1. Ph.D Dissertation defense, Jason Price, Indiana University, Bloomington, Sept. 17 2002
The potential for evolution of resistance to
Coleosporium asterum leaf rust in the clonal
perennial herb, Euthamia graminifolia

2. The higher plant as a series of niches for natural
enemies

3. Trust’s rust busts bridal lust

4. Goal of my research
• Assess the potential for evolution of quantitative
resistance in a clonal plant species
To do so, I’ll address the 3 necessary and sufficient conditions for
evolution of resistance by natural selection:
1) Variation in resistance
1) Inheritance of resistance
1) Association of resistance variation with fitness

5. Evolution of disease resistance
through vegetative reproduction
(1) Does resistance vary among host genotypes?
(2) Is resistance heritable by vegetative offspring?
(3) Does disease affect vegetative reproduction?

6. Euthamia graminifolia infected
with Coleosporium asterum

7. Pathosystem

8. Qualitative
resistance
Quantitative
resistance
Stops
Pathogen growth
Slows
pathogen growth
Race specific
(single gene)
Race non-specific
(multiple gene)

9. Taken directly from Burdon 1987
Quantitative resistance -- stages of action

10. • Resistance inverse of infection intensity
• infection intensity integrates many variables
– plant, pathogen, epidemiological, environment
• allows all heritable traits that lead to low disease
levels to be considered (Alexander 1992)
Relationship between resistance and
infection intensity

11. 1) Does resistance vary among host genotypes?
DESIGN

12. Resistance assessment plot (in 1998)

13. Measuring infection intensity

14. Genotypes within populations
vary in resistance level - 1
0
20
40
60
80
100
1 2 3 4 5 6 7 8 9 10 11 12
c) Bayles field 1998
*** *****
Genotype
%Leafareainfected Bayles field 1998
* = p < .05, ** = p < .01, *** = p < .001

15. Genotypes within populations vary in resistance level - 2
Genotype
0
20
40
60
80
100
1 2 3 4 5 6 7 8 9 10 11 12
a) Hilltop field 1997
* *** *
1 2 3 4 5 6 7 8 9 10 11 12
0
20
40
60
80
100
b) Hilltop field 1998
**
100
0
20
40
60
80
1 2 3 4 5 6 7 8 9 10 11 12
d) Bayles field 1999
0
20
40
60
80
100
1 2 3 4 5 6 7 8 9 10 11 12
e) Hilltop pots 2000
*** ** ***
0
20
40
60
80
100
1 2 3 4 5 6 7 8 9 10 11 12
f) Hilltop pots 2001
***
%Leafareainfected
Hilltop field Hilltop pots
2000
20011998
1997
1999
0
20
40
60
80
100
1 2 3 4 5 6 7 8 9 10 11 12
c) Bayles field 1998
*** *****
Bayles field
1998
* = p < .05, ** = p < .01, *** = p < .001

16. Genotype resistance level is consistent across datasets

17. Two genotypes stand out as being most resistant

18. Evolution of disease resistance
through vegetative reproduction
(1) Does resistance vary among host genotypes?
(2) Is resistance heritable by vegetative offspring?
(3) Does disease affect vegetative reproduction?

19. (2) Is resistance heritable by vegetative offspring?
• Sources of variation
Vp = Vg + Ve
Vg = Va + Vd + Vi
• Heritability
h2
= Va / Vp for sexual offspring
H2
= Vg / Vp for vegetative offspring
• Clonal repeatability = estimate of H2 (Vg / Vp)

20. Resistance was heritable by vegetative offspring
Survey
Clonal
Repeatabilit y
df Model df Error F Ratio p Value
Hillt op field 1997 0.265 11 225 8.11 <0.001
Hillt op field 1998 -- 11 100 1.30 0.234
Bayles field 1998 0.389 11 226 13.65 <0.001
Bayles field 1999 -- 11 169 0.74 0.696
Hillt op pots 2000 0.276 11 138 5.78 <0.001
Hillt op pots 2001 0.120 11 113 2.42 0.010

21. Genotypic selection for resistance
Increased representation of resistant
genotypes in ramet population =
Genotypic selection for resistance
High
R
genotype
Low
R
genotype
Sig.
Heritablility
of
Genotypic
variation
in resistance
(R)
Ramets

22. Evolution of disease resistance
through vegetative reproduction
(1) Does resistance vary among host genotypes?
(2) Is resistance heritable by vegetative offspring?
(3) Does disease affect vegetative reproduction?

23. (3) Does disease affect vegetative reproduction?
DESIGN
Treatment
Dataset Fungicide Water
Outdoor pots
Low
infection
n = 144
High
infection
n = 144
Experimental
field
Low
infection
n = 84
High
infection
n = 84
Greenhouse
‘control’
Uninfected
n = 24
Uninfected
n = 24

24. Fitness assessment
field design

25. Fungicide reduced infection intensity
Outdoor pots Experimental field
0
10
20
30
40
50
60
70
80
90
100
hbInf080901
2001
F W
0
10
20
30
40
50
60
70
80
90
100
hbinf092100
2000
F W
Leafareainfected(%)
p < .0001 p < .0001
-10
0
10
20
30
40
50
60
70
80
90
100
meanhbinf092000
2000
-10
0
10
20
30
40
50
60
70
80
90
100
110199hbinf
1999
F W F W
[puiyui p < .0001p =.0019

26. High infection plants had lower rhizome mass
in outdoor pots
Photo of potted plants
0
1
2
3
4
5
6
7
8
2000Stemmass(g)
AGDW00(g)
p = .3080
F W
(a)
0
1
2
3
4
5
6
7
8
9
10
2001Stemnumber
Stemnum01
p = .9530
F W
0
5
10
15
20
25
30
35
40
2001Rhizomemass(g)
•(est)bgdrywt
p <.0001
F W
0
10
20
30
40
50
60
70
2001Totalbasalarea(cm2)
•BA01
p = .0309
F W


27. High infection reduced rhizome mass
of high infection replicates of all genotypes
(b)
0
5
10
15
20
25
30
35
40
2001Rhizomemass(g)
1 2 3 4 5 6 7 8 9 10 11 12
Genotype
Water
Fungicide
*
**
*
*
*
*
*
p < .0001
2001Rhizomemass(g)
Outdoor pots 

28. No detectable effect of disease on above ground
measures of vegetative reproduction in the
experimental field
p=.0608 p=.8288p=.4350p=.1088 p=.1130
0
200
400
600
800
1000
1200
1400
1600
1800
2001Totalbasalarea(mm2)
•BA01(mm2)F W
0
20
40
60
80
100
120
140
2001Stemnumber
numsht01>1.4mmF W
0
100
200
300
400
500
600
700
800
2000Totalbasalarea(mm2)
•BA00F W
0
5
10
15
20
25
30
2000Stemnumber
finstm#00F W
10
15
20
25
30
35
40
45
1999Stemmass(g)
AGDW99(g)F W
Treatment


29. A single fragment in the experimental field

30. Rhizome ‘excavation’

31. Fungicide greatly reduced focal
fragment infection intensity in 2000
-10
0
10
20
30
40
50
60
70
80
90
100
Fragmentleafareainfected(%)
hbinf01
-10
0
10
20
30
40
50
60
70
80
90
100
Fragmentleafareainfected(%)
FragHBinf99
-10
0
10
20
30
40
50
60
70
80
90
100
Fragmentleafareainfected(%)
FragHBinf00
Treatment
Fungicide Water Fungicide Water Fungicide Water
p = .1089 p < .0001 p = .1211
(c) (d) (e)1999 2000 20011999 20012000
p =.1089 p =.1211p < .0001
F W F WF W
Leafareainfected(%)

32. Rhizome mass relative to above ground size was lower
in high infection fragments in the experimental field
0
200
400
600
800
1000
1200
2001Rhizomemass(g)
0 500 1000 1500 2000 2500
2001 Total Basal Area (mm2)
Water-sprayed fragments
0
200
400
600
800
1000
1200
0 500 1000 1500 2000 2500
Fungicide-sprayed fragments
Slopes differ p < .001
280
285
290
295
300
305
310
315
320
2000SenescenceDate(DOY)
0 10 20 30 40 50 60 70 80 90 100
2000 Fragment leaf area infected (%)
(b) n = 75, r^2 = -.241x + 299.99, p < .0001
r2 = .31, p<.0001

33. 0
1
2
3
4
5
6
2000Stemmass(g)
F W
Cell
0
5
10
15
20
25
30
35
40
2001Stemmass(g)
F W
Treatment
0
1
2
3
4
5
6
7
8
9
2001Stemnumber
F W
Cell
0
10
20
30
40
50
60
2001Rhizomenumber
F W
Cell
0
10
20
30
40
50
60
2001Rhizomemass(g)
F W
Cell
p = .5324 p = .0346 p = .0367 p = .1020 p = .2892
No effect of fungicide in the
absence of disease (greenhouse)


34. Recap
(1) Do host genotypes vary in resistance? Yes.
(2) Is resistance heritable by vegetative offspring? Yes.
(3) Does disease affect vegetative reproduction? Yes,
through decreased rhizome biomass
(4) Does disease affect sexual reproduction, and how
important is seedling recruitment in established
populations ?

35. No fungicide effect on seed production
in the absence of disease (greenhouse)
0
20
40
60
80
100
120
140
Viableseednumber(Thousands)
Fungicide Water
Treatment
p = .2211

36. Disease also reduced seed production
0
500
1000
1500
2000
2500
Viableseednumber
Est. Total Seed Num
Water
Fungicide
Treatment
p = .0002
Potted plants
0
200
400
600
800
1000
1200
1400
Viableseednumber
EstSeedNum
Water
Fungicide
Treatment
p = .0047
(a)
Experimental field

37. Recruitment plots and quadrats

38. Seedling recruitment was extremely
low in established populations
Environment Census area
(m2
)
Seed number Recruitment
(%)
Notes
Growth chamber/
Greenhouse
n/a 3 836 81
90% germination,
90% survival under
ideal conditions
Colonization plot 220 Å197 000 .06 Field recruitment with
reduced competition
and shading
Established plot 1760 Å787 000 .002 Very dense vegetation,
no effect of tilling
Natural
populations
480 Å1 181 000 .006 Vegetation was much
less dense than
established plot

39. High
R
Low
R
Genotypic selection within populations affects
genetic makeup of new populations
H2
Genotypic
variation
in
resistance
12,000
seeds
1500
seeds
Higher
likelihood of
colonization
of disturbed
area
Lower
likelihood of
colonization
of disturbed
area
Greater
representation
of genes of
resistant
genotypes in
colonizing
seed pool
For discussion see
Pan & Price 2001
Evol. Ecol. 15:583
Number of ramets
after a few years of
population growth

40. Synthesis
• All three conditions necessary for evolution of
resistance through differential vegetative
reproduction can occur in this pathosystem
• Seed recruitment is very low in established
populations, suggesting that vegetative reproduction
will be of primary importance for changes in gene
frequency within populations
• Changes in gene frequency within populations are
likely to affect the genetic makeup of new
populations

41. Maintenance of genotypic variation?
Relativerhizomemass
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Relativeresistancelevel
1 9 12 4 2 10 5 6 8 7 3 11
Genotype
Rhizome mass (pre sence of disease)
Rhizome mass (absence of dise ase )
Resistance level

42. Maintenance of genotypic variation?
Relativerhizomemass
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Relativeresistancelevel
1 9 12 4 2 10 5 6 8 7 3 11
Genotype
Rhizome mass (pre sence of disease)
Rhizome mass (absence of dise ase )
Resistance level

43. Maintenance of genotypic variation?
Relativerhizomemass
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Relativeresistancelevel
1 9 12 4 2 10 5 6 8 7 3 11
Genotype
Rhizome mass (pre sence of disease)
Rhizome mass (absence of dise ase )
Resistance level

44. Maintenance of genotypic variation?
Relativerhizomemass
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Relativeresistancelevel
1 9 12 4 2 10 5 6 8 7 3 11
Genotype
Rhizome mass (pre sence of disease)
Rhizome mass (absence of dise ase )
Resistance level

46. Acknowledgements
Committee
Jim Bever
Lynda Delph
Michael Tansey
Maxine Watson
Undergraduate L490’s
J. Paul
T. Pawlowski
L. Lasky
R. Lemaster
Kara Kitch
Claylab folks
Jean Pan
Paula Kover
Alissa Packer
Janice Alers-Garcia
Tammy Johnston
Jen Koslow
Jenn Rudgers
Funding sources:
Indiana Academy of Science
B.F. Floyd Memorial Fellowship
Undergraduate assistants to numerous to mention,
But esp. Scott Hovis and Amber Fullenkamp
Advisor: Keith Clay
Kneehigh Cooperative Daycare
Jonathan Mollenkopf and Nathan Murphy