First Report of Agroathelia rolfsii Causing Southern Blight on Alpinia hainanensis in China.

Abstract

Katsumada galangal seed (Alpinia hainanensis K. Schum) is an important member of the Zingiberaceae family, with both medicinal value and culinary applications (Park et al. 2020). In May 2023, a stem disease of Al. hainanensis was detected in a 100-acre plantation located in Danzhou City, Hainan Province in China, with an average incidence of 15% to 20%. The disease primarily targeted the basal stems, with the pathogen spreading by mycelia in the soil around the rhizome, and reaching neighboring plants. The infection caused the stem base to undergo necrosis, turning the tissue brown to black, and leading to a loosely attached epidermis. As the disease advanced, the necrosis progressed upward along the stem, resulting in curling of the leaves. A large number of white mycelia, as well as white immature sclerotia and brown mature sclerotia, were also observed on the surfaces of the leaves, stems and soil. The diameter of the mature sclerotia ranged from 0.7 to 1.6 mm (aver. ± SD = 1.2 ± 0.3 mm, n = 50). To identify the pathogen, eleven tissue samples were collected from symptomatic stems. The sclerotia on the surface of the tissue were sterilized with 75% (V/V) ethanol for 30 s, followed by three rinses with sterile water, and finally incubated on potato dextrose agar (PDA) at 28°C in darkness. Eleven isolates were obtained and subcultured at 28°C in darkness. After 48 hours, the colonies of these isolates had an average daily radial growth of 39.5 ± 0.4 mm (n = 11) and were white, featuring entire margins and abundant aerial mycelia. By the seventh day, spherical or oval white sclerotia began to form on the surface of their colonies. These sclerotia eventually turned brown, with a diameter ranging from 0.6 to 1.5 mm (aver. ± SD = 1.3 ± 0.3 mm, n = 50). The morphological characteristics of these isolates were similar to those of Agroathelia rolfsii (Sacc.) Redhead & Mullineux (basionym: Sclerotium rolfsii Sacc.; Amylocorticiaceae) (Redhead and Mullineux 2023). DNA of a representative isolate DZAS-01 was extracted for amplification of its internal transcribed spacer (ITS) rDNA region, the translation elongation factor 1-alpha (TEF1) gene and the large subunit (LSU) region using the primers ITS1/ITS4, EF1-983F/EF1-2218R and LROR/LR5, respectively (Moncalvo et al. 2000, Rehner and Buckley 2005, White et al. 1990). The three sequences have been deposited in GenBank (accession nos. PP659527 for ITS, 607 bp; PP976301 for TEF1, 1,040 bp; PP968104 for LSU, 876 bp). The BLASTn results showed that the similarity of the three sequences with the known sequences PP908473 (ITS), OL416131 (TEF1) and KY446370 (LSU) of Ag. rolfsii was 100%, 99.81% and 100%, respectively. Pathogenicity tests were conducted on ten potted, 6-week-old healthy Al. hainanensis plants. The stem bases of ten plants were wounded with sterile needles and divided into two groups with five plants in each group. Each plant in the first group was inoculated by placing three mature sclerotia of DZAS-01 near the wound (Urbina et al. 2024). Plants in the other group served as controls and were not inoculated. Ten plants were covered with plastic bags, placed at 25°C to 28°C with a 12-h/12-h light/dark cycle, and sprayed with sterile water daily. After seven days, all inoculated plants exhibited symptoms that were consistent with those in the field, while non-inoculated control plants remained symptom-free. This pathogenicity test was repeated three times with identical results. A fungus was re-isolated from symptomatic tissues of inoculated plants in each repeat trial and had identical morphological characteristics and ITS, TEF1 and LSU sequences to those of isolate DZAS-01. Ag. rolfsii can infect more than 500 species of cultivated and wild plants in tropical and subtropical regions (Daunde et al. 2020). To the best of our knowledge, this is the first report of Ag. rolfsii causing southern blight on Al. hainanensis in China. The results obtained from this study are going to provide guidance for preventing and treating this disease.