Field Evaluation and Greenhouse Screening of Canola Breeding Lines for Blackleg Resistance

1
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 AM University P.O. Box
1208, Normal, AL 35806
Abstract Field evaluation and greenhouse
screening of early maturing lines of canola were
conducted to determine their susceptibility/resist
ance to blackleg disease. Field evaluations were
done at Alabama AM Universitys 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 (Contd.) 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 30-40 x 10 4 propagules/ml. Fiel
d 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, 2005. 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 (Contd.) 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.

• 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 AM
  Universitys Winfred Thomas Agricultural Research
  Station located in Meridianville, AL during
  2005-06 cropping season.
• This study was conducted to
• Select canola from national accessions and early
  maturing lines that are best adapted to north
  Alabama
• Determine susceptibility of canola lines to
  blackleg in the field
• Evaluate resistance/susceptibility of early
  maturing lines of canola to Georgia and Canada
  blackleg isolates in the greenhouse

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.
Canola Lines Georgia Isolate Canada Isolate
92 23.59 29.27
36 20.55 30.25
116 19.44 31.78
137 10.22 14.21
77 17.32 31.58
17 20.18 18.32
Westar 32.20 45.50
Cyclone 4.99 11.43

Table 2. Average percent plants infected with
Georgia and Canada blackleg isolates (n36).
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 AM 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.
12 day old seedlings Wounded Unwounded
Georgia isolate 2.32 2.5
Canada isolate 55 10
33-day old seedlings
Georgia isolate 16.66 41.66
Canada isolate 75.00 16.00

METHODS Field establishment of blackleg.
Diseased stubble provided by USDA-ARS, Griffith,
GA, were scattered on rows of canola seedlings
in January 2006. 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).
Fig. 2. Test seedlings grown in Promix potting
soil.
Table 3. Percent infection of wounded and
unwounded 12- and 33-day old canola seedlings (n
36).
Photo from National Research Council, Canada
LITERATURE CITED West, J. S., D. P. Kharbanda, J.
M. Barbetti and L. D. B. Fitt. 2001. Epidemiology
and management of Leptosphaeria maculans (phoma
stem canker) on oilseed rape in Australia, Canada
and Europe. Plant Pathology 5010-27.
Table 1. Field infection (percent) of canola
accessions and breeding lines by blackleg.
Fig. 4. Canola with lesions caused by blackleg
disease.