BG Journal Club

2009 Fall, BG Journal Club2009 Fall, BG Journal ClubA multi-locus phylogeny for A multi-locus phylogeny for PhytophthoraPhytophthora utilizing markers derived from complete genome utilizing markers derived from complete genome sequencessequences

9/9/2009

Bongsoo Park,

Department of Plant Pathology

The Pennsylvania State University

Irish Potato Famine (1845-Irish Potato Famine (1845-1852)1852)

Courtesy D. Inglis, apsnet.org

Potato tuber with late blightPotato tuber with late blight

Grunwald - Blighted potato field Cochabamba

Tomato leaf and step blightTomato leaf and step blight

Black/Brown lesions on leaves

Water-soaked or chlorotic bordersExpanding rapidly and necrotic!

www.phytophthoradb.org

www.phytophthoradb.org

Disease cycle of late blight of Disease cycle of late blight of potato and tomato caused by P. potato and tomato caused by P. infestansinfestans

Plant Pathology, Agrios, 5ed

A. Sporangiophore; B. Sporangiophore branch showing swellings at successive sites of sporangium formation; C. Sporangia germination by zoospore

Figures-Phytophthora Disease Worldwide, 1997, Donald C. Erwin and Olaf K. Ribeiro

Sporangiophore and zoosporesSporangiophore and zoospores

Phytophthora genusPhytophthora genus Taxonomy

Eukaryota > Chromista (Kingdom)> Oomycota >

Oomycetes(Class)> Peronosporales > Pythiaceae > Phytophthora

Facultative parasitesSoil- and water-borne

Father of Plant PathologyFather of Plant Pathology

Heinrich Anton de Bary (1831-1888)http://en.wikipedia.org/wiki/Heinrich_Anton_de_Bary

Sudden Oak Death (SOD) in CA and OR

Phytophthora ramorumSymptoms include bleeding cankers on the tree’s trunk and dieback of the foliage.

Sudden Oak Death in Sudden Oak Death in CaliforniaCalifornia

Tanoak cankerTip necrosis on Rhododendron leaves

Ivors, K. http://www.phytophthoradb.org/

Ivors, K. http://www.phytophthoradb.org/

Sudden Oak Death in Sudden Oak Death in CaliforniaCalifornia

82 species(Phytophthora)82 species(Phytophthora)2 outgroup(Pythium)2 outgroup(Pythium)

Why it is important?Why it is important?

1) Diagnostic and species identification

Why it is important?Why it is important?

1) Diagnostic and species identification

2) Evolutionary history and hypothesis

Why it is important?Why it is important?

1) Diagnostic and species identification

2) Evolutionary history and hypothesis

3) Utilizing available genome sequences

Phytophthora InfestansPhytophthora ramorumPhytophthora sojae

Why it is important?Why it is important?

1) Diagnostic and species identification

2) Evolutionary history and hypothesis

3) Utilizing available genome sequences

Phytophthora InfestansPhytophthora ramorumPhytophthora sojae

4) Species complex studies initiated.

ITS RegionsITS Regions

ITS RegionsITS Regions

Highly conserved region, coding region

ITS RegionsITS Regions

Highly conserved region, coding region

Variation, Key for diagnostics

COXII RegionsCOXII Regions

COXII RegionsCOXII Regions

COXII RegionsCOXII Regions

Example)Phytophthora infestansPhytophthora mirabilisPhytophthora phaseoli

Evolution of PhytophthoraEvolution of Phytophthora

B

A

C D E

More complex, evolutionary More complex, evolutionary questionsquestions

More complex, evolutionary More complex, evolutionary questionsquestions

More complex, evolutionary More complex, evolutionary questionsquestions

Using full genome sequences, Alignment, EST sequence

More complex, evolutionary More complex, evolutionary questionsquestions

Using full genome sequences, Alignment, EST sequence

Criteria for choosing potential Criteria for choosing potential markersmarkers

P. ramorum (66.7Mbp)

P. sojae (86.0Mbp)

High similarity coding regions

Regions of lower similarity or

Predicted Introns

Similarity to ORF in other

eukaryotes

Presence of corresponding EST

sequences

Full genome sequence in 2006

at JGI

Full genome sequence in 2006

at JGI

229 72 27 7

Potential Informative

Homologous from other

eukaryotes

Regions of high

conservation identified

Primer design

Criteria for choosing potential Criteria for choosing potential markersmarkers

Tested by PCR

More loci, More accurate?More loci, More accurate?

(82+2) * (1+7) = 672672 * 2 = 1,344 (sequences)

What is hypothesis on this?

8,700 nucleotide sites

60S B-tublin EnolaseHeat shock

proteinLarge subunit TigA Ef-1a

DNA amplification and DNA amplification and sequencingsequencing

http://www.phytophthoradb.org

Phylogenetic analyses and Phylogenetic analyses and programsprograms

Maximum Likelihood

Maximum Parsimony

Bayesian Analyses

PAUP, PHYLIP MrBayes

7 loci sequences

Multiple sequence alignment with clustalX

Preliminary phylogenetic tree using NJ with a Kimura two parameter nucleotide substitution model

Modeltest, Akaike Information Criterion, GARLI

Flat Dirichlet, Tracer version, BEASTAlternative topological hypotheses in PAML

ResultResult

ResultResult

ResultResult

Phytophthora DatabasePhytophthora Database

Global Phytophthora NetworkGlobal Phytophthora Network

Plant Pathogen Database Plant Pathogen Database

and Genomic Initiativeand Genomic Initiative

at Penn State University since at Penn State University since 20052005

Thank you.Thank you.