TITLE: Baiting Pythium myriotylum from Infested Soil 
AUTHORS: Nimalka Weerasuriya

[DESCRIPTION]
Baiting Pythium from seedlings in infested soil or infected hulls.

[STEPS]
SECTION: Preparation
1. Prep P5ARP Plates 1-2 days before plating.
Prepare working culture plates (CMA, PDA + amp) up to 1 week before plating.


SECTION: Transfer Culture
11. Check plates after 24-48 h.
SECTION: Oospore Check
13. Check for "gold coin" oospores at plate edges to indicate Pythium myriotylum. 
Use CMA for oospore production
Use full strength PDA to increase hyphal growth for DNA extraction
Use V8 to increase hyphal growth for DNA extraction (untested)
SECTION: Harvest and Surface-Sterilization
8. Select appropriate samples with black rotted segments.
 

SECTION: Harvest and Surface-Sterilization
9. Try with or without 95 % (v/v) ethanol soak for 4 min or rinse in sterile RO water. Use a sieve in a small beaker.
Do not use bleach due to Pythium sensitivity. [2]
SECTION: Harvest and Surface-Sterilization
10. Plate on P5ARP or other media with bacterial inhibition (CMA or PDA + amp) [4].

SECTION: Transfer Culture
12. Hyphal tip transfer using sterile pin to clean plates of CMA (V8 or PDA).
SECTION: Harvest and Surface-Sterilization
8.1. Cut up segments into 1/4 inch (1-2 cm) pieces.
SECTION: Seed Pod or Seedling Baiting
2. Select whether you want to bait from diseased hulls in soil or from seedlings grown in diseased soil.
SECTION: Seed Pod or Seedling Baiting
3. Germinate susceptible seedlings by wrapping in RO-dampened paper towels and incubating for 2-4 days at 28 °C in the dark.
Check daily to re-wet paper towels as necessary.

SECTION: Surface Sterilization and Plating
3. Cut up hull segments into 1/4 inch (1-2 cm) pieces.
SECTION: Surface Sterilization and Plating
4. Blot completely dry on sterile paper towels.
SECTION: Surface Sterilization and Plating
5. Plate on P5ARP or other media with bacterial inhibition (CMA or PDA + amp).
 

SECTION: Preparing Working Culture
6. Check plates after 24-48 h. Pythium growth is quick, with white-cream hyphae.
SECTION: Preparing Working Culture
7. Do a hyphal tip transfer pf the growing mycelial edge using sterile loop to clean plates of CMA (V8 or PDA).
SECTION: Preparing Working Culture
8. Check for "gold coin" oospores at plate edges to likely indicate Pythium myriotylum. 
If so, continue to DNA sequencing protocol.
SECTION: Seed Pod or Seedling Baiting
4. Plant germinated seeds in infested soil, in triplicate, in 4" diameter pots with a drip tray. Maintain greenhouse at   humidity with temperatures between 24 °C and 30 °C.

SECTION: Seed Pod or Seedling Baiting
4.1. Water to maintain a level of 1-2" in drip tray for the first 2 days, and then to maintain soil moisture without excessive water until seedlings reach 4-6" in height.
SECTION: Harvest and Surface-Sterilization
7. Harvest seedlings. Rinse plants thoroughly in RO water to remove soil particles.
SECTION: Surface Sterilization and Plating
3.1. Try with or without 95% ethanol soak for 4 min. 
Do not use bleach due to Pythium sensitivity.

 

 

SECTION: Surface Sterilization and Plating
3.2. Rinse in sterile RO water for 4 min using a sieve in a small beaker.
SECTION: Seed Pod or Seedling Baiting
5. At 4 DAP, trim to 1 seed/pot, or carefully repot in separate pots.

SECTION: Seed Pod or Seedling Baiting
6. Grow for 15-20 days and check for rot symptoms. Brown rot will start at ~4-6 DAP, wilt at ~6-18 DAP.
Those exposed to higher temperatures may be more susceptible to rot.
SECTION: Harvest and Surface-Sterilization
9.1. Blot dry on sterile paper towels.