Translocations of eight species of burrow-nesting seabirds (genera Pterodroma, Pelecanoides, Pachyptila and Puffinus: Family Procellariidae)

Development of seabird translocation techniques is required to meet species recovery objectives, to improve conservation status, and to restore ecological processes. During 1997-2008 we undertook translocation trials on eight petrel species of four genera within the New Zealand region: common diving petrel (Pelecanoides urinatrix), fairy prion (Pachyptila turtur), grey-faced petrel (Pterodroma macroptera gouldi), Pycroft’s petrel (Pterodroma pycrofti), Chatham petrel (Pterodroma axillaris), Chatham Island taiko (Magenta petrel; Pterodroma magentae), fluttering shearwater (Puffinus gavia), and Hutton’s shearwater (Puffinus huttoni). A total of 1791 chicks within 5 weeks of fledging were moved up to 240 km, placed in artificial burrows and hand-fed until they fledged. Of these, 1546 fledged, and so far at least 68 have returned as adults to the translocation sites. Most birds were crop-fed a puree based on tinned sardines and fresh water. This diet worked well for all species regardless of their typical natural diet (planktonic crustaceans, squid, or fish) with all species fledging above or close to mean natural fledging weights.     

Effects of Lumbricus terrestris, Allolobophora chlorotica and Eisenia fetida on microbial community dynamics in oil-contaminated soil

Oil spills are one of the most common types of soil pollution. Bioremediation has become an attractive alternative to physicochemical methods of remediation, where feasible. Earthworms have been shown to stimulate the degradation of petroleum hydrocarbons in soil, and it was hypothesized that the role of earthworms in remediation lies in the enhancement of an oil degrading microbial community. The aim of this study was to characterize microbial activity and community dynamics in oil-contaminated soil incubated with or without earthworms. Three earthworm species (Eisenia fetida, Allolobophora chlorotica and Lumbricus terrestris) were incubated in crude oil polluted soil (ca. 10,000 mg/kg total petroleum hydrocarbons (TPH)) and a reference soil for 28 d. Control treatments with manual mixing and/or cattle dung amendment were also included. In the oil-contaminated soil, respiration and concentration of microbial biomass was significantly enhanced by earthworm amendment, and TPH concentrations decreased significantly. These effects were less evident in treatments with A. chlorotica, possibly due to a difference in behavior, since individuals of this endogeic species were found in a state of inactivity (aestivation). Microbial community dynamics were described by phospholipid fatty acid (PLFA) analyses. After 28 d, similar shifts in the soil PLFA composition were observed in the oil-contaminated soil irrespective of worm species. Fungal:bacterial ratios were increased in the presence of worms, but also by addition of dung as a food source, indicating a non-specific effect of metabolizable substrates. In contrast, the fatty acids 17:1ω8 (=Δ9-heptadecenoic acid) and 20:4ω6c (arachidonic acid) were specifically stimulated by the presence of earthworms in the oil-contaminated soil. The results showed that earthworms can contribute positively to bioremediation of oil-contaminated soil, but that the effect may be species-dependent.     

Population dynamics of the mycoparasite, sporidesmium sclerotivorum, and its host, sclerotinia minor, in soil

The infection and survival of sclerotia of Sclerotinia minor and the production ofmacroconidia of the mycoparasite, Sporidesmium sclerotivorum, were studied in vitro when each fungus was added to soil at various initial inoculum densities. The rate at which S. sclerotivorum invaded host sclerotia and caused their decay varied with the amount of the mycoparasite added to soil. The results suggest that approximately 5 macroconidia of the mycoparasite g^?1 of soil are needed to successfully infect sclerotia and bring about their decay, when soils are sampled and mixed every 2 weeks. The rate at which S. sclerotivorum infects sclerotia of S. minor and causes their decay is also dependent on the initial inoculum density of the host. Each infected sclerotium supports the production of about 15,000 new macroconidia in soil regardless of the initial inoculum density of the host. It is concluded that successful biological control by S. sclerotivorum is dependent on the soil population of both the host and the mycoparasite.     

Rapid identification and discrimination among Egyptian genotypes of Rhizobium leguminosarum bv. viciae and Sinorhizobium meliloti nodulating faba bean (Vicia faba L.) by analysis of nodC, ARDRA, and rDNA sequence analysis

Twenty-eight Rhizobium strains were isolated from the root nodules of faba bean (Vicia faba L.) collected from 11 governorates in Egypt. A majority of these strains (57%) were identified as Rhizobium leguminosarum bv. viciae (Rlv) based on analysis of a nodC gene fragment amplified using specific primers for these faba bean symbionts. The strains were characterized using a polyphasic approach, including nodulation pattern, tolerance to environmental stresses, and genetic diversity based on amplified ribosomal DNA-restriction analysis (ARDRA) of both 16S and 23S rDNA. Analysis of tolerance to environmental stresses revealed that some of these strains can survive in the presence of 1% NaCl and a majority of them survived well at 37 °C. ARDRA indicated that the strains could be divided into six 16S rDNA genotypes and five 23S rDNA genotypes. Sequence analysis of 16S rDNA indicated that 57% were Rlv, two strains were Rhizobium etli, one strain was taxonomically related to Rhizobium rubi, and a group of strains were most closely related to Sinorhizobium meliloti. Results of these studies indicate that genetically diverse rhizobial strains are capable of forming N₂-fixing symbiotic associations with faba bean and PCR done using nodC primers allows for the rapid identification of V. faba symbionts.     

Density loss and respiration rates in coarse woody debris of Pinus radiata, Eucalyptus regnans and Eucalyptus maculata

This study compared field and laboratory decomposition rates of coarse woody debris (CWD) (>10 cm diameter) from three tree species: Pinus radiata, Eucalyptus regnans, and Eucalyptus maculata. For this purpose, the density loss of logs on the ground sampled from chronosequences of sites following harvesting was determined using the water replacement technique. P. radiata logs were sampled 1, 2.5, 6, and 9 years following harvesting, and logs of E. regnans and E. maculata were collected from sites that were harvested 1, 3.5, 6.5, and 12 and 1.5, 6.5, and 11.5 years ago, respectively. In addition, the C/N ratio of wood was determined and current respiration rates of logs from these different age classes were measured through laboratory incubation. The times for loss of 95% of material (t_0.95) determined from density loss for these species were 24 years for P. radiata, 43 years for E. regnans, and 62 years for E. maculata. The decomposition rates of CWD derived from laboratory respiration were 6.1, 5.9 and 11.9 times higher than the decay rates from density loss in P. radiata, E. regnans, and E. maculata, respectively. This points to severe constraints of decomposition through adverse conditions in the field. The changes in respiration rates and C/N ratio with age of decaying logs indicated that the single component, negative exponential decay model could be applied satisfactorily only to P. radiata. In the case of the eucalypt species, substrate quality (expressed through respiration rates) declined in the oldest samples. This may be explained by the loss of rapidly decomposing sapwood and the retention of more decay-resistant heartwood. In these cases, a two-component model will be more suitable to describe the density loss of decaying wood.     

The invasion of pea roots, pisum sativum l., by soil microorganisms, Acanthamoeba palestinensis (reich) and pseudomonas SP

Pea roots (Pisum sativum L. cultivar Tall Sugar White) were inoculated with Pseudomonas sp., isolated from the roots of Timothy grass (Phleum pratensis L.). Microscopic examination showed that the bacterium had invaded many epidermal and outer cortical cells of the root. The presence of the soil amoeba, Acanthamoeba palestinensis (Reich), which readily ingests Pseudomonas sp., did not alter the root damage. The amoebae were also found in the epidermis and outer cortex of pea roots. No amoebae were found inside pea roots when Pseudomonas sp. was absent. Bacterial invasion also occurred in pea roots grown in garden soil previously sterilized by γ-irradiation and inoculated with Pseudomonas sp., but not in the same soil inoculated and unsterilized. Timothy grass roots were similarly uninfected in inoculated unsterilized garden soil.     

Rhizobium inoculation of Calopogonium caeruleum

The shade-tolerant cover legume Calopogonium caeruleum is promiscuous in its nodulating habits. In sand culture, symbiotic effectiveness of the strains tested was variable; 6 strains of rhizobia markedly improved shoot yields and 20 increased shoot N content. In pot experiments using cultivated and non-cultivated soils, inoculation gave no significant increase in shoot yields. When grown under rubber in plantation conditions at four localities, shoot dry matter yields, N content and nodulation also were not different from uninoculated plants when sampled for up to 2 yr after planting. This occurred despite the low numbers (< 10 g^?1 soil) of native rhizobia at some sites and an appreciable establishment (> 70% recovery in nodules) by the inoculant strains.     

Biological control of the take-all fungus, Gaeumannomyces graminis, by Phialophora radicicola and similar fungi

Phialophora radicicola is an avirulent fungal root-parasite of grasses and cereals, with runner hyphae like those of Gaeumannomyces graminis. Weakly and non-pathogenic varieties of these fungi control the pathogens, G. graminis vars. tritici and avenue. Biology of these fungi is considered and the evidence for biological control and possible mechanisms reviewed; control is probably widespread in natural plant communities, and host-mediated, perhaps by induction of plant resistance mechanisms.Prospects for application of biological control seem best for P. radicicola var. graminicola established on grass crops, as this is already exploited in British agriculture. New evidence is presented on the effects of grassland factors on this fungus, especially sward composition, age, mineral nutrition and management practices: its population might often be limited by the rate of new root production to replace those with cortices already colonized. Prospects for control by seed inoculation with P. radicicola var. radicicola and G. graminis var. graminis also seem good, but possible dangers of introducing them into cereal cropping are emphasized. The weak pathogens might be used also for indirect control by establishing hyper-parasites or inducing disease suppression (like take-all decline) in soils, but there is no evidence for ‘Phialophora decline’, at least in well-managed grasslands. Finally, different biocontrols of take-all might be combined, and biological with chemical ones for ophiobolus patch disease of turf.P. radicicola var. graminicola has a slight beneficial effect on grass yield, even when the pathogens, G. graminis vars. tritici and arenae arc absent; this probably contributes to its abundance in natural grasslands in Britain. The scale of biological control by this and similar fungi might explain why, in their absence, effective plant resistance to G. graminis is uncommon in the Gramineae.     

Soil temperature,mycorrhizal infection and nodulation of Medicago truncatula and Trifolium subterraneum

Establishment of vesicular-arbuscular mycorrhizal fungi in plant roots involves a pre-infection phase of propagule germination, hyphal growth and appressorium formation, followed by growth of the fungus within the root. The effect of soil temperature on the pre-infection stage was examined by counting the numbers of fungal “entry-points” on the main roots of Medicago truncatula and Trifolium subterraneum, grown at soil temperatures of 12°, 16°, 20° and 25°C for periods up to 12 days. Increased root temperature was positively associated with increased numbers of “entry-points”. This effect was more marked between 12° and 16°C than at higher temperatures, as shown by comparing plants at the same stage of development (emergence of spade leaf) and by calculating the results as entry points per cm root.The first root nodules appeared sooner at higher temperatures (20° and 25°), but subsequent development of nodules (measured as nodule number and aggregate volume of nodules per plant, up to 21 days) was best at 16°C for both host Rhizobium combinations in non-sterile and autoclaved soil. There was no evidence that competition between mycorrhizal fungi and Rhizobium for infection sites occurred.A method of obtaining numbers of infective propagules of vesicular-arbuscular mycorrhizal fungi in soil is described.     

Lotononis angolensis forms nitrogen fixing, lupinoid nodules with phylogenetically unique, fast-growing, pink-pigmented bacteria, which do not nodulate L. bainesii or L. listii

Root-nodule bacteria that nodulate the legume genus Lotononis are being investigated to develop new forage species for agriculture. Bacteria isolated from nodules of Lotononis angolensis were fast-growing, highly mucoid and pink-pigmented, and on the basis of 16S rRNA phylogeny <94% related to other genera in the Alphaproteobacteria. Root-nodule bacteria isolated from other Lotononis species (L. bainesii, L. solitudinis and L. listii) resembled the more common dry, slow-growing, pink-pigmented rhizobia previously described for L. bainesii. These isolates could be attributed to the Methylobacterium genus, although not to the type species Methylobacterium nodulans. Further differences were uncovered with nodulation studies revealing that nodule isolates from L. angolensis were effective at nitrogen fixation on their host plant, but could nodulate neither L. bainesii nor L. listii. Reciprocal tests showed isolates from L. bainesii, L. listii and L. solitudinis were incapable of nodulating L. angolensis effectively. Nodule morphology for L. bainesii, L. angolensis and L. listii was characteristically lupinoid, with little structural divergence between the species, and with nodules eventually enclosing the entire root.     

Numbers of nesting frigatebirds, Fregata minor and F. ariel, on Aldabra Atoll nature reserve,Seychelles

Aldabra Atoll, a strict Nature Reserve and major breeding station of lesser and greater frigatebirds, may be developed for tourism. Determining whether this affects the frigate breeding populations is complicated by their extended annual laying seasons and the biennial breeding periodicity of individuals rearing fledgelings. This study, using a census method incorporating adjustments for seasonal changes in nest numbers, changes in nest ownership and replacement laying, indicates that there are at least 4000 pairs of greater frigatebirds and 6000 pairs of lesser frigatebirds annually involved in breeding activities. There is some evidence that more male than female greater frigatebirds attempt to pair each season. Comparison with an earlier census suggests that severe human disturbance induces curtailment of laying seasons, major changes in nest distribution and prevents many birds from breeding.     

Distribution of monoterpenes between organic resources in upper soil horizons under monocultures of Picea abies, Picea sitchensis and Pinus sylvestris

The aim of this study was to compare the monoterpene content and distribution in litters and roots of three conifer species: Picea abies (L.) Karst, Picea sitchensis (Bong.) Carr. and Pinus sylvestris (L.). We analysed the monoterpene content of green needles, needle litter, F (fermentation) layer material and roots collected from monoculture plots. The rate of loss of monoterpenes from freshly fallen litter in the field was also studied at two monthly intervals over 10 months, to assess the length of time that monoterpenes entering the litter layer remain. Monoterpene analysis was carried out by extracting homogenised samples in hexane and identifying and quantifying the resulting monoterpenes using gas chromatography with flame ionisation detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS). Mean total monoterpene concentrations varied significantly between the three species examined (e.g. in freshly fallen litter 1531 ± 96, 100 ± 5 and 1175 ± 122 μg g⁻¹ d. wt for P. abies, P. sitchensis and P. sylvestris); each species had distinctive and consistent monoterpene profiles associated with each type of tissue, and total monoterpene concentrations in green needles varied between individual trees of the same species, particularly for P. sitchensis. A substantial proportion of the monoterpene content of green needles remained in the needles after litter fall for P. abies (42%), P. sitchensis (11%) and P. sylvestris (30%). Although rates of monoterpene loss from needle litters varied initially (P. sylvestris > P. abies > P. sitchensis), the majority of the monoterpene content was lost after 4-6 months. Maximum monoterpene emission rates from decaying litter were calculated of 39 (P. abies), 1.7 (P. sitchensis) and 39 μg m⁻² h⁻¹ (P. sylvestris). Monoterpene concentrations in F layer material were very low (<10 μg g⁻¹ d. wt). Roots, particularly in P. sylvestris, represented a significant pool of monoterpenes (185 ± 16, P. abies; 258 ± 54, P. sitchensis; 2133 ± 200 μg g⁻¹ d. wt, P. sylvestris). The monoterpene profile was similar between roots and litter of P. sylvestris (α-pinene most abundant), and for P. sitchensis, (limonene and α-pinene most abundant), although a different pattern was observed between needle litter (most abundant β-pinene) and roots (most abundant myrcene) of P. abies. The relatively high concentrations and different profiles of monoterpenes characterised in upper organic soil horizons here emphasise the need for their influence on soil ecological processes to be assessed.     

In vitro effects of Fusarium blight-controlling fungicides on pathogens of Poa pratensis

An experimental iprodione fungicide,3-(3,5dichlorophenyl)-N-(1-methylethyl)-2,4-dioxo-1-imidazolidinecarboxamide, controls Fusarium blight of Kentucky bluegrass (Poa pratensis L.), but it also amplifies the proportion of crowns colonized by Fusarium and the number of its propagules in soils. In contrast, the disease, the proportion of infected crowns, and the numbers of propagules in soil are generally suppressed by benomyl, methyl 1-(butylcarbamoyl)-2-benzimidazolecarbamate. Triadimefon, 1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)-2-butanone, also controls the disease but is not stimulatory or inhibitory of fusaria. Iprodione and benomyl were studied for their effects on growth and sporulation of Fusarium acuminatum isolated from diseased crowns; iprodione had no or slightly stimulatory effects, and benomyl greatly suppressed these processes, except in a benomyl-tolerant strain.Toxicities of iprodione and benomyl to 1555 identified Fusarium isolates from Kentucky bluegrass turf were determined, as were the toxicities of iprodione to 23 turfgrass pathogens. Of the Fusarium spp, only F. solani was significantly inhibited by iprodione, whereas all were inhibited by benomyl. Iprodione-sensitive fungi included species of Bipolaris, Corticium, Curvularia, Drechslera, Rhizoctonia, Sclerotinia, and Typhula. Insensitive fungi included Colletotrichum, Fusarium, Gaeumannomyces, and Pythium.Investigations with selective fungicides indicate that the primary causal agent of Fusarium blight is not among the fusaria, and that re-interpretation of the disease and its etiology is necessary.     

Non-pathogenic Fusarium strains protect the seedlings of Lepidium sativum from Pythium ultimum

Two root-colonizing Fusarium strains, Ls-F-in-4-1 and Rs-F-in-11, isolated from roots of Brassicaceae plants, induced the resistance in Lepidium sativum seedlings against Pythium ultimum. These strains caused an increase in the content of benzyl isothiocyanate, and of its precursor glucotropaeolin, in the roots of the host plants. The increased isothiocyanate content is one of the factors contributing to the resistance of L. sativum against P. ultimum. To be transformed into the fungitoxic compound benzyl isothiocyanate, glucotropaeolin has to be hydrolyzed by myrosinase, which can be produced either by plants or microorganisms. The Fusarium strain Ls-F-in-4-1 has a myrosinase activity but the strain Rs-F-in-11 has not. These results suggest that both strains are able to trigger the metabolic pathway leading to benzyl isothiocyanate production in the plant. In the case of the myrosinase-negative strain Rs-F-in-11, hydrolyzation into isothiocyanate is only due to the myrosinase activity of the plant, and in the other case, the myrosinase produced by the strain Ls-F-in-11 also would contribute to the production of isothiocyanate. This paper reports a new mode of action of non-pathogenic Fusarium strains in controlling P. ultimum.     

Improved zinc tolerance in Eucalyptus globulus inoculated with Glomus deserticola and Trametes versicolor or Coriolopsis rigida

The potential of interactions between saprophytic and arbuscular mycorrhizal (AM) fungi to improve Eucalyptus globulus grown in soil contaminated with Zn were investigated. The presence of 100 mg kg ⁻¹ Zn decreased the shoot and root dry weight of E. globulus colonized with Glomus deserticola less than in plants not colonized with AM. Zn also decreased the extent of root length colonization by AM and the AM fungus metabolic activity, measured as succinate dehydrogenase (SDH) activity of the fungal mycelium inside the E. globulus root. The saprophytic fungi Trametes versicolor and Coriolopsis rigida increased the shoot dry weight and the tolerance of E. globulus to Zn when these plants were AM-colonized. Both saprophytic fungi increased the percentage of AM root length colonization and elevated G. deserticola SDH activity in the presence of all Zn concentrations applied to the soil. In the presence of 500 and 1000 mg kg⁻¹ Zn, there were higher metal concentrations in roots and shoots of AM than in non-AM plants; furthermore, both saprophytic fungi increased Zn uptake by E. globulus colonized by G. deserticola. The higher root to shoot metal ratio observed in mycorrhizal E. globulus plants indicates that G. deserticola enhanced Zn uptake and accumulation in the root system, playing a filtering/sequestering role in the presence of Zn. However, saprophytic fungi did not increase the root to shoot Zn ratio in mycorrhizal E. globulus plants. The effect of the saprophytic fungi on the tolerance and the accumulation of Zn in E. globulus was mediated by its effect on the colonization and metabolic activity of the AM fungi.     

Fusarium species in soil,thatch and crowns of Poa pratensis turfgrass treated with fungicides

Little information exists concerning the effects of long-term fungicide programs on the qualitative and quantitative characteristics of fusaria in turfgrasses and in turfgrass soils. These effects were studied on Poa pratensis L, turfgrasses at three locations, using 14 fungicides, one nematicide, and five mixed fungicide programs. Some fungicides increased the numbers of fusaria in soil and thatch, some had no effect, and others greatly reduced the numbers. Changes in Fusarium species' compositions occurred independently from the changes in propagule numbers. The proportion of Fusarium-colonized turfgrass crowns was generally higher in fungicide-treated than in nontreated turfgrasses.     

Potential gene exchange between Bacillus thuringiensis subsp. kurstaki and Bacillus spp. in soil in situ

The possible transfer of genes from Bacillus thuringiensis subsp. kurstaki (Btk) to indigenous Bacillus spp. was investigated in soil samples from stands of cork oak in Orotelli (Sardinia, Italy) collected 5 years after spraying of the stands with a commercial insecticidal preparation (FORAY 48B) of Btk. Two colonies with a morphology different from that of Btk were isolated and identified as Bacillus mycoides by morphological and physiological characteristics and by 16S rDNA analysis. Amplification by the polymerase chain reaction (PCR) of the DNA of the two isolated B. mycoides colonies with primers used for the identification of the Btk cry genes showed the presence of a fragment of 238 bp of the cry1Ab9 gene that had a similarity of 100% with the sequence of the cry1Ab9 gene present in GenBank, indicating that the isolates of B. mycoides acquired part of the sequence of this gene from Btk. No cells of Btk or B. mycoides carrying the 238-bp fragment of the cry1Ab9 gene were isolated from samples of unsprayed control soil. However, the isolates of B. mycoides were not able to express the partial Cry1Ab protein. Hybridization with probes for IS231 and the cry1Ab9 gene suggested that the inverted repeated sequence, IS231, was probably involved in the transfer of the 238-bp fragment from Btk to B. mycoides. These results indicate that transfer of genes between introduced Btk and indigenous Bacillus spp. can occur in soil under field conditions.     

Effects of Medicago sativa, Taraxacum officinale and Bromus sterilis on the density and diversity of Collembola in grassy arable fallows of different ages

The density and diversity of Collembola of nine grassy arable fallows of different ages were investigated in a factorial design with the factors ‘plant species’ (legume: Medicago sativa, herb: Taraxacum officinale, grass: Bromus sterilis) and ‘age class’ (2-3, 6-8 and 12-15 years) including the random effect ‘site’ (1-9). In May 2008, four plots were selected randomly at each fallow. Within each plot five M. sativa, T. officinale and B. sterilis plants were extracted with their associated soil using steel cylinders. The material from each plant species was used for extraction of Collembola and for determination of environmental parameters. Thus, the new aspect of the present study compared to other field studies investigating the relationships between plant and Collembolan communities is the focus on the “micro-scale”, investigating the Collembolan communities of the soil associated with single plants.We found that species richness and density of total and euedaphic Collembola were significantly higher in B. sterilis than in T. officinale samples with the M. sativa samples being intermediate. Fine-root feeding euedaphic Collembola particularly benefited from the higher amount of fine roots in B. sterilis samples. We also discovered that the age of the fallows had no significant influence on the number of Collembolan species and the density of the Collembolan groups. Notably, however, species of the epedaphic genera Lepidocyrtus and Sminthurinus were associated with the 12-15 year-old fallows and presumably benefited from the high number of plant species in the old fallows. Finally, canonical correspondence analysis (CCA) indicated that the soil organic matter content and the microbial biomass, both potential food sources for many Collembolan species, were important structuring forces for the Collembolan communities.     

Accumulation of specific flavonoids in soybean (Glycine max (L.) Merr.) as a function of the early tripartite symbiosis with arbuscular mycorrhizal fungi and Bradyrhizobium japonicum (Kirchner) Jordan

This study is the first report assessing the effect of soil inoculation on the signalling interaction of Bradyrhizobium japonicum, arbuscular mycorrhizal fungi (AMF) and soybean plants throughout the early stages of colonisation that lead to the tripartite symbiosis. In a study using soil disturbance to produce contrasting indigenous AMF treatments, the flavonoids daidzein, genistein and coumestrol were identified as possible signals for regulating the establishment of the tripartite symbiosis. However, it was unclear whether soil disturbance induced changes in flavonoid root accumulation other than through changing the potential for AMF colonization. In this study, soil treatments comprising all possible combinations of AMF and B. japonicum were established to test whether (1) modifications in root flavonoid accumulation depend on the potential for AMF colonization, and (2) synthesis and accumulation of flavonoids in the roots change over time as a function of the early plant-microbial interactions that lead to the tripartite symbiosis. The study was comprised of two phases. First, maize was grown over 3-week periods to promote the development of the AM fungus Glomus clarum. Second, the interaction between soybean, G. clarum and B. japonicum was evaluated at 6, 10, 14 and 40 days after plant emergence. Root colonization by G. clarum had a positive effect on nodulation 14 days after emergence, producing, 30% more nodules which were 40% heavier than those on roots solely inoculated with B. japonicum. The tripartite symbiosis resulted in 23% more N₂ being fixed than did the simpler symbiosis between soybean and B. japonicum. The presence of both symbionts changed accumulation of flavonoids in roots. Daidzein and coumestrol increased with plant growth. However, development of the tripartite symbiosis caused a decrease in coumestrol; accumulation of daidzein, the most abundant flavonoid, was reduced in the presence of AMF.