Evolution of plant resistance to a fungal pathogen


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  • From flwrs and seeds to leaves and roots, Paths and Herbs attack all parts of their plant hosts as seen in this Fig from Harper’s 77 book on pop biol of plants
    Roots are attacked by rotters and gallers
    Smuts infect anthers, Ergots attack ovaries and fungal endophytes can completely sterilize their hosts
    Fungi, bacteria and viruses also attack leaf meristems, produce leaf galls and infect leaf mesophyll
    Nearest and dearest to my heart are the rust fungi which are usually found on leaves and stems
    Given their large economic impact on crops and ubiquity in natural plant pops, it is not surprising that resistance to natural enemies is a major focus of research
  • Path freq found to decrease host fitness and abundance: humans have found ways to use these fitness effects to their advantage
    The Australian national trust is distributing a rust to curb the spread of a noxius invasive called Bridal creeper prompting the press release headline toungue twister ” “
    This species produces mats of underground tubers that strangle less competative native spp & prevent potential competitors from germinating
    The rust causes plants to lose their leaves and use reserves from tubers, eventually killing existing plants, also causeing up to 75% reduction is seed production, greatly limiting its spread
    This system provides a dramatic example of a rust that could exert strong selective pressure for resistance in its clonal host
  • The goal of my research was to…
    Assessment of evolution in clonal plant species requires a very different approach than assessement of evolution in non-clonal species
    In non-clonal species seed production is of primary importance b/c it is the only way that new individuals can be produced
    For Euth and the majority of other clonal plants, vegetative ramet production is the primary means of producing new individuals within populations, so I focus on addressing the components of natural selection through vegetative reproduction
    Because clonal species are dominant members of many plant communities, it seems important to assess the potential for evolution of resistance through veg repro
    Although I adressed these comp in a single system, my results should be broadly applicable to the evol of R in clonal plant species that rely largely on veg reproduction for population establishment and maintenance

  • Phrased in terms of evolution through vegetative reproduction, questions addressing the three components of NS provide an outline of the majority of my talk:

    After addressing each of them in turn, I’ll assess importance of seed production in this system
  • My system involves a rust whose infection often reaches epidemic levels late in the season in natural populations: in any given year it is common to see Euth plants that are lit up like florescent orange Xmas trees
    On the left are stems of E gram from a natural populations showing a range of infection intensities
    Pictures on the right show details uredial pustules of C ast, which are the repeating stage of the rust
  • E.g. clonal perennial herb, Close relative of common g-rod
    Disease is introduced to pops each year by spores of C ast from 2 need pines
    Fungal hyphae grow within Euth leaves and then produce uredial pustules, which is the repeating stage of the disease
    Individual pustules produce 1000s of spores, which infect oth leaves same and Lvs other plants
    Underground connections break down at about this time each year so each stem is independent before the disease peaks
    Dis peak during seed prod & rhiz growth and then begins to produce spores that reinfect the alternate host
    Above grnd stems of Euth die back each fall & plants Overwinter as uninfected rhizomes that send up mult new shoots next spring
  • Most studies of resistance address qualitative ‘all or nothing’ resistance that Stops pathogen growth and is effective against specific pathogen races

    Euth exhibts QUANT res that slows pathogen growth and is effective against a range of pathogen races

    Mechs of QR are not well understood--may be due to variation in leaf cuticle thickness, leaf hair density, stomatal density or function, and/or availability of resources to pathogen
  • Since the mechs of QR are usually unkown, it is described by its effect on pathogen gro & repro as depicted in this figure from burdons 87 book
    time is represented on the Horizontal axis
    And the Vertical axis shows a # of pathogen spores
    Quantitative res can slow pathogen growth:
    By Reducing the number of spores that penetrate the leaf as shown y arrows that end here
    By Preventing some colonies from reproducing after they have penetrated the leaf
    By increasing the time to reproduction as indicated by curved line so this colony…
    And by Reducing pustule fecundity by decreasing their size or longevity

  • Quantitative resistance is defined as…
    (READ), plant resist characters
    As well as non-plant based vars, such
    Var. virul. in path pop.,
    Stochastic effects due to epidem. Of disease spread
    And environmental factors that might affect inf lvl
    All Traits
  • To address the first question (READ), I collected
    3 geno from each of 4 pops around MC
    After 2 generations of greenhouse propagation to reduce maternal environmental effects I had 40 clones of each genotype--
    20 clones of each of 12 geno were planted in a randomized block design in each of two exp fld sites on opposite sides of town: Hilltop field & Bayles field
    After further propagation (not depict here) 12 more replicates of each geno planted in pots & grown outdoors at hilltop field
  • Picture of one of the exp fld plots the 2nd year after planting
    Natural vegetation was left as undisturbed as poss to facilitate nat dis sprd
    Each pole marks sep replicate made up of 1-many stems derived from single stem in yr 1

  • Surveys of infection intensity due to natural disease spread were performed over a 3 yr period in the experimental field and later over two years in potted plants
    Infection intensity was estimated on a 0-10 scale for each ramet and these values were used to calculate average infection intensity for each group of ramets derived from a single replicate (which I’ll refer to as a frag)
    If there is GENOTYPIC variation in resistance in this system, surveys should show that randomly located replicate frags of a geno have inf intensities that were more similar to each other than they were to other neighboring fragments of different genos

  • These data show geno within origin pops vary in Res Lev
    X-axis geno, arranged by pop origin (sets of 3 bars with same shading came from same origin pop)
    Y-axis, mean % LAI for each genotype, so short bars represent greater resistance, error bars indicate 1 SE
    For origin pops with starred bars, at least one pair of genotypes differed in inf intens.
    In this site/year comb, frag had >50% of LA cov, & genotypes from within 3 of 4 original pops showed sig var. in infection level,
    No sig diffs in inf intensity among populations, since they were tested over geno(pop), this suggests that Var within POPS was greater than var between them

  • These are graphs on the same axes for each of the infection intensity surveys
    Each panel a diff site/year combo, (point out each)
    Nested ANOVAs again showed no sig effect of origin pop
    There were hwvr, sig geno (pop) effects for 5 of 6 site/yr comb
    Perhaps the most striking thing about these data is the major diffs in overall INF level between BF in 98 & 99 and the reason I disnt include data from 97 is because there weren’t any---this site remained completely uninfected in this year, adding to the evidence that inf level can fluctuate greatly across years
    It is also clear that potted plants tended to be more infected than those in the field, although the epidemic at Bayles in 98 shouws that their infection intensities were not unnaturally high
    Furthermore, data from both years at HF show that detectable differences in INF INT do occur at lower overall infection levels
    So...within-pop genotypic variation in resistance does occur under a variety of dis and envtl conditions, and can fluctuate greatly from year to year
  • In order to combine data across sites and years, fragment inf int was plotted with respect to the mean infection intensity of neighbor fragments across all experimental field datasets
    Logistic curves were fitted to these data, representing the infection intensities of focal fragments as a function of local population growth of the pathogen
    These data are for 3 fragments from a single origin population, and show that genotypes have fairly consistent infection intensity across environments and years, here genotype 9 (shown in green) is the most resistant, 8 (shown in blue) is intermediate and 7 (in red) is least resistant
  • This is a graph including all 12 genotypes across exp fld sites and years
    Genotypes 1 and 9 stand out as being more resistant across environments, we’ll see these genotypes highlighted again later…
    Thus genotypes regularly vary in resistance in high and low infection conditions as was seen in the individual datasets, and a couple of genotypes are more resistant across a variety of disease and environmental condtions
  • Now that we have seen that genotypes vary in resistance to disease, I’ll address the 2nd question:

  • The data I just showed you demonstrates that Vg is responsible for a sig port of the pheno var in inf intens (Vp)
    For evol of res to occur through genetic changes in sexual off, it must be further demonstrated that a sig portion of Vg is additive (Va), I.e. due to eff of indiv alleles, rather than due to interactions between alleles (such as dominance…) b/c mixis & recomb that occurs during sex repro will make interaction effects unpred. Thus heritability of sexual off..
    Vegetative offspring, however, inherit intact genotypes from their parent, including both individual and interaction effects of alleles affecting resistance. For clonally-derived offspring then, its is the size of Vg that matters.
    Clonal repeatability values quantify Vg, providing an estimate of the degree to which clonal offspring will resemble their parent under a partic set of cond, often referred to as broad-sense heritability (H2)
    CR is a measure of the proportion of pheno var that is explained by genotype
  • (Read) for 4 of the 6 plot/yr combo, showing that for these 4 datasets, genotype explained 10 to 40% of var in res
    R was More likely to be sig in high infection years, but also quite high in 1 low infection year at HF

  • This significant heritability of resitance will lead to evolution of resistance if resistant genotypes have greater rates of vegetative reproduction.
    I call this form of selection genotypic selection because it results from differential reproduction of genotypes
    This diagram provides an eg of GS based on a highly resistant genotype that reproductes twice as fast as a less resistant genotype.
    Both genotypes start off as a single ramet. Rhizomes of ramets of the high R geno produce 4 new ramets before ramets die each year, resulting in 16 ramets in the 3rd year. Rhiz of ramets of the low R geno produce 2 new ramets before ramets die each year, resulting in 4 ramets in the 3rd year.
    Thus, after two ramet generations, the resistant genotype makes up 80% of the population. This (READ)…is an eg of
    When evol is defined as a change in allele frequency in the ramet population, genotypic selection can be said to have caused evolution of resistance in this population.
  • So far, I’ve shown you that there is Geno var in res…
    And that it can be strong enough to significantly effect RES LEV of offspring ramets
    Furthermore Ive shown that this will lead to evolution of resistance through GS within populations if disease affects vegetative reproduction
  • To test for effects of disease on vegetative reproduction, I sprayed half of the experimental plants with fungicide and the other half with water. Genotypes were evenly represented between treatment groups.
    This design was used in outdoor pots and the experimental field to test for effects in the presence of disease and in the greenhouse to determine whether the fungicide effected vegetative reproduction in the absence of disease
    Plants were grown for a min of 2yr to allow detection of delayed of disease, which maybe esp important in this system since the disease peaks late in the season
  • These data show that the Fung was effective in reducing infection intensity
    Treatment (Fungicide and water) is on the x axis and infect int on y
    Fitness diffs would have been easier to detect if FS plants were inf free, but the actual effect of the treatment is more realistic, because QR also reduces rather than prevents infection
    In potted plants, inf was reduced by about 1/2, whereas field differences were lower in mag
    Thus Although Inf Ints differed sig in every case, diffs greater in potted plants than in field
    If disease does reduce vegetative reproduction, then we would expect to see lower stem number, stem mass or rhizome mass in WS (that is) Higher infection plants
  • READ
    There are a number of other graphs like this one so I’ll explain this one in detail
    On x axis are 2 treatment groups (fung and water-sprayed)
    Y-axes various measures of growth or veg repro arranged in chronological order, thus the first measurement was…
    Box plots depict the distribution of values of each variable:
    median, notch 95% conf interv of med, box ends 75%le, bar ends 90th %le
    A MANOVA, showed a significant effect of treatment on the mutivariate distribution of all 4 variables
    When looking at individial response variables, I frequently lowered alpha (THE value p had to be below to detect sig effects ) to correct for multiple tests on related variables… alpha value is displayed whenever it is below .05
    First 3 panels show that there were no effects of high infection on above-ground size during the 2 yrs of treatment, but high infection did result in lower rhizome mass by the end of the experiment as seen in the last panel, rhizomes of high infection plants weighed about half as much as those of low infection plants
    Next slide shows effects of high inf on rhiz mass of individual genotypes…
  • (Read)
    That is rhiz mass of FS plants (rep by striped bars) was always higher than rhiz mass of WS plants (rep by solid bars), & significantly so in 8 of 12 genotypes
    Assuming that rhizome mass is a good predictor of fragment size in the next year, then these results show that high disease levels cause decreased vegetative reproduction in potted plants
  • In exp fld there was no detectable effect of disease on AG veg repro aft 2 yrs of treat
    three years shown here b/c field plants were harvested midway through the 3rd year to allow for detection of delayed effects of disease
    Weak trends toward an effect dissappeared as time went on
    Absence of effect:
    lower Overall inf level of Exp fld plnts compared to potted plants, and smaller diffs in inf int among treat groups
    Also Extensive variation in frag size w/in treat groups, prob due to microenvironmental soil var
    Also note Rhizmass not included, b/c…
  • Fragments were large
    (This is a Sing frag and as you can see it is made up of dozens of stems)
  • So extensive labor was required to dig up rhiz of even a single fragment
    So I selected 16 focal frags from extremes of inf intensity in 00, Treated them for another year and dug up at end of 01
  • For these focal fragments, trt grps did not differ in Inf int in 99 or 01, but diffs in 2000 were almost 2x the size of the greatest diff among treat groups in the potted plants
    So, any diffs in rhiz mass among focal frags would be largely due to inf diffs in 2000
  • fig on left shows results from these 16 frags
    x axis is TBA (a measure of AG frag size)
    and y axis final rhiz biomass
    The solid line is a regression for water-sprayed (higher infection) fragments and the dotted line represents fungicide-sprayed, lower infection frags
    Lower slope of solid line shows that higher infection frags had lower rhiz biomass rel to AG size than low infection frags
    Fig on right shows that earlier senescence of more highly infected fragments is a potential mech for this result:
    Infection intensity of WS plants is on the x axis, and sen date is on the Y (from earlier to later); the negative slope of the regression line shows that plants with higher inf int senesced earlier, prob reducing the total resources avail for rhiz growth
    Thus field data produced similar results to potted plant data but to a lesser degree:
    High infection level reduced vegetative reproduction through decreased final rhizome biomass
  • no signficant effect of Fung Treat on veg repro in disease-free g-hse plants,
    If there was any effect of fungicide, it was in the opposite direction from that expected from reduced infection intensity, as indicated by trends toward DECREASED growth in stem number and mass in FS plants
    So we can be conf that diffs in veg repro in the presence of disease are due to infect intensity diffs rather than direct effects of fung

  • Te recap, host genos do…
    The affirmative answers to these questions suggest that resistance level can evolve in this system through differential reproduction of genotypes that vary in resistance.
    The large increase in stem number of frags in pots and field show that veg repro is an important determinant of rep of genotypes in established pops, and in the introduction I used the observation that seedling recrutiment is often limited in established clonal populations to justify a focus on selection through veg repro
    but the genetic structure of established populations could also be effected by seed recruitment, and seed recrutment is critical for estab of new pops since rhiz cannot disperse over large dist
    Next, I’ll show you the effects of disease on seed prod and then present data from a final experiment show that Seed Recr is rare in estab pops

  • X axis
    Y axis estimated
    The data from the same GH plants used to assess effects of fungicide treatment on veg repro and show that there was no effect of fungicide treat on seed production in the absence of disease
  • These data from the outdoor potted and experimental field tests of disease effects showed major reductions in seed prod

    Both in potted plants and exp field high infection produced about half as many seeds as low infection plants
  • To address recruitment in established populations, I added known quant of seeds to quadrats in an open plot, an estab exper field and nat pops
    Open plot shown here on L, and tilled and untilled quadrats in expt’l field shown on right
    Approx 40K seeds were added to each quad in winter = 12x natural seed density (based on seed rain of other g-rod spp)
  • (Read) Indeed
    Recruitment under controlled cond showed that 8 of 10 seeds were viable, germinated and survived to adulthood
    Under ‘natural’ conditions, however, recruitment was much lower
    Recruitment in the open ‘colonization’ plot was about 6 seedlings per 10,000 seeds and t in established plots and nat pops recruitment was an order of magnitude lower still
    Thus seed production should have a minimal impact on established populations
  • As differences in vegetative reproduction lead to increased representation of more resistant genotypes within populations, the probability that they will continue to do so increases, as does the proportion of their genes in the colonizing seed pool
    This diagram shows an abbreviated version of the genotypic selection diagram I showed earlier: after a few years of population growth, the majority of the ramets will be higher resistance genotypes
    Given that lower R ramets produce half as many seeds as higher R ramets, both genotypic selection and effects of disease on seed production will lead to a higher likelihood that seeds that colonize disturbed areas were produced by R genos, leading to greater representation of the genes of resistant genotypes in the colonizing seed pool
    If resistance is heritable by seed offspring then evon within pops will affect resistance level of new pops, if not resistant genotypes will still have greater lifetime reproductive success because more of their genes are represented in new populations
  • In summary, I’ve shown you that:
    And finally,
  • If genotypic selection for resistance commonly occurs in natural populations of Euthamia, we would expect the majority of individuals to have high resistance
    The first set of data I showed you, however, showed that resistance variation is common in natural populations and suggested that there may be extreme variation in disease pressure from year to year
    One way that genotypic variation in resistance can be maintained in the face of periodic selection for greater resistance is when there is a cost to disease resistance
    This figure synthesizes 3 kinds of data from potted plants, so I’ll walk you through it
    Genotypes are arranged on x-axis from most to least resistant, based on the bars which show resistance level on the L axis relative to the least resistant genotype, so geno 1 had 75% lower infection than the least resistant geno, geno 9 had 50% lower than genotype 11 and so on
    The lines show rhizome mass of potted plants in the presense and absence of disease, and again these are relative meas, so the solid line shows that geno 1 had highest rhiz mass in presense of disease, geno 9 had next highest, etc., and the dotted line shows that geno 11 had the highest biomass in the absence of disease, geno 3 the next highest, and so on
  • The left side of the figure shows that more resistant genos benefit from higher resistance level through higher relative rhiz mass in the presence of disease
    An its particularly intruiging that genos 1 & 9
  • The right side shows that, less resistant genoypes benefit from lower resistance level through higher rhiz mass in the absence of disease, suggesting a cost of resistance through decreased rhizome growth when the disease is absent
  • If rhizome mass is a good indicator of future vegetative reproduction, the frequency of resistant genotypes should increase after high infection years, whereas susceptible genotypes may increase in freqency after low infection level years, potentially maintaining variation in resistance in this system
  • I’d be happy to take questions after I acknowledge the large number of people without whom I’d have been unable to complete my research
    Since he’s pictured here,
    I’ll start will Dave Campbell
    who listened to listened to me ramble until I figured out the answer to my question on what seemed like a weekly basis for 5 summers,
    Watered my plants more than 2000x! Plowed this or mowed that on short notice, and frequntly fixed things that I broke or smoothed things over when I did unorthodox things that ruffled the feathers of the greenhouse establishment
  • No E. gram plants recruited in quads in open plot or established exp fld plot
    Here photos one quadrat in each treatment in estab plot or pop
    Show variation in density of other species
    Census areas were increased to the entire plot in the experimental field and 2.5m radius circles around each quad in nat pops to include seeds that may have been dispersed outsid e of the quads
  • Evolution of plant resistance to a fungal pathogen

    1. 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. 2. The higher plant as a series of niches for natural enemies
    3. 3. Trust’s rust busts bridal lust
    4. 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. 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. 6. Euthamia graminifolia infected with Coleosporium asterum
    7. 7. Pathosystem
    8. 8. Qualitative resistance Quantitative resistance Stops Pathogen growth Slows pathogen growth Race specific (single gene) Race non-specific (multiple gene)
    9. 9. Taken directly from Burdon 1987 Quantitative resistance -- stages of action
    10. 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. 11. 1) Does resistance vary among host genotypes? DESIGN
    12. 12. Resistance assessment plot (in 1998)
    13. 13. Measuring infection intensity
    14. 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. 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. 16. Genotype resistance level is consistent across datasets
    17. 17. Two genotypes stand out as being most resistant
    18. 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. 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. 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. 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. 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. 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. 24. Fitness assessment field design
    25. 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. 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. 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. 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. 29. A single fragment in the experimental field
    30. 30. Rhizome ‘excavation’
    31. 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. 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. 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. 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. 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. 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. 37. Recruitment plots and quadrats
    38. 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. 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. 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. 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. 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. 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. 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
    45. 45. 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