Salicylic acid affects mycorrhizal features, antioxidant enzyme activities and seed yield of linseed under water-deficit stress in open-field conditions.

The research aims were to study salicylic acid (SA) effects on mycorrhiza [hyphal width (HW), vesicle diameter (VD) and mycorrhizal colonization (MC)] and interaction between them on greenness index (GI), drought tolerance index (DTI), antioxidant enzymes activities, and seed yield of linseed under drought. A factorial experiment was conducted in an open-field place with mycorrhiza [non-inoculation, Funneliformis mosseae (FM), and Rhizoglomus intraradices (RI)], SA (250 µM and non-SA), and irrigation levels [100%, 70%, and 40% field capacity (FC)] as treatments. Severe drought increased VD, MC, superoxide dismutase (SOD), ascorbate peroxidase (APX), and peroxidase activities while decreased GI, DTI, and yield. The RI-linseed had higher MC, GI, SOD, and glutathione reductase (GR) activities, but FM-linseed had greater VD and yield under drought. Inoculated linseed with both mycorrhiza showed a reduction in DTI and yield under SA than non-SA. In RI-linseed, SA increased GI, MC, HW, VD, catalase and GR, but decreased in FM-plants. Mycorrhiza (particularly RI) alleviated drought (40% FC)-caused negative effects on linseed via the improvement of SOD, APX, and GI. Regardless of other treatments, SA had negative effects on HW and VD, but SA effects varied depending on mycorrhizal species so that SA increased HW, VD, and MC in RI. Due to the positive correlation between MC and HW, SA reduces FM colonization by reducing the HW of FM. Totally, SA along with RI species can mitigate the harmful effects of drought and improve tolerance via increasing MC, HW, VD, catalase, peroxidase, and GR activities.

Stress amelioration response of glycine betaine and Arbuscular mycorrhizal fungi in sorghum under Cr toxicity.

Chromium toxicity is a major problem in agricultural soils that negatively affects a plant's metabolic activities. It reduces biochemical and antioxidant defence system's activities. In search of the solution to this problem a two-year pot experiment (completely randomized design with three replications), in three genetically different varieties of sorghum (SSG 59-3, HJ 513 and HJ 541) under Cr toxicity (2 and 4 ppm) was conducted to determine the effect of glycine betaine (50 and 100mM) and Arbuscular mycorrhizal fungi (AMF) on the antioxidant system (enzymes viz. superoxide dismutase, ascorbate peroxidase, catalase, glutathione reductase, peroxidase and metabolites viz. glutathione, ascorbate, proline, ß-carotene) along with Cr accumulation and indices of oxidative stress parameters (polyphenol oxidase, hydrogen peroxide and malondialdehyde) at two growth stages (vegetative and grain filling). According to results; Cr stress (2 & 4 ppm) increased its accumulation and indices of oxidative stresses significantly (p≤0.05) in all varieties of sorghum at both growth stages. However, soil application of glycine betaine (GB) and AMF decreased Cr accumulation and indices of oxidative stress by increasing antioxidant enzymes and metabolites activities at both growth stages in all varieties. The combination of 100mM GB with AMF was observed most significant (p≤0.05) in decreasing oxidative stress and improved the antioxidant system's activities. The SSG 59-3 cultivar showed the lowest Cr accumulation (1.60 and 8.61 ppm), indices of oxidative stress and highest antioxidant system's activity among these three cultivars at both growth stages. Thus, SSG 59-3 was found most tolerant cultivars followed by HJ 513 and then HJ 541. These findings suggest that both GB and AMF, either individually or combined can play a positive role to reduce oxidative stress and increased antioxidant attributes under Cr toxicity in sorghum.

Cascading effects of moth outbreaks on subarctic soil food webs.

The increasing severity and frequency of natural disturbances requires a better understanding of their effects on all compartments of biodiversity. In Northern Fennoscandia, recent large-scale moth outbreaks have led to an abrupt change in plant communities from birch forests dominated by dwarf shrubs to grass-dominated systems. However, the indirect effects on the belowground compartment remained unclear. Here, we combined eDNA surveys of multiple trophic groups with network analyses to demonstrate that moth defoliation has far-reaching consequences on soil food webs. Following this disturbance, diversity and relative abundance of certain trophic groups declined (e.g., ectomycorrhizal fungi), while many others expanded (e.g., bacterivores and omnivores) making soil food webs more diverse and structurally different. Overall, the direct and indirect consequences of moth outbreaks increased belowground diversity at different trophic levels. Our results highlight that a holistic view of ecosystems improves our understanding of cascading effects of major disturbances on soil food webs.

Comprehensive Phytotoxicity Assessment Protocol for Engineered Nanomaterials.

In order for nanotechnology to be sustainably applied in agriculture, emphasis should be on comprehensive assessment of multiple endpoints, including biouptake and localization of engineered nanomaterials (ENMs), potential effects on food nutrient quality, oxidative stress responses, and crop yield, before ENMs are routinely applied in consumer and agronomic products. This chapter succinctly outlines a protocol for conducting nanophytotoxicity studies focusing on nanoparticle purification and characterization, arbuscular mycorrhizal fungi (AMF)/symbiont inoculation, biouptake and translocation/localization, varied endpoints of oxidative stress responses, and crop yield.

Nitrogen and phosphorus fertilization consistently favor pathogenic over mutualistic fungi in grassland soils.

Ecosystems across the globe receive elevated inputs of nutrients, but the consequences of this for soil fungal guilds that mediate key ecosystem functions remain unclear. We find that nitrogen and phosphorus addition to 25 grasslands distributed across four continents promotes the relative abundance of fungal pathogens, suppresses mutualists, but does not affect saprotrophs. Structural equation models suggest that responses are often indirect and primarily mediated by nutrient-induced shifts in plant communities. Nutrient addition also reduces co-occurrences within and among fungal guilds, which could have important consequences for belowground interactions. Focusing only on plots that received no nutrient addition, soil properties influence pathogen abundance globally, whereas plant community characteristics influence mutualists, and climate influence saprotrophs. We show consistent, guild-level responses that enhance our ability to predict shifts in soil function related to anthropogenic eutrophication, which can have longer-term consequences for plant communities.

Evaluation of the Effect of Strigolactones and Synthetic Analogs on Fungi.

Strigolactones (SLs) are components of root exudates as a consequence of active release from the roots into the soil. Notably, they have been described as stimulants of seed germination in parasitic plants and of the presymbiotic growth in arbuscular mycorrhizal (AM) fungi, which are a crucial component of the plant root beneficial microbiota. SLs have therefore the potential to influence other microbes that proliferate in the soil around the roots and may interact with plants. A direct effect of SL analogs on the in vitro growth of a number of saprotrophic or plant pathogenic fungi was indeed reported.Here we describe a standardized method to evaluate the effect of SLs or their synthetic analogs on AM and filamentous fungi. For AM fungi, we propose a spore germination assay since it is more straightforward than the hyphal branching assay and it does not require deep expertise and skills. For filamentous fungi that can grow in axenic cultures, we describe the assay based on SLs embedded in the solid medium or dissolved in liquid cultures where the fungus is inoculated to evaluate the effect on growth, hyphal branching or conidia germination. These assays are of help to test the activity of natural SLs as well as of newly designed SL analogs for basic and applied research.

Controlled Assays for Phenotyping the Effects of Strigolactone-Like Molecules on Arbuscular Mycorrhiza Development.

Arbuscular mycorrhiza is an ancient symbiosis between most land plants and fungi of the Glomeromycotina, in which the fungi provide mineral nutrients to the plant in exchange for photosynthetically fixed organic carbon. Strigolactones are important signals promoting this symbiosis, as they are exuded by plant roots into the rhizosphere to stimulate activity of the fungi. In addition, the plant karrikin signaling pathway is required for root colonization. Understanding the molecular mechanisms underpinning root colonization by AM fungi, requires the use of plant mutants as well as treatments with different environmental conditions or signaling compounds in standardized cocultivation systems to allow for reproducible root colonization phenotypes. Here we describe how we set up and quantify arbuscular mycorrhiza in the model plants Lotus japonicus and Brachypodium distachyon under controlled conditions. We illustrate a setup for open pot culture as well as for closed plant tissue culture (PTC) containers, for plant-fungal cocultivation in sterile conditions. Furthermore, we explain how to harvest, store, stain, and image AM roots for phenotyping and quantification of different AM structures.

Pb Stress and Ectomycorrhizas: Strong Protective Proteomic Responses in Poplar Roots Inoculated with Paxillus involutus Isolate and Characterized by Low Root Colonization Intensity.

The commonly observed increased heavy metal tolerance of ectomycorrhized plants is usually linked with the protective role of the fungal hyphae covering colonized plant root tips. However, the molecular tolerance mechanisms in heavy metal stressed low-colonized ectormyocrrhizal plants characterized by an ectomycorrhiza-triggered increases in growth are unknown. Here, we examined Populus × canescens microcuttings inoculated with the Paxillus involutus isolate, which triggered an increase in poplar growth despite successful colonization of only 1.9% ± 0.8 of root tips. The analyzed plants, lacking a mantle-a protective fungal biofilter-were grown for 6 weeks in agar medium enriched with 0.75 mM Pb(NO3)2. In minimally colonized 'bare' roots, the proteome response to Pb was similar to that in noninoculated plants (e.g., higher abundances of PM- and V-type H+ ATPases and lower abundance of ribosomal proteins). However, the more intensive activation of molecular processes leading to Pb sequestration or redirection of the root metabolic flux into amino acid and Pb chelate (phenolics and citrate) biosynthesis coexisted with lower Pb uptake compared to that in controls. The molecular Pb response of inoculated roots was more intense and effective than that of noninoculated roots in poplars.

Health-Promoting Compounds in Stevia: The Effect of Mycorrhizal Symbiosis, Phosphorus Supply and Harvest Time.

This work aimed to establish the synergic role of arbuscular mycorrhizal fungi (AMF) symbiosis, phosphorus (P) fertilization and harvest time on the contents of stevia secondary metabolites. Consequently, steviol glycosides (SVglys) concentration and profile, total phenols and flavonoids as well as antioxidant assays, have been assessed in inoculated and no-inoculated plants, grown with or without P supply and collected at different growth stages(69, 89 and 123 days after transplanting).The obtained results suggest that the synthesis of stevia secondary metabolites is induced and/or modulated by all the investigated variability factors. In particular, AMF symbiosis promoted total SVglys content and positively influenced the concentration of some minor compounds (steviolbioside, dulcoside A and rebaudioside B), indicating a clear effect of mycorrhizal inoculation on SVglys biosynthetic pathway. Interestingly, only the mycorrhizal plants were able to synthesize rebaudioside B. In addition, P supply provided the highest levels of total phenols and flavonoids at leaf level, together with the maximum in vitro antioxidant activities (FRAP and ORAC). Finally, the harvest time carried out during the full vegetative phase enhanced the entire composition of the phytocomplex (steviolbioside, dulcoside A, stevioside, rebaudioside A, B, C. total phenols and flavonoids). Moreover, polyphenols and SVglys appeared to be the main contributors to the in vitro antioxidant capacity, while only total phenols mostly contributed to the cellular antioxidant activity (CAA). These findings provide original information about the role played by AMF in association with P supply, in modulating the accumulation of bioactive compounds during stevia growth. At the cultivation level, the control of these preharvest factors, together with the most appropriate harvest time, can be used as tools for improving the nutraceutical value of raw material, with particular attention to its exploitation as functional ingredient for food and dietary supplements and cosmetics.

Symbiotic and Asymbiotic Germination of Dendrobium officinale (Orchidaceae) Respond Differently to Exogenous Gibberellins.

Seeds of almost all orchids depend on mycorrhizal fungi to induce their germination in the wild. The regulation of this symbiotic germination of orchid seeds involves complex crosstalk interactions between mycorrhizal establishment and the germination process. The aim of this study was to investigate the effect of gibberellins (GAs) on the symbiotic germination of Dendrobium officinale seeds and its functioning in the mutualistic interaction between orchid species and their mycobionts. To do this, we used liquid chromatograph-mass spectrometer to quantify endogenous hormones across different development stages between symbiotic and asymbiotic germination of D. officinale, as well as real-time quantitative PCR to investigate gene expression levels during seed germination under the different treatment concentrations of exogenous gibberellic acids (GA3). Our results showed that the level of endogenous GA3 was not significantly different between the asymbiotic and symbiotic germination groups, but the ratio of GA3 and abscisic acids (ABA) was significantly higher during symbiotic germination than asymbiotic germination. Exogenous GA3 treatment showed that a high concentration of GA3 could inhibit fungal colonization in the embryo cell and decrease the seed germination rate, but did not significantly affect asymbiotic germination or the growth of the free-living fungal mycelium. The expression of genes involved in the common symbiotic pathway (e.g., calcium-binding protein and calcium-dependent protein kinase) responded to the changed concentrations of exogenous GA3. Taken together, our results demonstrate that GA3 is probably a key signal molecule for crosstalk between the seed germination pathway and mycorrhiza symbiosis during the orchid seed symbiotic germination.

Effects of sodium sulfide application on the growth of Robinia pseudoacacia, heavy metal immobilization, and soil microbial activity in Pb-Zn polluted soil.

Sodium sulfide (Na2S) is usually used as an amendment in industrial sewage treatment. To evaluate the effects of Na2S on the growth of Robinia pseudoacacia (black locust), heavy metal immobilization, and soil microbial activity, the R. pseudoacacia biomass and nutrient content and the soil heavy metal bioavailability, enzyme activity, and arbuscular mycorrhizal (AM) fungal community were measured by a single-factor pot experiment. The Pb-Zn-contaminated soil was collected from a Pb-Zn mine that had been remediated by R. pseudoacacia for five years. Three pollution levels (unpolluted, mildly polluted, and severely polluted) were evaluated by the pollution load index. Na2S application increased the shoot biomass under severe and mild contamination. In soil, Na2S application decreased the bioavailable Pb and Zn contents under severe and mild contamination, which resulted in a decrease in the Pb and Zn content in R. pseudoacacia. However, Na2S application did not affect the total Pb content per plant and enhanced the total Zn content per plant because of the higher biomass of the plants under Na2S application. Increased phosphatase activity and increased available phosphorous content may promote the uptake of phosphorus in R. pseudoacacia. Moreover, Na2S application is beneficial to the diversity of AM fungi under mild and severe pollution. Overall, Na2S application has great potential for enhancing soil heavy metal immobilization, enhancing soil microbial activity, and improving the growth of R. pseudoacacia in polluted soils. Therefore, Na2S is suitable for use in Pb-Zn remediation to ameliorate environmental heavy metal pollution.

Global metabolic changes induced by arbuscular mycorrhizal fungi in oregano plants grown under ambient and elevated levels of atmospheric CO2.

Symbiotic plant-microorganisms interaction is a promising approach to avoid the environmental hazards of synthetic fertilizers and pesticides. Among these, arbuscular mycorrhizal fungi (AMF) are known to improve the growth and quality of many plant species; however the detailed metabolic mechanisms behind such beneficial effects are far from complete. Further, elevated levels of atmospheric CO2 (eCO2) could affect such AMF-plant association. Herein, we have investigated the individual and synchronous impact of AMF and eCO2 (620 ppm) on nutrient uptake, growth, photosynthesis, respiration, and levels of primary and secondary metabolites in oregano (Oreganum vulgare), an economically important herbal plant. Enhanced AMF colonization rate and a better mycelial growth were observed in roots of oregano grown under eCO2. Both AMF and eCO2 treatments significantly enhanced the growth and photosynthesis of oregano plants, however much improvements were observed by their synchronous application. eCO2 further increased the AMF-induced dark respiration and accumulation of macro and microelements. Hierarchical clustering analysis of individual primary and secondary metabolites revealed a metabolite-dependent response toward AMF and eCO2. The synchronous application of AMF and eCO2 resulted in promoted accumulation of the majority of the detected sugars, organic acids, amino acids, unsaturated fatty acids, phenolic acids and flavonoids, as compared with the sole treatments. Moreover, AMF and eCO2 acted synergistically in improving the antioxidant capacity and anti-lipid peroxidation activity of oregano. Therefore, this study suggests that AMF treatment induces a global metabolic change in oregano, the effect that is strengthened under eCO2.

Residual effects of biochar and phosphorus on growth and nutrient accumulation by maize (Zea mays L.) amended with microbes in texturally different soils.

The purpose of study was to examine the residual effects of two types of biochar amendments, two phosphorus (P) fertilizer levels, phosphorus solubilizing bacteria (PSB) and arbuscular mycorrhizal fungs (AMF) on plant growth, nutrients absorption and root architecture of Zea mays L. in texturally different soils. Biochar signficantly increased nutrients absorption and plant biomass production with P-fertilization and microbial inoculantion. Texturally different soils enhanced the plant biomass and nutrients absorption in their independent capacity on addition of biochar, microbial inoculants and P-fertilization. It was shown that mycorrhizal inoculation had positive influence on plant root and shoot biomass in both soils irrespective to the biochar type used. Root colonization was notably increased in biochar + mycorrhizae (B + M) inocultaed plants. It was shown that mycorrhizal inoculation had positive influence on nutrients absorption by plant roots and it had high content of P, potassium, calcium and magnesium in plants at all biochar and P levels. Without P fertilization, biochar amendments significantly promoted shoot P content and root colonization. The P application significantly influenced soil microbial activity in terms of nutrient concentration and plant growth. Root attributes were significantly inclined by microbial inoculation. Residual effects of biochar and P significantly enhanced the nutreints absorption and maize plant growth. Thus, we concluded that residual biochar and P fertilizer showed positive effects on nutrients absorption and maize plant growth promotion in differently textured soils. Microbial inoculants further stimulated the plant biomass production and nutrients absorption due to effective root colonization.

Arsenic toxicity and its mitigation in ectomycorrhizal fungus Hebeloma cylindrosporum through glutathione biosynthesis.

Arsenic (As) contamination is one of the most daunting environmental problem bothering the whole world. Exploring a suitable bioremediation technique is an urgent need of the hour. The present study focusses on scrutinizing the ectomycorrhizal (ECM) fungus for its potential role in As detoxification and understanding the molecular mechanisms responsible for its tolerance. When exposed to increasing concentrations of external As, the ECM fungus H. cylindrosporum accumulated the metalloid intracellularly, inducing the glutathione biosynthesis pathway. The genes coding for GSH biosynthesis enzymes, γ-glutamylcysteine synthetase (Hcγ-GCS) and glutathione synthetase (HcGS) were highly regulated by As stress. Arsenic coordinately upregulated the expression of both Hcγ-GCS and HcGS genes, thus resulting in increased Hcγ-GCS and HcGS protein expressions and enzyme activities, with substantial increase in intracellular GSH. Functional complementation of the two genes (Hcγ-GCS and HcGS) in their respective yeast mutants (gsh1Δ and gsh2Δ) further validated the role of both enzymes in mitigating As toxicity. These findings clearly highlight the potential importance of GSH antioxidant defense system in regulating the As induced responses and its detoxification in ECM fungus H. cylindrosporum.

Gibberellin Promotes Fungal Entry and Colonization during Paris-Type Arbuscular Mycorrhizal Symbiosis in Eustoma grandiflorum.

Arbuscular mycorrhizas (AMs) are divided into two types according to morphology: Arum- and Paris-type AMs. Gibberellins (GAs) mainly inhibit the establishment of Arum-type AM symbiosis in most model plants, whereas the effects of GAs on Paris-type AM symbiosis are unclear. To provide insight into the mechanism underlying this type of symbiosis, the roles of GAs were investigated in Eustoma grandiflorum when used as the host plant for Paris-type AM establishment. Eustoma grandiflorum seedlings were inoculated with the model AM fungus, Rhizophagus irregularis, and the effects of GA and the GA biosynthesis inhibitor uniconazole-P on the symbiosis were quantitatively evaluated. Exogenous GA significantly increased hyphopodium formation at the epidermis, thus leading to the promotion of fungal colonization and arbuscule formation in the root cortex. By contrast, the suppression of GA biosynthesis and signaling attenuated fungal entry to E. grandiflorum roots. Moreover, the exudates from GA-treated roots strongly induced the hyphal branching of R. irregularis. Our results show that GA has an contrasting effect on Paris-type AM symbiosis in E. grandiflorum compared with Arum-type AM symbiosis. This finding could be explained by the differential regulation of the early colonization stage, where fungal hyphae make contact with and penetrate the epidermis.

Understanding interaction effect of arbuscular mycorrhizal fungi in rice under elevated carbon dioxide conditions.

Arbuscular mycorrhizal fungi (AMF), particularly the Glomerales group, play a paramount role in plant nutrient uptake, and abiotic and biotic stress management in rice, but recent evidence revealed that elevated CO2 concentration considerably reduces the Glomerales group in soil. In view of this, the present study was initiated to understand the interaction effect of native Glomerales species application in rice plants (cv. Naveen) under elevated CO2 concentrations (400 ± 10, 550 ± 20, and 700 ± 20 ppm) in open-top chambers. Three different modes of application of the AMF inoculum were evaluated, of which, combined application of AMF at the seedling production and transplanting stages showed increased AMF colonization, which significantly improved grain yield by 25.08% and also increased uptake of phosphorus by 18.2% and nitrogen by 49.5%, as observed at 700-ppm CO2 concentration. Organic acids secretion in rice root increased in AMF-inoculated plants exposed to 700-ppm CO2 concentration. To understand the overall effect of CO2 elevation on AMF interaction with the rice plant, principal component and partial least square regression analysis were performed, which found both positive and negative responses under elevated CO2 concentration.

Brachypodium distachyon tar2lhypo mutant shows reduced root developmental response to symbiotic signal but increased arbuscular mycorrhiza.

Auxin is a major phytohormone that controls root development. A role for auxin is also emerging in the control of plant-microbe interactions, including for the establishment of root endosymbiosis between plants and arbuscular mycorrhizal fungi (AMF). Auxin perception is important both for root colonization by AMF and for arbuscule formation. AMF produce symbiotic signals called lipo-chitooligosaccharides (LCOs) that can modify auxin homeostasis and promote lateral root formation (LRF). Since Brachypodium distachyon (Brachypodium) has a different auxin sensitivity compared to other plant species, we wondered whether this would interfere with the effect of auxin in arbuscular mycorrhizal (AM) symbiosis. Here we tested whether tar2lhypo a Brachypodium mutant with an increase in endogenous auxin content is affected in LRF stimulation by LCOs and in AM symbiosis. We found that, in contrast to control plants, LCO treatment inhibited LRF of the tar2lhypo mutant. However, the level of AMF colonization and the abundance of arbuscules were increased in tar2lhypo compared to control plants, suggesting that auxin also plays a positive role in both AMF colonization and arbuscule formation in Brachypodium.

Rice straw biochar and phosphorus inputs have more positive effects on the yield and nutrient uptake of Lolium multiflorum than arbuscular mycorrhizal fungi in acidic Cd-contaminated soils.

The purpose of the study was to examine biochar amendment, phosphorus (P) fertilizer and arbuscular mycorrhizal fungi (AMF) on the yield, nutrient and cadmium (Cd) absorption of Lolium multiflorum in acidic soil. It was shown that mycorrhizal inoculation had no positive influence on the plant shoot biomass and the contents of nitrogen (N), P, potassium (K), calcium (Ca) and magnesium (Mg) in plants at all biochar and P level treatments. Irrespective of mycorrhizal inoculation and P level, biochar amendments markedly elevated the soil available P and K uptake in plant tissues. In contrast, biochar significantly decreased the translocation factor of plants, soil exchangeable Cd, and acid and neutral phosphatase activities, regardless of the mycorrhizal inoculation and P fertilizer. Without P fertilization, biochar amendments significantly promoted shoot P content, while biochar amendments significantly reduced shoot P content when P fertilizer was applied. Without biochar application, P fertilizer application significantly promoted the biomass and N uptake of shoots in both AMF inoculation treatments, while P fertilizer increased these only in the presence of biochar and mycorrhizal inoculation. The increased N content induced by the biochar amendment elevated the shoot N:P ratio and alleviated the N deficiency with P fertilizer input. Thus, we concluded that the addition of biochar and P fertilizer showed more positive effects on the promotion of growth and nutrient uptake of L. multiflorum than AMF grown in acidic Cd-contaminated soils.

Impacts of long-term elevated atmospheric CO2 concentrations on communities of arbuscular mycorrhizal fungi.

The ecological impacts of long-term elevated atmospheric CO2 (eCO2 ) levels on soil microbiota remain largely unknown. This is particularly true for the arbuscular mycorrhizal (AM) fungi, which form mutualistic associations with over two-thirds of terrestrial plant species and are entirely dependent on their plant hosts for carbon. Here, we use high-resolution amplicon sequencing (Illumina, HiSeq) to quantify the response of AM fungal communities to the longest running (>15 years) free-air carbon dioxide enrichment (FACE) experiment in the Northern Hemisphere (GiFACE); providing the first evaluation of these responses from old-growth (>100 years) semi-natural grasslands subjected to a 20% increase in atmospheric CO2 . eCO2 significantly increased AM fungal richness but had a less-pronounced impact on the composition of their communities. However, while broader changes in community composition were not observed, more subtle responses of specific AM fungal taxa were with populations both increasing and decreasing in abundance in response to eCO2 . Most population-level responses to eCO2 were not consistent through time, with a significant interaction between sampling time and eCO2 treatment being observed. This suggests that the temporal dynamics of AM fungal populations may be disturbed by anthropogenic stressors. As AM fungi are functionally differentiated, with different taxa providing different benefits to host plants, changes in population densities in response to eCO2 may significantly impact terrestrial plant communities and their productivity. Thus, predictions regarding future terrestrial ecosystems must consider changes both aboveground and belowground, but avoid relying on broad-scale community-level responses of soil microbes observed on single occasions.

Stimulation of asymbiotic sporulation in arbuscular mycorrhizal fungi by fatty acids.

Arbuscular mycorrhizal (AM) fungi are obligate symbionts that depend on living host plants to complete their life cycle1,2. This feature, which leads to their unculturability in the absence of plants, strongly hinders basic research and agricultural application of AM fungi. However, at least one AM fungus can grow and develop fertile spores independently of a host plant in co-culture with the bacterium Paenibacillus validus3. The bacteria-derived substances are thought to act as stimulants or nutrients for fungal sporulation, but these molecules have not been identified. Here, we show that (S)-12-methyltetradecanoic acid4,5, a methyl branched-chain fatty acid isolated from bacterial cultures, stimulates the branching of hyphae germinated from mother spores and the formation of secondary spores in axenic culture of the AM fungus Rhizophagus irregularis. Extensive testing of fatty acids revealed that palmitoleic acid induces more secondary spores than the bacterial fatty acid in R. irregularis. These induced spores have the ability to infect host plant roots and to generate daughter spores. Our work shows that, in addition to a major source of organic carbon6-9, fatty acids act as stimulants to induce infection-competent secondary spores in the asymbiotic stage and could provide the key to developing the axenic production of AM inoculum.