Occurrence and Characterization of Verticillium alfalfae Causing Alfalfa Verticillium Wilt in Inner Mongolia, China, with Preliminary Fungicide Sensitivity Assessment

Alfalfa Verticillium wilt, caused by Verticillium alfalfae, is a globally significant disease with increasing incidence and expanding epidemic areas. This study surveyed six major alfalfa-producing regions in Inner Mongolia, China-Chifeng, Tongliao, Ulanqab, Ordos, Bayannur, and Hohhot-and successfully isolated V. alfalfae exclusively from samples collected in Hohhot and Bayannur. Based on morphological characterization, multi-locus phylogenetic analysis (act, tef1-α, gapdh, and ts genes), and pathogenicity tests fulfilling Koch's postulates, all 33 isolates were consistently identified as V. alfalfae, with disease severity levels ranging from 3.04 to 4.79 on the susceptible cultivar Zhongmu No. 1. As a preliminary assessment, the in vitro sensitivity of a representative strain, Va8, to eight commercial fungicides was evaluated using the mycelial growth inhibition method. Among the tested fungicides, 30% difenoconazole-propiconazole exhibited the strongest inhibitory effect (EC₅₀ = 0.14 μg/mL), followed by 10% trifloxystrobin & 20% tebuconazole (EC₅₀ = 0.20 μg/mL). However, given the substantial virulence variation observed among isolates, these sensitivity data should be interpreted with caution, as population-level differences may exist. These findings represent the first confirmed report of V. alfalfae in Inner Mongolia and provide a preliminary yet critical reference for prioritizing candidate fungicides for future multi-isolate and field evaluations.