The legume Rhizosphere

Summary Examination of the root surfaces of Medicago tribuloides Desr. with phase contrast microscopy or electron microscopy using thin sections revealed the presence of a layer of material outside the root surface. In thin sections of KMnO₄ fixed roots this layer was composed of a thin electron dense layer, an electron dense granular matrix of varying width and an enclosing electron dense membrane. After inoculation with an effective Rhizobium strain, rhizobia were found aggregated in a definite zone adjacent to the root surface when either living roots were examined by phase microscopy or thin sections by electron microscopy. This layer was also found in inoculated and uninoculated roots of Trifolium fragiferum and T. pratense. The bacteria were packed with inclusion granules and lay enclosed by a membrane layer adjacent to the granular matrix seen in uninoculated roots. The ultrastructural organisation of root hairs is essentially similar to that of other differentiated root cells. The replicated surface of the uninoculated root hair wall is largely amorphous with a few sculptured portions resembling a cuticle layer. The inoculated root hair wall often shows areas of exposed, open microfibrillar meshwork with rhizobia sitting on the microfibrils. The rhizobia resemble a flagellated, coccoid swarmer form of Rhizobium which is found in the barrel medic rhizosphere.     

Scanning Electron Microscopy of Rhizobium trifolii Infection Sites on Root Hairs of White Clover

White clover root hairs which were inoculated with Rhizobium trifolii 4S (infectious strain) contained infection threads which were observed by light microscopy and scanning electron microscopy. Three morphological types of root hairs retaining infection threads were recognized. The bacteria were strongly attached between the surfaces of two plant cell walls as follows: between surfaces of a root hair tip curled back on itself, between a protuberance from a root hair and its cell surface, or between two root hair tips clinging together. An anatomical analysis documented the attachment site of the infection thread sheath from the inside of the root hair cell.     

INITIATION AND DEVELOPMENT OF ROOT NODULES OF CASUARINA (CASUARINACEAE)

Roots of seedlings of the “beefwood” tree, Casuarina cunninghamiana Miq. grown in nitrogen-free nutrient solution were inoculated with a suspension prepared from crashed root nodules taken from mature plants. Marked deformation of root hairs was evident but no infection threads were observed in root hairs. The mode of infection remains undetermined. Root nodules were initiated within three weeks and thereafter numerous upward-growing nodule roots developed from each nodule. Nodules in this symbiotic nitrogen-fixing plant resulted from an infection caused by an unidentified actinomycete-like soil microorganism. Anatomical analysis of nodule formation showed that nodules are the result of repeated endogenous lateral root initiations, one placed upon another in a complexly branched and truncated root system. The endophyte-infected cortical tissues derived from successive root primordia form the swollen nodular mass. Nodule roots develop from nodule lobes after escaping from the initial inhibitory effects of the endophyte. Included is a discussion of the anatomical similarities between nodules of Casuarina which produce nodule roots and those of Alnus which form coralloid nodules usually lacking nodule roots.     

Early cytological events in the infection of the root hairs ofTrifolium repens byRhizobium leguminosarum biovartrifolii observed by glass slide culture in living seedlings

This report describes the early cytological events in the infection byRhizobium leguminosarum biovartrifolii of the root hairs ofTrifolium repens seedlings kept alive on agar medium in glass slide culture experiment. The infection threads bearing rhizobia were formed as soon as the epidermal cells began to emerge as root hairs. On the top of some of these infected emerging root hairs, there were smoky, cell-debris-like bodies, which appeared to be derived from the cell wall dug by rhizobia. Similar bodies were also observed in longer root hairs. None of the root hair cells along the length of the roots which contained infection threads were curled or distorted. A substantial number of pink-colored nodules were later formed on the roots with non-curled infected root hairs.     

Colonization of Wheat Root Hairs and Roots by Agrobacteria

Formation of extracellular structures in pure culture and in interaction with wheat root surface was studied by scanning and transmission electron microscopy. The effects of various factors (growth temperature as well as pretreatment of agrobacteria with kalanchoe extract, acetosyringone, and centrifugation) on formation of extracellular structures was tested. The data on Agrobacterium tumefaciens (wild-type strain C58 and mutants LBA2525 (virB2::lacZ) and LBA288 (without the Ti plasmid)) adhesion to wheat root surface and root hairs after pretreatment of agrobacteria with inducer of virulence genes (vir) acetosyringone were obtained. Formation of agrobacterial cell aggregates on wheat root hair tips was demonstrated. The proportion of root hairs with agrobacterial aggregates on the root hair tip insignificantly changed after pretreatment with acetosyringone but considerably increased after treatment of A. tumefaciens C58 and LBA2525 with kalanchoe leaf extract. The most active colonization of root hairs and formation of agrobacterial aggregates on hair root tips was observed at 22°C. The capacity of agrobacteria for adhesion on monocotyledon surface could be changed by pretreatment of bacteria with various surface-active substances. Bacterial cells subjected to centrifugation had a decreased capacity for attachment to both wheat root surface and root hairs. The relationship between the capacity for adhesion and pilus production in agrobacteria was considered.     

Role of root hairs in phosphorus depletion from a macrostructured soil

One rape (Brassica napus cv. Wesroona) plant and four cotton (Gossypium hirsutum cv. Sicot 3) plants were grown in plastic cells containing soil labelled with 407 kBq of³³P g⁻¹ soil. After 5–8 days of growth, the³³P depletion zones of all plants were autoradiographed and³³P uptake by plants was measured. The autoradiographs were scanned with a microdensitometer and the optical densities at several places within the³³P depletion zones of roots were obtained. The volume of soil explored by root hairs was estimated from measurements of root diameters and lengths of roots and root hairs. About half of the total³³P depleted by cotion roots came from outside the root hair cylinder whereas most of³³P taken up by rape was from within the root hair cylinder. Plants grown in a macrostructured soil may have roots growing in voids, within aggregates or on the surfaces of aggregates. The results of this study demonstrate that root hairs have a strong influence on the accessibility of phosphorus to roots in such a soil, and thus on the phosphorus nutrition of plants.     

Gram-positive rhizobacterium <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> FZB42 colonizes three types of plants in different patterns

The colonization of three types of different plants, Zea mays, Arabidopsis thaliana, and Lemna minor, by GFP-labeled Gram-positive rhizobacterium Bacillus amyloliquefaciens FZB42 was studied in gnotobiotic systems using confocal laser scanning microscopy and electron microscopy. It was demonstrated that FZB42 was able to colonize all the plants. On one hand, similar to some Gram-negative rhizobacteria like Pseudomonas, FZB42 favored the areas such as the concavities in root surfaces and the junctions where lateral roots occurred from the primary roots; on the other hand, we clearly demonstrated that root hairs were a popular habitat to the Gram-positive rhizobacterium. FZB42 exhibited a specific colonization pattern on each of the three types of plants. On Arabidopsis, tips of primary roots were favored by FZB42 but not so on maize. On Lemna, FZB42 accumulated preferably along the grooves between epidermal cells of roots and in the concave spaces on ventral sides of fronds. The results suggested L. minor to be a promising tool for investigations on plant-microbial interaction due to a series of advantages it has. Colonization of maize and Arabidopsis roots by FZB42 was also studied in the soil system. Comparatively, higher amount of FZB42 inoculum (∼10⁸ CFU/ml) was required for detectable root colonization in the soil system, where the preference of FZB42 cells to root hairs were also observed.     

Root hair deformation in the white clover/Rhizobium trifolii symbiosis

Rhizobium trifolii most frequently infects its host white clover (Trifolium repens L.) by means of infection threads formed in markedly curled root hairs. Rhizobium infections are classified as either lateral or apical based on whether they originate in the branches or at the apex of the root hairs. A quantitative estimate of lateral and apical infection in the region of the host root (Trifolium repens L. cv. Regal Ladino) that possessed mature and immature root hairs at the time of inoculation with Rhizobium trifolii TAI (CSIRO, Canberra City, Australia) indicated that lateral infection occurred more frequently in the mature root hair region of the root. Apical infections were more common in the immature root hair region. Cell free filtrates collected from R. trifolii cultured in association with the host roots induced branching in white clover root hairs. A partially purified preparation of the branching factor was obtained from freeze-dried filtrates by ethanol extraction and ion exchange chromatography. Preliminary studies on the characteristics of these substances suggest that some are dialyzable and heat stable white others are non-dialyzable and heat labile. The dialyzable, heat-stable compounds contain neutral sugars and range between 1200 to 10000 daltons in size. In roots that were exposed to low concentrations (6–25 μg-ml^?1) of these partially purified deformation factors before inoculation, the developmentally mature root hairs were deformed at the time of inoculation. Nodules appeared in the mature and immature root hair region of these plants at the same time. In plants exposed to water, nodules were observed in the immature root hair region and mature root hair regions 3 and 5 days after inoculation, respectively. Based on these results, we conclude that the nodule development was hastened in the plants exposed to the root hair-deforming substances because the mature root hairs of these plants were made infectible at the time of inoculation by this exposure.     

INTERACTION BETWEEN THE ECTOMYCORRHIZAL FUNGUS PISOLITHUS TINCTORIUS AND ROOT HAIRS OF PICEA MARIANA (PINACEAE)

The ectomycorrhizal fungus Pisolithus tinctorius interacts with roots of Picea mariana to form a typical mantle and Hartig net. Hyphae alter their growth pattern when in contact with susceptible root hairs in the mycorrhizal infection zone and grow acropetally, gradually covering the length of the hair to form a mantlelike structure. Initial contact with the hair may be influenced by a fibrillar material on the root hair surface. Although many root hairs become surrounded by fungal hyphae, they are not penetrated, and therefore are not entry points for this symbiotic fungus.     

INFLUENCE OF NITROGEN SOURCE ON MORPHOLOGY OF RIVULARIACEAE (CYANOPHYTA)1

Thirty-four heterocyst-producing strains of Rivulariaceae (29 Calothrix, 1 Dichothrix, 2 Gloeotrichia, 2 Rivularia), which produced tapered trichomes in medium minus combined nitrogen, were grown in the presence of nitrate. One strain was unchanged in morphology under this condition. The remaining 33 strains developed trichomes lacking heterocysts. In 19 strains almost all the trichomes became untapered and in the other 14, similar untapered trichomes were produced, but also many tapered trichomes resembling Homoeothrix or Hammatoidea. Similar results were obtained when representative strains were incubated with ammonia as the source of combined N. Only five strains formed colorless hairs in the control medium (minus combined N). The presence of combined N did not diminish hair development in the two strains which had only a few short hairs, but hair frequency and length were both reduced considerably in the three strains with many long hairs in the control medium. Two strains of the non-heterocystous genus Homoeothrix were incubated in medium without combined N. Neither strain showed any growth or heterocyst development, indicating that neither is simply a growth form of a heterocystous genus.     

Roles of cottony hairs in directed seed dispersal in riparian willows

Willows usually establish on wet substrates with fine sediments at sites that are created by large disturbances, but suitable microsites are spatially and temporally limited. Thus, we hypothesized that willow seeds are selectively dispersed to suitable microsites, such as those with a wet substrate, rather than unsuitable microsites, such as those with a dry substrate, with seedling establishment mediated by the cottony hairs attached to seeds (directed dispersal). To test our hypothesis, we compared several recruitment-related traits, including buoyancy, germination, and trapping at favorable microsites, in seeds of the riparian willows Salix sachalinensis and S. integra with and without cottony hairs in laboratory and field experiments. In both field and laboratory experiments, more seeds with cottony hairs were trapped in water and wet sand than in dry sand, in which no seeds of either species germinated. These results indicate that cottony hairs facilitate the recruitment of seeds to microsites favorable for seed germination and help seeds avoid unfavorable microsites. On the water surface, 17.6% of S. sachalinensis seeds and 68.0% S. integra seeds with cottony hairs floated for more than 6 days, whereas all seeds without cottony hairs sank immediately after being placed on the water surface. These results suggest that cottony hairs facilitate long-distance dispersal via flowing water and also help avoid germination under water, where willow seedlings fail to establish. Seeds of the two willow species were released from the cottony hairs and germinated immediately after the seeds were placed on wet sand, but not after placement on water or dry sand. These results suggest that the seeds are released from the cottony hairs when the hairs become wet and the seeds are striking to a suitable microsite for seedling establishment, such as wet sand. In riparian willows, the cottony hairs promote directed dispersal by moving seeds to discrete and predictable microsites where the seedling establishment is disproportionately high.     

Morphological synergism in root hair length,density, initiation and geometry for phosphorus acquisition in Arabidopsis thaliana: A modeling approach

Low phosphorus availability regulates root hair growth in Arabidopsis by (1) increasing root hair length, (2) increasing root hair density, (3) decreasing the distance between the root tip and the point at which root hairs begin to emerge, and (4) increasing the number of epidermal cell files that bear hairs (trichoblasts). The coordinated regulation of these traits by phosphorus availability prompted us to speculate that they are synergistic, that is, that they have greater adaptive value in combination than they do in isolation. In this study, we explored this concept using a geometric model to evaluate the effect of varying root hair length (short, medium, and long), density (0, 24, 48, 72, 96, and 120 root hairs per mm of root length), tip to first root hair distance (0.5, 1, 2, and 4 mm), and number of trichoblast files (8 vs. 12) on phosphorus acquisition efficiency (PAE) in Arabidopsis. SimRoot, a dynamic three-dimensional geometric model of root growth and architecture, was used to simulate the growth of Arabidopsis roots with contrasting root hair parameters at three values of phosphorus diffusion coefficient (D _e=1×10^–7, 1×10^–8, and 1×10^–9 cm² s^–1) over time (20, 40, and 60 h). Depzone, a program that dynamically models nutrient diffusion to roots, was employed to estimate PAE and competition among root hairs. As D _e decreased from 1×10^–7 to 1×10^–9 cm² s^–1, roots with longer root hairs and higher root hair densities had greater PAE than those with shorter and less dense root hairs. At D _e=1×10^–9 cm² s^–1, the PAE of root hairs at any given density was in the order of long hairs > medium length hairs > short hairs, and the maximum PAE occurred at density = 96 hairs mm^–1 for both long and medium length hairs. This was due to greater competition among root hairs when they were short and dense. Competition over time decreased differences in PAE due to density, but the effect of length was maintained, as there was less competition among long hairs than short hairs. At high D _e(1×10^–7 cm² s^–1), competition among root hairs was greatest among long hairs and lowest among short hairs, and competition increased with increasing root hair densities. This led to a decrease in PAE as root hair length and density increased. PAE was also affected by the tip to first root hair distance. At low D _e values, decreasing tip to first root hair distance increased PAE of long hairs more than that of short hairs, whereas at high D _e values, decreasing tip to first root hair distance increased PAE of root hairs at low density but decreased PAE of long hairs at very high density. Our models confirmed the benefits of increasing root hair density by increasing the number of trichoblast files rather than decreasing the trichoblast length. The combined effects of all four root hair traits on phosphorus acquisition was 371% greater than their additive effects, demonstrating substantial morphological synergy. In conclusion, our data support the hypothesis that the responses of root hairs to low phosphorus availability are synergistic, which may account for their coordinated regulation.     

The effect of aluminium concentration on root hairs in white clover (Trifolium repens L.)

The effect of aluminium (Al) on root hair length and number is quantified using solution culture techniques with genotypes from white clover cultivar Tamar, that had previously been selected for long and short root hairs. The population differences were maintained in control (0 Al) treatments, with the long-haired population having hairs three times longer than the short-haired population. At an activity of 2.2 µM Al³⁺, root hair length decreased in both populations, the magnitude of the decrease being greater for the long-haired population. Root hair numbers decreased in a similar manner for both populations. At an activity of 4.4 µM Al³⁺ or higher, root hairs virtually disappeared and root growth was very stunted. The effect of Al on root hair development has not been previously quantified, however other workers have observed reduced root hair development in other species at activities of Al greater than 2.5 µM Al³⁺.     

Development and persistence of sandsheaths of Lyginia barbata (Restionaceae): relation to root structural development and longevity

Background and Aims

Strongly coherent sandsheaths that envelop perennial roots of many monocotyledonous species of arid environments have been described for over a century. This study, for the first time, details the roles played by the structural development of the subtending roots in the formation and persistence of the sheaths.

Methods

The structural development of root tissues associated with persistent sandsheaths was studied in Lyginia barbata, native to the Western Australian sand plains. Cryo-scanning electron microscopy CSEM, optical microscopy and specific staining methods were applied to fresh, field material. The role of root hairs was clarified by monitoring sheath development in roots separated from the sand profile by fine mesh.

Key Results and Conclusions

The formation of the sheaths depends entirely on the numerous living root hairs which extend into the sand and track closely around individual grains enmeshing, by approx. 12 cm from the root tip, a volume of sand more than 14 times that of the subtending root. The longevity of the perennial sheaths depends on the subsequent development of the root hairs and of the epidermis and cortex. Before dying, the root hairs develop cellulosic walls approx. 3 µm thick, incrusted with ferulic acid and lignin, which persist for the life of the sheath. The dead hairs remain in place fused to a persistent platform of sclerified epidermis and outer cortex. The mature cortex comprises this platform, a wide, sclerified inner rim and a lysigenous central region – all dead tissue. We propose that the sandsheath/root hair/epidermis/cortex complex is a structural unit facilitating water and nutrient uptake while the tissues are alive, recycling scarce phosphorus during senescence, and forming, when dead, a persistent essential structure for maintenance of a functional stele in the perennial Lyginia roots.     

ROOT GLOCHIDS AND ROOT SPURS OF OPUNTIA ARENARIA (CACTACEAE)

Root glochids of Opuntia arenaria Engelm. are produced by adventitious buds (areoles) that arise endogenously on fleshy roots. If they remain active, root areoles become dwarf shoots that initiate only glochids and trichomes unless the roots are uncovered or the shoots bearing the roots are removed. Then they will expand, producing aerial shoot joints. Root spurs are formed in great numbers on the profusely branched system of small roots. They consist of clusters of rootlets about one mm long that are initiated in sympodial sequence, each new rootlet arising endogenously near the base of the preceding one. On older spurs this results in the formation of a short axial peg. Rootlet primordia lack a root cap from the beginning and appear to mature quickly, losing all their meristematic characteristics and becoming completely covered with root hairs, even over the tip. They may represent a mechanism for the rapid production of root hairs during the infrequent periods when moisture is available. In any case, they seem to be transitory structures because cork soon forms beneath the older ones on a spur.     

Correlation between infection by Rhizobium leguminosarum and lectin on the surface of Pisum sativum L. roots

The lectin on the surface of 4- and 5-dold pea roots was located by the use of indirect immunofluorescence. Specific antibodies raised in rabbits against pea seed isolectin 2, which crossreact with root lectins, were used as primary immunoglobulins and were visualized with fluorescein- or tetramethylrhodamine-isothiocyanate-labeled goat antirabbit immunoglobulin G. Lectin was observed on the tips of newly formed, growing root hairs and on epidermal cells located just below the young hairs. On both types of cells, lectin was concentrated in dense small patches rather than uniformly distributed. Lectin-positive young hairs were grouped opposite the (proto)xylematic poles. Older but still-elongating root hairs presented only traces of lectin or none at all. A similar pattern of distribution was found in different pea cultivars, as well as in a supernodulating and a non-nodulating pea mutant. Growth in a nitrate concentration which inhibits nodulation did not affect lectin distribution on the surface of pea roots of this age. We tested whether or not the root zones where lectin was observed were susceptible to infection by Rhizobium leguminosarum. When low inoculum doses (consisting of less than 10⁶ bacteria·ml^-1) were placed next to lectin-positive epidermal cells and on newly formed root hairs, nodules on the primary roots were formed in 73% and 90% of the plants, respectively. Only a few plants showed primary root nodulation when the inoculum was placed on the root zone where lectin was scarce or absent. These results show that lectin is present at those sites on the pea root that are susceptible to infection by the bacterial symbiont.Abbreviations FITC fluorescein isothiocyanate - TRIC tetramethylrhodamine isothiocyanate     

Association of Azospirillum with Grass Roots

The association between grass roots and Azospirillum brasilense Sp 7 was investigated by the Fahraeus slide technique, using nitrogen-free medium. Young inoculated roots of pearl millet and guinea grass produced more mucilaginous sheath (mucigel), root hairs, and lateral roots than did uninoculated sterile controls. The bacteria were found within the mucigel that accumulated on the root cap and along the root axes. Adherent bacteria were associated with granular material on root hairs and fibrillar material on undifferentiated epidermal cells. Significantly fewer numbers of azospirilla attached to millet root hairs when the roots were grown in culture medium supplemented with 5 mM potassium nitrate. Under these growth conditions, bacterial attachment to undifferentiated epidermal cells was unaffected. Aseptically collected root exudate from pearl millet contained substances which bound to azospirilla and promoted their adsorption to the root hairs. This activity was associated with nondialyzable and proteasesensitive substances in root exudate. Millet root hairs adsorbed azospirilla in significantly higher numbers than cells of Rhizobium, Pseudomonas, Azotobacter, Klebsiella, or Escherichia. Pectolytic activities, including pectin transeliminase and endopolygalacturonase, were detected in pure cultures of A. brasilense when this species was grown in a medium containing pectin. These studies describe colonization of grass root surfaces by A. brasilense and provide a possible explanation for the limited colonization of intercellular spaces of the outer root cortex.     

In-situ analysis of pectic polysaccharides in seed mucilage and at the root surface of Arabidopsis thaliana

Pectic polysaccharides are a complex set of macromolecules of the primary cell wall matrix with distinct structural domains. The biosynthesis, organisation and function of these domains within cell wall matrices are poorly understood. An immersion immunofluorescence labelling technique was developed for the in-situ analysis of pectic polysaccharides at the surface of seeds and seedlings of Arabidopsis thaliana (L.) Heynh., and used to investigate the occurrence of pectic homogalacturonan (HG) and rhamnogalacturonan-I (RG-I) epitopes. Seed mucilage appeared to consist of two regions: a highly methyl-esterified HG was a major component throughout the mucilage, while an inner region with relatively low porosity was stabilized by calcium-based HG cross-linking. The small size and transparency of Arabidopsis roots allowed the occurrence of pectic HG and RG-I epitopes at root surfaces to be directly determined on whole-mount preparations. Pectic epitopes were not distributed evenly over root surfaces and were notably absent from lateral root apices and from the surface of root hairs. The use of defined antibody probes in the immersion immunolabelling protocol will be useful for the analysis of the influence of growth conditions and genetic factors on pectic polysaccharides in Arabidopsis. Received: 13 July 2000 / Accepted: 15 September 2000