1. A clearing picture of
Phytophthora evolution: from
the wide-angle to the zoom lens
for optimal phylogenetic focus
David Cooke, SCRI

2. • 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

3. Genomics in the oomycetes
P. infestans
P. ramorum
P. sojae
H. parasitica
P. capsici
P. andina
P. mirabilis
P. phaseoli
Albugo
Pythium?
Saprolegnia?
Aphanomyces
High throughput sequencing technologies
providing many new opportunities

4. Eukaryote tree of life
Lane & Archibald, TREE 2008

5. Contributions of genomics
Tyler et al., 2006 Science
P. ramorum and P. sojae genomes
Found 855 Phytophthora
genes with similarity to red
algae or cyanobacteria
Whisson et al 2007 Nature
Shared mechanisms of
effector transport into host
cells – oomycete and
Apicomplexans
Lane & Archibald, TREE 2008
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…

6. Long history of parasitism in oomycetes
Gordon Beakes – Chapter in “Oomycete
Genetics 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

7. Basal oomycetes
rDNA 18S tree
The Development,
Ultrastructural Cytology, and
Molecular Phylogeny of the
Basal Oomycete
Eurychasma 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

8. Cooke et al 2000
c.f. Blair et al 2007
• Multigene
approach
more robust
• Clades shared
• Basal branch
structure
similar

9. Pquininea
Pmacrochl
Prichard
EU301150Psp.TIB20079
Pfallax
Pcaptiosa
EU644726Psp.BFA
EU196368Psp.167008
EU196369Psp.167011
Pgallica
Pboehmeriae
DQ066921Psp.Drimys winteri
DQ002008Psp.cc2124
Pkernoviae 9 & 10
ITS Tree 2008
Ppolonica
Pinsol cooke
EF590254Pcuyabensis
EF680320Pnapoensis
EU419612Psp.6216900
EU419615Psp.8021391
EU334634Pirrigata23J7
EU644721Psp.BF3
EU644715Psp.BF4
EU644714Psp.BFK
EF680321Pthermophilum
EU644725Psp.BF1
EU583798Phydropathica
SCRP237
Rf6
EU164426.1Pzentmyerii
EF590256Plagoariana
EU644720Psp.BFR
EU644722Psp.BF36
Rf17
• Scan of GenBank Taxonomy
Pbrassicae
Pfoliorum
Pramorum
Pprimulae
Pporri
Phibernalis
browser for ITS seq.
Plateralis
Pmedicaginis
Ptrifolii
Psansomea
Psyringae
P. austrocedrae
Pdrechsleri 8 • 146 seq but some redundancy
Pkelmania
Pcryptogea
Perythroseptica
P. fragaefolia
Psojae
Pniederhauserii
Ppistaciae
Pmelonis • Clade 8 position of P.
7
Pcajani
Pvignae
Pcinnamomi
Pfvr
Pcinnparvi
Puliginosa
Peuropaea
cryptogea & drechsleri
Palnimult
Pcambivora
Pquercina
Pilicis
Ppsychrophila
Ppseudosyringae
Pnemorosa
Pheveae
3 • Clade 7 P. fragaefolia on deep
Pkatsurae
EF067922Psp.NZICMP 5
P. novaeguinee
Ppseudotsugae
Pcact
Pidaei
root
Phedraiandra
Pnicotianae
Ptentaculata
Pclandestina
Piranica
Pinfestans & andina
Pipomoeae
Pmirabilis
1 • Clade 5 P. novaeguinee and a
Pphaseoli
P. frigida
EU644723Psp.BLW71
Pmultivesiculata
EU301125Psp.TIB20072 strain
Pbisheria
NZ isolate
Pcolocasiae
Pbotryosa
EU419614Psp.204B
Pcitrophthora
EU419613Psp.1211
EF523387Psiskiyouensis
Ptropicalis
Pcapsici
Pglovera
DQ821185Psp.BR514
2 • Clade 2 P. frigida in basal
EU301132Psp.TIB20074
Pcitricola
Pinflata
EU877749Psp.59
position
EU877753.1Psp.63
EU877755Psp.65
EU877754Psp.64
Pmegakarya
Pelitch vogl
EU419617Psp.1931
• Clade 4 ‘tropical and
P. alticola
P. quercetorum
EU301118Psp.TIB20071 strain
Psp banksia
Ppalmivora
Parecae
4 australasian flavour?’
Paspara
EF590257Psulawesiensis
EU593265Psp.IMI329669
EU301170Psp.TIB20076
Pinundata
Phumicola
EU000108Psp.77231
EU594601Psp.1FFL2008
EF153672Psp.P12745
• Clade 6 dramatic expansion &
Pmegasperma
EU301174Psp.TIB20077
DQ396412Psp.AG34UASWS0200
EU000143Psp.AG43 appearance of aerial habit
6
EU106591Psp.92209C
EU594600Psp.2FFL2008
EU106590Psp.92207
EU301176Psp.TIB20078
EU869199PaustralisVHS
EU301158.1Psp.TIB20073 strai
EF152514Psp.GD2f
EF522140Psylvatica
EU725809Ppinifolia
EU594598Phungarica
EU594599Psp.4FFL2008
Pgonapodyides
EU419616Psp.B6
EU000124Psp.91112
AY787023Psp.AG52
0.1

10. Expansion of clade 9 & 10
P. quininea
P. macrochlamydospora
P. richardiae
EU301150Psp.TIB20079 • Discrete lineage from
P. fallax
P. captiosa
9 majority of genus
EU644726Psp.BFA
EU196368Psp.167008
EU196369Psp.167011
• No obvious common
P. gallica
10 morphological/behaviour
P. boehmeriae
al traits
P. kernoviae
P. polonica • Distinct geographical
P. insolita
P. cuyabensis origin but blurred by
P. napoensis
EU419612Psp.6216900 migration?
EU419615Psp.8021391
P. irrigata
EU644721Psp.BF3
EU644715Psp.BF4
EU644714Psp.BFK
P. thermophilum
EU644725Psp.BF1
P. hydropathica 9
P. parsiana
P. zentmyerii
P. lagoariana
EU644720Psp.BFR
EU644722Psp.BF36
Rf17

11. 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)

12. Oomycete pathogen population analysis
• Mutation
• Neutral markers • Recombination
• Natural selection
AFLPs • Gene flow
SSRs • Random genetic drift
P. infestans, P. ramorum, Pl. viticola • Migration
Accelerated discovery examined by Schena and
Cooke (BMC Genomics submitted)
Sequencing
Coalescent analysis
• Functional markers
Carbone web page
Host range
Virulence
Aggressiveness & Fitness
Fungicide resistance
Sequencing genes under strong selection
effector genes e.g. RxLRs P. infestans, Hy. parasitica

13. Simple Sequence Repeats – P. infestans
Marker G11
Isolate 1
Isolate 2
ABI 3730 capillary sequencer
Isolate 3
Isolate 4
Multiplex PCR – protocols
on www.eucablight.org

14. Changes in British P. infestans population
• Eucablight
Scotland database - powerful
• Increase in A2
mating type seen in
GB
• What about
genotypes?
England

15. Derived data plotted on web
• Differences in
allele frequency
between
countries
• Expansion to
South and
Central America
complete
• Further
expansion
under
discussion

16. Great Britain SSR genotypes 1982-2007
GB genotypes
100%
90%
3
80%
10
70% 13
71%
60%
Fre que nc y
US1
old A2
50% 7,8
40%
misc.
6
30%
2
20%
A1
10% 4 5 10%
12
0%
1982 1995 1996 1997/8 2003 2004 2005 2006 2007
Isolate no. 34 85 188 264 290 581 73 899 1452
• Major change in 2006 and 2007
• Some genotypes very durable, others not. Why?

17. GB genotype frequency within 2006 season
100%
90%
80%
70%
60%
50%
40%
30%
20%
Number of 10%
outbreaks
0%
May(1-31) June(1-15) June(16-30) July(1-15) July(16-31) Aug(1-15) Aug(16-31) Sep(1-30)
5 44 27 20 13 10 18 18
31 249 152 116 82 60 112 87
Number
of isolates

18. GB genotype frequency within 2007 season
100%
90%
80%
70%
60% What drives this change?
50%
40%
30%
20%
10%
Number of
outbreaks 0%
May(1-15) May(16-31) June(1-15) June(16-30) July(1-15) July(16-31) Aug(1-15)
4 4 22 80 139 45 14
29 23 144 387 574 220 52
Number
of isolates

19. Lesion area
•
•
6_
A1
_4
0
2
4
6
8
10
12
14
16
10
0A
N
13 L0
_A 62
2_ 69
6_ 388
A1 4
_3 B
92
0A
N
13 L0
_A 42
2_ 46
13 39
_A 64
2_ A
17 39
_A 2
2_ 8A
43
7_ 88
A1 D
_4
7_ 16
A1 8C
10 _41
_A 6
2_ 8B
13 44
_A 40
C
10 2_4
_A 13
2_ 2
39 B
36
C
S 2
E0
30
S 58
E
2_ 030
A1 8
_3 7
3b 88
_A 8A
2_
42
2_ 44
A1 E
_4
06
LD 8B
15
1
P
8a M
_A P6
1_ 22
42
3_ 56
A2 B
_4
01
2F
M
P
On average, genotype 13 isolates result in
61
8_ 8
larger lesions than other genotypes at 13oC
2a
Genotype 6 largest lesions of A1 genotypes
_A
1_ C4
42
32
E
C
Lesion area (mean of all varieties)
2
13 oC

20. Days
13
_A
0
1
2
3
4
5
6
7
8
2_
•
•
38
84
N B
L0
4 24
N 6
L0
13oC
6_ 6 26
A1 9
_ 41
13 00
_A A
2_
13 39
_A 64
2_ A
17 41
_A 32
2_ B
13 43
_A 88 D
2_
39
6_ 28
A1 A
_3
3b 92
_A 0A
2_
10 42
_A 44
2_ E
44
2_ 4
A1 0C
10 _4
_A 06
2_ 8B
39
2_ 36
A1 C2
_3
7_ 88
A1 8A
_4
16
8
LD C
3 _ 15 1
A2 P
_4
7_ 01
A1 2F
_4
16
8
SE B
sooner than other genotypes at 13oC
03
05
8
M
P6
22
Genotype 6 shortest LP of A1 genotypes
8_ SE
2a 03
_A 08
1_ 7
On average, genotype 13 isolates sporulate
8a 42
Latent period
_A 32E
1_
42
56
B
M
P6
18

21. Field trial results
100.00%
Percentage of each genotype
90.00%
80.00%
70.00% x
60.00% 7
50.00% 2
40.00% 8
6
30.00%
13
20.00%
10.00%
0.00%
D1 D2 D3 D4 D5 D6
No. lesions 67 67 99 100 17 20
• All isolates were pathogenic in lab test at D0
• Domination of genotype 13 clear
• Four other genotypes (1 alien) rare

22. Virulence of genotype 8- A1
R1 R2 R3 R4 R5 R6 R7 R8 R10 R11
BPC_06_4256B = 8_A1 1,4,7,10,11
Less complex and less aggressive (note 7,10&11)

23. Virulence of genotype 13 - A2
R1 R2 R3 R4 R5 R6 R7 R8 R10 R11
BPC_06_3928A = 13_A2 1,2,3,4,5,6,7,9,10,11
Complex virulence and aggressive
Resistant to metalaxyl

24. Structured screening of RxLR diversity
• Study of evolutionary forces on key pathogen traits
3 D Panel of 30 GB isolates
3_A2
2 B • Avr 3a
10_A2
• 3 SNPs (3 replacement changes)
• 23 EM homozygous (virulent)
• 6 EM/KI heterozygous (avirulent)
13_A2 • 1 KI homozygous (avirulent)
4 A
• 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
2 C
1_A1 • 9 SNPs (1 outside ORF, 2 silent, 6 replacement)
2 A • 5 haplotypes
6_A1 • 7 TTGAATCGC A
3 D • 2 YTGMWWYRY B
2_A1
• 2 YWRMWWYRY C
6 D • 14 YWRAAWYRY D
7&8_A1
• 1 CAAAAATAT E
• Haplotypes correspond to SSR lineages
7/1A/B/D/E
rare

25. 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.

26. Acknowledgments
• Eucablight team Jens Hansen, Poul Lassen & Alison Lees
• SCRI Team: Alison Lees, Naomi Williams and Louise
Sullivan,
• Italian team: Silvia Scibetta, Leonardo Schena, Santina
Cacciola 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