First Report of Ilyonectria coprosmae Causing Root Rot of Bletilla striata in Yunnan province of China.

Abstract

Bletilla striata is a valuable medicine in China, belonging to the Orchidaceae family, and is used for treating various ailments such as hemoptysis, pyocutaneous disease, and anal fissure by preventing blood flow, reducing swelling, and promoting granulation. In June 2022, a disease with symptoms similar to root rot was observed on B. striata in the pineland (the area was 0.4 hectare) of Lancang County (22°48'17" N, 99°46'58"22 E), Yunnan Province, China. The root rot incidence rate reached 16% (Table S1). The root rot incidence was calculated as follows: root rot incidence (%) = (number of root rot seedlings/total number of seedlings investigated) × 100. In May 2023, the similar symptoms were observed in the field, and the disease incidence was 17% (Table S1). Initially, there were no obvious symptoms on the leaves. Subsequently, the leaves wilted and brown spots appeared. Later, the entire leaf browned, withered and eventually died (Fig. S1A, B). The roots were brown and the browning spread from the root edge to the center, causing vascular bundle browning and dead lignified fibers in the cortex (Fig. S1C, D). To isolate the causal pathogen, 20 symptomatic root tissues were collected from 20 plants. Cutting the diseased tissues into small pieces (0.5 × 0.5 cm). After surface sterilization (30s with 75% ethanol and 3 min with 2% sodium hypochlorite, rinsed three times with sterile water), the disinfected root tissues were plated onto potato dextrose agar (PDA) and incubated at 25℃ for 4 to 6 days with 12 h light/dark photoperiod. A total of 10 single-spore isolates with similar morphology and conidial characteristics were obtained. one representative isolate BJG6 was selected for identification and further study. The fungal colony was reddish-brown or orange-white on PDA after 8 days of incubation at 25℃. The mycelium was like carpet or cotton, and the edge of colony was uniform (Fig. S1E). Large conidia were formed on simple conidial peduncles (Fig. S1F, G). The conidia with 1~3 septates and 1 mostly, with cylindrical shapes and narrow tops but sharp bases (Fig. S1H-J). Conidia with 1 septate measured as 5.5 (4.3-6.7) × 20.7 (16.0-25.4) μm (n=30), while those with 2 septates measured as 6.6 (5.8-7.4) × 26.5 (21.7-31.3) μm (n=30), and those with 3 septates was 6.9 (6.2-7.8) × 31.8 (29.3-34.3) μm (n=30). Ellipsoidal microconidia could be formed on conidiophore and measured as 2.4 (1.9-2.9) × 4.9 (5.9-3.9) μm to 2.7 (2.2-3.2) × 5.4 (4.3-6.5) μm (n=30). Spherical or subspherical chlamydospores were produced on low-nutrient agar, with an average size of 5.8(5.0-6.6) μm×5.3 (4.4-6.2) μm (n=30) (Fig. S1K, L). According to the morphology and conidial features, the pathogen was consistent with the description of Ilyonectria coprosmae (Cabral et al. 2012). The total genomic DNA was extracted, and primer pairs ITS4/ITS5 were used to amplify and sequence the rDNA-ITS region (ITS1-5.8 S rRNA-ITS2 gene regions) (White et al. 1990). The sequences were deposited in GenBank (SUB13905750 for ITS). BLAST searches revealed BJG6 showed 98% homology with corresponding sequences of Ilyonectria coprosmae in GenBank (JF735260). A phylogenetic tree (MEGA 7.0) was constructed using maximum-likelihood methods (Fig. S2). To identify pathogenicity, a cultured medium in a size of 6mm containing isolate BJG6 was inoculated onto ten healthy roots of B. striata, PDA plugs alone were used as the uninoculated controls. All samples were placed in a dark inoculation chamber at 25℃. The pathogenicity test was replicated three times. After two weeks, all inoculated roots appeared similar symptoms identical to those observed on field plants (Fig. S1M, N-P), while control plants remained healthy (Fig. S1Q, R). The same pathogenic fungus was reisolated from the symptomatic root rot, and the characteristics of colony and conidia were the same as the original isolates (Fig. S1S, T). These results confirmed I. coprosmae as the causal pathogen of root rot disease on B. striata in China by Koch's postulates tests for the first time. Further exploration should be conducted to understand the occurrence and migration of this disease, so as to develop specific and efficient disease management strategies in the future.