Biology of Phytophthora
Experiment 1 - Prezygotic isolating mechanisms
Crosses
Data Collection
Instructor Hints

Potato (left) and tomato (right) blight
Pictures from David Shaw's page: http://biology.bangor.ac.uk/~bss081/

Sexual Reproduction in the genus Phytophthora.

• Historical importance of Phytophthora.
• The blight disease of potatoes in the 1840's ( Phytophthora infestans).
• deBary and the germ theory of disease. (nice link:  http://www.eat-online.net/english/education/irish_potato.htm )
• Other ecological disasters caused by Phytophthora (Phytophthora cinnamomi in Australia).
• Modern removal of geographical barriers to genetic exchange.
• Repeated introduction of Phytophthora infestans to North America and Europe (A1 in 1840’s).
• Worldwide transport of plant and plant products (and plant pathogens).
• Importance of mating between previously independent lineages of Phytophthora.
• More virulent potato pathogens (asexual to sexual populations of Phytophthora infestans).
• Origin of new pathogens by interspecific hybridization.
• The anatomy of mating in Phytophthora.
• Oogonium, female structure (see right)
• Anteridium, male structure (see right)
  
                                
  

  Figure 1 - Male and Female structures of Phytophthora
  

  
  Oogonium
  

  
  

  Anteridium
  

  
  
  
    
• Bisexual fungus-like oomycete
• Mating types A1 and A2
• Life cycle (see figure below).

Experiment to investigate pre-zygotic isolating mechanisms

• Goal: to investigate pre-zygotic barriers to reproduction by experimentally crossing isolates of Phytophthora that differ in their degree of phylogenetic relatedness.
• Relatedness of  isolates (below)

Crossing design

Preparing mating crosses:

• STEP 1: Label bottoms of 7 small petri plates with:
• Two parallel lines that are 1.2 cm apart (see below)
• One line identified as A2-P. infestans.
• The other line identified as A1-“name of other species”.
• The 7th plate get two A2-P. infestans as a control.

• STEP 2: Cut out strips of agar containing Phytophthora
• The strips should be about 1.0 x 0.3 cm.
• Cut the strips from the nearer the edge of the plate.
•  You’ll need 8 strips of the A2-P. infestan s and
• 1 strip of each of the A1-isolates.
• STEP 3: Place the strips upside-down on the agar surface above the lines you drew and labeled.
• STEP 4: Wrap the plates with a strip of Parafilm and store them at 18°C for 1 week.

Reproductive Isolation in Phytophthora - Data Collection

• STEP 5: View the crosses
•  Invert the petri dishes on the microscope stage
• STEP 6: Quantify mating
• Transect the petri dish between the two samples of Phytophthora using the 10x objective of your microscope.
• Count all the oogonium/antheridium pairs that enter the field of view.
• Be sure to discriminate between oogonium/antheridium pairs and sporangia.