Use a minimal medium like in this publication : Correll JC, Klittich CJR, Leslie JR. Nitrate nonutilizing mutants of Fusarium oxysporum and their use in vegetative compatibility tests. Phytopathology 1987;77:1640–6. Then use different water immiscible solvent and do a liquid liquid extraction by phase separation. Start with ethyl acetate you'll have a lot of different chemicals. See this publications on A. brassicicola that are relevant for F. oxysporum : Pedras, M.S.C., Chumala, P.B., Jin, W., Islam, M.S., and Hauck, D.W. (2009). The phytopathogenic fungus Alternaria brassicicola: Phytotoxin production and phytoalexin elicitation. Phytochemistry 70, 394–402.
Calmes, B., Guillemette, T., Teyssier, L., Siegler, B., Pigné, S., Landreau, A., Iacomi, B., Lemoine, R., Richomme, P., and Simoneau, P. (2013). Role of mannitol metabolism in the pathogenicity of the necrotrophic fungus Alternaria brassicicola. Front. Plant Sci. 4.
The first paper is more relevant for the principle and the second is more precise on technical description.
I wish I help you
Lény
Hi, Kamlesh
It depends on which Fusarium species you are working with. Different Fusarium species produce different mycotoxins and their solubility is highly variable. For instance, deoxynivalenol, produced by F. graminearum among other species, is soluble in water and can be easily extracted.
Perhaps if you could provide more information regarding the origin of the Fusarium sp. you are working with and the culture media you're using I could be able to help you.
Regards,
Ismael
Hi, Kamlesh
Pl try this method and hope this will serve the purpose.
Culture the fusarium in to potato broath for 10 days at 27°C ±2°C. Ten days old broth filtered through whatman filter paper no. 1 and extracted with chloroform (25:25 v/v) in separating funnels. The bottom aqueous layer of chloroform was passed through an anhydrous sodium sulphate column to dry the remaining chloroform layer, which contains the dissolve mycotoxins. The eluted solution/chloroform was evaporated at 60°C in a vacuum evaporator. The residue dissolved in 500 µl (0.5 ml) acetonitrile. Aliquots of the acetonitrile extract (20 µl) were spotted in duplicate on TLC silica gel plate you can identify the toxins with the help of various stranded.
Hi,
I am a bit skeptical about the culture parameter of Kamlesh. Probably, it is for an other species than Fusarium oxysporum. For F. oxysporum, culture parameter must be 24-25°C, 5 days (because after F oxysporum start to die) and 125-150 rpm for 150 ml of culture in a flask of 500 ml. I have an other doubt, I think it's an error to use a PDB for toxin analysis because (1) depending on the batch of PDB you'll have variation of toxin production and (2) PDB is a very complex medium and the resulting extract could be more complex. From my point of view, synthetic medium is more simple but you can have less toxin secretion...
regards
Isolates from Fusarium genera were evaluated for their
mycotoxin producing potential. A rapid method of Bragulat et al., (2001) for
extraction of mycotoxin were adopted .The isolates were grown on Czapek
yeast autolysate agar (CYA) medium incubated at 25ºC for 7 days .Three plugs
(7-mm diameter) were removed from the inner, middle, and outer areas of each
colony. Plugs were weighed and introduced into 3-ml vials; 1 ml methanol was
added and the vials were shaken and incubated at 25ºC for 60 min. The extracts
were centrifuged three times for 5 min at 4000 rpm. The supernant was passed
through a filtration membrane Millipore filter 0.22 μm . Then the extracts were tested
What is the kind of medium ?
The best medium for testing the toxins production by fusarium sp. are cereal grains as crushed yellow corn
50 gm of crushed yellow corn in Erlenmeyer flask were autoclave for i hr at 120 psi. then left overnight, 25% of distilled water were added to elevate the moisture content up to 25% and inoculated with the spores of fusarium culture of 1 week old. The content were mixed in flask and incubated at 28-30 degree for i month then transferred to incubated at 8-10 degree for another 7-10 days.
after the end of incubation, the content of flask were dried in oven at 60 degree for 3-5 days , then the samples each finely ground in electric miller .
Th grind sample weighed and equal level of water: methanol reagent were added (45% water:55% methanol) , mixed for 10 min and left for overnight at room temperature to give the chance to extract all toxins in water: :methanol,
The content were filtrated by what-man paper to discard the the yellow corn that now free from toxins
To the filtrate add equal amount of ether or hexan for def-fating the extract (fate bind with toxin and interfere with the results) , shaking for 5 minutes, then the extract was separated using separator funnel ( the upper layer of hexan containing fate was discarded)
- Th extract was added to equal amount of chloroform and shacked for 10 minute and by separatory funnel take the chloroform layer containing toxins , the water methanol layer extracted again for other 2 times by chloroform to extract all toxin contents of samples . Then THE OBTAINED CHLOROFORM +TOXINS SUBJECTED TO EVAPORATION IN WATER BATH AT 60 DEGREE .
NOW WE HAVE THE RESIDUE AFTER EVAPORATION OF CHLOROFORM, IT IS THE SUGGESTED PARTICLES OF TOXINS
WE CAN APPLY ANY AVAILABLE METHOD IN OUR LABORATORY FOR MEASUREMENT THE OBTAINED AMOUNT AND KIND OF TOXIN OR NO
AS:
1- THIN LAYER CHROMATOGRAPHY WITH USING SPECIFIC STANDARD OF SUGGESTED TOXIN ,
2- MASS-SPECTROPH.
3- FLOROUMETRIC METHOD
SEE THE ATTACHED PAPER OF AUTHOR AND SUMMARY OF METHOD
I used pdb media to culture the fungus, let it 7_10 days to ensure it exite the toxin then Extracted it with classic method Extraction
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