First Report of Polygonatum cyrtonema Root Rot Caused by Colletotrichum spaethianum in Hunan province

Polygonatum cyrtonema Hua, also known as Huangjing, is a perennial herbaceous plant of the Polygonatum genus, belonging to the Liliaceae family, with significant medicinal and nutritional value (Lu et al. 2023). In June 2022, a root rot disease was observed on P.cyrtonema plants in Xinhua County (27°44′13″N, 111°18′29″E), Hunan Province, China. Disease incidence varied from 25 to 40% in the survey area of 2 hectares. The underground roots P.cyrtonema exhibited red brown spots that gradually expanded and sunken areas appeared, and the diseased plants presented leaf chlorosis and red-brown discoloration of the tubers, eventually leading to plant death. To identify the pathogen, symptomatic root tissues (0.5 × 0.5 cm) from the lesion margin surface were sterilized with 75% ethanol for 30 s and 2% NaClO for 3 min, rinsed three times with sterile water, air dried, the sterile root pieces were placed on potato dextrose agar (PDA) and incubated at 25°C. Three pure fungal isolates with the same morphological characteristics were obtained by monosporic isolation from seven pieces of roots, and the representative isolate Psi1101 was used for morphological studies and phylogenetic analyses. After 4 days of growth on the PDA at 25°C, the colony on PDA appeared white with cotton-shaped aerial hyphae, continued cultivation for 3 days, hyphae turned light gray to gray. Conidia were hyaline, curved or slightly curved, aseptate, unicellular, with a truncate base and acute apex, and measured (9.2-16.5)×(4.2-6.8)µm (n=50). These morphological characteristics are consistent with the description of a Colletotrichum species (Damm et al. 2009). For molecular identification, the primers ITS1/ITS4 (White et al. 1990), Bt2a/Bt2b (Glass and Donaldson 1995), and GPD1/GPD2 (Berbee et al. 1999) were used to amplify the partial region of rDNA-ITS, b-tubulin (TUB2), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from fresh mycelia of isolate Psi1101, respectively. BLAST analysis showed that the rDNA-ITS, TUB2, and GAPDH sequences of the isolates Psi1101 (GenBank accession nos. OQ711774.1, PP213778.1, and PP429927.1) had 98 to 100% identity with multiple GenBank sequences of Colletotrichum spaethianum. A phylogenetic tree based on concatenated sequences of rDNA-ITS, TUB2, and GAPDH using maximum-likelihood analyses revealed that the isolate Psi1101 was in the same clade with C. spaethianum strains. The isolate Psi1101 was identified as C. spaethianum based on the morphological characteristics and molecular phylogenetic. To test pathogenicity, three healthy tuber of 1-year-old P. cyrtonema plants were pin-pricked with a sterile needle, one tuber each, three mycelial plugs inoculated in wounds. Three tubers inoculated with fresh PDA plugs were used as controls. All inoculated tubers were maintained in a manual climatic box (12 h/12 h light/dark, 25 ± 3°C, 80% relative humidity). After 7 days, symptoms were observed similar to those of the original infected plants in the field, whereas control tubers were asymptomatic C. spaethianum was reisolated from the diseased tubers, whereas no pathogenic fungus was isolated from the control tubers. Colletotrichum mainly causes anthracnose in crop leaves(Yin et al. 2023), and root rot in roots(Fealko et al. 2019). To our knowledge, this is the first report of P. cyrtonema root rot caused by C. spaethianum in China. This result may expand the etiological study of C. spaethianum and the control strategy of P. cyrtonema root rot.