First Report of Macrophomina phaseolina Causing Leaf Spot of Kersting's Groundnut [Macrotyloma geocarpum (Harms) Maréchal et Baudet] in Benin.

Kersting's groundnut [Macrotyloma geocarpum (Harms) Maréchal et Baudet] is a neglected and underutilized nutrient-rich crop grown in Benin and in other West African countries. During a survey of Kersting's groundnut production areas between September and October 2021, leaf spot disease was observed in the West Atacora, Central Benin cotton, and clay soil zones with incidence values of 12%, 7%, and 5.4%, respectively. These leaf spots were present on young plants and became more severe as the plants grew older. Symptoms appeared as small round, brown spots surrounded by yellow halos. The center of these spots was light brown and visible on the surfaces of both the lower and upper leaves. As the disease worsened, the halo became less visible. The infected tissues were cut into small pieces, disinfected in 0.35% sodium hypochlorite for 1 min, followed by 70% alcohol for 1 min, and rinsed thrice with sterile distilled water. Each sample with 2-5 mm2 of leaf area placed in a Petri dish containing Potato Dextrose Agar (PDA). The Petri dishes were then incubated for 72h at 25°C. Pure cultures of the isolated fungus were obtained by removing the mycelial fragments and transferring them to new Petri dishes containing PDA. The Petri dishes were then incubated for 5-7 days at 25°C. Initially, the colonies looked whitish gray, but as the culture progressed, the color of the fungal colonies darkened. Thirty sclerotia from a representative isolate measured 45-165 μm in length x 35-103 μm in width (average), and the isolated fungus was identified as Macrophomina phaseolina. Conidia are unicellular, hyaline, and cylindrical. For further identification, the DNA regions of the internal transcribed spacer (ITS), translation elongation factor 1-α (TEF1) region, and partial β-tubulin (TUB) gene from one representative isolate were sequenced using the primer sets ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and T1/T22 (O'Donnell and Cigelnik, 1997). These sequences have been deposited in GenBank (accession numbers OR064031 for ITS, PV344496 for TEF-1α, and PV155704 for β-tubulin). BLAST in the NCBI database showed 99.61%, 97.50%, and 99.62% identity with M. phaseolina extracted from GenBank (ITS: OR501533; TEF-1α: KF531804; and TUB: MW592282). A phylogenetic tree was constructed using the maximum likelihood method and Tamura-Nei model with 1000 bootstrap replicates in MEGA 11, which showed that the isolate belonged to the same clade as M. phaseolina. To confirm the pathogenicity of the fungus, surface of the medium containing the mycelium and conidia of M. phaseolina was dissolved in sterile distilled water and adjusted to a concentration of 2.10⁴ conidia/ml. Kersting's groundnut seeds were soaked in this conidial suspension for 1 h before being dried in the shade and sown, whereas the controls were soaked in sterile distilled water. After flowering, typical leaf spot symptoms were observed in all artificially inoculated Kersting's groundnut plants. In contrast, the control plants showed no symptoms. The fungi were systematically re-isolated from the artificially developed symptoms. M. phaseolina has been reported to cause leaf spots on Vigna radiata in Australia (Fuhlbohm et al. 1996) and on Artocarpus hirsutustus in India (Prakash et al. 2007). In Benin, it has been reported on cowpea (Afouda et al. 2009). In addition, in Benin and Nigeria, Msikita et al. (1998) reported the disease as causing pre-harvest root rot in cassava. To our knowledge, this is the first report of Macrophomina phaseolina causing leaf spots on Kersting's groundnut in Benin and worldwide. This soil-borne fungus is responsible for a multitude of plant diseases that affect a range of economically significant species, including mung beans, soybeans, chickpeas, cotton, sunflowers, and maize. Yield losses of up to 70% have been reported in the semi-arid regions of Kenya in the common bean (Songa and Hillocks 1996). This identification could provide useful information for developing effective methods for controlling the disease.