Cultivation and detection of unculturable fungi in soil using soil infusion agar(SIA)‎

Abstract EN

Unculturable and non sporulating fungi represent a great challenge in studying biotrophic ,endotrophic and mycorrhizal fungal groups.

In this study a novel culture dependent technique complemented with PCR was used to detect and identify of unculturable fungi in soil .

To develop techniques for cultivation of unculturable fungi for the purpose of expanding studies on fungal biodiversity in soil , Soil Infusion Agar (SIA) was developed .

By this technique, from higher dilutions of soil five tiny colonies (less than 1mm in diameter) had grown on SIA after incubation for 1-3 months at 280C , but had not shown growth when replicated on traditional mycological media ,were selected , purified studied culturally and microscopically and identified by molecular methods .

Growth of the five clones on PDA, but not Sabouraud Dextrose Agar and Czpak Dox Agar of incubation when a loopful of a colony grown after several successive subculturing on SIA was transferred to PDA.

The growth on PDA has been empirically proven due to the inoculums size effect and the period of incubation at 280C.

Two fungal specific primer sets (EF4F/ITS4r and EF60F/ITS4r) were used to amplify partial sequences of fungal r RNA gene included ITS sequences .The five partial sequences of the five clones were aligned through the BLASTN phylogenetic analysis against NCBI database which revealed higher identities.

The blast tree showed that all clones are neighbor joined to Aspergillus ssp and Pichia sp.

These results led us to consider the clones as viable but non culturable fungi (VBNC fungi) like the common phenomenon found in some bacterial species.

Keywords: Unculturable fungi , Soil , Soil Infusion Agar( SIA) , PCR ,VBNC fungi.