First Report of <i>Colletotrichum fioriniae</i> Causing Bitter Rot of Apple in Massachusetts

In the summer of 2024, the UMass Extension Fruit Team was contacted due to reports of apple (<i>Malus domestica</i>) plants showing fruit rot symptoms. Infested fruits were found on ~70% of orchard trees and developed brown water-soaked lesions exhibiting sporulating concentric rings as are typical with bitter rot of apple. Symptomatic apples, cv. Cortland, were collected from a commercial orchard in Hampshire County. Diseased tissue was sterilized in 10% bleach for 1 min, rinsed with sterile water, and plated on potato dextrose agar amended with 100 μg/ml chloramphenicol. Hyphal tips of fungi were transferred to fresh plates which formed fluffy, dark gray mycelial mats with prominent pink undersides from all symptomatic apples. Orange spore masses formed near the center of the colonies, each of which contained numerous fusiform and cylindrical straight conidia, matching spores of the genus <i>Colletotrichum</i> (de Silva et al. 2019). Average conidia (n=169) length was 11.4 ± 1.9 µm and width was 4.9 ± 0.5 µm. Areal hyphae were collected from two isolates, CFF1-1 and APO1-1, and DNA was extracted for further molecular characterization. PCR was performed using primers targeting actin (<i>ACT</i>), calmodulin (<i>CAL</i>), GAPDH (<i>gpdA</i>), and ITS, followed by Oxford Nanopore sequencing (CFF1-1 Accessions numbers: PV288758, PV288759, PV288760; APO1-1 Accessions numbers: PV641589, PV641590, PV641591) (Carbone and Kohn 1999; Hassan et al. 2018; Templeton et al. 1992). Sequences were queried through NCBI's RefSeq Genome Database with BLASTN. Orthologous sequences from ten <i>Colletotrichum</i> genomes which appeared in all BLASTN queries were aligned, trimmed, and concatenated into super-genes using Mega11 (Tamura et al. 2021). Model selection was conducted using IQ-TREE and selected parameters were used to generate a maximum-likelihood tree from the concatenated sequence, placing the isolates in a high confidence cluster with <i>Colletotrichum fioriniae</i> (Nguyen et al. 2015). To confirm the virulence of the pathogen, apples (cv. Fuji) (n=8) were inoculated with spores, with or without injury to the cuticle. The inoculum consisted of 15 µL of spores at a concentration of 107 spores per mL. Injured apples (n=4) were penetrated 5 mm deep with a sterile 20 µL pipette tip before inoculation on the wound site while control plants (n=4) were inoculated with 10 µL of sterile water. Fruits were maintained under fluorescent lights for 18 hours/day (30°C) and in the dark for 6 hours/day (20°C) in a growth chamber and assessed after 9 days. All inoculated apples had sunken lesions consistent with field samples and bisected apples revealed the brown lesions penetrating 3-5 centimeters into the apple. Control plants had no clear lesions and bisected apples were visibly healthy. <i>C. fioriniae</i> re-isolated from infected tissue presented with identical hyphal /spore morphology. Fruit inoculations were repeated, yielding identical lesions. Bitter rot of apple has been inferred to several species in the <i>C. acutatum</i> species complex (including <i>C. fioriniae</i>) and is a prominent disease of apples in other apple growing regions. This disease has been sporadically reported throughout New England, though with the increasing frequency of warm and wet spells, occurrences are becoming more common. Future work should continue to evaluate <i>Colletotrichum</i> species present for disease management efficacy.