First report of Fusarium luffae causing soybean wilt in the USA.

During the 2023 soybean growing season in South Dakota, we scouted a farmer's field and observed soybean (Glycine max (L.) Merr.) plants with wilting symptoms and blighted leaves. Symptomatic stems and leaves were collected from the field to identify associated pathogens. 0.5 cm² size leaf and stem segments of the sample were surface sterilized by rinsing with 10% bleach for 5 minutes then dipping in 70% ethanol for one minute, and later placing in deionized sterile water for one minute. The sterilized segments were placed on wet filter paper and incubated under fluorescent light for three days. Fungal growth was observed, and the growing mycelia were transferred to potato dextrose agar plates amended with 50 µg/ml Ampicillin (PDA_a). Pure culture of the isolate was obtained using single sporing and transferring on new PDA_a plates. A dense aerial mycelial growth showing waxy yellow color with a pale orange tinge on the rear side covered the full plate after seven days of incubation at room temperature under fluorescent lights (Figure S1a and b). Developing macroconidia were falcate, curved, smooth to slightly rough, and hyaline with three-five septa (Figure S1c). For molecular identification, DNA of the recovered isolate was extracted and subjected to multiloci PCR (O'Donnell et al., 2010) to amplify and Sanger sequence the internal transcribed spacers region (ITS) (GenBank accession number PP393518), calmodulin (CAM-PP401978), RNA polymerase II second largest subunit (RPB2-PP401980), and translation elongation factor 1-α gene (TEF1-PP401979). The South Dakota isolate (SLSDF2) was identified as Fusairum luffae on NCBI and Fusarioid polyphasic identification databases with 99.40% similarity to Fusarium luffae strain NRRL31167. A phylogeny was inferred based on concatenated TEF1, RPB2, and CAM sequences to show species relatedness (Figure S3). The characterized isolate SDSLF2 was evaluated for soybean pathogenicity using spray inoculations on detached leaves and V₂ stage soybean plants (Figure S2a and b). The conidial suspension was prepared by growing the pathogen on mung bean agar for seven days. 2 ml of conidial suspensions (2.6 × 10⁴ conidia/ml) and mock control (sterilized water with 0.1% Tween-20) was sprayed on the detached leaves and whole plants. The experiment was repeated three times with four replicates in each. In the detached leaf assay, leaves were completely blighted (Figure S2a) within 96 hours. In whole plant assays, after two days of incubation, leaf blighting was visible and progressed with time. Four days post-inoculation, the infected plants showed extensive leaf symptoms, and ultimately defoliation occurred (Figure S2b). No symptoms were observed in mock controls of either of the experiments. The pathogen was reisolated from the infected tissues and its identity was confirmed as F. luffae by CAM sequencing fulfilling Koch's postulates. F. luffae has been reported to associated with soybeans in China (Zhao et al., 2022), however, to our knowledge, this is the first report of F. luffae pathogenic on soybeans in the USA, stressing the need to identify resistance sources to avoid any potential disease epidemic.