilling Koch's postulates. While C. cassiicola is well-documented for inducing leaf spots on various plant species, including Jasminum nudiflorum, Strobilanthes cusia, Acanthus ilicifolius, Syringa species (Hu et al., 2023; Liu et al., 2023; Xie et al., 2021; Wang et al., 2021), this study represents the first report of C. cassiicola causing leaf spots on Nanhaia speciosa in China. The identification of this pathogen in Nanhaia speciosa has significant implications for future epidemiological investigations and serves as a valuable reference for controlling leaf spot disease in Nanhaia speciosa.
First Report of Corynespora cassiicola Causing Septoria Leaf Spot on Scutellaria baicalensis in China
Scutellaria baicalensis Georgi. is a perennial herb in the Lamiaceae family, with a distribution in more than 10 provinces in China. At the current time, the cultivation area of S. baicalensis in China exceeds 58,000 hectares, with annual production approaching 28,000 tons. As a traditional Chinese herbal medicine, the root of S. baicalensis has many applications, such as anti-inflammatory, anti-neuroinflammatory and neuroprotective, anticancer, antiviral, antibacterial, and antioxidant activities, and is effective in treatment of colitis, hepatitis, pneumonia, respiratory infections, and allergic diseases. (Jang et al. 2023; Liu et al. 2023). From August to September 2022, septoria leaf spot symptoms were observed at the Institute of Medicinal Plant Development (40.04°N, 116.28°E), Beijing, China, and the incidence of this disease was up to 20% in the field through more than two weeks of continuous investigation. Initial symptoms on leaves were observed as small, dark-brown spots (0.5 to 2.0 mm), which then expanded to irregular lesions with a pale gray center surrounded by a black ring with a dark-brown edge and light brown halo (Fig. 1A1-A3). Plants were defoliated and withered in severe cases. Thirty-six symptomatic leaves of 12 diseased plants from three experimental sites were cut into 5 × 5 mm pieces, and surface sterilized with 75% ethanol for 30 s followed by 5% NaClO solution for 45 s, rinsed with sterile water three times, dried with sterile filter paper, and subsequently placed on potato dextrose agar (PDA) medium and incubated at 25°C in dark for two days. Isolates were purified by transferring hyphal tips to new PDA plates and incubated at 25°C in dark. Finally, eight isolates (A1, B3, D1, F2, E2, a4, e4 and f1) with similar colonial morphological characteristics were obtained. Colonies on PDA exhibited dense, downy, and white to grayish-green aerial mycelia and the reverse of colonies showed dark-brown in the center and grayish on the edge (Fig. 1D, E). Conidia were solitary or catenate, pale brown, obclavate to cylindrical, apex obtuse (Fig. 1B, C). The isolates were divided into two categories by examining 100 conidia (50 of each isolate), represented by isolates D1 and e4. Conidia of D1 measured 5.4 to 75.8 μm × 2.1 to 6.8 μm, mean 26.9 × 4.4 μm, had 0 to 6 pseudosepta, with 0 to 3 pseudosepta observed in 88% of conidia. Conidia of e4 measured 20.3 to 103.4 μm × 2.0 to 7.9 μm, mean 41.9 × 4.8 μm, had 0 to 6 pseudosepta, with 2 to 5 pseudosepta observed in 90% of conidia. These isolates were identified as Corynespora cassiicola based on morphology (Ellis 1971). DNA of the two isolates (D1 and e4) was extracted by the cetyltrimethylammonium bromide (CTAB) method, and internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1 alpha (TEF1-α), and beta-tubulin (TUB2) gene were amplified, using the primers ITS1/ITS4 (Bandi et al. 2022), EF1-728F/EF-986R (Wang et al. 2021), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. Sequences of ITS OQ991339 (524 bp) and OR044050 (533 bp) shared 99.8% identity to C. cassiicola, with a 99% coverage to MT228951 (536 bp) and OQ991340 (546 bp) in GenBank. Sequences of TEF1-α OR047441 (304 bp) and OR047443 (306 bp) shared 99.3% identity to C. cassiicola, with a 98% and 99% coverage to ON381927 (300 bp) and ON381933 (301 bp) in GenBank, respectively. Sequences of TUB2 OR047449 (427 bp) and OR047451 (427 bp) shared 99.53% identity to C. cassiicola, with a 99% and 98% coverage to MN604075 (442 bp) in GenBank, respectively. Phylogenetic trees were computed with ITS, TEF1-α, and TUB2 sequences in MEGA 11 using the Neighbor-Joining (NJ) method (Fig. 2). The results showed that the two isolates were C. cassiicola with more than 90% bootstrap support (1000 replicates). Nine 2-year-old seedlings of S. baicalensis were used for the pathogenicity assay. Three leaves from each plant were punctured with flame-sterilized needles, and inoculated with mycelial plugs (5 mm in diameter) of D1 and e4. Plants inoculated with sterile PDA plugs were used as control. All the inoculated seedlings were incubated at 25 oC and 90% relative humidity. About 3 to 4 days after inoculation, similar symptoms to those observed in the field were present on leaves inoculated with D1 and e4, while no symptoms were observed in the uninoculated control seedlings (Supplementary Fig. 1). Isolates with vigorous, downy, and white to grayish-green aerial mycelia were reisolated from the diseased leaves inoculated with D1 and e4 and identified as C. cassiicola by DNA sequencing, fulfilling Koch’s postulates. Based on morphological and multilocus phylogenetic results, these isolates were identified as C. cassiicola, a pathogen that threatens several important crops (Dixon et al. 2009; Zhang et al. 2018; Xie et al. 2021). To our knowledge, this is the first report of C. cassiicola as the causal pathogen of septoria leaf spot on S. baicalensis in China, which poses a potential threat to the production of S. baicalensis.
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