First report of Alfalfa root rot caused by Fusarium commune in China.

Abstract

Alfalfa (Medicago sativa) is the most important perennial forage legume worldwide, with high yields and nutrient quality. Diseases are significant threats to alfalfa and cause substantial yield losses (Fang et al. 2021). In March 2022, a disease with symptoms similar to fusarium wilt was observed in an alfalfa (cultivar Aurora) field in Baima teaching and research base (119°18'07″E, 31°61'47″N) in Nanjing, China. Reddish-brown discoloration of the roots, stele, basal stems and the withering symptoms on the aerial portions are the specific symptoms of fusarium wilt. The disease incidence varied from 3 to 6% in around 0.3 hectares of alfalfa fields surveyed. Fresh tissues from symptomatic alfalfa were cut into pieces (3-5 mm) and surface sterilized with 75% ethanol for 30 seconds, followed by 1% sodium hypochlorite (NaClO) for 120 seconds, and three rinses with sterile distilled water. Tissue pieces were placed on selective potato dextrose agar (PDA) containing 50 mg/L rifampicin and ampicillin. Plates were sealed and incubated at 25°C for 48 to 72 hours. Over 70 tissue fragments plated, 20 isolates from different fragments showed similar colonies. These isolates were purified by single spore isolation and grown on PDA and mung bean medium for morphology identification, molecular identification and pathogenicity test. The colony showed white to pink, abundant, densely aerial mycelium, while its backside was light violet. Macroconidia were hyaline, falcate with septa, and ranged in size from 25.5 to 61.5 × 3.8 to 6.2 μm (n=50). Microconidia were non-septate, hyaline, oval, straight to slightly curved, and were 5.6 to 10.7 × 2.4 to 3.2 μm (n=50). Spherical chlamydospores were 10.6 ± 1.3 μm in size. The rDNA internal transcribed spacers (ITS), RNA polymerase II subunit 2 (RPB2) and translation elongation factor 1-α (TEF) genes were amplified and sequenced (White et al. 1990; Carbone et al. 1999; Wang et al. 2019). Blast analysis showed 99.41% to 100% identity to F. commune sequences in the GenBank database (KU341324.1, MN892350.1, MH341219.1). The pathogenicity test was conducted using the dipping method in roots. Fresh F. commune hyphae were cut into 3 x 3mm agar plugs from a 7 cm PDA plate and inoculated in 200 mL mung bean medium on a shaker at 160 rpm, 25°C for 5 days. Spores were filtered through a cheese cloth, adjusted to 1 × 106 spores/mL with distilled water and used immediately. Roots in two-week-old alfalfa seedlings were soaked in spore suspension for 30 mins before being transplanted to sterile vermiculate with four repeats, while those in sterile water were used as non-inoculated control. The plants were placed in the green house at 25°C with 16 h of light and 8 h of darkness. The symptoms were observed two weeks following the inoculation. 30% of the seedlings withered, and reddish-brown discoloration symptoms were visible on the roots and stem of the unwithered plant. F. commune was isolated from these lesions again, and no disease symptoms were observed in the control plant. F. commune has been reported to cause root and stem rot in many plants worldwide, including tobacco (Zhong et al. 2021), maize (Mezzalama et al. 2021; Xi et al. 2019), soybean (Detranaltes et al. 2022) and sugarcane (Wang et al. 2018). Our results show that F. commune was the causative agent of alfalfa root rot. To our knowledge, this is the first report of F. commune causing root rot in alfalfa. The finding provides insights for disease diagnosis of alfalfa root rot disease and management in the region.