Apr 05, 2024

Public workspaceBaiting Pythium myriotylum from Infested Soil V.2

DOI
https://dx.doi.org/10.17504/protocols.io.yxmvm3726l3p/v2
  • Nimalka Weerasuriya1
  • 1USDA, Oklahoma State University
Nimalka Weerasuriya
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DOI: https://dx.doi.org/10.17504/protocols.io.yxmvm3726l3p/v2
Protocol Citation: Nimalka Weerasuriya 2024. Baiting Pythium myriotylum from Infested Soil . protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm3726l3p/v2Version created by Nimalka Weerasuriya
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 05, 2024
Last Modified: April 05, 2024
Protocol Integer ID: 97857
Keywords: pythium myriotylum from infested soil, baiting pythium from seedling, baiting pythium myriotylum, baiting pythium, pythium myriotylum, infested soil, seedling, infected hull
Abstract
Baiting Pythium from seedlings in infested soil or infected hulls.
Materials
P5ARP plates
CMA/PDA+amp plates
Soil with Pythium infestation signs
Seeds of susceptible peanut cultivar; we used PI 378012 from the USDA Germplasm Collection
Fume hood and tools for sterile plating technique
Sieve and beakers for surface sterilization
RO water
Forceps and scalpel/blade
95% ethanol
Flame

For seedling baiting:
4" pots with catch tray
greenhouse or adequate outdoor conditions
Mature banker plants (Alyssum and Peppers) for IPM
Preparation
Prep P5ARP Plates 1-2 days before plating.
Prepare working culture plates (CMA, PDA + amp) up to 1 week before plating.


Select whether you want to bait from diseased hulls in soil or from seedlings grown in diseased soil.
Step case

Peanut Seedlings
14 steps

To bait from soil infested with Pythium using susceptible germinated seedlings.
Germinate susceptible seedlings by wrapping in RO-dampened paper towels and incubating for 2-4 days at Temperature28 °C in the dark.
Check daily to re-wet paper towels as necessary.

Incubation
Plant germinated seeds in infested soil, in triplicate, in 4" diameter pots with a drip tray. Maintain greenhouse at 70 % humidity with temperatures between Temperature24 °C and Temperature30 °C .

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.
At 4 DAP, trim to 1 seed/pot, or carefully repot in separate pots.

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.
Harvest and Surface-Sterilization
Harvest seedlings. Rinse plants thoroughly in RO water to remove soil particles.
Select appropriate samples with black rotted segments.
Rotted segments of peanut seedling suitable for plating

Cut up segments into 1/4 inch (1-2 cm) pieces.
Try with or without Concentration95 % (v/v) ethanol soak for Duration00:04:00 or rinse in sterile RO water. Use a sieve in a small beaker.
Do not use bleach due to Pythium sensitivity. [2]
4m
Blot dry on sterile paper towels.
Plate on P5ARP or other media with bacterial inhibition (CMA or PDA + amp) [4].

Transfer Culture
Check plates after 24-48 h.
Overnight
Hyphal tip transfer using sterile pin to clean plates of CMA (V8 or PDA).
Oospore Check
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)
Overnight
Protocol references
1. Singleton, L. L., Mihail, J. D., & Rush, C. M. (Charles M. (1992). Methods for research on soilborne phytopathogenic fungi. APS Press.
2. Stanghellini, M. E., & Kronland, W. C. (1985). Detrimental effect of surface sterilization on isolation of Pythium spp. from feeder roots (Abstr.). Phytopathology, 75(11), 1333–1334. [link]
3. Garren, K. H. (1966). Peanut (Groundnut) Microfloras and Pathogenesis in Peanut Pod Rot. Journal of Phytopathology, 55(4), 359–367. https://doi.org/10.1111/j.1439-0434.1966.tb02238.x
4. Jeffers, S. N., & Martin, S. B. (1986). Comparison of Two Media Selective for Phytophthora and Pythium Species. Plant Disease, 70(11), 1038–1043. https://doi.org/10.1094/PD-70-1038