First Report of Neocosmospora pisi (syn. Fusarium vanettenii ) Causing Root Rot on Astragalus membranaceus in China

The dried roots of Astragalus membranaceus, a leguminous herb, are extensively used in traditional Chinese medicine (Zhang et al. 2025). Root rot is a prevalent soil-borne disease primarily caused by Fusarium spp., that poses a serious threat to the development of Astragalus industry. In September 2023, samples exhibiting typical root rot symptoms were collected from cultivated fields of A. membranaceus located in Ningxia Province, China. The diseased plants exhibited a brownish root rot, accompanied by foliar chlorosis, wilting and death of the entire plant in severe cases (Figure 1). Diseased roots were surface-disinfected and plated on potato dextrose agar and incubated at 25°C under dark conditions for seven days. A total of 39 isolates were obtained from diseased roots. Three representative isolates, designated NWYQ-27, NGYS-04 and NGYS-42, were selected for morphological and molecular characterisation, as well as pathogenicity tests. Colonies of the three isolates showed dense, radially proliferating mycelium, with colour varying from pale orange (NWYQ-27, NGYS-04) to red-pigmented (NGYS-42) (Figure 2a). Macroconidia were slightly curved with barely notched foot cells, with 2–4 septa, and measured 21.8–40.5 × 4.7–7.6 µm, while microconidia were ovoid, with 0 to 1 septa, measuring 6.2–14.7 × 3.6–5.8 µm (Figure 2b). All three isolates shared morphological characteristics consistent with the description of Neocosmospora pisi (syn. Fusarium vanettenii) (Sandoval-Denis et al. 2019; Jones 1923). Genomic DNA was extracted and three loci were amplified and sequenced: the internal transcribed spacer (ITS) region using primers ITS1/ITS4; the translation elongation factor 1-alpha (TEF-1α) gene using primers EF1/EF2; and the RNA polymerase second largest subunit (RPB2) gene using primers RPB2-5f2/RPB2-7cr (White et al. 1990; O'Donnell et al. 1998; Parihar et al. 2025). The sequences were deposited in GenBank under the original name Fusarium vanettenii with Accession Nos. PX122088–PX122090 (ITS), PX278692–PX278694 (TEF-1α), and PX240730–PX240732 (RPB2). BLASTn analysis revealed that the sequences exhibited 96.02%–100% identity to N. pisi sequences for ITS, TEF-1α and RPB2. A phylogenetic tree was constructed using the maximum likelihood method based on the concatenated TEF-1α and RPB2 gene sequences, placed the representative isolate NGYS-42 clustered within a monophyletic clade with N. pisi (Figure 3). To assess pathogenicity, roots of 1-month-old healthy seedlings were immersed in a 5 × 106 CFU/mL spore suspension for 30 min, while control plants were treated with sterile water. For each isolate, 50 seedlings were inoculated and incubated at 25°C with 85% relative humidity and 12 h light/12 h dark in a growth chamber. All inoculated plants developed characteristic root rot symptoms after three days’ incubation, whereas controls remained symptomless (Figure 4). Three independent assays demonstrated the consistent pathogenicity of all three isolates to A. membranaceus. The pathogen was re-isolated from symptomatic tissues and identified via sequencing as the original inoculant, thereby fulfilling Koch's postulates. Neocosmospora pisi has been reported to infect various plants, primarily causing root rot, fusarium wilt and blight. This study represents the first confirmed report of N. pisi as the causal agent of root rot in A. membranaceus.