Multiple species of Colletotrichum can cause bitter rot disease of apple, but the identities and relative representation of the species causing the disease in Kentucky are unknown. In total, 475 Colletotrichum isolates were collected from diseased apple fruit in 25 counties and characterized both morphologically and by using various molecular approaches. Multigene sequence analyses revealed that sample isolates belonged to several newly erected species within the Colletotrichum acutatum and C. gloeosporioides species complexes. The isolates were identified as C. fioriniae and C. nymphaeae, which reside within the C. acutatum species complex, and C. siamense, C. theobromicola, and C. fructicola, which are placed within the C. gloeosporioides species complex. C. fioriniae was the most common species causing bitter rot in Kentucky, comprising more than 70% of the isolates. Infectivity tests on detached fruit showed that C. gloeosporioides species-complex isolates were more aggressive than isolates in the C. acutatum species complex. However, isolates within the C. acutatum species complex produced more spores on lesions compared with isolates within the C. gloeosporioides species complex. Aggressiveness varied among individual species within a species complex. C. siamense was the most aggressive species identified in this study. Within the C. acutatum species complex, C. fioriniae was more aggressive than C. nymphaeae, causing larger, deeper lesions. Apple cultivar did not have a significant effect on lesion development. However, Colletotrichum spp. produced more spores on ‘Red Stayman Winesap’ than on ‘Golden Delicious’. Fungicide sensitivity tests revealed that the C. acutatum species complex was more tolerant to thiophanate-methyl, myclobutanil, trifloxystrobin, and captan compared with the C. gloeosporioides species complex. The study also revealed that mycelial growth of C. siamense was more sensitive to tested fungicides compared with C. fructicola and C. theobromicola. These research findings emphasize the importance of accurate identification of Colletotrichum spp. within each species complex, because they exhibit differences in pathogenicity and fungicide sensitivity.

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Azoxystrobin is the only synthetic, systemic fungicide labeled in the United States for management of frogeye leaf spot (FLS) of tobacco (Nicotiana tabacum L.), caused by Cercospora nicotianae. Though traditionally considered a minor disease in the United States, FLS has recently become yield and quality limiting. In 2016 and 2017, 100 C. nicotianae isolates were collected from symptomatic tobacco from eight counties in Kentucky, United States. Prior to azoxystrobin sensitivity testing, some C. nicotianae isolates were found to utilize the alternative oxidase pathway and, after assay comparisons, conidial germination was utilized to evaluate sensitivity in C. nicotianae as opposed to mycelial growth. Azoxystrobin sensitivity was determined by establishing the effective concentration to inhibit 50% conidial germination (EC50) for 47 (in 2016) and 53 (in 2017) C. nicotianae isolates. Distributions of C. nicotianae EC50 values indicated three qualitative levels of sensitivity to azoxystrobin. Partial cytochrome b sequence, encompassing the F129L and G143A mutation sites, indicated single-nucleotide polymorphisms (SNPs) conferring the F129L mutation in C. nicotianae of moderate resistance (azoxystrobin at 0.177 ≤ EC50 ≤ 0.535 µg/ml) and the G143A mutation in isolates with an azoxystrobin-resistant phenotype (azoxystrobin EC50 > 1.15 µg/ml). Higher frequencies of resistant isolates were identified from greenhouse transplant (4 of 17) and conventionally produced (58 of 62) tobacco samples, as compared with field-grown tobacco (<4 weeks prior to harvest; 4 of 62) or organically produced samples (1 of 7), respectively. Together, these results suggest that resistance to azoxystrobin in C. nicotianae occurs broadly in Kentucky, and generate new hypotheses about selection pressure affecting resistance mutation frequencies.