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First Report of Bitter Rot of Apple Caused by a Colletotrichum sp. in the C. kahawae Clade in Kentucky

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    Authors and Affiliations
    • M. J. McCulloch
    • N. W. Gauthier
    • L. J. Vaillancourt
    1. University of Kentucky, Lexington, KY

    Apple bitter rot causes average annual yield losses of 30% in Kentucky, with individual losses as high as 100% in some orchards (Gauthier et al. 2017). Five Colletotrichum spp. were previously identified as causal agents of bitter rot in Kentucky: C. fioriniae and C. nymphaeae in the C. acutatum species complex; and C. siamense, C. fructicola, and C. theobromicola in the C. gloeosporioides complex (Munir et al. 2016). Three of these species, C. fioriniae, C. siamense, and C. fructicola, have also been reported causing bitter rot in other states (Chechi et al. 2019; Kou et al. 2014). It is important to know which species are present in an orchard, because they vary in pathogenicity and fungicide sensitivity (Chechi et al. 2019; Munir et al. 2016). A sixth Colletotrichum species was isolated in 2013 from typical bitter rot lesions on ‘Honeycrisp’ apples in a commercial orchard in Bourbon County, Kentucky. Six isolates were collected from two apples on the same tree and single-spored for further study. Colonies were smooth and light to dark gray on top with a light orange border, and dark brown to black with an orange border on the reverse, when grown on potato dextrose agar (PDA) at 23°C with constant light. Conidia of two representative isolates were harvested from 10-day-old PDA plates. Conidia were hyaline and cylindrical with rounded ends, with some narrowing slightly at the base or center. Spore sizes for the two strains were (15.1 to) 17.3 to 22.3 (to 28.9) by (4.8 to) 5.1 to 6.5 (to 6.8) µm, and (14.7 to) 15.9 to 20.9 (to 21.4) by (4.9 to) 5.3 to 7.1 (to 7.5) µm. Hyphopodia on potato-carrot agar varied from rounded and smooth to oval with small midpoint lobes. Pathogenicity of two representative isolates was confirmed in detached fruit assays. ‘Fuji’ apples were surface sterilized, wound inoculated with a spore suspension (1 × 105 spores/ml), and placed in humidity chambers for 2 weeks. Typical bitter rot lesions resulted from inoculation with the two apple isolates but not from negative control treatments that consisted of mock-inoculated fruit, or fruit inoculated with C. graminicola, which is pathogenic to maize but not apples. The morphology of the fungus recovered from the inoculated apples matched the original strains, fulfilling Koch’s postulates. Sequences of seven genes were used for species identification: actin (ACT), partial mating type protein 1-2-1 gene and Apn2-Mat1-2 intergenic spacer (ApMat), calmodulin (CAL), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), internal transcribed spacer region (ITS), and beta-tubulin 2 (TUB2) (Liu et al. 2015; Weir et al. 2012) (accession numbers in the supplementary materials). Individual NCBI and Q-bank BLAST reports indicated conflicting species identities, but all were in the C. kahawae clade of the C. gloeosporioides species complex. Phylogenetic trees were generated from concatenated multigene sequences using the method of Liu et al. (2015) and Weir et al. (2012). Trees using all sequences except ApMat, or only ApMat and GS (Liu et al. 2015), confirmed a close affinity of the unknown apple isolates with C. kahawae but could not assign them to an identified species within the clade. C. kahawae is the only member of this clade that has previously been reported to cause bitter rot, in a single study from Belgium (Grammen et al. 2019). That strain differed in pathogenicity and fungicide sensitivity from other bitter rot strains in the same study. Given the strong support for distinction within the trees, the isolates from Kentucky may represent a new species, but more research is necessary to determine if that status is warranted. Meanwhile, it is important to publish this report because all previously identified bitter rot pathogens in the United States are only distantly related to members of the C. kahawae clade. Thus, the response of these strains to current bitter rot management regimes is unpredictable and requires further study.

    The author(s) declare no conflict of interest.

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    The author(s) declare no conflict of interest.