Evidence of the Ability of Microsatellite Method to Distinguish Cannabis Strains with High Cannabinoid Content.

Introduction: Cannabis is a plant with high potential for use in several sectors of the industry; however, it is also a controversial crop due to its tetrahydrocannabinol (THC) content. Moreover, the plant has a rather unclarified classification. Traditionally, two types of Cannabis have been distinguished, hemp as a source of fiber and low THC content, and marijuana with high THC levels, which is used as a drug. With the increasing use of CBD strains and wide range of commercially used THC strains, it is becoming paramount to be able to develop an easy and reliable method for Cannabis strain differentiation. The use of simple sequence repeat markers, or microsatellites, seems to be an applicable choice. Materials and Methods: In this study, 52 strains of Cannabis with variable cannabinoid content were collected from growers from different geographical regions and analyzed using 17 different microsatellite markers. For more precise differentiation, five strains were selected and a higher number of individuals of each were analyzed. Results: Fragment analysis and cluster analysis showed that when one to three individual plants per strain were analyzed, the method was able to classify these samples into distinguishable groups with similar gene structure. They also revealed that when a larger sample set was used (10 individual plants per strain), highly specific strain clusters could be fully discriminated. Conclusion: Our study involved the highest number of cannabinoid-rich strains up to now and showed that the microsatellite method can be used to reliably differentiate Cannabis strains and show their relationships.

Enhanced production of select phytocannabinoids in medical Cannabis cultivars using microbial consortia.

The root microbiome of medical cannabis plants has been largely unexplored due to past legal restrictions in many countries. Microbes that live on and within the tissue of Cannabis sativa L. similar to other plants, provide advantages such as stimulating plant growth, helping it absorb minerals, providing protection against pathogen attacks, and influencing the production of secondary metabolites. To gain insight into the microbial communities of C. sativa cultivars with different tetrahydrocannabinol (THC) and cannabidiol (CBD) profiles, a greenhouse trial was carried out with and without inoculants added to the growth substrate. Illumina MiSeq metabarcoding was used to analyze the root and rhizosphere microbiomes of the five cultivars. Plant biomass production showed higher levels in three of five cultivars inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis and microbial suspension. The blossom dry weight of the cultivar THE was greater when inoculated with R. irregularis and microbial suspension than with no inoculation. Increasing plant biomass and blossom dry weight are two important parameters for producing cannabis for medical applications. In mature Cannabis, 12 phytocannabinoid compounds varied among cultivars and were affected by inoculants. Significant differences (p ≤ 0.01) in concentrations of cannabidivarinic acid (CBDVA), cannabidivarin (CBDV), cannabigerol (CBG), cannabidiol (CBD), and cannabigerolic acid (CBGA) were observed in all Cannabis cultivars when amended with F, K1, and K2 inoculants. We found microbes that were shared among cultivars. For example, Terrimicrobium sp., Actinoplanes sp., and Trichoderma reesei were shared by the cultivars ECC-EUS-THE, CCL-ECC, and EUS-THE, respectively. Actinoplanes sp. is a known species that produces phosphatase enzymes, while Trichoderma reesei is a fungal train that produces cellulase and contributes to organic matter mineralization. However, the role of Terrimicrobium sp. as an anaerobic bacterium remains unknown. This study demonstrated that the use of inoculants had an impact on the production of phytocannabinoids in five Cannabis cultivars. These inoculants could have useful applications for optimizing cannabis cultivation practices and increasing the production of phytocannabinoids.

Establishing a quality management framework for commercial inoculants containing arbuscular mycorrhizal fungi.

Microbial inoculants containing arbuscular mycorrhizal (AM) fungi are potential tools in increasing the sustainability of our food production systems. Given the demand for sustainable agriculture, the production of such inoculants has potential economic value and has resulted in a variety of commercial inoculants currently being advertised. However, their use is limited by inconsistent product efficacy and lack of consumer confidence. Here, we propose a framework that can be used to assess the quality and reliability of AM inoculants. First, we set out a range of basic quality criteria which are required to achieve reliable inoculants. This is followed by a standardized bioassay which can be used to test inoculum viability and efficacy under controlled conditions. Implementation of these measurements would contribute to the adoption of AM inoculants by producers with the potential to increase sustainability in food production systems.

Antioxidant response in arbuscular mycorrhizal fungi inoculated wetland plant under Cr stress.

Arbuscular mycorrhizal fungi (AMF) provide a positive effect on antioxidant mechanisms in terrestrial plants under heavy metal stress. This study investigated the effects of AMF on wetland plant (Iris wilsonii) growth and antioxidant response under Cr stress at different water depths. Results showed that AMF inoculated I. wilsonii had higher antioxidant response than non-inoculated controls, with shoot superoxide dismutase (SOD), root SOD, shoot peroxidase (POD), and root POD contents increased by 4.7-39.6%, 7.5-29.5%, 11.2-68.6%, 16.8-50.3%, respectively. Meanwhile, shoot (root) proline, malondialdehyde (MDA) and superoxide anion (O2.-) contents in the AMF inoculated I. wilsonii were 10.2-44.3% (2.8-37.2%), 11.5-35.4% (16.9-28.2), and 14.9-30.5% (-0.9-26.3%) lower than those in the non-inoculated controls, respectively. Besides, AMF improved the growth of I. wilsonii with biomass, height, chlorophyll, K, and P contents in the shoots increased by 10.5-32.5%, 17.4-44.9%, 4.7-37.7%, 12.0-30.7%, 13.5-20.6%, respectively. Moreover, the I. wilsonii tolerance to Cr stress was also enhanced under the water depth of 6-3 cm. Therefore, AMF play an important role in wetland plant growth and antioxidant response under Cr stress, and it can improve wetland plants' tolerance to Cr stress at fluctuating water depth.

Phytochemical Composition and In Vitro Biological Activity of Iris spp. (Iridaceae): A New Source of Bioactive Constituents for the Inhibition of Oral Bacterial Biofilms.

The inhibition and eradication of oral biofilms is increasingly focused on the use of plant extracts as mouthwashes and toothpastes adjuvants. Here, we report on the chemical composition and the antibiofilm activity of 15 methanolic extracts of Iris species against both mono-(Pseudomonas aeruginosa, Staphylococcus aureus) and multi-species oral biofilms (Streptococcus gordonii, Veillonella parvula, Fusobacterium nucleatum subsp. nucleatum, and Actinomyces naeslundii). The phytochemical profiles of Iris pallida s.l., Iris versicolor L., Iris lactea Pall., Iris carthaliniae Fomin, and Iris germanica were determined by ultra-high performance liquid chromatography-high-resolution tandem mass spectroscopy (UHPLC-HRMS/MS) analysis, and a total of 180 compounds were identified among Iris species with (iso)flavonoid dominancy. I. pallida, I. versicolor, and I. germanica inhibited both the quorum sensing and adhesion during biofilm formation in a concentration-dependent manner. However, the extracts were less active against maturated biofilms. Of the five tested species, Iris pallida s.l. was the most effective at both inhibiting biofilm formation and disrupting existing biofilms, and the leaf extract exhibited the strongest inhibitory effect compared to the root and rhizome extracts. The cytotoxicity of the extracts was excluded in human fibroblasts. The inhibition of bacterial adhesion significantly correlated with myristic acid content, and quorum sensing inhibition correlated with the 7-ß-hydroxystigmast-4-en-3-one content. These findings could be useful for establishing an effective tool for the control of oral biofilms and thus dental diseases.

Alien ectomycorrhizal plants differ in their ability to interact with co-introduced and native ectomycorrhizal fungi in novel sites.

Alien plants represent a potential threat to environment and society. Understanding the process of alien plants naturalization is therefore of primary importance. In alien plants, successful establishment can be constrained by the absence of suitable fungal partners. Here, we used 42 independent datasets of ectomycorrhizal fungal (EcMF) communities associated with alien Pinaceae and Eucalyptus spp., as the most commonly introduced tree species worldwide, to explore the strategies these plant groups utilize to establish symbioses with EcMF in the areas of introduction. We have also determined the differences in composition of EcMF communities associated with alien ectomycorrhizal plants in different regions. While alien Pinaceae introduced to new regions rely upon association with co-introduced EcMF, alien Eucalyptus often form novel interactions with EcMF species native to the region where the plant was introduced. The region of origin primarily determines species composition of EcMF communities associated with alien Pinaceae in new areas, which may largely affect invasion potential of the alien plants. Our study shows that alien ectomycorrhizal plants largely differ in their ability to interact with co-introduced and native ectomycorrhizal fungi in sites of introduction, which may potentially affect their invasive potential.

Arbuscular mycorrhizal fungi colonization and physiological functions toward wetland plants under different water regimes.

Arbuscular mycorrhizal fungi (AMF) have been widely reported to occur in the association with wetland plants. However, the factors that affect AMF colonization in wetland plants and physiological functions in AMF inoculated wetland plants are poorly studied. This study investigated the effects of four water regimes (below the surface of sands: water levels of 5 cm, 9 cm, 11 cm, and fluctuating water depth (9-11 cm)) on AMF root colonization in two wetland plants (Phalaris arundinacea and Scirpus sylvaticus) which are commonly used in constructed wetland. Results showed that two lower water regimes were the most suitable for the formation of root colonization by AMF. Plant species did not show any significant difference in AMF colonization. The AMF colonization of 15.6-23.3% in the roots of both wetland plants were determined under the water regimes of 11 cm and 9-11 cm. In comparison to the non-inoculated plants, root length, shoot height, biomass, shoot total phosphorus and chlorophyll contents of both wetland plants under the fluctuating water regimes (9-11 cm) were increased by 35.4-46.2%, 13.1-26.6%, 33.3-114.3%, 25.7-80% and 14.3-24%, respectively. Although malondialdehyde (MDA) contents in both AMF inoculated wetland plants were decreased under the lower water levels, the MDA contents under the water regime of 11 cm were still high. Therefore, these results indicated that the physiological functions in wetland plants with high AMF colonization might be improved under a specific water regime condition (e.g. depth of fluctuating water regime).

Using microbial seed coating for improving cowpea productivity under a low-input agricultural system.

BACKGROUND: Plant-growth-promoting rhizobacteria (PGPR) and arbuscular mycorrhizal (AM) fungi have the ability to enhance the growth, fitness, and quality of various agricultural crops, including cowpea. However, field trials confirming the benefits of microbes in large-scale applications using economically viable and efficient inoculation methods are still scarce. Microbial seed coating has a great potential for large-scale agriculture through the application of reduced amounts of PGPR and AM fungi inocula. Thus, in this study, the impact of seed coating with PGPR, Pseudomonas libanensis TR1 and AM fungus, Rhizophagus irregularis (single or multiple isolates) on grain yield and nutrient content of cowpea under low-input field conditions was evaluated. RESULTS: Seed coating with P. libanensis + multiple isolates of R. irregularis (coatPMR) resulted in significant increases in shoot dry weight (76%), and in the number of pods and seeds per plant (52% and 56%, respectively) and grain yield (56%), when compared with non-inoculated control plants. However, seed coating with P. libanensis + R. irregularis single-isolate (coatPR) did not influence cowpea grain yield. Grain lipid content was significantly higher (25%) in coatPMR plants in comparison with control. Higher soil organic matter and lower pH were observed in the coatPMR treatment. CONCLUSIONS: Our findings indicate that cowpea field productivity can be improved by seed coating with PGPR and multiple AM fungal isolates under low-input agricultural systems. © 2019 Society of Chemical Industry.

Seed Coating: A Tool for Delivering Beneficial Microbes to Agricultural Crops.

Plant beneficial microbes (PBMs), such as plant growth-promoting bacteria, rhizobia, arbuscular mycorrhizal fungi, and Trichoderma, can reduce the use of agrochemicals and increase plant yield, nutrition, and tolerance to biotic-abiotic stresses. Yet, large-scale applications of PBM have been hampered by the high amounts of inoculum per plant or per cultivation area needed for successful colonization and consequently the economic feasibility. Seed coating, a process that consists in covering seeds with low amounts of exogenous materials, is gaining attention as an efficient delivery system for PBM. Microbial seed coating comprises the use of a binder, in some cases a filler, mixed with inocula, and can be done using simple mixing equipment (e.g., cement mixer) or more specialized/sophisticated apparatus (e.g., fluidized bed). Binders/fillers can be used to extend microbial survival. The most reported types of seed coating are seed dressing, film coating, and pelleting. Tested in more than 50 plant species with seeds of different dimensions, forms, textures, and germination types (e.g., cereals, vegetables, fruits, pulses, and other legumes), seed coating has been studied using various species of plant growth-promoting bacteria, rhizobia, Trichoderma, and to a lesser extent mycorrhizal fungi. Most of the studies regarding PBM applied via seed coating are aimed at promoting crop growth, yield, and crop protection against pathogens. Studies have shown that coating seeds with PBM can assist crops in improving seedling establishment and germination or achieving high yields and food quality, under reduced chemical fertilization. The right combination of biological control agents applied via seed coating can be a powerful tool against a wide number of diseases and pathogens. Less frequently, studies report seed coating being used for adaptation and protection of crops under abiotic stresses. Notwithstanding the promising results, there are still challenges mainly related with the scaling up from the laboratory to the field and proper formulation, including efficient microbial combinations and coating materials that can result in extended shelf-life of both seeds and coated PBM. These limitations need to be addressed and overcome in order to allow a wider use of seed coating as a cost-effective delivery method for PBM in sustainable agricultural systems.

Abiotic contexts consistently influence mycorrhiza functioning independently of the composition of synthetic arbuscular mycorrhizal fungal communities.

The relationship between mycorrhiza functioning and composition of arbuscular mycorrhizal (AM) fungal communities is an important but experimentally still rather little explored topic. The main aim of this study was thus to link magnitude of plant benefits from AM symbiosis in different abiotic contexts with quantitative changes in AM fungal community composition. A synthetic AM fungal community inoculated to the model host plant Medicago truncatula was exposed to four different abiotic contexts, namely drought, elevated phosphorus availability, and shading, as compared to standard cultivation conditions, for two cultivation cycles. Growth and phosphorus uptake of the host plants was evaluated along with the quantitative composition of the synthetic AM fungal community. Abiotic context consistently influenced mycorrhiza functioning in terms of plant benefits, and the effects were clearly linked to the P requirement of non-inoculated control plants. In contrast, the abiotic context only had a small and transient effect on the quantitative AM fungal community composition. Our findings suggest no relationship between the degree of mutualism in AM symbiosis and the relative abundances of AM fungal species in communities in our simplified model system. The observed progressive dominance of one AM fungal species indicates an important role of different growth rates of AM fungal species for the establishment of AM fungal communities in simplified systems such as agroecosystems.

Nano Zero-Valent Iron Mediated Metal(loid) Uptake and Translocation by Arbuscular Mycorrhizal Symbioses.

Nano zero-valent iron (nZVI) has great potential in the remediation of metal(loid)-contaminated soils, but its efficiency in metal(loid) stabilization in the plant-microbe continuum is unclear. This study investigated nZVI-mediated metal(loid) behavior in the arbuscular mycorrhizal (AM) fungal-maize ( Zea mays L.) plant association. Plants with AM fungal inoculation were grown in metal(loid)- (mainly Zn and Pb) contaminated soils (Litavka River, Czech Republic) amended with/without 0.5% (w/w) nZVI. The results showed that nZVI decreased plant metal(loid) uptake but inhibited AM development and its function in metal(loid) stabilization in the rhizosphere. AM fungal inoculation alleviated the physiological stresses caused by nZVI and restrained nZVI efficiency in reducing plant metal(loid) uptake. Micro proton-induced X-ray emission (µ-PIXE) analysis revealed the sequestration of Zn (possibly through binding to thiols) by fungal structures in the roots and the precipitation of Pb and Cu in the mycorrhizal root rhizodermis (possibly by Fe compounds originated from nZVI). XRD analyses further indicated that Pb/Fe mineral transformations in the rhizosphere were influenced by AM and nZVI treatments. The study revealed the counteractive effects of AM and nZVI on plant metal(loid) uptake and uncovered details of metal(loid) behavior in the AM fungal-root-nZVI system, calling into question about nZVI implementation in mycorrhizospheric systems.

Plant growth promotion of Miscanthus × giganteus by endophytic bacteria and fungi on non-polluted and polluted soils.

Putative endophytes of Miscanthus × giganteus were isolated, and screened in the laboratory, greenhouse and field for their plant growth promoting properties in this host. Pantoea ananatis and Pseudomonas savastanoi were the predominant bacteria in leaves whereas other pseudomonads prevailed in roots. Almost all fungal endophytes belonged to the Pezizomycotina and most were isolated from roots; Fusarium oxysporum was most abundant, followed by the genera Periconia, Exophiala, Microdochium and Leptodontidium. All endophytic groups produced phytohormones and some bacteria also produced siderophores, solubilised P and exhibited ACC-deaminase activity in vitro. In subsequent pot experiments with pre-selected endophytes, several isolates including pseudomonads, Variovorax paradoxus, Verticillium leptobactrum, Halenospora sp. and Exophiala sp. enhanced Miscanthus growth in gamma-sterilised soil. These promising Miscanthus-derived isolates were tested either as single or mixed inocula along with a mixed bacterial inoculum originating from poplar. No significant effects of inocula were detected in a pot experiment in non-sterilised soil. On two marginal field sites the mixture of bacterial endophytes from poplar had a consistently negative effect on survival and growth of Miscanthus. Contrarily, mixtures consisting of bacteria or fungi originating from Miscanthus promoted growth of their host, especially on the heavy metals-polluted site. The combination of bacteria and fungi was inferior to the mixtures consisting of bacteria or fungi alone. Our observations indicate extensive potential of mixed bacterial and fungal endophytic inocula to promote establishment and yield of Miscanthus grown on marginal and polluted land and emphasise the necessity to test particular microbial-plant host combinations. Morphotypes of fungi isolates from Miscanthus × giganteus.

Combined effects of fungal inoculants and the cytokinin-like growth regulator thidiazuron on growth, phytohormone contents and endophytic root fungi in Miscanthus × giganteus.

Aim of this study was to investigate main effects and interactions between symbiotic fungi and the cytokinin-like growth regulator thidiazuron (TDZ) in Miscanthus × giganteus. The arbuscular mycorrhiza fungus Rhizophagus intraradices (AMF) and the endophyte Piriformospora indica (PI) were chosen as model symbionts. The fungal inoculants and TDZ had no significant effect on plant growth but modulated phytohormone levels in the leaves. TDZ induced accumulation of salicylic acid in controls, but not in plants inoculated with fungi. Leaf concentrations of abscisic acid (ABA) derivatives, auxin (indole-3-acetic acid) precursors and catabolites and numerous cytokinins were increased by R. intraradices but lowered by P. indica. TDZ raised concentrations of ABA compounds, the non-indole auxin phenylacetic acid, jasmonate and some cytokinins, but decreased cis-zeatin and N6-(Δ2-isopentenyl)adenine levels. Inoculation with AMF reduced abundance of endogenous clampless endophytes. TDZ application strongly reduced formation of arbuscular mycorrhiza and increased occurrence of clamped mycelia (i.e. basidiomycetous endophytes). Our study provides a thorough outline of the phytohormone homeostasis under the combined influence of beneficial inoculants and a growth regulator, highlighting the necessity to study their interaction in the whole plant-microbial context.

Asymmetric response of root-associated fungal communities of an arbuscular mycorrhizal grass and an ectomycorrhizal tree to their coexistence in primary succession.

The arbuscular mycorrhizal (AM) grass Calamagrostis epigejos and predominantly ectomycorrhizal (EcM) tree Salix caprea co-occur at post-mining sites spontaneously colonized by vegetation. During succession, AM herbaceous vegetation is replaced by predominantly EcM woody species. To better understand the interaction of AM and EcM plants during vegetation transition, we studied the reciprocal effects of these species' coexistence on their root-associated fungi (RAF). We collected root and soil samples from three different microenvironments: stand of C. epigejos, under S. caprea canopy, and contact zone where roots of the two species interacted. RAF communities and mycorrhizal colonization were determined in sampled roots, and the soil was tested for EcM and AM inoculation potentials. Although the microenvironment significantly affected composition of the RAF communities in both plant species, the effect was greater in the case of C. epigejos RAF communities than in that of S. caprea RAF communities. The presence of S. caprea also significantly decreased AM fungal abundance in soil as well as AM colonization and richness of AM fungi in C. epigejos roots. Changes observed in the abundance and community composition of AM fungi might constitute an important factor in transition from AM-dominated to EcM-dominated vegetation during succession.

Inoculation effects on root-colonizing arbuscular mycorrhizal fungal communities spread beyond directly inoculated plants.

Inoculation with arbuscular mycorrhizal fungi (AMF) may improve plant performance at disturbed sites, but inoculation may also suppress root colonization by native AMF and decrease the diversity of the root-colonizing AMF community. This has been shown for the roots of directly inoculated plants, but little is known about the stability of inoculation effects, and to which degree the inoculant and the inoculation-induced changes in AMF community composition spread into newly emerging seedlings that were not in direct contact with the introduced propagules. We addressed this topic in a greenhouse experiment based on the soil and native AMF community of a post-mining site. Plants were cultivated in compartmented pots with substrate containing the native AMF community, where AMF extraradical mycelium radiating from directly inoculated plants was allowed to inoculate neighboring plants. The abundances of the inoculated isolate and of native AMF taxa were monitored in the roots of the directly inoculated plants and the neighboring plants by quantitative real-time PCR. As expected, inoculation suppressed root colonization of the directly inoculated plants by other AMF taxa of the native AMF community and also by native genotypes of the same species as used for inoculation. In the neighboring plants, high abundance of the inoculant and the suppression of native AMF were maintained. Thus, we demonstrate that inoculation effects on native AMF propagate into plants that were not in direct contact with the introduced inoculum, and are therefore likely to persist at the site of inoculation.

Real-time PCR quantification of arbuscular mycorrhizal fungi: does the use of nuclear or mitochondrial markers make a difference?

Root colonization by arbuscular mycorrhizal fungi (AMF) can be quantified by different approaches. We compared two approaches that enable discrimination of specific AMF taxa and are therefore emerging as alternative to most commonly performed microscopic quantification of AMF in roots: quantitative real-time PCR (qPCR) using markers in nuclear ribosomal DNA (nrDNA) and mitochondrial ribosomal DNA (mtDNA). In a greenhouse experiment, Medicago truncatula was inoculated with four isolates belonging to different AMF species (Rhizophagus irregularis, Claroideoglomus claroideum, Gigaspora margarita and Funneliformis mosseae). The AMF were quantified in the root samples by qPCR targeted to both markers, microscopy and contents of AMF-specific phospholipid fatty acids (PLFA). Copy numbers of nrDNA and mtDNA were closely related within all isolates; however, the slopes and intercepts of the linear relationships significantly differed among the isolates. Across all isolates, a large proportion of variance in nrDNA copy numbers was explained by root colonization intensity or contents of AMF-specific PLFA, while variance in mtDNA copy numbers was mainly explained by differences among AMF isolates. We propose that the encountered inter-isolate differences in the ratios of mtDNA and nrDNA copy numbers reflect different physiological states of the isolates. Our results suggest that nrDNA is a more suitable marker region than mtDNA for the quantification of multiple AMF taxa as its copy numbers are better related to fungal biomass across taxa than are copy numbers of mtDNA.

Arbuscular Mycorrhiza Stimulates Biological Nitrogen Fixation in Two Medicago spp. through Improved Phosphorus Acquisition.

Legumes establish root symbioses with rhizobia that provide plants with nitrogen (N) through biological N fixation (BNF), as well as with arbuscular mycorrhizal (AM) fungi that mediate improved plant phosphorus (P) uptake. Such complex relationships complicate our understanding of nutrient acquisition by legumes and how they reward their symbiotic partners with carbon along gradients of environmental conditions. In order to disentangle the interplay between BNF and AM symbioses in two Medicago species (Medicago truncatula and M. sativa) along a P-fertilization gradient, we conducted a pot experiment where the rhizobia-treated plants were either inoculated or not inoculated with AM fungus Rhizophagus irregularis 'PH5' and grown in two nutrient-poor substrates subjected to one of three different P-supply levels. Throughout the experiment, all plants were fertilized with 15N-enriched liquid N-fertilizer to allow for assessment of BNF efficiency in terms of the fraction of N in the plants derived from the BNF (%NBNF). We hypothesized (1) higher %NBNF coinciding with higher P supply, and (2) higher %NBNF in mycorrhizal as compared to non-mycorrhizal plants under P deficiency due to mycorrhiza-mediated improvement in P nutrition. We found a strongly positive correlation between total plant P content and %NBNF, clearly documenting the importance of plant P nutrition for BNF efficiency. The AM symbiosis generally improved P uptake by plants and considerably stimulated the efficiency of BNF under low P availability (below 10 mg kg-1 water extractable P). Under high P availability (above 10 mg kg-1 water extractable P), the AM symbiosis brought no further benefits to the plants with respect to P nutrition even as the effects of P availability on N acquisition via BNF were further modulated by the environmental context (plant and substrate combinations). As a response to elevated P availability in the substrate, the extent of root length colonization by AM fungi was reduced, the turning points occurring at about 8 and 10 mg kg-1 water extractable P for M. sativa and M. truncatula, respectively. Our results indicated competition for limited C resource between the two kinds of microsymbionts and thus degradation of AM symbiotic functioning under ample P supply.