An alternative to the Phytophthora lab

Background:

 

Silene caroliniana with Microbotryum infection (center)
(photo Michael Hood)

Obtaining Microbotryum

Laboratory Exercise

• Choose one of the four tester strains. 
• This tester strain is then paired with every other strain of opposite mating type 
• include its own species as a "positive control"
• Example S. alba A1 x S. alba A2 
• include its own mating type as a "negative control"
• Example S. alba A1 x S. alba A1
  
• You will be given 14 stock tubes, each with an equal concentration of sporidia (mating-ready cells).
• Take 8 tubes and mix your focal tester with every other strain of opposite mating type.
• Label all tubes intelligently
• Put 10 μl  of focal tester in each tube
• Add 10 μl  of  strain to be tested
• Vortex
• Take two replicate water agar plates (20g agar / 1L distilled water)
• Divide on bottom into 8 wedges, 
• Put a dot in the middle of each wedge to mark "the spot" that the mix will be dropped on to, 
• Label each wedge intelligently.  
• These two plates are replicates and should be labeled identically.
• Plate out by putting 2 μl of mixture onto water agar plate at each dot in appropriate wedge. 
• Put into incubator in at 15 C until they are used.

Data Collection

• Lift a piece of agar containing the mating cells
• Place face-up on a microscope slide and place a cover slip on it.  
• Put it under a microscope. BE CAREFUL. USE LOW POWER FIRST TO GET APPROX. FOCUS. ONLY THEN move to high power (x20 or x40) - DO NOT go to x100.
• Count number of single and  number of conjugating sporidia, up to or just over a hundred total (count conjugating pairs as two each).
• Use some objective method, e.g. start at one corner of the view field, and count, or find a less dense area and do a transect moving the stage and covering several fields of view etc.
• IN ANY CASE make sure you are not differentially counting conjugating and non-conjugating cells.
• Calculate the frequency of conjugation between each pair and bring your results for next week's class.

GenBank assignment with Microbotryum

• Work out geographical distances between all strains using atlas by generating a geographic distance matrix.
• Go to GenBank (search on NCBI or GenBank) and work out genetic distances between both parasite strains and host species.  Use ITS sequences.
• Isolate sequences where possible.
• Generate a genetic distance matrix for host and parasite (i.e. blast each against all others).

Methods - For Instructors

• All samples are cultured on Potato Dextrose Agar (PDA - purchased from Sigma or Fisher) at room temperature. (Note:  M. violaceum is transferred between Petri plates using any standard yeast or bacteriological techniques)
• It would be great if the full crossing matrix could be competed by  the class, but it is not practical. So, we had the students divide up into four groups and gave each group a focal culture that they were going to cross against all other cultures. For example, one group had M.v- Silene alba A1 and A2 and M.v - Silene caroliniana A1 and  A2 as the four focal strains  (using the A1 and A2 and M.v strains from the same sample added a nice reciprocal test to the design.
• The expectation was that mating would be most frequent when it crosses with itself. Here 's how the crosses were performed.
• All the cultures had been transferred to new PDA plates within seven days of the lab so that the cells would all be viable.
• The day before the lab 1.5% water agar plates were prepared, enough for two per student
• The morning of the lab  each of the cultures were used to make sporidial suspensions in sterile deionized water.
• We suspended a glob of the culture about 4 times the size of a pinhead into 1 mL of water.  
• We then used a hemacytometer  to quantify the concentration of sporidia in each suspension (first diluting a small sample by 100 fold so that there were few enough cells to count. We weren't too exacting about these counts.
• We then made the sporidial concentrations equal by diluting down to about 2500 sporidia per μl .  This number is arbitrary; any number will do as long as the suspension looks slightly cloudy with sporidia.
• For their focal strains, we aliquotted  100 μl of the sporidial suspensions into labeled microcentrifuge tubes.
• For each strain to be crossed, we aliquotted 10 μl  into a labeled tube.
• We made a bunch of these, enough for each student to pick up one focal strain tube and one each of the seven other tubes.
• The students then pipetted 10 μl from their focal strain tube into each of the seven tubes (which already contained 10 μl of the other strains) and mixed by tapping the tubes.
• Then they labeled two 1.5% water agar Petri plates on the bottom in the following manner.
• Each pie wedge was labeled with one of the seven crosses, and the last wedge was a "focal strain alone " control.

• The sporidial mixtures were placed on the water agar Petri plates by pipetting  2 μl  drops in the center of the wedge. This forms a spot of sporidia is a liquid is absorbed into the agar.
• The water agar plates were then incubated at between 5 and 15 C
• One of the two plates was examined at 24 hours and the other after 72 hours.
• The frequency of mating was determined by microscopic examination. First a cover glass was placed down on one of the spots of sporidia. The cover glass was tapped  down lightly to make good contact with the sporidia, and then it was lifted off using forceps. A small (5-10 μl) drop of water was placed on the cover glass and mixed with the sporidia. This was then inverted onto a microscope slide. Mating sporidia are connected by a conjugation and can be seen with a 20 or 40x objective.
• The students counted the number of sporidia involved in mating and those that were not. They used either random fields of view or transects.

Sample Class Data (UVA 2002)

Hints on GenBank Exercise