Fungal morphology can be observed under a microscope, and chitin, a specific fungal cell wall component, can be stained with dyes to generally identify fungal species. For a more specific identification, fungal genomic DNA can be isolated using a standard bacterial DNA isolation protocol (Sambrook et al. 1989) or commercial kits, such as the DNeasy Plant Mini Kit (Qiagen).

For identification, the internal transcribed spacer (ITS) regions of fungal ribosomal DNA are widely used because the regions are highly variable (Ghimire et al. 2011). The specific primers ITS1 (5′-TCC GTA GGT GAA CCT TGC GG-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) are used to PCR-amplify highly variable ITS1 and ITS2 regions surrounding the 5.8S coding region (Martin and Rygiewicz 2005). However, the primers do not effectively exclude host plant sequences in mixed samples so ITS1-F (5′-CTT GGT CAT TTA GAG GAA GTA A-3′) and ITS4-B (5′-CAG GAG ACT TGT ACA CGG TCC AG-3′) were designed to amplify fungal ITS regions, and a pair of ITS1F and ITS4 resulted in strong PCR amplification from both ascomycetes and basidiomycetes (Gardes and Bruns 1993).

In general, PCR reactions should include 1X reaction buffer containing Mg++, 1 gl of 10 gM of each primer, 1 gl of 10 mM dNTPs, 0.5-1.0 unit Taq DNA polymerase, and 5O-20o ng genomic DNA to total 25-50 gl. PCR can be performed in any thermal cycler with program like 95°C for 2-4 min, then 95°C for 30 sec, 55°C for 30 sec, 72°C for 45 sec for 35 cycles, finally 72°C for 10 min. PCR products are checked in agarose gel first to make sure only one clear band exists, then the product is either cloned into pGEM-T vector (Promega) or similar kits, such as TA cloning kits for sequencing. Direct sequencing of the PCR product after purification using Qiagen’s PCR purification kit is also an option.

Once PCR product sequences are obtained, BLASTN searches can be performed to compare similar sequences from gene bank to identify the species of the target microorganism. A phylogenetic tree can also be constructed to further clarify its evolutionary relationship among other species. In addition, PCR-RFLP, Length Heterogeneity PCR, and Terminal Restriction Fragment Length Polymorphism can be used to characterize microbial communities (Martin and Rygiewicz 2005).