I think if you put 100 ml or more of solution with 10^5 zoospores/ml and let them to settle down, and the remove water you can get the needed concentration.

Try and cultivate the isolate on V8 for one week, withou light, repicate to sterile water and let it under fluorescent light for another week. Then change the water to a sterile potasium nitrate solution ( 0.001%) for one day and extract the solution. You should reach such concentration without problems.

Good luck with your work.

D. J.L. Andrés Ares

I advise you to try José Luis Andrés Ares method too.

We use a number of methods to get zoospores. Typically we grow the culture on half-strength V8, then cut plugs from the margin of colonies and place them in either 1% soil extract solution or MES buffer up to the top of the plug but not submerging it in a Petri dish and incubating overnight. To get a higher concentration of zoospores, we add more plugs, or mash the mycelium to increase surface area, or use a different soil for the soil extract. Sometimes our isolate is just not a good producer and we switch to a better one or we pass the poor isolate back through its host to reinvigorate it.

Thank you all for your input, I'm going to try a few different salt solutions and see which one gives me the best yield.

Nina Shishkoff How do you make your 1% soil solution? Do you simply mix your soil with distilled water, filter it and then autoclave it?

Yes, that's pretty much it. Sometimes we mix the soil with water and let it steep overnight, and then filter (with a coarse Whatman filter in a funnel) and autoclave.

Nina Shishkoff Thank you very much, I will try that.

You can always spin down the spore suspension in the centrifuge as well to increase the number of spores per ml

Radwan Barakat Hi Radwan thank you for your answer. I already tried to centrifuge them at 3000 rpm for 10 minutes but the zoospores seemed to burst as I could not find many in the pellet. Has this method worked for you?

Yes you are right Emmanuelle. Extreme agitation can cause that. I suggest that after growing the culture on V-8 juice broth, you replace distilled water with an autoclaved solution of 10-4 M 2-(N morpholino)-ethane-sulfonic acid (MES) adjusted to pH 6.2 with 1 N KOH for optimum production of sporangia. Sporangia may be induced to produce zoospores by chilling mycelial mats suspended in MES at 5-10 C for 15-30 min. and returning the cultures to 25 C for 1 hr.