First report of Colletotrichum grossum causing apple bitter rot worldwide (Italy)

Apple bitter rot is a globally widespread disease that is observed on bothpreharvest and postharvest fruits, contributing to considerable economiclosses. While the Colletotrichum acutatum species complex is predominantin Europe (Baroncelli et al. 2014; Amaral Carneiro and Baric 2021),in recent years, the Colletotrichum gloeosporioides species complex isemerging, raising many concerns (Amaral Carneiro et al. 2023). Circular,slightly sunken, brown lesions with acervuli produced in concentric spotswere observed on the cultivar Story Inored harvested in September2022 from an organic orchard in Masi (Padova Province, Italy), with adisease incidence close to 30%. Tissue samples were excised under asepticconditions from 10 surface-cleaned diseased fruits at the margin betweenhealthy and diseased pulp tissues, transferred to potato dextrose agar me-dium, and incubated in the dark at 25°C for 7 days, whereafter five single-spore cultures were obtained. Pure colonies grown at 25°C for 7 daysappeared light gray-white on the upper side with floccose aerial mycelium,whereas the reverse side was dark gray with a distinct margin. Conidia werehyaline, cylindrical in shape with both ends rounded or one end acute, andmeasured 16.6 ± 1.4 × 6.1 ± 0.5 µm (mean ± SD) (n = 50) as described byDiao et al. (2017). To identify the species, genomic DNA of a represen-tative isolate (C38) was extracted, and beta-tubulin (TUB2), calmodulin(CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutaminesynthetase (GS), and Apn2-Mat1-2 intergenic spacer (ApnMat) genes andthe internal transcribed spacer (ITS) region were amplified by PCR andSanger sequenced (Rojas et al. 2010; Weir et al. 2012). The obtainedDNA sequences of TUB2, CAL, GAPDH, GS, ApnMat, and ITS weresubmitted to GenBank under the accession numbers OR025589, OR025586,OR025587, OR025588, OR025585, and OR004800, respectively.A MegaBLAST analysis resulted in 100% identity to the epitype CAUG7 ofColletotrichum grossum (Diao et al. 2017) for GAPDH (KP890159) andTUB2 (KP890171), 99.85% for CAL (KP890147), and 99.5% for ITS(KP890165). The phylogenetic tree constructed by concatenation withthe obtained sequences, as well as references, revealed that the isolateC38 clustered within C. grossum, confirming the BLAST approach.Pathogenicity tests were performed on 40 ‘Story Inored’ apples. The appleswere cleaned, wounded with a sterilized needle, and exposed to two differentconditions: 20 apples (10 inoculated with 20 µl of a spore suspension [104spores/ml] and 10 inoculated with sterile water as controls) were incubated at20°C with a 12-h photoperiod for 14 days, whereas the remaining 20 apples,prepared with the same approach, were placed at 1°C for 3 months and thenat room temperature for 14 days. Symptoms appeared after 6 days on applesincubated at 20°C, whereas those stored at 1°C displayed symptoms at11 days after being placed at room temperature. In both conditions, lesionswere similar to those observed on the original fruits, whereas the controlsremained asymptomatic. Identity of reisolated fungal colonies was confirmed byCAL, GAPDH, and GS region sequence analysis. C. grossum has been reportedrarely: in 2017 on Capsicum annuum var. grossum in China, in 2018 onMangifera indica leaves in Cuba, and in 2021 on Rhyncospermum jasminoidesin Italy (Diao et al. 2017; Guarnaccia et al. 2021; Manzano Le´on et al. 2018). Tothe best of our knowledge, this is the first report of apple bitter rot caused byC. grossum worldwide.