Pythium Zoospore Production Soaking Solution

Nimalka M Weerasuriya

Published: 2023-07-31 DOI: 10.17504/protocols.io.e6nvwdkndlmk/v1

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Abstract

Creation of soaking solutions for Pythium myriotylum to be used for large-scale zoospore production.

Steps

Preparation

Have mature colonies of verified Pythium myriotylum growing on CMA or 1.5-2% WA. Colony maturity ~7 days, with visible oospores.

Citation

Jones, B. L., & Woodard, K. E 1986 A Technique for Evaluating Peanut Germ Plasm for Resistance to Pythium myriotylum Plant Disease, 70(11), 1038–1043 https://doi.org/10.1094/PD-70-1038

Soaking Solutions

Make soaking solutions 1, 2 and 3:

Citation

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

2.1.

Soaking Solution #1 , 0.01Molarity (M) (1 L):

1L RO water at 7 in 1 L beaker

Add 1g CaCO₃

Filter through Whatman #1

pH is adjusted from 8.5 to 10.5 with 1 to duplicate soaking solution in original methods (Pacumbaba, cite)

2.2.

Soaking Solution #2 , 0.01Molarity (M)+0.001Molarity (M) (500 mL):

pH is adjusted to 7 with 1

Note

(1 M (mol/l) = 1 N for an acid that releases 1 proton* when dissolved in water)

2.3.

Soaking Solution #3 , 0.001Molarity (M) (500 mL):

pH is adjusted to 7 with 1

Autoclave for 20 minutes liquid cycle

Sterilizer (Consolidated)

Testing Zoospore Production

Take 30mL of each Soaking Solution onto surface of 7-day cultures in plate.

Incubate under light at Room temperature for 24h 0m 0s.

Check for abundant sporangia that will appear after immersion.

At 1.5 up to 4 h every 30 minutes:

Take 80µL from each immersion to a microcentrifuge tube with 20µL. Gently invert to stain (or gently vortex) and immobilize zoospores.

Take 10µL of liquid into haemocytometer and examine under 40x to quantify.

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