A soil-free root observation system for the study of root-microorganism interactions in maize

Background and aims

The root surface of a plant usually exceeds the leaf area and is constantly exposed to a variety of soil-borne microorganisms. Root pathogens and pests, as well as belowground interactions with beneficial microbes, can significantly influence a plants' performance. Unfortunately, the analysis of these interactions is often limited because of the arduous task of accessing roots growing in soil. Here, we present a soil-free root observation system (SF-ROBS) designed to grow maize (Zea mays) plants and to study root interactions with either beneficial or pathogenic microbes.

Methods

The SF-ROBS consists of pouches lined with wet filter paper supplying nutrient solution.

Results

The aspect of maize grown in the SF-ROBS was similar to soil-grown maize; the plant growth was similar for the shoot but different for the roots (biomass and length increased in the SF-ROBS). SF-ROBS-grown roots were successfully inoculated with the hemi-biotrophic maize fungal pathogen Colletotrichum graminicola and the beneficial rhizobacteria Pseudomonas putida KT2440. Thus, the SF-ROBS is a system suitable to study two major belowground phenomena, namely root fungal defense reactions and interactions of roots with beneficial soil-borne bacteria.

Conclusions

This system contributes to a better understanding of belowground plant microbe interactions in maize and most likely also in other crops.     

Inter-root movement of Azospirillum brasilense and subsequent root colonization of crop and weed seedlings growing in soil

Inter-root movement and dispersion of the beneficial bacterium Azospirillum brasilense were monitored in root systems of wheat seedlings growing in the field and in growth chamber soil trays. Two strains were used, a motile wild-type strain (Cd, mot⁺) and a motility deficient strain (mot^–), which was derived from the Cd strain. Root colonization by two wild-type strains (Cd and Sp-245) was studied in 64 plant species growing in pots in the greenhouse. The two wild-type strains of A. brasilense were capable of colonizing all tested plant species. In soil trays and in the field, mot⁺ cells moved from inoculated roots to non-inoculated roots of either wheat plants or weeds growing in the same field plot, but the mot^– strain did not move toward non-inoculated roots of either plant species. In the field, both mot⁺ and mot^– strains of A. brasilense survived well in the rhizosphere of wheat for 30 days, but only mot⁺ moved between different weeds, regardless of the species, botanical family, or whether they were annuals or perennials. In plant-free, water-saturated soils, either in columns or in the field, both strains remained at the inoculation site and did not move.It is proposed (a) that A. brasilense is not a plant-specific bacterium and that (b) colonization of the entire root system in soil is an active process determined by bacterial motility; it is not plant specific, but depends on the presence of plants. Correspondence to: Y. Bashan     

Changes in cucumber hypocotyl cell wall dynamics caused by Azospirillum brasilense inoculation

We previously reported that Azospirillum brasilense induced a more elastic cell wall and a higher apoplastic water fraction in both wheat coleoptile and flag leaf. These biophysical characteristics could permit increased growth. Knowledge of the biochemical effects the bacteria could elicit in plant cell walls and how these responses change plant physiology is still scarce. The objective of this work was to analyze whether A. brasilense Sp245 inoculation affected elongation and extensibility of growing cucumber (Cucumis sativus) hypocotyls and ionically bound cell wall peroxidase activities. Hypocotyl tip and basal segments were excised from A. brasilense Sp245-inoculated cucumber seedlings growing in darkness under hydroponic conditions. Elongation, cell wall extensibility, cell wall peroxidase activities against ferulic acid and guaiacol and NADH oxidase activities were analyzed. Azospirillum-inoculated cucumber seedlings grew bigger than non-inoculated ones. Dynamic cell wall differences were detected between inoculated and non-inoculated hypocotyls. They included greater acid-induced cell wall extension and in vivo elongation when incubated in distilled water. Although there was no difference between treatments in either region of the hypocotyl NADH oxidase and ferulic acid peroxidase activities were lower in both regions in inoculated seedlings. These lesser activities could be delaying the stiffening of cell wall in inoculated seedlings. These results showed that the cell wall is a target for A. brasilense growth promotion.     

Active attachment ofAzospirillum brasilense to root surface of non-cereal plants and to sand particles

The rhizosphere bacteriumAzospirillum brasilense Cd adsorbed strongly to light-textured and heavy-textured soils, but only slightly to quartz sand. Bacterial attachment to sand particles was mediated by a network made up of various sizes and shapes of fibrillar material. Inoculation of sand with an aggregate-deficient mutant resulted in no detectable fibrillar formation. Rinsing or agitating the sand, colonized by the wild-type and the mutant, had a greater effect on the mutant than on the parental strain. We propose that bacterial fibrils are essential for anchoring ofA. brasilense to sand. A. brasilense Cd was capable of efficiently colonizing the elongation and root-hair zones of tomato, pepper, cotton and soybean plants as well as of wheat plants. All inoculated plants demonstrated: (i) larger amounts of a mucigel-like substance on the root surface than non-inoculated plants, and (ii) fibrillar material which anchored the bacterial cells to the root surface. These fibrils established also connections between cells within bacterial aggregates. On non-water stressed soybean roots, mostA. brasilense Cd cells occurred as vibroid forms. Whereas, those on roots of water-stressed plants.(wilting) were cyst-like. A lower rhizosphere bacterial population was observed on water-stressed plants. When water stress conditions were eliminated, cells reverted to the vibroid form. A concomitant increase in the bacterial population was observed. We suggest that cyst-like formation is a natural response forA. brasilense Cd in the rhizosphere of water-stressed plants.     

Root-to-Root Travel of the Beneficial Bacterium Azospirillum brasilense

The root-to-root travel of the beneficial bacterium Azospirillum brasilense on wheat and soybean roots in agar, sand, and light-textured soil was monitored. We used a motile wild-type (Mot⁺) strain and a motility-deficient (Mot^-) strain which was derived from the wild-type strain. The colonization levels of inoculated roots were similar for the two strains. Mot⁺ cells moved from inoculated roots (either natural or artificial roots in agar, sand, or light-textured soil) to noninoculated roots, where they formed a band-type colonization composed of bacterial aggregates encircling a limited part of the root, regardless of the plant species. The Mot^- strain did not move toward noninoculated roots of either plant species and usually stayed at the inoculation site and root tips. The effect of attractants and repellents was the primary factor governing the motility of Mot⁺ cells in the presence of adequate water. We propose that interroot travel of A. brasilense is an essential preliminary step in the root-bacterium recognition mechanism. Bacterial motility might have a general role in getting Azospirillum cells to the site where firmer attachment favors colonization of the root system. Azospirillum travel toward plants is a nonspecific active process which is not directly dependent on nutrient deficiency but is a consequence of a nonspecific bacterial chemotaxis, influenced by the balance between attractants and possibly repellents leaked by the root.     

Restoration of Growth of Durum Wheat (Triticum durum var. waha) Under Saline Conditions Due to Inoculation with the Rhizosphere Bacterium Azospirillum brasilense NH and Extracts of the Marine Alga Ulva lactuca

Inoculation with the rhizosphere bacterium Azospirillum brasilense NH, originally isolated from salt-affected soil in northern Algeria, greatly enhanced growth of durum wheat (Triticum durum var. waha) under saline soil conditions. Important plant parameters like the rate of germination, stem height, spike length, dry weight of roots and shoots, chlorophyll a and b content, proline and total sugar contents, 1000-seed weight, seed number per spike, and weight of seeds per spike were measured. At salt stress conditions (160 and 200 mM NaCl) A. brasilense NH restored almost completely vegetative growth and seed production. The combination with extracts of the marine alga Ulva lactuca resulted in even more improved salt tolerance of durum wheat. Proline and total sugar accumulation, a sign of physiological plant stress under inhibitory salt conditions, was reduced in plants inoculated with A. brasilense NH with and without addition of algal extracts. Inoculation with the salt-sensitive A. brasilense strain Sp7 could not restore salt-affected plant growth at 200 mM NaCl. Furthermore, it could be demonstrated by fluorescence in situ hybridization and confocal laser scanning microscopy that A. brasilense NH is able to colonize roots of durum wheat endophytically under salt-stressed conditions. Thus, the salt-tolerant rhizobacterium A. brasilense NH could effectively provide alone or in combination with extracts of U. lactuca a promising solution to overcome salt inhibition which is a major threat hindering productive wheat cultivation in arid saline soils.     

Microorganisms and nematodes increase levels of secondary metabolites in roots and root exudates of Plantago lanceolata

Plant secondary metabolites play an important role in constitutive and inducible direct defense of plants against their natural enemies. While induction of defense by aboveground pathogens and herbivores is well-studied, induction by belowground organisms is less explored. Here, we examine whether soil microorganisms and nematodes can induce changes in levels of the secondary metabolites aucubin and catalpol (iridoid glycosides, IG) in roots and root exudates of two full-sib families of Plantago lanceolata originating from lines selected for low and high constitutive levels of IG in leaves. Addition of soil microorganisms enhanced the shoot and root biomass, and the concentration of aucubin in roots of both Plantago lines without affecting IG levels in the rhizosphere. By contrast, nematode addition tended to reduce the root biomass and enhanced the stalk biomass, and increased the levels of aucubin and catalpol in root exudates of both Plantago lines, without affecting root IG concentrations. The Plantago lines did not differ in constitutive levels of aucubin and total IG in roots, while the concentration of catalpol was slightly higher in roots of plants originally selected for low constitutive levels of IG in leaves. Root exudates of “high IG line” plants contained significantly higher levels of aucubin, which might be explained by their higher root biomass. We conclude that soil microorganisms can induce an increase of aucubin concentrations in the roots, whereas nematodes (probably plant feeders) lead to an enhancement of aucubin and catalpol levels in root exudates of P. lanceolata. A potential involvement of secondary metabolites in belowground interactions between plants and soil organisms is discussed.     

Enhanced Mineral Uptake by Zea mays and Sorghum bicolor Roots Inoculated with Azospirillum brasilense

Inoculation of corn (Zea mays) seeds with Azospirillum brasilense strain Cd or Sp 7 significantly enhanced (30 to 50% over controls) the uptake of NO₃⁻, K⁺, and H₂PO₄⁻ into 3- to 4-day- and 2-week-old root segments. No gross changes in root morphology were observed; altered cell arrangement in the outer four or five layers of the cortex was seen in photomicrographs of cross sections of inoculated corn roots. The surface activity involved in ion uptake probably increased, as shown by the darker staining by methylene blue of the affected area. Shoot dry weight increased 20 to 30% in inoculated plants after 3 weeks, presumably by enhancement of mineral uptake. Corn and sorghum plants grown to maturity on limiting nutrients in the greenhouse showed improved growth from inoculation approaching that of plants grown on normal nutrient concentrations. Enhanced ion uptake may be a significant factor in the crop yield enhancement reported for Azospirillum inoculation.     

Root endophyte symbiosis in vitro between the ectomycorrhizal basidiomycete Tricholoma matsutake and the arbuscular mycorrhizal plant Prunus speciosa

We previously reported that Tricholoma matsutake and Tricholoma fulvocastaneum, ectomycorrhizal basidiomycetes that associate with Pinaceae and Fagaceae, respectively, in the Northern Hemisphere, could interact in vitro as a root endophyte of somatic plants of Cedrela odorata (Meliaceae), which naturally harbors arbuscular mycorrhizal fungi in South America, to form a characteristic rhizospheric colony or “shiro”. We questioned whether this phenomenon could have occurred because of plant–microbe interactions between geographically separated species that never encounter one another in nature. In the present study, we document that these fungi formed root endophyte interactions and shiro within 140 days of inoculation with somatic plants of Prunus speciosa (=Cerasus speciosa, Rosaceae), a wild cherry tree that naturally harbors arbuscular mycorrhizal fungi in Japan. Compared with C. odorata, infected P. speciosa plants had less mycelial sheath surrounding the exodermis, and the older the roots, especially main roots, the more hyphae penetrated. In addition, a large number of juvenile roots were not associated with hyphae. We concluded that such root endophyte interactions were not events isolated to the interactions between exotic plants and microbes but could occur generally in vitro. Our pure culture system with a somatic plant allowed these fungi to express symbiosis-related phenotypes that varied with the plant host; these traits are innately programmed but suppressed in nature and could be useful in genetic analyses of plant–fungal symbiosis.     

Azospirillum and arbuscular mycorrhizal colonization enhance rice growth and physiological traits under well-watered and drought conditions

The response of rice plants to inoculation with an arbuscular mycorrhizal (AM) fungus, Azospirillum brasilense, or combination of both microorganisms, was assayed under well-watered or drought stress conditions. Water deficit treatment was imposed by reducing the amount of water added, but AM plants, with a significantly higher biomass, received the same amount of water as non-AM plants, with a poor biomass. Thus, the water stress treatment was more severe for AM plants than for non-AM plants. The results showed that AM colonization significantly enhanced rice growth under both water conditions, although the greatest rice development was reached in plants dually inoculated under well-watered conditions. Water level did not affect the efficiency of photosystem II, but both AM and A. brasilense inoculations increased this value. AM colonization increased stomatal conductance, particularly when associated with A. brasilense, which enhanced this parameter by 80% under drought conditions and by 35% under well-watered conditions as compared to single AM plants. Exposure of AM rice to drought stress decreased the high levels of glutathione that AM plants exhibited under well-watered conditions, while drought had no effect on the ascorbate content. The decrease of glutathione content in AM plants under drought stress conditions led to enhance lipid peroxidation. On the other hand, inoculation with the AM fungus itself increased ascorbate and proline as protective compounds to cope with the harmful effects of water limitation. Inoculation with A. brasilense also enhanced ascorbate accumulation, reaching a similar level as in AM plants. These results showed that, in spite of the fact that drought stress imposed by AM treatments was considerably more severe than non-AM treatments, rice plants benefited not only from the AM symbiosis but also from A. brasilense root colonization, regardless of the watering level. However, the beneficial effects of A. brasilense on most of the physiological and biochemical traits of rice plants were only clearly visible when the plants were mycorrhized. This microbial consortium was effective for rice plants as an acceptable and ecofriendly technology to improve plant performance and development.     

Enhanced Growth of Wheat and Soybean Plants Inoculated with Azospirillum brasilense Is Not Necessarily Due to General Enhancement of Mineral Uptake

The capacity of Azospirillum brasilense to enhance the accumulation of K⁺, P, Ca²⁺, Mg²⁺, S, Na⁺, Mn²⁺, Fe²⁺, B, Cu²⁺, and Zn²⁺ in inoculated wheat and soybean plants was evaluated by using two different analytical methods with five A. brasilense strains originating from four distinct geographical regions. A Pseudomonas isolate from the rhizosphere of Zea mays seedlings was included as a control. All A. brasilense strains significantly improved wheat and soybean growth by increasing root and shoot dry weight and root surface area. The degree of plant response to inoculation varied among the different strains of A. brasilense. All strains were capable of colonizing roots, but the best root colonizer, Pseudomonas sp., had no effect on plant growth. The numbers of organisms of Brazilian strains Sp-245 and Sp-246 colonizing roots were similar regardless of the host plant. Numbers of organisms for the other strains were directly dependent on the host plant. The main feature characterizing mineral accumulation in inoculated plants was that all inoculation treatments changed the mineral balance of the plants, but in an inconsistent manner. Enhancement of mineral uptake by plants also varied among strains to a great extent and was directly dependent on the strain-plant combination; i.e., a strain capable of increasing accumulation of a particular ion in one plant species or cultivar often lacked the ability to do so in another. Minerals in inoculated plants were not evenly distributed in different plant tissues, and the changes varied among groups of plants within each bacterial strain inoculation treatment. We suggest that, although A. brasilense strains are capable of changing the mineral balance and content of plants, it is unlikely that this ability is a general mechanism responsible for plant improvement by A. brasilense.     

Effects of Temperature, Nitrogen Fertilization, and Plant Age on Nitrogen Fixation by Setaria italica Inoculated with Azospirillum brasilense (strain cd)

The association between the nitrogen-fixing bacterium Azospirillum brasilense (strain cd) and the grass Setaria italica was studied under different environmental and soil conditions. Highest acetylene reduction rates in intact plants were observed at the booting stage of Setaria (2350 nmol ethylene produced hour⁻¹ plant⁻¹) at 27 C. Higher temperatures, up to 32 C, enhanced ethylene reduction. Significant increases in shoot dry weight, panicle weight, and length were obtained in inoculated plants fertilized with suboptimal NH₄NO₃ levels. The increase in nitrogen content of plants inoculated with A. brasilense was shown to be due to N₂ fixation. This was demonstrated by growing plants in washed quartz sand with no combined nitrogen. The bacteria also increased branching and development of roots. It was concluded that inoculation of Setaria with A. brasilense may lead both to increases in plant yield and saving of nitrogen fertilizer.     

Inoculation of seed-borne fungus in the rhizosphere of Festuca arundinacea promotes hydrocarbon removal and pyrene accumulation in roots

Background and aims

The selective inoculation of specific hydrocarbon-degrading microbes into the plant rhizosphere offers a useful means for remediating hydrocarbon-contaminated soils. The effect of inoculating a seed-borne filamentous fungus (Lewia sp.) on hydrocarbon removal by Festuca arundinacea and its growth was studied on perlite (model soil) and soil, both spiked with hydrocarbons.

Methods

A hydrocarbon mixture (1,500 mg kg^?1) of two polycyclic aromatic hydrocarbons (PAH), phenanthrene and pyrene, blended with hexadecane (1.0:0.5:0.5 weight) was used. Greenhouse experiments were carried out for 45 days. Inoculated and non-inoculated plants were grown in dark cylindrical glass pots containing perlite or soil.

Results

Inoculation with Lewia sp. stimulated (100 %) root growth in spiked perlite. Inoculated plants showed higher phenanthrene removal (100 %) compared to non-inoculated plants in perlite and soil. Pyrene removal by inoculated plants was 37-fold higher than that by non-inoculated plants in perlite; in soil, pyrene removal by inoculated plants (97.9 %) differed significantly from that of non-inoculated plants (91.4 %). Accumulation of pyrene in roots (530.9 mg kg^?1 of dry roots) was promoted in perlite.

Conclusions

Our results demonstrate that Lewia sp. (endophytic fungus) improved the efficiency of PAH removal by F. arundinacea, on both perlite and soil, stimulating pyrene accumulation in roots.     

Wheat lectin as a factor in plant-microbial communication and a stress response protein

Wheat lectin (wheat germ agglutinin, WGA), a representative of a broad group of cereal lectins, is excreted by plant roots into the surrounding medium and interacts with both pathogenic microflora and growth-stimulating rhizobacteria. WGA was found to serve as a molecular signal for the rhizobacterium Azospirillum brasilense, which forms endophytic and associative symbioses with wheat plants. The bacterial response to the lectin was pleiotropic: WGA at concentrations from 10^?10 to 10^?6 M exerted a dose-dependent effect on a range of processes in the bacterium that are important for the establishment and functioning of symbiosis. Plants with different WGA content differed in their responses to severe nitrogen starvation and to seed treatment with Azospirillum.     

Sharing of Diverse Mycorrhizal and Root-Endophytic Fungi among Plant Species in an Oak-Dominated Cool–Temperate Forest

Most terrestrial plants interact with diverse clades of mycorrhizal and root-endophytic fungi in their roots. Through belowground plant–fungal interactions, dominant plants can benefit by interacting with host-specific mutualistic fungi and proliferate in a community based on positive plant–mutualistic fungal feedback. On the other hand, subordinate plant species may persist in the community by sharing other sets (functional groups) of fungal symbionts with each other. Therefore, revealing how diverse clades of root-associated fungi are differentially hosted by dominant and subordinate plant species is essential for understanding plant community structure and dynamics. Based on 454-pyrosequencing, we determined the community composition of root-associated fungi on 36 co-occurring plant species in an oak-dominated forest in northern Japan and statistically evaluated the host preference phenotypes of diverse mycorrhizal and root-endophytic fungi. An analysis of 278 fungal taxa indicated that an ectomycorrhizal basidiomycete fungus in the genus Lactarius and a possibly endophytic ascomycete fungus in the order Helotiales significantly favored the dominant oak (Quercus) species. In contrast, arbuscular mycorrhizal fungi were generally shared among subordinate plant species. Although fungi with host preferences contributed to the compartmentalization of belowground plant–fungal associations, diverse clades of ectomycorrhizal fungi and possible root endophytes were associated not only with the dominant Quercus but also with the remaining plant species. Our findings suggest that dominant-ectomycorrhizal and subordinate plant species can host different subsets of root-associated fungi, and diverse clades of generalist fungi can counterbalance the compartmentalization of plant–fungal associations. Such insights into the overall structure of belowground plant–fungal associations will help us understand the mechanisms that facilitate the coexistence of plant species in natural communities.     

Early changes in root characteristics of maize (Zea mays) following seed inoculation with the PGPR Azospirillum lipoferum CRT1

The effect of direct inoculation of seeds with the plant growth promoting rhizobacteria (PGPR) Azospirillum lipoferum CRT1 was assessed on maize (Zea mays) grown for 35 days after sowing (d.a.s.) in controlled conditions (greenhouse) in a luvisol soil from south-eastern France. WhinRhizo^® software was used to describe the following changes in the root system morphology for each plant: distribution and average root diameter, root surface and the number of tips. The stress at breakage and stiffness of the roots in tension were also determined. Evaluation of biochemical components of roots was achieved by direct Attenuated Total Reflectance (or reflection) (ATR)-Fourier transform infrared (FTIR) on root section. Inoculated roots exhibited significantly larger numbers of tips and extending surface to rhizosphere when compared to controls. Measured mechanical parameters of inoculated roots showed a slight increase in rupture stress up to the largest diameter (1.2 mm) when compared to controls. Stiffness (Young’s modulus) values were nearly constant for inoculated plants with higher values than for non-inoculated plants at day 26 and day 35. Using Principal Components Analysis of ATR-FTIR profiles, the polysaccharide enrichment of inoculated roots compared to controls was found at day 35. Noticeable absorbance at wavenumber specific to aromatic ether (lignin) was observed in control plants. All these data had a pattern of immature root properties, when maize was inoculated with Azospirillum lipoferum CRT1. Observed modifications of root development are possibly conducive to unseen beneficial effects, like water retention, resistance to mechanical stress, or root litter quality. Studies on more mature plants are required to assess if the differences between inoculated and control plants would persist or become accentuated with time until harvest.     

Bidirectional plant‐mediated interactions between rhizobacteria and shoot‐feeding herbivorous insects: a community ecology perspective

1. Plants interact with various organisms, aboveground as well as belowground. Such interactions result in changes in plant traits with consequences for members of the plant‐associated community at different trophic levels. Research thus far focussed on interactions of plants with individual species. However, studying such interactions in a community context is needed to gain a better understanding.
2. Members of the aboveground insect community induce defences that systemically influence plant interactions with herbivorous as well as carnivorous insects. Plant roots are associated with a community of plant‐growth promoting rhizobacteria (PGPR). This PGPR community modulates insect‐induced defences of plants. Thus, PGPR and insects interact indirectly via plant‐mediated interactions.
3. Such plant‐mediated interactions between belowground PGPR and aboveground insects have usually been addressed unidirectionally from belowground to aboveground. Here, we take a bidirectional approach to these cross‐compartment plant‐mediated interactions.
4. Recent studies show that upon aboveground attack by insect herbivores, plants may recruit rhizobacteria that enhance plant defence against the attackers. This rearranging of the PGPR community in the rhizosphere has consequences for members of the aboveground insect community. This review focusses on the bidirectional nature of plant‐mediated interactions between the PGPR and insect communities associated with plants, including (a) effects of beneficial rhizobacteria via modification of plant defence traits on insects and (b) effects of plant defence against insects on the PGPR community in the rhizosphere. We discuss how such knowledge can be used in the development of sustainable crop‐protection strategies.

     

Interaction of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> and <Emphasis Type="Italic">Glomus intrarradix</Emphasis> in Sugar Cane Roots

Fifteen-day-old variety NA 56-79 sugar cane seedlings were inoculated with Azospirillum brasilense and Glomus intrarradix. This article aims at examining changes in sugar cane root seedlings inoculated with Glomus intrarradix and Azospirillum brasilense, the increase in microbial biomass and the acetylene reduction process as well. The internal root colonization was studied 20 days after inoculation using scanning and a transmission electron microscope. Both microorganisms entered the sugar cane root through the emergent lateral roots. The microorganisms were capable of coexisting both intra and intercellularly, producing changes in the cell wall, thus allowing colonization and interaction between the organisms. These changes increased the number of microorganisms inside the root as well as acetylene nitrogen reduction. Sugar cane plant biomass increased with joint-inoculation. The number of endophytic microorganisms and nitrogen fixing activity increased when they were colonized by Azospirillum and Glomus together.