Effects of pollination distance on reproduction and offspring performance in Hypochoeris radicata: Experiments with plants from three European regions

Fragmentation of habitats has resulted in increased inbreeding for many plant species, while the introduction of foreign seed material for ecological restoration has resulted in crosses between plants from distant populations. Both processes may reduce plant fitness and increase the risk of extinction. Variation in the expression of inbreeding and outbreeding depression has been found among different genotypes and among populations, but little is known about large scale geographical patterns within species. We studied the effects of cross-proximity on seed production and offspring performance in the perennial meadow plant Hypochoeris radicata (Asteraceae) from five populations in each of three European regions (Bohemia in northwest Czechia, Northern Hesse in central Germany, and Salland in the central Netherlands). Five artificial cross types were conducted with varying proximity of mates: selfing (self), within family crosses (WF), within population crosses (WP), between population crosses (BP), and between region crosses (BR), and the offspring were grown in a common garden. Independent of the region of origin of the maternal plant, selfing, WF and BP crosses resulted in lower seed set and germination than WP crosses, indicating partial self-incompatibility, inbreeding depression and reduced performance in the F1 progeny resulting from outbreeding. However, crosses between regions resulted in similar seed set and germination as within population crosses. For late traits, the effects of inbreeding and interpopulation crosses differed among regions. WP crosses exhibited the highest survival, flowering and multiplicative fitness only in progeny from Czech maternal plants. Our results suggest that the sensitivity of populations to introgression may vary among regions and that outbreeding depression does not necessarily increase with interpopulation distance. However, the current study investigated only effects in the F1 in a common garden and outbreeding depression may be stronger in the F2 and in field populations.     

Reforestation of burned stands: The effect of ectomycorrhizal fungi on Pinus pinaster establishment

The area occupied by Pinus pinaster in Portugal is rapidly diminishing because of forest fires. Ectomycorrhizal fungi form obligate, mutually beneficial associations with P. pinaster which improve plant growth and resistance to adverse conditions. The aim of this work was to assess whether native ectomycorrhizal fungi could be a useful tool in the reforestation of burned areas. The work was conducted in a forest nursery greenhouse, where P. pinaster seedlings were inoculated with compatible ectomycorrhizal fungal isolates: Suillus bovinus, Pisolithus tinctorius, Rhizopogon roseolus, and a mixture of the three fungi, using burned and unburned forest soil as substrate. Inoculation significantly enhanced the growth of P. pinaster, with R. roseolus proving to be the most effective in burned soil, with an 8-fold increase in plant fresh weight. Overall, inoculation stimulated growth most in burned than in unburned soil.This study suggests that inoculation with selected ectomycorrhizal fungi in containerised nurseries can be an advantageous approach for the successful establishment of P. pinaster in burned soil. The obtained results point out to the interest of extending these studies into fire-impacted areas, using ectomycorrhizal fungi as a biological tool.     

Colonization of the rhizosphere, rhizoplane and endorhiza of garlic (Allium sativum L.) by strains of Trichoderma harzianum and their capacity to control allium white-rot under field conditions

Five strains (C4, C13, C17, C30, C44) of Trichoderma harzianum that efficiently antagonise Sclerotium cepivorum in vitro, were used in central México to inoculate field grown garlic (Allium sativum L.). Endorhiza, rhizosphere and soil colonization were evaluated at different times and at various root and soil depths. Experiments were conducted in both the 2000 and 2001 crop cycles to evaluate the capacity of five strains as biological control agents (BCA) against S. cepivorum. Significant differences were observed among the strains in terms of their ecological behavior. Strain C4 showed the best endorhizal and rhizosphere colonization. Strain C44, besides being a good rhizosphere colonizer, dispersed widely in the soil. In terms of all three variables, strain C30 had poor colonization ability, whereas the strains C4, C17 and C44 were best able to control white rot in the field. Of these, strain C4 showed the best performance over 2 years when it was applied as a pre-colonized substrate at planting time. High early endorhizal colonization was positively correlated with the BCA capacity of the strains. These data suggest that the ecological behavior of Trichoderma strains should be included as a criterion for strain selection for biological control purposes.     

Survival and reproductive performance of Hawksbill turtles Eretmochelys imbricata L. on Cousin Island,seychelles

Hawksbill turtles have been tagged on Cousin Island from 1973 to the present day. Recently therefore the breeding females in a season have included turtles not previously tagged and remigrants, turtles known from their tags to have nested on Cousin in at least one past season. This paper details some differences in the two groups. Remigrants produced larger clutches (176 v. 153), laid more clutches on Cousin in a season (3·68 v. 2·50) and were about five times more likely to be recaptured there in a subsequent season. Overall 25–30% of the hawksbills tagged were recaptured in a later season. The mean interval between the successive breeding seasons of individual females was 2·67 years.     

非寄主蔬菜及其提取物对菜粉蝶的驱避作用

在室内和田间测定了番茄、茴香、荆芥、莴苣、香菜和丝瓜6种非寄主蔬菜对菜粉蝶的驱避作用。室内测定结果表明,用非寄主蔬菜粗提物处理甘蓝后,菜粉蝶成虫对甘蓝的选择反应率降低,尤其是交配雌蝶对荆芥、茴香、番茄和莴苣提取物处理的甘蓝的选择反应率最低,分别仅为3.67%、4.30%、5.27%和8.26%。在非寄主蔬菜粗提物处理过的甘蓝苗上,成虫的产卵量明显减少,其中以番茄、茴香和荆芥粗提物的驱避活性最强,处理后1d的非选择性产卵驱避率分别为91.04%、75.54%和71.67%,选择性产卵驱避率分别为94.64%、79.66%和78.96%。田间试验结果表明,甘蓝田套种番茄、茴香和荆芥后,可使田间落卵量分别减少68.80%、62.62%和55.09%,这3种非寄主蔬菜对菜粉蝶的有效驱避距离分别为4m、2m和2m左右。因此,可以通过在甘蓝田套种非寄主蔬菜或喷洒其粗提物驱避菜粉蝶,减少幼虫发生为害。     

Transfer and loss of naturally-occurring plasmids among isolates of Rhizobium leguminosarum bv. viciae in heavy metal contaminated soils

Plasmid transfer among isolates of Rhizobium leguminosarum bv. viciae in heavy metal contaminated soils from a long-term experiment in Braunschweig, Germany, was investigated under laboratory conditions. Three replicate samples each of four sterilized soils with total Zn contents of 54, 104, 208 and 340 mg kg⁻¹ were inoculated with an equal number (1×10⁵ cells g⁻¹ soil) of seven different, well-characterized isolates of R. leguminosarum bv. viciae. Four of the isolates were from an uncontaminated control plot (total Zn 54 mg kg⁻¹) and three were from a metal-contaminated plot (total Zn 340 mg kg⁻¹).After 1 year the population size was between 10⁶ and 10⁷ g⁻¹ soil, and remained at this level in all but the most contaminated soil. In the soil from the most contaminated plot no initial increase in rhizobial numbers was seen, and the population declined after 1 year to <30 cells g⁻¹ soil after 4 years. One isolate originally from uncontaminated soil that had five large plasmids (no. 2-8-27) was the most abundant type re-isolated from all of the soils. Isolates originally from the metal-contaminated soils were only recovered in the most contaminated soil. After 1 year, four isolates with plasmid profiles distinct from those inoculated into the soils were recovered. One isolate in the control soil appeared to have lost a plasmid. Three isolates from heavy metal contaminated soils (one isolate from the soil with total Zn 208 mg kg⁻¹ and two isolates from the soil with total Zn 340 mg kg⁻¹) had all acquired one plasmid. Plasmid transfer was confirmed using the distinct ITS-RFLP types of the isolates and DNA hybridization using probes specific to the transferred plasmid. The transconjugant of 2-8-27 which had gained a plasmid was found in one replicate after 2 years of the most contaminated soil but comprised more than 50% of the isolates. A similar type appeared in a separate replicate of the most contaminated soil after 3 years and persisted in both of these soils until the final sampling after 4 years. After 2 years isolates were recovered from four of the soil replicates with the chromosomal type of 2-8-27 which appeared to have lost one plasmid, but these were not recovered subsequently.Isolate 2-8-27 was among the isolates most sensitive to Zn in laboratory assays, whereas isolate 7-13-1 showed greater zinc tolerance. Acquisition of the plasmid conferred enhanced Zn tolerance to the recipients, but transconjugant isolates were not as metal tolerant as 7-13-1, the putative donor. Laboratory matings between 2-8-27 and 7-13-1 in the presence of Zn resulted in the conjugal transfer of the same small plasmid from 7-13-1 to isolate 2-8-27 and the transconjugant had enhanced metal tolerance. Our results show that transfer of naturally-occurring plasmids among rhizobial strains is stimulated by increased metal concentrations in soil. We further demonstrate that the transfer of naturally-occurring plasmids is important in conferring enhanced tolerance to elevated zinc concentrations in rhizobia.     

Effects of interactions between arbuscular mycorrhizal fungi and bacteria on the growth of Lotus corniculatus L.: From the perspective of regulating rhizosphere fungal community

Arbuscular mycorrhizal fungi (AMF) provide essential nutrients to crops and are affected by fertilizers. Phosphate-solubilizing bacteria (PSB), nitrogen-fixing bacteria (NFB), and AMF have mutually beneficial relationships with plants, but the effects of their interactions on plant growth by regulating rhizosphere fungal community have not been sufficiently studied. In this study, a greenhouse pot experiment was conducted to investigate the interactions between AMF and bacteria (PSB and NFB) on the growth of Lotus corniculatus L. Specifically, the role of rhizosphere fungal community in the growth of Lotus corniculatus L. was explored using Illumina MiSeq high-throughput sequencing. The results showed that combined inoculation of AMF with PSB and NFB increased plant biomass, plant height, and fungal colonization rate. The richness, complexity, and stability of rhizosphere fungal community also increased after combined inoculation of AMF with PSB and/or NFB, particularly with PSB. In addition, combined inoculation of AMF with PSB and NFB enriched the abundance of beneficial microorganisms, with Chaetomium and Humicola showing the greatest alterations. The structural equation model showed that the interactions of AMF with PSB and NFB promoted plant growth by affecting fungal network structure and soil enzyme activities involved in carbon, nitrogen, and phosphorus cycling. These findings provide evidence for the effects of interactions of AMF with PSB and NFB on rhizosphere fungal community and plant growth.     

Quantification of Wautersia [Ralstonia] basilensis in the mycorrhizosphere of Pinus thunbergii Parl. and its effect on mycorrhizal formation

The bacterium Wautersia [Ralstonia] basilensis has been shown to enhance the mycorrhizal symbiosis between Suillus granulatus and Pinus thunbergii (Japanese black pine). However, no information is available about this bacterium under field conditions. The objectives of this study were to detect W. basilensis in bulk and mycorhizosphere soils in a Japanese pine plantation in the Tottori Sand Dunes, determine the density of W. basilensis in soil, and determine the optimal cell density of W. basilensis for mycorrhizal formation in pine seedlings. We designed and validated 16S rRNA gene-targeted specific primers for detection and quantification of W. basilensis. SYBR Green I real-time PCR assay was used. A standard curve relating cultured W. basilensis cell density (10³-10⁸ cells ml⁻¹) to amplification of DNA showed a strong linear relationship (R = 0.9968). The specificity of the reaction was confirmed by analyzing DNA melting curves and sequencing of the amplicon. The average cell density of W. basilensis was >4.8 × 10⁷ cells g⁻¹ of soil in the mycorrhizosphere and 7.0 × 10⁶ cells g⁻¹ in the bulk soil. We evaluated the W. basilensis cell density required for mycorrhizal formation using an in vitro microcosm with various inoculum densities ranging from 10² to 10⁷ cells g⁻¹ soil (10⁴-10⁹ cells ml⁻¹). Cell densities of W. basilensis of >10⁶ cells g⁻¹ of soil were required to stimulate mycorrhizal formation. In vivo and in vitro experiments showed that W. basilensis was sufficiently abundant to enhance mycorrhizal formation in the mycorrhizosphere of Japanese black pine sampled from the Tottori Sand Dunes.     

The genetic diversity of Rhizobium leguminosarum bv. viciae in cultivated soils of the eastern Canadian prairie

Soil populations of Rhizobium leguminosarum bv. viciae (Rlv) that are infective and symbiotically effective on pea (Pisum sativum L.) have recently been shown to be quite widespread in agricultural soils of the eastern Canadian prairie. Here we report on studies carried out to assess the genetic diversity amongst these endemic Rlv strains and to attempt to determine if the endemic strains arose from previously used commercial rhizobial inoculants. Isolates of Rlv were collected from nodules of uninoculated pea plants from 20 sites across southern Manitoba and analyzed by plasmid profiling and PCR-RFLP of the 16S-23S rDNA internally transcribed spacer (ITS) region. Of 214 field isolates analyzed, 67 different plasmid profiles were identified, indicating a relatively high degree of variability among the isolates. Plasmid profiling of isolates from proximal nodules (near the base of the stem) and distal nodules (on lateral roots further from the root crown) from individual plants from one site suggested that the endemic strains were quite competitive relative to a commercial inoculant, occupying 78% of the proximal nodules and 96% of the distal nodules. PCR-RFLP of the 16S-23S rDNA ITS also suggested a relatively high degree of genetic variability among the field isolates. Analysis of the PCR-RFLP patterns of 15 selected isolates by UPGMA indicated two clusters of three field isolates each, with simple matching coefficients (SMCs) ≥0.95. However, to group all field isolates together, the SMC has to be reduced to 0.70. Regarding the origin of the endemic Rlv strains, there were few occurrences of the plasmid profiles of field isolates being identical to the profiles of inoculant Rlv strains commonly used in the region. Likewise, the plasmid profiles of isolates from nodules of wild Lathyrus plants located near some of the sites were all different from those of the field isolates. However, comparison of PCR-RFLP patterns suggested an influence of some inoculant strains on the chromosomal composition of some of the field isolates with SMCs of ≥0.92. Overall, plasmid profiles and PCR-RFLP patterns of the isolates from endemic Rlv populations from across southern Manitoba indicate a relatively high degree of genetic diversity among both plasmid and chromosomal components of endemic strains, but also suggest some influence of chromosomal information from previously used inoculant strains on the endemic soil strains.     

Fruiting failure and limited recruitment in remnant populations of the hawkmoth-pollinated tree Oxyanthus pyriformis subsp. pyriformis (Rubiaceae)

Plants with highly specialized pollination systems may be particularly vulnerable to the effects of habitat fragmentation. The sub-canopy tree Oxyanthus pyriformis subsp. pyriformis has deep tubular flowers (c. 90 mm in length) that are specialized for pollination by long-tongued hawkmoths. Existing natural populations of this taxon appear to be confined to two suburban habitat fragments in the city of Durban, South Africa. Breeding system experiments showed that the trees are self-incompatible and thus completely reliant on pollen vectors for sexual reproduction. Hawkmoths were rarely observed to visit flowers, despite intensive monitoring over four seasons from 1997 to 2003. Fruit set as determined from examination of more than 150,000 flowers at the two sites was extremely low (0.04% and 0.07% of flowers), but increased to more than 70% in flowers that were hand-pollinated with pollen from another tree. This >1000-fold increase in the likelihood of fruit production following pollen-supplementation indicates that trees experience severe pollen-limitation of fruit set. Natural fruit set is so low that only a few dozen fruits were recorded in each population during two years of monitoring. This is of concern as O. pyriformis trees are dependent solely on seeds for recruitment. Demographic surveys showed that remaining natural populations have fewer saplings than adult trees and only two seedlings for every adult tree. The long-term viability of reintroduced populations of O. pyriformis is also in doubt, as we observed an almost complete lack of recruitment in a population that was established in a seemingly suitable forest site 20 years ago. Regular hand-pollination and, in some instances, further planting of saplings, may be the only way to safeguard these populations.     

Local perceptions of jaguars (Panthera onca) and pumas (Puma concolor) in the Iguaçu National Park area, south Brazil

Jaguars (Panthera onca) have been killed by local residents within the boundaries and lands surrounding Iguaçu National Park (INP), Brazil. Both jaguars and pumas (Puma concolor) occur in the region, however, livestock predation by pumas has rarely been reported. Our objective was to assess the local perceptions about jaguars and pumas. We identified two major factors that distinguished the perceptions towards the two species: less people feared the puma than the jaguar; and most people believed that jaguars, but not pumas, were released into INP by local authorities. Interestingly, despite those major differences in these perceptions, feelings towards the two species tended to be the same. Perceptions towards jaguars were not influenced by the predation history of the properties, suggesting that the predation impact was not remarkable enough to influence local perceptions towards carnivores. This is apparently the first study on local perceptions towards large carnivores in Brazil.     

Estimation of nitrogen fixation by Trifoliumsubterraneum L. and Medicago truncatula gaertn. grown in pots using a non-destructive acetylene reduction assay

Non-destructive acetylene reduction assays were successfully performed using small (1.21) incubation chambers and a 1 h incubation at 20°C. The concentration of C₂H₂ substrate used in the assays reached saturation at a partial pressure of 10 kPa for nodulated subterranean clover and barrel medic grown in a sandy loam. The optimum rates of C₂H₂ reduction associated with both species occurred within the range of 25–50% of the soil moisture content at field capacity (33 kPa). The ratio of moles N₂ fixed to moles C₂H₂ reduced was calculated to be 1:2.9 for the subterranean clover—Rhizobium symbiosis and 1:3.3 for the barrel medic—Rhizobium symbiosis.     

嘉陵江与乌江重要渔业功能区水生态环境质量研究

研究结果表明,嘉陵江与乌江重要渔业功能区渔业水生态环境质量综合评价均为轻污染,部分采样点重金属和有机物均有超标现象,单项污染指数表明水质主要污染物为总P和石油类,总P超标严重,水质pH值、总P、挥发酚、石油类、Cr~(6 )、非离子氨具有相关性。并提出改善该渔业功能区水生态环境质量的对策。     

The effect of acidity on the production of signal molecules by Medicago roots and their recognition by Sinorhizobium

The Medicago sativa-Sinorhizobium symbiosis is challenged by acidity, resulting in generally poor nodulation and production. Medicago murex, however, can nodulate and grow at low pH. The effect of low pH on signal exchange in the Sinorhizobium-Medicago symbiosis was studied to gain a greater understanding of the basis for poor nodulation of M. sativa compared to M. murex. Root exudates from M. sativa and M. murex grown in buffered nutrient solution at pH 4.5, 5.8 and 7.0, were collected to measure the expression of nodB induction in Sinorhizobium. A nodB-gusA fusion was constructed and inserted into Sinorhizobium medicae strains WSM419 (acid tolerant) and CC169 (acid sensitive). We identified greater induction by root exudates from both Medicago spp. collected at pH 4.5 than at pH 5.8 and 7.0, less induction by M. murex than M. sativa and less induction of WSM419 than CC169. The same major inducing compounds, 4′,7-dihydroxyflavanone (liquiritigenin), 4′,7-dihydroxyflavone, and 2′,4′,4-trihydroxychalcone (isoliquiritigenin), were identified in exudates of M. murex and M. sativa at all pH values, although in increasing amounts at lower pH. Poor nodulation of M. sativa relative to M. murex under acid conditions is not the consequence of decreased induction of Sinorhizobium nodB by chemical inducers present in the root exudates of both species at low pH.     

The bioprotective effect of AM root colonization against the soil-borne fungal pathogen Gaeumannomyces graminis var. tritici in barley depends on the barley variety

The systemic effect of root colonization by the arbuscular mycorrhizal fungus (AMF) Glomus mosseae on the susceptibility of old and modern barley varieties to the soil-borne fungal pathogen Gaeumannomyces graminis var. tritici (Ggt) was studied in a split-root system. Plants were precolonized on one side of the split-root system with the AMF and thereafter the other side of the split-root system was inoculated with the pathogen. At the end of the experiment the level of bioprotection was estimated by quantifying lesioned roots and the determination of the root fresh weight. AM root colonization provided protection in some of the barley genotypes tested, but not in others. This protective effect seemed to vary in the oldest and the most modern barley variety tested.     

The establishment of heathland vegetation on ex-arable land: the response of Calluna vulgaris to soil acidification

The UK Biodiversity Action Plan has identified the creation of lowland heathland as an important objective. Heathland restoration studies have identified soil pH, elevated soil nutrients and large weed seed banks as major problems in the restoration of heathland vegetation on ex-arable land. Heathland vegetation is usually found on nutrient-poor acidic soils. Creating acidic soil conditions on ex-arable sites thus may produce a suitable environment for the establishment of heath vegetation. Soil acidification by the addition of sulphur has been shown to reduce the soil pH and the availability of nutrients in arable soils. A series of experiments was established to investigate the effects of soil acidification using sulphur on the establishment of Calluna vulgaris and the development of weed vegetation. The application of sulphur at 0.24 kg m⁻² to an arable soil was found to increase the survival rate of C. vulgaris cuttings planted in it. The mechanism of increased C. vulgaris survival appeared to be by sulphur application significantly reducing the cover of arable weeds arising from the soil seed bank. Higher rates of sulphur application (0.36 and 0.48 kg m⁻²) resulted in the death of many C. vulgaris plants. However C. vulgaris seedlings were able to establish successfully on these ex-arable soils within 18-24 months following the addition of these levels of sulphur. The application of sulphur appears to offer a practical solution to heathland creation on ex-arable land. However, it may be necessary to provide an interval of between 18 and 24 months between the application of sulphur and the addition of C. vulgaris plants or seeds for the successful establishment of heathland vegetation.     

The natural environment of Cupressus sempervirens in Greece as a basis for its use in the Mediterranean region

This paper studies the natural stands of Cupressus sempervirens L. in Greece. C. sempervirens stands are located on the islands of Crete, Samos, Rhodes, Kos, Symi, and Milos, while populations on the islands of Chios and Kalymnos were discovered recently. Stands in the Peloponnese and in the Ionian Islands are considered introduced and naturalized. Data on natural populations show that C. sempervirens is indifferent to physiographic factors, substrate material, and physicochemical soil properties. The species grows on sites ranging from the semiarid bioclimatic zone with warm winters, to the wet zone with cold and fairly harsh winters. According to the xerothermic index in the taxon's natural distribution area, all Mediterranean bioclimate characters may appear, from intense thermo‐Mediterranean to weak meso‐Mediterranean. Cypress participates in all the vegetation zones from the thermo‐Mediterranean to mountainous Mediterranean and a cluster analysis (TWINSPAN) of sixty‐two cypress relevés led to the recognition of six woodland types defined by species composition, and assigned to three alliances: Oleo‐Ceratonion, Quercion ilicis, and Acero‐Cupression. This great plasticity of the species with regard to natural environmental factors, together with its relative resistance to fire, renders cypress one of the most valuable forest species for a large part of the Mediterranean region. Copyright © 2006 John Wiley & Sons, Ltd.     

The adverse effects of soil salinization on the growth of Trifolium alexandrinum L. and associated microbial and biochemical properties in a soil from Iran

The aim of this study was to determine the effects of increasing concentrations of salt solutions (including 0.12, 2, 6, and 10 dS m⁻¹) on the growth of berseem clover (Trifolium alexandrinum L.) and related soil microbial activity, biomass and enzyme activities. Results showed that the dry weights of root and shoot decreased with an increase in the concentrations of salt solutions. Soil salinization depressed the microbiological activities including soil respiration and enzyme activities. Substrate-induced respiration was consistently lower in salinized soils, whereas microbial biomass C did not vary among salinity levels. Higher metabolic quotients (qCO₂) and unaffected microbial biomass C at high EC values may indicate that salinity is a stressful factor, inducing either a shift in the microbial community with less catabolic activity or reduced efficiency of substrate utilization. Acid phosphatase and alkaline phosphatase activities decreased with increasing soil salinity. We found significant, positive correlations between the activities of phosphatase enzymes and plant's root mass, suggesting that any decrease in the activities of the two enzymes could be attributed to the reduced root biomass under saline conditions.     

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.