Development of an Assessment Method for Host Resistance of Alfalfa to Root Rot Caused by <i>Rhizoctonia solani</i>

ABSTRACT Alfalfa ( Medicago sativa ) is the most important forage legume crop in China. Root rot disease caused by Rhizoctonia solani is associated with severe yield and economic losses in alfalfa. Using alfalfa varieties with resistance is considered the most effective and sustainable way to manage the disease in alfalfa. However, there is still a lack of effective methods to evaluate the host reactions of alfalfa to R. solani . Ensuring consistent and effective disease assessment through visual estimation of host resistance is often challenging when studying soil‐borne plant pathogen infections. This study firstly developed an assessment method based on the treatment of inoculum density at 0.1%, 0.25%, and 0.5% (w/w), inoculation methods of millet seed‐based and mycelial suspension inoculums, and seedling ages at two and eight weeks. Nine alfalfa varieties were then used to confirm the assessment method. Results showed that the millet‐seed‐based inoculation method, combined with a 0.5% inoculum density, resulted in the highest disease severity of 78% in roots and 70% in shoots, with a biomass reduction of 86% in roots and 74% in shoots. Seedlings inoculated two weeks post‐germination exhibited higher susceptibility than those inoculated eight weeks post‐germination. The selected inoculation method, inoculum density and seedling age effectively differentiated the resistance of alfalfa varieties, with Gannong 9 and WL366 exhibiting strong resistance to R. solani , while Gongnong 1 and Common were the most susceptible. Besides, both harvesting time points (four weeks and eight weeks after pathogen inoculation) effectively differentiated the resistance levels among varieties. The method includes preparing millet‐seed‐based inoculum, infecting two‐week‐old alfalfa seedlings with 0.5% inoculum density, and harvesting at four or eight weeks by assessing disease severity and plant growth parameters. This study provides a method for evaluating the host resistance of alfalfa to R. solani under controlled environmental conditions with the aim to screen and breed alfalfa varieties with resistance for effective management of root rot.