Differential Responses of Arbuscular Mycorrhizal Fungal Communities to Long-Term Fertilization in the Wheat Rhizosphere and Root Endosphere.

Arbuscular mycorrhizal fungi (AMF) provide essential nutrients to crops and are critically impacted by fertilization in agricultural ecosystems. Understanding shifts in AMF communities in and around crop roots under different fertilization regimes can provide important lessons for improving agricultural production and sustainability. Here, we compared the responses of AMF communities in the rhizosphere (RS) and root endosphere (ES) of wheat (Triticum aestivum) to different fertilization treatments, nonfertilization (control), mineral fertilization only (NPK), mineral fertilization plus wheat straw (NPKS), and mineral fertilization plus cow manure (NPKM). We employed high-throughput amplicon sequencing and investigated the diversity, community composition, and network structure of AMF communities to assess their responses to fertilization. Our results elucidated that AMF communities in the RS and ES respond differently to fertilization schemes. Long-term NPK application decreased the RS AMF alpha diversity significantly, whereas additional organic amendments (straw or manure) had no effect. In contrast, NPK fertilization increased the ES AMF alpha diversity significantly, while additional organic amendments decreased it significantly. The effect of different fertilization schemes on AMF network complexity in the RS and ES were similar to their effects on alpha diversity. Changes to AMF communities in the RS and ES correlated mainly with the pH and phosphorus level of the rhizosphere soil under long-term inorganic and organic fertilization regimes. We suggest that the AMF community in the roots should be given more consideration when studying the effects of fertilization regimes on AMF in agroecosystems. IMPORTANCE Arbuscular mycorrhizal fungi are an integral component of rhizospheres, bridging the soil and plant systems and are highly sensitive to fertilization. However, surprisingly little is known about how the response differs between the roots and the surrounding soil. Decreasing arbuscular mycorrhizal fungal diversity under fertilization has been reported, implying a potential reduction in the mutualism between plants and arbuscular mycorrhizal fungi. However, we found opposing responses to long-term fertilization managements of arbuscular mycorrhizal fungi in the wheat roots and rhizosphere soil. These results suggested that changes in the arbuscular mycorrhizal fungal community in soils do not reflect those in the roots, highlighting that the root arbuscular mycorrhizal fungal community is pertinent to understand arbuscular mycorrhizal fungi and their crop hosts' responses to anthropogenic influences.

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.

Diversity and community structure of ectomycorrhizal fungi in Pinus thunbergii coastal forests bordering the Yellow Sea of China.

Ectomycorrhizas play a fundamental role in the function of forest ecosystems, being essential for plant nutrition absorption and soil quality. Many afforestation and reforestation programmes have begun to recover and maintain coastal forests in China, using pine species including Pinus thunbergii. We investigated the ectomycorrhizal colonization status of P. thunbergii in coastal pine forests of the Yellow Sea of China. We identified a total of 53 ectomycorrhizal fungal species in 74 soil samples collected from three sites and found that Thelephoraceae (10 spp.) and Russulaceae (8 spp.) were the most species-rich ectomycorrhizal fungal lineages. Russula sp. 1 was the most abundant species, accounting for 15.3% of the total ectomycorrhizal tips identified. Most of the remaining species were rare. At this small scale, host identity had no significant effect on the ectomycorrhizal fungal community composition (A = 0.036, P = 0.258), but sampling sites did (A = 0.135, P = 0.041). In addition, Na+ and K+ content and soil pH had significant effects on the ectomycorrhizal fungal community. The ectomycorrhizal fungal community associated with different host plants will become an important new direction for research, as ectomycorrhiza may have the potential to improve host capacity to establish in salt-stressed environments. This will provide a theoretical basis and technical support for saline soil reforestation and rehabilitation using pine species with compatible, native ectomycorrhizal fungi in Yellow Sea coastal areas.

Propagation and characterization of viable arbuscular mycorrhizal fungal spores within maize plant (Zea mays L.).

BACKGROUND: The harmful effect of chemical fertilizer application on human health and the environment as a modern method of meeting the food demand of the increasing world population demands an urgent alternative that is environmentally friendly, which will pose no harm to human health and the environment. Arbuscular mycorrhizal fungi (AMF) are beneficial soil microorganisms that provide various ecological functions in increasing soil fertility and enhancing plant growth. This present study aimed to propagate, characterize and examine the effect of viable arbuscular mycorrhizal fungal spores on maize (Zea mays L) hosts using molecular methods. The propagation of AMF in the host plant using sterile soil and vermiculite was conducted in the greenhouse. RESULT: The effect of AMF inoculation revealed a significant difference (P > 0.05) in maize growth, root colonization and AMF spore count when compared with the control. In all the parameters measured in this study, all the AMF spores propagated had a positive effect on the maize plant over the control, with the highest value mostly recorded in Rhizophagus irregularis AOB1. The molecular characterization of the spore using a specific universal primer for Glomeromycota established the success of the propagation process, which enhanced the classification of the AMF species into Rhizophagus irregularis OAB1, Glomus mosseae OAB2 and Paraglomus occultum OAB3. CONCLUSION: This finding will be a starting point in producing arbuscular mycorrhizal inoculum as a biofertilizer to enhance plant growth promotion. © 2021 Society of Chemical Industry.

Dispersal of Arbuscular Mycorrhizal Fungi: Evidence and Insights for Ecological Studies.

Dispersal is a critical ecological process that modulates gene flow and contributes to the maintenance of genetic and taxonomic diversity within ecosystems. Despite an increasing global understanding of the arbuscular mycorrhizal (AM) fungal diversity, distribution and prevalence in different biomes, we have largely ignored the main dispersal mechanisms of these organisms. To provide a geographical and scientific overview of the available data, we systematically searched for the direct evidence on the AM fungal dispersal agents (abiotic and biotic) and different propagule types (i.e. spores, extraradical hyphae or colonized root fragments). We show that the available data (37 articles) on AM fungal dispersal originates mostly from North America, from temperate ecosystems, from biotic dispersal agents (small mammals) and AM fungal spores as propagule type. Much lesser evidence exists from South American, Asian and African tropical systems and other dispersers such as large-bodied birds and mammals and non-spore propagule types. We did not find strong evidence that spore size varies across dispersal agents, but wind and large animals seem to be more efficient dispersers. However, the data is still too scarce to draw firm conclusions from this finding. We further discuss and propose critical research questions and potential approaches to advance the understanding of the ecology of AM fungi dispersal.

Bacillus subtilis CP4, isolated from native soil in combination with arbuscular mycorrhizal fungi promotes biofortification, yield and metabolite production in wheat under field conditions.

AIMS: The aim of this study was to identify the best combination of plant growth promoting bacteria (PGPB) and arbuscular mycorrhizal fungi (AMF) for biofortification and enhancing yield in wheat as well as improve soil health under field conditions. Another aim was to get insights into metabolite dynamics in plants treated with PGPB and AMF. METHODS AND RESULTS: Different combinations of PGPB and AMF that gave good results in greenhouse study were used in a field study. The combined application of Bacillus subtilis CP4 (native PGPB) and AMF gave the best results with a significant increase in biomass, macronutrient and micronutrient content in wheat grains and improvement in yield-related parameters relative to the untreated control. PGPB and AMF treatment increased antioxidant enzymes and compounds and decreased the level of an oxidation marker. Metabolite profiling performed using Gas Chromatography-Mass Spectrometry (GC-MS) showed significant upregulation of specific organic acids, amino acids, sugars and sugar alcohols in plants treated with CP4 and AMF. The altered pathways due to CP4 and AMF inoculation mainly belong to carbohydrate and amino acid metabolism. A positive correlation was observed between some organic acids, sugars and amino acids with wheat growth and yield parameters. The activities of soil enzymes increased significantly with the best results shown by native PGPB and AMF combination. CONCLUSIONS: A native bacterial isolate Bacillus subtilis CP4 in combination with AMF showed exceptional ability for biofortification and yield enhancement under field conditions. The upregulation of a number of metabolites showed correlation plant growth promotion and nutrients. SIGNIFICANCE AND IMPACT OF THE STUDY: The combined application of native B. subtilis CP4 and AMF could offer a more sustainable approach for the development of a biofertilizer to enhance wheat nutrient content and production and soil health thereby advancing agriculture.

New species of Tulasnella associated with Australian terrestrial orchids in the Cryptostylidinae and Drakaeinae.

Many orchids have an obligate relationship with Tulasnella mycorrhizal fungi for seed germination and support into adulthood. Despite the importance of Tulasnella as mycorrhizal partners, many species remain undescribed. Here, we use multiple sequence locus phylogenetic analyses to delimit and describe six new Tulasnella species associated with Australian terrestrial orchids from the subtribes Cryptostylidinae and Drakaeinae. Five of the new species, Tulasnella australiensis, T. occidentalis, T. punctata, T. densa, and T. concentrica, all associate with Cryptostylis (Cryptostylidinae), whereas T. rosea associates with Spiculaea ciliata (Drakaeinae). Isolates representing T. australiensis were previously also reported in association with Arthrochilus (Drakaeinae). All newly described Tulasnella species were delimited by phylogenetic analyses of four loci (nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 [ITS], C14436 [ATP synthase], C4102 [glutamate synthase], and mt 16S rDNA [mtLSU]). The pairwise sequence divergence between species for the ITS region ranged from 5.6% to 25.2%, and the maximum sequence divergence within the newly described species ranged from 1.64% to 4.97%. There was a gap in the distribution of within- and between-species pairwise divergences in the region of 4-6%, with only one within-species value of 4.97% (for two T. australiensis isolates) and one between-species value of 5.6% (involving an isolate of T. occidentalis) falling within this region. Based on fluorescence staining, all six new Tulasnella species are binucleate and have septate, cylindrical hyphae. There was some subtle variation in culture morphology, but colony diameter as measured on 3MN+vitamin medium after 6 wk of growth did not differ among species. However, T. australiensis grew significantly (P < 0.02) slower than others on ½ FIM and » potato dextrose agar (PDA) media. Formal description of these Tulasnella species contributes significantly to documentation of Tulasnella diversity and provides names and delimitations to underpin further research on the fungi and their relationships with orchids.

High-throughput sequencing analysis of the rhizosphere arbuscular mycorrhizal fungi (AMF) community composition associated with Ferula sinkiangensis.

BACKGROUND: Ferula sinkiangensis is an increasingly endangered medicinal plant. Arbuscular mycorrhiza fungi (AMF) are symbiotic microorganisms that live in the soil wherein they enhance nutrient uptake, stress resistance, and pathogen defense in host plants. While such AMF have the potential to contribute to the cultivation of Ferula sinkiangensis, the composition of AMF communities associated with Ferula sinkiangensis and the relationship between these fungi and other pertinent abiotic factors still remains to be clarified. RESULTS: Herein, we collected rhizosphere and surrounding soil samples at a range of depths (0-20, 20-40, and 40-60 cm) and a range of slope positions (bottom, middle, top). These samples were then subjected to analyses of soil physicochemical properties and high-throughput sequencing (Illumina MiSeq). We determined that Glomus and Diversispora species were highly enriched in all samples. We further found that AMF diversity and richness varied significantly as a function of slope position, with this variation primarily being tied to differences in relative Glomus and Diversispora abundance. In contrast, no significant relationship was observed between soil depth and overall AMF composition, although some AMF species were found to be sensitive to soil depth. Many factors significantly affected AMF community composition, including organic matter content, total nitrogen, total potassium, ammonium nitrogen, nitrate nitrogen, available potassium, total dissolvable salt levels, pH, soil water content, and slope position. We further determined that Shannon diversity index values in these communities were positively correlated with total phosphorus, nitrate-nitrogen levels, and pH values (P < 0.05), whereas total phosphorus, total dissolvable salt levels, and pH were positively correlated with Chao1 values (P < 0.05). CONCLUSION: In summary, our data revealed that Glomus and Diversispora are key AMF genera found within Ferula sinkiangensis rhizosphere soil. These fungi are closely associated with specific environmental and soil physicochemical properties, and these soil sample properties also differed significantly as a function of slope position (P < 0.05). Together, our results provide new insights regarding the relationship between AMF species and Ferula sinkiangensis, offering a theoretical basis for further studies of their development.

Isolation and identification of beneficial orchid mycorrhizal fungi in Bletilla striata (Thunb.) Rchb.f.(Orchidaceae).

Orchid mycorrhizal fungi (OMF) are essential for orchids growth. Bletilla striata (Thunb.) Rchb.f.(Orchidaceae) has high ornamental and medicinal value. Beneficial OMF isolation is crucial to improve the survival rate of B. striata tissue culture and transplanting. In this study, we isolated and identified the beneficial OMF in B. striata from the roots of sterilized wild B. striata seedlings by culturing in four different mediums. The germination states of B. striata seeds inoculated with diverse OMF were classified and calculated. Fresh and dry weight increments of B. striata seedlings inoculated with diverse OMF were recorded after 90 d of culturing on 1/2 MS medium. ITS sequences of beneficial fungi were amplified by PCR and taxonomically identified using BLAST against the GenBank nucleotide database. Ten kinds of OMF strains were isolated from B. striata and named R1 to R10. R6 significantly promoted B. striata seeds germination (p < .01). R3 and R6 significantly promoted both the fresh and dry weight increments of B. striata seedlings (p < .05). The ITS sequence of R6 was most similar to the sequence of Serendipita. R3 was identified as Schizothecium fimbriatum by 100% ITS identity. R6 and R3 were beneficial OMF in B. striata.

Molecular evidence supports simultaneous association of the achlorophyllous orchid Chamaegastrodia inverta with ectomycorrhizal Ceratobasidiaceae and Russulaceae.

BACKGROUND: Achlorophyllous orchids are mycoheterotrophic plants, which lack photosynthetic ability and associate with fungi to acquire carbon from different environmental sources. In tropical latitudes, achlorophyllous forest orchids show a preference to establish mycorrhizal relationships with saprotrophic fungi. However, a few of them have been recently found to associate with ectomycorrhizal fungi and there is still much to be learned about the identity of fungi associated with tropical orchids. The present study focused on mycorrhizal diversity in the achlorophyllous orchid C. inverta, an endangered species, which is endemic to southern China. The aim of this work was to identify the main mycorrhizal partners of C. inverta in different plant life stages, by means of morphological and molecular methods. RESULTS: Microscopy showed that the roots of analysed C. inverta samples were extensively colonized by fungal hyphae forming pelotons in root cortical cells. Fungal ITS regions were amplified by polymerase chain reaction, from DNA extracted from fungal mycelia isolated from orchid root samples, as well as from total root DNA. Molecular sequencing and phylogenetic analyses showed that the investigated orchid primarily associated with ectomycorrhizal fungi belonging to a narrow clade within the family Ceratobasidiaceae, which was previously detected in a few fully mycoheterotrophic orchids and was also found to show ectomycorrhizal capability on trees and shrubs. Russulaceae fungal symbionts, showing high similarity with members of the ectomycorrhizal genus Russula, were also identified from the roots of C. inverta, at young seedling stage. Ascomycetous fungi including Chaetomium, Diaporthe, Leptodontidium, and Phomopsis genera, and zygomycetes in the genus Mortierella were obtained from orchid root isolated strains with unclear functional role. CONCLUSIONS: This study represents the first assessment of root fungal diversity in the rare, cryptic and narrowly distributed Chinese orchid C. inverta. Our results provide new insights on the spectrum of orchid-fungus symbiosis suggesting an unprecedented mixed association between the studied achlorophyllous forest orchid and ectomycorrhizal fungi belonging to Ceratobasidiaceae and Russulaceae. Ceratobasidioid fungi as dominant associates in the roots of C. inverta represent a new record of the rare association between the identified fungal group and fully mycoheterotrophic orchids in nature.

Impact of land use history on the arbuscular mycorrhizal fungal diversity in arid soils of Argentinean farming fields.

Arbuscular mycorrhizal fungi (AMF) are a key soil functional group, with an important potential to increase crop productivity and sustainable agriculture including food security. However, there is clear evidence that land uses, crop rotations and soil features affect the AMF diversity and their community functioning in many agroecosystems. So far, the information related to AMF biodiversity in ecosystems like the Argentinean Puna, an arid high plateau where plants experience high abiotic stresses, is still scarce. In this work, we investigated morphological and molecular AMF diversity in soils of native corn, bean and native potato Andean crops, under a familiar land use, in Chaupi Rodeo (Jujuy, Argentina), without agrochemical supplements but with different histories of crop rotation. Our results showed that AMF morphological diversity was not only high and variable among the three different crop soils but also complemented by Illumina MiSeq data. The multivariate analyses highlighted that total fungal diversity is significantly affected by the preceding crop plants and the rotation histories, more than from the present crop species, while AMF communities are significantly affected by preceding crop only in combination with the effect of nitrogen and calcium soil concentration. This knowledge will give useful information on appropriate familiar farming.

The endangered Hastings River mouse (Pseudomys oralis) as a disperser of ectomycorrhizal fungi in eastern Australia.

Rodents are the most widespread and diverse order of vertebrate mycophagists and are key to the dispersal of mycorrhizal fungi. Rodents consume and subsequently disperse fungi through their feces on every continent except Antarctica. This study examines the fungal taxa consumed by the Hastings River mouse (Pseudomys oralis), an endangered Australian endemic rodent from the family Muridae. We analyzed 251 fecal samples collected over a 19-year period between 1993 and 2012 at sites throughout the distribution of the animal in New South Wales and Queensland. We show that at least 16 genera of mycorrhizal fungi are eaten by this species and that it is therefore playing an important role as a vector of ectomycorrhizal truffle-like fungi in eastern Australia. Similar to the fungal diets of other mammals in eastern Australia, seasonal fungal consumption was greatest in autumn and winter. The dietary diversity of P. oralis also appeared to follow a geographic trend from south to north; samples collected at sites in the southern part of the species' range had greater diversity than those from sites in the northern part of the range, and overall, diets from southern sites yielded more fungal taxa than did northern sites. This study provides novel insights into the diet of P. oralis and highlights the importance of previously overlooked ecosystem services this species provides through its dispersal of mycorrhizal fungi.

Into the wild blueberry (Vaccinium angustifolium) rhizosphere microbiota.

The ability of wild blueberries to adapt to their harsh environment is believed to be closely related to their symbiosis with ericoid mycorrhizal fungi, which produce enzymes capable of organic matter mineralization. Although some of these fungi have been identified and characterized, we still know little about the microbial ecology of wild blueberry. Our study aims to characterize the fungal and bacterial rhizosphere communities of Vaccinium angustifolium (the main species encountered in wild blueberry fields). Our results clearly show that the fungal order Helotiales was the most abundant taxon associated with V. angustifolium. Helotiales contains most of the known ericoid mycorrhizal fungi which are expected to dominate in such a biotope. Furthermore, we found the dominant bacterial order was the nitrogen-fixing Rhizobiales. The Bradyrhizobium genus, whose members are known to form nodules with legumes, was among the 10 most abundant genera in the bacterial communities. In addition, Bradyrhizobium and Roseiarcus sequences significantly correlated with higher leaf-nitrogen content. Overall, our data documented fungal and bacterial community structure differences in three wild blueberry production fields.

Histochemical Staining and Quantification of Arbuscular Mycorrhizal Fungal Colonization.

Histochemical staining and light microscopy-based techniques have been widely used to detect and quantify arbuscular mycorrhizal fungi (AMF) in roots. Here we describe a standardized method for staining of AMF in colonized roots, and we provide possible modifications to adjust the protocol according to particular requirements, such as the type of root material or the reduction of toxic products. In addition, we also summarize some of the most common ways to quantify arbuscular mycorrhizal colonization.

Fluorescent Staining of Arbuscular Mycorrhizal Structures Using Wheat Germ Agglutinin (WGA) and Propidium Iodide.

The colonization of a host plant root by arbuscular mycorrhizal (AM) fungi is a progressive process, characterized by asynchronous hyphal growth in intercellular and intracellular spaces, leading to the coexistence of diverse intraradical structures, such as hyphae, coils, arbuscules, and vesicles. In addition, the relative abundance of intercellular and intracellular fungal structures is highly dependent on root anatomy and the combination of plant and fungal species. Lastly, more than one fungal species may colonize the same root, adding a further level of complexity. For all these reasons, detailed imaging of a large number of samples is often necessary to fully assess the developmental processes and functionality of AM symbiosis. To this aim, the use of rapid and efficient staining methods that can be used routinely is crucial.We herein present a simple protocol to obtain high detail images of both overall intraradical fungal colonization pattern and fine morphology, in AM root sections of Lotus japonicus. The procedure is based on tissue clearing, fluorescent staining of fungal cell walls with fluorescein isothiocyanate-conjugated wheat germ agglutinin (FITC-WGA), and the combined counterstaining of plant cell walls with propidium iodide (PI). The resulting images can be acquired using traditional or confocal fluorescence microscopes and used for qualitative and quantitative analyses of fungal colonization, of particular interest for the comparison of mycorrhizal phenotypes between different experimental conditions or genetic backgrounds.

Molecular Quantification of Arbuscular Mycorrhizal Fungal Root Colonization.

Specific quantification of root-colonizing arbuscular mycorrhizal fungi (AMF) by quantitative real-time PCR is a high-throughput technique, most suitable for determining abundances of AMF species or isolates in previously characterized experimental systems. The principal steps are the choice and validation of an appropriate assay to specifically amplify a gene fragment of the target AMF, preparation of templates from root samples, and quantification of the fungal gene copy numbers in these templates. The use of a suitable assay is crucial for a correct data collection but also highly specific for each experimental system and is therefore covered by general recommendations. Subsequently, specific steps are described for the validation of the assay using a standard dilution series, the determination of appropriate dilutions of DNA extracts from roots, and the quantification of the gene copy numbers in samples including calculations.

Analysis of Arbuscular Mycorrhizal Fungal Communities by Terminal Restriction Fragment Length Polymorphism (TRFLP).

Terminal restriction fragment length polymorphism (TRFLP) approaches enable the detection and identification of microbial taxa into samples coming from root or soil material DNA extraction. The low taxonomic diversity of arbuscular mycorrhizal (AM) fungi makes this technique a cheap and adequate method for fingerprinting their communities. Here, I describe the TRFLP database approach, a version of the technique in which the AM fungal taxa present in the sample pool is identified for, later, match their presence in the different samples contained in the experiment. A final AM fungal operational taxonomic unit x sample presence-absence matrix is obtained, which allows the subsequent multivariate statistical analysis of the experimental results.

Laser Microdissection as a Useful Tool to Study Gene Expression in Plant and Fungal Partners in AM Symbiosis.

Laser microdissection (LMD) technology has been widely applied to plant tissues, offering novel information on the role of different cell-type populations during plant-microbe interactions. In this chapter, protocols to apply the LMD approach to study plant and fungal transcript profiles in different cell-type populations from arbuscular mycorrhizal (AM) roots are described in detail, starting from the biological material preparation to gene expression analyses by RT-PCR and RT-qPCR.

Detection of Arbuscular Mycorrhizal Fungal Gene Expression by In Situ Hybridization.

The complexity of the obligate symbiotic interaction of arbuscular mycorrhizal (AM) fungi and their host roots requires sophisticated molecular methods. In particular, to capture the dynamic of the interaction, cell-specific methods for gene expression analysis are required. In situ hybridization is a technique that allows to determine the location of transcript accumulation within tissues, being of special interest for these fungi that cannot be genetically modified. The method requires proper fixation and embedding methods as well as specific probes for the hybridization allowing detection of specific transcripts. In this chapter, we present a method to prepare roots, which have established a symbiosis with an arbuscular mycorrhizal fungus for the detection of fungal transcripts. This includes chemical fixation, subsequent embedding in a suitable medium, sectioning and pretreatment of sections, the hybridization procedure itself, as well as the immunological detection of RNA-RNA hybrids.

Isotope Labeling to Study Phosphorus Uptake in the Arbuscular Mycorrhizal Symbiosis.

Isotope labeling enables the detection and quantification of nutrient fluxes between soil and plants through arbuscular mycorrhizal (AM) fungi. Here we describe the use of radioactive isotopes, 33P and 32P, to study the uptake of P from soil by AM fungal mycelium and its transfer to the host plant through the mycorrhizal pathway.