Taxon-specific oligonucleotide primers for detection of Glomus etunicatum

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 I TS1Kpn and ITS4Pst as primers. The amplification products (597, 599, 598, a nd 613 bp, respectively) were cloned and sequenced. The similarity among IT S region sequences from MD107, MD127, and TN101 was 99%, whereas the sequen ce similarity between the ITS regions of these three DNAs and that from FL3 29 was 91%. The 5.8S rDNA sequences of all four G. etunicatum isolates were identical. In contrast, major dissimilarities in the corresponding rDNA, s equence regions of other glomalean taxa were observed. Oligonucleotide sequ ences unique to G. etunicatum were tested for their specificity in PCR ampl ification of genomic DNA from spores of 55 isolates comprising 29 glomalean fungi: 18 isolates of G. etunicatum, five G. intraradices, three G. claroi deum, 16 other Glomus isolates, and 11 other glomalean taxa from each of fo ur other genera. The G. etunicatum isolates were from a broad range of geog raphic regions and soils. The oligonucleotide pair GETU1:GETU2 primed speci fic amplification of an oligonucleotide sequence (approximately 400 bp) pre sent 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 non-m ycorrhizal controls, including roots of corn (Zea mays). In addition, the p air successfully detected G. etunicatum in nested PCR using a primary PCR p roduct amplified from highly diluted extracts of colonized corn roots using modified ITS1:ITS4 primers. In the phylogenetic analysis of Glomus 5.8S an d ITS2 rDNA region sequences, which included 500 bootstrap data sets, confi dence in the G. etlunicatum branch was very strong (90%) and clearly indepe ndent of G. claroideum and G. intraradices, to which it is very closely rel ated.