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 The entire genome was sequenced in less than 3 years since discovery of organism less than 3 years since discovery of organism

* * 12 SSR loci (di- and tri- repeats identified) 12 SSR loci (di- and tri- repeats identified)

* Loci selected to be polymorphic both between * Loci selected to be polymorphic both between and within continental populationsand within continental populations

* 500+ representative isolates analyzed* 500+ representative isolates analyzed

CCGAAATCGGACCTTGAGTGCGGAGAGAGAGAGAGACTGTACGAGCCCGAGTCTCGCAT

We found We found same same genotypes in genotypes in nurseries and nurseries and forests proving forests proving origin of wild origin of wild outbreakoutbreak

Bay/OakBay/Oak association association

Bleeding cankerBleeding canker

Canker margin in phloemCanker margin in phloem

Bay Coast Live Oak (no sporulation)

SporangiaSporangia

Infectious diseases spread not randomly but around initialinfections

Duglas-fir sapling branch tip wilted by Duglas-fir sapling branch tip wilted by P. P. ramorumramorum

Symptoms on Buckeye leaves and petiolesSymptoms on Buckeye leaves and petioles

Scorching of mapleScorching of mapleleaves caused by leaves caused by P. P. ramorumramorum

AutumnAutumn

SpringSpring

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

Dna probes (plus/minus)Dna probes (plus/minus)

DNADNAfingerprintsfingerprints

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Environmental sampleEnvironmental sample

DNA extractionDNA extraction

Designed 2 sets of Designed 2 sets of P. ramorumP. ramorum specific primers specific primers (www primer3 software)(www primer3 software)

• phyto1-phyto4 (1st round PCR)phyto1-phyto4 (1st round PCR)• highly specific for highly specific for P. ramorumP. ramorum

• 687 bp fragment 687 bp fragment (in between red arrows)(in between red arrows)

• phyto2-phyto3 (2nd round PCR)phyto2-phyto3 (2nd round PCR)• nested in phyto1-4 amplicon; specific for nested in phyto1-4 amplicon; specific for PhytophthoraPhytophthora spp spp..

• 291 bp fragment (in between yellow arrows)291 bp fragment (in between yellow arrows)

DNA-based diagnosticsDNA-based diagnostics

Phyto1 Phyto4Phyto3Phyto2

ITS1 ITS25.8S

Culture versus nested PCRCulture versus nested PCR

0.00.10.20.30.40.50.60.70.80.91.0

Total(N=216)

Foliar: Not Bay(N=116)

Foliar: Bay(N=36)

Wood(N=65)

PCR PosCulture Pos

Fra

ctio

n P

osi

tive

Fra

ctio

n P

osi

tive

Significant effect of diagnostic type (P <0.001) and sample type (P=0.0036)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

Jul Oct

Ja

n Apr

Jul Oct

Ja

n Apr

Jul Oct

Ja

n Apr

Jul Oct

Ja

n Apr

Jul Oct

Ja

n Apr

Jul

Total precipitation (mm

)0

100

200

300

400

500

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Spores in rainwater 2001-06

month

Mean cfu/L

+ S

E

0

50

100

150

200

250

Davidson et al. 2005; unpublished

2002 2003 2004 2005

Disease cycleDisease cycle

0

20

40

60

80

Mar-03 May-03 Jul-03 Sep-03 Nov-03 Jan-04 Mar-04

Susceptibility of oaksSusceptibility of oaks(lesion size)(lesion size)

0

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80

0 6 12 18 24 30 36 42 48 54

Time (h)

Ave

rag

e le

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mm

_)

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15 17 19 21 23 25 27 29Temperature (C)

Lesio

n a

rea (

mm

2 )

Wetness > 12 h

Temp >19 C

Bay Laurel / Tanoak SOD Spore Survey

Date

Temp (C)

Rain (mm)

How to control emergent How to control emergent exotic diseasesexotic 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 Forest pathogens can never be eradicatederadicated

PREVENT: DiagnosePREVENT: Diagnose

Symptoms relatively generic, veryvariable, and pathogen not always culturable

LAB CULTURESDNA TESTS

AgriFos and AgriFos and PentraBark PentraBark

Topical Topical ApplicationApplication

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0

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Azomite Positive Control Agrifos

Agrifos vs. Azomite Treatments(efficacy 1 - 24 months)

Can

ker

Siz

e (m

m)

aa aa

bb

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