2. CLASS REVIEW 2010 Lectures

3. Understanding of nature, an essential part of culture Forests essential for life on the planet Fungi essential for survival of forests

4. 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

5. 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

6. 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

7. 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

8. New host pathogen combinations Pathogen stays/Plant moves: invasive plant Pathogen moves/Plant stays: exotic epidemic Pathogen moves/Plant moves: biological control

9. 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

10. 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)

11. 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

12. 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

13. 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 that genetic variability

14. DNA polymorphisms can be diagnostic –Mutations/Sex/Barriers to mating Plant Diseases can be biotic (interaction between host and causal agent ), or abiotic Many organisms can cause plant diseases, but fungi are the No.1 cause Diversity of fungi, but all have ideal structure for plant infection: –hypha/cord/rhizomorph/infection peg/appressorium –Sexual vs. asexual reproduction: can do both

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

16. Dominant vs. codominant genetic markers Concept of “genotype” Alternatively fixed allele vs.difference in frequencies PLANT HOST INTERACTION: timing, physical/chemical interaction, basic genetic compatibility leads to virulence, gene for gene hypothesis, pathogenicity

17. Categories of wild plant diseases Seed decay Seedling diseases Foliage diseases Systemic infections Parasitic plants Cankers, wilts, and diebacks Root and butt rots Floral diseases

18. 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

19. The biology of the organism drives an epidemic Autoinfection vs. alloinfection Primary spread=by spores Secondary spread=vegetative, clonal spread, same genotype. Completely different scales (from small to gigantic) Coriolus Heterobasidion Armillaria Phellinus

21. OUR ABILITY TO: Differentiate among different individuals (genotypes) Determine gene flow among different areas Determine allelic distribution in an area

22. WILL ALLOW US TO DETERMINE: How often primary infection occurs or is disease mostly chronic How far can the pathogen move on its own Is the organism reproducing sexually? is the source of infection local or does it need input from the outside

23. Important fungal genetic systems: Intersterility genes Somatic (vegetative) compatibility Mating system

24. Summary AFLP, RAPDs, RFLPs, microsatellites Repeatability Test for power (PID and test progeny) Have we sampled enough? Rarefaction curves, resampling, need to be ob flat portion of curve

25. Summary From raw data to genetic distance Distance distribution AMOVA PHIst Distance based trees Number of polymorphic alleles

26. The “scale” of disease Dispersal gradients dependent on propagule size, resilience, ability to dessicate, NOTE: not linear Important interaction with environment, habitat, and niche availability. Examples: Heterobasidion in Western Alps, Matsutake mushrooms that offer example of habitat tracking Scale of dispersal (implicitely correlated to metapopulation structure)---

27. The scale of disease Curves of spore dispersal (rapid dilution effect, e.g most spores fall near source, but a long low tail, a few spores will travel long distances Genetic structure of species: the more structure the more fragmented the less dispersal Mantel tests, spatial autocorrelation: plot the genetic distance against the geographic distance

30. Using DNA sequences Obtain sequence Align sequences, number of parsimony informative sites Gap handling Picking sequences (order) Analyze sequences (similarity/parsimony/exhaustive/bayesian Analyze output; CI, HI Bootstrap/decay indices

31. Population genetics concepts Gene flow, migration Lack of gene flow, genetic substructuring=differentiation Hardy Weinberg= for diploid or dikaryotic organims predicts levels of heterozygosity Inbreeding coefficient Fst

32. How do we know that we are sampling a population? We actually do not know Mostly we tend to identify samples from a discrete location as a population, obviously that’s tautological Assignment tests will use the data to define population, that is what Grubisha et al. did using the program STRUCTURE

33. CLASS REVIEW 2010 Research papers

34. Hayden et al paper describes how PCR assay is designed: 1- primers only match target species and not relatives: PCR product = pathogen is there 2-nested approach 3-control primers amplify all plants

36. Molecular Ecology (2008) doi: 10.1111/j.1365-294X.2008.03773.x Blackwell Publishing Ltd Reconstruction of the Sudden Oak Death epidemic in California through microsatellite analysis of the pathogen Phytophthora ramorum S. MASCHERETTI,* P. J. P. CROUCHER,* A. VETTRAINO, † S. PROSPERO ‡ and M. GARBELOTTO* * Department of Environmental Science, Policy and Management, 137 Mulford Hall, University of California, Berkeley, CA 94720-3114, USA, †Department of Plant Protection, University of Tuscia, I-01100 Viterbo, Italy, ‡INRA, UMR 1202 Biodiversité Gènes et communités, Equipe de pathologie Forestiere, BP 81, 33883 Villenave d′ Omon Cedex, Fra nc e Abstract The genetic structure of the clonally reproducing Sudden Oak Death (SOD) pathogen in California was investigated using seven variable microsatellites. A total of 35 multilocus genotypes were identified among 292 samples representative of populations from 14 forest sites and of the nursery trade. AMOVA indicated significant genetic variability both within (44.34%) and among populations (55.66%). Spatial autocorrelation analyses indicated that Moran ’ s index of similarity reached a minimum of 0.1 at 350m, increased to 0.4 at 1500 m and then decreased to zero at 10km. These results suggest a bimodal pattern of spread, with medium range dispersal (1500 – 10000m) putatively attributed to the presence of strong winds. Lack of genetic structure was identified for three groups of populations. One group notably included the nurseries ’ population and two forest populations, both linked to early reports of the pathogen. A neighbour-joining analysis based on pairwi s e ΦST values indicated that the clade inclusive of the nurseries ’ populations is basal to all California populations. A network analysis identified three common genotypes as the likely founders of the California infestation and proposes a stepwise model for local evolution of novel genotypes. This was supported by the identification in the same locations of novel genotypes and of their 1- or 2-step parents. We hypothesize that the few undifferentiated population groups indicate historical human spread of the pathogen, while the general presence of genetically structured populations indicates that new infestations are currently generated by rare medium or long-range natural movement of the pathogen, followed by local generatio

37. Key points Organism is exotic, why? How does it kill oaks? How far does it spread? Is it in equilibrium, How does it attain diversity? What ecological conditions are necessary? What can be done?

39. Key points Pine mortality near Rome, never reported before suggesting something new Heterobasidion root rot basidiocarps found at base of trees Multiple loci analysis indicates pathogen is from North America Likely to have been brought into Europe by US Army with untreated lumber

40. Nature 394, 137 - 138 (1998) © Macmillan Publishers Ltd. Cause of sea fan death in the West Indies

41. Humongous fungus The Fungus Armillaria bulbosa is among the largest and oldest living organisms

42. Key points Armillaria does not reproduce via asexual spores Same genotype found in a large area RAPDs and RFLP of mitochondrion and mating alleles Tested sensitivity of RAPDs on full sibs Age estimated by dividing maximum distance within clone by annual growth rate

44. Key points Native fungus, host specialized How does it infest stands? Does it need stumps? How was research done? Sampling and analysis What type of forests will enhance secondary spread? Is source of inoculum local or not? How was it shown that nuclei can rearrange themselves

46. Key points Wood decay fungus, generalist Sexually reproducing hence lots of local diversity Easily airborne, easy to find hosts, no genetic structure within Sweden Structure between Sweden and Finland Methods: RAPDS and AMOVA

48. Key points Pathogen, very host-specific Infection is mostly primary by airborne meiospores Method: AFLP analysis on haploid meiospores AMOVA indicated significant genetic diversity both within and among populations Lack of host= barrier to migration

50. Key points Mycorrhizal fungus, obligate symbiont Symbiont with most conifers, air dispersed Japanese market buys some species, rejects others Species accepted by market are monophyletic At least 3 species: circumboreal, mexican, and west coast North America= center of diversity Oldest species is in North America Methods: DNA sequencing and AFLPs Isolation by distance: distant populations more different genetically

51. Isolation by landscape in populations of a prized edible mushroom Tricholoma matsutake

53. Key points Specific mycorrhizal symbiont, underground mushrooms, animal dispersed Islands in islands Compare genetics of fruitbodies and of seed banks Genetic structure indicate low gene flow among sites, but similar genetic structure between two islands