Transcript Lecture #4 - College of Natural Resources, UC Berkeley

SUMMARY
• Sudden Oak Death
– Deemed introduced because disease was never seen
before, mortality rates were very high, and distribution did
not match range of hosts
– Genetic studies reveal simple genetic structure in forests.
Only one lineage of clonally reproducing individuals. AFLPs
and microsatellites indicate forest lineage is different from
european nursery lineage. In US nurseries three very
different lineages are found
– Symptoms vary depending on host: in oaks the pathogen
causes a girdling necrosis with bleeding on the trunk, on
rhodies and bays it cause leaf blight
– Epidemiologically important vs. dead-end hosts
•US forest isolates
clearly distinct from EU
nursery isolates, also
have different mating
type
•Isolates from nurseries
in WA, OR, & BC both
of the US and EU types
•Potential for XXX sex
and recombination in
US nurseries
•US forest population
is genetically very
homogeneous,
trademark of an
introduced species
The entire genome was sequenced in
less than 3 years since discovery of organism
* 12 SSR loci (di- and tri- repeats identified)
* Loci selected to be polymorphic both between
and within continental populations
* 500+ representative isolates analyzed
CCGAAATCGGACCTTGAGTGCGGAGAGAGAGAGAGACTGTACGAGCCCGAGTCTCGCAT
We found
same
genotypes in
nurseries and
forests proving
origin of wild
outbreak
Bay/Oak association
Bay
Coast Live Oak (no sporulation)
Canker margin in phloem
Bleeding canker
Sporangia
Infectious diseases spread not randomly but around initial
infections
Duglas-fir sapling branch tip wilted by P.
ramorum
Symptoms on Buckeye leaves and petioles
Scorching of maple
leaves caused by P.
ramorum
Spring
Autumn
More problems
• Host lists started expanding ( now over
100) in all plant families and ferns
• Symptoms looked extremely different on
different hosts
• Isolation of organism from symptomatic
tissue often not possible
• Isolation success extremely different in
different seasons
Environmental sample
DNA extraction
Dna probes (plus/minus)
++-
DNA
fingerprints
DNA-based diagnostics
Designed 2 sets of P. ramorum specific primers
(www primer3 software)
•
phyto1-phyto4 (1st round PCR)
• highly specific for P. ramorum
• 687 bp fragment (in between red arrows)
•
phyto2-phyto3 (2nd round PCR)
• nested in phyto1-4 amplicon; specific for Phytophthora spp.
• 291 bp fragment (in between yellow arrows)
ITS1
Phyto1
5.8S
ITS2
Phyto2
Phyto3
Phyto4
Fraction Positive
Culture versus nested PCR
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
PCR Pos
Culture Pos
Total
(N=216)
Foliar: Foliar: Bay Wood
Not Bay
(N=36)
(N=65)
(N=116)
Significant effect of diagnostic type (P <0.001) and sample type (P=0.0036)
The assay we developed
became the first DNA assay to
diagnose non viral plant
pathogens. Now diagnosis of
most microbes will be DNA
based
Synchrony pathogen-host
Spores in rainwater 2001-06
600
250
500
Disease cycle
200
SE
+
400
150
300
100
200
50
l ct n r l ct n r l ct n r l ct n r l ct n r0 l
Ju O Ja Ap Ju O Ja Ap Ju O Ja Ap Ju O Ja Ap Ju O Ja Ap Ju
2002
2003
month
2004
2005
Total precipitation (mm)
Mean cfu/L
0
100
80
60
Davidson et al. 2005; unpublished
Susceptibility of oaks
(lesion size)
40
20
0
Mar-03 May-03 Jul-03 Sep-03 Nov-03 Jan-04 Mar-04
Average lesion (mm_)
80
60
40
Wetness > 12 h
20
0
0
6
12
18
24
30
36
42
48
54
Time (h)
2
Lesion area (mm)
50
40
30
Temp >19 C
20
10
0
15
17
19
21
23
25
Temperature ( C)
27
29
Bay Laurel / Tanoak SOD Spore Survey
35
Temp (C)
30
Rain (mm)
25
20
15
10
5
0
Date
How to control emergent
exotic diseases
• PREVENT THEIR INTRODUCTION
• LIMIT THE HUMAN-SPREAD OF
PATHOGENS (infected plants, plant
parts, dirty tools)
• EMPLOY HOST RESISTANCE
• CHEMICAL AND OTHER MITIGATION
STRATEGIES
Forest pathogens can never be
eradicated
PREVENT: Diagnose
Symptoms relatively generic, very
variable, and pathogen not always
culturable
DNA TESTS
LAB CULTURES
AgriFos and
PentraBark
Topical
Application
+
Agrifos vs. Azomite Treatments
(efficacy 1 - 24 months)
a
14
a
Canker Size (mm)
12
10
8
6
b
4
2
0
Azomite
Positive Control
Agrifos
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
• Invasion driven by ecological conditions
• Enemy release hypothesis
• Resource availability
(pathogenicity/virulence)
Pathogenicity
• Qualitative: ability to cause disease
• Often regulated by a single gene
• Avr genes in pathogen and resistance
genes in host
Gene for gene
• Resistance in host is dominant
• Virulence is recessive
ar aR
Ar AR
Gene for gene
• Resistance in host is dominant
• Virulence is recessive
ar aR
Ar AR
Resistance: no
disease
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)
Avr/R genes matches are
specific
• Race of the pathogen (avr1) matched
by variety of the crop (R1).
• At the base of crop breeding science
• If R genes target avr genes linked to
important housekeeping functions, they
are more durable
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
Plants immune response
• Plants do not possess an immune system
such as that of animals
• They do recognize pathogens
• Recognition initiates secondary metabolic
processes that produce chemicals that will
stop or slow microbial infections: thickening of
cell wall, premature cell death (HR response),
systemic resistance
Virulence: quantitative
response
• Multiple genes controlling:
– Phenotypic traits conferring virulence
– Production of plant detoxifying enzymes
– Production of plant toxins
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
than genetic variability
CAN WE PREDICT:
• Selection of increased R in host?
– Host: R to exotic may be significantly present because it
identifies native pathogen.
– R may be absent.
– R may be present at low frequency. If host does not
exchange genes long distance, but only in areas already
infested there is a stronger selection process. Otherwise
locally selected R genes may be swamped by genes coming
from outside the area of infestation
– Shorter generation times favor pathogen
The red queen hypothesis
• Coevolutionary arm race
• Dependent on:
– Generation time has a direct effect on rates of evolutionary
change
– Genetic variability available
– Rates of outcrossing (Hardy-weinberg equilibrium)
– Metapopulation structure
CAN WE PREDICT:
• Selection of increased virulence in pathogen?
– It depends on the presence or absence of trade-off
– Does increased virulence make pathogen more fit?
– It has been shown that in some cases (but not always), there
is a trade-off between virulence and transmission
• Rapid generation time of pathogens. Reticulated
evolution very likely. Pathogens will be selected for
INCREASED virulence if no trade-offs are present
• In the short/medium term with long lived trees a
pathogen is likely to increase its virulence
• In long term, selection pressure should result in
widespread resistance among the host
Frequency-, or density
dependent, or balancing
selection
• New alleles, if beneficial because linked to a
trait linked to fitness e.g. by conferring
advantageous heterozygosity will be
positively selected for.
– Example: two races of pathogen are present, but
only one resistant host variety, suggests second
pathogen race has arrived recently
– Mating alleles: two mating alleles indicate a single
founder individual and high relatedness among
genotypes. In a varied natural population you
expect multiple mating alleles