Transcript Field Evaluation and Greenhouse Screening of Canola

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 35806
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 (Cont’d.)
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 2005-06 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
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.
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, 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.
Photo by A. Ananga
METHODS
Field establishment of blackleg. Diseased stubble provided by USDAARS, 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. 1. Blackleg
fungal culture in
PDA.
Fig. 2. Test seedlings grown
in Promix potting soil.
Photo from National Research Council,
Canada
Fig. 3. Infected canola leaf
showing chlorosis (early
symptom).
Fig. 4. Canola with lesions
caused by blackleg disease.
RESULTS (Cont’d.)
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, 12H17, 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.
1B-4
IE-8
IG-7
NSL167291
NSL80306
2A-3
6A-7
5.00E-01
5C-3
5C1
3A-8
3A-7
6A-11
6D-9
6K-3
7H-3
A8-7
NSL44750
12J-18
NSL34575
10D-2
10B-1
NSL6105
12M-9
12K-8
12K-13
13C-4
18A-1
17D-1
17C-2
16A-1
45A71
18B3
14A1
30C1
7
4
4
2
2
2
1
11
3
9
1
1
1
2
1
5
2
7
3
3
1
3
1
1
9
18
3
5
1
1
1
4
1
6
2
Table 1. Field
infection (percent)
of canola accessions
and breeding lines
by blackleg.
Canola
Lines
Georgia
Isolate
Canada
Isolate
92
36
23.59
20.55
29.27
30.25
116
19.44
31.78
137
77
17
10.22
17.32
20.18
14.21
31.58
18.32
Westar
32.20
45.50
Cyclone
4.99
11.43
Table 2. Average percent plants infected
with Georgia and Canada blackleg isolates
(n=36).
12 –day old seedlings Wounded
Georgia isolate 2.32
Canada isolate 55
Unwounded
2.5
10
33-day old seedlings
Georgia isolate 16.66
41.66
Canada isolate 75.00
16.00
Table 3. Percent infection of wounded and unwounded
12- and 33-day old canola seedlings (n= 36).
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
50:10-27.
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.
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.