First Report of Phytophthora nicotianae causing Root Rot on Medicago polymorpha in China.

Medicago polymorpha (commonly known as Jin hua cai) is a nutritious and palatable forage and vegetable crop in China. From November 2023 to March 2024, symptoms of root rot and foliage blight were observed on M. polymorpha in a vegetable production base in Nanjing (31.7960°N, 118.8902°E), Jiangsu Province, China. Typical symptoms included water-soaked lesions, brown to dark-brown discoloration of roots, and wilting. Infected plants eventually collapsed and died as the disease progressed. A survey conducted in 0.75 hectares of M. polymorpha fields revealed disease incidence ranging from 20% to 40%. Symptomatic root tissues were surface-sterilized (75% ethanol for 30 s, 1% NaClO for 120 s, rinsed thrice with sterile water), plated on 10% V8 agar amended with 50 mg/L rifampicin and ampicillin, and incubated at 25°C for 3 days. Phytophthora-like colonies with aseptate hyphae were isolated. Twelve pure cultures (PN-1 to PN-12) were obtained by hyphal tipping and grown on V8 medium for morphological characterization, molecular identification, and pathogenicity testing. All isolates produced colonies with loose, fluffy aerial mycelia. Morphological characterization revealed papillate, noncaducous sporangia (35-60 × 30-53 µm; n=30), spherical chlamydospores (21-33 µm; n=30), and ovoid zoospores (6-9 µm; n=30). Three isolates (PN-1 to PN-3) were selected for molecular identification. The internal transcribed spacer (ITS) region of rDNA and the cytochrome c oxidase subunit 1 (COX1) gene were amplified and sequenced (White et al. 1990; Robideau et al. 2011). ITS sequences (GenBank PV077295, PV413032, PV413033) showed 98.6-99.9% identity with P. nicotianae (OR342796.1, KJ494913.1, MT065865.1). COX1 sequences (GenBank PV102957, PV424230, PV424231) matched P. nicotianae (MH760233.1, MT216343.1) with 99.9-100% identity. Pathogenicity of P. nicotianae (PN-1 to PN-3) was confirmed using the mycelial agar plug inoculation method. Briefly, V8 medium containing fresh mycelium was chopped and mixed with vermiculite, and one dish (9 cm diameter) of the mycelium was added to each pot (7 cm upper diameter, 5 cm lower diameter). As a control, sterile V8 medium without mycelium was similarly processed and mixed into vermiculite. Pathogenicity tests for each of the three isolates were conducted in three independent replicates. Seeds of M. polymorpha (cultivar Huaiyang) were soaked in sterilized water for 12 h and then planted in sterilized vermiculite mixed with mycelial pieces (10 seeds/pot). Plants were maintained in a greenhouse at 25°C under a 14 h/10 h light/dark cycle for 14 days. Seed germination commenced four days after planting. Inoculated plants began to exhibit root rot symptoms 10 days post-inoculation, which were morphologically consistent with field-observed symptoms. P. nicotianae was re-isolated from the lesions, while no symptoms were observed in control plants. P. nicotianae has been reported to cause root rot in various crops worldwide, including tobacco (Csinos et al. 2021), citrus (Xiao et al. 2023) and pineapple (Shen et al. 2013). Our results confirm that P. nicotianae is the causal agent of root rot on M. polymorpha. To our knowledge, this is the first report of P. nicotianae causing root rot on M. polymorpha. This report will help identify M. polymorpha root rot disease based on field symptoms and provide a basis for disease management strategies in the region.