© 2012 Plant Management Network.
Outbreak of Blackleg in Canola in North Dakota is Caused by New Pathogenicity Groups
Luis E. del Rio Mendoza, Achala Nepal, and Samuel Markell, Department of Plant Pathology, North Dakota State University, Fargo, ND 58102
del Rio Mendoza, L. E., Nepal, A., and Markell, S. 2012. Outbreak of blackleg in canola in North Dakota is caused by new pathogenicity groups. Online. Plant Health Progress doi:10.1094/PHP-2012-0410-01-RS.
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.
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.
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.
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 CaCO3; the pH of the medium was adjusted to 7.2 and, after sterilization by autoclave, amended with 7.5 ml streptomycin (10 µg/ml) 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
x Inoculations made when seedlings were at the cotyledon
y Resistance genes present in differentials are included in
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.
Table 2. Prevalence and incidence of blackleg (Leptosphaeria maculans) in canola (Brassica napus L.) fields in ten North Dakota counties in 2009.
x Values not calculated.
Pathogenicity Groups Associated with Highest Blackleg Incidences
Five different L. maculans pathotypes were identified among 59 isolates retrieved from residues collected from 20 fields in eight North Dakota counties (Table 3). The most frequently identified pathotype was PG-4, which accounted for 51% of all isolates evaluated, and was retrieved from samples from all ten counties. PG-T was the second most commonly identified pathotype accounting for 25% of all isolates evaluated. PG-T was identified from residues from five counties. Approximately 8% of isolates belong to PG-3, but only two of the 59 isolates evaluated belong to PG-2. The virulence profile of six other isolates could not be assigned to any of the five pathotypes; this group accounted for less than 12% of all isolates.
Table 3. Frequency of occurrence of Leptosphaeria maculans pathogenicity groups 1, 2, 3, 4, and T in 15 canola fields that had blackleg incidences >30% in 2009 in North Dakota.
x ND = Pathogenicity group not determined.
Discussion and Conclusions
Blackleg has re-emerged as a major production problem for canola producers in North Dakota. Data presented in this document provides evidence that the L. maculans population is increasing in virulence and new pathogenicity groups are frequently occurring in canola growing areas of the state. These new pathogenicity groups are able to overcome the resistance mechanisms present in most commercially available cultivars (13). Furthermore, these changes in the pathogen population may be responsible for the re-emergence of the disease, and may lead to more frequent and/or severe blackleg epidemics in the future. Additional resistance genes deployed into commercial genotypes and/or other management strategies will need to be used to manage the disease.
Historically, the first and most significant blackleg epidemic occurred on the widely planted cultivar Westar that was not known to have resistance to L. maculans. Westar was released in 1984 in Canada (9). Soon after its release, Westar replaced most other cultivars in Canada, where canola was well-established, but also in North Dakota, where canola hectarage was just starting to expand. The extensive cultivation of this cultivar, combined with short or absent crop rotation with non-host crops facilitated the development of blackleg epidemics in Canada (8) and North Dakota (10). The quick introduction of resistant cultivars reduced the immediate importance of blackleg, but simultaneously put pressure on the pathogen population to adapt.
The first evidence of virulence changes in the L. maculans populations in North Dakota came in 2003 with the recovery of isolates from two counties with virulence on the differential cultivars Glacier and Quinta. These isolates were later identified as PG-3, PG-4, and PG-T (3,4). In this study, PG-3, 4, and T were identified from ten of the 16 largest canola-producing counties in the state. Since only samples from heavily affected fields were processed for this study, one can only speculate that it is likely these pathotypes are already present in counties from which samples were not processed. Likewise, the near absence of PG-2 in samples from these fields could indicate the replacement of PG-2 by more virulent pathotypes is occurring. With the absence of commercial resistance to these new pathotypes, utilization of other disease-management strategies, such as crop-rotation and fungicide application, will become very important for canola production.
Since crop residues are the main source of inoculum (14), growers need to move away from practices that facilitate survival of L.maculans and exposure of plants to overwintering inoculum. Author observation indicates that planting canola in consecutive years or every other year is common in some areas of North Dakota. Since production of L. maculans inoculum is highest in the first two years the stubble residues are in the field (18), canola under these rotation schemes is exposed to high amounts of inoculum. However, lengthening crop rotations alone may not be enough to prevent blackleg epidemics from occurring as inoculum can produced in residues older than two years (18), and L. maculans spores can be transported long distances and from adjacent fields (12). Consequently, when planning crop sequences, growers should also try to maximize distances between fields with canola residues and newly planted canola. Marcroft et al. (12) estimated that, under Australian conditions, the minimum safe distance between a winter canola field and a field with canola residues is approximately 100 m. Furthermore, most canola fields in North Dakota are planted either under reduced or no-tillage conditions, practices that leave most residues on the soil surface. While the burial of crop residues reduces the rate of pathogen survival (7), tillage practices that invert the upper layer of soil are no longer popular and it is unlikely that growers would return to them due to concerns with soil erosion and higher cost.
Only two fungicides, azoxystrobin and pyraclostrobin, are currently registered for use against blackleg in North Dakota. Azoxystrobin has been registered since 2000 (15); however, the product has been seldom used because blackleg outbreaks had not been observed until now. Pyraclostrobin was registered for use in the 2010 growing season (16). While these fungicides can increase yield in the presence of disease, they represent a production expense that could be avoided if effective genetic resistance is available. In addition, both fungicides belong to FRAC group 11, thus judicious use of these chemicals will be needed to avoid fungicide-resistance development. Research aimed at expanding the list of registered compounds and determining the sensitivity of L. maculans to these fungicides is in progress (unpublished data).
The increasing prevalence of PG-3, PG-4, and PG-T reported in this study may recreate, to a point, the conditions that existed prior to the 1991 outbreak; that is, large areas planted to blackleg-susceptible canola genotypes with limited crop rotation. Moreover, the increased utilization of conservation tillage practices and higher statewide canola-acreage may increase inoculum loads to levels far higher than in 1991. In the absence of effective resistance to these emerging PG-types among commercial cultivars, better crop residue management and use of fungicides will be necessary to avoid blackleg epidemics as severe as in 1991.
1. Balesdent, M. H., Barbetti, M. J., Li, H., Sivasithamparam, K., Gout, L., and Rouxel, T. 2005. Analysis of Leptosphaeria maculans race structure in a worldwide collection of isolates. Pop. Biol. 95:1061-1071.
2. Berglund, D. R. 2003. 2002 canola variety trials. Coop. Ext. Bull. A-1124. North Dakota State Univ., Fargo, ND.
3. Bradley, C. A., Parks, P. S., Chen, Y., and Fernando, W. G. D. 2005. First report of pathogenicity groups 3 and 4 of Leptosphaeria maculans on canola in North Dakota. Plant Dis. 89:776.
4. Chen, Y., and Fernando, W. G. D. 2006. Prevalence and pathogenicity groups of Leptosphaeria maculans in western Canada and North Dakota, USA. Can. J. Plant Pathol. 28:533-539.
5. Fitt, B. D. L., Brun, H., Barbetti, M. J., and Rimmer, S. R. 2006. World-wide importance of phoma stem canker (Leptosphaeria maculans and L. biglobosa) on oilseed rape (Brassica napus). Eur. J. Plant Pathol. 114:3-15.
6. Gugel, R. K., and Petrie, G. A. 1992. History, occurrence, impact, and control of blackleg of rapeseed. Can. J. Plant Pathol. 14:36-45.
7. Huang, Y. J., Fitt, B. D. L., and Hall, A. M. 2003. Survival of A-group and B-group Leptosphaeria maculans (phoma stem canker) ascospores in air and mycelium on oilseed rape stem debris. Ann. Appl. Biol. 143:359-369.
8. Juska, A., Busch, L., and Tanaka, K. 1997. The blackleg epidemic in Canadian rapeseed as a "normal agricultural accident". Ecol. Appl. 7:1350-1356.
9. Klassen, A. J., Downey, R. K., and Capcara, J. J. 1987. Westar summer rape. Can. J. Plant Sci. 67:491–493.
10. Lamey, H. A. 1995. Blackleg and Sclerotinia disease of canola in North Dakota in 1991 and 1993. Plant Dis. 79:322-324.
11. Li, H., Sivasithamparam, K., and Barbetti, M. J. 2003. Breakdown of a Brassica rapa ssp. sylvestris single dominant blackleg resistance gene in B. napus rapeseed by Leptosphaeria maculans field isolates in Australia. Plant Dis. 87:752.
12. Marcroft, S. J., Sprague, S. J., Pymer, S. J., Salisbury, P. A., and Howlett, B. J. 2004. Crop isolation, not extended rotation length, reduces blackleg (Leptosphaeria maculans) severity of canola (Brassica napus) in south-eastern Australia. Australian J. Exp. Agric. 44:601-606.
13. Marino, D., and del Rio, L. E. 2010. Screening of plant introduction materials from Brassica species for resistance against PG3 and PG4 isolates of blackleg. (Abstr.) Phytopathology 100:S77.
14. McGee, D. C. 1977. Black leg (Leptosphaeria maculans (Desm.) Ces. et de Not.) of rapeseed in Victoria: sources of infection and relationships between inoculum, environmental factors and disease severity. Aust. J. Agric. Res. 28:53-62.
15. McMullen, M. P., and Lamey, H. A. 2000. 2000 Field crop fungicide guide. Coop. Ext. Serv. Publication PP-622 (revised). North Dakota State Univ., Fargo, ND.
16. McMullen, M. P., and Markell, S. G. 2009. 2010 North Dakota field crop fungicide guide. Coop. Ext. Serv. Publ. PP-622 (revised). North Dakota State Univ., Fargo, ND.
17. Mengistu, A., Rimmer, S. R., Kock, E., and Williams, P. H. 1991. Pathogenicity grouping of isolates of Leptosphaeria maculans on Brassica napus cultivars and their disease reaction profiles on rapid-cycling Brassica. Plant Dis. 75:1279-1282.
18. Petrie, G. A. 1995. Long-term survival and sporulation of Leptosphaeria maculans from blackleg-infected rapeseed/canola stubble in Saskatchewan. Can. Plant Dis. Surv. 75:23-34.
19. Williams, P. H., and Delwiche, P. A. 1980. Screening for resistance to blackleg of crucifers in the seedling stage. Pages 164-170 in: Proc. Eucarpia Cruciferae 1979 Conference. Wageningen, Netherlands.