Sword bean (Canavalia gladiata DC.), in the legume family Fabaceae, is an annual vine cultivated mainly in central India. The unripe pods are used as a vegetable in Africa and Asia (Ekanayake et al. 2000; Vadivel et al. 1998). Although not grown commercially, it is used as food in south-central Korea (Lee 2003).

In 2018, withering and drying out were observed in sword beans cultivated at the experimental research field of Gyeongsangnam-do Agricultural Research and Extension Services (disease incidence, 10%). The disease mainly affected the stems and pods of sword beans that touched the ground (Fig. 1a). The stems that touched the ground were water-soaked at the start of the infection and rotted, slowly withered, and eventually died (Fig. 1b and c). Numerous small white and brownish circular sclerotia (1 − 3 mm in diameter) formed on the white mycelial mats (Fig. 1d). To date, there have been no reports of sclerotium rot on sword beans elsewhere in the world (Farr and Rossman 2022).

To isolate the causative fungus from diseased sword beans (n = 3), tissues at the boundary between the healthy and lesion parts of the diseased stems were cut into 5 × 5 mm pieces, sterilized with 1% NaOCl for 1 min, and then washed 3 times with sterile distilled water. The pieces were transferred to sterile filter paper, dried, and placed on water agar plates, which were incubated at 25 °C for 3 days, and the edges of the growing mycelia were transferred to potato dextrose agar (PDA) plates and incubated at 25 °C for 4 days. All three fungal isolates (MHGNU F134a–c) were obtained, one from each sample.

The morphological characteristics of the isolates was examined by light microscopy. The colonies observed were white and numerous white sclerotia were observed, which later turned brown and were 1–3 mm in size. For scanning electron microscopy (SEM), 7-day-old PDA cultures were pretreated as described previously (Kwon et al. 2017). Clamp connections of the mycelia were observed and mycelia were 4–9 μm wide (Fig. 1e).

Since disease symptoms were observed on plant parts that were in direct contact with the ground, we performed a pathogenicity test using soil inoculum according to Kwon et al. (2015, 2017). Briefly, 7-day-old PDA cultures of the representative isolate MHGNU F134 were used to prepare a soil inoculum. Harvested mycelium mats containing sclerotia from 10 PDA plates were mixed thoroughly with 1 kg of autoclaved soil and the soil mixture was used as the inoculum. The soil inoculum (200 g) was scattered over topsoil in a plastic box (32 × 26 × 2 cm) and three sword bean pods were placed directly on top. As negative controls, three sword bean pods were placed in plastic boxes containing uninfected soil. All pots were kept separately in a greenhouse and symptom development was observed. Four days after inoculation, the sword bean pods began to rot, and sclerotia formed on the silky white mycelial mats which grew vigorously on the lesions (Fig. 1f). The causative fungus was re-isolated from the lesions of all three inoculated pods to satisfy Koch’s postulates. The morphological characteristics of the fungus re-isolated from the inoculated fruit were identical to those of the original isolate. The negative control remained asymptomatic.

Fig. 1
figure 1

Sword bean showing sclerotium rot and mycological characteristics of the causative fungus, Athelia rolfsii: a infected sword beans became chlorotic and blighted eventually; b stem rot with white mycelia; c fruit rot with a mycelial mat and sclerotia; d sclerotia formed on the lesion; e scanning electron micrograph of a clamp connection (arrow), bar = 5 μm; f pod rot induced 7 days after artificial inoculation

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To identify of the causative fungus, the complete internal transcribed spacer (ITS) rRNA gene region of three isolates (MHGNU F134a–c) was amplified by PCR using the primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) (White et al. 1990). The PCR conditions were a final concentration of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 nM dNTPs, 10 pmol of primer, and 0.1 U of rTaq DNA polymerase (Takara) in a 50 µL reaction volume. Each PCR program consisted of predenaturation (98℃, 2 min), followed by 30 cycles of denaturation (98℃, 30 s), annealing (60℃, 30 s), and extension (72℃, 30 s), with a final extension (72℃, 4 min). The amplified product was electrophoresed on a 0.8% agarose gel, stained with ethidium bromide, and the band was confirmed with a UV transilluminator. The identified band was separated and purified using QIAquick PCR Purification Kit (Qiagen), cloned into the pGEM-T Easy Cloning Vector (Promega), and then sequenced using primers M13F and M13R (Macrogen). The ITS rRNA (684 bp) sequences of the three fungal isolates were identical. The sequence of a representative isolate was deposited in GenBank (acc. no. ON129555) and analyzed using BLASTN; it showed 100% homology with Athelia rolfsii. We performed the neighbor joining method (NJ) using MEGA ver. 6.0 (http://www.megasoftware.net/mega4/mega41.html) (Tamura et al. 2013) together with the sequences of related fungal strains downloaded from GenBank. Sclerotium cepivorum was used as an outgroup (Mahadevakumar et al. 2015; Kwon et al. 2017). The Tamura − Nei substitution model and nearest neighbor-interchange search options with 1000 bootstrap replicates were used. In previous studies, the ITS sequences of A. rolfsii and S. delphinii strains formed three clusters, designated ITS-RFLP subtypes r-1, r-2, and r-3, based on the RsaI restriction sites in the phylogenetic tree (Harlton et al. 1995; Okabe and Matsumoto 2003). Cluster r-1 consisted of both S. delphinii and A. rolfsii strains, cluster r-2 included only A. rolfsii strains and cluster r-3 contained S. delphinii strains (Okabe and Matsumoto 2003). In the phylogenetic tree, the fungus isolated from sword beans was in a clade r-1 containing the reference strains of A. rolfsii and S. delphinii (Fig. 2). In addition to phylogenetic analysis, we performed the A. rolfsii-specific PCR analysis using forward primer SCR-F (5’-CGTAGGTGAACCTGCGGA-3’) and reverse primer SCR-R (5’-CATACAAGCTAGAATCCC-3’) according to a previous report (Jeeva et al. 2010). The 540-bp PCR amplicons corresponding to A. rolfsii-specific amplification were produced in all three isolates MHGNU F134a–c (Fig. 3).

Fig. 2
figure 2

Phylogenetic tree of the ITS region of Athelia rolfsii and Sclerotium species strains constructed by neighbor joining method according to a previous study (Okabe and Matsumoto 2003); the tree was rooted with Sclerotium cepivorum. The Tamura − Nei substitution model and nearest neighbor-interchange search options with 1000 bootstrap replicates were used. The arrow indicates the ITS region of A. rolfsii isolated in this study. r-1, r-2, and r-3, are ITS types (Harlton et al. 1995)

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Fig. 3
figure 3

Atheilia rolfsii-specific PCR analysis using the SCR-F and SCR-R primers. M:1-kbp ladder, lane 1–3: MHGNU F134a–c isolates of A. rolfsii isolated from sword beans, and lane 4: no DNA template. The arrow indicates the 540-bp PCR amplicons corresponding to A. rolfsii-specific amplification

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The representative A. rolfsii MHGNU F134a isolated from sword beans was registered with the Microbial Bank (KACC 48539) of the Korean Agricultural Culture Collection, National Institute of Agricultural Sciences, Rural Development Administration.

Based on the morphological characteristics, pathogenicity, ITS sequence results, and the species-specific PCR analysis, the causative fungal pathogen was identified as A. rolfsii reported by Mordue (1974). To the best of our knowledge, this is the first report of sclerotium rot caused by A. rolfsii in sword bean plants in the world. This disease is highly dependent on environmental conditions, including warm weather and high humidity (Punja 1985; Punja and Damiani 1996). Recent outbreaks of this disease suggest that A. rolfsii is a threat to sword bean production.