Total DNA was extracted from about 20-50 mg of a trap culture of mycorrhizal fungal mycelium using the DNeasy PlantMini Kit (Qiagen). Partial ribosomal SSU DNA fragments were then amplified using a universal eukaryotic primer NS31 (5′-TTG GAG GGC AAG TCT GGT GCC-3′) (Simon et al. 1992) and the primer AM1 (5′-GTT TCC CGT AAG GCG CCG AA-3′), which only amplifies AM fungal SSU sequences but not plant sequences (Helgason et al. 1998). Basically, the PCR reaction follows the protocol described above. PCR products were run on agarose gel to ensure that only one band amplified, and then they were purified with Qiagen PCR purification kit for direct sequencing with either NS31 or AM1 primer. Also, PCR products can be cloned to the pGEM-T vector and then sequenced with T7 and /or SP6 primers.

Visual identification can be carried out on AM fungal spores, as they are larger than other fungal spores; most spores are between 100 to 200 pm in diameter and can be easily observed under a dissecting microscope (Jarstfer and Sylvia 2002). AM fungi in roots can also be observed after chemical staining by using the staining agent 0.05% trypan blue in lactophenol reported by Phillips and Hayman (1970). The chemical trypan blue is considered a carcinogen but is still used in some laboratories (Utobo et al.

2011) . Alternatively, a simple and inexpensive method has been developed (Vierheilig et al. 1998) with ink and vinegar as the staining agent, which is not toxic. Although it is very easy to use, cheap, quick, and can be used for large number of samples, not all inks stain all AM fungi. In general, almost all black inks give good staining, and the structures are clearest under a dark field illumination with a stereomicroscope (Vierheilig et al. 2005).