First report of <i>Fusarium incarnatum</i> causing leaf spot of fenugreek in Iran

Fenugreek (Trigonella foenum-graecum) is an annual herbaceous plant belonging to the Fabaceae family. In Iran, fenugreek is an important edible and medicinal leafy vegetable with a long history of cultivation. In December 2024, a leaf spot disease was observed on five fenugreek farms in Hormozgan province, Iran. The disease incidence varied from 5 to 20% amongst the farms. The spots were circular, 5–10 mm in diameter, and dark brown to black in colour which developed over the leaf surface and caused blight symptoms (Fig. 1). To determine the causal agent, leaf pieces of 5 × 5 mm were taken from the border between healthy and necrotic tissues, surface sterilised with 1% sodium hypochlorite for five minutes, rinsed thrice with sterile distilled water, placed on potato dextrose agar (PDA) and incubated at 27°C for one week. Fusarium colonies were predominantly isolated from the pieces. On PDA, the colonies grew rapidly and produced abundant dense aerial mycelia that initially were off-white and became beige with age. Light orange sporodochia were produced by some isolates. Macroconidia were relatively slender with a curved dorsal surface and a straighter ventral surface, measuring 10–14 µm (Fig. 2) (Leslie & Summerell, 2006). Total genomic DNA of three representative isolates (FLS14, FLS24 and FLS84) was extracted from seven-day-old cultures and ITS1/ITS4, EF1/EF2 and 5f2/7cr primers were used for PCR amplification and Sanger sequencing of the internal transcribed spacer region (ITS1–5.8S-ITS2), translation elongation factor-1 alpha gene (tef-1α) and RNA polymerase second largest subunit (rpb2) (White et al., 1990; O'Donnell et al., 1998; Lombard et al., 2019). The ITS (Genbank Accession No. PQ897996, PQ897997 and PQ897998), tef-1α (PQ902079, PQ902080 and PQ902081) and rpb2 (PV602127, PV602128 and PV602129) sequences had 99–100% identity to F. incarnatum strains in GenBank (GQ505769, MN170410, GQ505793 and GQ505794). The phylogenetic analyses of the aligned sequences were carried out using the software MrBayes v. 3.2.1. Based on Bayesian inference of combined ITS, tef-1α and rpb2 sequences, the isolates obtained in this study were identified as F. incarnatum (Fig. 3). To confirm pathogenicity, 30 healthy 25-day-old fenugreek seedlings grown in 15-cm diameter plastic pots containing an autoclaved peat/soil mixture were inoculated through a foliar spray application with conidial suspensions (1 × 106 conidia ml−1) prepared from the F. incarnatum isolates. Control plants were treated with sterile distilled water. All plants were kept in a greenhouse at 30 ±2°C with a relative humidity of 55%. The experiments were conducted twice with four replicates. Inoculation led to the appearance of small, brown lesions, measuring 2–5 mm in diameter which became more widespread and coalesced with each other. No disease symptoms appeared on the leaves of the control seedlings (Fig. 4). Fusarium incarnatum was re-isolated only from symptomatic leaves. Recent reports describe F. incarnatum infecting foxtail millet and muskmelon in China (Cao et al., 2019; Zheng et al., 2019) and Cucumis trigonus in India (Mali et al., 2015). To our knowledge, this is the first confirmed report of F. incarnatum on fenugreek worldwide.