Congruence of ribosomal DNA sequencing, fatty acid methyl ester profiles and morphology for characterization of the genus Rhizophagus (arbuscular mycorrhiza fungus)

Difficulties in obtaining sterile axenic cultures and heterogeneity in nuclear-encoded ribosomal DNA (n-rDNA) sequences within a single arbuscular mycorrhizal spore make genetic analysis of arbuscular mycorrhizal fungi (AMF) a complicated task, and currently available methods of genotyping are inadequate for identification to the species level. Therefore, we applied a multipronged approach on different isolates grown in root organ culture (ROC) belonging to the genus Rhizophagus which were not characterized at species level. Each strain was characterized using the fatty acid methyl ester profile (FAME), partial sequencing of a small subunit-internal transcribed spacer (SSU-ITS) and a large subunit (LSU) region of n-rDNA, and morphological examination of spores. Neighbor-joining trees obtained from the SSU-ITS rDNA sequences were broadly similar to those obtained from the LSU rDNA sequences. FAME profiles of the same isolates used for molecular characterization were obtained using fatty acid datasets, and results were compared to a neighbor-joining tree of n-rDNA sequence. Based on the results of these studues, a combination of morphology, biomarkers (FAME), and molecular sequencing (of highly variable D1-D2 of LSU and ITS) is recommended for phylogenetic analysis and characterization of species/strain of Glomeromycota. © Springer-Verlag Berlin Heidelberg and the University of Milan 2013.