Characterization of root colonization profiles by a microcosm community of arbuscular mycorrhizal fungi using 25S rDNA‐targeted nested PCR

Abstract

The aim of the present work was to study colonization patterns in roots by different arbuscular mycorrhizal fungi developing from a mixed community in soil. As different fungi cannot be distinguished with certainty in planta on the basis of fungal structures, taxon-discriminating molecular probes were developed. The 5' end of the large ribosomal subunit containing the variable domains D1 and D2 was amplified by PCR from Glomus mosseae (BEG12), G. intraradices (LPA8), Gigaspora rosea (BEG9) and Scutellospora castanea (BEG1) using newly designed eukaryote-specific primers. Sequences of the amplification products showed high interspecies variability and PCR taxon-discriminating primers were designed to distinguish between each of these four fungi. A nested PCR, using universal eukaryotic primers for the first amplification and taxon-discriminating primers for the second, was performed on individual trypan blue-stained mycorrhizal root fragments of onion and leek, and root colonization by four fungi inoculated together in a microcosm experiment was estimated. More than one fungus was detected in the majority of root fragments and all four fungi frequently co-existed within the same root fragment. Root colonization by G. mosseae and G. intraradices was similar from individual mixed inoculum, whilst the frequency of S. castanea and Gig. rosea increased in the presence of the two Glomus species, suggesting that synergistic interactions may exist between some arbuscular mycorrhizal fungi.

Identification of Bacterial Groups Preferentially Associated with Mycorrhizal Roots of Medicago truncatula

“…AM are ancient; the first fossil evidence of this symbiosis dates back 400 million years (33). Several authors have proposed that AM have contributed to the colonization of early land plants (32,38).AM are generally assumed to be nonspecific associations, since Glomeromycota are able to colonize roots of several host plants and are themselves colonized by different AM fungal species (12,14,35,44). Despite this lack of host specificity, the diversity of AM fungi has been shown to affect the plant community composition under field conditions (43), and the genetic structure of the AM fungal community was shown to differ significantly according to the plant species…”

“…AM are generally assumed to be nonspecific associations, since Glomeromycota are able to colonize roots of several host plants and are themselves colonized by different AM fungal species (12,14,35,44). Despite this lack of host specificity, the diversity of AM fungi has been shown to affect the plant community composition under field conditions (43), and the genetic structure of the AM fungal community was shown to differ significantly according to the plant species (B. Pivato, P. Lemanceau, S. Siblot, G. Berta, C. Mougel, and D. van Tuinen, submitted for publication).…”

“…Furthermore, the corresponding studies did not take into account the natural diver-sity of AM fungi, since they were based on the inoculation of selected strains of Glomeromycota. However, under field conditions, plants are generally colonized by several types of functionally different Glomeromycota (12,13,14,44; Pivato et al, submitted). Therefore, the inoculation of specific strains of Glomeromycota to assess their impact on the indigenous bacterioflora may have induced bias.…”

Determining a minimum detection threshold in terminal restriction fragment length polymorphism analysis

“…Spores were crushed in 40 µl of 1x TE buffer by micropestle and boiled for two minutes. Samples were centrifuged at 13,000 rpm for one minute in the presence of 10 µl of 20% Chelex 100 (BioRad) to pellet cell wall fragments and 20 µl of supernatant was transferred to new tubes (Van Tuinen et al 1998). The extracts were subjected to an ethanol precipitation to purify DNA and resuspended in 1x TE buffer for downstream applications.…”

Semi-quantitative analysis of arbuscular mycorrhizal colonization of onion roots inoculated with single or mixed species based upon PCR-RFLP

“…Since then, many reports using molecular techniques have been published for the identification of AM fungi colonizing roots. These include taxon-or group-specific PCR (Millner et al 1998;van Tuinen et al 1998), and PCR specific to AM fungi followed by restriction fragmenl length polymorphism (RFLP) (Redecker et al 1997) or by sequencing (Helgason et al 1998). These I To whom correspondence should be addressed.…”