Outbreak of Blackleg in Canola in North Dakota is Caused by New Pathogenicity Groups

In 2009, a blackleg outbreak was detected in canola fields of North Dakota. The disease, which is caused primarily by the fungus Leptosphaeria maculans, was observed in 88 of 99 fields scouted with 39 fields having incidences >30%. The mean blackleg incidence in these 39 fields was 61% (range 50 to 84%). Fifty nine L. maculans isolates were retrieved from 20 of these 39 fields and classified in five pathogenicity groups (PG) using a set of three differentials cultivars. PG-4 was the pathotype most commonly isolated from these fields with 51% of all isolates, followed by PG-3 with 25% and PG-T with 8% of isolates. Only 3% of the isolates belong to PG-2, a pathotype that was previously considered the most prevalent in the region. Increased prevalence of these new pathogenicity groups represents a threat to the canola industry in the state. Introduction Blackleg is a disease that affects crucifer plants worldwide (5). The disease is caused by the fungus Leptosphaeria maculans (Desm.) Ces. & de Not. [anamorph Phoma lingam (Tode ex. Schw.)]. Annual yield losses attributed to this disease in other parts of the world have been estimated in several millions of dollars (5,8). In North Dakota, there are no official estimates of the impact of this disease, although anecdotal information by growers suggests that yield losses of up to 45% have been observed. Variability in virulence of L. maculans isolates were characterized first as pathogenicity groups using cultivars Westar, Quinta, and Glacier as differentials (17), and more recently as races (1). Change in virulence of L. maculans populations in a relatively short period of time has been recorded (11) and is one of the reasons why blackleg is considered one of the most important diseases affecting canola production. It is uncertain when L. maculans arrived to North Dakota; however, blackleg was readily observed by 1991, when canola production in the state was below 8,000 ha (~19,000 acres) (10). That year resulted in the first and most significant blackleg outbreak reported in the state. Lamey (10) reported that the outbreak was caused by strains belonging to pathogenicity group 2 (10). By 2002, most canola cultivars planted in the state were considered either resistant or moderately resistant to pathogenicity group 2 (2). In 2003, strains from pathogenicity groups 3, 4, and T were detected in canola residues from two North Dakota counties (3,4). The discovery of these new strains was the first indication that the virulence profile of L. maculans populations in North Dakota may be changing. In 2009, an end-of-season field survey indicated several fields in different parts of the state had unusually high levels of blackleg. Thus, a study was conducted to determine whether the outbreaks were associated with the recently discovered pathotypes. 10 April 2012 Plant Health Progress Disease Data and Sample Collection In 2009, 176 fields distributed in 25 North Dakota counties were scouted in late August for presence of blackleg. While the survey was part of a larger effort that estimated prevalence of other canola pests in the state (data not published), we are reporting results from counties with fields where blackleg incidence was >30%. Fields were scouted immediately after canola was cut and wind-rowed and the stems were still fresh. Efforts were made to scout roughly one field for every 2,025 ha (~5,000 acres) of canola planted in each county and, in counties with multiple samples, to space sampled fields at least 8 km (5 miles). Disease incidence in each field was estimated by visually inspecting the lower 20 cm of 50 stems for signs and/or symptoms of the disease. The most diagnostic features were the presence of cankers (Fig. 1), usually at the base of the stem that had pycnidia on them (6). Symptomatic samples composed mainly of up to five stems from some of these fields were brought to the laboratory to confirm the presence of the pathogen. Fig. 1. Stem canker caused by Leptosphaeria maculans on canola. L. maculans pycnidia are visible in area pointed by arrow. Stem tissues were washed in running tap water to eliminate soil particles and other debris. Stem pieces containing the cankered area were separated from the rest of tissues and split in halves. One half was stored for future reference and the other was surface disinfested by immersion in either commercial bleach (Clorox Regular bleach, The Clorox Company, Oakland, CA) for 10 sec or in a 30% aqueous solution of the bleach for 10 to 30 sec, depending on the condition of the sample. After the bleach treatment, the samples were lightly blotted with a sterile filter paper to remove excess moisture from its surface. Small pieces of infected tissues, usually containing pycnidia, were scraped with a sterile scalpel and mixed with several droplets of sterile distilled water (~0.05 ml each). Once in the water, tissues were finely chopped to suspend spores. A 0.5-ml sample of this suspension was streaked onto a 16% V8 medium amended with streptomycin and penicillin [837 ml distilled water, 163 ml V8 juice (Campbell Soup Co., Camden, NJ), 15 g agar (Bacto-Agar, Becton Dickinson and Co., Sparks, MD), and 3 g CaCO ; the pH of the medium was adjusted to 7.2 and, after sterilization by autoclave, amended with 7.5 ml streptomycin (10 μg/ml) 3 10 April 2012 Plant Health Progress and 7.5 ml of penicillin (10 μg/ml)]. Inoculated medium was incubated 3 to 4 days at 21°C in constant white fluorescent light. Single-spore colonies were transferred into dishes containing clean V-8 medium and incubated for two weeks as described. Spores from these colonies were collected by suspending the contents of each Petri dish in 5 ml sterile distilled water and transferring the suspension into a vial. Spore concentrations were adjusted to 107 spores/ml and stored at -20°C until used. Identification of Pathogenicity Groups The virulence phenotype of isolates retrieved from samples collected from 20 fields with the highest incidence were characterized using the North American standard differential set, comprised of three genotypes; Westar, Quinta, and Glacier (17). The inoculation method used was similar to that described by Chen and Fernando (4). Briefly, each cotyledon of ten-day-old seedlings was lightly pricked once with sharp tweezers and a 10-μl aliquot of a spore suspension was deposited on it. Inoculated seedlings were incubated for 24 h in a mist chamber to facilitate infection and then returned to the greenhouse for incubation at 22/18°C day/night temperature and 14-h photoperiod. Each isolate was inoculated on triplicate sets of six seedlings each. Disease reaction was evaluated 14 days after inoculation using a 0-9 scale developed by Williams and Delwiche (19). The median of 36 observations per isolate was calculated using the univariate procedure of SAS (Proc Univariate, SAS version 9.2, SAS Institute Inc., Cary, NC) and characterized as 0-2 resistant (R), 3-6 intermediate resistant (I), and 7-9 susceptible (S). Based on this reaction, the pathogenicity group (PG) of each isolate was established (Table 1). Table 1. Phenotypic reaction of Brassica napus differential cultivars to inoculation with Leptosphaeria maculans strains of different pathogenicity groups. x Inoculations made when seedlings were at the cotyledon stage using 107 spores/ml pycnidiospores suspensions. y Resistance genes present in differentials are included in parentheses. Table modified after Mengistu et al. (17). Blackleg Prevalence in 2009 Blackleg prevalence, proportion of scouted fields with blackleg symptomatic plants was high in ten North Dakota counties surveyed in 2009. Blackleg was observed in 88 of the 99 fields scouted, with 39 fields having blackleg incidences >30% (Table 2). The mean blackleg incidence in fields with ≤30% blackleg was 12% (range 7 to 20%), whereas in fields with >30% the mean was 61% (range 50 to 84%). The highest frequencies of fields with blackleg incidence >30% were observed in Renville, Mountrail, Ward, McHenry, and McLean counties. In these counties, between 47 and 78% of scouted fields had mean blackleg incidence >30%. These counties are located in North Central North Dakota, with the exception of McLean Co., which is located in the south central part of the state. Pathogenicity group Differential cultivarsy Westar Glacier Quinta