1. Overview Armillaria bulbosa (gallica) Known as the Humungous Fungus, or honey mushroom Form rhizomorphs, which make up much of the “humungous” part Basidiocarp: cap 6 cm in diameter, stem is 5-10 cm tall Facultative tree root pathogen

2. Life cycle: Reproduction Sexual –Basidiocarps release spores (n) after karyogamy and meiosis –2 mating-type loci, each with multiple alleles in the population –Isolates (n) must have different alleles at two mating type loci to be sexually compatible Asexual –vegetative spreading of rhizomorph The large mass of rhizomorph that is genetically isolated is called a clone

3. Building up the question… “By extending the areas sampled in subsequent years, we were finally able to delimit the large area occupied by this genotype and then go on to show that this genotype likely represents and ‘individual’” - Myron Smith

4. Researcher’s Question The clonal “individual” is especially difficult to define because the network of hyphae is underground How do you unambiguously identify an individual fungi within a local population?

5. Approach 1. Collect samples 2. Check mating type - Somatic compatibility test - Distrubution of mating-type alleles 3. Molecular testing - RFLP - RAPD 4. Statistics 5. More testing

6. Methods and Materials 1 1. Collecting samples Researcher collected samples over a 30 hectare area by baiting Armillaria with poplar stakes and taking tissues and spores They then grew the successfully colonized stakes in soil taken from the study site Each fungal colony cultured was called an isolate.

7. Methods and Materials 2 2. Checking mating type - Somatic incompatibility For two fungal isolates to fuse, all somatic compatibility loci must be the same. Fusion means they’re clones  Example (not Armillaria)

8. Methods and Materials 2 2. Checking mating type - Distrubution of mating alleles -Mating occurs only when coupled isolates have different alleles at two unlinked, multiallelic loci: A and B. (They have an incompatibility system) -If fruit bodies had the same alleles at A and B, and were collected from the same area, they were assumed to be from the same clone

9. Result 1 Somatic compatilbilty: –isolates from vegetative mycelium from a large sampling area fused Mating alleles –They had the same mating type

10. Result 1 “Clone 1” was found to exceed 500 m in diameter –Used previously collected mtDNA restriction fragment patterns

11. Sensitivity of Approach Problem: These tests alone are not enough to distinguish a clone from closely related individuals

12. Why? Q: The first two tests were not sensitive enough to tell a clone from a close relative…Why? A: Spores from same point source have the same mating-type alleles, but the offspring they produce after inbreeding are genetically distinct.

13. Methods and Materials 3 3. Molecular Testing - RFLP analysis at 5 polymorphic, heterozyg. loci of mtDNA from “Clone 1” - RAPD analysis at 11 loci

14. RAPDS vs. RFLPs Use 1 short PCR primer When it finds match on template at a distance that can be amplified (primer binds twice within 50 to 2000 bp) RAPD amplicon Dominant, annoymous Total genomic, vs single locus Use endonuclease to digest DNA at specific restriction site Run digest and see how amplicon was cut Single locus is co-dominant

15. Result 2 RFLP – All 5 loci from Clone 1 were heterozygous and identical (both alleles present at loci: 1,1) RAPD –All 11 RAPD products were present in all vegetative isolates”

16. Statistical Analysis The probability of retaining heterozygosity at each parental locus in an individual produced by mating of sibling monospore isolates… = 0.0013 So they were pretty confident that cloning was responsible for their results, not inbreeding

17. More testing, just in case To be completely confident, they tested: –1) that nearby Clone 2 was different and lacked 5 of the Clone 1 heterozyg. RAPD fragments, –2) more loci, totaling 20 RAPD fragments 27 nuclear DNA RFLP fragments ** all were identical in Clone 1

18. Sensitivity of RAPDs Tested on subset of spores from same basidiocarp RAPDs differentiated among full sibs

19. Conclusions Somatic compatibility, mating allele loci, mtDNA, RFLP, and RAPD tests all indicate that a single organism could indeed occupy a 15 hectare area

20. Conclusions The larger individual, Clone 1 was estimated to weigh 9700 kg and be over 1500 years old

21. Implications ????? Fungi are one of the oldest and largest organisms on the planet Recycle nutrients…very important! Armillaria bulbosa also a pathogen; its effects on forest above may be huge as well.

22. Index of association Ia= if same alleles are associated too much as opposed to random, it means sex is not occurring Association among alleles calculated and compared to simulated random distribution

23. Evolution and Population genetics Positively selected genes:…… Negatively selected genes…… Neutral genes: normally population genetics demands loci used are neutral Loci under balancing selection…..

24. RAPDS use short primers but not too short Need to scan the genome Need to be “readable” 10mers do the job (unfortunately annealing temperature is pretty low and a lot of priming errors cause variability in data)

25. RAPDS use short primers but not too short Need to scan the genome Need to be “readable” 10mers do the job (unfortunately annealing temperature is pretty low and a lot of priming errors cause variability in data)

26. RAPDS can also be obtained with Arbitrary Primed PCR Use longer primers Use less stringent annealing conditions Less variability in results

27. Result: series of bands that are present or absent (1/0)

29. Root disease center in true fir caused by H. annosum

30. Ponderosa pineIncense cedar

31. Yosemite Lodge 1975 Root disease centers outlined

32. Yosemite Lodge 1997 Root disease centers outlined

33. WORK ON PINES HAD DEMONSTRATED INFECTIONS ARE MOSTLY ON STUMPS Use meticulous field work and genetics information to reconstruct disease from infection to explosion On firs/sequoia if the stump theory were also correct we would find a stump within the outline of each genotype