Give stress under UV light .. and use less media for isolation

Thank you Swamy Gowda M R

Thank you Kouadri Mohamed El Amine

Culture it on PDA and incubate it for 5-6 days for sporulation.

You can as well place sterile filter paper into the PDA media before inoculation. This technique also helps with spores formation or sporulation.

I wish you all the very best.

Thank you Osabutey Samuel

You can replace the PDA medium with V8 medium, it gave best results when fungal colonies were grown in it at 25 C in the dark with agitation for 7 days, the mycelium mass was ground, poured in plates which contain PDA and incubated at 25 C under near ultraviolet light and 12 hour photoperiod . This method induces the fungus to produce conidia.

I don't prefer exposure the plates to UV light, because UV light may causes mutation. Therefor we must find other methods to induce sporulation. One of them using V8, it's effective medium.

Also, you can growing the fungus on PDA medium to allow mycelium to grow by incubating plates on 25 C for about 4 days, then transfer the plates to the freezer with 0° C for few hours, after that you must remove the plates from the freezer and put them in the room temperature. This method stimulate the fungus to produce conidia.

You can see the paper named: In vitro production of conidia of Alternaria solani. By: Tatiana T.M.S. et al. Tropical Plant Pathology, 35 (4), 203-212 (2010).

I attached this paper in order to read it carefully, it may be helpful.

The medium V8 agar-CaCO3 was considered as a standard medium for Alternaria solani sporulation, because it induces the fungus to produce spores. This medium contains 200 ml of V8 Vegetable Juice, 3 g of CaCO3, 16 g of Agar agar and 800 ml of Distilled water, with Streptomycin sulfate (50 µg/ml) and Chloramphenicol (75 µg/ml) .

Thank you so much for the time you have given sir, the Best answer so far I have got. I will follow the procedure Yehya A. Salih

You are most welcome dear Navya Botlagunta,

I recommend the above research to be noticed carefully and applying it. It's very useful as I see.

Hope you are achieving good results.

With cordial regards.

V-8 Agar medium consists of the following contents:

They must be autoclaved together at 121 C and 15 Pounds. Inch-1 for about 30 minutes in autoclave.

To distinguish between the spores of Alternaria solani and Alternaria alternata which always isolated from the leaf spotting diseases in order to avoid the confusion between them, A. solani spores have long peaks reach to two third the conidium long or more, while A. alternata spores have very short peaks.

I had the same problem and tried many different type of medium, temperature, light conditions etc. from the past papers. Nothing worked.

Finally, I accidentally found that black light (or UV) worked. For the purpose of my experiment, the effect of black light on the spores didn't matter.