Intraradical Dynamics of Two Coexisting Isolates of the Arbuscular Mycorrhizal Fungus Glomus intraradices Sensu Lato as Estimated by Real-Time PCR of Mitochondrial DNA

Abstract

bReal-time PCR in nuclear ribosomal DNA (nrDNA) is becoming a well-established tool for the quantification of arbuscular mycorrhizal (AM) fungi, but this genomic region does not allow the specific amplification of closely related genotypes. The large subunit of mitochondrial DNA (mtDNA) has a higher-resolution power, but mtDNA-based quantification has not been previously explored in AM fungi. We applied real-time PCR assays targeting the large subunit of mtDNA to monitor the DNA dynamics of two isolates of Glomus intraradices sensu lato coexisting in the roots of medic (Medicago sativa). The mtDNA-based quantification was compared to quantification in nrDNA. The ratio of copy numbers determined by the nrDNA-and mtDNA-based assays consistently differed between the two isolates. Within an isolate, copy numbers of the nuclear and the mitochondrial genes were closely correlated. The two quantification approaches revealed similar trends in the dynamics of both isolates, depending on whether they were inoculated alone or together. After 12 weeks of cultivation, competition between the two isolates was observed as a decrease in the mtDNA copy numbers of one of them. The coexistence of two closely related isolates, which cannot be discriminated by nrDNA-based assays, was thus identified as a factor influencing the dynamics of AM fungal DNA in roots. Taken together, the results of this study show that real-time PCR assays targeted to the large subunit of mtDNA may become useful tools for the study of coexisting AM fungi. Communities of arbuscular mycorrhizal (AM) fungi are an integral component of the soil-plant system, colonizing the roots of the majority of land plants, facilitating plant nutrient uptake, and constituting an important channel of carbon movement from plants to soil (35). The PCR amplification of AM fungal DNA with taxon-specific primers enables the targeted detection of AM fungal taxa within a community and detailed studies of their coexistence involving the screening of large sample sets (21,37,47). This approach is promising, particularly if applied in conjunction with quantitative real-time PCR (qPCR) (17), which allows for the determination of the abundance of the fungal taxa in samples. Studies using qPCR with taxon-specific primers demonstrated that qPCR is a more sensitive tool to monitor community changes than commonly used nested endpoint PCR (3,19,29), and it corroborated the callings for the quantitative evaluation of AM fungal communities (11, 32).The qPCR assays that have been developed so far for the quantification of AM fungal DNA in roots or in soil rely, with one exception (12), on nuclear markers in the relatively well-characterized nuclear ribosomal DNA (nrDNA). This region, however, lacks the resolution power to discriminate among closely related species, such as Glomus claroideum and G. etunicatum (8,44), or even isolates of the same species. It is known that high developmental and functional diversity exists at these lower taxonomic levels in AM fungi (20,28), but interactions amon...

Effects of Inoculum Additions in the Presence of a Preestablished Arbuscular Mycorrhizal Fungal Community

“…The plateau level is a feature of microscopically determined root colonization dynamics after nonvital staining of all fungal structures. In contrast, vital staining and quantification of fungal nuclei based on nrDNA copy numbers often reveal a peak of fungal vitality after a few weeks of cultivation, followed by a decline (19,22,35,42). Assuming acceleration of the root colonization process by higher propagule levels, the negative abundance responses to intraspecific inoculation with G. intraradices and G. claroideum may in fact reflect shifts in colonization dynamics, with an earlier vitality peak and onset of the vitality decline, both occurring before the first harvest after 6 weeks of cultivation.…”

“…Two specific primer pairs were therefore developed, each amplifying only a part of the G. mosseae ribotypes, and applied as a duplex assay. For the G. intraradices isolate, previously designed primers (22) were used, as they were found suitable for the present study. The specificity of these qPCR assays was tested in silico using the Fast PCR software (28) as well as experimentally by cross-amplification experiments using different templates (plasmid standards, medic DNA extracted from roots and leaves, fungal DNA extracted from spores, and DNA extracted from medic roots colonized by each fungal isolate).…”

Inoculation of drought-stressed strawberry with a mixed inoculum of two arbuscular mycorrhizal fungi: effects on population dynamics of fungal species in roots and consequential plant tolerance to water deficiency

“…Nevertheless, individual AMF species may differ in their effects on plant growth. The multiple occupancy of a single root fragment by AMF from different genera or species is now widely accepted (Alkan et al 2006;Reddy et al 2005;Van Tuinen et al 1998;Wagg et al 2011a), although this cooccupation is currently poorly understood (Alkan et al 2006;Krak et al 2012), and it remains unclear if the relationship is competitive, synergistic or antagonistic; indeed, their relationship may vary with plant hosts and/or environmental conditions (Wagg et al 2011a).…”

Arbuscular Mycorrhizal Fungal Composition Affects the Growth and Nutrient Acquisition of Two Plants from a Karst Area

“…Kiers et al (2011) have suggested that individual AM species might use different strategies to supply resources and benefits to host plants when multiple AM species share an individual root system. The relationship between different AM fungal species, such as synergy, competition or antagonist and the effect of that relationship on a plant species is difficult to determine (Krak et al 2012), especially in karst habitats. Mixed inoculation was better than single inoculation for enhancing the plant traits for nutrient acquisition and biomass accumulation of B. papyrifera seedlings (Figure 1), which suggested that there could be a synergistic relationship among the AM fungi species.…”