TITLE: Evaluating Peanut Germ Plasm for Resistance to Pythium myriotylum using Sorghum Seeds
AUTHORS: Nimalka Weerasuriya

[DESCRIPTION]
Using sorghum colonized by Pythium myriotylum to evaluate the peanut germ plasm resistance of peanut seedlings in a greenhouse.

[STEPS]
SECTION: Prep and Seed Planting
1. Make a 1:1 sand-soil (v/v) potting mixture for the required number of pot replicates. Include 1-2 additional replicates for each treatment, in case seedling emergence is inconsistent.
Place pots in trays and water to soak medium. 
Let pots soak overnight.
SECTION: Planting (Day 0)
2. Check that the potting mixture is thoroughly hydrated.
Use a clean plastic or bamboo/plastic rod to create two holes in the center of each hemisphere, at a depth of 1".
Plant 2 seeds per pot, with the radicle (pointy end) facing down. 
Cover holes and water as needed.
Label pots with planting date and seed variety.

 

SECTION: Sorghum Prep and Inoculation (~2 DAP)
3. At ~2 DAP, weigh out sorghum seeds for each pot, where each pot receives 0.5-1 g
Place sorghum seeds in two labeled flasks, one for Pythium inoculation, one for control.
SECTION: Planting (Day 0)
2.1. Place pots with trays under fluorescent lights at recommended greenhouse conditions.
Water as needed until emergence (typically 7-12 DAP).
SECTION: Sorghum Prep and Inoculation (~2 DAP)
4. Decant water and cover opening with two layers of aluminum foil. Add autoclave tape and label flasks with initials and date.
Autoclave on liquid cycle at 121 °C for 20 minand let cool overnight.
SECTION: Sorghum Prep and Inoculation (~2 DAP)
3.1. Add water to completely soak seeds overnight for 1440 min in flasks, covering tops with aluminum foil.
SECTION: Sorghum Prep and Inoculation (~2 DAP)
4.1. Autoclave a second time and let cool overnight.
SECTION: Sorghum Prep and Inoculation (~2 DAP)
5. Under a sterilized workbench, inoculate with Pythium myriotylum strain(s).
SECTION: Sorghum Prep and Inoculation (~2 DAP)
5.1. Place flasks in an incubator at 25 °C and monitor growth until the oomycete has fully colonised the substrate (7-10 days). 
Check for white mycelial growth in Pythium flasks and ensure the Control flasks remain clean.
SECTION: Seedling Preparation
6. Monitor seedlings for emergence and growth until the V-1 vegetative stage, where the first pair of tetrafoliate leaves are unfurling or completely unfurled.
Remove the second seedling from each pot by clipping just below the cotyledons above the root. If there are pots without viable seedlings, remove them and replace with the extra replicate pots.

 

SECTION: Sorghum Preparation
7. Prepare Pythium inoculant in flasks by mixing the media with a sterile metal double-ended spatula. Spatulas can be autoclaved in advance or the morning-of, or thoroughly sterilized using 100% EtOH and a flame.
SECTION: Sorghum Preparation
7.1. Mix by breaking up sorghum mats so that individual seeds can be separated. Try not to puncture seeds excessively.

SECTION: Sorghum Preparation
7.2. Weigh required amount of inoculum for each pot in clean plastic weigh boats. A recommended range is between 0.5-1 g per 500 mL potting mixture.
Adding the same total weight of sand into the flask may help break up mycelial mats.
 

SECTION: Seedling Inoculation with Pythium
8. Inoculate seedlings in pots by dropping sorghum seeds down each of the four holes until all holes are evenly filled.
SECTION: Seedling Preparation
6.1. Using a clean plastic rod, create 4 holes at a depth of 3" around the perimeter of each pot, slightly wider than the size of a sorghum seed.

 

SECTION: Seedling Inoculation with Pythium
9. Cover holes and water gently. Bottom watering is recommended to keep the top of the soil from being disturbed, and it helps moves Pythium up towards the roots with the flow of water.
SECTION: Monitoring Growth
10. Maintain a wet-moist watering schedule, where plants are kept wet for 4 days, and moist for 3 days (watering every day vs watering every other day).
SECTION: Monitoring Growth
11. Monitor plant growth for 15-20 days, counting the appearance of wilt or necrosis. Maintain greenhouse temperatures >24 °C.
Brown rot may appear from 4 to 6 days after infection.
Wilt symptoms may appear from 6 to 18 days after infection.
Note V-# vegetative stages or any R-# reproductive stages if visible.

SECTION: Monitoring Growth
10.1. For the first 4 days of watering, keep a tray below the plants to maintain a flooded root zone.
SECTION: Seedling Wilt and Necrosis
12. At harvest, carefully remove plants from potting mix without pulling on the stem.
SECTION: Seedling Wilt and Necrosis
13. Bait Pythium from infected parts using protocol: Baiting Pythium myriotylum from infested soil.