Summary of third class

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Summary of third class

• Unique DNA sequence can be used to identify
  target organism by
• Designing a taxon specific assay (usullay
  employing primers that will only bind to target)
• Using BLAST search engine
• Placing sequence in own database and perfor
  phylogeographic analysis
• DNA loci need to be variable among species and
  relatively similar among individuals of the same
  species. If sequence (allele) is completely
  fixed within one species and variable between
  species we call that an alternatively fixed
  allele

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Summary of third class

• DNA is amplified through the PCR reaction before
  being analyzed (sequenced, electrophoresed,
  RFLPed)
• With TS-PCR presence of amplified fragment
  (amplicon) is a sign that target is present
• However size of amplicon is still and normally
  used to confirm this
• Size is determined through gel or capillary
  electrophoreses, by running a size standard

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Summary of third class

• DNA sequences are co-dominant, a single locus
  may be sufficient for species determination, but
  multiple independent loci are needed to define
  individual strains or genotypes
• When using DNA information to build trees we
  need to know the statistical support for the
  clades we see
• Too much variation is not necessarily good
  because of homoplasy.

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Summary of third class

• P. ramorum case European and North American
  individuals are not directly connected because
  they are placed on two distinct and strongly
  supported clades. If one of them was derived
  from the other it would be nested within it
• P. ramorum limited genetic diversity in
  California when compared to worldwide nursery
  pops indicates it is introduced in California
  remember that term of comparison is needed as
  some microbes display almost no variation
  worldwide
• Exotic microbes go through a genetic bottleneck
  and a strong founder effect.

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• 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

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What about the two readings?

• Heterobasidion
• Armillaria

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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
CCGAAATCGGACCTTGAGTGCGGAGAGAGAGAGAGACTGTACGAGCCCGA
GTCTCGCAT
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Mating Type A1 A2 A2
Growth Rate Fast Slow Fast
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Terminology Genotype Lineage Population
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Results of 1st microsatellite study

• There actually three distinct (genotypically and
  phenotypically) lineages of P. ramorum
• Very low diversity in US forests (microsats
  cannot discriminate among individuals, clonality
  confirmed), only one lineage
• Several genotypes but only one lineage in EU
  nurseries
• Three lineages in US nurseries

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Was the pathogen first in US forests or in US
nurseries?
Slide 12
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Was the pathogen first in US forests or in US
nurseries?
Slide 12
nurseries
forests
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From the genome we learned

• P. ramorum in the US comes from a sexually
  reproducing population (heterozygosity rate in
  its 2n genome)
• P. ramorum appears to have sex only periodically
  and then reproduces clonally

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Co-linearity of P. ramorum and P. sojae
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Further application to track invasion

• First microsatellites could not differentiate
  among genotypes, because of clonality
• We checked in more variable areas (areas with no
  colinearity for instance ) for microsatellites
  that would differentiate genotypes
• Ability to differentiate even between very
  closely related clonal genotypes would allow us
  to study patterns of introduction, potential for
  local differentiation or evolution, understand
  pathogen spread by looking at spread of genes

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We found hypervariable microsatellites that could
do the jobso what did we do?
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Genotyped approximately 300 isolates from 17
forest sites and from a number of nurseries
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Distribution of Phytophthora ramorum in December
2002
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Distribution of Phytophthora ramorum in December
2006
Map from www.suddenoakdeath.org M. Kelly,
UC-Berkeley
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We stratified our sampling in the following
way1- Sites gt 10 years2- Sites infested for
10-5 years 3- New infestations
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Infestations differ in composition of genotypes,
even if they are in the same county. ( 13 sites,
Contingency analysis Plt0.0001)
2
1
6
2
2
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High diversity (high Fst) indicates migration
among sites is limited. Dynamics of medium range
spread based on rare successful movement of
infectious propagules, followed by local
differentiation
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Overlap of genotypes between nursery and old
wild populations
Marin
P0.16, Fst almost 0
Santa Cruz
Sonoma
Populations are at tens of miles from one another
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When we constructed an unrooted cladogram using
PHIst
New sites
Intermediate sites
Nurseries and old sites
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Mantel test among all individuals. Morans I vs
ln (geographic distance)
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DNA applications to study ecology of pathogen

• Culturing and direct observation unreliable
• DNA based test doe not mean organism is alive or
  active
• Reverse transciptase PCR targets messanger RNA
  and allows for quantification of signal, we can
  actually study whether organism is active (many
  copies of mRNA), alive but dormant (few copies),
  or dead no copies

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Mt DNA

• gtP. ramorum COXI (925bp)
• TTAATTTTATTTCAACTATTTATAATATGCGAGCTCCTGGTTT
  AAGTTTCCATAGATTACCTTTATTTGTTTGGTCTGTATTAATTACAGCTT
  TTCTTTTATTATTAACATTACCTGTTTTAGCTGGTGCAATTACTATGTTA
  TTAACTGATAGAAATTTAAATACTTCTTTTTATGATCCATCAGGCGGAGG
  TGATCCTGTGTTATATCAACATTTATTTTGGTTTTTTGGTCACCCTGAAG
  TTTATATTTTAATTTTACCAGCATTTGGTATTATTAGTCAAGTTTCTGCA
  GCTTTTGCTAAAAAAAATGTATTTGGTTATTTAGGTATGGTTTATGCTAT
  GTTATCTATAGGTTTATTAGGTTCTATTGTTTGGGCACATCATATGTTTA
  CTGTAGGTTTAGATGTAGATACTAGAGCTTATTTTTCTGCAGCTACTATG
  ATTATTGCTGTACCTACAGGTATTAAAATTTTTAGTTGGTTAGCTACTTT
  ATGGGGTGGTTCATTAAAATTTGAAACACCTTTATTATTTACCTTAGGTT
  TTATTTTATTATTTGTTATGGGTGGAGTAACA

Ram Reverse
COX I 925 bp
Ram Forward
Amplicon 138 bp
Cytochrome c oxidase is large transmembrane
protein, found in the mitochondrion, which acts
as the terminal enzyme of respiratory chain.
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RT-PCR from symptomatic bay leaves (China Camp
Oct-2005)
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EFFECTS OF SOD

• Ecological disaster tanoaks at risk of
  extinction change in forest structure and
  composition
• Biota linked to vanishing trees highly impacted
• Hydrogeological impact
• Climate and nutrient cycle impact
• Hazard to humans and properties
• Added cost because of intense regulation of
  pathogen cleanisng, prescriptions, testing
• Affecting trade

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Emergent Disease

• Previously unknown pathogen
• Unknown Biology and Epidemiology
• Ecology of California coastal forests little
  understood

ALL RESEARCH USEFUL FOR MGMT.
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Adaptive management

• Need to act even with imperfect knowledge
• Mgmt. changes as understanding increases
• Adapt strategies from similar situations
• Strategies may vary depending on situation
• Real world testing?

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Scale Dependent Research and Management

• Individual tree protect individuals but do
  little to slow epidemic, timing and understanding
  of specific pathways essential
• Landscape protect a stand, make local
  environment unfavorable to pathogen, scaling
  essential
• Regional to continental change distribution and
  composition of species, prevention and modeling
  essential

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Introduction phase 1- Escape of pathogen
from Infected nursery plants at two locations
Mount Tamalpais (Marin County), and
Scotts Valley (Santa Cruz County) 2- Nurseries
and two sites have identical strain composition,
but distance between sites is impossible for
natural spread of organism
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Rhododendron In EU mostly a nursery issue, but
also present in nurseries in US and Canada
Stem canker
Leaf necrosis
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Bay/Oak association
Coast Live Oak (no sporulation)
Bay (highly infectious)
Canker margin in phloem
Bleeding canker
Sporangia
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Bimodal spread pattern50 reduction at 10 m200
m in absence of wind1-5 km in presence of
strong winds
Scale of dispersal essential to scale control
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ERADICATION in OREGON

• PLANTS
• SOIL
• WATER

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Jay Smith Road

• 50 acres, 4 units
• Thinning
• Thinning Burning
• Monitor sprouts and new seedlings over 2 years
• Monitor rainwater and soil populations of P.
  ramorum

Source Y. Valachovic, C. Lee
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Before
After
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Thinning and inoculum load
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AgriFos and PentraBark Topical Application

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Zoospore Inoculator for P. Ramorum
Zoospores are in Direct Contact with Tree for 24
hrs.
Grafting Wax Creates a Water Tight Seal
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Agrifos vs. Azomite Treatments (efficacy 1 - 24
months)
a
a
Canker Size (mm)
b
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Statewide tanoak resistance program
Tanoak Petiole Plug Assay
Less susceptible
More susceptible
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Some conclusions

• Understanding of biology and ability to detect
  pathogen essential for individual treatment of
  trees and early eradication
• Scale of dispersal makes landscape level control
  and eradication difficult because spread from
  hotspots can coalesce (a couple of miles)
• Regional level management difficult because
  costly in these mostly unmanaged forests disease
  management has to fit with overall goals of
  landowner

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Bicycles for Cargo Transport
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