Apple trees (Malus domestica L. Borkh.) exhibiting extensive and unknown tree fruit decline symptoms were observed in commercial orchards over the past 5 years in Ontario, Canada. The trees exhibited shoot dieback, attached wilted leaves and cankers on the main trunk. Trees with rapid development of cankers upward from the graft union developed extensive vascular discoloration that resulted in sudden collapse of the entire tree. In 2018-19, up to 42% mortality was observed on 2- to 8-year old apple trees. Nine symptomatic trees were collected from two orchards located in southwestern and one in southcentral Ontario. Samples (1 cm length) were collected from symptomatic trunk and shoot tissue, surface sterilized with 70% ethanol for 30 sec, followed by 1% NaClO for 20 min and three rinses in sterile water. The samples were air-dried and placed on a 2% potato dextrose agar (PDA, Difco) culture media supplemented with kanamycin (50 mg L-1). The PDA plates were incubated at 22°C for 5 days in the dark. All fungal colony-forming units that developed were hyphal-tip transferred to individual PDA plates and incubated at 22°C for 7 days in the dark. Purified mycelial isolates were classified into morphotypes prior to molecular identification. One morphotype showed white to olive-green colonies that developed on a moderately dense mycelial mat with aerial hyphae. Several solitary and globose black pycnidia that contained a single ostiole were produced on pine needles on PDA after incubation at 22°C for 14 days in the dark. Conidia were hyaline, fusiform, aseptate with an average size of 3.9 - 5.2 x 21.4 - 26.6 μm (N=50). Genomic DNA was extracted from 5-day old culture of a representative isolate, #M68-17, grown on PDA using the Plant/Fungi DNA Isolation Kit (Norgen Biotech Corp., Thorold, ON, Canada). The rDNA internal transcribed spacer (ITS), translation elongation factor 1- alpha gene (TEF-1α), and β-tubulin gene (TUB2) were amplified using the primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (O'Donnell et al. 1998), respectively, and sequenced. The nucleotide sequences obtained (GenBank # MZ926850, MZ934654, MZ934655) were 100.00% similar to those of Botyrosphaeria dothidea (Moug. ex Fr.) Ces. & De Not. isolates from other hosts in several countries in the NCBI database (MT111097, MT309401, MN515421, respectively). Randomized Accelerated Maximum Likelihood (RAxML) analysis using the three gene sequence data was completed (Stamatakis et al. 2008). The isolate #M68-17 was clustered with high bootstrap support values with the B. dothidea isolates from the fungal biodiversity centre (CBS) collection, including the ex-epitype (CBS 115476, CBS 110302) (Fig. 1). A living culture of the representative isolate, #M68-17, was deposited in the Canadian Collection of Fungal Cultures (DAOMC 252246). Pathogenicity tests were conducted in the lab on wood cuttings and in planta. Ten 20 cm apple cuttings and five potted one year old apple seedlings were surface sterilized, wounded and inoculated with 4 mm mycelium agar plug from a 5-day old culture of isolate #M68-17 and wrapped with Parafilm. Three control apple cuttings and seedlings were inoculated with PDA plugs and incubated in the same environment. Cuttings were placed inside a clear plastic container with moist paper towels and incubated at room temperature in the dark. Seedlings were placed in an open-air area between two greenhouses and watered as needed. Twelve days post-inoculation, the average length of the developed necrotic lesions on cuttings was 8.8 ± 0.4 cm. Necrotic and sunken canker symptoms appeared at 10 days, spread upward from the inoculation point and by 6 weeks the upper portion of the seedling was dead (Fig. 2). B. dothidea was isolated from all the inoculated cuttings and seedlings, thus fulfilling Koch's postulates. Control cuttings and seedlings showed no symptoms, and the fungus was not isolated from the wood. B. dothidea was reported as a pathogen causing cankers on a wide range of woody crop plants including apples, almonds, pistachios, hazelnut, walnut, olive and grapes in the United States, China, Uruguay, Spain, Tunisia and Turkey (Chebil et al. 2014; Delgado-Cerrone et al. 2016; Moral et al. 2019; Tang et al. 2012; Türkölmez et al. 2016). However, this is the first report of B. dothidea causing stem canker and death of young apple seedlings in Ontario, Canada. The findings suggest that B. dothidea has the potential to severely affect apple production in Ontario. Accurate identification of pathogen(s) associated with apple decline will support management of the disease.
Keywords: Causal Agent; Crop Type; Fruit; Fungi; Pathogen detection; Subject Areas; tree fruits.