Dear Ma'am,U can  culture both the organisms on a PDA by spread plate method by making the spore suspension......Select the Phytophthora by  morphological identification and sub culture it....I think this would help u to remove the contaminants

Thanks Mohan, how do I make spore suspension of mixed culture. I am not sure, if I can make spore suspension of Phytophthora, though I  have made zoospore suspension before. Do you have any reference for how to do spread plate method plating fungi.

Try sub culturing them in rye PARP agar to clean them

Should be able to use an antifungal as a selective agent in the media. There are several that are active against true fungi only (e.g., Acremonium) and not oomycetes (e.g., Phytophthora). But, you'll have to make sure that Phytophthora doesn't have some cross-sensitivity either. This might work.

Thanks, any suggestions, which fungicides could be used against true fungi, I have been plating on PARPH CMA but I will check on rye PARP now.

You can purify these fungi by hyphal tip culture method. Phytophthora is fast grwing where as Acremonium slow..  Place a bit of mixed culture in the centre of a petridish containing any agar medium.. Incubate. After a few hours the hyphal tips of Phytophthora are cut and  planted in fresh tube of agar slant.

Thanks, Acermonium is actually the fast grower on my plate, and I tried sub culturing. But I never tried after few hours.

I would also try hyphal tipping.  First transfer to plain water agar.  Then the hyphae will grow far apart from one another.  In a good dissecting scope you should be able to distinguish hyphae of the two species.  Then you need an extremely fine tool, like a broken sliver of razor blade or piece glass drawn out to a point in a hot flame.  Cut out single hyphae and transfer to regular medium.

The fungicide approach should also work.  Look for semi-selective media used to isolate Phytophthora species from field material.  They will likely contain the right fungicides at the right concentrations.

But . . . are you sure the Phytophthora is still in there?

Try plating the mixed culture on 5 mg/L benomyl (add to PARPH or just in CMA).  I've found most Acremonium type fungi to be sensitive to benzimidazoles and P. infestans, P. capsici, and P. drechsleri to be insensitive.  

Be sure to test sensitivity of your Phytophthora to benomyl first.

Phytophthora is pretty abundant growing at the bottom where as Acermonium is growing on the top. I tried benomyl, both are growing though slowly.

Mix 3-5 mg / L of any of these products in the growth medium: triadimenol, propiconazol, ciproconazol, difenoconazole, tebuconazol, flusilazol, miclobutanil, hexaconazol, bitertanol, penconazol, epoxiconazol, fuconazol, etc.

Acremoniun (Cephalosporium) is a mold.

Note: Apply product (fungicide) when your medium being cooled to prevent degradation fungicide.

Thanks, this is very helpful info, I surely modify my media with one of the amendments.

Thanks, that is the approach I am going to take now.

I I think the best way is subculture of Phytophthora isolates on the PARPH-CMA that includes some fungicides such as Benomyl and PCNB and antibiotics such as Rifampicin, Hymixazole and so on.

Look literature for Phytophthora and Acremonium fungicide resistance in your geographic area first.

Phytophthora is a phycomycete and grows fast where as  Acremonium is slow growing. Hyphal tip culture method is the best for purification. Select proper medium for better growth of  Phytophthora.

Actually Acermonium for some reason is growing fast also, it is outgrowing Phytophthora. I am waiting for the Triazole based fungicide to test on, right now  I am adding benlate to slow it down and trying to subculture on water agar. The later is a little bit effiective.

try v-8 juice agar, subculture by single hyphal tip method and amend media with hexaconazole

How about metconazole, should that work.

Dear Shima Hello

You can use biocontrol ,by adding  exracted oil of  Lantana camara . that passed through millipore 0.22 then added to your media

warm regards

Thanks Nihad, that is interesting. I have tried to control by adding an azole based fungicide. So far it controls but still not 100%. It seem like the fungi contaminating my culture is a mycoparasite. It is my guess, but could be wrong.

Thanks 

I tested this method and display agood result ,you can treat it.

Warm Regards

Thanks Nihad, I will try it.

Dear colleague, 

An easy way to obtain pure cultures again is to grow the mixed culture at different temperatures. Fungi can differ greatly in their ability to tolerate low and high temperatures and may show very different optimum growth temperatures.

You can inoculate several plates with your mixed culture using the streaking technique, and then incubate them, for example, at 6, 12, 24, 30, 36, and 40ªC. Even if your isolate is contaminated with a mycoparasite, you have a very good chance to recover your fungus.

Good luck with your research!

Hugo

well if selective media, seective fungicides and different temperatures are not working then you can use the fungus to infect its host plant. if it causes disease u can reisolate from diseased tissue. 

Dear Shima

First:

You have to make sure from the following points:

1.     If you have seen the contamination on non-inoculated growth medium,  the contaminant fungi may came from incorrectly sterilized media and/or from equipment.

2.     If the contamination occurred in inoculated plates, that mean the contaminations are either in the inoculum you grown or on the tools that you are inoculating with.

3.     If you observed the contamination on the surface of the plates, but not on point that you inoculated, so the contamination may came in from the air around.

Would you please think about the above expectations and let me know whether  the contamination problems come from these points. Thank you and good luck.

Second:

Use streak and dilution methods during isolation of Phytophthora plus chloramphenicol could minimize the contamination. The attached file are quit useful for answer your question.

Good luck

Dear Shima,

Do use the fungicides, different temperatures, dilutionand plating method and possibly reinoculate onto plant and isolate againe.

Thanks, Mr Boris,

I was able to isolate and purify by manipulating the temperature.

Hi Shima,

Thank you for querying this issue because I am facing exactly the same problem. Majority of my Phytophthora cultures have this weird looking fungus overgrowing Phytophthora. I also use PARP (isolation), V8, PDA  (sub) as my culture media. Any success or advice you can share? Thank you

Hello Musan,

I have always seen this contaminating fungi, which produce this bubble like structure with mycelium inside along with my Phytophthora. I have been somewhat successful, when I raise single zoospore culture, or grow colony above 350C . Fusarium species are specially nuisance when you are trying to isolate from roots or bark. As it seems like it is an endophyte and always grow even when your surface sterlize the plant tissue. Send the picture of the contaminating fungi, does it look like the one I have?

Hi Shima,

Thank you for your response with that useful information. This is the water bubble like looking fungi that comes in all sorts of sizes (small, media or big bubbles). I've spent so much time trying to figure our what or how to get rid of and prevent it from reappear ing in my future cultures.  

Hi shima

You can read this paper

https://www.fungaldiversity.org/fdp/sfdp/FD_3_29-38.pdf

2

http://www.level.com.tw/html/ezcatfiles/vipweb20/img/img/20297/contamination-COR.pdf

3

https://www.researchgate.net/post/Any_suggestions_for_eliminating_fungal_contamination_on_petri_dishes_and_liquid_cultures

Good Luck

You can use some fungicide such as Benomyl, chlorothalonil,

difenoconazole, E2752, fluazinam, mancozeb, prochloraz and

terbuconazole, with the addition of a wetting agent against Acremonium

Read this paper

https://www.nzpps.org/journal/49/nzpp_491570.pdf

Reduce the strength of the media. I try mostly 1/4 strength media for the isolation of zoosporic fungi.