Sustainable alternatives to manage crop
diseases: Potato late blight management
in Nariño, Colombia – a case study
Present...
Seguridad Alimentaria y Nutrición
Third parties
Food Security Narino, Colombia
Improving Potato Production for
Increased F...
Project Philosophy
We have our own
way of life, but we
welcome your
help to improve
our food security
1. Nutrition
and health
2. Potato
breeding
3. Nutrients
and functional
foods in
potato
4. Resistance
to late blight
of pot...
Potato late blight (gota)
Phytophthora infestans
P. infestans
Late blight causes annual losses of
potatoes that would be s...
Indigenous community concerns
 Gota (late blight) causes
significant losses
 We apply fungicides
more than once a week
...
Sporulation surrounds lesion area
in initial infection state
Sporulation is not on leaflets in
advanced infection states S...
Qualitative Resistance
R genes reported = 21
(Vleeshouwers et al., 2011)
Pathogen produces
Effector protein (AVR)
Host pro...
Quantitative Resistance: QTLs = 211
Resistance mechanisms unknown (Black Box)
(Danan et al. 2011)
So far, 211 QTLs have be...
Potato genome sequence 2011
Use of Genome sequencing
To feed the worlds poor
Quantitative Resistance
improvement (durable)
 Step I: Identification of disease resistance
QTLs and genes
• Molecular ma...
Potato cultivar improvement
 Conventional breeding
• Field evaluation: yield, tuber qualities,
resistance to selected dis...
OMICs: Forward and Reverse genetics
Kushalappa and Gunnaiah 2013
Inoculation and incubation
(spot inoculation to reduce expt. error)
Resistance phenotype:
Disease severity assessment
Lesion diameter
Area
Lesion length
Area
Sample collection
Metabolomics & proteomics
 Leaves & Stems
• 72 h post inoculation
• Ground in liquid
nitrogen
Extraction - Metabolites
Problems and progress
• No single solvent can extract all
the metabolites
• Methanol + water
• Me...
ESI LTQ (MS/MS) C-Trap
Orbitrap (AME < 5 ppm)
Biochemical analysis: LC-ESI-LTQ-Orbitrap
(High resolution mass spectrometer...
Peak deconvolution: dataset: XCMS-Output
Information extraction:
Metabolite ID (Based on MS1)
Resistance related
constitutive and induced metabolites
• Statistical analysis: t-test (data reduction)
• Fold change in a...
Metabolic pathway regulation
Cell wall thickness – prevent pathogen spread
RR metabolite
Cell wall thickening
Hydroxycinnamic acid amides (HCAAs) = high fold change
Resistant cultivar
Pathogen inoc...
Cell wall thickening
Multiple disease/pest resistance?
Cell
Metabolic pathway: Diploid potato
(S. tuberosum Group phureja)
TranscriptabundanceTranscriptabundance
Transcriptabundance
Candidate genes involved in Cell wall thickening
Amino acid substitution leads to instability of
4-coumaryl ligase protein
Substitution of Alanine (A) with
Valine (V) at p...
Gene resistance function
validation based on VIGS
RR Gene not
silenced
RR Gene
silenced
RR Metabolites
Disease severity
Pa...
Knowledge application:
Plant resistance improvement
 Transfer of RR genes to elite cultivars
•Marker Assisted Breeding
•C...
Elite potato
cultivar
Tissue culture
Potato plant
Gene
replacement
Cisgenic and transgenic to improve potato resistance to...
Thank you all
FACE BOOK
McGill University
Universidad Nacional de Colombia
Kushalappa Yogendra Pushpa Mosa
Sarmiento Rodri...
Sustainable Food Production: Sustainable alternatives to manage crop diseases: Potato late blight management in Nariño, Co...
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Sustainable Food Production: Sustainable alternatives to manage crop diseases: Potato late blight management in Nariño, Colombia – a case study

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Sustainable Food Production: Sustainable alternatives to manage crop diseases: Potato late blight management in Nariño, Colombia – a case study

  1. 1. Sustainable alternatives to manage crop diseases: Potato late blight management in Nariño, Colombia – a case study Presented at the International Food Security Dialogue 2014 “Enhancing Food Production, Gender Equity and Nutritional Security in a Changing World.” Sponsored By: Hosted By: Kushalappa AC, Mosquera T & collaborators May 1, 2014
  2. 2. Seguridad Alimentaria y Nutrición Third parties Food Security Narino, Colombia Improving Potato Production for Increased Food Security of Indigenous Communities in Colombia
  3. 3. Project Philosophy We have our own way of life, but we welcome your help to improve our food security
  4. 4. 1. Nutrition and health 2. Potato breeding 3. Nutrients and functional foods in potato 4. Resistance to late blight of potato 6. Family roles and gender 5. Educational program - ECAs Food security and nutrition THEMES
  5. 5. Potato late blight (gota) Phytophthora infestans P. infestans Late blight causes annual losses of potatoes that would be sufficient to feed Several millions of people (Fisher et al., 2012).
  6. 6. Indigenous community concerns  Gota (late blight) causes significant losses  We apply fungicides more than once a week  Most of our profits go for fungicides  Our children eat lots of pesticides and they get sick
  7. 7. Sporulation surrounds lesion area in initial infection state Sporulation is not on leaflets in advanced infection states Sporulation is present on stem lesions Qualitative & Quantitative Resistance MONOGENIC (R-genes) POLYGENIC (RR-genes)
  8. 8. Qualitative Resistance R genes reported = 21 (Vleeshouwers et al., 2011) Pathogen produces Effector protein (AVR) Host produces R proteins by R-genes Effecter triggered immunity Qualitative resistance Hypersensitive reaction Not durable
  9. 9. Quantitative Resistance: QTLs = 211 Resistance mechanisms unknown (Black Box) (Danan et al. 2011) So far, 211 QTLs have been identified, based on 29 QTL maps for resistance to foliage, stem and tubers, with phenotypic variance of 4-63%.
  10. 10. Potato genome sequence 2011 Use of Genome sequencing To feed the worlds poor
  11. 11. Quantitative Resistance improvement (durable)  Step I: Identification of disease resistance QTLs and genes • Molecular marker assisted selection (MAS) • Genome wide association studies (GWAS) • Metabolo-genomics of potato genotypes  Step II: Gene function elucidation - OMICs  Step III: Transfer to elite cultivars - Cisgenics
  12. 12. Potato cultivar improvement  Conventional breeding • Field evaluation: yield, tuber qualities, resistance to selected diseases/pests • Nutritional qualities: UPLC • A new cultivar released in July 2014  Advanced technologies • Genome wide association mapping • OMICs approach to identify Candidate genes
  13. 13. OMICs: Forward and Reverse genetics Kushalappa and Gunnaiah 2013
  14. 14. Inoculation and incubation (spot inoculation to reduce expt. error)
  15. 15. Resistance phenotype: Disease severity assessment Lesion diameter Area Lesion length Area
  16. 16. Sample collection Metabolomics & proteomics  Leaves & Stems • 72 h post inoculation • Ground in liquid nitrogen
  17. 17. Extraction - Metabolites Problems and progress • No single solvent can extract all the metabolites • Methanol + water • Methanol + water (semipolar)
  18. 18. ESI LTQ (MS/MS) C-Trap Orbitrap (AME < 5 ppm) Biochemical analysis: LC-ESI-LTQ-Orbitrap (High resolution mass spectrometer) LC C-18 Kinetex MS/MS MS1 Detects more than 10 000 peaks in one run
  19. 19. Peak deconvolution: dataset: XCMS-Output
  20. 20. Information extraction: Metabolite ID (Based on MS1)
  21. 21. Resistance related constitutive and induced metabolites • Statistical analysis: t-test (data reduction) • Fold change in abundance of compound in resistant relative to susceptible genotype • RRC = RM/SM • RRI = (RP/RM)/(SP/SM)
  22. 22. Metabolic pathway regulation Cell wall thickness – prevent pathogen spread
  23. 23. RR metabolite Cell wall thickening Hydroxycinnamic acid amides (HCAAs) = high fold change Resistant cultivar Pathogen inoculated Susceptible cultivar Pathogen inoculated
  24. 24. Cell wall thickening Multiple disease/pest resistance? Cell
  25. 25. Metabolic pathway: Diploid potato (S. tuberosum Group phureja)
  26. 26. TranscriptabundanceTranscriptabundance Transcriptabundance Candidate genes involved in Cell wall thickening
  27. 27. Amino acid substitution leads to instability of 4-coumaryl ligase protein Substitution of Alanine (A) with Valine (V) at position 158 leads to decrease in protein stability by ∆∆G= -0.80 (negative value) Substitution of Histidine (H) with Aspartic acid (D) at position 298 leads to decrease in protein stability by ∆∆G= -0.70 158 4CL-RP F06037 4CL-SP Shepody 4CL-RP F06025 4CL- Gene bank 298 4CL-RP F06037 4CL-SP Shepody 4CL-RP F06025 4CL- Gene bank Where as, ∆∆G- Free energy ∆∆G: ∆G(New Protein)-∆G(Wild Type) in Kcal/mol ∆∆G<0: Decrease Stability ∆∆G>0: Increase Stability (Capriotti et al. 2006)
  28. 28. Gene resistance function validation based on VIGS RR Gene not silenced RR Gene silenced RR Metabolites Disease severity Pathogen biomass Resistant Susceptible
  29. 29. Knowledge application: Plant resistance improvement  Transfer of RR genes to elite cultivars •Marker Assisted Breeding •Cisgenics (gene transfer between sexually compatible genotypes) Second phase?
  30. 30. Elite potato cultivar Tissue culture Potato plant Gene replacement Cisgenic and transgenic to improve potato resistance to late blight Wild potato Cisgenic potato plants Transgenic potato plants Elite potato cultivar Bacteria or Other organisms (http://www.glogster.com) Tissue culture Potato plant Late blight resistance gene
  31. 31. Thank you all FACE BOOK McGill University Universidad Nacional de Colombia Kushalappa Yogendra Pushpa Mosa Sarmiento Rodriguez Mosquera Liyao-JiSarkar

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