CLASS REVIEW 2010
Lectures
• Understanding of nature, an essential
part of culture
• Forests essential for life on the planet
• Fungi essential for survival of forests
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DNA mutates, evolves, and different DNA sequences can be assigned to
different individuals, populations from different provenances, closely related
species, different species, different microbial pathovars
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DNA-based phylogeography allowed to discover pine pathogen in Italy was
of North American origin
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DNA based genealogies allowed to identify hybridization between native
and exotic pathogen
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DNA allows to identify new species and to determine whether they are
exotic or not
Definitions
• Propagule= structure used by an
organism to spread or survive
• Locus= a physical portion of a
chromosome,a gene
• Intron= a portion of DNA , a locus that
does not code for a protein
• Exon= a coding gene
Definitions-2
• Alleles= different DNA sequences at the
same locus
• If a locus has variation in sequence it is
polymorphic (many forms)
• Polymorphisms are differences in DNA
among organisms, the more polymorphisms
the easier it is to differentiate organisms
• There are more polymorphisms in introns
Definitions-3
• Invasive organisms: exotic organism that reproduces
and occupies progressively a larger area:
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Fast reproductive cycle
Vectored
Hardy
Occupy unoccupied niches
Different drain on natural resources
Make environment favorable for itself and other invaders
Linked to disturbances
If pathogen , more changes because top of pyramid
May hybridize with native species: new taxon is created
New host pathogen
combinations
• Pathogen stays/Plant moves: invasive
plant
• Pathogen moves/Plant stays: exotic
epidemic
• Pathogen moves/Plant moves:
biological control
Success. The “1:10” rule
• Can exotic withstand new environment
• Can it withstand attacks of predators
• Can it outcompete similar native
organisms by accessing resources
– Can a pathogen be pathogenic
– Can a pathogen be sufficiently virulent
Functions of avr/R genes
• Avr genes may help detoxify plant
enzymes, secure necessary aminoacids
or proteins, plant toxins, promoting
pathogen growth. Normally they are
mobile, wall-bound products
• R genes normally recognize multiple avr
genes and start hypersensitive
response (programmed cell death)
Can be R genes
accumulated?
• There is a cost associated with R genes
• Mostly R genes initiate costly defense
processed, often even when challenged
by innocuous microbes
• Some evidence that in absence of
specific avr, R are lost
CAN WE PREDICT:
• Success of an exotic microbe?
– Survival structures such as cysts, spores, etc
– Saprotrophic ability (ability to feed on dead matter)
– Degree of host specialization, the more specialized the
harder it may be to establish
– Phylogenetic distance of hosts (the closertive and new hosts
are, the easier the establishment)
– Similar ecology
CAN WE PREDICT:
• Levels of the epidemic?
– Density dependance: abundance of susceptible
hosts
– Genetic variation in host. In general it is assumed
that genetic variation in host populations slows
down epidemics, however backing data from
natural ecosystems is missing. It could be that low
genetic diversity associated with widespread
presence of resistance may be more beneficial
that genetic variability
• DNA polymorphisms can be diagnostic
– Mutations/Sex/Barriers to mating
• Plant Diseases can be biotic (interaction between
host and causal agent ), or abiotic
• Many organisms can cause plant diseases, but fungi
are the No.1 cause
• Diversity of fungi, but all have ideal structure for plant
infection:
– hypha/cord/rhizomorph/infection peg/appressorium
– Sexual vs. asexual reproduction: can do both
Definitions
• Alternatively fixed alleles
• Dominant vs. co-dominant markers
• Genotype
• Dominant vs. codominant genetic markers
• Concept of “genotype”
• Alternatively fixed allele vs.difference in frequencies
• PLANT HOST INTERACTION: timing, physical/chemical
interaction, basic genetic compatibility leads to virulence, gene for
gene hypothesis, pathogenicity
Categories of wild plant
diseases
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Seed decay
Seedling diseases
Foliage diseases
Systemic infections
Parasitic plants
Cankers, wilts , and diebacks
Root and butt rots
Floral diseases
• Janzen-Connol hypothesis; explanation of why diseases lead to
spatial heterogeneity
• Diseases also lead to heterogeneity or changes through time
– Driving succession
– The Red Queen Hypothesis: selection pressure will increase number of
resistant plant genotypes
• Co-evolution: pathogen increase virulence in short term, but in long
term balance between host and pathogen
• Density dependance
The biology of the organism
drives an epidemic
• Autoinfection vs. alloinfection
• Primary spread=by spores
• Secondary spread=vegetative, clonal spread, same
genotype . Completely different scales (from small to
gigantic)
Coriolus
Heterobasidion
Armillaria
Phellinus
OUR ABILITY TO:
• Differentiate among different individuals
(genotypes)
• Determine gene flow among different areas
• Determine allelic distribution in an area
WILL ALLOW US TO
DETERMINE:
• How often primary infection occurs or is
disease mostly chronic
• How far can the pathogen move on its own
• Is the organism reproducing sexually? is the
source of infection local or does it need input
from the outside
Important fungal genetic
systems:
• Intersterility genes
• Somatic (vegetative) compatibility
• Mating system
Summary
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AFLP, RAPDs, RFLPs, microsatellites
Repeatability
Test for power (PID and test progeny)
Have we sampled enough? Rarefaction
curves, resampling, need to be ob flat
portion of curve
Summary
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From raw data to genetic distance
Distance distribution
AMOVA PHIst
Distance based trees
Number of polymorphic alleles
The “scale” of disease
• Dispersal gradients dependent on propagule size,
resilience, ability to dessicate, NOTE: not linear
• Important interaction with environment, habitat, and
niche availability. Examples: Heterobasidion in
Western Alps, Matsutake mushrooms that offer
example of habitat tracking
• Scale of dispersal (implicitely correlated to
metapopulation structure)---
The scale of disease
• Curves of spore dispersal (rapid dilution effect, e.g
most spores fall near source, but a long low tail, a
few spores will travel long distances
• Genetic structure of species: the more structure the
more fragmented the less dispersal
• Mantel tests, spatial autocorrelation: plot the genetic
distance against the geographic distance
8
y = 0.2452x + 0.5655
r 2 = 0.0266
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6
Φ ST/(1-Φ ST)
5
4
3
2
1
0
1.5
2
2.5
3
3.5
4
4.5
Ln Geographic Distance (m)
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5.5
6
6.5
1
0.6
0.5
0.4
Moran's I
0.3
0.2
0.1
0
-0.1
-0.2
1
10
100
1000
10000
Mean Geographical Distance (m)
2
100000
1000000
Using DNA sequences
• Obtain sequence
• Align sequences, number of parsimony informative
sites
• Gap handling
• Picking sequences (order)
• Analyze sequences
(similarity/parsimony/exhaustive/bayesian
• Analyze output; CI, HI Bootstrap/decay indices
Population genetics concepts
• Gene flow, migration
• Lack of gene flow, genetic
substructuring=differentiation
• Hardy Weinberg= for diploid or dikaryotic organims
predicts levels of heterozygosity
• Inbreeding coefficient
• Fst
How do we know that we are
sampling a population?
• We actually do not know
• Mostly we tend to identify samples from
a discrete location as a population,
obviously that’s tautological
• Assignment tests will use the data to
define population, that is what Grubisha
et al. did using the program
STRUCTURE
CLASS REVIEW 2010
Research papers
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Hayden et al paper
describes how PCR
assay is designed: 1primers only match target
species and not relatives:
PCR product = pathogen
is there
2-nested approach
3-control primers amplify
all plants
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Molecular Ecology (2008) doi: 10.1111/j.1365-294X.2008.03773.x
Blackwell Publishing Ltd
Reconstruction of the Sudden Oak Death epidemic in
California through microsatellite analysis of the pathogen
Phytophthora ramorum
S. MASCHERETTI,* P. J. P. CROUCHER,* A. VETTRAINO,† S. PROSPERO‡ and M. GARBELOTTO*
*Department of Environmental Science, Policy and Management, 137 Mulford Hall, University of California, Berkeley, CA 94720-3114,
USA, †Department of Plant Protection, University of Tuscia, I-01100 Viterbo, Italy, ‡INRA, UMR 1202 Biodiversité Gènes et
communités, Equipe de pathologie Forestiere, BP 81, 33883 Villenave d′ Omon Cedex, France
Abstract
The genetic structure of the clonally reproducing Sudden Oak Death (SOD) pathogen in
California was investigated using seven variable microsatellites. A total of 35 multilocus
genotypes were identified among 292 samples representative of populations from 14 forest
sites and of the nursery trade. AMOVA indicated significant genetic variability both within
(44.34%) and among populations (55.66%). Spatial autocorrelation analyses indicated that
Moran’s index of similarity reached a minimum of 0.1 at 350m, increased to 0.4 at 1500 m
and then decreased to zero at 10km. These results suggest a bimodal pattern of spread, with
medium range dispersal (1500–10000m) putatively attributed to the presence of strong
winds. Lack of genetic structure was identified for three groups of populations. One group
notably included the nurseries’ population and two forest populations, both linked to early
reports of the pathogen. A neighbour-joining analysis based on pairwise ΦST values
indicated that the clade inclusive of the nurseries’ populations is basal to all California
populations. A network analysis identified three common genotypes as the likely founders
of the California infestation and proposes a stepwise model for local evolution of novel
genotypes. This was supported by the identification in the same locations of novel genotypes
and of their 1- or 2-step parents. We hypothesize that the few undifferentiated population
groups indicate historical human spread of the pathogen, while the general presence of
genetically structured populations indicates that new infestations are currently generated
by rare medium or long-range natural movement of the pathogen, followed by local generatio
Key points
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Organism is exotic, why?
How does it kill oaks?
How far does it spread?
Is it in equilibrium, How does it attain
diversity?
• What ecological conditions are
necessary?
• What can be done?
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Key points
• Pine mortality near Rome, never reported
before suggesting something new
• Heterobasidion root rot basidiocarps found at
base of trees
• Multiple loci analysis indicates pathogen is
from North America
• Likely to have been brought into Europe by
US Army with untreated lumber
Nature 394, 137 - 138 (1998) ©
Macmillan Publishers Ltd.
•Cause of sea fan death in the West Indies
Humongous fungus
• The Fungus Armillaria bulbosa is
among the largest and oldest living
organisms
Key points
• Armillaria does not reproduce via asexual
spores
• Same genotype found in a large area
• RAPDs and RFLP of mitochondrion and
mating alleles
• Tested sensitivity of RAPDs on full sibs
• Age estimated by dividing maximum distance
within clone by annual growth rate
Key points
• Native fungus, host specialized
• How does it infest stands? Does it need
stumps?
• How was research done? Sampling and
analysis
• What type of forests will enhance secondary
spread?
• Is source of inoculum local or not?
• How was it shown that nuclei can rearrange
themselves
Key points
• Wood decay fungus, generalist
• Sexually reproducing hence lots of local
diversity
• Easily airborne, easy to find hosts, no
genetic structure within Sweden
• Structure between Sweden and Finland
• Methods: RAPDS and AMOVA
Key points
Pathogen, very host-specific
• Infection is mostly primary by airborne
meiospores
• Method: AFLP analysis on haploid
meiospores
• AMOVA indicated significant genetic diversity
both within and among populations
• Lack of host= barrier to migration
Key points
• Mycorrhizal fungus, obligate symbiont
• Symbiont with most conifers, air dispersed
• Japanese market buys some species, rejects
others
• Species accepted by market are
monophyletic
• At least 3 species: circumboreal, mexican,
and west coast
• North America= center of diversity
• Oldest species is in North America
• Methods: DNA sequencing and AFLPs
• Isolation by distance: distant populations
more different genetically
Isolation by landscape in populations of a prized edible
mushroom Tricholoma matsutake
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Key points
• Specific mycorrhizal symbiont, underground
mushrooms, animal dispersed
• Islands in islands
• Compare genetics of fruitbodies and of seed
banks
• Genetic structure indicate low gene flow
among sites, but similar genetic structure
between two islands