First report of an aggressive species of <i>Neopestalotiopsis</i> affecting strawberry in Canada

In October 2020, Pestalotiopsis-like disease symptoms were observed in a one-acre field of strawberry (Fragaria × ananassa cv. Albion) located in Ontario, Canada. Symptoms appeared as light brown necrotic lesions surrounded by darker red-brown haloes on leaves, black fruiting bodies, and wilt. Disease incidence in the field was 40% and a further 5% of the plants were dead (Figure 1, 2 & 3). One lesion was isolated from the crown tissue of six actively collapsing plants. Lesions were sterilized with 0.5% sodium hypochlorite for one minute, rinsed twice for five minutes in 50 ml sterile water, and placed on 1.5% water agar. Following two days of incubation at 24°C, single hyphal tips were transferred to potato dextrose agar and incubated at 24°C for seven days. Fungi were isolated from four of the six plants. Cultures had a white, cottony mycelium upper surface and a white to pale orange lower surface. Black viscous acervuli appeared on the upper surface of the colonies before day 14. Conidia were five-celled, fusiform to ellipsoid and 23.0-25.1 μm long and 7.2-7.4 μm wide (n = 30). DNA was extracted from seven-day old cultures and the internal transcribed spacer (ITS) region, β-tubulin, and translation elongation factor 1-alpha were amplified using PCR (Maharachchikumbura et al., 2014). Amplified products were sequenced in both directions and sequences deposited in GenBank (Accession Nos. OQ731784, OQ731785 and OR002051, respectively). Amplified products from all four cultures shared 100% nucleotide identity. The genomic sequence data shared 100% nucleotide identity with the aggressive Neopestalotiopsis species that was first reported in North America (Baggio et al., 2021). Sequence identities include 375/375 bp for the ITS, 381/381 bp β-tubulin, and 588/588 bp for the translation elongation factor 1-alpha. To fulfill Koch's postulates, three four-week-old strawberry cv. Montana plants were spray inoculated until run-off with 1×104 conidia/ml from the fungus isolated from a crown sample or sterile distilled water. Spore suspensions were obtained from seven-ten-day old colonies and all experiments were repeated three times. Experimental plants were grown under controlled environmental conditions at 85% humidity, 12 hour photoperiod of 24°C day/22°C night and light levels of 100μE.m2s−1. After 5–14 days following inoculation, all of the plants inoculated with the fungus exhibited foliar lesions and fruiting bodies with conidia (Figure 4). Plants sprayed with sterile distilled water did not show any symptoms. Fungi were re-isolated from a single spore in all plants inoculated with fungi but not from those plants sprayed with sterile distilled water. DNA was extracted from the re-isolated fungi and subject to PCR amplification and sequencing, as described above. The DNA sequence from all three loci had 100% sequence identity to the original ITS, β-tubulin, and translation elongation factor 1-alpha loci. This is the first time that the aggressive species of Neopestalotiopsis, previously reported in the United States (Baggio et al., 2021; Rotondo et al., 2022; Guan et al., 2023) has been reported in Canada. We thank the Berry Growers of Ontario and Ontario Ministry of Agriculture, Food and Rural Affairs for supporting this work.

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