First report of Apple bitter rot caused by Colletotrichum godetiae in Ontario.

Bitter rot is an emerging disease of apple ( Malus domestica ) fruit in Ontario in part due to changing weather conditions. The disease was mostly documented in warm and humid regions such as the southern USA, and Central and South America. Thirteen Ontario orchards in the fall of 2019 and 15 in 2020 were scouted for bitter rot based on their previous history of the disease. 100 fruit were collected from ten asymptomatic trees per cultivar and two susceptible cultivars, 'Empire' and 'Ambrosia' were scouted per orchard. If an orchard did not have either one of these cultivars, 'Honeycrisp' or 'Gala' were used. The fruit was stored at 4-5 o C for five months and then left at 22 o C for two weeks and assessed thereafter for bitter rot symptoms. Monoconidial cultures of Colletrotrichum spp. were established from the symptomatic fruit using potato dextrose agar media with antibiotics at 22 o C 14-hour light cycles. The fungal isolates were divided into two groups based on colony morphology observations seven days after culturing. The first group showed light grey, cottony, aerial mycelium on top and a pink to dark red color on the reverse of the plate. The second group showed light to dark grey, cottony mycelium on top and dark green colonies on the reverse of the plate. It is difficult to identify Colletrotricuhum species solely based on morphology, therefore the representative isolates from each group were used for multilocus gene sequencing and species identification. Genomic DNA was extracted using the Qiagen DNeasy Plant Mini Kit according to the manufacturer's protocols. The ITS region was amplified and sequenced using the primers ITS-1F (Grades & Bruns 1993) and ITS-4 (White et al. 1990). Primers T1 and T2 (O'Donnell & Cigelnik 1997) were used to amplify and sequence the 750 bp region of the TUB gene. Primers GDF1 and GDR1 (Guerber et. al. 2003) were used to amplify a 280 bp region of the GADPH gene. Following an NCBI nucleotide blast search, the isolates from group 1 were identified as C. fioriniae .The ITS sequences of group 2 isolates were matched 100% to the Colletotrichum godetiae type strain CBS133.44 and they were 99% matched to the closest species C. Johnstonii CBS128532. The TUB sequences matched 100% identity to 20 sequences belonging to C. godetiae , 99.59 % identity to C. godetiae type strain CBS133.44, and 97.75% to C. Johnstonii CBS128532. The GADPH sequences matched with 100% identity to C. godetiae ON241087.1 or MT816329.1 and 99-99.5% identity to the type strain CBS133.44 and 98.61-99% identity to CBS128532. Based on the blast results the group 2 isolates were identified as C. godetiae and their sequences were submitted to GenBank with ID OP702962 for ITS and OP972240 and OP972241 for GADPH and OP972242 for the TUB gene. Out of 50 isolates collected in this work, 94% belonged to group 1 and 6% belonged to group 2. Koch's postulates were performed on selected isolates by artificial inoculation of 5 healthy detached fruits of the cultivar, 'Empire.' Fruit surfaces were wiped with 70% ethanol, dried, wounded with a sterile needle, and then inoculated with a 10 µl of spore suspension containing 1x10^6 spores /ml. Inoculated fruits were incubated in a humid chamber at 22°C in dark. Symptoms started to appear at 5 days post-inoculation and looked like small brown circular lesions which developed orange spore masses as they grew into larger lesions. The non-inoculated control fruit did not develop lesions. Fungal colonies were established from the spores collected from the inoculated fruit and found to have identical morphological characteristics to the original isolates. C. godetiae has previously been reported to cause bitter rot in apples from various countries in Europe (Baroncelli et al. 2014; Munda, 2014; Wenneker et al. 2016). C. fioriniae has previously been reported as the dominant species causing bitter rot in apples although other species including C. chrysophilum and C. noveboracense have also been reported as causal agents from the Eastern USA (Khodadadi et al. 2020). To the best of our knowledge, this is the first report of Colletotrichum godetiae causing the bitter rot of apples in Ontario, Canada.