Prevalence of Carbendazin Resistance in Field Populations of the Rice False Smut Pathogen Ustilaginoidea virens from Jiangsu, China, Molecular Mechanisms, and Fitness Stability

Rice false smut (RFS), caused by Ustilaginoidea virens, is an important fungal disease of rice. In China, Methyl Benzimidazole Carbamates (MBCs), including carbendazim, are common fungicides used to control RFS and other rice diseases. In this study, resistance of U. virens to carbendazim was monitored for three consecutive years during 2018 to 2020. A total of 321 U. virens isolates collected from Jiangsu Province of China were tested for their sensitivity to carbendazim on PSA. The concentration at which mycelial growth is inhibited by 50% (EC50) of the carbendazim-sensitive isolates was 0.11 to 1.38 mu g/mL, with a mean EC50 value of 0.66 mu g/mL. High level of resistance to carbendazim was detected in 14 out of 321 isolates. The resistance was stable but associated with a fitness penalty. There was a statistically significant and moderate negative correlation (r= -0.74, p < 0.001) in sensitivity between carbendazim and diethofencarb. Analysis of the U. virens genome revealed two potential MBC targets, Uv beta 1Tub and Uv beta 2Tub, that putatively encode beta-tubulin gene. The two beta-tubulin genes in U. virens share 78% amino acid sequence identity, but their function in MBC sensitivity has been unclear. Both genes were identified and sequenced from U. virens sensitive and resistant isolates. It is known that mutations in the beta 2-tubulin gene have been shown to confer resistance to carbendazim in other fungi. However, no mutation was found in the Uv beta 2Tub gene in either resistant or sensitive isolates. Variations including point mutations, non-sense mutations, codon mutations, and frameshift mutations were found in the Uv beta 1Tub gene from the 14 carbendazim-resistant isolates, which have not been reported in other fungi before. Thus, these results indicated that variations of Uv beta 1Tub result in the resistance to carbendazim in field isolates of Ustilaginoidea virens.