Towards growth of arbuscular mycorrhizal fungi independent of a plant host

When surface-sterilized spores of the arbuscular mycorrhizal fungus (AMF) Glomus intraradices Sy167 were germinated on agar plates in the slightly modified minimum mineral medium described by G. Bécard and J. A. Fortin (New Phytol. 108:211-218, 1988), slime-forming bacteria, identified as Paenibacillus validus, frequently grew up. These bacteria were able to support growth of the fungus on the agar plates. In the presence of P. validus, hyphae branched profusely and formed coiled structures. These were much more densely packed than the so-called arbuscule-like structures which are formed by AMF grown in coculture with carrot roots transformed with T-DNA from Agrobacterium rhizogenes. The presence of P. validus alone also enabled G. intraradices to form new spores, mainly at the densely packed hyphal coils. The new spores were not as abundant as and were smaller than those formed by AMF in the monoxenic culture with carrot root tissues, but they also contained lipid droplets and a large number of nuclei. In these experiments P. validus could not be replaced by bacteria such as Escherichia coli K-12 or Azospirillum brasilense Sp7. Although no conditions under which the daughter spores regerminate and colonize plants have been found yet, and no factor(s) from P. validus which stimulates fungal growth has been identified, the present findings might be a significant step forward toward growth of AMF independent of any plant host.     

Phosphate uptake, transport and transfer by the arbuscular mycorrhizal fungus Glomus intraradices is stimulated by increased carbohydrate availability

* The influence of carbohydrate availability to mycorrhizal roots on uptake, metabolism and translocation of phosphate (P) by the fungus was examined in axenic cultures of transformed carrot (Daucus carota) roots in symbiosis with Glomus intraradices. * 14C-labelled carbohydrates, 33P-phosphate and energy dispersive X-ray microanalysis were used to follow the uptake and transfer of C and P in the arbuscular mycorrhizal (AM) symbiosis. * The uptake of P by the extraradical mycelium (ERM) and its translocation to the mycorrhizal roots was stimulated and the metabolic and spatial distribution of P within the fungus were altered in response to increased carbohydrate availability. Sucrose supply resulted in a decrease of polyphosphates and an increased incorporation into phospholipids and other growth-related P pools and also caused elevated cytoplasmic P levels in the intraradical mycelium (IRM) within the root and higher cytoplasmic P levels in the root cortex. * These findings indicate that the uptake of P by the fungus and its transfer to the host is also stimulated by the transfer of carbon from plant to fungus across the mycorrhizal interface.     

A phosphate transporter gene from the extra-radical mycelium of an arbuscular mycorrhizal fungus Glomus intraradices is regulated in response to phosphate in the environment

The majority of vascular flowering plants are able to form symbiotic associations with arbuscular mycorrhizal fungi. These symbioses, termed arbuscular mycorrhizas, are mutually beneficial, and the fungus delivers phosphate to the plant while receiving carbon. In these symbioses, phosphate uptake by the arbuscular mycorrhizal fungus is the first step in the process of phosphate transport to the plant. Previously, we cloned a phosphate transporter gene involved in this process. Here, we analyze the expression and regulation of a phosphate transporter gene (GiPT) in the extra-radical mycelium of the arbuscular mycorrhizal fungus Glomus intraradices during mycorrhizal association with carrot or Medicago truncatula roots. These analyses reveal that GiPT expression is regulated in response to phosphate concentrations in the environment surrounding the extra-radical hyphae and modulated by the overall phosphate status of the mycorrhiza. Phosphate concentrations, typical of those found in the soil solution, result in expression of GiPT. These data imply that G. intraradices can perceive phosphate levels in the external environment but also suggest the presence of an internal phosphate sensing mechanism.     

Mucoid mutants of the biocontrol strain pseudomonas fluorescens CHA0 show increased ability in biofilm formation on mycorrhizal and nonmycorrhizal carrot roots

Extracellular polysaccharides play an important role in the formation of bacterial biofilms. We tested the biofilm-forming ability of two mutant strains with increased production of acidic extracellular polysaccharides compared with the wild-type biocontrol strain Pseudomonas fluorescens CHA0. The anchoring of bacteria to axenic nonmycorrhizal and mycorrhizal roots as well as on extraradical mycelium of the arbuscular mycorrhizal fungus Glomus intraradices was investigated. The nonmucoid wild-type strain P. fluorescens CHA0 adhered very little on all surfaces, whereas both mucoid strains formed a dense and patchy bacterial layer on the roots and fungal structures. Increased adhesive properties of plant-growth-promoting bacteria may lead to more stable interactions in mixed inocula and the rhizosphere.     

Branched absorbing structures (BAS): a feature of the extraradical mycelium of symbiotic arbuscular mycorrhizal fungi

The present work describes the morphogenesis and cytological characteristics of 'branched absorbing structures' (BAS, formely named arbuscule-like structures, ALS), small groups of dichotomous hyphae formed by the extraradical mycelium of arbuscular mycorrhizal (AM) fungi. Monoxenic cultures of the AM fungus Glomus intraradices Smith & Schenck and tomato ( Lycopersicum esculentum Mill.) roots allowed the continuous, non-destructive study of BAS development. These structures were not observed in axenic cultures of the fungus under different nutritional conditions or in unsuccessful (asymbiotic) monoxenic cultures. However, extraradical mycelium of G. intraradices formed BAS immediately after fungal penetration of the host root and establishment of the symbiosis. The average BAS development time was 7 d under our culture conditions, after which they degenerated, becoming empty septate structures. Certain BAS were closely associated with spore formation, appearing at the spore's substending hypha. Branches of these spore-associated BAS (spore-BAS) usually formed spores. Electron microscopy studies revealed that BAS and arbuscules show several ultrastructural similarities. The possible role of BAS in nutrient uptake by the mycorrhizal plant is discussed.     

Temperature constraints on the growth and functioning of root organ cultures with arbuscular mycorrhizal fungi

In this study we investigated the effects of temperature on fungal growth and tested whether the differences in fungal growth were related to the effects of temperature on carbon movement to, or within, the fungus. Growth curves and C uptake-transfer-translocation measurements were obtained for three arbuscular mycorrhizal fungi (AMF) isolates cultured within a 6-30 degrees C temperature range. A series of experiments with a model fungal isolate, Glomus intraradices, was used to examine the effects of temperature on lipid body and 33P movement, and to investigate the role of acclimation and incubation time. Temperature effects on AMF growth were both direct and indirect because, despite clear independent root and AMF growth responses in some cases, the uptake and translocation of 13C was also affected within the temperature range tested. Root C uptake and, to a lesser extent, C translocation in the fungus, were reduced by low temperatures (< 18 degrees C). Uptake and translocation of 33P by fungal hyphae were, by contrast, similar between 10 and 25 degrees C. We conclude that temperature, between 6 and 18 degrees C, reduces AMF growth, and that C movement to the fungus is involved in this response.     

Tit for tat? A mycorrhizal fungus accumulates phosphorus under low plant carbon availability

The exchange of carbohydrates and mineral nutrients in the arbuscular mycorrhizal (AM) symbiosis must be controlled by both partners in order to sustain an evolutionarily stable mutualism. Plants downregulate their carbon (C) flow to the fungus when nutrient levels are sufficient, while the mechanism controlling fungal nutrient transfer is unknown. Here, we show that the fungus accumulates nutrients when connected to a host that is of less benefit to the fungus, indicating a potential of the fungus to control the transfer of nutrients. We used a monoxenic in vitro model of root organ cultures associated with Glomus intraradices, in which we manipulated the C availability to the plant. We found that G. intraradices accumulated up to seven times more nutrients in its spores, and up to nine times more in its hyphae, when the C pool available to the associated roots was halved. The strongest effect was found for phosphorus (P), considered to be the most important nutrient in the AM symbiosis. Other elements such as potassium and chorine were also accumulated, but to a lesser extent, while no accumulation of iron or manganese was found. Our results suggest a functional linkage between C and P exchange.     

Evidence for specificity of cultivable bacteria associated with arbuscular mycorrhizal fungal spores

Bacteria associated with arbuscular mycorrhizal (AM) fungal spores may play functional roles in interactions between AM fungi, plant hosts and defence against plant pathogens. To study AM fungal spore-associated bacteria (AMB) with regard to diversity, source effects (AM fungal species, plant host) and antagonistic properties, we isolated AMB from surface-decontaminated spores of Glomus intraradices and Glomus mosseae extracted from field rhizospheres of Festuca ovina and Leucanthemum vulgare. Analysis of 385 AMB was carried out by fatty acid methyl ester (FAME) profile analysis, and some also identified using 16S rRNA gene sequence analysis. The AMB were tested for capacity to inhibit growth in vitro of Rhizoctonia solani and production of fluorescent siderophores. Half of the AMB isolates could be identified to species (similarity index 0.6) within 16 genera and 36 species. AMB were most abundant in the genera Arthrobacter and Pseudomonas and in a cluster of unidentified isolates related to Stenotrophomonas. The AMB composition was affected by AM fungal species and to some extent by plant species. The occurrence of antagonistic isolates depended on AM fungal species, but not plant host, and originated from G. intraradices spores. AM fungal spores appear to host certain sets of AMB, of which some can contribute to resistance by AM fungi against plant pathogens.     

Increased spore production by Glomus intraradices in the split-plate monoxenic culture system by repeated harvest,gel replacement,and resupply of glucose to the mycorrhiza

Monoxenic culture of Glomus intraradices Schenck and Smith with Ri T-DNA transformed roots in two-compartment Petri dishes is a very useful technique for physiological studies and the production of clean fungal tissues. Experiments were conducted to increase the efficiency of this method for the production of arbuscular mycorrhizal fungus spores. Approximately 20,000 spores could be harvested every 2 months from the distal (fungus only) compartment of a 9-cm-diameter divided Petri dish. The method requires replacement of the gelled media in the distal compartment and resupply of 200 mg glucose to the proximal (root) compartment coincident with harvest of spores. These modifications resulted in an approximate threefold increase in spore production per unit time over the standard split-plate culture technique.     

Phosphorus availability influences elemental uptake in the mycorrhizal fungus Glomus intraradices, as revealed by particle-induced X-ray emission analysis

We investigated element accumulation in the arbuscular mycorrhizal fungus Glomus intraradices. Fungal spores and mycelia growing in monoxenic cultures were analyzed. The elemental composition was quantified using particle-induced X-ray emission (PIXE) in combination with scanning transmission ion microscopy. In the spores, Ca and Fe were associated mainly with the spore wall, while P and K showed patchy distributions and their concentrations were correlated. Excess of P in the hyphal growth medium increased the P and Si concentrations in spores and increased the K/Ca ratio in spores. Increased P availability decreased the concentration of Zn and Mn in spores. We concluded that the availability of P influences the uptake and accumulation of several elements in spores. It is demonstrated that PIXE analysis is a powerful tool for quantitative analysis of elemental accumulation in fungal mycelia.     

Phialophora graminicola, a dark septate fungus, is a beneficial associate of the grass Vulpia ciliata ssp. ambigua

The influence of three organic compounds and bakers' dry yeast on growth of external mycelium and phosphorus uptake of the arbuscular mycorrhizal fungus Glomus intraradices Schenck & Smith (BEG 87) was examined. Two experiments were carried out in compartmentalized growth systems with root-free sand or soil compartments. The sand and soil in the root-free compartments were left untreated or uniformly mixed with one of the following substrates (0.5 mg g⁻¹ soil): bakers' dry yeast, bovine serum albumin, starch or cellulose. Effects of the organic substrates on biomass and hyphal length density of the arbuscular mycorrhizal fungus were examined by using specific fatty acid signatures in combination with direct microscopy. Micro-organisms other than the arbuscular mycorrhizal fungus were measured by fatty acid signatures, and radioactive ³³P labelling of the root-free soil was used to determine arbuscular mycorrhizal hyphal phosphorus uptake. In general, hyphal growth of G. intraradices was enhanced by yeast and bovine serum albumin, whereas the carbon sources, starch and cellulose, depressed fungal growth. By analysing the fatty acid 16:1ω5 from phospholipids (indicating mycelium) and neutral lipids (indicating storage structures) it was shown that increased fungal growth due to yeast was mainly in vegetative hyphae and less in storage structures. Arbuscular mycorrhizal hyphal phosphorus uptake was decreased by cellulose, but unaffected by the other substrates compared with the control. This means that both growth and phosphorus transport by the arbuscular mycorrhizal fungus were decreased under cellulose treatment. However, the composition of the microbial community varied under different substrate conditions indicating a possible interactive component with arbuscular mycorrhizal hyphal growth and phosphorus uptake.     

Detection of an arbuscular mycorrhizal fungus in roots of different plant species with the PCR.

PCR was used with the primer pair VANS1-NS21 to detect the arbuscular mycorrhizal fungus Glomus intraradices (commercial inoculum source) on roots of lettuce, zinnia, leek, pepper, and endive plants. The appropriate amplification product was obtained directly from roots without DNA extraction and purification.     

Genetic diversity of the arbuscular mycorrhizal fungus Glomus intraradices as determined by mitochondrial large subunit rRNA gene sequences is considerably higher than previously expected

Glomus intraradices is a widespread arbuscular mycorrhizal fungus (AMF), which has been found in an extremely broad range of habitats, indicating a high tolerance for environmental factors and a generalist life history strategy. Despite this ecological versatility, not much is known about the genetic diversity of this fungal species across different habitats or over large geographic scales. A nested polymerase chain reaction (PCR) approach for the mitochondrial rRNA large subunit gene (mtLSU), distinguished different haplotypes among cultivated isolates of G. intraradices and within mycorrhizal root samples from the field. From analysis of 16 isolates of this species originating from five continents, 12 mitochondrial haplotypes were distinguished. Five additional mtLSU haplotypes were detected in field-collected mycorrhizal roots. Some introns in the mtLSU region appear to be stable over years of cultivation and are ancestral to the G. intraradices clade. Genetic diversity within G. intraradices is substantially higher than previously thought, although some mtLSU haplotypes are widespread. A restriction fragment length polymorphism approach also was developed to distinguish mtLSU haplotypes without sequencing. Using this molecular tool, intraspecific genetic variation of an AMF species can be studied directly in field plants.     

Phosphorus effect on phosphatase activity in endomycorrhizal maize

Success of a mycorrhizal symbiosis is influenced by the availability of phosphorus (P) in the soil. Maize ( Zea mays L. cv. Great Lakes 586) plants were grown under five different levels of soil P, either in the presence or absence of formononetin or the vesicular‐arbuscular mycorrhizal (VAM) fungus Glomus intraradices Schenck and Smith. We detected physiological differences in mycorrhizal roots very early in the development of symbiosis, before the onset of nutrient‐dependent responses. Under low P levels, VAM roots accumulated a greater shoot dry weight (13%), root P concentration (15%) and protein concentration (30%) than non-VAM roots, although root growth was not statistically significantly different. At higher P levels, mycorrhizal roots weighed less than non-VAM roots (10%) without a concomitant host alteration of growth or root P concentration. Mycorrhizal colonization decreased as soil P increased. Formononetin-treatment enhanced colonization of the root by G. intraradices and partially overcame inhibition of VAM colonization by high soil P concentrations. This is the first report that formononetin improves root colonization under high levels of soil P. Acid phosphatase (ACP) and alkaline phosphatase (ALP) activities were closely related to the level of fungal colonization in corn roots. ACP activity in corn roots responded more to soil P availability than did ALP activity (38% more). These results suggest that ACP was involved in the increased uptake of P from the soil, while ALP may be linked to active phosphate assimilation or transport in mycorrhizal roots. Thus, soil P directly affected a number of enzymes essential in host-endophyte interplay, while formononetin enhanced fungal colonization.     

Functional diversity of arbuscular mycorrhizal fungal isolates in relation to extraradical mycelial networks

We investigated the functional significance of extraradical mycorrhizal networks produced by geographically different isolates of the arbuscular mycorrhizal fungal (AMF) species Glomus mosseae and Glomus intraradices. A two-dimensional experimental system was used to visualize and quantify intact extraradical mycelium (ERM) spreading from Medicago sativa roots. Growth, phosphorus (P) and nitrogen (N) nutrition were assessed in M. sativa plants grown in microcosms. The AMF isolates were characterized by differences in extent and interconnectedness of ERM. Phenotypic fungal variables, such as total hyphal length, hyphal density, hyphal length per mm of total or colonized root length, were positively correlated with M. sativa growth response variables, such as total shoot biomass and plant P content. The utilization of an experimental system in which size, growth rate, viability and interconnectedness of ERM extending from mycorrhizal roots are easily quantified under realistic conditions allows the simultaneous evaluation of different isolates and provides data with a predictive value for selection of efficient AMF.     

丛枝菌根真菌对枳根净离子流及锌污染下枳苗矿质营养的影响

盆栽实验研究了不同施Zn水平(0、300 mg/kg和600 mg/kg)下,接种丛枝菌根真菌Glomus intraradices对枳苗生长、Zn、Cu、P、K、Ca、Mg分布的影响,并采用非损伤微测技术测定分析了菌根化与非菌根化枳根净Ca²⁺、H⁺、NO₃^-离子流动态。结果表明:(1)在不同施Zn水平下,接种菌根真菌显著提高了枳苗地上部及根部鲜重;随着施Zn水平的提高,菌根侵染率呈降低趋势,枳苗地上部与根部Zn含量呈增加趋势,且接种株根部Zn含量显著高于未接种株。(2)接种株未施Zn处理的地上部Cu、P、K、Mg和根部Cu含量、施600 mg/kg Zn处理的根部Cu及施300 mg/kg Zn处理的根部P含量均显著高于对照,而菌根真菌侵染对枳苗Ca含量并无显著性影响。(3)接种株未施Zn处理的根部距根尖端0 μm和600 μm处净Ca²⁺流出速率、600 μm处净H⁺流入速率、2400 μm处净NO₃^-流入速率均显著高于未接种株。     

Suppression of the biocontrol agent trichoderma harzianum by mycelium of the arbuscular mycorrhizal fungus glomus intraradices in root-free soil

Trichoderma harzianum is an effective biocontrol agent against several fungal soilborne plant pathogens. However, possible adverse effects of this fungus on arbuscular mycorrhizal fungi might be a drawback in its use in plant protection. The objective of the present work was to examine the interaction between Glomus intraradices and T. harzianum in soil. The use of a compartmented growth system with root-free soil compartments enabled us to study fungal interactions without the interfering effects of roots. Growth of the fungi was monitored by measuring hyphal length and population densities, while specific fatty acid signatures were used as indicators of living fungal biomass. Hyphal 33P transport and beta-glucuronidase (GUS) activity were used to monitor activity of G. intraradices and a GUS-transformed strain of T. harzianum, respectively. As growth and metabolism of T. harzianum are requirements for antagonism, the impact of wheat bran, added as an organic nutrient source for T. harzianum, was investigated. The presence of T. harzianum in root-free soil reduced root colonization by G. intraradices. The external hyphal length density of G. intraradices was reduced by the presence of T. harzianum in combination with wheat bran, but the living hyphal biomass, measured as the content of a membrane fatty acid, was not reduced. Hyphal 33P transport by G. intraradices also was not affected by T. harzianum. This suggests that T. harzianum exploited the dead mycelium but not the living biomass of G. intraradices. The presence of external mycelium of G. intraradices suppressed T. harzianum population development and GUS activity. Stimulation of the hyphal biomass of G. intraradices by organic amendment suggests that nutrient competition is a likely means of interaction. In conclusion, it seemed that growth of and phosphorus uptake by the external mycelium of G. intraradices were not affected by the antagonistic fungus T. harzianum; in contrast, T. harzianum was adversely affected by G. intraradices.     

Cadmium accumulation and buffering of cadmium-induced stress by arbuscular mycorrhiza in three Pisum sativum L. genotypes

The role of arbuscular mycorrhiza in reducing Cd stress was investigated in three genotypes of Pisum sativum L. (cv. Frisson, VIR4788, VIR7128), grown in soil/sand pot cultures in the presence and absence of 2-3 mg kg(-1) bioavailable Cd, and inoculated or not with the arbuscular mycorrhizal fungus Glomus intraradices. Shoot, root and pod biomass were decreased by Cd in non-mycorrhizal plants. The presence of mycorrhiza attenuated the negative effect of Cd so that shoot biomass and activity of photosystem II, based on chlorophyll a fluorescence, were not significantly different between mycorrhizal plants growing in the presence or absence of the heavy metal (HM). Total P concentrations were not significantly different between mycorrhizal and non-mycorrhizal plants treated with Cd. From 20-50-fold more Cd accumulated in roots than in shoots of Cd-treated plants, and overall levels were comparable to other metal-accumulating plants. Genetic variability in Cd accumulation existed between the pea genotypes. Concentration of the HM was lowest in roots of VIR4788 and in pods of VIR4788 and VIR7128. G. intraradices inoculation decreased Cd accumulation in roots and pods of cv. Frisson, whilst high concentrations were maintained in roots and pods of mycorrhizal VIR7128. Shoot concentrations of Cd increased in mycorrhizal cv. Frisson and VIR4788. Sequestration of Cd in root cell walls and/or cytoplasm, measured by EDS/SEM, was comparable between non-mycorrhizal pea genotypes but considerably decreased in mycorrhizal cv. Frisson and VIR7128. Possible mechanisms for mycorrhiza buffering of Cd-induced stress in the pea genotypes are discussed.