© 2011 Plant Management Network.
Identification of Brassica napus Lines with Partial Resistance to Sclerotinia sclerotiorum
S. D. Khot, V. N. Bilgi, and L. E. del Río, Department of Plant Pathology, North Dakota State University, Fargo, ND 58108; and C. A. Bradley, Department of Crop Sciences, University of Illinois, Urbana, IL 61801
Khot, S. D., Bilgi, V. N., del Río, L. E., and Bradley, C. A. 2011. Identification of Brassica napus lines with partial resistance to Sclerotinia sclerotiorum. Online. Plant Health Progress doi:10.1094/PHP-2010-0422-01-RS.
A collection of Brassica napus plant introduction (PI) lines was evaluated in a series of research trials to identify lines with resistance to Sclerotinia sclerotiorum, causal agent of Sclerotinia stem rot of canola. Five PI lines (169080, 286418, 436554, 458940, and 633119) were identified that consistently had SSR resistance levels equal to or greater than the partially resistant check cultivar Hyola 357. In addition, two of these lines (436554 and 458940) were found to exhibit SSR field resistance levels similar to Hyola 357. The PI lines identified from our research studies could be used in canola breeding programs to develop cultivars with improved resistance to S. sclerotiorum.
Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum, is endemic to canola (Brassica napus) production areas in North Dakota, and can cause severe yield reductions (6). Canola growers in North Dakota are limited to applying fungicides and rotating with non-host crops for management of SSR. Efficacious fungicides are available for control of SSR on canola (7). However, the cost of a fungicide application is more than most growers are willing to spend, especially considering that it takes at least a 17% incidence of SSR for the yield reduction to equal the cost of a fungicide (9). Crop rotation as a solo SSR management practice is not very effective due to the persistent nature of S. sclerotiorum sclerotia in soil (15). If canola cultivars with resistance to S. sclerotiorum were available, growers would be able to manage SSR more effectively and less expensively.
While no B. napus canola or rapeseed cultivar has shown complete resistance to S. sclerotiorum, differences in the response of some cultivars or breeding lines have been reported (2,5,12,16,17,18,21,23,27), thus encouraging further efforts to screen B. napus germplasm for resistance to S. sclerotiorum.
The objective of this research was to screen the B. napus accessions maintained by the United States Department of Agriculture-Agricultural Research Service (USDA-ARS) National Plant Germplasm System (NPGS) and identify accessions with resistance to S. sclerotiorum.
Brassica napus Plant Introduction Lines, Canola Cultivars, and Sclerotinia sclerotiorum Isolate
A collection of B. napus plant introduction (PI) lines maintained by the USDA-ARS NPGS at the North Central Regional PI Station (Ames, IA) was used in this research. In addition, the canola cultivars Hyola 357 and Pioneer 44A89 were used as partially resistant and susceptible check cultivars, respectively (4). Isolate WM009 of S. sclerotiorum, obtained from canola growing in a field in Benson County, ND, was used in this research; this isolate had been used previously to screen B. napus lines and canola cultivars for resistance to S. sclerotiorum in other studies (5,27). The WM009 isolate was shown to be the most aggressive on canola of four S. sclerotiorum isolates from canola in a greenhouse test (C. A. Bradley, unpublished). In addition, research by Zhao et al. (27) demonstrated that among four S. sclerotiorum isolates used to inoculate B. napus lines, the WM009 isolate (referred to as "Benson" in the article) did not differ in overall virulence compared to the other isolates.
Greenhouse and Growth Chamber Screening for Resistance to Sclerotinia sclerotiorum
B. napus PI lines and canola cultivars evaluated in greenhouse and growth chamber trials were inoculated with S. sclerotiorum using a petiole inoculation technique. This technique was found to differentiate B. napus lines and canola cultivars for their reaction to S. sclerotiorum in previous research (5,27). To perform inoculations, the larger opening of a sterile 200-µl pipette tip was pushed into the margin of a 3- to 4-day-old S. sclerotiorum colony growing on potato dextrose agar (PDA; Becton, Dickinson and Company, Sparks, MD) to acquire a 6- to 8-mm-thick plug. Petioles of the third fully expanded leaf (growth stage 2.3) (13) were selected for inoculation. The petioles were severed approximately 2.5 cm from the main stem using a razor blade. Holding the tapered end of each inoculum-filled pipette tip, the petiole was forced through the agar plug within the pipette. Plants were evaluated for a 10-day period and mortality for each day was recorded on the day that a plant exhibited irreversible wilt (27) or exhibited a girdling lesion so severe that the stem portion above the lesion toppled over. The area under disease progress curve (AUDPC) was calculated using the daily cumulative percentage of dead plants (25).
The screening process was conducted in three stages, with only the more resistant materials being advanced to the next stage. Eight to nine seeds of each accession were planted in a potting mix (Sunshine mix no. 1, SunGro Horticulture Canada Ltd., Seba Beach, AB, Canada) in 266 cm³ plastic drinking cups with holes in the bottom for drainage. Cups were watered daily. The plants were thinned to 5 per cup after they had reached growth stage 1 (13). Each cup was considered to be an experimental unit. In the greenhouse, high-pressure sodium lamps (1,000 µmol/m²/s) were used to supplement natural sunlight for 16 h per day, and temperature in the greenhouse ranged between 20 to 25°C. The growth chamber was set to maintain a 14-h light and 10-h dark cycle with day and night temperature at 25 and 23.5°C, respectively.
Stage 1. Stage 1 was conducted to identify and discard susceptible PI lines. Because of greenhouse space constraints, stage 1 was composed of 3 trials in which 144, 145, and 232 different accessions were evaluated without replicates. In each trial, the partially-resistant and susceptible checks were included. The checks have been identified above (5). PI lines with a mean AUDPC value at least 1.0 standard deviation below the grand mean for all PI lines were advanced to stage 2 (4,14,19,20). To determine if data from the 3 trials could be combined, the data from the check cultivars were analyzed by analysis of variance (ANOVA) using the general linear model procedure (PROC GLM) in SAS (version 9.2, SAS Institute Inc., Cary, NC) to determine if there was a significant (P ≤ 0.05) trial × cultivar interaction.
No significant trial × cultivar interaction was found for the check cultivars; therefore, data from all three trials were combined. Of the 521 B. napus PI lines planted, only 447 were inoculated and evaluated due to germination and emergence problems. The AUDPC values ranged from 0 to 900 in the lines (Fig. 1). The partially resistant and susceptible check cultivars, Hyola 357 and Pioneer 44A89, had AUDPC values of 25 and 448, respectively. The overall AUDPC mean was 367, and the standard deviation was 320; therefore, 141 PI lines which had AUDPC values ≤ 47 (1 standard deviation below the overall mean) were advanced to stage 2.
Stage 2. In stage 2 of screening in the greenhouse, a randomized complete block (RCB) design with three replications was used to evaluate 47 of the 141 PI lines and the two checks. Seeds from the other lines did not germinate due to unknown reasons. The trial was repeated once. Data were analyzed by ANOVA using PROC GLM in SAS. Fisher’s protected least significant difference (LSD, α = 0.05) was used to compare AUDPC means.
The two trials were combined and analyzed together since no significant trial × PI line interaction was observed. The AUDPC values of the partially resistant and susceptible check cultivars, Hyola 357 and Pioneer 44A89, were 0 and 875, respectively (Table 1). Twenty PI lines did not differ significantly from the partially resistant check cultivar Hyola 357. These twenty lines were advanced to stage 3.
Table 1. Reactions of forty-nine Brassica napus plant introduction lines or canola cultivars to Sclerotinia sclerotiorum inoculated in the greenhouse using a petiole inoculation techniquex.
x Combined data from two repeated trials.
y Area under disease progress curve.
z Fisher’s protected least significant difference where α = 0.05.
Stage 3. Stage 3 screening was conducted in the growth chamber in two replicated trials repeated over time. The two trials were arranged in a RCB design with three replications in each trial. In addition to Hyola 357 and Pioneer 44A89 being included as check cultivars, the partially resistant check cultivar Major was included (27). Data were analyzed using PROC GLM in SAS, and Fisher’s protected LSD was used to compare AUDPC means.
The two trials were combined and analyzed together since no significant trial × PI line interaction was observed. The AUDPC values of the check cultivars were 614, 306, and 58 for Pioneer 44A89, Major, and Hyola 357, respectively (Table 2). Seven of the 20 PI lines had AUDPC values that did not differ significantly from Hyola 357, and thirteen PI lines had AUDPC values that either were significantly lower than or not different than Major.
Table 2. Reactions of twenty Brassica napus plant
x Combined data from two repeated trials.
y Area under disease progress curve.
z Fisher’s protected least significant difference where α = 0.05.
Field Screening for Resistance to Sclerotinia sclerotiorum
Field trials were conducted during the summers of 2004 and 2006 at the North Dakota State University Agricultural Experiment Station in Fargo, ND. S. sclerotiorum inoculum was prepared by infesting sterilized seeds of pearl millet (Pennisetum typhoides). To do this, pearl millet seeds were placed in 25 × 38 cm aluminum pans, soaked in distilled water for 24 h, drained, covered with aluminum foil, and autoclaved for 1 h at 121°C on two consecutive days. Three-day-old cultures of S. sclerotiorum growing on PDA were cut into approximately 1 cm² strips and mixed into the sterilized seeds. The newly infested pearl millet seeds were incubated at 21°C for approximately 11 days. Pans were shaken daily to allow S. sclerotiorum to evenly colonize the sterilized seed. The inoculum was stored at 4°C until used.
Eight PI lines (169075, 436554, 458939, 458940, 469758, 469863, 469898, and 649141) and the check canola cultivars Hyola 357 and Pioneer 44A89 were planted in small hill plots using a seed drill modified to plant hill plots (Hege seed drill, Hege Equipment Company, Colwich, KS). These eight PI lines were chosen for field testing because of their advancement to stage 3 growth chamber screening (described above). In addition, these PI lines demonstrated a high level of seed germination and plant emergence in greenhouse and growth chamber tests, and adequate seed supply was available for these PI lines for use in the field trials. Twenty seeds of each PI line or cultivar were planted into each hill and hills were spaced 30 cm apart. Seeds were planted on 27 and 23 May in 2004 and 2006, respectively. The experimental design was a RCB with three replications in 2004 and four replications in 2006.
Plants were inoculated with S. sclerotiorum infested pearl millet seeds when the majority of the lines and cultivars had reached the mid to late flowering stage (growth stage 4.2) (13) on 8 and 19 July in 2004 and 2006, respectively. Approximately 10 ml of inoculum was evenly sprinkled onto the plants in each hill plot by hand. A mist-irrigation system was set up in the field each year to provide constant leaf wetness for approximately three weeks post-inoculation. The mist-irrigation system used misting nozzles spaced 9 m apart with risers 1.2 m tall and delivered 7 liters/min for 3 min every 30 min. Prior to inoculation, plants were briefly misted with water so that the inoculum would adhere to the plants. Three weeks after inoculation, SSR severity was measured using a lesion phenotype (LP) index on a 0 to 4 scale, where: 0 = no symptom, no lesion, no water-soaking, and no wilt; 1 = small lesion at junction of petiole and stem, no water-soaking, and no wilt; 3 = expanded, sunken, water-soaked lesion and no wilt; and 4 = expanded, sunken, water-soaked lesion resulting in irreversible wilt of foliage (27). LP index values were compared using Friedman’s non-parametric test. In preparing the data, the median values and respective mean ranks for all PI lines and cultivars were calculated using PROC RANK in SAS. Then PROC MIXED with the ANOVAF option was used to calculate the ANOVA-type statistic treatment estimated relative effects as described by Shah and Madden (24). Estimated relative effects and confidence intervals for relative effects were calculated using the ld_ci.sas macro from E. Brunner (University of Gottingen, Germany).
When comparing relative treatment effects in the 2004 trial, the moderately resistant check cultivar Hyola 357 and all PI lines except 469758 differed significantly from the susceptible check cultivar Pioneer 44A89 (Table 3). In addition, the PI lines that were significantly different from Pioneer 44A89 were not significantly different than the moderately resistant check cultivar Hyola 357. In 2006, only Hyola 357 and PI 649149 had relative treatment effects that were significantly different from Pioneer 44A89. The PI lines 436554, 458939, 458940, 469898, and 649149 were not significantly different from Hyola 357.
Table 3. Reactions of ten Brassica napus plant introduction lines or canola cultivars to Sclerotinia sclerotiorum inoculated in field trials at Fargo, ND, in 2004 and 2006.
x Treatment mean rank = (Σ Ri /n), where Ri represent each rank value given to a particular treatment (one per replication) and n represents the number of replications.
y Estimated relative treatment effect, where R is the treatment mean rank, and N is the total number of observations in the study.
Discussion and Conclusions
Several PI lines that consistently had SSR resistant levels equal to or greater than the partially resistant check cultivar Hyola 357 were identified through these series of screening, and included the following five PI lines: 169080, 286418, 436554, 458940, and 633119. Two of these PI lines were evaluated in field trials (436554 and 458940), and were shown to have SSR resistant levels similar to Hyola 357 in the field. The sources of these PI lines were Turkey, Nepal, China, Japan, and the United States, respectively. Other researchers have reported SSR partially resistant B. napus germplasm lines from Australia, China, and France (17,18,27).
In our research trials reported here, only one isolate of S. sclerotiorum was used to identify B. napus lines with partial resistance to S. sclerotiorum. The S. sclerotiorum isolate used in our research was shown to be highly-aggressive on B. napus in comparison to three other S. sclerotiorum isolates in a preliminary experiment (C. A. Bradley, unpublished), and Zhao et al. (27) found this isolate to be similar to three other S. sclerotiorum isolates in overall virulence on B. napus. In a preliminary growth chamber study (C. A. Bradley, unpublished) with seven B. napus lines and three S. sclerotiorum isolates from canola, sunflower (Helianthus annuus), and soybean (Glycine max), no significant (P ≤ 0.05) B. napus line × S. sclerotiorum isolate interaction was observed for AUDPC values, which indicated that B. napus lines reacted similarly to each S. sclerotiorum isolate. In addition, Zhao et al. (27) reported no differential interaction with specific B. napus accessions among the four S. sclerotiorum isolates, suggesting that the isolates had not developed specificity for B. napus accessions. Similarly, Pratt and Rowe (22) reported that alfalfa (Medicago sativa) cultivars responded similarly to five S. sclerotiorum isolates, and Auclair et al. (1) reported no significant interactions between five soybean cultivars and four S. sclerotiorum isolates when measuring disease severity. These previous research results (1,22,27) along with our preliminary results suggest that using only one S. sclerotiorum isolate to screen for resistance to SSR in B. napus accessions is appropriate; however, results of recent research conducted in Australia reported by Garg et al. (11) indicated that significant differences in pathogenicity were observed between different S. sclerotiorum isolates across different Brassica genotypes and that more than one S. sclerotiorum isolate should be used in germplasm screening programs. In light of the results reported by Garg et al. (11), it is important that B. napus lines identified to have partial resistance to S. sclerotiorum be further subjected to additional S. sclerotiorum isolates to verify their reactions across isolates.
The PI lines identified from our research studies could be used in canola breeding programs to develop cultivars with improved resistance to S. sclerotiorum. Zhao and Meng (26) and Zhao et al. (28) identified loci associated with partial resistance to SSR in B. napus lines. Studies to evaluate the genetics of resistance to S. sclerotiorum in the PI lines identified in our research need to be conducted. This would allow for the development of markers that could be used to incorporate these genetics into a canola breeding program more efficiently.
Germplasm accessions of other Brassica species maintained by the USDA-ARS NPGS need to be evaluated for their resistance to SSR, such as B. rapa and B. juncea, as canola-quality B. rapa and B. juncea cultivars also can be developed. B. juncea lines from Australia, China, and India with partial resistance to SSR have been identified by other researchers (16,17,18). In addition, partial resistance to SSR also has been identified in B. carinata and B. oleracea lines (3,10,23). Accessions of non-Brassica species of crucifers also should be evaluated. Chen et al. (8) and Garg et al. (12) reported that lines derived from B. napus, B. juncea, and B. rapa crosses with other crucifer species (Capsella bursa-pastoris, Diplotaxis tenuisiliqua, Erucastrum abyssinicum, and E. cardaminoides) had improved levels of resistance to SSR.
This research was supported by grants from the USDA-ARS Sclerotinia Initiative, the USDA-ARS Crucifer Crop Germplasm Committee, the Northern Canola Growers Association, and the North Dakota State Board of Agricultural Research and Education. We thank C. Chesrown for assistance in the field, and S. Neate and P. Gross (Barley Pathology Program) for lending the hill plot planter.
1. Auclair, J., Boland, G. J., Kohn, L. M., and Rajcan, I. 2004. Genetic interactions between Glycine max and Sclerotinia sclerotiorum using a straw inoculation method. Plant Dis. 88:891-895.
2. Bailey, D. J. 1987. Screening for resistance to Sclerotinia sclerotiorum in oilseed rape using detached leaves. Tests Agrochem. Cult. 8:152-153.
3. Baswana, K. S., Rastogi, K. B., and Sharma, P. P. 1991. Inheritance of stalk rot resistance in cauliflower (Brassica oleracea var. Botrytis L.). Euphytica 57:93-96.
4. Bradley, C. A., Hartman, G. L., Nelson, R. L., Mueller, D. S., and Pedersen, W. L. 2001. Response of ancestral lines and commercial cultivars to Rhizoctonia root and hypocotyl rot. Plant Dis. 85:1091-1095.
5. Bradley, C. A., Henson, R. A., Porter, P. M., LeGare, D. G., del Rio, L. E., and Khot, S. D. 2006. Response of canola cultivars to Sclerotinia sclerotiorum in controlled and field environments. Plant Dis. 90:215-219.
6. Bradley, C. A., and Lamey, H. A. 2005. Canola disease situation in North Dakota, U.S.A., 1993-2004. Pages 33-34 in: Proc. 14th Australian Research Assembly on Brassicas. Australia, Port Lincoln, SA.
7. Bradley, C. A., Lamey, H. A., Endres, G. J., Henson, R. A., Hanson, B. K., McKay, K. R., Halvorson, M., LeGare, D. G., and Porter, P. M. 2006. Efficacy of fungicides for control of Sclerotinia stem rot of canola. Plant Dis. 90:1129-1134.
8. Chen, H. F., Wang, H., and Li, Z. Y. 2007. Production and genetic analysis of partial hybrids in intertribal crosses between Brassica species (B. rapa, B. napus) and Capsella bura-pastoris. Plant Cell Rep. 26:1791-1800.
9. del Río, L. E., Bradley, C. A., Henson, R. A., Endres, G. J., Hanson, B. K., McKay, K., Halvorson, M., Porter, P. M., LeGare, D. G., and Lamey, H. A. 2007. Impact of Sclerotinia stem rot on yield of canola. Plant Dis. 91:191-194.
10. Dickson, M. H., and Petzoldt, R. 1996. Breeding for resistance to Sclerotinia sclerotiorum in Brassica oleracea. Acta Hort. 408:103-108.
11. Garg, H., Kohn, L. M., Andrew, M., Li, H., Sivasithamparam, K., and Barbetti, M. J. 2010. Pathogenicity of morphologically different isolates of Sclerotinia sclerotiorum with Brassica napus and B. juncea genotypes. Eur. J. Plant Pathol. 126:305-310.
12. Garg, H., Atri, Chhaya, Sandhu, P. S., Kaur, B., Renton, M., Banga, S. K., Singh, H., Singh, C., Barbetti, M. J., and Banga, S. S. 2010. High level of resistance to Sclerotinia sclerotiorum in introgression lines derived from hybridization between wild crucifers and the crop Brassica species B. napus and B. juncea. Field Crops Res. 117:51-58.
13. Harper, F. R., and Berkenkamp, B. 1975. Revised growth-stage key for Brassica campestris and B. napus. Can. J. Plant Sci. 55:657-658.
14. Hartman, G. L., Huang, Y. H., Nelson, R. L., and Noel, G. R. 1997. Germplasm evaluation of Glycine max for resistance to Fusarium solani, the causal organism of sudden death syndrome. Plant Dis. 81:515-518.
15. Huang, H. C., and Kozub, G. C. 1994. Longevity of normal and abnormal sclerotia of Sclerotinia sclerotiorum. Plant Dis. 78:1164-1166.
16. Li, C. X., Li, H., Siddique, A. B., Sivasithamparam, K., Salisbury, P., Banga, S. S., Banga, S., Chattopadhyay, C., Kumar, A., Singh, R., Singh, D., Agnihotri, A., Liu, S. Y., Li, Y. C., Tu, J., Fu, T. D., Wang, Y. F., and Barbetti, M. J. 2007. The importance of the type and time of inoculation and assessment in the determination of resistance in Brassica napus and B. juncea to Sclerotinia sclerotiorum. Austral. J. Agric. Res. 58:1198-1203.
17. Li, C. X., Li, H., Sivasithamparam, K., Fu, T. D., Li, Y. C., Liu, S. Y., and Barbetti, M. J. 2006. Expression of field resistance under Western Australian conditions to Sclerotinia sclerotiorum in Chinese and Australian Brassica napus and Brassica juncea germplasm and its relation with stem diameter. Austral. J. Agric. Res. 57:1131-1135.
18. Li, C. X., Liu, S. Y, Sivasithamparam, K., and Barbetti, M. J. 2009. New sources of resistance to Sclerotinia stem rot caused by Sclerotinia sclerotiorum in Chinese and Australian Brassica napus and B. juncea germplasm screened under Western Australian conditions. Australas. Plant Pathol. 38:149-152.
19. Mueller, D. S., Hartman, G. L., Nelson, R. L., and Pedersen, W. L. 2002. Evaluation of Glycine max germ plasm for resistance to Fusarium solani f. sp. glycines. Plant Dis. 86:741-746.
20. Mueller, D. S., Nelson, R. L., Hartman, G. L., and Pedersen, W. L. 2003. Response of commercially developed soybean cultivars and the ancestral soybean lines to Fusarium solani f. sp. glycines. Plant Dis. 87:827-831.
21. Newman, P. L., and Bailey, D. J. 1987. Screening for resistance to Sclerotinia sclerotiorum in oilseed rape in the glasshouse. Tests Agrochem. Cult. 8:150-151.
22. Pratt, R. G., and Rowe, D. E. 1995. Comparative pathogenicity of isolates of Sclerotinia trifoliorum and S. sclerotiorum on alfalfa cultivars. Plant Dis. 79:474-477.
23. Sedun, F. S., Seguin-Swartz, G., and Rakow, G. F. W. 1989. Genetic variation in reaction to Sclerotinia stem rot in Brassica species. Can. J. Plant Sci. 69:229-232.
24. Shah, D. A., and Madden, L. V. 2004. Nonparametric analysis of ordinal data in designed factorial experiments. Phytopathology 94:33-43.
25. Tooley, P. W., and Grau, C. R. 1984. Field characterization of rate-reducing resistance to Phytophthora megasperma f. sp. glycinea in soybean. Phytopathology 74:1201-1208.
26. Zhao, J., and Meng, J. 2003. Genetic analysis of loci associated with partial resistance to Sclerotinia sclerotiorum in rapeseed (Brassica napus L.). Theor. Appl. Genet. 106:759-764.
27. Zhao, J., Peltier, A. J., Meng, J., Osborn, T. C., and Grau, C. R. 2004. Evaluation of Sclerotinia stem rot resistance in oilseed Brassica napus using a petiole inoculation technique under greenhouse conditions. Plant Dis. 88:1033-1039.
28. Zhao, J., Udall, J. A., Quijada, P. A., Grau, C. R., Meng, J., and Osborn, T. C. 2006. Quantitative trait loci for resistance to Sclerotinia sclerotiorum and its association with a homeologous non-reciprocal transposition in Brassica napus L. Theor. Appl. Genet. 112:509-516.