First report of Fusarium clavum causing root rot on foxtail millet in Gansu, China

Foxtail millet (Setaria italica), a nutritious, drought-resistant crop, originated from the Yellow River Basin in China (Jia and Diao 2022). In August 2023, root rot symptoms were observed on foxtail millet in Zhongchuan and Zhaijiasuo towns, Huining County, Gansu, with an incidence rate of approximately 10%. The diseased plants exhibited gray-white mold on the stem nodes, with dark brown necrosis in the vascular tissues. The stem bases and roots were cut into small pieces (5 mm × 5 mm × 5 mm) that included both infected and healthy tissues and surface sterilized with 75% ethanol for 2 min, followed by 10% sodium hypochlorite for 5 min, then rinsed three times in sterilized water. Finally, these small pieces were inoculated on a potato dextrose agar (PDA) medium for culture. Grown fungal colonies were inoculated onto PDA plates, and isolates HNZCGF-2 and HNZJSGF-3 were obtained using the single-spore isolation technique. The isolates were cultured on PDA at 25°C for 5 days for morphological observation. Colonies reached 80–90 mm in diameter, characterized bywhite to cream-colored, floccose aerial mycelium. Conidia were sickle-shaped, with an elongated apical cell and a more pronounced basal cell heel, containing three to seven transverse septa, and in a size range of 20.8-39.2 × 4.1-6.7 µm (mean 30.3 × 5.2 µm, n = 50). Several tandem, yellowish-brown, spherical, and smooth to rough chlamydospores were formed on the aerial hyphae. These morphological features are consistent with those of Fusarium incarnatum-equiseti species complex, as described by O’Donnell et al. (2009). The mycelia were collected to extract genomic DNA using the CTAB method (Rogers and Bendich 1994). Amplification of the RNA polymerase II second largest subunit (RPB2) and calmodulin (CAM) genes employed primers RPB2-5f2/RPB2-7cr and CL1/CL2A, respectively. The obtained sequences were compared to those in the GenBank database using BLASTn analysis. The RPB2 and CAM sequences from two isolates showed more than 99% homology with sequences of Fusarium clavum. Sequence data were deposited in GenBank under accession numbers PX577122 to PX577123 for RPB2 and PX577124 to PX577125 for CAM. The aligned sequences were concatenated in the order of RPB2 and CAM using Phylosuite v1.2.1, and substitution models and optimal partitioning strategies were determined by PartitionFinder2 for maximum likelihood analysis. The phylogenetic tree showed that isolates HNZCGF-2 and HNZJSGF-3 were clustered in the same clade with F. clavum. When seedlings (cultivar Yingu 2) reached the 4–5 leaf stage, the non-wounded roots were inoculated with HNZCGF-2 and HNZJSGF-3 conidial suspensions at 1×105 conidia/mL using the root irrigation method. Each plant received 3 mL, with twenty plants per pot and three pots per treatment (isolate HNZCGF-2, isolate HNZJSGF-3, and a sterile water control). After inoculation, all plants were maintained in a greenhouse at 25°C for observation. Within 7 days post-inoculation, both isolates induced wilt symptoms in approximately 90% of inoculated plants, including black-brown discoloration, rot, and brittleness at the stem bases and roots, accompanied by leaf yellowing and wilting. The pathogen reisolated from symptomatic roots was morphologically and molecularly identical to the original isolate. All control plants remained asymptomatic, and no pathogen was reisolated. In this study, the isolates HNZCGF-2 and HNZJSGF-3 caused root rot of foxtail millet, which was identified as F. clavum by morphological characteristics, molecular biology, and pathogenicity. Fusarium nygamai and Fusarium oxysporum have been reported to cause root rot in foxtail millet (Xu et al, 2023; Du et al, 2024). This is the first report of F. clavum causing root rot on foxtail millet.