First Report of Phomopsis Stem Canker of Sunflower (Helianthus annuus) Caused by Diaporthe gulyae in Canada.

During September 2012, Phomopsis stem canker was observed on sunflowers (Helianthus annuus L.) in a production field during seed filling with an average incidence of 15% in Morden, Manitoba (approximately 49°11'N and 98°09'W). The infected plants had elongated, brown-black lesions surrounding the leaf petiole, with numerous pycnidia, pith damage, and mid-stem lodging. Twenty sunflower plants were randomly sampled from the field. Isolations were made from the margins of the necrotic stems lesions by plating small pieces (5 mm) on potato dextrose agar (PDA) amended with 0.02% streptomycin sulfate. Plates were incubated at 25°C for 14 days under a 12-h photoperiod, and hyphal tips of white to grey colonies were transferred to PDA. Five isolates producing black pycnidia (occasionally with ostiolate beaks) and alpha conidia were tentatively identified as a Diaporthe sp. Alpha conidia were ellipsoidal, hyaline, and 6.5 to 8.5 × 2.5 to 3.5 μm. DNA was extracted from the mycelium of five isolates, and the ITS region was amplified and sequenced using primers ITS5 and ITS4 (4). BLASTn analysis of the 600-bp fragment (GenBank Accession Nos. KM391960 to KM391964) showed that the best match was Phomopsis sp. AJY-2011a strain T12505G (Diaporthe gulyae R.G. Shivas, S.M. Thompson & A.J. Young [3], Accession No. JF431299) from H. annuus with identities = 540/540 (100%) and gaps = 0/540 (0%). The five D. gulyae isolates were tested for pathogenicity on a sunflower confection inbred cv. HA 288 using the stem-wound method (2). Four-week-old sunflower plants (10 plants per isolate) were inoculated by wounding the stems on the second internode with a micropipette tip and placing a Diaporthe-infested mycelial plug on the wound. All plugs were attached to the wound with Parafilm. The pots were placed on the greenhouse benches at 25°C under a 16-h light/dark cycle. At 3 days after inoculation, dark brown lesions were observed on the stems extending upward from the inoculation site. Stem and leaves wilted, causing plant death 14 days after inoculation. Disease severity was calculated as a percentage of stem lesion (lesion length/stem length × 100%) at 14 days after inoculation. Significant differences (P ≤ 0.05) in disease severity were observed among D. gulyae isolates, which ranged from 34.9 to 100.0% (n = 5). Ten control plants similarly treated with sterile PDA plugs did not display symptoms. To complete Koch's postulates, D. gulyae was re-isolated from the inoculated stems, and the pathogen's identity was confirmed via sequencing of the ITS regions using primers ITS5 and ITS4 (4). The pathogen was not isolated from the control plants. D. gulyae was first reported as a pathogen on H. annuus in Australia and United States in 2011 (1,3). The pathogen was determined to be as or more aggressive than the other causal agents of Phomopsis stem canker (2,3), and its identification in both countries was circumstantially associated with increased incidence and yield loss in commercial production fields (1,3). In Canada, Phomopsis stem canker has been observed in sunflower fields over the last 10 years at low incidences, especially in years with above-normal temperatures during the sunflower growing season; however, the causal agent was not confirmed. To the best of our knowledge, this is the first report of D. gulyae causing Phomopsis stem canker on sunflowers in Canada. Since there is currently no known resistance to D. gulyae in sunflower, this newly discovered pathogen may become a threat to sunflower production in Canada. References: (1) F. Mathew et al. Phytopathology 101:S115, 2011. (2) F. Mathew et al. Phytopathology 103:S2.91, 2013. (3) S. M. Thompson et al. Persoonia 27:80, 2011. (4) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, San Diego, 1990.