Saskatchewan, Canada
In vitro screening of barley coleoptiles against mycotoxins produced by Fusarium species causing Fusarium head blight
T.S. Grewal, B.G. Rossnagel and G.J. Scoles. Department of Plant Sciences/ Crop Development Centre, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK S7N 5A8 CANADA
Introduction
Barley Fusarium head blight (FHB) caused by Fusarium spp., especially F. graminearum Schwabe is of great economic importance in Canada and many areas of the world (Tekauz et al. 2000, Steffenson 2003) mostly because this fungus produces mycotoxins (especially trichothecenes) that make infected barley unsuitable for malting or feed. Deoxynivalenol (DON) is the most common mycotoxin in barley and other mycotoxins such as nivalenol, zearalenone, 3-aceltyl DON, 15-acetyl DON, and T-2 have also been observed (Campbell et al. 2000). Trichothecene is a pathogenicity factor in FHB of wheat (Eudes et al. 2001) as strains of F. graminearum null for the enzyme trichodiene synthetase demonstrated reduced virulence. Coleoptile tissue of FHB resistant wheat cultivars was 10 times more resistant to DON and other metabolites than that of susceptible cultivars (Wang and Miller 1988). Therefore, in-vitro screening of wheat coleoptiles for sensitivity to trichothecene may serve as a useful tool for screening (Eudes et al. 2000). The present study was conducted to determine the relationship between tolerance in barley at the coleoptile stage to the trichothecenes and disease at heading stage under field conditions.
Material and Methods
Purified mycotoxins (DON, 3-Acetyldeoxynivalenol (ADON), Nivalenol, T-2 Toxin) were purchased from Sigma-Aldrich, Inc. For preliminary experiments, four barley cultivars/lines with susceptible (S) and moderately resistant (MR) reaction to FHB viz. CDC Bold (S), CI4196 (MR), Stander (S), Chevron (MR) were used. Seeds were sterilized with 0.12 % formalin. Seeds were placed on the wet Whatman filter papers in Petriplates and incubated in the dark at 25°C for 3 days. After 3 days, two 6 mm segments of coleoptiles were cut with a knife, 3 mm below the apex. Four coleoptile segments were placed in each well of a 24 well Falcon® MultiwellTM plate with 0.5 mL of buffer (1.794g/L KH2PO4, 1.019g/L of citric acid, 2% sucrose, pH = 5.6) plus the trichothecenes mixture (DON 0.7 mg/L, 3-ADON 0.7 mg/L, Nivalenol 1.4 mg/L, T-2 Toxin 2.1 mg/L) (Eudes et al. 2000). There were 4 reps each for control (buffer only) and treatments (toxins with buffer). Each rep consisted of 4 coleoptile segments. Plates were incubated in the dark at 25°C for 24 h. Coleoptile segment length was measured under a stereo-microscope using an ocular micrometer. An experiment was conducted to evaluate the differences between 24 h and 48 h incubations and 1st and 2nd coleoptile segments. Subsequently, coleoptile segments of several barley lines with S and MR FHB reaction were screened against Fusarium mycotoxins using the standardized protocol.
Results and discussion
In preliminary experiments with four cultivars, coleoptile segment elongation in the control varied from 15.0 - 60.0% after 24 h of incubation indicating variability between lines for coleoptile elongation (data not shown). Per cent inhibition in treatments versus the control was greater for S than MR lines. There was no further coleoptile elongation when the plates were incubated for 48 h. Elongation in the first segment was generally greater than that of second segment. In further experiments, the 1st coleoptile segment was used and incubated for 24 h.
Several lines with S and MR field FHB reaction were evaluated. Initially we used ocular units and calculated % inhibition versus control (Table 1). The majority of MR lines had 10% or less inhibition versus control whereas inhibition in susceptible lines was more than 10%. Ocular units were then converted into mm and elongation in the control and treatments and % inhibition versus control was calculated (Table 2). In a majority of the MR lines, the growth of coleoptile segments in the control was much less than that of susceptible lines. This greatly influenced the % inhibition versus control as there was little growth in the control itself. When % inhibition versus control was calculated after removing the initial segment length (6 mm), only a few MR lines viz. 2ND16092, CDC Freedom, AC Metcalfe, CDC Kendall, Chevron showed significantly less inhibition versus the control and this was not consistent with field rating of S and MR lines. The overall mean inhibition in MR lines was numerically less than that of susceptible lines but statistically non-significant.
Trichothecenes have no role in the initial infection (Type I resistance) but
are a virulence factor for the spread of disease (Type II). Most barley lines have 'acceptable' levels of Type II resistance (Steffenson 2003) and breeders are looking for Type I resistance. While there was a general tendency for MR lines in the 2-rowed to demonstrate lower inhibition than S lines, in conclusion, resistance to mycotoxins at the coleoptile stage was not correlated to Type I resistance in barley.
References
Campbell, H., Choo, T. M., Vigier, B. and Underhill, L. 2000. Can. J. Plant Sci. 80: 977-980.
Eudes, F., Comeau, A., Rioux, S. and Collin, J. 2000. Can. J. Plant Pathol., 22: 286-292.
Eudes, F., Comeau, A., Rioux, S. and Collin, J. 2001. Can. J. Plant Pathol., 23: 318-322.
Steffenson, B.J. 2003. In: Leonard, K.J., Bushnell, W.R. (eds.): Fusarium Head Blight of Wheat and Barley. The American Phytopathological Society, St. Paul, Minnesota, USA. : 241-295.
Tekauz, A., McCallum, B., and Gilbert, J. 2000. Can. J. Plant Pathol., 22: 9-16.
Wang, Y. Z. and Miller, J. D. 1988. J. Phytopathology 122: 118-125.
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Length of coleoptile segments (ocular units)* |
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Cultivar |
Control |
Treatment |
% inhibition versus control |
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2 - rows |
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|
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CI4196 |
90.3 |
81.0 |
10 |
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HDE84194 |
87.4 |
79.6 |
9 |
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2ND16092 |
98.5 |
97.7 |
1 |
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|
TR306 |
112.6 |
96.2 |
15 |
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CDC Freedom |
109.3 |
98.7 |
10 |
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CDC McGwire |
106.4 |
88.9 |
16 |
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AC Metcalfe |
109.5 |
99.5 |
9 |
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CDC Kendall |
90.5 |
87.2 |
4 |
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MC9813-08 |
130.5 |
100.3 |
23 |
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TR360 |
89.6 |
80.2 |
11 |
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TR340 |
103.5 |
85.9 |
17 |
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Harrington |
116.3 |
91.9 |
21 |
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CDC Bold |
117.3 |
93.2 |
21 |
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HB353 |
128.3 |
100.6 |
22 |
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CDC Helgason |
98.7 |
86.9 |
12 |
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BM9203-74 |
101.8 |
88.6 |
13 |
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6- rows |
|
|
|
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Chevron |
99.6 |
91.8 |
8 |
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CDC Sisler |
116.9 |
94.8 |
19 |
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Stander |
113 |
91.6 |
19 |
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CDC Silky |
94.4 |
86.8 |
8 |
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Control refers to phosphate-citrate buffer only |
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Treatment refers to buffer + mycotoxins |
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* 6 mm = 75 on ocular micrometer. |
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The lines in bold are moderately resistant to FHB in field. |
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Control |
Treatment |
|
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Cultivar/Line |
Growth (mm)* |
Elongation** |
Growth (mm) |
Elongation |
% inhibition versus control*** |
|
2 - rows |
|
|
|
|
|
|
CI4196 |
1.2 |
20.4 |
0.5 |
8.0 |
61 |
|
HDE84194 |
1.0 |
16.5 |
0.4 |
6.1 |
63 |
|
2ND16092 |
2.2 |
37.3 |
1.5 |
25.2 |
32 |
|
TR306 |
3.0 |
50.1 |
1.7 |
28.3 |
44 |
|
CDC Freedom |
2.7 |
45.7 |
1.9 |
31.6 |
31 |
|
CDC McGwire |
2.5 |
41.9 |
1.1 |
18.5 |
56 |
|
AC Metcalfe |
2.8 |
46.0 |
2.0 |
32.7 |
29 |
|
CDC Kendall |
1.2 |
20.7 |
1.0 |
16.3 |
21 |
|
MC9813-08 |
4.4 |
74.0 |
2.0 |
33.7 |
55 |
|
TR360 |
1.2 |
19.5 |
0.4 |
6.9 |
65 |
|
TR340 |
2.3 |
38.0 |
0.9 |
14.5 |
62 |
|
Harrington |
3.3 |
55.1 |
1.4 |
22.5 |
59 |
|
CDC Bold |
3.4 |
56.4 |
1.5 |
24.3 |
57 |
|
HB353 |
4.3 |
71.1 |
2.0 |
34.1 |
52 |
|
CDC Helgason |
1.9 |
31.6 |
1.0 |
15.9 |
50 |
|
BM9203-74 |
2.1 |
35.7 |
1.1 |
18.1 |
49 |
|
6- rows |
|
|
|
|
|
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Chevron |
2.0 |
32.8 |
1.3 |
22.4 |
32 |
|
CDC Sisler |
3.4 |
55.9 |
1.6 |
26.4 |
53 |
|
Stander |
3.0 |
50.7 |
1.3 |
22.1 |
56 |
|
CDC Silky |
1.6 |
25.9 |
0.9 |
15.7 |
39 |
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LSD at 0.05 |
|
|
|
|
20 |
|
Control refers to phosphate-citrate buffer only |
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Treatment refers to buffer + mycotoxins |
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The lines in bold are moderately resistant to FHB in field. |
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* Length after 24 hours - initial growth (6 mm). |
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**Elongation as a % of initial length. |
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*** Elongation in control - Elongation in treatment/Elongation in control x 100 |
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