A clearing picture of Phytophthora evolution: from

the wide-angle to the zoom lens for optimal phylogenetic focus

David Cooke, SCRI

• Introduction – aims of paper

• Early evolution of oomycetes

• Lessons learned from genomics

• Current status of Phytophthora phylogeny

• Tools and drivers: fine-scale population shifts

• Conclusions

• Acknowledgements

Genomics in the oomycetes

P. infestansP. ramorumP. sojaeH. parasiticaP. capsici

P. andinaP. mirabilisP. phaseoliAlbugoPythium?Saprolegnia?Aphanomyces

High throughput sequencing technologies providing many new opportunities

Eukaryote tree of life

Lane & Archibald, TREE 2008

Contributions of genomics

Lane & Archibald, TREE 2008

Tyler et al., 2006 ScienceP. ramorum and P. sojae genomesFound 855 Phytophthoragenes with similarity to red algae or cyanobacteria

Whisson et al 2007 NatureShared mechanisms of effector transport into host cells – oomycete and Apicomplexans

RxLR class of effectors considered key to pathogenicity: a reduced repertoire noted in Hy. parasitica genome (Warwick HRI). Also an absence of genes involved in zoospore formation…

Long history of parasitism in oomycetes

Gordon Beakes – Chapter in “OomyceteGenetics and Genomics: Diversity, Plant and Animal Interactions, and Toolbox”(in press, 2008)

Long history of parasitism in oomycetes– diverse and elegant range of infection devices & abundance of marine species.

Tree of Life upgrade planned

Basal oomycetesThe Development, Ultrastructural Cytology, and Molecular Phylogeny of the Basal OomyceteEurychasma dicksonii …

Satoshi Sekimoto, Gordon W. Beakes, Claire M.M. Gachon, Dieter G. Müller, Frithjof C. Küpper, Daiske Honda Protist, 2008

Parasites of plants across oomycete lineages

rDNA 18S tree

Cooke et al 2000 c.f. Blair et al 2007

• Multigeneapproach more robust

• Clades shared

• Basal branch structure similar

ITS Tree 2008• Scan of GenBank Taxonomy

browser for ITS seq.

• 146 seq but some redundancy

• Clade 8 position of P. cryptogea & drechsleri

• Clade 7 P. fragaefolia on deep root

• Clade 5 P. novaeguinee and a NZ isolate

• Clade 2 P. frigida in basal position

• Clade 4 ‘tropical and australasian flavour?’

• Clade 6 dramatic expansion & appearance of aerial habit

0.1

PquinineaPmacrochl

PrichardEU301150Psp.TIB20079PfallaxPcaptiosaEU644726Psp.BFAEU196368Psp.167008EU196369Psp.167011Pgallica PboehmeriaeDQ066921Psp.Drimys winteriDQ002008Psp.cc2124Pkernoviae PpolonicaPinsol cookeEF590254PcuyabensisEF680320PnapoensisEU419612Psp.6216900EU419615Psp.8021391EU334634Pirrigata23J7EU644721Psp.BF3EU644715Psp.BF4EU644714Psp.BFKEF680321PthermophilumEU644725Psp.BF1EU583798PhydropathicaSCRP237Rf6EU164426.1PzentmyeriiEF590256PlagoarianaEU644720Psp.BFREU644722Psp.BF36Rf17 PbrassicaePprimulaePporriPfoliorum PhibernalisPramorumPlateralisPsyringaeP. austrocedraePdrechsleriPmedicaginisPtrifoliiPsansomeaPkelmaniaPcryptogeaPerythrosepticaP. fragaefoliaPsojaePniederhauseriiPpistaciaePmelonis

PcajaniPvignae

PcinnamomiPcinnparvi

PfvrPuliginosa

PeuropaeaPalnimultPcambivora

PquercinaPilicisPpsychrophila

PpseudosyringaePnemorosa

PheveaePkatsurae

EF067922Psp.NZICMPP. novaeguineePpseudotsugae

PcactPidaei

PhedraiandraPnicotianae

PtentaculataPclandestinaPiranica

Pinfestans & andinaPipomoeae

PmirabilisPphaseoli

P. frigidaEU644723Psp.BLW71

PmultivesiculataEU301125Psp.TIB20072 strain

PbisheriaPcolocasiae

PbotryosaEU419614Psp.204BPcitrophthoraEU419613Psp.1211

EF523387PsiskiyouensisPtropicalis

PcapsiciPglovera

DQ821185Psp.BR514EU301132Psp.TIB20074

PcitricolaPinflataEU877749Psp.59EU877753.1Psp.63EU877755Psp.65EU877754Psp.64

PmegakaryaPelitch vogl

EU419617Psp.1931PpalmivoraParecae

P. alticolaP. quercetorumEU301118Psp.TIB20071 strain Psp banksia

PasparaEF590257Psulawesiensis

EU593265Psp.IMI329669EU301170Psp.TIB20076

PinundataPhumicola

EU000108Psp.77231EU594601Psp.1FFL2008EF153672Psp.P12745

PmegaspermaEU301174Psp.TIB20077

DQ396412Psp.AG34UASWS0200EU000143Psp.AG43EU106591Psp.92209C

EU594600Psp.2FFL2008EU106590Psp.92207EU301176Psp.TIB20078

EU869199PaustralisVHSEU301158.1Psp.TIB20073 strai

EF152514Psp.GD2fEF522140Psylvatica

EU725809PpinifoliaEU594598PhungaricaEU594599Psp.4FFL2008

PgonapodyidesEU419616Psp.B6EU000124Psp.91112AY787023Psp.AG52

6

4

2

153

7

8

9 & 10

Expansion of clade 9 & 10

• Discrete lineage from majority of genus

• No obvious common morphological/behavioural traits

• Distinct geographical origin but blurred by migration?

P. quinineaP. macrochlamydospora

P. richardiaeEU301150Psp.TIB20079

P. fallaxP. captiosa

EU644726Psp.BFAEU196368Psp.167008EU196369Psp.167011

P. gallicaP. boehmeriae

P. kernoviae

P. polonicaP. insolita

P. cuyabensisP. napoensis

EU419612Psp.6216900EU419615Psp.8021391

P. irrigataEU644721Psp.BF3

EU644715Psp.BF4EU644714Psp.BFK

P. thermophilumEU644725Psp.BF1

P. hydropathicaP. parsiana

P. zentmyeriiP. lagoarianaEU644720Psp.BFR

EU644722Psp.BF36Rf17

10

9

9

Status of Phytophthora phylogenetics?

• Move from single gene to multigene phylogenies

• ITS remains good starting point

• Number of species increasing but main structure unchanged

• Expansion of clades 9 & 10 most marked

• Ever expanding range of taxa in clade 6

• Monitoring methods by Silvia Scibetta – powerful tool

• Still issues with GenBank – reference collection better in independent source (Phytophthora database)

Oomycete pathogen population analysis

• Neutral markersAFLPsSSRs

P. infestans, P. ramorum, Pl. viticolaAccelerated discovery examined by Schena and Cooke (BMC Genomics submitted)

SequencingCoalescent analysis

• Functional markersHost rangeVirulenceAggressiveness & FitnessFungicide resistanceSequencing genes under strong selection

effector genes e.g. RxLRs P. infestans, Hy. parasitica

Carbone web page

• Mutation • Recombination • Natural selection • Gene flow• Random genetic drift • Migration

Simple Sequence Repeats – P. infestans

ABI 3730 capillary sequencer

Marker G11

Isolate 2

Isolate 1

Isolate 4

Isolate 3

Multiplex PCR – protocols on www.eucablight.org

Changes in British P. infestans population

• Eucablightdatabase - powerful

• Increase in A2 mating type seen in GB

• What about genotypes?

Scotland

England

Derived data plotted on web

• Differences in allele frequency between countries

• Expansion to South and Central America complete

• Further expansion under discussion

Great Britain SSR genotypes 1982-2007

• Major change in 2006 and 2007• Some genotypes very durable, others not. Why?

A2

A1

misc.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Freq

uenc

y

1982 1995 1996 1997/8 2003 2004 2005 2006 2007

GB genotypes

7,8

6

512

2

4

3

10

1371%

10%

US1old

85 581 73 899Isolate no. 145229026418834

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

May(1-31) June(1-15) June(16-30) July(1-15) July(16-31) Aug(1-15) Aug(16-31) Sep(1-30)

GB genotype frequency within 2006 season

Number of outbreaks

Number of isolates

87112608211615224931181810132027445

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

May(1-15) May(16-31) June(1-15) June(16-30) July(1-15) July(16-31) Aug(1-15)

GB genotype frequency within 2007 season

Number of outbreaks

Number of isolates

5222057438714423291445139802244

What drives this change?

Lesion area (mean of all varieties)

0

2

4

6

8

10

12

14

16

6_A1

_410

0ANL0

6269

13_A

2_38

84B

6_A1

_392

0ANL0

4246

13_A

2_39

64A

13_A

2_39

28A

17_A

2_43

88D

7_A1_

4168

C7_

A1_41

68B

10_A

2_44

40C

13_A

2_41

32B

10_A

2_39

36C2

SE0305

8SE0

3087

2_A1_

3888

A3b

_A2_

4244

E2_

A1_40

68B

LD 15

1 PMP62

2

8a_A

1_42

56B

3_A2

_401

2FMP61

8 C4

8_2a

_A1_

4232

E C2Le

sion

are

a

13 oC

• On average, genotype 13 isolates result in larger lesions than other genotypes at 13oC

• Genotype 6 largest lesions of A1 genotypes

Latent period

• On average, genotype 13 isolates sporulatesooner than other genotypes at 13oC

• Genotype 6 shortest LP of A1 genotypes

012345678

13_A2_

3884B

NL042

46NL0

6269

6_A1_

4100

A13

_A2_

3964

A13_

A2_413

2B17_

A2_438

8D13

_A2_

3928

A6_

A1_392

0A3b

_A2_42

44E

10_A2_4

440C

2_A1_

4068

B10

_A2_

3936

C22_

A1_388

8A7_

A1_41

68C

LD 15

1 P3_

A2_401

2F7_

A1_416

8BSE030

58MP62

2SE03

087

8_2a_

A1_423

2E8a_

A1_425

6BMP61

8D

ays

13oC

Field trial results

• All isolates were pathogenic in lab test at D0• Domination of genotype 13 clear• Four other genotypes (1 alien) rare

0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

90.00%

100.00%Pe

rcen

tage

of e

ach

geno

type

D1 D2 D3 D4 D5 D6

x728613

No. lesions 67 67 99 100 17 20

Virulence of genotype 8- A1

R1 R2 R6R3 R5R4 R7 R8 R10 R11

BPC_06_4256B = 8_A1 1,4,7,10,11

Less complex and less aggressive (note 7,10&11)

Virulence of genotype 13 - A2

R1 R2 R6R3 R5R4 R7 R8 R10 R11

BPC_06_3928A = 13_A2 1,2,3,4,5,6,7,9,10,11Complex virulence and aggressive

Resistant to metalaxyl

• Study of evolutionary forces on key pathogen traits Panel of 30 GB isolates• Avr 3a

• 3 SNPs (3 replacement changes)• 23 EM homozygous (virulent) • 6 EM/KI heterozygous (avirulent)• 1 KI homozygous (avirulent)

• avirulent strains confined to a specific lineage which explains decline of this avirulence• R3a not deployed widely (3.5% area) – low selection pressure implies no ‘cost’ to virulence for avr3a

• PEX 161• 9 SNPs (1 outside ORF, 2 silent, 6 replacement)• 5 haplotypes

• 7 TTGAATCGC A• 2 YTGMWWYRY B• 2 YWRMWWYRY C• 14 YWRAAWYRY D• 1 CAAAAATAT E

• Haplotypes correspond to SSR lineages

6

2

2

4

3

3

2

7/1

3_A210_A2

13_A2

1_A1

6_A1

2_A1

7&8_A1

rare

Structured screening of RxLR diversity

C

B

A

D

D

A

A/B/D/E

D

Conclusions

• Genomics

High throughput, affordable (£5K per 100 Mb) DNA sequencing offering insights into mechanisms of pathogenicity, host range and ultimately host resistance responses. Post-genomics is going to be a ‘long haul’.

• Phylogenetics

Exciting prospects for environmental monitoring of Phytophthora diversity in support of conventional approaches. Evaluating risks. Exploring what happens in ‘balanced’ natural ecosystems as an aid to understanding invasive species. Phylogeography to improve our understanding of origins of species. Phylogenomics to understand mechanisms conserved across genus.

• Population analysis

Tracking populations – co-ordinated responses needed – tracking using standardised tools – sharing ‘intelligence’ on local problems. Predicting risk? How does population structure influence management? Examining selection pressures on key genes.

Acknowledgments

• Eucablight team Jens Hansen, Poul Lassen & Alison Lees

• SCRI Team: Alison Lees, Naomi Williams and Louise Sullivan,

• Italian team: Silvia Scibetta, Leonardo Schena, SantinaCacciola and others

• BPC team: David Shaw, Ruairidh Bain, Nick Bradshaw, Moray Taylor

• Many, many people who have contributed inspiration, friendship & cultures

• Potato Council & RERAD for funding