Impact of two Erwinia sp. on the response of diverse Pisum sativum genotypes under salt stress.

Currently, salinization is impacting more than 50% of arable land, posing a significant challenge to agriculture globally. Salt causes osmotic and ionic stress, determining cell dehydration, ion homeostasis, and metabolic process alteration, thus negatively influencing plant development. A promising sustainable approach to improve plant tolerance to salinity is the use of plant growth-promoting bacteria (PGPB). This work aimed to characterize two bacterial strains, that have been isolated from pea root nodules, initially called PG1 and PG2, and assess their impact on growth, physiological, biochemical, and molecular parameters in three pea genotypes (Merveille de Kelvedon, Lincoln, Meraviglia d'Italia) under salinity. Bacterial strains were molecularly identified, and characterized by in vitro assays to evaluate the plant growth promoting abilities. Both strains were identified as Erwinia sp., demonstrating in vitro biosynthesis of IAA, ACC deaminase activity, as well as the capacity to grow in presence of NaCl and PEG. Considering the inoculation of plants, pea biometric parameters were unaffected by the presence of the bacteria, independently by the considered genotype. Conversely, the three pea genotypes differed in the regulation of antioxidant genes coding for catalase (PsCAT) and superoxide dismutase (PsSOD). The highest proline levels (212.88 µmol g-1) were detected in salt-stressed Lincoln plants inoculated with PG1, along with the up-regulation of PsSOD and PsCAT. Conversely, PG2 inoculation resulted in the lowest proline levels that were observed in Lincoln and Meraviglia d'Italia (35.39 and 23.67 µmol g-1, respectively). Overall, this study highlights the potential of these two strains as beneficial plant growth-promoting bacteria in saline environments, showing that their inoculation modulates responses in pea plants, affecting antioxidant gene expression and proline accumulation. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01419-8.

Characterization of endophytic bacteria isolated from root nodules of lentil in intercropping with durum wheat.

Legumes improve soil fertility by interacting symbiotically with nitrogen-fixing rhizobia allocated in root nodules. Some bacterial endophytes can coexist with rhizobia in nodules and might help legumes by enhancing stress tolerance, producing hormones stimulating plant growth, and increasing plant nutrient intake. Twenty-six bacterial endophytes from Lens culinaris root nodules cultivated in intercropping with Triticum durum were identified and characterized molecularly and biochemically. Potential plant growth-promoting strains have been selected according to the indole acetic acid and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase production, and for their inorganic phosphate solubilization ability. The presence of genes associated to ACC deaminase and nitrogenase was evaluated. Six selected strains were grown with varying NaCl and polyethylene glycol concentrations to test their salt and osmotic stress tolerance. Priestia megaterium 11NL3 and Priestia aryabhattai 19NL1, resulted to be tolerant to salinity and osmotic stress, were tested on four genotypes of T. durum seeds in different stress conditions. The effect of strain inoculation on seed germination, vigor, and root-to-shoot ratio varied depending on the type of stress and on the durum wheat genotypes. For future research, it will be necessary to test the selected bacterial strains at different plant phenological stages and to clarify the mechanisms involved in the different outcomes of plant-microbe interactions.

Above- and belowground interplay: Canopy CO2 uptake, carbon and nitrogen allocation and isotope fractionation along the plant-ectomycorrhiza continuum.

In forests, mycorrhizal fungi regulate carbon (C) and nitrogen (N) dynamics. We evaluated the interplay among ectomycorrhizas (ECM), ecosystem C fluxes, tree productivity, C and N exchange and isotopic fractionation along the soil-ECM-plant continuum in a Mediterranean beech forest. From bud break to leaf shedding, we monitored: net ecosystem exchange (NEE, a measure of the net exchange of C between an ecosystem and the atmosphere), leaf area index, stem growth, N concentration, δ13 C and δ15 N in rhizosphere soil, ectomycorrhizal fine root tips (ERT), ECM-free fine root portions (NCR) and leaves. Seasonal changes in ERT relative biomass were strictly related to NEE and mimicked those detected in the radial growth. The analysis of δ13 C in ERT, leaves and NCR highlighted the impact of canopy photosynthesis on ERT development and an asynchronous seasonal C allocation strategy between ERT and NCR at the root tips level. Concerning N, δ15 N of leaves was negatively related to that of ERT and dependent on seasonal 15 N differences between ERT and NCR. Our results unravel a synchronous C allocation towards ERT and tree stem driven by the increasing NEE in spring-early summer. Moreover, they highlighted a phenology-dependent 15 N fractionation during N transfer from ECM to their hosts. This evidence, obtained in mature beech trees under natural conditions, may improve the knowledge of Mediterranean forests functionality.

Diverse mycorrhizal maize inbred lines differentially modulate mycelial traits and the expression of plant and fungal phosphate transporters.

Food production is heavily dependent on soil phosphorus (P), a non-renewable mineral resource essential for plant growth and development. Alas, about 80% is unavailable for plant uptake. Arbuscular mycorrhizal fungi may promote soil P efficient use, although the mechanistic aspects are yet to be completely understood. In this study, plant and fungal variables involved in P acquisition were investigated in maize inbred lines, differing for mycorrhizal responsiveness and low-P tolerance, when inoculated with the symbiont Rhizoglomus irregulare (synonym Rhizophagus irregularis). The expression patterns of phosphate transporter (PT) genes in extraradical and intraradical mycelium (ERM/IRM) and in mycorrhizal and control maize roots were assessed, together with plant growth responses and ERM extent and structure. The diverse maize lines differed in plant and fungal accumulation patterns of PT transcripts, ERM phenotypic traits and plant performance. Mycorrhizal plants of the low-P tolerant maize line Mo17 displayed increased expression of roots and ERM PT genes, compared with the low-P susceptible line B73, which revealed larger ERM hyphal densities and interconnectedness. ERM structural traits showed significant correlations with plant/fungal expression levels of PT genes and mycorrhizal host benefit, suggesting that both structural and functional traits are differentially involved in the regulation of P foraging capacity in mycorrhizal networks.

Arbuscular Mycorrhizal Fungi Increase Nutritional Quality of Soilless Grown Lettuce while Overcoming Low Phosphorus Supply.

Lettuce is widely used for its healthy properties, and it is of interest to increase them with minimal environmental impact. The purpose of this work was to evaluate the effect of the arbuscular mycorrhizal fungus (AMF) Funneliformis mosseae in lettuce plants (Lactuca sativa L. cv. Salinas) cultivated in a soilless system with sub-optimal phosphorus (P) compared with non-inoculated controls at two different P concentrations. Results show that lettuce inoculation with the selected AMF can improve the growth and the nutritional quality of lettuce even at sub-optimal P. Leaf content of chlorophylls, carotenoids, and phenols, known as important bioactive compounds for human health, was higher in mycorrhizal lettuce plants compared with non-mycorrhizal plants. The antioxidant capacity in AMF plants showed higher values compared with control plants grown at optimal P nutrition level. Moreover, leaf gas exchanges were higher in inoculated plants than in non-inoculated ones. Nitrogen, P, and magnesium leaf content was significantly higher in mycorrhizal plants compared with non-mycorrhizal plants grown with the same P level. These findings suggest that F. mosseae can stimulate plants growth, improving the nutritional quality of lettuce leaves even when grown with sub-optimal P concentration.

Genetic variability assessment of 127 Triticum turgidum L. accessions for mycorrhizal susceptibility-related traits detection.

Positive effects of arbuscular mycorrhizal fungi (AMF)-wheat plant symbiosis have been well discussed by research, while the actual role of the single wheat genotype in establishing this type of association is still poorly investigated. In this work, the genetic diversity of Triticum turgidum wheats was exploited to detect roots susceptibility to AMF and to identify genetic markers in linkage with chromosome regions involved in this symbiosis. A tetraploid wheat collection of 127 accessions was genotyped using 35K single-nucleotide polymorphism (SNP) array and inoculated with the AMF species Funneliformis mosseae (F. mosseae) and Rhizoglomus irregulare (R. irregulare), and a genome-wide association study (GWAS) was conducted. Six clusters of genetically related accessions were identified, showing a different mycorrhizal colonization among them. GWAS revealed four significant quantitative trait nucleotides (QTNs) involved in mycorrhizal symbiosis, located on chromosomes 1A, 2A, 2B and 6A. The results of this work enrich future breeding activities aimed at developing new grains on the basis of genetic diversity on low or high susceptibility to mycorrhization, and, possibly, maximizing the symbiotic effects.

Appressoria and phosphorus fluxes in mycorrhizal plants: connections between soil- and plant-based hyphae.

Arbuscular mycorrhizal fungi (AMF) live in symbiosis with plant roots, facilitating mineral nutrient transfer from soil to hosts through large networks of extraradical hyphae. Limited data are available on the fungal structures (appressoria) connecting soil- to root-based mycelium, in relation to plant nutrition. Two in vivo systems were set up using three AMF, Funneliformis mosseae, Funneliformis coronatus and Rhizoglomus irregulare, grown in symbiosis with Cichorium intybus. The assessment of plant P content, number of appressoria, diameter of their subtending hyphae and length of colonized roots allowed calculation of the total cross-section area of appressorium-subtending hyphae, which differed among the three AMF and was correlated with plant P contents and with extraradical mycelium density. A conservative evaluation of P fluxes from soil- to plant-based hyphae occurring through appressoria gave values ranging from 1.7 to 4.2 × 10-8 mol cm-2 s-1 (moles per total cross-section area of the appressorium subtending hyphae per time elapsed), depending on AMF identity. This work suggests that, beyond intraradical colonization and extraradical mycelium extent, connections between extraradical and intraradical fungal mycelium through appressoria are important for mycorrhizal plant nutrition, as appressorium structural traits and density can be related to P transfer mediated by AMF.

A Whole-Plant Culture Method to Study Structural and Functional Traits of Extraradical Mycelium.

An in vivo whole-plant bi-dimensional experimental system has been devised and tested with different host plants, in order to obtain extraradical mycelium (ERM) produced by different arbuscular mycorrhizal fungi (AMF). In this system, a host plant germling is inoculated with AMF to establish mycorrhizal symbiosis, and, after colonization, newly formed extraradical hyphae and spores are removed. Then the mycorrhizal root system is wrapped in a nylon net and placed between membranes in a Petri dish, allowing ERM to grow on the membrane surface. Such extraradical hyphae may be used for in situ morphometric analyses or collected for molecular or biochemical assays: in the latter case, the plant with its root sandwich may be reassembled to renew mycelium production. In this experimental system, which was tested with diverse host plant species and lines, values of explored membrane surface areas and densities of ERM showed wide ranges of variation, and its length ranged from 9.7 ± 2.0 to 48.8 ± 9.9 m per plant, depending on host and AMF identity. Across the different plant-AMF combinations tested, the whole-plant system produced 2.0 ± 0.6 to 5.3 ± 0.3 mg of ERM fresh biomass per plant per harvest. This experimental system can be used for a wide range of AMF and host plants species, either establishing arbuscular mycorrhizas or other mycorrhizal interactions. ERM produced and collected in the whole-plant system is suitable for morphological, physiological, and molecular analyses, facilitating studies on the different aspects of mycorrhizal symbiotic interactions.

Gene expression in Rhizoglomus irregulare at two different time points of mycorrhiza establishment in Helianthus annuus roots, as revealed by RNA-seq analysis.

Arbuscular mycorrhizal fungi (AMF) play a fundamental role in plant growth and nutrition in natural and agricultural ecosystems. Despite the importance of such symbionts, the different developmental changes occurring during the AMF life cycle have not been fully elucidated at the molecular level. Here, the RNA-seq approach was used to investigate Rhizoglomus irregulare specific and common transcripts at two different time points of mycorrhizal establishment in Helianthus annuus in vivo. Four days after inoculation, transcripts related to cellular remodeling (actin and tubulin), cellular signaling (calmodulin, serine/threonine protein kinase, 14-3-3 protein, and calcium transporting ATPase), lipid metabolism (fatty acid desaturation, steroid hormone, and glycerophospholipid biosynthesis), and biosynthetic processes were detected. In addition to such transcripts, 16 days after inoculation, expressed genes linked to binding and catalytic activities; ion (K+, Ca2+, Fe2+, Zn2+, Mn2+, Pi, ammonia), sugar, and lipid transport; and those involved in vacuolar polyphosphate accumulation were found. Knowledge of transcriptomic changes required for symbiosis establishment and performance is of great importance to understand the functional role of AMF symbionts in food crop nutrition and health, and in plant diversity in natural ecosystems.

Mycorrhizal networks facilitate the colonization of legume roots by a symbiotic nitrogen-fixing bacterium.

Arbuscular mycorrhizal fungi (AMF) absorb and translocate nutrients from soil to their host plants by means of a wide network of extraradical mycelium (ERM). Here, we assessed whether nitrogen-fixing rhizobia can be transferred to the host legume Glycine max by ERM produced by Glomus formosanum isolate CNPAB020 colonizing the grass Urochloa decumbens. An H-bridge experimental system was developed to evaluate the migration of ERM and of the GFP-tagged Bradyrhizobium diazoefficiens USDA 110 strain across an air gap compartment. Mycorrhizal colonization, nodule formation in legumes, and occurrence of the GFP-tagged strain in root nodules were assessed by optical and confocal laser scanning microscopy. In the presence of non-mycorrhizal U. decumbens, legume roots were neither AMF-colonized nor nodulated. In contrast, G. formosanum ERM crossing the discontinuous compartment connected mycorrhizal U. decumbens and G. max roots, which showed 30-42% mycorrhizal colonization and 7-11 nodules per plant. Fluorescent B. diazoefficiens cells were detected in 94% of G. max root nodules. Our findings reveal that, besides its main activity in nutrient transfer, ERM produced by AMF may facilitate bacterial translocation and the simultaneous associations of plants with beneficial fungi and bacteria, representing an important structure, functional to the establishment of symbiotic relationships.

Diversity of a phosphate transporter gene among species and isolates of arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal fungi (AMF) are a key group of beneficial obligate biotrophs, establishing a mutualistic symbiosis with the roots of most land plants. The molecular markers generally used for their characterization are mainly based on informative regions of nuclear rDNA (SSU-ITS-LSU), although protein-encoding genes have also been proposed. Within functional genes, those encoding for phosphate transporters (PT) are particularly important in AMF, given their primary ability to take up Pi from soil, and to differentially affect plant phosphate nutrition. In this work, we investigated the genetic diversity of PT1 gene sequences and sequences of the taxonomically relevant SSU-ITS-LSU region in two isolates of the species Funneliformis coronatus, three isolates of the species Funneliformis mosseae and two species of the genus Rhizoglomus, originated from geographically distant areas and cultured in vivo. Our results showed that partial PT1 sequences not only successfully differentiated AMF genera and species like ribosomal gene sequences but also highlighted intraspecific diversity among F. mosseae and F. coronatus isolates. The study of functional genes related to the uptake of key mineral nutrients for the assessment of AMF diversity represents a key step in the selection of efficient isolates to be used as inocula in sustainable agriculture.

Two herbicides, two fungicides and spore-associated bacteria affect Funneliformis mosseae extraradical mycelium structural traits and viability.

The extraradical mycelium (ERM) produced by arbuscular mycorrhizal fungi is fundamental for the maintenance of biological fertility in agricultural soils, representing an important inoculum source, together with spores and mycorrhizal root fragments. Its viability and structural traits, such as density, extent and interconnectedness, which are positively correlated with the growth and nutrition of host plants, may be affected by different agronomic practices, including the use of pesticides and by different mycorrhizospheric communities. This work, carried out using a whole-plant experimental model system, showed that structural traits of ERM, such as length and density, were strongly decreased by the herbicides dicamba and glufosinolate and the fungicides benomyl and fenhexamid, while anastomosis frequency and hyphal branching were differentially modulated by singly inoculated mycorrhizospheric bacteria, depending on their identity.

Arbuscular mycorrhizal fungi induce the expression of specific retrotransposons in roots of sunflower (Helianthus annuus L.).

Retrotransposon expression during arbuscular mycorrhizal (AM) fungal colonisation of sunflower roots (Helianthus annuus) was analysed using Illumina RNA-Seq, in order to verify whether mycorrhizal symbiosis can activate retrotransposable elements. Illumina cDNA libraries were produced from RNAs isolated from the roots of sunflower plants at 4 and 16 days after inoculation with the AM fungus Rhizoglomus irregulare and from their respective control plants. Illumina reads were mapped to a library of reverse transcriptase-encoding sequences, putatively belonging to long terminal repeat retrotransposons of Gypsy and Copia superfamilies. Forty-six different reverse transcriptase sequences were transcribed, although at a low rate, in mycorrhizal or control roots and only four were significantly over-expressed at day 16, compared with control roots. Almost all expressed or over-expressed sequences belonged to low-copy elements, mostly, of the Copia superfamily. A meta-analysis, using publicly available Illumina cDNA libraries obtained from sunflower plants treated with different hormones and chemicals, mimicking stimuli produced by abiotic and biotic stresses, was also conducted. Such analyses indicated that the four reverse transcriptase sequences over-expressed in mycorrhizal roots were explicitly induced only by AM symbiosis, showing the specificity of AM stimuli compared to that of other fungal/plant interactions.

Designing the Ideotype Mycorrhizal Symbionts for the Production of Healthy Food.

The new paradigm in agriculture, sustainable intensification, is focusing back onto beneficial soil microorganisms, for the role played in reducing the input of chemical fertilizers and pesticides and improving plant nutrition and health. Worldwide, more and more attention is deserved to arbuscular mycorrhizal fungi (AMF), which establish symbioses with the roots of most land plants and facilitate plant nutrient uptake, by means of a large network of extraradical hyphae spreading from colonized roots to the surrounding soil and functioning as a supplementary absorbing system. AMF protect plants from biotic and abiotic stresses and are able to modulate the activity of antioxidant enzymes and the biosynthesis of secondary metabolites (phytochemicals), such as polyphenols, anthocyanins, phytoestrogens and carotenoids, that play a fundamental role in promoting human health. An increasing number of studies focused on the use of AMF symbionts for the production of functional food, with enhanced nutritional and nutraceutical value. Yet, while several plant species were investigated, only few AMF were utilized, thus limiting the full exploitation of their wide physiological and genetic diversity. Here, we will focus on AMF effects on the biosynthesis of plant secondary metabolites with health-promoting activity, and on the criteria for a finely tuned, targeted selection of the best performing symbionts, to be utilized as sustainable biotechnological tools for the production of safe and healthy plant foods.

Genetic markers associated to arbuscular mycorrhizal colonization in durum wheat.

In this work we investigated the variability and the genetic basis of susceptibility to arbuscular mycorrhizal (AM) colonization of wheat roots. The mycorrhizal status of wild, domesticated and cultivated tetraploid wheat accessions, inoculated with the AM species Funneliformis mosseae, was evaluated. In addition, to detect genetic markers in linkage with chromosome regions involved in AM root colonization, a genome wide association analysis was carried out on 108 durum wheat varieties and two AM fungal species (F. mosseae and Rhizoglomus irregulare). Our findings showed that a century of breeding on durum wheat and the introgression of Reduced height (Rht) genes associated with increased grain yields did not select against AM symbiosis in durum wheat. Seven putative Quantitative Trait Loci (QTLs) linked with durum wheat mycorrhizal susceptibility in both experiments, located on chromosomes 1A, 2B, 5A, 6A, 7A and 7B, were detected. The individual QTL effects (r2) ranged from 7 to 16%, suggesting a genetic basis for this trait. Marker functional analysis identified predicted proteins with potential roles in host-parasite interactions, degradation of cellular proteins, homeostasis regulation, plant growth and disease/defence. The results of this work emphasize the potential for further enhancement of root colonization exploiting the genetic variability present in wheat.

Lifespan and functionality of mycorrhizal fungal mycelium are uncoupled from host plant lifespan.

Arbuscular mycorrhizal fungi (AMF) are obligate symbionts, living in associations with the roots of most land plants. AMF produce wide networks of extraradical mycelium (ERM) of indeterminate length, spreading from host roots into the surrounding soil and establishing belowground interconnections among plants belonging to the same or to different taxa. Whether their lifespan and functionality are limited by host plant viability or can be extended beyond this limit is unknown. To address this issue, we performed time-course studies to investigate viability and functionality of ERM produced in an in vivo whole-plant system by Funneliformis mosseae and Rhizoglomus irregulare, after shoot detachment. Our data revealed that viability and functionality of F. mosseae and R. irregulare extraradical hyphae were uncoupled from host plant lifespan. Indeed, ERM spreading from roots of intact or shootless plants showed comparable levels of viability, similar structural traits and ability to establish mycorrhizal symbioses with new plants, as long as five months after shoot removal. Our findings expand the current knowledge on AMF biology and life cycle, providing data on ERM long-term survival in the soil of two Glomeracean species, functional to the prompt establishment of mycorrhizal symbioses and to the maintenance of soil biological fertility.

Divergence of Funneliformis mosseae populations over 20 years of laboratory cultivation, as revealed by vegetative incompatibility and molecular analysis.

Arbuscular mycorrhizal fungi (AMF) are widespread, important plant symbionts. They absorb and translocate mineral nutrients from the soil to host plants through an extensive extraradical mycelium, consisting of indefinitely large networks of nonseptate, multinucleated hyphae which may be interconnected by hyphal fusions (anastomoses). This work investigated whether different lineages of the same isolate may lose the ability to establish successful anastomoses, becoming vegetatively incompatible, when grown separately. The occurrence of hyphal incompatibility among five lineages of Funneliformis mosseae, originated from the same ancestor isolate and grown in vivo for more than 20 years in different European locations, was assessed by systematic detection of anastomosis frequency and cytological studies. Anastomosis frequencies ranged from 60 to 80% within the same lineage and from 17 to 44% among different lineages. The consistent detection of protoplasm continuity and nuclei in perfect fusions showed active protoplasm flow both within and between lineages. In pairings between different lineages, post-fusion incompatible reactions occurred in 6-48% of hyphal contacts and pre-fusion incompatibility in 2-17%. Molecular fingerprinting profiles showed genetic divergence among lineages, with overall Jaccard similarity indices ranging from 0.85 to 0.95. Here, phenotypic divergence among the five F. mosseae lineages was demonstrated by the reduction of their ability to form anastomosis and the detection of high levels of vegetative incompatibility. Our data suggest that potential genetic divergence may occur in AMF over only 20 years and represent the basis for detailed studies on the relationship between genes regulating anastomosis formation and hyphal compatibility in AMF.

Transcriptome changes induced by arbuscular mycorrhizal fungi in sunflower (Helianthus annuus L.) roots.

Arbuscular mycorrhizal (AM) fungi are essential elements of soil fertility, plant nutrition and productivity, facilitating soil mineral nutrient uptake. Helianthus annuus is a non-model, widely cultivated species. Here we used an RNA-seq approach for evaluating gene expression variation at early and late stages of mycorrhizal establishment in sunflower roots colonized by the arbuscular fungus Rhizoglomus irregulare. mRNA was isolated from roots of plantlets at 4 and 16 days after inoculation with the fungus. cDNA libraries were built and sequenced with Illumina technology. Differential expression analysis was performed between control and inoculated plants. Overall 726 differentially expressed genes (DEGs) between inoculated and control plants were retrieved. The number of up-regulated DEGs greatly exceeded the number of down-regulated DEGs and this difference increased in later stages of colonization. Several DEGs were specifically involved in known mycorrhizal processes, such as membrane transport, cell wall shaping, and other. We also found previously unidentified mycorrhizal-induced transcripts. The most important DEGs were carefully described in order to hypothesize their roles in AM symbiosis. Our data add a valuable contribution for deciphering biological processes related to beneficial fungi and plant symbiosis, adding an Asteraceae, non-model species for future comparative functional genomics studies.

An in vivo whole-plant experimental system for the analysis of gene expression in extraradical mycorrhizal mycelium.

Arbuscular mycorrhizal fungi (AMF) establish beneficial mutualistic symbioses with land plants, receiving carbon in exchange for mineral nutrients absorbed by the extraradical mycelium (ERM). With the aim of obtaining in vivo produced ERM for gene expression analyses, a whole-plant bi-dimensional experimental system was devised and tested with three host plants and three fungal symbionts. In such a system, Funneliformis mosseae in symbiosis with Cichorium intybus var. foliosum, Lactuca sativa, and Medicago sativa produced ERM whose lengths ranged from 9.8 ± 0.8 to 20.8 ± 1.2 m per plant. Since ERM produced in symbiosis with C. intybus showed the highest values for the different structural parameters assessed, this host was used to test the whole-plant system with F. mosseae, Rhizoglomus irregulare, and Funneliformis coronatus. The whole-plant system yielded 1-7 mg of ERM fresh biomass per plant per harvest, and continued producing new ERM for 6 months. Variable amounts of high-quality and intact total RNA, ranging from 15 to 65 µg RNA/mg ERM fresh weight, were extracted from the ERM of the three AMF isolates. Ammonium transporter gene expression was successfully determined in the cDNAs obtained from ERM of the three fungal symbionts by RT-qPCR using gene-specific primers designed on available (R. irregulare) and new (F. mosseae and F. coronatus) ammonium transporter gene sequences. The whole-plant experimental system represents a useful research tool for large production and easy collection of ERM for morphological, physiological, and biochemical analyses, suitable for a wide variety of AMF species, for a virtually limitless range of host plants and for studies involving diverse symbiotic interactions.