First Report of <i>Agroathelia rolfsii</i> Causing Stem Rot on Kersting's Groundnut ( <i>Macrotyloma geocarpum</i> ) in Benin

Kersting's groundnut (Macrotyloma geocarpum) is an opportunity crop cultivated in several West African countries, mainly Benin (Agossou et al. 2023). It is highly prized for its nutritional, cultural and economic value. Despite its potential, this legume is considered a neglected and underutilised species, subject to several constraints, including disease, which is a bottleneck in production. During September and October 2021, stem rot was detected in Kersting's groundnut growing in the central Benin cotton and clay soil zones, with incidence rates of 0.74% and 0.21%, respectively. Stem rot symptoms were mainly observed near the base of the stems. The infected stems exhibited slightly depressed, brown lesions covered with rapidly growing white mycelial mats (Figure 1). Dark brown sclerotia were also observed in these plants. In the laboratory, samples of infected plants were cut into small pieces, disinfected in 0.35% sodium hypochlorite for 1 min, then in 70% alcohol for 1 min, and rinsed three times with sterile distilled water. After air drying, they were placed in Petri dishes containing potato dextrose agar (PDA) medium and incubated at 25°C for 7 days. After this incubation period, white mycelia and sclerotia formed on PDA. Pure cultures were obtained by removing mycelial fragments and transferring them to new Petri dishes containing PDA. The plates were incubated for 5–10 days at 25°C. Sclerotia,1–2 mm in diameter, formed on the fungal colonies and gradually turned from white to dark brown with age (Figure 2). Clamp connections were also observed (Figure 3). For molecular identification of the pathogen, the internal transcribed spacer (ITS) and translation elongation factor 1-α (TEF1) gene from one representative isolate were sequenced using the ITS1/ITS4 (White et al. 1990) and EF1-983F/EF1-2212R primer sets (Rehner and Buckley 2005). BLASTn analysis showed 98.64%–96.70% nucleotide identity to those of the equivalent sequences of Agroathelia rolfsii retrieved from GenBank (ITS: OQ134754, EF1-α: MW322688). The sequences of the isolates from this study have been deposited in GenBank (Accession Nos. PV785768 for ITS and PV798299 for TEF-1α). Phylogenetic analysis using the maximum likelihood method and the Tamura-Nei model with 1000 bootstrap replicates in MEGA 11 revealed that the isolate shared a common clade with the A. rolfsii reference sequence in the GenBank database (Figure 4). Pathogenicity tests were done on 15 plants (6–7 weeks old) under greenhouse conditions (28 ± 2°C and 70% relative humidity). These plants were wounded at the base of their stems using a sterile needle and the plants were inoculated by placing four sclerotia from 15-day-old cultures near the wounds. These plants were finally covered with plastic bags, sprayed with sterile water daily, and kept under greenhouse conditions (28 ± 2°C and 70% relative humidity) with a 12/12 hour light/dark cycle. Seven to ten days after inoculation, all 15 plants were infected, whereas uninoculated plants remained healthy (Figure 5). Worldwide, this pathogen has been reported to cause stem rot in various plants, including Helianthus annuus (Zhang et al. 2023) and Lycopersicon esculentum (Dafny Yelin et al. 2024). In Benin, that pathogen has been observed on cowpea, causing seedling damping off, and crown and stem rot, resulting in yield losses with serious socioeconomic implications (Adandonon 2004). To our knowledge, this is the first report of stem rot caused by A. rolfsii on M. geocarpum in Benin and globally. This study will be beneficial for the prevention and control of stem rot caused by A. rolfsii in the future. This work was supported by funding from the Intra-Africa Academic Mobility Program (GENES) of the European Union to the first author as part of a PhD scholarship to Omar Toure at the University of Abomey-Calavi (Republic of Benin).