First Report of <i>Fusarium avenaceum</i> Causing Apple ( <i>Malus domestica</i> ) Fruit Storage Rot in Canada

In May 2023, fruit rot lesions were observed in 'Honeycrisp' apple (Malus domestica), fruit in commercial storage conditions in Nova Scotia, Canada. Rot lesions were observed on 10% of the fruits from some orchards and were brown, soft, and of irregular shape. To isolate causal pathogens, symptomatic apples were surface-sterilized in 70 % ethanol for 2 min and 1% NaOCl for 3 min, and then rinsed three times with sterilized water. A sterile scalpel was used to cut 5 mm x 5mm pieces from the margin between diseased and healthy tissue which were then plated on potato dextrose agar (PDA; Difco) amended with Novobiocin (100 mg/l) and incubated at 22℃ under light for 3-4 days. Two fungal colonies that consistently grew on PDA plates were hyphal-tip transferred to fresh PDA plates. Colonies were pale yellow to golden in color, becoming lighter at the margins, and produced cottony aerial mycelium after 7 days on PDA in the dark at 22℃. Microconidia were ovoid to fusiform with 0-1 septa (mean length 12 ± 1.8 μm, mean width 3.8 ±0.6 μm, n=50). Macroconidia were slightly curved, with 3-5 transverse septa (mean length 26 ± 4.9 μm, mean width 5.1 ±0.92 μm, n=50). Based on morphological characteristics, the two isolates resemble F. avenaceum (Fr.) Sacc., as described by Booth (1971) in Descriptions of Fungi and Bacteria (C. Booth, Ed.). To identify the species of Fusarium, genomic DNA from two isolates was subjected to PCR amplification and sequencing of TEF1α region (O’Donnell et al. 1998), ITS region (de Hoog and Van den End 1998; Masclaux et al. 1995), and RPB2 region (O’Donnell et al. 2022). The sequences were deposited in GenBank (TEF1α: accession numbers, PP549540 and PP549541, ITS: accession numbers, PP496676 and PP496677 and RPB2: accession numbers, PP525085 and PP525086) for isolate 187-1A and 898-2B respectively. BLASTn analyses of TEF-1-α partial gene sequences established both isolates’ identity as F. avenaceum with 100 % identity and 97% query coverage to isolate (Accession number KM189442). RPB2 sequences were 99.8 % identical with 100% query coverage to F. avenaceum isolate (Accession number MFG80214). To further confirm the identification of the causal pathogen, 1000 bootstrap searches were performed to obtain the best-scoring Maximum Likelihood (ML) tree. The phylogenetic tree was rooted to F. lateritium. Phylogenetic analysis of concatenated sequences of ITS, RPB2, and TEF-1-α confirmed that the isolates were F. avenaceum. Koch’s postulates were tested using pathogenicity assay, five 'Honeycrisp ' apple fruit were wounded/not wounded on the skin and inoculated with a 6 mm PDA disc containing mycelium from the margin of 7 days old F. avenaceum colonies, and five fruit were inoculated with sterile PDA disc and five with non- apple pathogenic F. avenaceum isolate. Fruit were stored in trays lined with sterile paper towels moistend with sterile water, covered with domed lids to maintain high humidity, and incubated in the dark at 22℃ . Rot lesions were observed after 7 days, non-pathogenic isolate did not produce any symptoms up to three weeks post-inoculation. Lesions with a mean diameter of 14.7±2.86 mm were observed on F. avenaceum inoculated fruit, while no lesions were observed on fruit inoculated with sterile PDA or with non- apple pathogenic F. avenaceum isolate. This experiment was repeated twice with similar results. F. avenaceum was successfully reisolated from the lesion of an inoculated apple both times and confirmed by sequencing of the RPB2 and TEF1α regions. In another assay unwounded apples fruits were placed in contact with inoculated apples. Symptoms appeared on the unwounded fruits approximately four weeks later, confirming that lesion development is delayed without wounding. F. avenaceum has been identified as a rot pathogen affecting apple fruit during postharvest storage in the United States, Croatia, and Morocco (Khadiri et al. 2024; Sever et al. 2012). This could become a serious problem for Canada’s apple industry during storage, therefore further studies are required to understand the epidemiology of the disease and recommend a phytosanitary program for its control. This report will help to better understand F. avenaceum causing apple fruit rot during post harvest storage in Canada.

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