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Rootstock based management of disease in vineyard crops Eric Stafne ICABR-AAWE Pre-Conference Workshop: Technology and Innovation in the Grape and Wine.

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Presentation on theme: "Rootstock based management of disease in vineyard crops Eric Stafne ICABR-AAWE Pre-Conference Workshop: Technology and Innovation in the Grape and Wine."— Presentation transcript:

1 Rootstock based management of disease in vineyard crops Eric Stafne ICABR-AAWE Pre-Conference Workshop: Technology and Innovation in the Grape and Wine Industries Feudi di San Gregorio – Localita’ Cerza Grossa 83050 June 24, 2012, 9:30 am “Using Biotechnology to extend the rootstock concept” Plant Science Department Abhaya M. Dandekar

2 DHS ‘Most Destructive’ List of Plant Pathogens How do we generate resistance to destructive diseases when none exists in the germplasm? How do we manage the disease while maintaining production and the livelihood of growers?

3 Key Problem: 1900ft radius citrus canker eradication scars Pest and Pathogen Populations and their ability to cause disease –Lower Productivity –Lower Quality Chemical pesticides control the insect vector but do not target the pathogen directly. Once the pathogen infects the plants, no treatments are available to eliminate it except vine/tree removal

4 Disease Triangle for Orchard/Vineyard Crops HOST Disease/ Pest Complex Pathogen Vector & Environment Disease is the exception rather than the rule! Disease/Pest Resistance: - Genetic - Epigenetic Chemical Warfare: - Eradicate target pest - Pheromone confusion Early Disease Detection: - Eradication - Replant with clean stock

5 “Cloning” Varietal vines are clones –Vegetative propagation –Genetic uniformity All vines are composite genetic systems –Genetically distinct scion Unique wine quality –Genetically distinct rootstock Pest/disease resistant Nutrient or abiotic stress tolerant Eric Stafne Varietal vine Locally Adapted Rootstock “Grafting”

6 American Rootstocks fight the Phylloxera Plague in Europe Phylloxera is native to N America Damages the very young roots Resistance to phylloxera evolved/selected in the natural environment among the wild grapevine relatives Phylloxera feasting on the best wines Europe had to offer

7 Anatomy of the “Graft-Union” Adhesion of rootstock and scion tissues Proliferation of a bridge callus at the graft interface Vascular differentiation across the graft interface allowing the flow of water, nutrients and growth regulators including pathogens Can we move proteins and siRNA? Rootstock Scion Callus

8 Strategy: Genetic resistance mediated by transgenic expression of therapeutic proteins and/or siRNA to control the multiplication of pests and pathogens and to counteract their virulence Applications: “Transgenic Rootstocks”: To limit pests and pathogens in grafted scions. “Transgenic trap crop”: Trap crops to reduce pathogen inoculums and to attract the insect vector thus reducing pesticide applications. Deliver Therapeutics via Rootstocks Therapeutic Proteins

9 Ann-1 Xf strain Temecula 9a5c Dixon M12 M23 Grape: Pierce’s Disease Citrus: Variegated Chlorosis Almond: Leaf Scorch Oleander: Leaf Scorch Xylella fastidosa (Xf) causes serious diseases In many economically important plants

10 Colonization of Xf in the plant xylem and the clogging of the xylem is the primary cause of the disease Plant Vascular System

11 Chatterjee et al., Ann Rev Phytopathol. (2008) 46: 243-271. Long distance movement Type 4 pili Degradation of pit pore membranes Polygalacturonase (PG) Surface Adhesion/attachment Biofilm formation Vessel clogging Disease symptoms Complex lifestyle of Xylella fastidiosa the causative agent of Pierce’s Disease Asymptomatic Symptomatic

12 Testing two Promising Protein-Based Therapeutics Expression of PGIP to counteract the virulence and movement of the pathogen Xylella fastidiosa Expression of (Chimeric Antimicrobial Proteins) to improve pathogen destruction and inoculum reduction blocking transmission of PD in the field Xf PG PGIP CAP binds to mopB on Xf surface Pore formation Surface binding Goutam Goupta

13 Interfering with Xylella movement Pit-pore membranes connect one xylem vessel to another Pit membranes are made of pectin Xylella expresses a polygalacturonase (PG) to degrade the pectin PGIP (polygalacturonase inhibitory protein) inhibits the activity of PG Lindow Cook

14 45-77TS Leaf symptoms and stem MRI of PGIP expressing Grapevines Round 3 chi-PGIP 45-77 TS Round 3 chi-PGIP Ana Maria Ibanez, Hossein Gouran

15 Degrading the inoculum: Express a chimeric antimicrobial protein (CAP) in xylem tissues. CAP binds to bacteria surface then destroys the bacteria

16 051096-004051096-003 2 months Uninfected Control 2 months Infected Control 1 month 2 months Construction and testing of binary vectors for Agrobacterium-mediated transformation expressing a synthetic gene encoding chimeric antimicrobial protein

17 Reduced Leaf Scorching in Transgenic Grapevines expressing CAP Leaf number 8 above the point of inoculation was harvested 10 weeks post infection with 20 million Xf cells

18 Measuring xylem blockage using MRI Transgenic expressing CAP show less xylem blockage as compared to the wild type controls 10 cm above the site of inoculation with 20 million Xf cells Transgenic Wild Type

19 Riverside and Armstrong Vineyards Riverside Yolo Human Sharpshooters

20 Xylem Sap from HNE-CecB transgenic lines display antimicrobial activity against Xf

21 Strategies to Control Crown Gall Escobar and Dandekar, TRENDS in Plt Sci. 8(8): 380-386 (2003) Contaminated Nursery Stock Infection of Crown and roots Young trees do not establish Tree decline Loss of productivity Paradox leading rootstock is very susceptible Contaminated orchards cannot be replanted

22 Inhibiting Genetic Colonization of Agrobacterium with RNAi Induced Silencing iaaM iaaHipt Wild type T-DNA EXPRESSION Crown Gall Cells AUXIN CYTOKININ 35S MaaiiaaM 35S ipt tpi pDE00.0201 Dicer siRNA 01/6 WT iaaM 01/6 Vctr. Wt 21bp 41bp 01/6 Vctr. Wt ipt

23 Crown Gall Resistance by Silencing Gall Formation Arabidopsis: root bundle assay Wild-Type Line 01/33 Escobar et al., Plant Sci 163: 591-597 (2002) Wild-Type Line 01/258 Walnut: shoot inoculation Tomato: stem inoculation Wild-Type Line 01/6 Model Systems Escobar et al., PNAS 98: 13437-13442 (2001)

24 Transgenic Paradox Rootstocks Resistant to Crown Gall Site of Infection Charles Leslie

25 Stacking Resistance to Crown Gall and Nematodes in Walnut Rootstocks -Strong vigor -High yield efficiency -Resistance to pests and disease -Graft compatibility -Transplantability CA Walnut orchards –Nematodes- 90% Crown gall - 85%

26 Life cycle of lesion nematode (Dr. G.N. Agrios). Pratylenchus vulnus

27 Binary vector in Agrobacterium rhizogenes used for P. vulnus gene silencing Binary vector in Agrobacterium tumefaciens used for induction of oncogene silencing GUS nptII selectable marker gene GFP no selectable marker gene Binary vectors used for co-transformation Monica Britton

28 Transformed plantlets of J1 genotype with Pv010 gene Transgenic-nematode damage is minimal Transgenic-nematode damage is significant High PV010 expressionVery low PV010 expression Lalani Walawage

29 Pv010 gene expression level in different J1 transgenic lines in co- transformation Inhibition of root lesion nematodes in transformed walnut plantlets Lalani Walawage

30 1) Promote Vigor 2) Stress tolerance -Biotic -Abiotic 3) Mineral nutrition 4) Precocity 5) Dwarfing 1)Disease resistance No linkage drag Rapid genotype development 2)Pest resistance 3)Secretion of therapeutic proteins Destroy pathogens Control their virulence 4)Synthesis of sRNA Control Pathogens Control Pests 5)No Gene Flow No Pollen No Seeds Conventional Transgenic Why Transgenic Rootstocks? Can Save an Industry French wine industry was saved from phylloxera with American native rootstocks Rangpur lime rootstocks saved citrus in Brazil from CTV decline on sour orange

31 1)Rootstocks in new orchards 2)Inarched grafts within existing orchards 3)Trap crops around existing orchards 4)No transgenic fruit 5)Reduce pesticide use 6)Compatible with IPM 7)Destroys pathogen 8)Easy to stack resistances Benefit to the Industry In-arch grafting of ‘Rangpur Lime Rootstock’ to save CTV infect trees on ‘Sour Orange Rootstocks’ from ‘quick decline’ in Brazil

32 Acknowledgments Dandekar Lab UCD Sandra Uratsu Matthew Escobar Federico Martinelli Ana Maria Ibanez Hossein Sadeghi-Gouran My Phu Russell Reagan Rafael Nascimento Sarah McFarland Lalani Walawage Mary Lou Mendum Kevin Quach David Dolan Gale McGranahan Chuck Leslie Cristina Davis Weixiang Zhao Alexander Aksenov William Cheung Goutam Gupta LANL Anu Choudhry Paige Pardington Cecilia Aguero George Bruening UCD Luiz Goulart Rodrigo Almeida Raissa D’Souza UCD Soumen Roy Elizabeth Leicht Tim Spann UFL Kim Bowman USDA-ARS Ute Albrecht Oliver Feihn Kirsten Skogerson Special Services Transformation David Tricoli Metabolomics Valdimir Tolstikov DNA Sequencing Charles Nicolet Bioinformatics Core Dawei Lin Monica Britton Joseph Fass Vince Buffalo Funding Sources Citrus Research Board California Walnut Board UC Discovery FCPRAC/CDRF CDFA-PD UC-PD

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