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DETECTION OF QUARANTINE AND BLACKLEG DISEASE-CAUSING BACTERIAIN POTATO SEEDS BY PCR
Christophe Debonneville, Denise Altenbach, and Dijana Stolz
EAPR Symposium, NeuchâtelSeptember 2, 2019.
BIOREBA AGYour Partner in Agro-Diagnostics
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BIOREBA AG
• Family-owned company (12 collaborators)
• Partner in Agro-diagnostics (plant pathogens) since 1980
• Export in > 100 countries
• ISO 9001 certified
• ISO/IEC 17025 accredited lab
• R+D, production, logistic and salesin Switzerland
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Blackleg disease testing on seed potatoes
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Partners in Switzerland
Contractor
-Development of PCR-based diagnostic tests (CTI project)
-Sample preparation
-DNA extraction and analysis
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• Testing is done mainly on imported seed potato lots
Blackleg disease in Switzerland
• Mostly classes SE, S (A).
• We test the following pathogens:
• Dickeya sp. • Pectobacterium atrosepticum• Pectobacterium wasabiae (P. parmentieri on potato)• Pectobacterium carotovorum subsp. brasiliense
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300 tubers = 1 sample
Step 1: Sample preparation
Subsamples 6 x 50
50 50 50 50 50 50
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Step 2: DNA Extraction
6 x 50: 48h bei 26°C
6 x 50: DNA Extraktion
Enrichment:
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6 x qPCR: - Dickeya sp. (Dsp) - Pectobacterium atrosepticum (Patr)
6 x PCR: Pectobacterium wasabiae (parmentieri) (Pwas)6 x PCR: Pectobacterium carotovorum ssp. brasiliense (Pcbr)
Step 3: PCR Analysis
Total: 18 PCR Reactions per seed potato lot
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Results in 2017 and 2018
2018: 47 imported seed lots
0%
≥ 3.5%
0.4 - 0.8%1.4 – 2.2%
Dickeya sp. P. atrosepticum P. wasabiae(parmentieri)
P.c.subsp.brasiliense
2017: 46 imported seed lots
Dickeya sp. P. atrosepticum P. wasabiae(parmentieri)
P.c.subsp.brasiliense
0%
≥ 3.5%
0.4 - 0.8%1.4 – 2.2%
2 2 2 17
3
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Quarantine bacteria testing
Clavibacter michiganensis subsp. sepedonicus (Cms)(causal agent of bacterial ring rot)
Ralstonia solanacearum (Rs)(causal agent of bacterial brown rot)
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Customers Collection of the seed lots (200 tubers each)
Seed potatocompany
Lab in Germany
Sample preparation
BIOREBATestservice
DNA extraction and qPCR
Lab-bill to seed company
BIOREBA-bill to seed company
BIOREBA – reporting direct to customers
Quarantine bacteria: workflow
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200 tubers = 1 sample
Quarantine bacteria: sampling
DNA extraction
qPCR analysis
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Quarantine bacteria: qPCR
Triplex qPCR:
Clavibacter michiganensis subsp. sepedonicus (Cms)
Ralstonia solanacearum (Rs)
Internal control gene (Cox)
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Quarantine bacteria: standards
Cms/Rs double-infected samples: dilution serie from bacteria spiked into healthy tuber sap.
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Quarantine bacteria: example
Cms Rs
Rs phylotypes I, II, and III are detected
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Quarantine bacteria: reporting
Vador DarkSchattenseite 531724 Schwarzesterne
Vador
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Thank you
Your BIOREBA Team