TAXON SPECIFIC OLIGONUCLEOTIDE PRIMERS FOR DETECTION OF TWO ANCIENT ENDOMYCORRHIZAL FUNGI, GLOMUS OCCULTUM AND GLOMUS BRASILIANUM

Authors: Millner, Patricia; Mulbry, Walter; Reynolds, Sara

Submitted to: Federation of European Microbiological Societies Microbiology Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/31/2001
Publication Date: N/A
Citation: N/A

Interpretive Summary: The most common, natural root-fungus symbionts known are present throughout the world in intensively and marginally managed landscapes. They aid in plant nutrient uptake, root disease control, and soil quality. There are about 150 species, all of which have been differentiated by classically recognized morphological characteristics that do not reflect functional attributes. The need for spores to determine species has hampered ecological studies of these fungi on the surface of plant roots. This report describes molecular tools that can be used to detect and differentiate very closely related species of this ancient group of plant symbiotic fungi. Readily available PCR and blotting methods are used to accomplish the detection and differentiation. This will enable investigators to undertake ecological studies of these symbionts in field collected roots and to ascertain the purity of maintained pot cultures.

Technical Abstract: The 5.8S subunit and flanking internal transcribed spacer (ITS) regions in rDNA from spores of five purported strains of Glomus occultum and two strains of the closely related species, Glomus brasilianum (WV224 and ITH43), were amplified by PCR using ITS1Kpn and ITS4Pst as primers, then cloned and sequenced. The 5.8 rDNA sequences from the five purported G. occultum strains were relatively homogeneous (94-99% sequence similarity), whereas ITS regions were typically more heterogenous (71-99% sequence similarity). Alignment of G. occultum sequences with those from two G. brasilianum strains revealed a greater degree of homology between 5.8 rDNA regions (93-99% similarity) than between ITS regions (72-79% similarity). The high degree of similarity among ITS regions of GR582 and those from the two G. brasilianum strains indicates that GR582 is more likely a strain of G. brasilianum than of G. occultum. Sequences unique to G. occultum were tested for specificity in PCR with genomic DNA from spores from 28 isolate of 20 glomalean fungi. Primer pair GOCC56:GOCC427 amplified a 370 bp sequence from all strains of G. occultum and G. brasilianum and did not prime with DNA template from other glomalean fungi or nonmycorrhizal controls, including roots of Z. mays. This primer pair detected G. occultum and G. brasilianum DNA in nested PCR using highly diluted extracts of colonized corn (Zea mays) roots. A second primer pair, GBRAS186:GBRAS388, amplified a 200bp sequence present only in G. brasilianum and strain GR582. These primer pairs, based on sequence data of multiple strains of glomalean taxa, provides new molecular tools to detect and differentiate recently characterized ancestral lineages of the symbiotic Glomales, G. occultum and G. brasilianum, that are undetectable by standard staining procedures in