Development of a Loop-Mediated Isothermal Amplification ... · - Banana tissue. Sensitivity Up to 1...
Transcript of Development of a Loop-Mediated Isothermal Amplification ... · - Banana tissue. Sensitivity Up to 1...
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Eppo Meeting Angers; Dec 1, 2015
Development of a Loop-Mediated Isothermal Amplification assay for rapid detection of Fusarium oxysporum f.sp. cubensetropical race 4 through Diversity Array Technology sequencing
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Banana: plant at risk
Banana is the most important food in terms of production
value after rice, wheat and maize
About 107 million tons of bananas produced / year
>150 countries
• 13% is exported (>95% Cavendish)
• 87% is consumed
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Non-pathogenic strains>150 plant pathogenic,
divided into formae speciales
Pathogenic forms
Fusarium oxysporum complex
f.sp. cubense
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causal agent of Fusarium wilt/Panama disease
soil-borne (30-50 years in soil)
asexual fungus (asexual spores)
Guo et al. Plos One, Vol 9:4. April 2014
27-55 x 3.3-5.5µm 5-16 x 2.4-3.5µm 7-11µm
Fusarium oxysporum f.sp. cubense (Foc)
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Races, based on specific banana cultivars that they can infect
Banana cultivar Race 1 Race 2Race 4
ST4 TR4Gros Michel (AAA) Susceptible Resistant ? Susceptible
Bluggoe (ABB)Cooking type Resistant Susceptible ? Susceptible
Cavendish (AAA) Resistant Resistant Susceptible Susceptible
Diversity: Races & Vegetative Compatibility Groups (VCG)
Fusarium oxysporum diversity
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Banana cultivarGros Michel -> Cavendish
Threat to Gros Michel Banana Production
Global distribution of Fusarium wilt (Race 1) disease
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Banana producing countries TR4 occurrence
Global distribution of Foc TR4
2015
Threat to Cavendish Banana Production
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Photos credit to B.N. Corcolon
Foc TR4 devastation in the Philippines
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RacesVegetative
Compatibility groups24 reported VCGs
01213/16
0120, 0121, 0124, 0129
0120, 0124/5, 0125, 0126, 01210
0123, 0124
0122, 0128Race 2 -Plantain ABB
& cooking banana
ST4
Race 4 -Cavendish AAA
TR4
Race 1 -Gros Michel AAA
Fusarium oxysporum f.sp. cubense classification
VCG
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Vegetative Compatibility Groups
Visual inspection Molecular based probes
Disadvantage- Time consuming ~1 month- nitM tester not always available- Self-incompatible isolates
DisadvantageNot accurate diagnosis due to similar symptoms produce by other factors (e.g. Moko disease)
Source: Gasparotto, L.
Source: Gasparotto, L.
Source: Ordonez, N.
Disadvantage- Laboratory based-methods- Highly skilled workers- DNA extraction inhibitors
Diagnosis
Foc TR4 diagnostics
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Aim: Detection of unique genomic regions using DArTseqfor on-site TR4 specific probe development
DArTseq● Whole-genome fingerprint tool, without relying on prior
sequence data information (e.g. 15% genome coverage for Foc) ● Steps includes genome complexity reduction concept, via
combination of Restriction Enzymes and sequencing of the representations on Illumina
● Selection of unique and low-copy sequences (e.g. 69bp), by in silico hybridisations
Data #Markers
DArTseq 15 905
DNA extraction on:29 Foc isolates
All 24 VCGs and 4 races
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DArTseq principle
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Aim: Detection of unique genomic regions using DArTseqfor on-site TR4 specific probe development
DArTseq● Whole-genome fingerprint tool, without relying on prior
sequence data information (e.g. 15% genome coverage for Foc) ● Steps includes genome complexity reduction concept, via
combination of Restriction Enzymes and sequencing of the representations on Illumina
● Selection of unique and low-copy sequences (e.g. 69bp), by in silico hybridisations
Data #Markers
DArTseq 15 905
DNA extraction on:29 Foc isolates
All 24 VCGs and 4 races
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On-Site detection
80 Unique DArTseq fragments (69bp each) for TR4 from Silicon data
After blasting against all Foc and Fo genomes 12 Unique DArTseq TR4
LAMP primer design relies on a genomic region of around 400 bp
200bp 200bp
TR4 genome
sequence
Selection of unique DArTseq TR4 fragments
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500bp
500bp
M
M M
M MWat
er (
-)
Wat
er (
-)
Wat
er (
-)
0121
3
Foc 24 VCGs
0121
3
Foc 24 VCGs
0121
3
Foc 24 VCGs
Seq A Seq B
Seq C
Selection of unique DArTseq TR4 fragments
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Validation:Specificity
23 TR4 isolates45 non target DNA:
- Other Foc VCGs- Other F. oxysporum- Moko bacteria- Banana tissue
SensitivityUp to 1 pg pure TR4 DNA detected
After 7 days, TR4 was detected on the corm of Gran Naine banana plants
Loop-mediated Isothermal Amplification-LAMP - DNA amplification takes place at an
isothermal condition- Specific amplification to recognize 6
distinct regions on the target region- Costly and time effective - Not highly skilled personnel required
On-site diagnosis development on unique TR4 region
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Foc TR4 on-site field diagnosis
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Foc TR4 on-site field diagnosis
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DArTseq appeared to be a valuable tool for selection of unique fragments without relying on prior sequence data information
An unique TR4 region (~500bp) was targeted and used for diagnostic development
LAMP TR4 primer validation was successfully completed on a range of pure DNAs and banana tissue samples
LAMP TR4 validation under field conditions is ongoing
Conclusion
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Plant Research International &Phytopathology groupWageningen, The Netherlands
- Nadia Ordóñez Román- Maricar Salacinas- Odette Mendes- Michael Seidl- Cees Waalwijk- Gert Kema- Cor Schoen
Diversity Arrays Technology Pty. Ltd.Canberra, Australia
- Andrzej Kilian
Acknowledgements
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Discovery of a ~500bp unique TR4 area highly conservedand most likely an intergenic region
Source: Ordonez, N & Seidl, M., et al . PLOs Pathogens. in Press