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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
The higher plant as a series of niches for natural enemies
Trust’s rust busts bridal lust
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
2) Inheritance of resistance
3) Association of resistance variation with fitness
Evolution of disease resistancethrough vegetative reproduction
(1) Does resistance vary among host genotypes?
(2) Is resistance heritable by vegetative offspring?
(3) Does disease affect vegetative reproduction?
Euthamia graminifolia infected with Coleosporium asterum
Overwinter asrhizomes
Spores from2-needle pines
Growth of fungal hyphae within leaf
Repeatingstage
Spores to2-needle pines
Above-ground growthdies each winter Disease peaks during flowering
Pathosystem
Qualitativeresistance
Quantitativeresistance
Stops Pathogen growth
Slows pathogen growth
Race specific (single gene)
Race non-specific(multiple gene)
Taken directly from Burdon 1987
Quantitative resistance -- stages of action
• Resistance º 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
1) Does resistance vary among host genotypes? DESIGN
4 Populations
3 genotypes/population11
Hardin RidgeGriffy
FriendshipKent Farm
14
9 1276
532
108
Clonal Propagation (40 plants/genotype)
1 2 3HR
4 5 6
GR7 8 9
FS10 1211
KF
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
... 20 replicates of each genotype at Bayles Rd. plot
... 20 replicates of each genotype at Hill Top plot
Resistance assessment plot (in 1998)
Measuring infection intensity
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
% L
eaf
area
infe
cted
Bayles field 1998
* = p < .05, ** = p < .01, *** = p < .001
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 11 120
20
40
60
80
100b) 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***
% L
eaf a
rea
infe
cted
Hilltop field Hilltop pots2000
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 field1998
* = p < .05, ** = p < .01, *** = p < .001
Genotype resistance level is consistent across datasets
Two genotypes stand out as being most resistant
Evolution of disease resistancethrough vegetative reproduction
(1) Does resistance vary among host genotypes?
(2) Is resistance heritable by vegetative offspring?
(3) Does disease affect vegetative reproduction?
(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)
Resistance was heritable by vegetative offspring
SurveyClonal
Repeatabilitydf Model df Error F Ratio p Value
Hilltop field 1997 0.265 11 225 8.11 <0.001
Hilltop 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
Hilltop pots 2000 0.276 11 138 5.78 <0.001
Hilltop pots 2001 0.120 11 113 2.42 0.010
Genotypic selection for resistance
Increased representation of resistantgenotypes in ramet population =Genotypic selection for resistance
HighR
genotype
LowR
genotype
Sig. Heritablility
of Genotypic variation
in resistance
(R)
Ramets
Evolution of disease resistancethrough vegetative reproduction
(1) Does resistance vary among host genotypes?
(2) Is resistance heritable by vegetative offspring?
(3) Does disease affect vegetative reproduction?
(3) Does disease affect vegetative reproduction?
DESIGN
Treatment
Dataset Fungicide Water
Outdoor potsLow
infectionn = 144
High infectionn = 144
Experimental field
Low infectionn = 84
High infectionn = 84
Greenhouse‘control’
Uninfectedn = 24
Uninfectedn = 24
Fitness assessment field design Control recruitment plot
3 N
2
7 12
65
4
10.5 m
28.5 m
5.0 m
30.5 m 75°
95°
85°
105°
Paired Tilled/Untilled quadratsControl quadrats
Sub-blocksFungicide-sprayed fragment
Water-sprayed fragment
Phenology Fragment
Fitness assessmentplot
Growth phenology plot
7
18m
Resistance assessment plot
Focal fragment
Fungicide reduced infection intensity
Outdoor pots Experimental field
0
10
20
30
40
50
60
70
80
90
100
hbInf080901
2001
F W0
1020
3040506070
8090
100
hbinf092100
2000
F W
Lea
f are
a in
fect
ed (
%) p < .0001 p < .0001
-10
0
10
20
30
40
50
60
70
80
90
100
meanhbinf092000
2000
-100
102030405060708090
100
110199hbinf
1999
F W F W
[puiyui p < .0001p =.0019
High infection plants had lower rhizome mass in outdoor pots
Photo of potted plants
0
1
2
3
4
5
6
7
8
2000 S
tem
mass (
g)
AGDW00(g)
p = .3080
F W
(a)
0
1
2
3
4
5
6
7
8
9
10
2001 S
tem
num
ber
Stemnum01
p = .9530
F W0
5
10
15
20
25
30
35
40
2001 R
hiz
om
e m
ass (
g)
(est)bgdrywt
p <.0001
F W0
10
20
30
40
50
60
70
2001 T
ota
l basal are
a (
cm2)
BA01
p = .0309
F W
High infection reduced rhizome massof high infection replicates of all genotypes
(b)
0
5
10
15
20
25
30
35
40
2001 R
hiz
om
e m
ass (
g)
1 2 3 4 5 6 7 8 9 10 11 12
Genotype
Water
Fungicide
***
*
*
*
**
p < .0001
2001
Rhi
zom
e m
ass
(g) Outdoor pots
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
2001 T
ota
l b
asal are
a (
mm
2)
BA01(mm2)F W0
20
40
60
80
100
120
140
2001 S
tem
nu
mb
er
numsht01>1.4mmF W0
100
200
300
400
500
600
700
800
2000 T
ota
l b
asal are
a (
mm
2)
BA00F W0
5
10
15
20
25
30
2000 S
tem
nu
mb
er
finstm#00F W10
15
20
25
30
35
40
45
1999 S
tem
mass (
g)
AGDW99(g)F W
Treatment
A single fragment in the experimental field
Rhizome ‘excavation’
Fungicide greatly reduced focal fragment infection intensity in 2000
-10
0
10
20
30
40
50
60
70
80
90
100
Fra
gm
en
t le
af
are
a in
fecte
d (
%)
hbinf01-10
0
10
20
30
40
50
60
70
80
90
100
Fra
gm
en
t le
af
are
a in
fecte
d (
%)
FragHBinf99-10
0
10
20
30
40
50
60
70
80
90
100
Fra
gm
en
t le
af
are
a in
fecte
d (
%)
FragHBinf00
Treatment
Fungicide Water Fungicide Water Fungicide Water
p = .1089 p < .0001 p = .1211
(c) (d) (e)1999 2000 20011999 20012000p =.1089 p =.1211p < .0001
F W F WF W
Leaf
are
a in
fect
ed
(%
)
Rhizome mass relative to above ground size was lower in high infection fragments in the experimental field
0
200
400
600
800
1000
1200
20
01
Rh
izo
me
ma
ss (
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
2000 S
enesc
ence
Date
(D
OY
)
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
0
1
2
3
4
5
6
2000 S
tem
mass (
g)
F WCell
0
5
10
15
20
25
30
35
40
2001 S
tem
mass (
g)
F W
Treatment
0
1
2
3
4
5
6
7
8
9
2001 S
tem
num
ber
F WCell
0
10
20
30
40
50
60
2001 R
hiz
om
e n
um
ber
F WCell
0
10
20
30
40
50
60
2001 R
hiz
om
e m
ass (
g)
F WCell
p = .5324 p = .0346 p = .0367 p = .1020 p = .2892
No effect of fungicide in the absence of disease (greenhouse)
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 ?
No fungicide effect on seed production in the absence of disease (greenhouse)
0
20
40
60
80
100
120
140
Via
ble
seed
nu
mb
er
(Th
ou
san
ds)
Fungicide WaterTreatment
p = .2211
Disease also reduced seed production
0
500
1000
1500
2000
2500
Via
ble
se
ed
nu
mb
er
Est. Total Seed Num
WaterFungicide
Treatment
p = .0002
Potted plants
0
200
400
600
800
1000
1200
1400
Via
ble
se
ed
nu
mb
er
EstSeedNum
Water
Fungicide
Treatment
p = .0047(a)
Experimental field
Recruitment plots and quadrats
Seedling recruitment was extremely low in established populations
Environment Census area(m2)
Seed number Recruitment(%)
Notes
Growth chamber/ Greenhouse
n/a 3 836 8190% germination,90% survival underideal conditions
Colonization plot 220 Å 197 000 .06 Field recruitment withreduced competitionand shading
Established plot 1760 Å 787 000 .002 Very dense vegetation,no effect of tilling
Natural populations
480 Å 1 181 000 .006 Vegetation was muchless dense thanestablished plot
HighR
LowR
Genotypic selection within populations affects genetic makeup of new populations
H2 Genotypic variation
in resistance
12,000seeds
1500seeds
Higher likelihood of colonization of disturbed area
Lower likelihood of colonization of disturbed area
Greater representation of genes of resistantgenotypes in colonizing seed pool
For discussion seePan & Price 2001Evol. Ecol. 15:583
Number of rametsafter a few years of population growth
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
Maintenance of genotypic variation?
Rela
tive rh
izom
e m
ass
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
1R
ela
tive r
esis
tance
level
1 9 12 4 2 10 5 6 8 7 3 11
Genotype
Rhizome mass (presence of disease)
Rhizome mass (absence of disease)
Resistance level
Maintenance of genotypic variation?
Rela
tive rh
izom
e m
ass
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
1R
ela
tive r
esis
tance
level
1 9 12 4 2 10 5 6 8 7 3 11
Genotype
Rhizome mass (presence of disease)
Rhizome mass (absence of disease)
Resistance level
Maintenance of genotypic variation?
Rela
tive rh
izom
e m
ass
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
1R
ela
tive r
esis
tance
level
1 9 12 4 2 10 5 6 8 7 3 11
Genotype
Rhizome mass (presence of disease)
Rhizome mass (absence of disease)
Resistance level
Maintenance of genotypic variation?
Rela
tive rh
izom
e m
ass
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
1R
ela
tive r
esis
tance
level
1 9 12 4 2 10 5 6 8 7 3 11
Genotype
Rhizome mass (presence of disease)
Rhizome mass (absence of disease)
Resistance level
Acknowledgements
CommitteeJim BeverLynda DelphMichael TanseyMaxine Watson
Undergraduate L490’sJ. PaulT. PawlowskiL. LaskyR. LemasterKara Kitch
Claylab folksJean PanPaula KoverAlissa PackerJanice Alers-GarciaTammy JohnstonJen KoslowJenn Rudgers
Funding sources:Indiana Academy of ScienceB.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