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