Three Clonal Lineages of Phytophthora cinnamomi in Australia Revealed by Microsatellites M.P Dobrowolski et al., Phyto

1. Three Clonal Lineages of Phytophthora cinnamomi in Australia Revealed by Microsatellites M.P Dobrowolski et al., Phytopathology 2002

2. Today’s talk: • Overview of P. cinnamomi and it’s impact in Western Australia • General biology • How does an introduced pathogen evolve • Use of microsatellites • Reveals limited introductions, but changes are occurring through mitosis

3. Phytophthora cinnamomi • Oomycete (Kingdom Chromista, brown algae) • Genus Phytophthora has ~50 spp. • Heterothallic (requires 2 mating types for sex) • Soilborne pathogen- infects roots/stem collars • Present in >67 countries • Isolated from >1000 plant spp. • Introduced pathogen to Australia in early 1900’s

4. P. cinnamomi in Western Australia • Causes disease “jarrah dieback” • 1921 first jarrah deaths; 1964 deaths shown to be caused by P. cinnamomi. • Predominantly A2 mating type

5. Impact Natural Ecosystems • Between 8-9,000 plant species in south-west of Western Australia • Approximately 2000 species are susceptible to Phytophthora cinnamomi • Indirect effects of P. cinnamomi on plant and animal communities is unknown

6. P. cinnamomi distribution

10. How is it spread? • Natural – root contact, free draining water (warm & moist; spring, summer & early autumn) • Artificial – transport of infested soil (tyres, road making) – hikers (boots, tent pegs & toilet trowels) – planting infected nursery stock

11. Goals of the study • Previously reported using isozymes that there is limited genetic variability • Authors developed microsatellite markers (different alleles identified by different length of PCR amplicon) • Does microsatellite analysis confirm low genetic diversity • Is there sex? • Is there variation without sex, and if so what does it tell us

12. Materials and Methods • Sample intensively in Western Australia • Run microsatellites • How many genotypes they found in Western Aiustralia • Are alleles in linkage disequilibrium • Are Western Australia genotypes found in the rest of the world? Is linkage disequilibrium the same • What kind of allelic variation is there and what does it imply

13. Results-1 Only three genotypes found (limited variability) Three genotypes occupy distinct areas but with some verlap They carry distinct mating alleles There are a variants, with alleles slightly different Variants are clustered

14. Results-2 Index of association indicates that alleles are co-segregating Cosegregation indicates no sex, but mitotic recombination Clustering of variants suggests these are arising locally (if they were separate introductions they would be all over) There are a variants, with alleles slightly different Mitotic crossing over explains variants and LOH

15. Results-3 Same genotypes of WA found in Eastern Australia and in other parts of the world Other genotypes, clearly different and generated by sex, also present, especially in PNG Clustering of variants suggests these are arising locally (if they were separate introductions they would be all over)

16. CONCLUSION Limited variability in spite the fact that both mating types are present indicates no sex (confirmed by Ia) • There is diversity created locally by mitotic recombination • Proof that it is being moved around the world

17. IMPLICATIONS Need to stop worldwide movement of this pathogen • In Western Australia need to prevent local movement, because both mating types are present • There is local adaptation through mitosis, one more reason to avoid even local movement of strains

18. Summary of first class • Undertanding of nature, an essential part of culture • Forests essential for life on the planet • Fungi essential for survival of forests

19. Summary of second class • DNA mutates, evolves, and different DNA sequences can be assigned to different individuals, populations from different provenances, closely related species, different species, different microbial pathovars • DNA-based phylogeography allowed to discover pine pathogen in Italy was of North American origin • DNA based genealogies allowed to identify hybridization between native and exotic pathogen • DNA allows to identify new species and to determine whether they are exotic or not

20. Definitions • Propagule= structure used by an organism to spread or survive • Locus= a physical portion of a chromosome,a gene • Intron= a portion of DNA , a locus that does not code for a protein • Exon= a coding gene

21. Definitions-2 • Alleles= different DNA sequences at the same locus • If a locus has variation in sequence it is polymorphic (many forms) • Polymorphisms are differences in DNA among organisms, the more polymorphisms the easier it is to differentiate organisms • There are more polymorphisms in introns

22. Definitions-3 • Invasive organisms: exotic organism that reproduces and occupies progressively a larger area: • Fast reproductive cycle • Vectored • Hardy • Occupy unoccupied niches • Different drain on natural resources • Make environment favorable for itself and other invaders • Linked to disturbances • If pathogen , more changes because top of pyramid • May hybridize with native species: new taxon is created

23. Summary of second class • DNA mutates, evolves, and different DNA sequences can be assigned to different individuals, populations from different provenances, closely related species, different species, different microbial pathovars • DNA-based phylogeography allowed to discover pine pathogen in Italy was of North American origin • DNA based genealogies allowed to identify hybridization between native and exotic pathogen • DNA allows to identify new species and to determine whether they are exotic or not

24. Definitions • Alternatively fixed alleles • Dominant vs. co-dominant markers • Genotype

25. Summary of fourth lesson • ASCOMYCETES, BASIDIOMYCETES, OOMYCETES • DISEASE TRIANGLE+ humans • Dominant/CO-Dominant/ Genotype

26. Summary of sixth lesson • Janzen-Connol hypothesis; explanation of why diseases lead to spatial heterogeneity • Diseases also lead to heterogeneity or changes through time • Driving succession • The Red Queen Hypothesis: selection pressure will increase number of resistant plant genotypes • Co-evolution: pathogen increase virulence in short term, but in long term balance between host and pathogen • Density dependance

27. Summary of fifth lesson • Disease as “disease triangle”, effect of humans, disease as pant-microbe interaction • Different types of disease of wild plants • True effect of disease: fertility+mortality+indirect effect on pollinators+unfair competitive advantage….but what about the “ carry over effect” • Density dependance

28. Summary of sixth lesson • Janzen-Connol hypothesis; explanation of why diseases lead to spatial heterogeneity • Diseases also lead to heterogeneity or changes through time • Driving succession • The Red Queen Hypothesis: selection pressure will increase number of resistant plant genotypes • Co-evolution: pathogen increase virulence in short term, but in long term balance between host and pathogen • Complexity of forest diseases: primary vs. secondaruy, modes of dispersal etc

29. Summary of seventh lesson • SEX; the great homogenizing force, and also ability to create new alleles • INTERSTERILITY/ MATING> SOMATIC COMPATIBILITY • NEED TO USE MULTIPLE MARKERS; SC does that, otherwise go to molecular markers • PCR/ RAPDS

30. Summary of 8th lesson • Exotic microbes have a reduced level of genetic variability • If genotypes fall on clades separated by long branches, it may be an indication there is no sex going on between individuals belonging to the two branches. Formal tests like the Index of association can test for that • Anonymous multilocus analysis can be done without any knowledge of the genome using markers such as RAPDs or AFLPs • Need to eliminate co-segregant markers and to use Jaccard;s

31. Molecular Markers • DNA & PROTEINS • mtDNA = often used in systematics; in general, no recombination = uniparental inheritance • cpDNA = often used in systematics; in general, no recombination = uniparental inheritance • Microsatellites = tandem repeats; genotyping & population structure • Allozymes = variations of proteins; population structure • RAPDs = short segments of arbitrary sequences; genotyping • RFLPs = variants in DNA exposed by cutting with restriction enzymes; genotyping, population structure • AFLPs = after digest with restriction enzymes, a subset of DNA fragments are selected for PCR amplification; genotyping

32. HOW CAN WE DETERMINE IF THEY ARE RELATED? • By using random genetic markers we find out the genetic similarity among these genotypes infecting adjacent trees is high • If all spores are generated by one individual • They should have the same mitochondrial genome • They should have one of two mating alleles

33. HOW TO DETERMINE WHETHER DIFFERENT SITES BELONG TO THE SAME POP OR NOT? • Sample the sites and run the genetic markers • If sites are very different: • All individuals from each site will be in their own exclusive clade, if two sites are in the same clade maybe those two populations actually are linked (within reach) • In AMOVA analysis, amount of genetic variance among populations will be significant (if organism is sexual portion of variance among individuals will also be significant) • F statistics: Fst will be over ) 0.10 (suggesting stongt structuring) • There will be isolation by distance