Fungicide Sensitivity of Sclerotinia sclerotiorum: A Thorough Assessment Using Discriminatory Dose, EC50, High-Resolution Melting analysis, and Description of New Point Mutation Associated with Thiophanate-Methyl Resistance

Abstract

Thiophanate-methyl (TM), fluazinam, and procymidone are fungicides extensively used for white mold control of common bean in Brazil. We assessed the sensitivity of Brazilian isolates of Sclerotinia sclerotiorum to these three fungicides using discriminatory doses and concentration that results in 50% mycelial growth inhibition (EC50) values. In total, 282 isolates from the most important production areas were screened and none was resistant to fluazinam or procymidone. The EC50 values varied from 0.003 to 0.007 and from 0.11 to 0.72 μg/ml for fluazinam and procymidone, respectively. One isolate was resistant to TM. The EC50 of the TM-resistant isolate was greater than 100 μg/ml, whereas the EC50 of the sensitive isolates varied from 0.38 to 2.23 μg/ml. The TM-resistant isolate had a L240F mutation in the β-tubulin gene. This is the first report of mutation at codon 240 causing resistance to a benzimidazole fungicide in S. sclerotiorum. The high-resolution melting analysis allowed the distinction of TM-sensitive and -resistant isolates by specific melting peaks and curves. The TM-resistant isolate had mycelial growth, sclerotia production, and aggressiveness comparable with that of the sensitive isolates, indicating that this genotype will likely compete well against sensitive isolates in the field. This study demonstrates that resistance to TM, fluazinam, and procymidone is nonexistent or rare. Resistance management practices should be implemented, however, to delay the spread of TM-resistant genotypes.