CiOs1, CiOs4 and CiOs5 modulate temperature-dependent growth and fludioxonil resistance in Calonectria ilicicola, causing soybean red crown rot.

Calonectria ilicicola causes soybean red crown rot (RCR), along with premature leaf aging, early defoliation, and necrosis, leading to seriously losses of yield and quality worldwide. Fludioxonil (Flu), a phenylpyrrole fungicide with a broad spectrum and high activity against phytopathogenic fungi. In this study, baseline sensitivity of 100 isolates collected from main soybean production areas to Flu was determined in vitro. The result showed that the EC values of the 100 isolates ranged from 0.071 to 0.20 μg/mL. A total of 15 fludioxonil-resistance (Flu) mutants were in-vitro domesticated from 5 fludioxonil sensitive (Flu) isolates, and the resistance factors (RF) of 15 Flu mutants were > 400. All the Flu mutants exhibited fitness penalty in conidiation, virulence, osmo-adaptation. Additionally, positive cross-resistance pattern was observed between fludioxonil and iprodione, but not pydiflumetofen, tebuconazole or fluazinam. Furthermore, sequence alignment results exhibited that four mutation types in CiOs1, CiOs4 and CiOs5 were classified in the Flu mutants. To reveal the biological roles of four genes (CiOs1 ∼ CiOs2 & CiOs4 ∼ CiOs5), all the single gene deletion mutants for four above-described genes exhibited reduced vegetative growth, conidiation, virulence, sensitivity against Flu, and increased sensitivity to the osmotic agent NaCl. Intriguingly, all the single gene deletion mutants shared the same shifted temperature-sensitivity regulation pattern on fungal mycelial linear growth at 20 °C when cultivated on the V8 medium at 4 °C ∼ 37 °C. Taken together, this study is first reported that baseline sensitivity of C. ilicicola to Flu, and biological and molecular characterizations of Flu mutants, providing further understanding of fludioxonil for controlling RCR caused by C. ilicicola.