Diversity in species composition and fungicide resistance profiles in Colletotrichum isolates from apples

Outbreaks of bitter rot were observed in three commercial apple orchards in Illinois despite best management efforts during the 2018 production season. Three isolates from symptomatic fruit from these orchards and two isolates from an orchard in South Carolina were identified to the species level using morphological tools and calmodulin, glyceraldehyde-3-phosphate dehydrogenase, and beta-tubulin gene sequences. The isolates from Illinois were identified as Colletotrichum siamense of the Colletotrichum gloeosporioides species complex and the ones from South Carolina as Colletotrichum fioriniae and Colletotrichum fructicola of the Colletotrichum acutatum and the C. gloeosporioides species complex, respectively. Two of the three C. siamense isolates from Illinois were resistant to azoxystrobin and thiophanate-methyl as determined in mycelial growth tests in vitro. EC₅₀ values were >100 μg/ml for both fungicides. One isolate was only resistant to azoxystrobin. None of the isolates from South Carolina was resistant to either of the two compounds. All five isolates were sensitive to fludioxonil (EC₅₀ values <0.1 μg/ml), propiconazole (EC₅₀ values ranged from 0.15 to 0.36 μg/ml), and benzovindiflupyr (EC₅₀ values ranged from <0.1 to 0.33 μg/ml). Resistance in C. siamense to azoxystrobin and thiophanate-methyl was confirmed in detached fruit studies using apples treated with label rates of registered product. Resistance to thiophanate-methyl in C. siamense was based on E198A mutation in b-tubulin gene, whereas resistance to azoxystrobin was based on G143A in cytochrome b (CYTB). One isolate resistant to azoxystrobin possessed no amino acid variation in CYTB. This study shows that quinone outside inhibitor fungicide resistance in Colletotrichum from apple has emerged and is being selected for in Illinois apple orchards by current spray strategies. Resistance monitoring may alert growers to potential threats, but the employment of molecular tools based on current knowledge of resistance mechanisms will provide incomplete results.