DNA barcoding of arbuscular mycorrhizal fungi

Plant beneficial microorganisms, such as arbuscular mycorrhiza fungi (AMF), increasingly attract
scientific and agronomic attention due to their capacity to increase nutrient accessibility for plants
and to reduce inorganic fertilizer requirements. AMF are thought to form symbioses with most land
plants, obtaining carbon from the autotrophic host whilst enhancing uptake of poorly available
nutrients.
The species of AMF are mainly identified by spore morphology, which is time consuming, requires
expertise and is rarely applicable to AMF identification in roots. Molecular tools such as analysis of
standardized DNA fragment sequences may allow the recognition of species through a ‘DNA
barcode’, which may partly overcome this problem. The focus of this study was to evaluate
different regions of widely used rDNA repeats for their use as DNA barcodes for AMF including the
small subunit rRNA gene (SSU), the internal transcribed spacer (ITS) and the large subunit rRNA
gene (LSU). Closely related species in the genus Ambispora, members of which have dimorphic
spores, could not be separated by analysis of the SSU region, but of the ITS region. Consequently,
the SSU was not used for subsequent analysis, but a DNA fragment covering a small part of the
SSU, the entire ITS region and about 800 bp of the LSU (SSUmCf-LSUmBr fragment) was
analysed, providing phylogenetic resolution to species. New AMF specific primers for these
potential barcoding regions were developed and can be applied, without amplification of non-target
organisms, for AMF species determination, including identification from field and root samples.
Analyses based on the application of the SSUmCf-LSUmBr fragment showed that the widely used
AMF model organism Glomus sp. DAOM197198 (formerly called Glomus intraradices) is not
conspecific with Gl. intraradices. The SSUmCf-LSUmBr fragment clearly provides a much higher
species resolution capacity when compared with the formerly preferred ITS and LSU regions.
Further study of several groups of AMF species using different regions of the SSUmCf-LSUmBr
fragment revealed that only the complete SSUmCf-LSUmBr fragment allowed separation of all
analysed species. Based on these results, an extended DNA barcode covering the ITS region and
parts of the LSU region is suggested as a DNA barcode for AMF. The complete SSUmCf-LSUmBr
fragment sequences can serve as a database backbone for also using smaller rDNA fragments as
barcodes. Although the smallest fragment (approximately 400 bp) analysed in this study was not
able to discriminate among AMF species completely, such short regions covering the ITS2 or LSU
D2 regions, respectively, would most likely be suitable for community analyses with 454 GS-FLX
Titanium sequencing, providing that the analyses is based on the longer DNA sequences.