Fitness cost and molecular regulation analysis of thifluzamide resistance in Rhizoctonia solani AG-5 inciting maize banded leaf and sheath blight in Shanxi Province of China

Rhizoctonia solani anastomosis group (AG)-5 is the predominant pathogen inciting maize banded leaf and sheath blight in Shanxi province, China, for which the sensitivity baseline to thifluzamide has been established. However, no risk assessment of thifluzamide resistance in R. solani AG-5 has been conducted. Consequently, thifluzamide-resistant mutants of R. solani AG-5 were generated by combined UV irradiation and fungicide selection in the laboratory. Fitness, genetic stability, intracellular structures, and fitness-related differentially expressed genes were subsequently analyzed. Ten resistant mutants were obtained, all of which exhibited moderate-to-high resistance that remained stable for 20 generations. These mutants showed a subtle expansion in their optimal growth temperature and pH ranges as their resistance to thifluzamide increased. Despite this, they exhibited reduced mycelial growth, sclerotia production, melanin formation, and virulence, whereas sclerotia germination rates were higher than in the parental sensitive strain. The mutant Y94.4R maintained vacuole numbers and size post-thifluzamide treatment, whereas the sensitive strain Y94 exhibited vacuole disappearance. Compared to Y94 strain, the expression levels of Atg22, Vps45, Ktr1_3, Aha1, DnaJ, Hikeshi, and Stip1 gene were less upregulated (1.81-3.65 folds) in Y94.4R strain under thifluzamide stress. No amino-acid substitutions were detected in SdhA, SdhB, and SdhD subunits; however, alanine (A) at position 46 of SdhC subunit was replaced by threonine (T) in thifuzamide-resistant mutants. This study showed that the resistant mutants exhibited reduced ecological fitness and competitiveness, suggested that the observed fitness costs might partially support the durability of thifluzamide under field conditions, and provided candidate genes for future disease-management strategies.