Identification of Leptosphaeria maculans Pathogenicity Group 4 Causing Blackleg on Winter Canola in Oklahoma

Winter canola (Brassica napus L.) is a relatively new crop to Oklahoma and other southern U.S. states where it is considered a desirable rotation crop with wheat. In 2009, approximately 15,000 ha of winter canola were harvested in Oklahoma (3); that area is expected to almost double in 2010. Blackleg, a potentially devastating canola disease, was detected in Oklahoma in 2009. Blackleg is caused by Leptosphaeria maculans (Desmaz.) Ces. & de Not (anamorph = Phoma lingam (Tode:Fr.) Desmaz.). In early 2010, leaf samples showing typical symptoms of blackleg were collected from four canola fields near the town of Enid in Garfield County, OK. Small portions of infected tissues were surface disinfested in an aqueous solution of NaOCl (0.5% a.i.) for 1 min, rinsed twice in sterile distilled water, and plated on V8 medium. Seven colonies were isolated and when grown in pure culture, all produced 2 × 4.5 μm guttulate, unicellular, hyaline spores in pycnidia that ranged from 200 to 480 μm in diameter. These morphological characteristics correspond with those of P. lingam (1). To verify the pathogenic nature of the isolates and establish the pathogenicity group (PG) to which they belong, a standard inoculation protocol was followed on a set of three differential cultivars, Quinta, Glacier, and Westar (2). Briefly, for each isolate, tiny puncture wounds were made with sterile needles on the cotyledons of six 10-day-old plants of each differential and a 10-μl aliquot of a pycnidiospore suspension (1 × 10⁷ spores ml^-1) was deposited on the wounds. Also, a set of differentials were inoculated with distilled water (mock inoculation). Inoculated plants were incubated overnight in a misting chamber at 21°C in the dark and returned the next day to the greenhouse. Disease severity was recorded 10 days after inoculation using a 0 to 9 scale in which 0 to 2 = resistant, 3 to 6 = intermediate, and 7 to 9 = susceptible. This process was repeated three times. Two of the seven isolates evaluated were highly virulent on all three differentials, an indication they belong to pathogenicity group 4 (2). The other five isolates produced small lesions on Westar (resistant reaction) but failed to develop symptoms on the other two differentials. This phenotypic reaction has been associated with strains of PG-1. Mock-inoculated plants did not develop lesions. To our knowledge, this is the first time blackleg isolates from Oklahoma have been identified to the PG level. While this information will assist breeders in the development of both spring and winter canola lines with resistance to blackleg, additional studies are necessary to determine the relative prevalence and diversity of the various PG in Oklahoma. References: (1) G. H. Boerema. Trans. Br. Mycol. Soc. 67:289, 1976. (2) A. Mengistu et al. Plant Dis. 75:1279, 1991. (3) USDA. National Agricultural Statistics Service. Retrieved from http://www.nass.usda.gov/Statistics_by_State/Ag_Overview/ AgOverview_OK.pdf , September 20, 2010.