Carbohydrate, protein and amino acid status of Glycine-Glomus-Bradyrhizobium symbioses

Soybean [ Glycine max (L.) Merr. cv. Amsoy 71] plants were inoculated with either the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus fasciculatum , with a strain of Bradyrhizobium japonicum . or with both endophytes together. Non-inoculated plants were fertilized with levels of N and P that resulted in plant growth similar to that following infection by Bradyrhizobium or Glomus , respectively. In general, plants colonized by Glomus contained more pinitol (1 d -3-O-methyl- chiro -inositol) but less starch than corresponding P-fertilized soybeans, while nodulated soybeans contained more pinitol and sucrose in the roots than corresponding N-fertilized plants. Compared to nutrient-amended soybeans, increases or decreases in free amino acids in symbiotically grown plants tended to follow transient changes in N content and endophyte activity. The exception to this was the elevated levels of methionine in nodulated roots or aspartate and arginine in Glomus -inoculated roots regardless of N level. There were both quantitative and qualitative changes in the proteins found in inoculated soybeans, especially in infected roots where nodulins (nodule-specific proteins) and endomycorrhizins (mycorrhiza-specific proteins) were detected by polyacrylamide gel eletrophoresis. Five nodulins (11.8% of root proteins) were detected with molecular weights of 14, 34, 40, 41 and 100 kDa. Five endomycorrhizins (5.1% of root proteins) were detected with molecular weights of 16, 17, 18, 22 and 30 kDa. Differences in nutrient uptake and allocation in symbiotic plants could not be duplicated by fertilizer input alone, although nutrient addition could lead to similar growth rates and dry weight accumulation. These findings suggest that the quality and quantity of plant carbohydrates, proteins and amino acids shift in response to the physiological changes resulting from infection by N₂-fixing bacteria or endomycorrhizal fungi.     

Nutrient transfer between the root zones of soybean and maize plants connected by a common mycorrhizal mycelium

The objective of the study was to determine whether nutrient fluxes mediated by hyphae of vesicular-arbuscular mycorrhizal (VAM) fungi between the root zones of grass and legume plants differ with the legume's mode of N nutrition. The plants, nodulating or nonnodulating isolines of soybean [ Glycine max (L.) Merr.], were grown in association with a dwarf maize ( Zea mays L.) cultivar in containers which interposed a 6-cm-wide root-free soil bridge between legume and grass container compartments. The bridge was delimited by screens (44 μm) which permitted the passage of hyphae, but not of roots and minimized non VAM interactions between the plants. All plants were colonized by the VAM fungus Glomus mosseae (Nicol. & Gerd.) Gerd. and Trappe. The effects of N input to N-sufficient soybean plants through N₂-fixation or N-fertilization on associated maize-plant growth and nutrition were compared to those of an N-deficient (nonnodulating, unfertilized) soybean control. Maize, when associated with the N-fertilized soybean, increased 19% in biomass, 67% in N content and 77% in leaf N concentration relative to the maize plants of the N-deficient association. When maize was grown with nodulated soybean, maize N content increased by 22%, biomass did not change, but P content declined by 16%. Spore production by the VAM fungus was greatest in the soils of both plants of the N-fertilized treatment. The patterns of N and P distribution, as well as those of the other essential elements, indicated that association with the N-fertilized soybean plants was more advantageous to maize than was association with the N₂-fixing ones.     

The Glycine–Glomus–Rhizobium symbiosis. IV. Interactions between the mycorrhizal and nitrogen-fixing endophytes

Abstract Soybean (Glycine max (L.) Men) plants were grown under controlled conditions in an experiment designed as a 4 × 4 factorial. The factors were N or P nutrition, with different strains of Rhizobium japonicum or N-fertilization as levels of the first factor and different species of vesicular-arbuscular mycorrhizal (VAM) fungi or P fertilization as levels of the other. Organisms used were R. japonicum strains USDA 110, USDA 136, and 61A118, and the VAM fungi Glomus versiforme (Karst.) Berch, Glomus fasciculatum (Thaxt. sensu Gerd.) Gerd. and Trappe, and Glomus mosseae (Nicol. & Gerd.) Gerd. and Trappe. There were 16 treatments: nine Rhizobium + Glomus combinations, three Rhizobium + V and three Glomus+ N combinations, and one non-symbiotic set of plants supplied with N + P. The tripartite symbioses were evaluated by analysis of variance against the Rhizobium + P and Glomus + N comparison treatments for effects on root and leaf dry mass, root N and P content, nodule mass and activity, and VAM colinization. Significant to highly significant main effects and interactions were found in virtually all evaluations due to both Rhizobium strain and VAM–fungal species. We conclude that different endophyte isolates affect not only the host plant, but also the development and function of their co-endophytes. These findings establish the existence of inter-endophyte compatibility, an important consideration when selecting or engineering for desirable endophyte traits.     

Influence of inoculation withAzospirillum brasilense andGlomus fasciculatum on sorghum nutrition

Sorghum (Sorghum bicolor (L.) Moench cv. Bok 8) plants were inoculated with either the vesicular-arbuscular mycorrhizal (VAM) fungusGlomus fasciculatum, with a strain ofAzospirillum brasilense, or with both endophytes together. Non-inoculated plants were fertilized with quantities of N and P that had been found to compensate for the input of nutrients following azospirillum or glomus colonization. Total plant dry weight in all treatments was statistically indistinguishable at harvest (10 weeks). In general, plants colonized by Glomus contained less P, Mn, starch and sucrose, but more Cu, Zn and proline than P-fertilized plants. Azospirillum-inoculated sorghum contained less N, glucose, threonine and glutamine, but more Fe and glutamate than N-amended plants. Mycorrhizal roots contained five specific fatty acids not found in non-VAM plants. Inoculated plants displayed altered nutrient requirements, membrane composition and metabolite levels, indicating that colonization by these endophytes influenced host physiology, even under conditions where N or P input was negligible.Contribution from the Western Regional Research Center, USDA-ARS (CRIS No. 5325-41000-008).     

Growth and nutrient uptake of sorghum cultivated with vesicular-arbuscular mycorrhiza isolates at varying pH

This study was conducted to determine the effects of different pH regimes on root colonization with four vesicular-arbuscular mycorrhiza (VAM) isolates, and VAM effects on host plant growth and nutrient uptake. Sorghum [Sorghum bicolor (L.) Moench] was grown at pH 4.0, 5.0, 6.0 and 7.0 (±0.1) in hydroponic sand culture with the VAM isolates Glomus etunicatum UT316 (isolate E), G. intraradices UT143 (isolate I), G. intraradices UT126 (isolate B), and an unknown Glomus isolate with no INVAM number (isolate A). Colonization of roots with the different VAM isolates varied differentially with pH. As pH increased, root colonization increased with isolates B and E, remained unchanged with isolate I, and was low at pH 4.0 and high at pH 5.0, 6.0, and 7.0 with isolate A. Isolates E and I were more effective than isolates A and B in promoting plant growth irrespective of pH. Root colonization with VAM appeared to be independent of dry matter yields or dry matter yield responsiveness (dry matter produced by VAM compared to nonmycorrhizal plants). Dry matter yield responsiveness values were higher in plants whose roots were colonized with isolates E and I than with isolates A and B. Shoot P concentrations were lower in plants colonized with isolates E and I than with isolates A and B or nonmycorrhizal plants. This was probably due to the dilution effect of the higher dry matter yields. Neither the VAM isolate nor pH had an effect on shoot Ca, Mg, Zn, Cu, and Mn concentrations, while the VAM isolate affected not only P but also S, K, and Fe concentrations. The pH x VAM interaction was significant for shoot K, Mg, and Cu concentrations.     

Development of two endomycorrhizal symbioses on soybean and comparison with phosphorus fertilization

Summary Soybean (Glycine max L. Merr. cv. Amsoy 71) plants were grown in a greenhouse in a soil very low in plant-available P, and plants were harvested 5 times over a 21-week growth period. Soybeans were inoculated with one of two species of VAM fungi or received daily one of three nutrient solutions of different P concentrations (0.0, 0.2, or 1.0mMP). Until week 9, the dry weights, leaf areas and developmental stage of soybeans inoculated withG. fasciculatum orG. mosseae were similar to the 1.0 or 0.2mMP-treated plants, respectively. Phosphorus concentrations were significantly lower in VAM plants at weeks 6 and 9 as compared to non-VAM soybeans given 1.0mMP, suggesting P input in VAM plants was immediately used for new growth. Total P input for VAM plants was linear over 21 weeks, and the average rate of P uptake for these plants was 0.19mg P d⁻¹. Estimated specific P uptake rates (SPUR) for the mycorrhizae (VAM roots) were twice that of the control (0.0mMP) roots. The calculated SPURs forG. fasciculatum andG. mosseae hyphae were 95 and 120μg P g⁻¹ VAM d⁻¹ respectively, a 4 to 5 fold increase over non-inoculated roots, indicating more attention must be paid to P assimilation by VAM fungi in P-fixing substrates. Contribution from the Western Regional Research Center, USDA-ARS (CRIS No. 5325-20580-003).     

Influence of temperature on colonization of spring barleys by vesicular arbuscular mycorrhizal fungi

The effects of three soil temperatures on growth of spring barleys (Hordeum vulgare L.) and on their root colonization by vesicular arbuscular mycorrhizal (VAM) fungi from agricultural soils in Montana (USA) or Syria at different inoculum concentrations were tested in soil incubators in the greenhouse. The number of mycorrhizal plants as well as the proportion and intensity of roots colonized increased with higher soil temperatures. VAM fungi from Montana, primarily Glomus macrocarpum, were cold tolerant at 11°C while those from Syria, primarily G. hoi, were heat tolerant at 26°C. Inoculum potential of Montana VAM fungi was higher than Syrian VAM fungi in cool soils. Harmal, selected from Syrian barley land races, had the highest colonization by mycorrhizal fungi of the cultivars tested.Journal Series Paper: J-2532 Montana Agricultural Experiment Station.     

Characterization and localization of plant phenolics likely involved in the pathogen resistance expressed by endomycorrhizal roots

Phytochemical characterization of the major phenolic compounds and their ultrastructural localization were carried out on onion roots (Allium cepa L.) colonized by two vesicular-arbuscular mycorrhizal (VAM) fungi: Glomus intraradix Schenck & Smith and G. versiforme (Karst.) Berch. Free and wall-bound forms of phenolic components were quantified in relation to the duration of symbiosis. Both ferulic and p-coumaric acids, as well as N-feruloyltyramine were identified as the major phenolic metabolites bound to the cell walls of VAM onion roots. Results from mycorrhized and control plants suggest the presence of a mechanism leading to the oxidative condensation of phenols, the latter process depending on the presence or absence of symbiosis. Bioassays reveal that N-feruloyltyramine induces the branching of hyphae and reduces total fungal development. The overall results lead us to suggest that the progressive binding of phenolic compounds in VAM roots is directly involved in the control of VAM endophytic establishment and development, as it gradually reduces the plasticity and elasticity of the symbiotic matrix. Phenolic compounds bound to cell walls could also be indirectly responsible for the resistance of VAM roots to pathogenic fungi, since they result in increased resistance by the cell wall to the action of digestive enzymes.Presented in part at the 31st Annual Meeting and Symposium of the Phytochemical Society of North America, Fort Collins, Colo., June 1991     

Studies on nodulated and mycorrhizal peanuts

Yields of fruits, plant size and chemical content of shoots, roots and seeds were higher in peanuts infected with Glomus mosseae. Infection with Glomus and Rhizobium stimulated nodulation and acetylene reduction rates. Dually infected plants given various phosphorus treatments, produced more fruits, dry matter, and nodules and contained more phosphorus and nitrogen compared with nodulated-only plants. Mycorrhizal infections were high in all treatments. Together these two endophytes contributed greatly to the vigour of the host.     

Rapid methods for quantifying VAM fungal infections in maize roots

Roots of maize (Zea mays cv W64A × W182E) infected by vesicular arbuscular mycorrhizal (VAM) fungi (Glomus versiforme (Karst) Berch or a Glomus species isolated from an alfalfa soil) exhibit a bright yellow pigmentation. The percentage of pigmented roots can be quantified by a rapid visual estimate or by a grid intersect method. Both methods gave similar estimates of VAM infection to those obtained using a grid intersect count on cleared roots stained with chlorazol black E. Thus for experimental or field evaluation where speed and quantity are important, the rapid visual estimate (less than one minute for each washed root system) yields reliable results. The yellow root intersect method takes longer (5–15 minutes per root system) but gives more reproducible results. The yellow root pigmentation is light sensitive However, root systems can be reliably assayed after 1 week when stored at 5°C in the dark or after 1 year if dried.     

Soil color and surface residue effects on seedling light environment

Three leafless pea cultivars (JI 1198, BS 142 and BS 4) with the same phenotype and similar patterns of development, were tested in a sterilized low-phosphate soil for their response to phosphate fertilizer and to vesicular-arbuscular mycorrhizal (VAM) infection by threeGlomus species. Cultivar JI 1198 was very responsive to phosphate but not to inoculation withGlomus mosseae, Glomus caledonium orGlomus epigaeum. Phosphate and VAM treatments increased growth of cultivar BS 142 but were ineffective with cultivar BS 4. Fungal infectivity could not be related with endophyte effectiveness at stimulating plant growth, although the percentage of root length infected by each one of the threeGlomus species did not vary between cultivars. Genetic differences among plant cultivars can thus markedly affect the symbiosis between the host root and VAM fungi; this suggests that potential host-endophyte combinations need to be evaluated before being tested in the field.     

Effect of soil liming and vesicular-arbuscular-mycorrhizal inoculation on the growth and micronutrient content of the teff plant

In a greenhouse experiment involving an acid soil teff [Eragrostis tef (Zucc.) Trotter] plants failed to grow unless the soil was limed or inoculated with either of two vesicular-arbuscular-mycorrhizal (VAM) fungi,Glomus mosseae orGlomus macrocarpum. Plant growth increased by liming and to a lesser extent by VAM fungal inoculation. Liming also enhanced root colonization by VAM fungi. Shoot micronutrient content generally increased as a result of inoculation, and decreased by increased lime applications.     

Effect of vesicular-arbuscular mycorrhizal fungi and ofagrobacterium radiobacter on maize growth

An influence of dual inoculation with the rhizosphere bacteriumAgrobacterium radiobacter, and the VAM fungi,Glomus mosseae andGlomus sp., on maize growth and mycorrhizal infection was observed. Separate inoculations of bacteria or fungi showed significant positive effects on the shoot biomass production of pot-cultured plants only at the last of three consecutive harvests. Plant biomass production was enhanced substantially after a dual inoculation with bacteria and fungi. Synergistic interaction of fungal and bacterial inoculation and growth stimulation was evident at all three harvests compared to uninoculated plants and also compared to plants inoculated with fungi or bacteria only. The dual inoculation increased the shoot biomass of plants by approximately 30% as compared with control. No significant differences were found in mycorrhizal infection between plants uninoculated and inoculated with bacteria.Agrobacterium radiobacter seems to be compatible with mycorrhizal symbiosis and can act a synergistic partner of some VAM fungi.     

Enhanced N-Transfer from a Soybean to Maize by Vesicular Arbuscular Mycorrhizal (VAM) Fungi

Using a split-root technique, roots of soybean plants were divided between two pots. In one of the two pots, two maize plants were grown and half of those pots were inoculated with the vesicular arbuscular mycorrhizal (VAM) fungus, Glomus fasciculatus. Fifty-two days after planting, ¹⁵N-labeled ammonium sulfate was applied to the pots which contained only soybean roots. Forty-eight hours after application, significantly higher values for atom per cent ¹⁵N excess were found in roots and leaves of VAM-infected maize plants as compared with the non-VAM-infected maize plants. Results indicated that VAM fungi did enhance N transfer from one plant to another.     

A fungal root symbiont modifies plant resistance to an insect herbivore

Vesicular-arbuscular mycorrhizal (VAM) fungi are common root-colonizing symbionts that affect nutrient uptake by plants and can alter plant susceptibility to herbivores. I conducted a factorial experiment to test the hypotheses that colonization by VAM fungi (1) improves soybean (Glycine max) tolerance to grazing by folivorous Mexican bean beetle (Epilachna varivestis), and (2) indirectly affects herbivores by increasing host resistance. Soybean seedlings were inoculated with the VAM fungus Glomus etunicatum or VAM-free filtrate and fertilized with high-[P] or low-[P] fertilizer. After plants had grown for 7 weeks first-instar beetle larvae were placed on bagged leaves. Growth of soybean was little affected by grazing larvae, and no effects of treatments on tolerance of soybeans to herbivores were evident. Colonization by VAM fungus doubled the size of phosphorus-stressed plants but these plants were still half the size of plants given adequate phosphorus. High-[P] fertilizer increased levels of phosphorus and soluble carbohydrates, and decreased levels of soluble proteins in leaves of grazed plants. Colonization of grazed plants by VAM fungus had no significant effect on plant soluble carbohydrates, but increased concentration of phosphorus and decreased levels of proteins in phosphorus-stressed plants to concentrations similar to those of plants given adequate phosphorus. Mexican bean beetle mass at pupation, pupation rate, and survival to eclosion were greatest for beetles reared on phosphorus-stressed, VAM-colonized plants, refuting the hypothesis that VAM colonization improves host plant resistance. VAM colonization indirectly affected performance of Mexician bean beetle larvae by improving growth and nutrition of the host plant. Received: 28 February 1997 / Accepted: 23 June 1997     

Occurrence and importance of mycorrhizae in aquatic trees of New South Wales,Australia

Vesicular arbuscular mycorrhizal (VAM) infection was found in KOH-cleared and lactophenolblue-stained roots of Salix babylonica, Melaleuca quinquenervia and Casuarina cunninghamiana. These are all trees growing on creeks and river banks, in stationary or slowly flowing fresh or brackish waters in swamps, creeks, drains and channels, and in seepage areas of New South Wales, Australia. Larger and older roots lacked VAM infection in the inner cortex, probably due to suberisation of cells, and the endophyte was restricted to the epidermal layers. Spores and sporocarps of the VAM fungi Glomus fasciculatus, G. mosseae, Sclerocystis rubiformis, Gigaspora margarita and an unidentified Scutellospora sp. were wet sieved and decanted from aquatic sediments and soils. The presence of similar VAM fungal spores in the aquatic sediments and terrestrial soil suggests that they probably enter the aquatic sediments through run off from the land ecosystem. All three plants formed vesicular arbuscular (VA) mycorrhizae almost exclusively in the marshy, periodically inundated soils, but the same plant species formed endo-/ ectomycorrhizae when growing in soil with higher redox potentials (E _h). Salix and Melaleuca tree roots possessed both VAmycorrhizae and ectomycorrhizae. VAM roots of Casuarina were equipped with both N-fixing Frankia nodules and proteoid roots. VAM endophytes did not invade nodular cortical tissues, suggesting the presence of an exclusion mechanism which needs further study. The highest VAM infection was found in nodulated specimens. Free-floating roots growing in water close to the banks were non-mycorrhizal but were mycorrhizal in the bottom-rooting state. VAM spore number and mycorrhizal infection seem to be associated with redox-potential, i.e. lower at sites such as swamps, water or sediments with lower E _h values than in terrestrial soils with higher E _h values. A relationship between soil moisture gradient and VAM infection pattern became apparent from the study of a C. cunninghamiana transect on a creek embankment, i.e. typical vesicles and arbuscules were found in roots from drier soils, there was a lack of arbuscules in relatively wet soils but large lipid-filled intracellular vesicles were present, and typical vesicles and arbuscules were absent in flooded creek beds where roots were associated with coenocytic intercellular hyphae with abundant lipid droplets. The importance of VA mycorrhiza, ectomycorrhizae, N-fixing root nodules and proteoid roots at the land-water interface is discussed with reference to the use of these trees as pioneering species for stabilising river and stream banks, reducing erosion, windbreaking, and as a long-term and inexpensive means of achieving biological control of aquatic weeds by shading waterways.     

Influence of three vesicular-arbuscular mycorrhizal fungi (Glomaceae) on the activity of specific enzymes in the root system of Cucumis sativus L.

The influence of three vesicular-arbuscular mycorrhizal (VAM) Glomus species on the activity of enzymes in the roots of Cucumis sativus was tested. Cucumber plants were grown in a split-root system, in which colonized and uncolonized roots of a single plant could be separated. The activity of the host root malate dehydrogenase (MDH), glucose 6-phosphate dehydrogenase (Gd), glutamate oxaloacetate transaminase (GOT) and glutamate dehydrogenase (GDH) was measured on a densitometer after separation of the host and fungal enzymes on polyacrylamide gels.The results showed that only minor changes in the activity of the host root enzymes occurred after VAM inoculation. Gd was stimulated by VAM and phosphorus, and one of the fungi decreased the activity of GDH in the host plant when both parts of the root system were colonized.     

Effect of the VAM fungus Glomus sp. on the growth and yield of soybean inoculated with Bradyrhizobium japonicum

The effect of two Bradyrhizobium japonicum strains (D344 and Urbana), on the frequency and intensity of infection by a VAM fungal Glomus sp. and the effect of VAM on biomass production by nodulating plants were tested in soybean growing in a soil containing low levels of accessible P and N. During the initial stage of vegetative growth, mycorrhiza frequency in roots inoculated with the two rhizobial strains did not differ. However, during flowering it was 178% higher in roots with the strain D344 than in the presence of the strain Ubrana. At final harvest (green pods) the VAM frequency did not differ in the presence of either strain. VAM positively affected biomass production, foliar concentrations of P, Zn and Cu, and number and dry matter yield of pods, but did not increase concentrations of total N and K. In nonmycorrhizal plants total nitrogenase activity (not nodule mass) and growth were higher with the rhizobial strain Urbana. The greatest nitrogenase activity, growth and yield occurred in the presence of the VAM fungus, and did not differ for plants with different strains of rhizobia.     

Dependence of Arbuscular-Mycorrhizal Fungi on Their Plant Host for Palmitic Acid Synthesis

Lipids are the major form of carbon storage in arbuscular-mycorrhizal fungi. We studied fatty acid synthesis by Glomus intraradices and Gigaspora rosea. [¹⁴C]Acetate and [¹⁴C]sucrose were incorporated into a synthetic culture medium to test fatty acid synthetic ability in germinating spores (G. intraradices and G. rosea), mycorrhized carrot roots, and extraradical fungal mycelium (G. intraradices). Germinating spores and extraradical hyphae could not synthesize 16-carbon fatty acids but could elongate and desaturate fatty acids already present. The growth stimulation of germinating spores by root exudates did not stimulate fatty acid synthesis. 16-Carbon fatty acids (16:0 and 16:1) were synthesized only by the fungi in the mycorrhized roots. Our data strongly suggest that the fatty acid synthase activity of arbuscular-mycorrhizal fungi is expressed exclusively in the intraradical mycelium and indicate that fatty acid metabolism may play a major role in the obligate biotrophism of arbuscular-mycorrhizal fungi.     

Effects of a vesicular-arbuscular mycorrhizal fungus and other soil microorganisms on growth,mineral nutrient acquisition and root exudation of soil-grown maize plants

Maize (Zea mays L. cv. Alize) plants were grown in a calcareous soil in pots divided by 30-m nylon nets into three compartments, the central one for root growth and the outer ones for hyphal growth. Sterle soil was inoculated with either (1) rhizosphere microorganisms other than vesicular-arbuscular mycorrhizal (VAM) fungi, (2) rhizosphere microorganisms together with a VAM fungus [Glomus mosseae (Nicol. and Gerd.) Gerdemann and Trappel], or (3) with a gamma-irradiated inoculum as control. Plants were grown under controlled-climate conditions and harvested after 3 or 6 weeks. VAM plants had higher shootroot ratios than non-VAM plants. After 6 weeks, the concentrations of P, Zn and Cu in roots and shoots had significantly increased with VAM colonization, whereas Mn concentrations had significantly decreased. Root exudates were collected on agar sheets placed on the interface between root and hyphal compartments. Six-week-old VAM and non-VAM plants had similar root exudate compositions of 72–73% reducing sugars, 17–18% phenolics, 7% organic acids and 3% amino acids. In another experiment in which root exudates were collected on agar sheets with or without antibiotics, the amounts of amino acids and carbohydrates recovered were similar in VAM and non-VAM plants. However, threeto sixfold higher amounts of carbohydrates, amino acids and phenolics were recovered when antibiotics were added to the agar sheets. Thus, the high microbial activity in the rhizosphere and on the rhizoplane limits the exudates recovered from roots.