You should use hemp-seed on CMA  + Phytophthora then transfer on water and incubate under fluorescence lamp for 24 hours.

Hi Sharifa: If you have grow the fungus on liquid medium and the fungus produce a good amount of sporangia, Just keep the fungus in 4C for 2-3 h and after that put it on (25 C) room Temp for 10 min you can see the sporangia release the zoospore under microscopy.

Good luck 

Hello!, Another technique is take out colonized fragments from the margin of the colony of each isolate grown on V8 juice agar medium, and place in Petri dishes with sterile soil extract solution. The fragments with sporangia (some germinating and release zoospores) are observed after 3 or 4 days directly under microscopy!! 

A technique that works well for me is:

Plug of active mycelium in an empty 9 cm Petri dish

Add 10-12 ml clarified V8 liquid medium (made by taking carton of Campbell's V8 juice, adding a teaspoon or two of calium carbonate, shaking vigorously then centrifuge out all particulates followed by filtering through Whatman no1 or similar then diluting 1 in 10 in distilled water which gives a straw-coloured liquid [liquid gold!])

Leave to grow mycelial mat (approx a week). Then while culture is still young and active, remove the V8 and wash the mycelial mat about 3 times in sterile pond water (or sterile soil extract water - Pond water = collect pond water, filter through Whatman no 1 and autoclave. Soil extract water = take a litre of distilled water, add a fist full of soil from a flower bed or similar shake up, leave to settle then filter and autoclave {I find it's better to autoclave, leave for two days then autoclave a second time}).  THen add 10-12 ml of sterile pond water and leave plate on bench in indirect daylight for 24 -48h, after which time there should be lots of sporangia - get these to synchronously release zoospores by popping plate in fridge at 4 deg C for 30-40 minutes then leaving in warm on lab bench for a further hour or so when you should have lots of lovely swimming zoospores! 

If cultures don't produce sporangia using this method - it sometimes helps to add a little bit of beta-sitosterol to the V8 medium.

Hope this works out for you

Cheers

TimP

Oops! forgot to add quantitation - simplest method is using a haemocytometer (e.g. 'Improved Neubauer'), problem is, zoospores don't keep still whilst you count them! You can slow them down for counting by adding a small amount of glycerol, but remember to factor this dilution by addition into your calculations!

Cheers T

Thanks everyone. I will try all these methods. Hopefully it works ;) Wish me luck!

Hello Sharifah

Use tomato agar or soybean agar for supporting the growth of Phytophthora and also for sexual and asexual reproduction. I enclosed relative references could be helpful for your question. Good luck