Michael SchmidtJason P. Bond

Ahmad FakhourySouthern 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 RP94M50 R S SSpencer S S SGH3983 S S RLS97-1610

S R R

97-1610 Spencer GH3983 Forrest P94M5005

10152025303540

Root colonization with aggressive Fv strain

Cultivar

ng f

unga

l DN

A

FungusFungus + SCNFungus +RKN

97-1610 Spencer GH3983 Forrest P94M500

0.51

1.52

2.53

3.54

4.55

SDS leaf scorch with aggressive Fv strain

Cultivar

Ratin

g

FungusFungus + SCNFungus +RKN

Cultivar H. glycines

M. incognita F. virguliforme

Forrest R R RP94M50 R S SSpencer S S SGH3983 S S RLS97-1610

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 RP94M50 R S SSpencer S S SGH3983 S S RLS97-1610

S R R

97-1610 Spencer GH3983 Forrest P94M500

0.51

1.52

2.53

3.54

4.5

SDS leaf scorch with non-aggressive strain

Cultivar

Ratin

g

FungusFungus + SCN

Cultivar H. glycines

M. incognita F. virguliforme

Forrest R R RP94M50 R S SSpencer S S SGH3983 S S RLS97-1610

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.