Gray mold in strawberries in the Paraná state of Brazil is caused by Botrytis cinerea and its isolates exhibit multiple-fungicide resistance

The objectives of this work were to investigate the Botrytis species that cause gray mold in strawberries in Paraná and to study their sensitivity to fungicides: procymidone (P), iprodione (I), boscalid (B), thiophanate-methyl (T), fluazinam (F), azoxystrobin (A) and difenoconazole (D). The identification of 150 isolates of Botrytis sp. was carried out by amplifying the DNA by PCR using specific primers. The sensitivity to fungicides was determined by in vitro assays with discriminatory doses and EC₅₀ (effective concentration to inhibit 50% of the growth of the fungus), molecular characterization of mutations and ex vivo assays for disease control efficacy. Thirty phenotypes were selected from combinations (S-sensitive and R-resistant) for the EC₅₀ studies. Molecular characterization was performed using specific primers for mutations in the cyt b, bos1 and β-tubulin genes to confirm the resistance genotype. Tests were carried out with fruit treated with the fungicides (P), (B), (F) and (A) x the different phenotypes. All isolates were confirmed as Botrytis cinerea. Forty-one isolates were classified as fungicide sensitive phenotypes and of these, six isolates showed resistance to all seven tested fungicides. The percentage of isolates resistant to the fungicides azoxystrobin, boscalid, difenoconazole, fluazinam, iprodione and procymidone was 90.7; 45.3; 33.3; 32.7; 44.0 and 43.3%, respectively. A total of 94.6% isolates were classified as highly resistant to thiophanate-methyl. The EC₅₀ for the fungicides procymidone, fluazinam and boscalid ranged from less than 0.1 to greater than 100 μg/ml. For azoxystrobin, most isolates had an EC₅₀ greater than 100 μg/ml. Isolates with more than one mutation for different fungicides were found. For the assay in fruits with infections from the field, only the fungicide fluazinam differed from the control treatment, showing lower final incidence of 33.3% and control efficiency of 55.6%. For the assay with inoculated fruits, the fungicide fluazinam best controlled the disease for the three isolates presenting an average final incidence of 33.3% and an average control efficiency of 61.9%. Therefore, disease management in the state of Paraná should be reviewed in terms of registering new fungicides to gray mold and the use of integrated management measures to reduce the reliance on fungicides.