Page 1 of 5 First report of bitter rot of apple caused by a Colletotrichum sp. in the C. kahawae clade in Kentucky M. J. McCulloch, N. W. Gauthier, L. J. Vaillancourt 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 KY: 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 (Kou et al., 2014; Chechi et al., 2019). It is important to know which species are present in an orchard because they vary in pathogenicity and fungicide sensitivity (Munir et al., 2016; Chechi et al., 2019). A sixth Colletotrichum species was isolated in 2013 from typical bitter rot lesions on ‘Honeycrisp’ apples in a commercial orchard in Bourbon County, KY. 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 23⁰C with constant light. Conidia of two representative isolates were harvested from ten-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-) 17.3 to 22.3 (-28.9) by (4.8-) 5.1 to 6.5 (-6.8) µm, and (14.7-) 15.9 to 20.9 (-21.4) by (4.9-) 5.3 to 7.1 (-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 surfacesterilized, wound-inoculated with a spore suspension (1 x 105 spores/mL), and placed in humidity chambers for two 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 Page 2 of 5 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 β-tubulin 2 (TUB2) (Liu et al. 2015; Weir et al. 2012) (See Supplemental Figure 2 for accession numbers). 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. Colletotrichum 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 KY 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 U.S. 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. Chechi, A., J. Stahlecker, M. E. Dowling, and G. Schnabel. 2019. Diversity in species composition and fungicide resistance profiles in Colletotrichum isolates from apples. Pesticide Biochemistry and Physiology. 158: 18-24 Gauthier, N. W., Leonberger, K., Bessin, R., Springer, M., Strang, J., and Wright, S. 2017. A profile of commercial apple production in Kentucky 2017. https://ipmdata.ipmcenters.org/documents/cropprofiles/KY_Apple_CropProfile.pdf Grammen, A., Wenneker, M., Van Campenhout, J., Pham, K. T. K., Van Hemelrijck, W., Bylemans, D., Geeraerd, A., and Keulemans, W. 2019. Identification and pathogenicity Page 3 of 5 assessment of Colletotrichum isolates causing bitter rot of apple fruit in Belgium. European Journal of Plant Pathology 153:47-63. Kou, L. P., Gaskins, V., Luo, Y. G., & Jurick, W. M. (2014). First report of Colletotrichum fioriniae causing postharvest decay on ‘Nittany’ apple fruit in the United States. Plant disease, 98(7), 993-993. Liu, F., Weir, B. S., Damm, U., Crous, P. W., Wang, Y., Liu, B., Wang, M., Zhang, M., and Cai, L. 2015. Unravelling Colletotrichum species associated with Camellia: employing ApMat and GS loci to resolve species in the C. gloeosporioides complex. Persoonia 35:63-86. Munir, M., Amsden, B., Dixon, E., Vaillancourt, L., and Ward Gauthier, N. A. 2016. Characterization of Colletotrichum species causing bitter rot of apple in Kentucky orchards. Plant Disease 100:2194-2203. Weir, B. S., Johnston, P. R., and Damm, U. 2012. The Colletotrichum gloeosporioides species complex. Stud Mycol 73:115-180. Page 4 of 5 Figure 1. A-H colony, conidium, and hyphopodium morphology of two unknown Colletotrichum kahawae clade isolates from apple. A and B upper colony surface on potato dextrose agar (PDA); C and D colony reverse on PDA; E and F morphology of conidia recovered from PDA; G and H hyphopodia produced on potato-carrot agar. A, C, E, and G isolate HC278; B, D, F, and H isolate HC292. Scale bar is 20 µm. I-L typical bitter rot lesions produced on two-week-old ‘Fuji’ apples inoculated with two unknown Colletotrichum kahawae clade isolates. I and J isolate HC278; K and L isolate HC292. 507x584mm (72 x 72 DPI) Page 5 of 5 Figure 3. PhyML Approximate Likelihood-Ratio Test tree of concatenated ITS, GAPDH, ACT, TUB2, CAL, and GS alignments indicating phylogenetic affinities for reference Colletotrichum spp. and the unknown apple Colletotrichum isolates HC278 and HC292 (green font). Accession numbers are in gray in the gene order ITS, GAPDH, ACT, TUB2, CAL, GS. Missing genes are denoted by “--”. Colletotrichum boninense was used as the outgroup. Bootstrap values are shown in red font. Previously reported Colletotrichum spp. on apple in KY are indicated in blue font. The C. kahawae clade is highlighted by the green box. The scale bar indicates 0.08 expected changes per site. 656x557mm (72 x 72 DPI)