Mycorrhizal Fungus Glomus Intraradices Research Articles

Effect ofGlomus intraradices isolated from Pb-contaminated soil on Pb uptake byAgrostis capillaris is changed by its cultivation in a metal-free substrate

Development and heavy metal tolerance of two cultivation lineages of the indigenous isolate of arbuscular mycorrhizal fungus (AMF)Glomus intraradices PH5 were compared in a pot experiment in soil from lead (Pb) smelter waste deposits. One lineage was sub-cultured in original Pb-contaminated soil; the second one was maintained for 13 months in an inert substrate (river sand) without Pb stress. The contribution of these cultivation lineages to the Pb uptake and accumulation by the host plantAgrostis capillaris was investigated. The experiment was conducted in a compartmented system where the lateral compartments withAgrostis seedlings were separated from the central pot containing 4-week olderAgrostis plants by a nylon mesh for allowing out-growing of extraradical mycelium (ERM) from the pot.

Combined effect of an arbuscular mycorrhizal fungus and a biocontrol bacterium againstPythium ultimum in soil

The combined effect of the arbuscular mycorrhizal (AM) fungusGlomus intraradices and the bacteriumBurkholderia cepacia onPythium ultimum was studied in a growth system with root-free soil compartments allowing examination of microbial interactions without direct interference from roots. Signature fatty acids were used to quantify individual microbial populations. Biomass and amount of energy reserves were estimated using specific phospholipid fatty acids (PLFA) and neutral lipid fatty acids (NLFA), respectively. Population density ofP. ultimum was estimated by colony forming units on selective media. Both biocontrol agents (BCAs) reduced population density ofP. ultimum. Biomass ofB. cepacia measured using signature fatty acids was inhibited by the presence of mycelium ofG. intraradices, which might explain why no additive effect onP. ultimum was found when combining the two BCAs. In conclusion, combiningG. intraradices andB. cepacia did not enhance the biocontrol efficacy againstP. ultimum.

Locally accumulated extractable compounds in mycorrhizal parts of maize roots suppress the growth ofHyphae ofGlomus intraradices

Extracts from mycorrhizal roots, non-mycorrhizal roots obtained from mycorrhizal root systems, and non-inoculated maize roots were fractionated by gel chromatography and their effects on the hyphal growth of arbuscular mycorrhizal fungusGlomus intraradices were studied under aseptic conditions. Low-molecular (MW lower than 3000) extracts obtained from mycorrhizal roots contained the fraction that most rapidly suppresses the hyphae growth. This was not observed for the fastest fraction obtained from non-mycorrhizal roots. The increased content of some inorganic ions, mainly potassium, was recorded in the fastest fraction of the mycorrhizal root extracts. At the same time, higher contents of two unidentified major organic compounds were revealed in this fraction of the mycorrhizal root extract compared to the non-mycorrhizal one. Some of these compounds, which are locally accumulated in the mycorrhizal parts of the root, are probably responsible for the effect observed. Potassium alone, even at a high concentration, cannot be responsible for the supression observed.

Quantification of Fusarium solani f. sp. phaseoli in Mycorrhizal Bean Plants and Surrounding Mycorrhizosphere Soil Using Real-Time Polymerase Chain Reaction and Direct Isolations on Selective Media

ABSTRACT The capacity of the arbuscular mycorrhizal fungus Glomus intraradices in reducing the presence of Fusarium solani f. sp. phaseoli in bean plants and the surrounding mycorrhizosphere soil was evaluated in a compartmentalized experimental system. Quantification of the pathogen and the symbiont in plant tissues, the soil regions of the mycorrhizosphere (rhizosphere and mycosphere), and the bulk soil was accomplished using specific polymerase chain reaction (PCR) primers in real-time PCR assays, culture-dependant methods, and microscopic determination techniques. Nonmycorrhizal bean plants infected with the pathogen had distinctive Fusarium root rot symptoms, while infected plants previously colonized by G. intraradices remained healthy. The amount of F. solani f. sp. phaseoli genomic DNA was significantly reduced in mycorrhizal bean plants and in each mycorrhizosphere soil compartment. The presence of G. intraradices in the mycorrhizosphere was not significantly modified, although the mycorrhizal colonization of roots was slightly increased in the presence of the pathogen. The results suggest that the reduced presence of Fusarium as well as root rot symptoms are caused by biotic and/or abiotic modifications of the mycorrhizosphere as a result of colonization with G. intraradices.

Interaction between foliar-feeding insects, mycorrhizal fungi, and rhizosphere protozoa on pea plants

Summary Pea plants ( Pisum sativum ) were grown in pots with and without the arbuscular mycorrhizal fungus (AMF) Glomus intraradices . Plants in each AM treatment were subjected to adult weevils ( Sitona lineatus ) during the periods 15–25 d or 23–39 d and were harvested at day 25 or day 39. Herbivory stimulated AMF root colonisation of mycorrhizal plants at the first harvest and stimulated rhizosphere protozoa in the absence of AMF. At the second harvest, herbivory decreased AMF colonization and had no effect on rhizosphere protozoa. The presence of AMF had no effect on herbivory at the first harvest but decreased herbivory at the second harvest. Below-ground respiration was stimulated by herbivory and this effect was most pronounced during vegetative growth. The results therefore suggest, that herbivory stimulated below-ground carbon transfer in young plants in the nutrient acquisition phase as opposed to the reproductive phase where herbivory had no such effect. This herbivory-induced carbon transfer will benefit the AMF symbiont when present and the free-living rhizosphere micro-organisms – here represented by the bacterivorous protozoa – in the absence of AMF.

Soil pasteurization and inoculation with Glomus intraradices alters flower production and bulb composition of Zephyranthes spp.

SummaryWe assessed whether adding inoculum of the vesicular-arbuscular mycorrhizal fungus (VAMF) Glomus intraradices into growing medium of three Zephyranthes spp (White Rain Lily [WRL], Z. candida; Pink Fairy Lily [PFL], Z. robusta; Yellow Zephyr Lily [YZL], Z. sulphurea) alters aspects of flower and bulb production. Shoots of inoculated plants emerged 7–13 d earlier than those of non-inoculated plants. Inoculation slightly delayed the emergence of flower buds on WRL and PFL, but did not delay the time of flower opening of WRL. Inoculated YZL flowered 4–11.d earlier than non-inoculated plants. The number of flowers produced by YZL was consistently increased by inoculation, while the inoculation with VAMF increased flower production by WRL and PFL only when plants were growing in pasteurized soil. Leaf biomass of inoculated WRL was larger than non-inoculated plants, while leaf biomass was generally smaller in inoculated PFL and YZL. Partitioning of biomass to bulbs and offsets varied with species, soil pasteurization, and inoculation. Inoculation increased the combined weight of bulbs and offsets at the end of the second growing cycle by 50–150%. Inoculated YZL and WRL consistently produced more offsets in the second growing season after inoculation. For all species, inoculation increased phosphorus and carbohydrates and decreased nitrogen and amino acids in bulbs. Adding VAMF into the growing medium of Zephyranthes altered aspects of plant development and biomass partitioning important to flower and bulb production during the first growing cycle after inoculation, and most effects of VAMF inoculation are more pronounced in the second growing cycle after inoculation. Of the three species examined, Z. sulphurea showed the most consistent responses to inoculation.

Mycorrhizal Fungi in Rooting Substrate Influences the Quantity and Quality of Roots on Stem Cuttings of Hick's Yew

In a commercial nursery propagation system for hick's yew (Taxu×media `Hicksii'), we assessed whether or not the addition of inoculum of the vesicular-arbuscular mycorrhizal fungus (VAMF) Glomus intraradices into the rooting substrate during cutting propagation increased rooting, and how the quantity of inoculum influenced rooting. At 15 and 22 weeks (108 and 156 d) after cuttings were treated with root hormones and stuck, root initiation was higher on cuttings stuck in the rooting substrate containing VAMF inoculum. Increasing the quantity of inoculum in the rooting substrate increased root growth during the early stages of rooting. However the highest level of inoculum tested increased adventitious root initiation without increased root growth. Our results indicate that if VAMF inoculum is used during propagation from cuttings, there are optimal levels required to alter the initiation and growth of roots. For hick's yew, 1:100 or 2:100 (by volume) rates of G. intraradices in the rooting substrate increased the number of primary roots and growth of adventitious roots on cuttings above that achieved by using rooting hormone alone.

Direct quantification of fungal DNA from soil substrate using real-time PCR

Detection and quantification of genomic DNA from two ecologically different fungi, the plant pathogen Fusarium solani f. sp. phaseoli and the arbuscular mycorrhizal fungus Glomus intraradices, was achieved from soil substrate. Specific primers targeting a 362-bp fragment from the SSU rRNA gene region of G. intraradices and a 562-bp fragment from the F. solani f. sp. phaseoli translation elongation factor 1 alpha gene were used in real-time polymerase chain reaction (PCR) assays conjugated with the fluorescent SYBR® Green I dye. Standard curves showed a linear relation ( r 2=0.999) between log values of fungal genomic DNA of each species and real-time PCR threshold cycles and were quantitative over 4–5 orders of magnitude. Real-time PCR assays were applied to in vitro-produced fungal structures and sterile and non-sterile soil substrate seeded with known propagule numbers of either fungi. Detection and genomic DNA quantification was obtained from the different treatments, while no amplicon was detected from non-seeded non-sterile soil samples, confirming the absence of cross-reactivity with the soil microflora DNA. A significant correlation ( P<0.0001) was obtained between the amount of genomic DNA of F. solani f. sp. phaseoli or G. intraradices detected and the number of fungal propagules present in seeded soil substrate. The DNA extraction protocol and real-time PCR quantification assay can be performed in less than 2 h and is adaptable to detect and quantify genomic DNA from other soilborne fungi.

The effect of fulvic acids on the toxicity of lead and manganese to arbuscular mycorrhizal fungus Glomus intraradices.

The effect of fulvic acids (FA) on arbuscular mycorrhizal (AM) fungi and on the toxicity of lead and manganese toward these symbionts were demonstrated in vitro. Incubation of root segments colonized with the AM fungus Glomus intraradices in undiluted fraction of FA (813 mg/L carbon) decreased an outgrowth of intraradical hyphae. Diluted FA solutions (< 271 mg/L C) did not influence the proportion of root segments bearing proliferating hyphae; solution containing 27.1 mg/L C even increased the proliferation. A decrease of heavy metal toxicity toward the fungus was observed when FA (81.3 mg/L C) were added to the solutions containing higher concentrations (< or = 100 mumol/L) of Mn and Pb; the positive effect of FA was not significant at higher concentrations of metals (0.5 mmol/L). A short-term cultivation of six different saprophytic microorganisms--three actinomycetes and three filamentous fungi--on the FA media (81.3 mg/L C) did not result in a modification of hyphal proliferation from the root segments subsequently incubated in these media.

MYCORRHIZAL FUNGI INCREASE CHROMIUM UPTAKE BY SUNFLOWER PLANTS: INFLUENCE ON TISSUE MINERAL CONCENTRATION, GROWTH, AND GAS EXCHANGE

ABSTRACT As a potential phytoremediation system for phytoextraction of chromium (Cr), we evaluated the influence of the arbuscular mycorrhizal fungus Glomus intraradices on leaf tissue elemental composition, growth and gas exchange of sunflower (Helianthus annuus L.). Sunflower seedlings were either inoculated with mycorrhizal fungi (AM) or non-inoculated (Non-AM) and then exposed to two Cr species: {12 mmol of trivalent cation (Cr+3) [Cr(III)] or 0.1 mmol of divalent dichromate anion (Cr2O7 −) [Cr(VI)]}. Both Cr species depressed plant growth, decreased stomatal conductance (g s ) and net photosynthesis (A). However, Cr(VI) was more toxic than Cr(III) as indicated by early reduction in g s , and greater reduction in plant growth and arbuscule formation. Mycorrhizal fungi helped to partially alleviate Cr toxicity as indicated by greater growth in plants treated with either Cr species, and increased gas exchange of Cr(III) plants. With Cr(III), Cr concentrations were the same in tissue of AM and Non-AM plants, while Non-AM Cr(VI) treatments had higher Cr concentration than AM plants; however with both Cr species, the larger-sized AM plants had greater total Cr accumulation on a whole-plant basis. Chromium reduced leaf elemental nitrogen (N), phosphorus (P), and potassium (K), and increased aluminum (Al), iron (Fe), and zinc (Zn) uptake. Chromium-treated AM plants had higher K and decreased levels of Fe and Al than Non-AM plants. Except for the depression of arbuscular formation in Cr(VI) treated plants, high colonization occurred with either Cr species. While higher P may have contributed to mycorrhizal benefits with Cr(III) plants, it does not account for any AM benefits observed with Cr(VI) plants.

Intra- and intersporal diversity of ITS rDNA sequences in Glomus intraradices assessed by cloning and sequencing, and by SSCP analysis

The level of molecular variability in the arbuscular mycorrhizal fungus (AMF) Glomus intraradices was assessed. We established several monoxenically growing single-spore cultures of the G. intraradices collected from a long-term soil tillage experiment in Tänikon (Switzerland). They were grown in symbiosis with Ri T-DNA transformed carrot roots. The ITS region was amplified from genomic DNA of fungal spores using ITS1 and ITS4 primers. The amplified ITS region of three of the fungal isolates was cloned and sequenced. All sequences obtained from our samples closely resembled those previously reported for G. intraradices, and no evidence for genetic exchange was found on the interspecific or intergeneric level. Phylogenetic analysis shøwed the presence of at least two different sequence families within a single-spore isolate. The single strand conformation polymorphism (SSCP) method detected different sequence types in a single PCR sample. This technique enabled us to detect several distinct ITS sequences within each of the single-spore isolates of the AMF. Estimates of intra- and intersporal sequence diversity were obtained by comparing diversity within and among the single-spore isolates. A higher level of diversity was found among spores than within a single spore.

Bacterial populations associated with mycelium of the arbuscular mycorrhizal fungus Glomus intraradices

The influences of the arbuscular mycorrhizal fungus Glomus intraradices on the culturable aerobic–heterotrophic bacterial communities in the rhizosphere and hyphosphere of cucumber plants ( Cucumis satvius) were investigated. Mycorrhizal and non-mycorrhizal plants were grown in compartmentalised growth units, each with a root compartment and two lateral root-free compartments. Samples representing rhizosphere, root-free soil, root-free sand and washed sand extract were collected 52 days after sowing from treatments both with and without mycorrhiza. No significant difference in total bacterial number was observed between the mycorrhizal and non-mycorrhizal treatment. Fourteen hundred bacterial colonies were isolated and identified by fatty acid methyl ester analysis using the Sherlock system (MIDI Inc.), 87 species within 48 genera were identified with a similarity index >0.30. Pseudomonas, Arthrobacter and Burkholderia were the genera most frequently encountered. Large differences in bacterial community structure were observed between rhizosphere soil, root-free soil/sand and washed sand extract, whereas major differences between mycorrhizal and non-mycorrhizal treatments were observed for a few bacterial species only. Isolates identified as Paenibacillus spp. were more frequently found in the mycorrhizal treatment and especially in the washed sand extract with mycelium of G. intraradices, indicating that bacteria within this genus may live in close association with mycelium of these fungi.

Bacterial populations associated with mycelium of the arbuscular mycorrhizal fungus Glomus intraradices

Abstract The influences of the arbuscular mycorrhizal fungus Glomus intraradices on the culturable aerobic-heterotrophic bacterial communities in the rhizosphere and hyphosphere of cucumber plants (Cucumis satvius) were investigated. Mycorrhizal and non-mycorrhizal plants were grown in compartmentalised growth units, each with a root compartment and two lateral root-free compartments. Samples representing rhizosphere, root-free soil, root-free sand and washed sand extract were collected 52 days after sowing from treatments both with and without mycorrhiza. No significant difference in total bacterial number was observed between the mycorrhizal and non-mycorrhizal treatment. Fourteen hundred bacterial colonies were isolated and identified by fatty acid methyl ester analysis using the Sherlock system (MIDI Inc.), 87 species within 48 genera were identified with a similarity index >0.30. Pseudomonas, Arthrobacter and Burkholderia were the genera most frequently encountered. Large differences in bacterial community structure were observed between rhizosphere soil, root-free soil/sand and washed sand extract, whereas major differences between mycorrhizal and non-mycorrhizal treatments were observed for a few bacterial species only. Isolates identified as Paenibacillus spp. were more frequently found in the mycorrhizal treatment and especially in the washed sand extract with mycelium of G. intraradices, indicating that bacteria within this genus may live in close association with mycelium of these fungi.

Copper release from chemical root-control baskets in hardwood tree production.

The City of Montreal, Canada, evaluated the environmental impact and usefulness of in-ground copper (Cu)-treated baskets in controlling root growth of hardwood trees in nursery culture. Using baskets planted with 5-yr-old Norway maple (Acer platanoides L.) trees, the amount and temporal pattern of Cu release from the basket surface into soil was determined for two copper formulations: Cu metal powder and Cu(OH)2. Release of both Cu formulations from the basket surface decreased exponentially over time, with Cu concentration at the basket surface dropping to 2% of the initial Cu applied by the end of the second field season. Total Cu content increased significantly in the soil around the baskets (from 7 to 28 mg Cu kg(-1) soil) and in the baskets (from 7 to 50-70 mg Cu kg(-1) soil) over the two years of the study. Three levels of phosphorus application (33, 66, and 100% of the regular nursery rate of 465 kg ha(-1) yr(-1)) did not affect release of Cu from the basket surface. The release of Cu metal at 28 and 105 d in the field was significantly increased by inoculation with the symbiotic arbuscular mycorrhizal fungus (AMF) Glomus intraradices Schenck & Smith; however, AMF inoculation had no affect on Cu(OH)2 release. Trees grown in Cu-treated baskets and inoculated with G. intraradices had similar colonization to non-inoculated trees, suggesting that inoculation was not very effective and that AMF inoculum was already present in the root ball of the trees at planting. After two years, copper basket-grown trees had significantly less root colonization than isolated control trees growing in the open field. This strongly suggests that conditions inside the baskets were not favorable to AMF.

A mycorrhiza-responsive protein in wheat roots.

A small protein, designated Myk15, was found to be strongly induced in wheat ( Triticum aestivum) roots colonized by the arbuscular mycorrhizal fungus Glomus intraradices. This protein, which is most abundant in root fractions characterized by strong mycorrhizal colonization, has been characterized using two-dimensional polyacrylamide gel electrophoresis and microsequencing. It has an apparent molecular mass of 15 kDa and an isoelectric point of 4.5. The N-terminal sequence has high similarity to a peptide sequence deduced from an expressed sequence tag (EST) clone derived from Medicago truncatula roots colonized by G. intraradices. This EST clone is predicted to code for a protein with a similar size and isoelectric point as Myk15. The N-terminus of the deduced M. truncatula protein contains a highly hydrophobic stretch of 24 amino acid residues preceding the region with high similarity to the Myk15 N-terminus. This hydrophobic stretch is predicted to form a transmembrane alpha-helix and may correspond to a cleavable targeting domain.

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.

Expression of nitrate transporter genes in tomato colonized by an arbuscular mycorrhizal fungus.

PCR amplifications using tomato DNA and degenerate oligonucleotide primers allowed identification of a new putative nitrate transporter, termed NRT2;3. Its sequence showed typical motifs of a high affinity nitrate transporter of the Major Facilitator Superfamily (MFS). The formation of its mRNA was positively controlled by nitrate, and negatively by ammonium, but not by glutamine. In situ hybridization experiments showed that this transporter was mainly expressed in rhizodermal cells. Results from expression studies with two other nitrate transporters, LeNRT1;1 and LeNRT2;1, were essentially in accord with data of the literature. In roots colonized by the arbuscular mycorrhizal fungus Glomus intraradices Sy167, transcript formation of NRT2;3 extended to the inner cortical cells where the fungal structures, arbuscules and vesicles, were concentrated. Northern analyses indicated that the expression of only NRT2;3 among the transporters assayed was higher in AMF colonized tomato roots than in non-colonized controls. AMF-colonization caused a significant expression of a nitrate reductase gene of G. intraradices. The results may mean that AMF-colonization positively affects nitrate uptake from soil and nitrate allocation to the plant partner, probably mediated preferentially by LeNRT2;3. In addition, part of the nitrate taken up is reduced by the fungal partner itself and may then be transferred, when in excess, as glutamine to the plant symbiotic partner.

Identification of a cDNA from the arbuscular mycorrhizal fungus Glomus intraradices that is expressed during mycorrhizal symbiosis and up-regulated by N fertilization.

A cDNA library was constructed with RNA from Glomus intraradices-colonized lettuce roots and used for differential screening. This allowed the identification of a cDNA (Gi-1) that was expressed only in mycorrhizal roots and was of fungal origin. The function of the gene product is unknown, because Gi-1 contained a complete open reading frame that was predicted to encode a protein of 157 amino acids which only showed little homology with glutamine synthetase from Helicobacter pylori. The time-course analysis of gene expression during the fungal life cycle showed that Gi-1 was expressed only during the mycorrhizal symbiosis and was not detected in dormant or germinating spores of G. intraradices. P fertilization did not significantly change the pattern of Gi-1 expression compared with that in the unfertilized treatment, whereas N fertilization (alone or in combination with P) considerably enhanced the Gi-1 transcript accumulation. This increase in gene expression correlated with plant N status and growth under such conditions. The possible role of the Gi-1 gene product in intermediary N metabolism of arbuscular mycorrhizal symbiosis is further discussed.

Differential and systemic alteration of defence-related gene transcript levels in mycorrhizal bean plants infected with Rhizoctonia solani

The role of arbuscular mycorrhizas in response of plants to soilborne root pathogens is unclear. A time course study was conducted to monitor disease development and expression of mRNA for the defence-related genes phenylalanine ammonia lyase, chalcone synthase, chalcone isomerase, and hydroxyproline-rich glycoprotein in bean (Phasoelus vulgaris L.) plants colonized by the arbuscular mycorrhizal fungus Glomus intraradices Schenck &amp; Smith and postinfected with the soilborne pathogen Rhizoctonia solani Kühn. Precolonization of bean plants by G. intraradices did not significantly reduce the severity of rot symptoms. RNA blot analysis of the defence-related genes revealed a systemic increase in the four defence genes in response to R. solani infections. On the other hand, precolonization of bean plants with G. intraradices elicited no change in phenylalanine ammonia lyase, chalcone synthase, and chalcone isomerase transcripts. A differential and systemic alteration in the expression of all four defence genes was observed in all tissues only during the pathogenic interaction of arbuscular mycorrhizal beans. Depending on the time after infection with R. solani and the tissue examined, varying responses from stimulation to suppression to no change in transcript levels were detected.Key words: induced resistance, defence-related genes, RNA analysis, Rhizoctonia solani, Glomus intraradices.

Copper Release from Chemical Root-Control Baskets in Hardwood Tree Production

The City of Montreal, Canada, evaluated the environmental impact and usefulness of in-ground copper (Cu)-treated baskets in controlling root growth of hardwood trees in nursery culture. Using baskets planted with 5-yr-old Norway maple (Acer platanoides L.) trees, the amount and temporal pattern of Cu release from the basket surface into soil was determined for two copper formulations: Cu metal powder and Cu(OH)2 Release of both Cu formulations from the basket surface decreased exponentially over time, with Cu concentration at the basket surface dropping to 2% of the initial Cu applied by the end of the second field season. Total Cu content increased significantly in the soil around the baskets (from 7 to 28 mg Cu kg−1 soil) and in the baskets (from 7 to 50–70 mg Cu kg−1 soil) over the two years of the study. Three levels of phosphorus application (33, 66, and 100% of the regular nursery rate of 465 kg ha−1 yr−1) did not affect release of Cu from the basket surface. The release of Cu metal at 28 and 105 d in the field was significantly increased by inoculation with the symbiotic arbuscular mycorrhizal fungus (AMF) Glomus intraradices Schenck & Smith; however, AMF inoculation had no affect on Cu(OH)2 release. Trees grown in Cu-treated baskets and inoculated with G. intraradices had similar colonization to non-inoculated trees, suggesting that inoculation was not very effective and that AMF inoculum was already present in the root ball of the trees at planting. After two years, copper basket–grown trees had significantly less root colonization than isolated control trees growing in the open field. This strongly suggests that conditions inside the baskets were not favorable to AMF.