First Report of Agroathelia delphinii Causing Rhizome Rot of Polygonatum cyrtonema Hua (Duohua Huangjing) in China

Polygonatum cyrtonema Hua (Duohua Huangjing, Asparagaceae in angiosperms) is a traditional medicinal and edible plant in China. Its rhizomes can potentially enhance immunity, reduce tumor growth and the effects of aging, improve memory, and even reduce blood sugar levels (Zhao et al. 2020). Many Polygonatum-derived health-care products have been developed, and annual demand in China for processed Polygonatum is up to 4000 tons (Su et al. 2018). However, in May 2024 there was an outbreak of rhizome rot in the P. cyrtonema Hua planting fields (N31°01'50″, E121°26'31″) located on the Shanghai Jiao Tong University campus, China. Approximately 40 % of the plants were affected. The surfaces of the rhizomes appeared rotten and were covered with white and turquoise hyphae. We also observed brown, basal stem necrosis, as well as leaf yellowing and wilting. To isolate the causal organism, affected rhizome tissues were disinfected with 75% ethanol for 1 min, rinsed 5 times with sterile water, placed on potato dextrose agar (PDA) medium containing 50 µg/ml of Chlortetracycline, and then incubated at 28 ℃. Hyphal tips of the resulting fungal colonies were sub-cultured after 3 days of incubation. The suspect pathogen had white, fluffy aerial mycelium, and grew radially at an average rate of 13.75 ± 0.55 mm/day. White sclerotia werebserved after 5 days of incubation, which subsequently turned yellow-brown, and ultimately reddish brown after 2 weeks. Mature sclerotia were of the globular or ellipsoidal type (0.6-2.1 mm in diameter with a mean of 1.23 mm; n = 100). Septal hyphae and clamp connections were observed with the Olympus microscope. These morphological features were similar to the description of Agroathelia delphinii (anamorph Sclerotium delphinii), a known pathogen of delphinium (Punja and Damiani 1996). Our isolated strain was denoted as HYW-1. For molecular identification, HYW-1 genomic DNA was extracted using the CTAB method (Mahadevakumar et al. 2018). The internal transcribed spacer region (ITS) and large subunit rRNA (LSU) gene were subsequently PCR-amplified with the primer pairs ITS1/ITS4 and LR5/LROR (White et al. 1990) and then sequenced at Sangon Biotech (Shanghai, China). The resulting HYW-1 ITS and LSU sequences were submitted to the NCBI nr database for BlastN analysis (GenBank accessions PP976559 and PP907792, respectively), and they were 99.69%, and 99.46% identical to the corresponding to the type strain of Agroathelia delphinii (Welch) Redhead (Amylocorticiaceae, Agaricomycotina) sequences (GenBank accessions JN241576 and MH866785, respectively). Based on the ITS and LSU sequences, a phylogenetic tree was generated using the the maximum likelihood method of MEGA 7.0. Strains HYW-1 and CBS 305.32 were members of the same clade. To complete Koch's Postulates, two HYW-1 hyphal discs (5 mm in diameter) were placed on each of the rhizomes of six healthy P. cyrtonema Hua seedlings. As a control, two uncolonized agar discs were placed on the rhizomes of six additional healthy seedlings. All seedlings were moved to a greenhouse with conditions of 90% RH and 28 °C. White hyphae were observed on the surface of inoculated plants after 3 days, and leaf wilt was observed after 10 days. The control seedlings showed no symptomatology. Infected tissues were collected for pathogen re-isolation, and subsequent ITS sequence analysis revealed that the causal agent was consistent with the original isolate HYW-1. To the best of our knowledge, this is the first report of A. delphinii causing rhizome rot on P. cyrtonema Hua in China.