Yes pathogenic fungus in culture, looses its virulence after many time subculturling so it is always advice to do pathogenicity test with fresh culture only

Yes. If it takes a long time to use the fungus, inoculate and re-isolate it. Or preserve at low temperature or even freeze.

Yes, this process is known as attenuation. Inoculate fungus on plant and re-isolate it.

Thank you, everyone. Ashish Singh Bisht i am doing the same now to inoculate and isolate from plant..

Bruno do Nascimento Silva can we store it directly at an ultra low temperature or do we have to add any other chemical to the media before freezing.. like Glycerol

I have had similar problems with a couple of fungi before knowing that pathogenicity could be lost. As mentioned by other colleagues, one way to recover the pathogenicity of your culture is to inoculate and reisolate it. Additionally, I would like to add that a very simple (and inexpensive) method to preserve your cultures long-term is to store them in water. For this, when you have a freshly isolated culture, transfer a small portion of agar (just like when making a subculture) into an Eppendorf tube (or any preferred tube) with a little bit of sterile distilled water. That's it. Cultures preserved in this way can remain viable for many years. To recover them, simply transfer the portion of agar from the Eppendorf tube to a routine culture medium (like PDA), and you will obtain a colony identical to the original, with its pathogenic properties intact.