Quantifying arbuscular mycorrhizal community dynamics in agricultural systems

Project: Research project (funded)Research

Project Details


Arbuscular mycorrhizal fungi (AMF) have been hypothesized to play a key role in soil and plant nutrient cycling and agricultural ecosystem sustainability, however, while a great deal is known about AMF root colonization, little is still known about the amount and dynamics of arbuscular mycorrhizas in soil. The aim of this project is therefore to develop novel molecular tools to quantify mycorrhizal abundance in soil. The techniques developed will then be used to test hypotheses about the impact of contrasting agricultural management strategies on AMF abundance and diversity. This knowledge will provide a greater insight into a pivotal soil organism and key determinant of plant productivity and hence aid in the development of sustainable agricultural systems.

Layman's description

Rapid development of molecular techniques in recent years made new genetic tools available for application in molecular ecology. Arbuscular mycorrhizal fungi (AMF), a group of common and widespread soil fungi associated with the roots of most land plants, are difficult to track in their soil environment, which makes them particularly attractive for using these new genetic quantification tools. In this project genetic tools were developed that enable quantification of particular groups and individual species of AMF. Five quantification assays, based on ribosomal nucleic acid sequence variation and three based on variation in the actin gene were developed, employing real-time polymerase chain reaction (PCR) technology. These assays were shown to measure the concentrations of nucleic acids in a highly sensitive, specific, and reproducible way, yielding good correlations between PCR quantified nucleic acids and the numbers of fungal spores from which the nuclei acids had been extracted. The assays’ specificity was confirmed by testing them against a large collection of reference fungal isolates that covered the whole group of AMF. However, total abundance of fungal hyphae in and outside of roots and the concentration of AMF-indicator fatty acids from soil samples were not correlated with results from genetic quantification. Using staining techniques, heterogeneity in abundance and distribution of live fragments and nuclei in hyphae were identified as being the most probable cause for these difficulties to relate traditional and genetic measurements of fungal abundance. Nonetheless, genetic quantification of nucleic acids might allow predictions about physiological activities of particular fungi or plant-fungal associations, which has not been possible due to a lack of morphological and biochemical characters to distinguish individual members of AMF.

Key findings

We successfully developed and validated TaqMan® real-time quantitative PCR (qPCR) assays to detect and quantify the abundance of eight AMF taxa, members of the phylogenetic clades Glomus group Aa, Ab, B, a group of Scutellospora spp., and G. hoi (UY110). For Glomus group Aa, Ab, and Scutellospora spp. there are now assays targeted to the nuclear ribosomal small subunit (nrSSU) RNA as well as the actin gene. This is important because it will enable confirmation of qPCR results from a high as well as a low-copy gene target and may also help to account for differences in gene copy numbers among strains belonging to the same species. Optimal primer and probe concentrations were established, after first confirming presence of only single PCR products and absence of primer dimers, using plasmid template and SYBR Green® I chemistry. The qPCR assays’ specificity for the target taxa was shown using a large and phylogenetically broad collection of reference AMF isolates in cross-amplification tests with genomic DNA extracts. It was confirmed that template abundance was accurately, precisely, and reproducibly measured over a broad range of concentrations, using serial dilutions of a plasmid of known molecule numbers and replicate DNA extracts from different numbers of fungal spores of single spore isolates of Glomus mosseae and G. claroideum. In a further validation step we established good correlations between the number of spores and PCR-quantifiable nrSSU loci with both the DNA and RNA fraction, isolated from entire mycelia of split-plate cultures of a reference isolate of G. intraradices, propagated on root organ cultures. However, by using this fungal material we were unable to disentangle the PCR signal of nuclei from spores and hyphae, although the signal form spores was overwhelming. Attempts to find correlations between total and vital hyphal length and qPCR results were unsuccessful using both an isolate of G. diaphanum with pronounced hyphal growth before sporulation under in vitro conditions and leek roots colonised by an isolate of G. mosseae, which did not produce vesicles. There was strong evidence that heterogeneity in fungal root colonisation and nuclear distribution was responsible for the lack of correlation between fungal hyphal length and nucleic acid measurements. This evidence was obtained from experiments in which nucleic acids from root fragments of different lengths were extracted and quantified and from studies using vital and nuclear staining. In summary, qPCR assays were developed that will for the first time enable specific quantification of the abundance of targeted AMF taxa that would otherwise be indistinguishable by morphological and biochemical characters.

Results and outputs - Resources generated
-Five new taxon-specific TaqMan® real-time PCR assays targeted to the SSU nrRNA gene and three targeted to the actin gene.
-Partial SSU, actin, and mtLSU sequences of AMF isolates
-DNA extraction procedure for AMF from soil-based growth substrates.
-In vitro cultures of five AMF strains on root organ cultures.
-A living culture collection of AMF reference strains in pots.
-19 new single spore isolates of Glomus hoi (UY110)

Effective start/end date10/01/059/01/07


  • BBSRC: £137,133.85