First Report of Verticillium Stripe of Canola Caused by Verticillium longisporum in North Dakota

North Dakota leads the U.S.A. in canola (Brassica napus L.) production (5) and approximately one-third of the acreage is located in the northeastern portion of the state. A field survey conducted at the end of the 2021 growing season in northeastern North Dakota revealed the presence of a single field with approximately 15% plants exhibiting whitish discoloration symptoms in the lower third of the stems. The epidermis on the discolored areas was peeling, and the exposed tissues were dark grey to dark-brown. Stem samples taken to the laboratory were surface disinfested with a 5% aqueous solution of NaOCl for 60 sec and rinsed thrice with sterile water. Under aseptic conditions, small stem pieces were plated on V-8 medium amended with 150 mg each of penicillin and streptomycin per liter of medium and incubated at 22 oC under 16 h light daily. Under the microscope, conidiophores were verticillate, hyaline, and had three branches. Conidia were single-celled, hyaline, and measured on average 9.2 + 1.8 µm. Microsclerotia were irregularly shaped. These features match the description of Verticillium longisporum (Stark) Karapapa Bainbr. & Heale, (4). Genomic DNA was extracted from a single-spore culture of an isolate as described by Azizi et al. (1). PCR assays were conducted twice on two independent DNA samples extracted from the same isolate using V. longisporum species-specific primer set VlspF1 and VlspR4 (2) with denaturation at 95 oC for 3 min, followed by 35 cycles of amplification at 94 oC for 1 min then 56 oC for 30 sec and 72 oC for 1 min, followed by a final period at 72 oC for 5 min. The sequenced PCR product, which had 100% homology with GenBank V. longisporum reference samples KY704097 and HE972063, was assigned GenBank accession number OR088215. Pathogenicity tests were conducted in greenhouse. Briefly, twenty seeds of the canola cv. Westar were incubated on a sterilized wet paper towel for five days at 22 oC. The seedlings were carefully lifted, and their root tips cut with scissors. Ten wounded seedlings were immersed in a V. longisporum spore suspension with 2.07 x 106 spores per ml for 30 minutes and the other ten in distilled water (controls). The plants were transplanted into pots (10x10x13 cm) containing Sunshine Mix # 1 potting mix (Fison Horticulture, Vancouver, B. C.). The study was conducted twice, with individual plants as replications. Three weeks later, 20% of inoculated plants had died and at physiological maturity, the rest of them had stunted growth and blackened internal stem tissues while external stem symptoms resembled those found in the field. All control plants reached maturity without symptoms. The pathogen re-isolated from inoculated plants were morphologically identical to the one retrieved from the field. These results confirmed the isolate as Verticillium longisporum. This is the first report of Verticillium stripe on canola in the US. In North America, the disease was first reported in Manitoba, Canada, in 2014 but subsequent surveys showed it is widespread in Canada (3). Identification of genetic resistance against this disease is required to ameliorate the threat this disease represents to US canola production.