Cultivation and Detection of Unculturable Fungi in Soil Using Soil Infusion Agar(SIA)

Unculturable and non sporulating fungi represent a great challenge in studying biotrophic ,endotrophic mycorrhizal fungal groups. In this study novel culture dependent technique complemented with PCR was used to detect identify of unculturable soil . To develop techniques for cultivation the purpose expanding studies on biodiversity , Soil Infusion Agar (SIA) developed By technique, from higher dilutions five tiny colonies (less than 1mm diameter) had grown SIA after incubation 1-3 months at 280C but not shown growth when replicated traditional mycological media ,were selected purified studied culturally microscopically identified by molecular methods Growth clones PDA, Sabouraud Dextrose Czpak Dox loopful colony several successive subculturing transferred PDA. The PDA has been empirically proven due inoculums size effect period 280C. Two specific primer sets (EF4F/ITS4r EF60F/ITS4r) were amplify partial sequences r RNA gene included ITS .The aligned through BLASTN phylogenetic analysis against NCBI database which revealed identities. blast tree showed that all are neighbor joined Aspergillus ssp Pichia sp. These results led us consider as viable culturable (VBNC fungi) like common phenomenon found some bacterial species.