A TAXON SPECIFIC OLIGONUCLEOTIDE PROBE FOR GLOMUS ETUNICATUM

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

Submitted to: Mycorrhiza
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/18/2000
Publication Date: N/A
Citation: N/A

Interpretive Summary: Nearly all agricultural and native plants form symbiotic associations with a group of soil microorganisms known as arbuscular mycorrhizae. The maintenance of an active community of these fungi in the soil is crucial for land and soil management. There are about 158 species of this group of fungi known worldwide. Because they cannot be grown without plants, and because they are difficult to identify, very little is known about how soil, crop and land management practices are affecting their diversity. Scientists need to have better ways of identifying these organisms when they are inside the plant roots and growing in soil. The work reported here describes the design and specificity of a genetically-base tool that can be used to detect and distinguish the presence of one of the most commonly encountered mycorrhizal fungi in agricultural soils, Glomus etunicatum. This tool, species-specific oligonucleotide primers, will enable scientists to locate and identify the presence of this fungus in roots and soils even when other mycorrhizal fungi and microorganisms are present. This will be immensely helpful in helping scientists learn more about how to prevent loss of species diversity in this unique and important group of fungi.

Technical Abstract: The 5.8S subunit and flanking internal transcribed spacer (ITS) regions in nuclear ribosomal DNA (rDNA) from spores of Glomus etunicatum MD107, MD127, TN101, and FL329 were amplified by polymerase chain reaction (PCR) using ITS1Kpn and ITS4Pst as primers. The amplification products (597, 599, 598, and 613 bp, respectively) were cloned and sequenced. The similarity among DNA ITS region sequences amplified by PCR from MD107 and TN101 and was 99%, whereas the sequence similarity between the ITS regions of these 3 DNAs and that from FL329 was 91%. The 5.8s rDNA sequences were 100% similar for all 4 G. etunicatum isolates. The sequences of the 5.8s rDNA for G. etunicatum TN101 and FL329 were 100%, similar to that of G. intraradices FL208, and 99% similar to that of G. claroideum MD125. There was a 92-93% sequence similarity in the ITS regions among the 4 G. etunicatum DNAs and those from the closely related taxa G. intraradices and G. claroideum. In contrast, major dissimilarities with sequences of other glomalean taxa were observed. Oligonucleotide sequences unique to G. etunicatum were tested for their specificity in PCR amplification of genomic DNA from spores from 54 isolates comprising 29 glomalean fungi: 18 isolates of G. etunicatum, 4 G. intraradices, 2 G. claroideum, 10 other Glomus isolates, and 11 other glomalean taxa from each of 4 other genera. The G. etunicatum isolates were from a broad range of geographic regions and soils. The oligonucleotide pair, GETU1:GETU2, primed specific amplification of an oligonucleotide sequence (approximately 400 base pairs) present in all G. etunicatum. This primer pair did not prime PCR when template consisted of DNA from any of the other glomalean fungi or any of the nonmycorrhizal controls, including roots of Z. mays.