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Michael SchmidtJason P. Bond
Ahmad Fakhoury
Southern Illinois University
New Approaches to Study the Cyst Nematode/Sudden Death
Syndrome Interaction in Soybean
Distribution of SCN and SDSSCN SCN & SDS
Soybean Cyst Nematode
• Heterodera glycines
• Many HG types classified
• Resistance is qualitative/quantitative
Root Knot Nematode Two species of concern in Illinois
Meloidogyne incognita (southern root-knot)Has over 500 different hosts including corn
Meloidogyne hapla (northern root-knot)More limited host range, corn is a non host
Both species are important in soybean, crops, orchards, vineyards, etc.
• Fusarium virguliforme (Fv).• Soilborne, root rotting fungus that colonizes tap root and
crown.• Produces a toxin causing foliar symptoms (scorch).
Sudden Death Syndrome
Components of SDS Resistance
• Foliar symptoms - resistance to toxin effects, measured as DX. DX=DS*DI/9. DS= disease severity valued on a 1-9 scale. DI=disease incidence as a percentage of plants in plot having disease.
• Root infection/colonization - resistance to the fungal pathogen
SCN Interactions with SDS
• Synergistic – • Roy et al., 1989- greenhouse• McLean and Lawrence, 1993 - greenhouse• Rupe et al., 1991; field trials• Hershman et al., 1990, field trials• Xing and Westphal, 2006 - microplots
• Additive • Gao et al., 2006 - greenhouse
Limitations - SCN/SDS Research
• Field studies were/are conducted in fields with SCN • SCN densities – generally the resolution at field
level.
• Providing an environment suitable for expression of SDS.
• Difficult y in quantifying Fv in soil and roots.
• Need for the right compliment of soybean isolines contrasting for SCN and SDS resistance.
New Tools to Study the Nematode/Fv/Soybean Interaction
• Develop a transformation system for Fv
• Generate fluorescing strains of the fungus – GFP = Green fluorescing protein.
• Identify GFP phenotypes differing in aggressiveness toward soybean. To study the infection & colonization To screen for resistance
Restriction Enzyme Mediated Integration (REMI)
Linearized Plasmid
Fungal DNA
Isolation & Characterization of
Regions Flanking the Integration
Integration of the Linearized Plasmid
Phenotypic Analysis
Characterizing the disrupted gene(s)
New Phenotypes Developed
GFP Strain in Soybean Roots
REMI Results
• An aggressive Fv isolate known as Mont1 (Monticello, IL) was chosen as source population for transformation.
• Several GFP strains were produced.
• An aggressive strain of Fv was identified.
• A non-aggressive strain was identified.
OBJECTIVE: To use the aggressive and non-aggressive strains to:
• Determine the role of SCN and RKN in the ability of Fv to penetrate and colonize soybean roots.
• Determine the role of SCN and RKN in the ability of Fv to cause SDS foliar symptoms.
Experimental Design
• Greenhouse experiment.• Five soybean cultivars different in their resistance to SCN,
RKN, and SDS. • Cultivar s were challenged with the GFP-expressing
aggressive strain and the GFP-expressing non-aggressive strain.
• Cultivars were co-challenged with SCN and RKN.• The experiment consisted of 30 treatments replicated 5
times.• Duration of the experiment was 15 days. • Fv root colonization quantified via QPCR.• SDS leaf scorch evaluated on a 1-9 scale.
Cultivar
H. glycines
M. incognita
F. virguliforme
Forrest R R R
P94M50 R S S
Spencer S S S
GH3983 S S R
LS97-1610
S R R
Cultivars
InoculumMock Inoculation
Aggressive strain
Non-aggressive strain
SCN + aggressive strain
SCN + non-aggressive strain
RKN + aggressive strain
Five cultivars x Six inoculum levels = 30 treatments.Inoculum: SCN=2300egs/plant, RKN=2400 eggs/plant, Fv=7,700,000spores.
Greenhouse Experiment
Waterbath system maintains constant temperature across all experimental units.Five replications, sample size of 3 plants. ~0.5 liter soil/container.
Cultivar H. glycines
M. incognita F. virguliforme
Forrest R R R
P94M50 R S S
Spencer S S S
GH3983 S S R
LS97-1610
S R R
97-1610 Spencer GH3983 Forrest P94M500
5
10
15
20
25
30
35
40
Root colonization with aggressive Fv strain
Cultivar
ng f
ungal D
NA
FungusFungus + SCNFungus +RKN
97-1610 Spencer GH3983 Forrest P94M500
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
SDS leaf scorch with aggressive Fv strain
Cultivar
Rati
ng
FungusFungus + SCNFungus +RKN
Cultivar H. glycines
M. incognita F. virguliforme
Forrest R R R
P94M50 R S S
Spencer S S S
GH3983 S S R
LS97-1610
S R R
97-1610 Spencer GH3983 Forrest P94M500
5
10
15
20
25
30
Root colonization with non-aggressive strain
Cultivar
Cultivar H. glycines
M. incognita F. virguliforme
Forrest R R R
P94M50 R S S
Spencer S S S
GH3983 S S R
LS97-1610
S R R
97-1610 Spencer GH3983 Forrest P94M500
0.5
1
1.5
2
2.5
3
3.5
4
4.5
SDS leaf scorch with non-aggressive strain
Cultivar
Rati
ng
FungusFungus + SCN
Cultivar H. glycines
M. incognita F. virguliforme
Forrest R R R
P94M50 R S S
Spencer S S S
GH3983 S S R
LS97-1610
S R R
Results
• Co-inoculation with Fv and either SCN or RKN lead to a significant increase in Fv root colonization and SDS foliar symptoms for all cultivars.
• Co-inoculating with the fungus and the nematode can overwhelm plant resistance to SDS under greenhouse conditions.