Microtiter method to monitor Corynespora cassiicola and sensitivity of the pathogen to carbendazim, prothioconazole and pyraclostrobin

The fungus Corynespora cassiicola causes target spot on soybeans in Brazil and one of the recommended controls for this disease involves the use of fungicides. Here, we evaluated the sensitivity of C. cassiicola isolates to fungicides, comparing the effective concentrations required to inhibit 50% of fungal growth (EC50) using a colorimetric microtiter method, with the EC50 determined by mycelial growth inhibition in a fungicide-amended medium obtained by Xavier et al. (2013), for 16 isolates for carbendazim and prothioconazole. The correlation between EC50 values for both methods were 0.90 and 0.88 for carbendazim and prothioconazole, respectively. After this, the sensitivities of 134 C. cassiicola isolates collected in soybean fields in Paran?a (PR) and Mato Grosso (MT) in the 2012/13 and 2013/14 crop seasons to carbendazim, prothioconazole, and pyraclostrobin fungicides were evaluated using microtiter method. The spores of the isolates were diluted in Yeast Bacto Acetate culture medium and added to fungicide solutions at concentrations of zero; 0.0032; 0.016; 0.08; 0.4; 2; 10; and 50 ?g mL-1. A microplate reader with a 540 nm wavelength was used to estimate the EC50 values. Isolates showed growth inhibition at concentrations higher than 50 ?g mL-1, showing a high frequency of resistant isolates, in total, 67% of the isolates from PR and 88% from MT (carbendazim); 99% from PR and 82% from MT (pyraclostrobin); and 11% from PR and 9% from MT (prothioconazole). The results showed that the lower control efficiency for target spot in soybeans in PR and MT to MBC and QoI fungicides could be associated with the presence of resistant isolates.