Overview

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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

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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

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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

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Researchers 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?

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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

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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.

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Methods and Materials 2
Example (not Armillaria)

• 2. Checking mating type
• - Somatic incompatibilityFor two fungal
  isolates to fuse, all somatic compatibility loci
  must be the same.
• Fusion means theyre clones ?

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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

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Result 1

• Somatic compatilbilty
• isolates from vegetative mycelium from a large
  sampling area fused
• Mating alleles
• They had the same mating type

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Result 1

• Clone 1 was found to exceed 500 m in diameter
• Used previously collected mtDNA restriction
  fragment patterns

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Sensitivity of Approach

• Problem These tests alone are not enough to
  distinguish a clone from closely related
  individuals

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Why?

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

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Methods and Materials 3

• 3. Molecular Testing
• - RFLP analysis at 5 polymorphic, heterozyg. loci
  of mtDNA from Clone 1
• - RAPD analysis at 11 loci

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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

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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

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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

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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

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Sensitivity of RAPDs

• Tested on subset of spores from same basidiocarp
• RAPDs differentiated among full sibs

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Conclusions

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

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Conclusions

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

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Implications

• ?????
• Fungi are one of the oldest and largest organisms
  on the planet
• Recycle nutrientsvery important!
• Armillaria bulbosa also a pathogen its effects
  on forest above may be huge as well.

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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

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Evolution and Population genetics

• Positively selected genes
• Negatively selected genes
• Neutral genes normally population genetics
  demands loci used are neutral
• Loci under balancing selection..

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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)

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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)

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RAPDS can also be obtained with Arbitrary Primed
PCR

• Use longer primers
• Use less stringent annealing conditions
• Less variability in results

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Result series of bands that are present or
absent (1/0)
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Root disease center in true fir caused by H.
annosum
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Ponderosa pine
Incense cedar
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Yosemite Lodge 1975 Root disease centers
outlined
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Yosemite Lodge 1997 Root disease centers
outlined
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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

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