Jan 31, 2024
Version 3
Pythium Zoospore Production Soaking Solution V.3
This protocol is a draft, published without a DOI.
- 1USDA, OSU Stillwater
Protocol Citation: Nimalka Weerasuriya 2024. Pythium Zoospore Production Soaking Solution. protocols.io https://dx.doi.org/Version 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: In development
We are still developing and optimizing this protocol
Created: January 31, 2024
Last Modified: January 31, 2024
Protocol Integer ID: 94530
Keywords: pythium, myriotylum, zoospore, soaking solutions, fungi, oospore, calcium enhanced zoospore production of pythium myriotylum, pythium zoospore production soaking solution creation, scale zoospore production for pythium myriotylum, calcium enhanced zoospore production, scale zoospore production, pythium myriotylum, journal of phytopathology, phytopathology, soaking solution
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Abstract
Creation and test of soaking solutions to be used for large-scale zoospore production for Pythium myriotylum. This is modified from methods in:
Nyochembeng, L. M., Pacumbaba, R. P., & Beyl, C. A. (2002). Calcium Enhanced Zoospore Production of Pythium myriotylum in vitro. Journal of Phytopathology, 150(7), 396–398. https://doi.org/10.1046/J.1439-0434.2002.00759.X
Materials
Soaking Solutions:
- Verified Pythium myriotylum culture
- 1.5-2% WA or CMA plates, 90 mm (4 * replicates)
- RO/DI water
- 1 L beaker
- 4 x 500 mL autoclavable bottles and lids
- Calcium carbonate (CaCO3)
- Sucrose
- Whatman #1 filter paper and funnel
- 1 N KOH – pH adjustment
- 1-5 N HCl – pH adjustment
- pH meter and standards
- Stir bars and stir plate
- Autoclave
Testing Zoospore Soaking Solutions:
- Haemocytometer
- Microscope 40X and slides
- Counter
- 0.08% Methylene blue
Preparation
Have mature colonies of verified Pythium myriotylum growing on CMA or 1.5-2% WA 90 mm plates. Colony maturity ~7-14 days, with visible oospores.
Note
Fungal growth rates may vary with ambient temperature and petri plate sizes (60 mm vs 90 mm), so wait until mycelium covers the petri plate and has visible oospores before proceeding. Oospores will form at the edges of the dish.
Citation
LINK
Soaking Solutions
Make Soaking Solutions 1, 2, 3, and Control.
- Prep 4 x 1 L autoclavable bottles for each Soaking Solutions (1-3) and Control.
Citation
LINK
Soaking Solution #1, 0.01 Molarity (M) Ca++ (1 L):
- 1 L RO water at 7 in 1 L beaker
- Add 1 g CaCO3
- Filter through Whatman #1 filter paper using a funnel into a 1 L bottle.
- Add a stir bar to the bottle.
- Adjust pH from 8.5 to 10.5 to 7.0 with 5 Mass Percent HCl and 1 Mass Percent KOH to duplicate the soaking solution in original methods.
Note
Normality is a measure of concentration that is equal to the gram equivalent weight of solute per litre of solution. Gram equivalent weight is a measure of the reactive capacity of a molecule.
The original methods use 1 N HCl, but it is sometimes too weak to induce pH changes unless large volumes are used.
Soaking Solution #2, 0.01 Molarity (M) Ca++ +0.001 Molarity (M) sucrose (500 mL):
- Transfer 500 mL of SS#1 into a clean 500 mL bottle labeled SS #2.
- Add 171 mg sucrose to make 0.001 Molarity (M) sucrose solution.
Note
Where Mass (g) = Concentration (mol/L; M) x Volume (L) x Molecular Weight (g/mol)
Sucrose (g) = 0.001 mol/L sucrose x 0.500 L x 342.3 g/mol
Sucrose (g) = 171.15 milligrams
- Check that pH is 7 .
Soaking Solution #3, 0.001 Molarity (M) sucrose (500 mL):
- Add 171 mg sucrose to make 0.001 Molarity (M) sucrose solution in 500 mL RO water
- Check that pH is 7 .
Autoclave for 20 minutes on liquid cycle. Remove from autoclave immediately to reduce water evaporation and concentration changes.
Testing Zoospore Production
1d
Take 30 mL of each Soaking Solution Soaking Solution Preparation onto surface of Pythium myriotylum plate cultures that have actively produced oospores.
Incubate under light at Room temperature for 24:00:00 .
Check for abundant sporangia that will appear after immersion.
1d
After 1.5 up to 4 h every 30 minutes:
Take 80 µL soaking liquid from each immersion to a microcentrifuge tube with 20 µL 0.08% methylene blue . Gently invert to stain (or gently vortex) and immobilize zoospores.
Note
Methylene blue (0.1% stock):
- Dissolve 0.1 g methylene blue in 100 mL dH2O
Methyene blue (0.08% working solution):
- Mix 8 mL MB stock solution with 92 mL RO water in a labeled dropper bottle. This solution is used for the viability staining.
Take 10 µL of liquid into haemocytometer and examine under 40x to quantify.
Protocol references
Nyochembeng, L. M., Pacumbaba, R. P., & Beyl, C. A. (2002). Calcium Enhanced Zoospore Production of Pythium myriotylum in vitro. Journal of Phytopathology, 150(7), 396–398. https://doi.org/10.1046/J.1439-0434.2002.00759.X
Citations
Step 1
Jones, B. L., & Woodard, K. E. A Technique for Evaluating Peanut Germ Plasm for Resistance to Pythium myriotylum
https://doi.org/10.1094/PD-70-1038Step 2
Nyochembeng, L. M., Pacumbaba, R. P., & Beyl, C. A. Calcium Enhanced Zoospore Production of Pythium myriotylum in vitro
https://doi.org/10.1046/J.1439-0434.2002.00759.X