First report of a Canadian isolate of <i>Phytopythium vexans</i> causing root rot disease on apple and peach under laboratory conditions

In the summer of 2019 and 2020, soil samples were collected from three commercial orchards (one apple, one peach and one apricot) located in southern Ontario, Canada. Each of the sites had a history of tree fruit root rot disease. Five soil samples were pooled to obtain one composite sample per orchard which was tested for the presence of oomycetes. The fruit baiting technique described by Jabiri et al. (2021) was used. Pear fruit were disinfected with 70% ethanol for 30 seconds and rinsed three times with sterile water. For each composite sample, 500 g of soil and one disinfected pear fruit were placed in a plastic box and sterile distilled water was added until the pear fruit was submerged. All boxes were incubated at 25°C for five days in the dark. Brown soft lesions were observed on the pear fruit surface two days following the incubation. Small sections of brown lesions (5×5 mm) from the infected pear fruit were plated onto cornmeal agar (BD Biosciences, USA) medium containing 0.25 g/l ampicillin and incubated at 25°C for five days in the dark. Oomycete-like hyphal-tips growing from the diseased tissues were transferred to V8 juice agar medium (Benfradj et al., 2017) and incubated at 25°C in the dark. After 10 days, eight isolates showed the same cultural and morphological characteristics and yielded a whitish radiate mycelial growth pattern (Fig. 1) with subglobose to ovoid and papillate or non-papillate sporangia (Fig. 2) measuring 26.59±2.3 μm × 16.57±1.9 μm (n = 7), the typical morphology of Phytopythium vexans (de Bary) Abad, de Cock, Bala, Robideau, Lodhi & Lévesque (de Cock et al., 2015). To confirm pathogen identity, total genomic DNA was extracted from a three-day-old culture of one representative isolate (SS21), grown on V8 broth medium, using a Plant/Fungi DNA Isolation Kit (Norgen Biotech Corp., Canada). The rDNA internal transcribed spacer (ITS) was amplified using the primer pairs ITS1/ITS4 (White et al., 1990) and sequenced. The nucleotide sequence obtained (GenBank Accession No. ON383216) was identical to those of P. vexans isolates from strawberry roots in China (MT192339 and MT192338). A living culture of P. vexans isolate SS21 was deposited in the Canadian Collection of Fungal Cultures (DAOMC 252529). Pathogenicity tests were performed using a perlite test system (Popp et al., 2019) on four-week-old healthy apple and peach seedlings. Root systems of five apple (cv. Gala) and five peach (cv. Loring) seedlings were rinsed carefully with sterile distilled water and placed in 9 cm diameter Petri dishes filled with sterilised perlite substrate amended with a 2% malt extract solution, and inoculated with 10 sterilised rye seeds infected artificially with P. vexans isolate SS21. Dishes were wrapped with aluminum foil to keep the roots in the dark (Fig. 3a, b). Seedlings were kept in a plant growth chamber under a day/night temperature regime of 26/24°C, photoperiod of 16 hours and watered as needed to keep the perlite wet. Five control apple and five peach seedlings were grown and handled in the same way, except that they were not inoculated with the pathogen. Root and crown rot, leaf curling, and decline occurred within nine days on peach (Fig. 3b, d) and six weeks post-inoculation on apple (Fig. 4b) on all inoculated plants, suggesting that P. vexans isolate SS21 was more virulent on peach than apples. Phytopythium vexans was re-isolated from all the inoculated seedlings, thus fulfilling Koch's postulates. Control plants remained symptom-free (Fig. 3a, c; Fig 4a), and P. vexans was not isolated from the root tissue. Phytopythium vexans was reported as a pathogen causing root and crown rot and decline on fruit trees and woody ornamentals including apple, avocado, citrus, durian, ginkgo, flowering cherry, grapevine, kiwi, pear and red maple in the Canary Islands, China, Italy, Morocco, South Africa, Tunisia, Turkey, Vietnam and the USA (Benfradj et al., 2017; Baysal-Gurel et al., 2021; Jabiri et al., 2021; Zhou et al., 2022). To the best of our knowledge, this is the first report of a Canadian isolate of P. vexans causing disease in apple and peach under laboratory conditions in Ontario. The findings suggest that P. vexans has the potential to severely affect apple and peach production in Ontario, where most of Canada's fruits are produced. This report will serve as a foundation for further studies on the extent of the P. vexans spread and the implementation of effective management strategies.

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