benomyl (or Benlate) has very low solubility in water (I think 50%). But if you have been given the dose of 10 mg/l which is 10 ppm just use it as such. First crush it in a weighing boat with a few drops of water to make a paste and gradually add more water. You can then add the suspension to your medium prior to autoclaving. As far as I know benomyl is not affected by autoclaving and you should get the same results by adding it before autoclaving as it would not be easy to filter-sterilize. Hope this helps.

Thanks so much for your explanation. I also found from CBS website that Dichloran can also be add before autoclaving.

There is just one thing I didn't understand. If you want to cultivate a basidiomycetes, why you want to use benomyl? Benomyl is a fungicide, so, it coul d eliminates your fungus. I think you need use an antibiotic such as erythromicine or other.

You have to dissolve fungicide in sterilized distilled water and then add it to the culture media at 45-50ºC before dispensing into petri dishes. To prepare the solution you have to take into account the percentage of active ingredient in the commercial product. For example 50 mL of water and 100 mg of fungicide at 50% purity, which gives a concentration of 1mg/mL. To a concentration of 10mg / L would have to add 10 mL / L in the culture medium.

I hope you find it useful!

Hi Alvin

I have used Benomyl in my medium , about Benomyl 100mg/l for fittonia leaf culture , leafs were very highly contaminated to a internal fungi , benomyl control fungus growth for maximum 20 days after culture and must of explants were show fungus growth after 20 days ,I autoclave benomyl with medium .

We used to make a stock solution in methanol. I think 1 g/l (=1 mg/ml if you want to use more reasonable volumes). and filter sterilise it (although there is not much that can grow on methanol). Then add it to the autoclaved medium at 45-50ºC as Laura said. You'll have to add 1 ml/l to get 1 mg/l final concentration, and keep in mind the purity indeed. You may want to play around a bit with the cnoncentrations, 10 mg/l is quite a lot, it won't even dissolve completely at this concentration.

There are actually some selective media out there where they use a lot of benomyl, just weighing in the powder before autoclaving and pouring everything as a slurry, by Kersies or Kirk, from the top of my head.........

Antonio Hernandez: Benomyl is a fungicide, you are right. However, Basidiomycetes show a particular tolerance to it.

See:

http://www.scielo.br/pdf/bjm/v37n4/v37n4a04.pdf

http://aem.asm.org/content/62/11/4288.full.pdf

just examples.

Cheers

I agree with Henk-Jan. it is often best to use a solute to dissolve the benomyl in as a stock solution such as methanol or ethanol and then use water to make a working solution at no more than 1 mg/L, which you can add to your media at the necessary concentration. If you want to also inhibit bacteria a combination of kanamycin and ampicilllin works well.

Thanks so much

As stated by others above benomyl is not antibiotic and many of the other broad spectum antibiotics can be used. It is not clear what kind of contamination i.e. fungal or bacterial contaminants are to be avoided

Yes. Benomyl is not fit your research on cultivation of Basidiomycetes (fungi). Usually, we add antibiotics to control bacterial growth during fungus cultivation.

I found that when you isolate basidios from their characteristic wood rotting symptoms, you get quite clean cultures if you take a VERY small piece from the center of the rot and plate it on a medium such as PDA. If you use a good technique in isolating the basidio, you might not even need a selective medium. Have a look at literature on ESCA disease of grapevines where a number of basidiomycetes is involved.

Use broad spectrum antibiotics like streptopenicillin or chloramphenicol 10mg / Lit

Make a stock solution with known concentration (n1), then to make a medium of v2 volume and n2 concentration, use the following equation:

(n1) (v1)= (n2) (v2) -> v1= (n2)(v2)/(n1)

namely, take v1 volumes of stock solution and add it to some part of medium so that the final medium is adjusted to v2. You shall add after autoclaving your medium when it is cool enough (ca. 45C).

Benomyl cannot be useful in the isolation of Rhizoctonia isolates if these basidiomycetous pathogens are included in your research program. I think it is better to check the impact of urea concentrations on the possibility of selective isolation of basidiomycetes which can degrade this toxic compound, while ascomycetes cannot do this.

Thanks so much for all your explanation. Thanks for Carlos Velez for the papers which explains why we use fungicide to isolate basidiomycetes, in particular the fungal pathogen on decayed wood.

I've used the following "antibiotic cocktail" to avoid bacterial contaminants with good results in PDA:

15 mg/L streptomycin sulfate

15 mg/L gentamicin sulfate

12 mg/L tetracycline hydrochloride

Chloramphenicol or Streptomycin (10mg/L) can also be used

I think that antibiotics for bacteria are known for all microbiologists... What Alvin ask is how to avoid growing of fungi others than Basidiomycota. Cheers

I have a question, I am in same situation for my experiments. Do you think benomyl is enough temperature resistance for autoclaving? or I should add it after autoclaving medium?

Based on a fast search, I wouldn't add it before autoclaving:

http://mcb.berkeley.edu/labs/koshland/Protocols/Media/Benomyl.html

http://labs.mcb.harvard.edu/murray/publications/Schuyler_2009_19475395.pdf

http://www.plantphysiol.org/content/126/2/613.full

Benomyl is effective against Ascomycetes but Basidiomycetes are usually resistant against it. To dissolve benomyl, you can either use DMSO or 70% Ethanol.

Hi

I have a question

Benomyl add to culture medium before Autoclave or after that?

Rahele Panahabadi after autoclaving media, wait for a while, just before the agar solidifies, add the antibiotic as antibiotics are sensitive to high temperatures. All the very best!