Field Evaluation and Greenhouse Screening of Canola Breeding Lines for Blackleg Resistance – Preliminary Results R. Ward, L. Nyochembeng and E. Cebert Department of Plant and Soil Science Alabama A&M University P.O. Box 1208, Normal, AL METHODS (Cont’d.) Water suspension of fungal mycelia and pseudothecia was prepared by macerating fungal culture in deionized water using a blender. Inoculum estimates for both isolates were determined by counting mycelial fragments and/or pseudothecia in 1ml of suspension using a hemacytometer. Inoculum levels used were x 10 4 propagules/ml. Field evaluation. In the field, sixty four (64) canola lines including five national accessions (NSL) were planted in 3-m single rows with 3 replications on October 6, Plants were examined for blackleg infection approximately three months after soil inoculation. Plants that showed symptoms such as chlorotic spots and lodging were recorded; severity of symptoms was not rated. Greenhouse screening. In the greenhouse, five high yielding, early maturing canola lines (92, 36, 137, 116, 77 and 17) were evaluated for their susceptibility/resistance to Canada and Georgia blackleg isolates; Westar (susceptible) and Cyclone (resistant) were used as controls. Test seedlings were grown in Promix potting soil in flats (Fig 2). Fungal/water suspension was sprayed on the test plants to saturation using a hand sprayer. Inoculated plants were misted for 8 hours on day 1 and twice daily thereafter. Infected plants showing symptoms (Fig. 3) were counted two weeks after inoculation. Another greenhouse test was conducted to compare infection of 12- and 33-day old seedlings of early maturing canola (breeding line # 82) by Georgia isolate. Comparison was also made between wounded and unwounded leaves; wounding was done by scoring the upper surface without cutting through the leaves using a dissecting needle. RESULTS (Cont’d.) Greenhouse Screening. Comparisons between Georgia and Canada blackleg isolates in their efficacy to infect selected high yielding early maturing lines of canola. Initial pathogenic symptoms in test plants were discernible 10 days after inoculation. Our preliminary results presented in Table 2 indicated higher virulence of Canada than Georgia isolate; likewise, there was a variation in susceptibility/ resistance expressed by test lines. Data showed that test lines possess some level of resistance to both blackleg isolates; all lines tested were comparatively less susceptible to both isolates compared to Westar (S control). Among lines evaluated, line 137 had the lowest percent infected plants. However, all test lines performed poorly compared to Cyclone (R control). Several other high yielding lines of early maturing canola will be screened for their ability to resist blackleg. Other strains of blackleg disease will be used in later evaluations. Canola line 36 was used to determine relative susceptibility/ resistance of early maturing canola at two age groups, i.e., 12 and 33 days old seedlings, in greenhouse tests. Additionally, susceptibility of wounded versus unwounded leaves to blackleg infection was also determined. Our preliminary results are presented in Table 3. Data showed that 33-day old seedlings were more susceptible to blackleg infection than 12-day old seedlings and that wounding did not seem to improve infection of treated plants. For 12-day old seedlings, higher infection rates were observed on wounded than on unwounded plants; results also showed higher infection from Canada isolate compared to Georgia. However, compared to 12-day old seedlings, 33-day old plants showed increased susceptibility to infection. Also, for Georgia isolate wounded plants had lower infection rate compared to their unwounded counterparts. Additional tests will be conducted to also include other promising early maturing lines. Abstract Field evaluation and greenhouse screening of early maturing lines of canola were conducted to determine their susceptibility/resistance to blackleg disease. Field evaluations were done at Alabama A&M University’s Winfred Thomas Agricultural Research Station located in Meridianville. Field evaluation included several USDA germplasm accessions and high yielding early maturing canola breeding lines developed at AAMU. Canola stubble from blackleg infected field in Georgia were introduced into the test area in January; plants showing disease symptoms (i.e., leaf lesions and lodging) were determined for each test cultivar in April. Preliminary field data suggested variability in susceptibility to blackleg among test lines. However, the percentage of infected plants was low across lines; the low infection rates could be attributed to the lateness of soil inoculation as well as to the low level of inoculum present in the soil. In the greenhouse, the efficacy of fungal isolates from Canada and Georgia in infecting selected early maturing test lines, and the effects of seedling age and leaf-surface wounding to infectivity of blackleg were also determined. Results from our greenhouse tests showed Canada isolate to be more virulent than the Georgia isolate. Our data also showed variation in susceptibility of test lines to blackleg infection. Comparison with the susceptible (Westar) and resistant (Cyclone) controls indicated some degree of resistance to blackleg in all lines (92, 36, 137, 116, 77 and 17) tested. Preliminary results on the effects of leaf wounding and age of seedlings on blackleg infection were also presented. METHODS Field establishment of blackleg. Diseased stubble provided by USDA- ARS, Griffith, GA, were scattered on rows of canola seedlings in January Canola lines were planted on October 6, 2005 and were 4-6 in tall at date of inoculation. The stubble were broken into smaller pieces (2-6 in) and were scattered uniformly on canola plots. No attempt to measure inoculum levels in the soil was made at any time. Fungal cultures and inoculum. Two blackleg isolates (i.e., Canada and Georgia) were used in this study. Isolates from Georgia were obtained from infected canola stubble following the method described by (year). Canada isolate (Leroy PG2) was obtained from Canada and cultured in potato dextrose agar (PDA) or V8 fortified agar in Petri dishes at room temperature (Fig. 1). INTRODUCTION Numerous studies have shown that canola can be profitably grown in southeastern United States. However canola production in the region is practically non-existent primarily due to lack of market. However, with the growing interest in canola as feedstock for biodiesel production, growing canola commercially in the region becomes a realistic possibility. At this time, soybean is generally the main feedstock for biodiesel production primarily due to the large acreages planted to this farm crop. Likewise, farmers have grown soybean for generations; they are very familiar with the agronomic aspects of growing soybean. Comparatively, however, canola is superior to soybean in terms of oil production; canola seeds have better quality and higher oil content (40% versus 20%) than soybean. Canola farming is not exclusive of growing soybean; winter canola as a substitute for winter wheat can double-crop with soybean. As a winter crop, canola promises more revenue to farmers than winter wheat in the region. In the south, soybean is planted in late May or early June. With early maturing canola cultivars, farmers will have adequate time to prepare land for planting summer crops such as soybean. Canola lines bred for early maturity are also being evaluated for their susceptibility/resistance to blackleg disease. Blackleg, Leptosphaeria maculans, is one of the most destructive diseases of Brassicas, and is a serious problem in Canada, Australia and Europe (West et al. 2001) and in other areas where canola is grown. Although there are cultural and chemical methods of controlling this disease, the use of host plant resistance is considered most effective. Studies to identify sources of seedling and adult plant resistance have intensified with the growing acreages planted to canola worldwide. In this study, several national accessions and early maturing lines of canola were evaluated at Alabama A&M University’s Winfred Thomas Agricultural Research Station located in Meridianville, AL during cropping season. This study was conducted to: 1)Select canola from national accessions and early maturing lines that are best adapted to north Alabama 2)Determine susceptibility of canola lines to blackleg in the field 3)Evaluate resistance/susceptibility of early maturing lines of canola to Georgia and Canada blackleg isolates in the greenhouse Table 1. Field infection (percent) of canola accessions and breeding lines by blackleg. 1B-4 IE-8 IG-7 NSL NSL A-3 6A E-01 5C-3 5C1 3A-8 3A-7 6A-11 6D-9 6K-3 7H-3 A8-7 NSL J-18 NSL D-2 10B-1 NSL M-9 12K-8 12K-13 13C-4 18A-1 17D-1 17C-2 16A-1 45A71 18B3 14A1 30C Canola Lines Georgia Isolate Canada Isolate Westar Cyclone Table 2. Average percent plants infected with Georgia and Canada blackleg isolates (n=36). SUMMARY Field evaluation and greenhouse screening were conducted primarily to select for resistant lines of agronomically superior early maturing lines of winter canola currently being developed as part of the canola breeding program at Alabama A&M University. Only high yielding lines were included in our tests particularly those conducted in greenhouse. In the field, parental lines (USDA accessions) were included. Results from both field and greenhouse studies revealed several promising lines of early maturing winter canola; several showed a certain degree of resistance to blackleg. For future field evaluations, we plan to increase the level of blackleg inoculum in our test area to increase selection pressure on test lines. In the greenhouse, additional virulent strains of blackleg will be used to select for high performing lines of early maturing canola. RESULTS Field Evaluation. Our preliminary data are presented in Table 1. Among 64 lines tested, only 35 showed symptoms of infection ranging between 1 and 18 percent. The following breeding lines were not infected: IJ-12, 2A-13, 192A-16, 172A-14, 9A-3, 9A-7, 12I-18, 12H- 17, 1.20E-10, 10A-6, 12L-3, 13C-5, 1.30E-02, 18A-2, NSL80311, 13E-5, 13E-4, 18B-1, 15H-1, 20A-1, 20B-3, 19A-2 and 30B-7. The relatively low level of infection maybe due to low level of blackleg inoculum present in the soil. Prolonged drought during the cropping season could have also contributed to low level of infection. Regardless, variation in the susceptibility/resistance of test lines was evident. Further field tests are necessary before any conclusive statements could be made. Soil analysis should also be conducted to assess level of blackleg inocula in the soil before and after each season and to determine build up (or degradation) of fungal density in the soil. Fig. 2. Test seedlings grown in Promix potting soil. Fig. 3. Infected canola leaf showing chlorosis (early symptom). Photo by A. Ananga Fig. 1. Blackleg fungal culture in PDA. 12 –day old seedlingsWoundedUnwounded Georgia isolate Canada isolate day old seedlings Georgia isolate Canada isolate Table 3. Percent infection of wounded and unwounded 12- and 33-day old canola seedlings (n= 36). Fig. 4. Canola with lesions caused by blackleg disease. Photo from National Research Council, Canada LITERATURE CITED West, J. S., D. P. Kharbanda, J. M. Barbetti and L. D. B. Fitt Epidemiology and management of Leptosphaeria maculans (phoma stem canker) on oilseed rape in Australia, Canada and Europe. Plant Pathology 50:10-27.