First Report of Corynespora cassiicola Causing Septoria Leaf Spot on Scutellaria baicalensis in China.

ABSTRACT

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.