Root Rot of Angelica sinensis Caused by Clonostachys rosea and Fusarium acuminatum in China.

Abstract

Angelica sinensis is a perennial plant in the Apiaceae and a source of one of the most important traditional medicines in China. This medicinal herb has been mainly cultivated in Gansu, Sichuan, Yunnan, Shanxi, Hubei, and Qinghai Provinces. In June 2020, angelica plants in a field in the village of Sada, Minhe County, Haidong city, in Qinghai Province (102°83'00.00″E, 36°31'93.00″N, elevation:2437 m), displayed symptoms of yellowing, stunting, root rot, and wilting（Fig. S1）. Severe brown discoloration of vascular tissue in the stems of infected plants was observed. Root rot seriously affected the quality and yield of A. sinensis. The incidence of root rot in the fields reached 15% in 2020 and 18% in 2021. Fungi were isolated from diseased roots using a single spore method. The identity of isolates was confirmed by sequencing the nuclear ribosomal internal transcribed spacer (ITS) using the primers ITS1/ITS4 (White et al, 1990) and the translation elongation factor 1-alpha (TEF1-α) gene using the primers EF-1/EF-2 (O'Donnell et al. 1998). BLAST analysis of the ITS and TEF1-α sequences of isolates DG-1, DG-1-1, DG-1-2, DG-2, DG-2-1, DG-5, and DG-5-1 in the NCBI database showed 99% to 100 % nucleotide identity to those of Clonostachys rosea (NCBI accession Nos. MZ424803-MZ424809 (ITS), and MZ451383-MZ451389 (TEF1-α)). BLAST analysis of the ITS and TEF1-α sequences of isolates, DG-3, DG-3-1, and DG-4 in the NCBI database produced matches of 99.1% or better to Fusarium acuminatum (NCBI accession Nos. MZ424810-MZ424812 (ITS), and MZ441148-MZ441150 (TEF1-α)). Conidia of C. rosea were hyaline, round or oval, aseptate, with an average length and width of 6.5-8.5×2.5-3.5 μm. Chlamydospores were round or oval, with an average length and width of 9.9-16.2×8.1-13.6 μm. Conidiophores of C. rosea were hyaline, septate, Verticillium-like, conidiogenous cells were slender and cylindrical. Macroconidia of F. acuminatum were hyaline, falciform, 0-5 septate, with an average length and width of 22.5-39.5 × 2.5-4.5 μm. Microconidia were rare. Chlamydospores were oval, with an average length and width of 9.5-14.3×8.1-11.8 μm. Conidiophores of F. acuminatum were hyaline, septate, branched (Fig. S2). A pathogenicity test was performed by inoculating a conidial suspension of 1×105 per ml in 0.025% Tween 20 onto roots of A. sinensis. Inoculated roots were incubated in a humid and dark chamber at 26°C for 24 h, and then with 12 h light/12 h dark for six days. The pathogenicity assay was repeated three times for each isolate. Root rot was observed seven days after inoculation and mycelia were observed on the surface of the lesions (Fig. S1). Two isolates were recovered from the lesions and named DG-2-D and DG-3-1-D. The colonial morphology of these two isolates and the original isolates DG-2 and DG-3-1 was identical after six days of inoculation on PDA (potato dextrose agar) (Fig. S2). The two isolates recovered were identified as C. rosea and F. acuminatum, respectively, by amplifying and sequencing a portion of the TEF1-α gene. To our knowledge, this is the first report of C. rosea causing root rot of A. sinensis in China. Fusarium acuminatum was reported to cause root rot of A. sinensis in Gansu province, China in 2021 (Niu et al. 2021). Clonostachys rosea has been previously reported to cause root rot of soybean in the United States (Bienapfl et al. 2012). The disease must be considered in existing management practices, and our findings provide a foundation for management this disease.