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Contact GEVES
Geoffrey ORGEUR – +33(0)2 41 22 58 56 – [email protected]
Laboratory of Pathology – 25 rue Georges Morel 49071 Beaucouzé – France
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VALIDATION OF DETECTION PROTOCOL OF PHOMA LINGAM ON BRASSICAE SEEDS BY BLOTTER OR MEDIA
G. ORGEUR1, M. McEWAN2, A. CHAMAILLE1, B. LITOU1, M. LARENAUDIE1, C. ANDRO1, M. ROLLAND1
and V. GRIMAULT1.
1-Laboratoire de Pathologie, GEVES, rue Georges Morel, 49071 BEAUCOUZE-France ; 2-SASA, Roddinglaw Road, Edinburgh, EH12 9FJ, UK
INTRODUCTION
Results show no significant difference on detection of Phoma lingam by media or blotters protocols. The qualitative analysis shows a good sensitivity, specificity, repeatability and reproducibility of each method with a better accuracy for malt agar and blotter deep freezer protocols. The participants validated the better capacity of deep freezer to suppress the germination of seeds compared to the toxic herbicide 2,4-D. Two confirmation methods are available to identify the aggressive and non aggressive Phoma lingam isolates.
OBJECTIFS
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COMPARISON OF PROTOCOLS
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COMPARATIVE TESTS
Three seed lots (healthy, medium and high infected) were tested for homogeneitiy and validated by qualititive and quantitative analysis (Hampel’s method). 10 participants from France, Germany, United Kingdom, USA, Japan, Netherland, Australia and Israel compared the methods :
Malt agar (5 participants) / Blotters 2,4-D (8 participants) / Blotters Deep freezer (10 participants).
- Compare and judge efficiency and limits of different detection protocols of seed borne disease Phoma stem canker - Determine the optimal conditions to establish a common testing protocol. - Replace the ISTA 2.4-D method by another method (a deep freezer step) in order to stop germination of seeds.
First symptoms can appear after germination on seedling and cause damping off. Different protocols are described in litterature to detect L. maculans on media or blotters. For a better assessment of the pathogen, a step to suppress germination of seeds is used in the blotter test (Mathur and Kongsdal, 2003). Two different techniques exist : the toxic herbicide 2,4-D (ISTA method 7-004) and a deep freezer step (ISF method).
Leptosphaeria maculans (anamorph Phoma Lingam), is a worldwide pathogen causing black leg, canker, and dry rot of Brassicas and other crucifers. This causal agent is present in many regions around the world and responsible of serious economical damages (Fitt et al. 2008). Initial infections are mainly caused by ascospores dispersed from pseudothecia on infected debris (Smith et al. 1988).
Samples Malt Agar Blotter
Deep freezer
Blotter
2,4-D
1 0.10% 0.10% 0.10%
2 0.10% 0.10% 0.10%
3 0.10% 0.10% 0.10%
4 0.10% 0.00% 0.10%
5 0.10% 0.10% 0.10%
6 0.10% 0.10% 0.10%
Detection limit Contamination rate Inhibition of germination
64%
0%
13% 20%
0% 3% 0%
20%
40%
60%
80%
100%
Malt Agar
Blotters (Deep
freezer)
Blotters (2,4D)
Malt Agar
Blotters (Deep
freezer)
Blotters (2,4D)
High infected seeds lot Low infected seeds lot
% o
f ge
rmin
atio
n r
ate
Confirmation methods
Pathogenicity Test PCR
Primers provided by Liu et al. and PRI
Method of detection 2,4-D vs Deep freezer
Proposed work flow
With no significant difference in detection, all methods were conserved for CT. Inhibition of germination by deep freezer was included in the CT
No significant difference (p=0,58) between the methods Significant difference (p=0,02) between 2,4-D vs DF
30.4%
0.03% 0.1% 0.01%
47.3%
0.00% 0.0%
20.0%
40.0%
60.0%
80.0%
100.0%
Blotters (2,4D)
Blotters (Deep freezer)
% o
f ge
rmin
atio
n r
ate
Healthy Medium High
Pathogenicity Test
PCR
Results
expected
positive
Results
expected
negative
Sensitivity Specificity Accuracy
Results
positive 20 0
100 % 100 % 100 % Results
negative 0 17
Tested on the Phoma collection 20 target and 17 non target isolates
Malt Agar Blotters (2,4D)
Blotters (Deep freezer)
Sensitivity 100,0% 100,0% 100,0%
Specificity 100,0% 95,8% 98,6%
Accuracy 100,0% 85,2% 95,1%
Reproductibility 100,0% 95,5% 93,3%
Repeatability 100,0% 97,7% 100,0%
Qualititive Analysis
Pathogenicity Test
PCR
Seed health testing
Negative
Positive
Leptospheria maculans
Non target isolate
Positive Negative
Leptospheria biglobosa
Suspect isolates (Visual characteristics)
CONCLUSION
7.50%
11.25% 11.25%
0.25% 0.75% 0.50%
12.50%
9.50% 10.00%
0.25% 0.25% 0.50% 0%
5%
10%
15%
20%
Malt Agar
Blotters (Deep
freezer)
Blotters (2,4D)
Malt Agar
Blotters (Deep
freezer)
Blotters (2,4D)
High infected seeds lot Low infected seeds lot
% o
f P
ho
ma
lin
ga
m
% Phoma lingam detected at SASA
% Phoma lingam detected at GEVES
1.7% 2.8% 1.9%
6.9%
12.8%
8.9%
0.0%
5.0%
10.0%
15.0%
20.0%
Malt Agar Blotters (2,4D)
Blotters (Deep freezer)
% o
f P
ho
ma
lin
ga
m Healthy Medium High
Work flow PCR
Pathogenicity Test
L. maculans specific PCR
Seed health testing
Negative
Positive
Leptospheria maculans
Non target isolate
Positive Negative
Leptospheria biglobosa
Suspect isolates (Visual characteristics) Tested on Accuracy
+ 11 100
% - 25
Tested on Accuracy
+ 9 100
% - 27
Tested on Accuracy
+ 11 100
% - 25
Liu et al. 2006 L. maculans L. biglobosa
PRI (real-time) L. maculans
Major repeatability issue
The assay developped by PRI targets only L.
maculans
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