Selection Pressure Pathways and Mechanisms of Resistance to the Demethylation Inhibitor-Difenoconazole in Penicillium expansum

Abstract

Penicillium expansum causes blue mold, the most economically important postharvest disease of pome fruit worldwide. Beside sanitation practices, the disease is managed through fungicide applications at harvest. Difenoconazole (DIF) is a new demethylation inhibitor (DMI) fungicide registered recently to manage postharvest diseases of pome fruit. Herein, we evaluated the sensitivity of 130 P. expansum baseline isolates never exposed to DIF and determined the effective concentration (EC50) necessary to inhibit 50% germination, germ tube length, and mycelial growth. The respective mean EC50 values of 0.32, 0.26, and 0.18 μg/ml indicate a high sensitivity of P. expansum baseline isolates to DIF. We also found full and extended control efficacy in vivo after 6 months of storage at 1°C. We conducted a risk assessment for DIF-resistance development using ultraviolet excitation combined with or without DIF-selection pressure to generate and characterize lab mutants. Fifteen DIF-resistant mutants were selected and showed EC50 values of 0.92 to 1.4 μg/ml and 1.7 to 3.8 μg/ml without and with a DIF selection pressure, respectively. Resistance to DIF was stable in vitro over a 10-week period without selection pressure. Alignment of the full CYP51 gene sequences from the three wild-type and 15 mutant isolates revealed a tyrosine to phenylalanine mutation at codon 126 (Y126F) in all of the 15 mutants but not in the wild-type parental isolates. Resistance factors increased 5 to 15-fold in the mutants compared to the wild-type-isolates. DIF-resistant mutants also displayed enhanced CYP51 expression by 2 to 14-fold and was positively correlated with the EC50 values (R2 = 0.8264). Cross resistance between DIF and fludioxonil, the mixing-partner in the commercial product, was not observed. Our findings suggest P. expansum resistance to DIF is likely to emerge in commercial packinghouse when used frequently. Future studies will determine whether resistance to DIF is qualitative or quantitative which will be determinant in the speed at which resistance will develop and spread in commercial packinghouses and to develop appropriate strategies to extend the lifespan of this new fungicide.

CD74 knockout protects against LPS‐induced myocardial contractile dysfunction through AMPK‐Skp2‐SUV39H1‐mediated demethylation of BCLB

“…In vitro study was then performed to examine the cell signalling mechanism in cardiomyocyte autophagy and contractile response upon LPS challenge in CD74 knockout mice. Neonatal murine cardiomyocytes from WT and Cd74 −/− mice were transfected with an adenovirus expressing GFP‐LC3 fusion protein for 24 hr prior to exposure of LPS (4 μg·ml −1 ) for 6 hr in the presence or absence of the AMPK activator AICAR (500 μM; Nyblom et al, ), the autophagy inducer rapamycin (5 μM; Yuan et al, ), the methylation inhibitor 5‐AzaC (10 μM; Liu et al, ), the demethylation inhibitor DIF (0.5 μg·ml −1 ; Ali & Amiri, ) or the SUV39H1 inhibitor chaetocin (50 nM; Yang et al, ). Data shown in Figure that LPS stimulated autophagosome formation was abolished by CD74 ablation.…”

“…Cardiomyocyte yield was approximately 65–75% which was unaffected by CD74 KO or LPS challenge. To assess the role of AMPK, autophagy, SUV39H1 and autophagy protein methylation in LPS‐induced cardiomyocyte dysfunction, cardiomyocytes from WT and Cd74 −/− mice were exposed to LPS (4 μg·ml −1 ; Ceylan‐Isik et al, ) for 6 hr in the presence or absence of the AMPK activator AICAR (500 μM; Nyblom, Sargsyan, & Bergsten, ), the autophagy inducer rapamycin (5 μM; Yuan et al, ), the methylation inhibitor 5‐aza‐20‐deoxycytidine (azacytidine or 5‐AzaC,) 10 μM; Liu et al, ; Onay, Yapici Eser, Ulasoglu Yildiz, Aslan, & Tali, ), the demethylation inhibitor difenoconazole (DIF, 0.5 μg·ml −1 ; Ali & Amiri, ), or the SUV39H1 (suppressor of variegation 3‐9 homolog 1) inhibitor chaetocin (50 nM; Yang et al, ) prior to mechanical assessment.…”

“…Fluorescence was calibrated using InSpeck microspheres (Molecular Probes) and was quantitated using an ImagePro analysis software (Media Cybernetics, Silver Spring, MD; Wang et al, ). To determine the cause–effect role of O 2 − production in various cell signalling pathways‐mediated cardiac contractile responses, cardiomyocytes from WT and Cd74 −/− mice were exposed to LPS (4 μg·ml −1 ; Ceylan‐Isik et al, ) for 6 hr in the presence or absence of the AMPK activator AICAR (500 μM; Nyblom et al, ), the autophagy inducer rapamycin (5 μM; Yuan et al, ), the methylation inhibitor 5‐aza‐20‐deoxycytidine (5‐AzaC, 10 μM; Liu et al, ; Onay et al, ), the demethylation inhibitor difenoconazole (DIF, 0.5 μg·ml −1 ; Ali & Amiri, ), or the SUV39H1 inhibitor chaetocin (50 nM; Yang et al, ) prior to the loading with DHE (5 μM) at 37°C for 30 min. Cardiomyocytes were then rinsed, and DHE fluorescence intensity was measured using a fluorescent microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 530 nm.…”

“…NCM were transfected with GFP‐LC3 adenovirus for 24 hr (Wang, Ge, et al, ) and were treated with LPS (4 μg·ml −1 ) at 37°C for 24 hr in the absence or presence of the AMPK activator AICAR (500 μM; Nyblom et al, ), the autophagy inducer rapamycin (5 μM; Yuan et al, ), the methylation inhibitor 5‐AzaC (10 μM; Liu et al, ; Onay et al, ), the demethylation inhibitor DIF (0.5 μg·ml −1 ; Ali & Amiri, ) or the SUV39H1 inhibitor chaetocin (50 nM; Yang et al, ). After treatment, cells were gently rinsed with PBS three times before being fixed in 4% paraformaldehyde (20 min at room temperature).…”

Characterization of difenoconazole resistance in Lasiodiplodia theobromae from papaya in Brazil

“… In that pathogen, expression could be induced with DMI fungicide exposure prior to RNA extraction . The positive correlation of CYP51 ‐expression was also observed in difenoconazole‐resistant and ‐sensitive Penicillium expansum , a causal agent of blue mold in pome fruit . In many other fungi, promoter sequences or other insertion sequences upstream of CYP51 are associated with overexpression, however, such variation was not identified upstream of Lt CYP51 in our isolates.…”