Symbiotic vesicle ultrastructure in high pressure-frozen,freeze-substituted actinorhizae

Summary Using tissue stained en bloc with chromic acid or tissue prepared by high pressure-freezing and freeze-substitution, it was possible to analyze quantitatively the ultrastructure of symbiotic vesicle envelopes (SVE) inAlnus serrulata, Ceanothus americanus, Elaeagnus umbellata, andMyrica cerifera. The lamina measured about 4.7 nm in thickness in thin section. Despite diverse symbiotic vesicle morphology, the SVE thickness was similar in all of these symbioses: 36–71 nm, which corresponded to 6–15 laminae based on counts of chromic acid-stained SVEs. This similarity in structure suggests that a similar environmental signal regulates envelope thickness in the different root nodules. Based on previous studies, this is likely to be pO₂. Three types of envelope morphologies were distinguished: (1) theAlnus-type (as inAlnus andElaeagnus), which had localized thickenings around the vesicle and had thickest dimensions over the stalk; (2) theCeanothus-type. characterized as a relatively uniform envelope over both vesicle and attached hypha, and (3) theMyrica-type, which had no stalk region and a basal SVE thickness of about six laminae throughout except where localized thickening occurred. Localized thickening of the SVE resulted from extra numbers of laminae being deposited, generally over regions where septa contacted the edge of the vesicle. Freeze-substituted symbiotic vesicles had a variety of novel structures that are poorly preserved in chemically-fixed tissue. A paracrystalline body inAlnus symbiotic vesicles may be composed of particles that also exist free in the symbiotic vesicle cytoplasm. In addition, a previously unknown complex at the base of theAlnus-type symbiotic vesicle and within its stalk was evident in freeze-substituted tissues.Abbreviations HPF/FS high pressure-frozen/freeze-substituted - SV symbiotic vesicle - SVE symbiotic vesicle envelope Dedicated to the memory of Professor John G. Torrey     

A comparison of cultural characteristics and infectivity ofFrankia isolates from root nodules ofCasuarina species

Summary The isolations of three new strains ofFrankia were made from root nodules ofCasuarina cunninghamiana growing aeroponically. Two strains, HFPCCI1 and HFPCcI2 isolated by Lopez are typicalFrankia strains, producing sporangia among filamentous mats in culture and, in the absence of combined nitrogen, forming vesicles and showing acetylene reduction. They are red-pigmented and, although failing to nodulateCasuarina hosts, effectively nodulatedElaeagnus andHippophae. A third strain HFPCcI3 isolated by Zhang from the same source, also a typicalFrankia, can form sporangia and vesicles in culture and reduce acetylene, is unpigmented, fails to nodulateElaeagnus but effectively nodulatesC. cunninghamiana andC. equisetifolia. Comparisons are made among all of theCasuarina isolates in our collection from around the world (twelve in all) with regard to their cultural characteristics and capacity to infect host plant species. Questions are raised about the specificity of the various isolates and their possible affinities. Opportunities are suggested for inoculation of seedlings for forestry and field application using the infective, effective strains now available.     

Immunogold localisation of hemoglobin inCasuarina root nodules

Summary Immunocytochemical localisation of hemoglobin on sections ofFrankia-infecledCasuarina glauca nodule tissue confirmed its presence in nitrogen-fixing infected cells. Using colloidal gold as the marker, hemoglobin was shown to be restricted to the cytoplasm and nucleus of infected cells. None was associated with endophyte hyphae or uninfected cells. As infection developed, with its associated thickening and modification of host cell walls, the level of label, and by implication, the level of hemoglobin increased.Abbreviations SPL semi-permeable layer - GA glutaraldehyde - PFA paraformaldehyde     

Production of growth hormones inCasuarina cunninghamiana root nodules induced byFrankia strain HFPCgI4

Summary Measurements of auxin and cytokinin activities in extracts ofCasuarina root nodules were made. The nodules were induced either by pure culture ofFrankia strain CgI4 or by crushed nodule inoculum. Levels of cytokinin activity were significantly higher in root nodules induced by pureFrankia culture than in those induced by crushed nodule inoculum. However, the reasons for this are unknown. Seasonal variation in levels of cytokinin activity inCasuarina nodules has also been detected.Dedicated to the memory of Professor John G. Torrey     

Cellulose and xylans in the interface capsule in symbiotic cells of actinorhizae

Summary Enzyme-gold affinity labeling was used to show that in mature infected cells of actinorhizal symbioses the capsule on the plant host side of the symbiotic interface contained cellulose and xylans. Host species examined for cellulose wereAlnus rubra, Casuarina equisetifolia, C. glauca, Ceanothus cuneata, C. velutinus, Elaeagnus pungens, andMyrica cerifera.. Cellulose was in the capsule throughout the infected cell, implying that during development cellulose synthase was present in the host cell membrane component of the symbiotic interface. Any possible degradation of capsule cellulose by the microsymbiont was either incomplete or transient, because the polymer was present in mature infected cells. Cellulose labeling inCeanothus andElaeagnus was less consistent than in the other species. Dual labeled capsules inCasuarina glauca andAlnus rubra showed a similar distribution of xylans and cellulose. Cytochemical studies indicate that the capsule contains three major classes of cell wall polysaccharides: cellulose, hemicellulose (xylans), and pectins (shown previously). This suggests that the capsule is essentially a thin, internal, tubular plant cell wall.Abbreviations Au₅ Au₁₅ colloidal gold particles with mean diameter of 5 and 15 nm, respectively - CBHI cellobiohydrolase I - CBHII cellobiohydrolase II - PBS phosphate-buffered saline     

Inoculation and production of container-grown red alder seedlings

Summary The inoculation ofAlnus rubra (red alder) withFrankia sp. can lead to a highly efficient symbiosis. Several factors contribute to the successful establishment of nitrogenfixing nodules: (1) quantity and quality ofFrankia inoculant; (2) time and method of inoculation; (3) nutritional status of the host plant.Frankia isolates were screened for their ability to nodulate and promote plant growth of container-grown red alder. Inoculations were performed on seedlings and seeds. Apparent differences in symbiotic performance could be seen when seeds or seedlings were inoculated. Plants inoculated at planting performed significantly better than those inoculated four weeks later in terms of shoot height, nodule number and shoot dry weight. If inoculation was delayed further, reduction in shoot height, nodule number and shoot dry weight resulted. The effect of fertilizer was also investigated with regard to providing optimal plant growth after inoculation. Plants receiving 1/5 Hoagland's solution minus nitrogen showed maximal plant growth with abundant nodulation. Plants receiving 1/5 Hoagland's solution with nitrogen showed excellent plant growth with significantly reduced nodulation.     

Effect of spring flooding on endophyte differentiation,nitrogenase activity,root growth and shoot growth inMyrica gale

Summary Spring flooding was investigated as a possible limiting factor in the development of nitrogenase activity, root growth, and shoot growth inMyrica gale. Dormant, one year oldMyrica gale plants were placed in a greenhouse in early April and given three treatments: control (not flooded), flooded-water (flooded with water to 2.5 cm above the soil level) and flooded-peat (flooded with water-saturated peat to 4.0 cm above the soil level). Nitrogenase activity was absent at budbreak but appeared concurrently with the differentiation of vesicles by theFrankia sp. endophyte. Flooding delayed the onset of nitrogenase activity, substantially reduced the specific nitrogenase activity of the nodules, and also severely limited the production of the new nodule biomass. Consequently by 67 days past budbreak nitrogenase activity was much greater in the control plants (5.55±0.42 mol C₂H₄/plant.h; ± SE; N=9) than in the flooded-water (1.18±0.29) and flooded-peat (0.15±0.05) plants. Production of new secondary roots was substantially reduced in the flooded plants but adventitious roots were rapidly produced along the flooded portion of the stem in the better aerated zone near the surface. New nodules formed on several adventitious roots by 67 days indicating that the plants are able to replace their largely nonfunctional deeply flooded nodules with new nodules in the aerobic zone. Initially shoot growth was unaffected by flooding but by 67 days the flooded plants had substantially less leaf biomass, lower leaf and stem nitrogen concentrations, and less total shoot nitrogen content than the control plants.     

The initiation,development and structure of root nodules inElaeagnus angustifolia L. (Elaeagnaceae)

Summary A correlated light and electron microscopic study was undertaken of the initiation and development of root nodules of the actinorhizal tree species,Elaeagnus angustifolia L. (Elaeagnaceae).Two pure culturedFrankia strains were used for inoculation of plants in either standing water culture or axenic tube cultures. Unlike the well known root hair infection of other actinorhizal genera such asAlnus orMyrica the mode of infection ofElaeagnus in all cases was by direct intercellular penetration of the epidermis and apoplastic colonization of the root cortex. Root hairs were not involved in this process and were not observed to be deformed or curled in the presence of the actinomyceteFrankia. In response to the invasion of the root, host cells secreted a darkly staining material into the intercellular spaces. The colonizingFrankia grew through this material probably by enzymatic digestion as suggested by clear dissolution zones around the hyphal strands. A nodule primordium was initiated from the root pericycle, well in advance of the colonizingFrankia. No random division of root cortical cells, indicative of prenodule formation was observed inElaeagnus. As the nodule primordium grew in size it was surrounded by tanninised cells of a protoperiderm. The endophyte easily traversed this protoperiderm, and once inside the nodule primordium cortex ramified within the intercellular spaces at multiple cell junctions. Invasion of the nodule cortical cells occurred when a hyphal branch of the endophyte was initiated and grew through the plant cell wall, again by apparent enzymatic digestion. The plant cell plasmalemma of invaded cells always remained intact and numerous secretory vesicles fused with it to encapsulate the advancingFrankia within a fibrous cell wall-like material. Once within the host cell some endophyte cells began to differentiate into characteristic vesicles which are the presumed site of nitrogen fixation. This study clearly demonstrates that alternative developmental pathways exist for the development of actinorhizal nitrogen-fixing root symbioses.     

Effects of the plant host on the detergent sensitivity and viability of Rhizobium bacteroids

Bacteroids prepared from different legume species showed large differences in detergent sensitivity as judged by changes in turbidity and the release of cytochrome c oxidase activity after detergent treatments. There was a strong correlation between the detergent sensitivity and non-viability of bacteroids. Differences in the detergent sensitivity of bacteroids were determined by the plant host rather than the Rhizobium strain or the effectiveness of the symbiosis. The most common level of detergent sensitivity observed amongst bacteroids from 34 legume species was intermediate between lupin bacteroids and brothcultured bacteria.     

Effect of carbon source on growth,nitrogenase and uptake hydrogenase activities of Frankia isolates from Casuarina sp.

The effect of different carbon sources on the growth of Frankia isolates for Casuarina sp. was studied. In addition, regulation of nitrogenase and uptake hydrogenase activity by carbon sources was investigated. For each of the three isolates, JCT287, KB5 and HFPCcI3, growth was greatest on the carbon sources pyruvate and propionate. In general the carbon sources which gave the greatest growth gave the highest levels of nitrogenase activity, but repressed the activity of uptake hydrogenase. The regulation of growth, uptake hydrogenase activity and nitrogenase activity is discussed.     

Effect of juglone on growthin vitro ofFrankia isolates and nodulation ofAlnus glutinosa in soil

Summary In vitro growth (total protein content) of 5Frankia isolates was significantly inhibited at 10^–4 M juglone (5-hydroxy-1, 4-napthoquinone) concentration, but the degree of inhibition varied with theFrankia isolate. Isolates fromAlnus crispa [Alnus viridis ssp.crispa (Ait.) Turril] were most tolerant of 10^–4 M juglone relative to controls, while an isolate fromPurshia tridentata (Pursh.) D.C. was most inhibited, displaying a dramatic decrease in growth and greatly altered morphology.Nodulation of black alder [Alnus glutinosa L. (Gaertn.)] in an amended prairie soil inoculated with aFrankia isolate from red alder (Alnus rubra Bong.) was significantly decreased by the addition of aqueous suspensions of 10^–3 M and 10^–4 M juglone. This decrease was partially independent of decreased plant growth. The addition of an equal volume of sand to the soil mixture further decreased nodulation of black alder.Frankia inoculation of the soil mixtures significantly increased the total number of nodules formed per seedling, and the degree of differences in seedling nodulation owing to juglone and soil treatments.     

Phylogenetic diversity of Bradyrhizobium strains nodulating Calicotome spinosa in the Northeast of Algeria

Fifty-two slow-growing strains were isolated from root nodules of Calicotome spinosa grown in the Northeast of Algeria and grouped in 24 rep-PCR clusters. One representative strain for each profile was further phylogenetically characterized. The nearly complete 16S rRNA gene sequence indicated that all strains were affiliated to Bradyrhizobium. Multi-Locus Sequence Analysis (MLSA) of the atpD, glnII and recA genes and of the 16S-23S rRNA internal transcribed spacer (ITS) showed that these strains formed four divergent clusters: one close to Bradyrhizobium canariense and Bradyrhizobium lupini and three others separate from all the described species, representing three putative new Bradyrhizobium species. A phylogenetic analysis based on the nodC gene sequence affiliated the strains to either of the two symbiovars, genistearum or retamae.     

Morphology,physiology and infectivity of twoFrankia isolates An 1 and An 2 from root nodules ofAlnus nitida

Summary Two different strains, An 1 and An 2, were obtained from root nodules ofAlnus nitida Endl., collected from one locality in the area of its natural habitat near Bahrin, District Swat, Pakistan. The light and electron microscopy of the isolates revealed the occurrence of septate and branched hyphae bearing sporangia and vesicles. The strains differed in their growth requirements, nitrogen-fixing ability and production of extracellular pigments, thus indicating the existence of more than oneFrankia strain in the same locality. In the absence of combined nitrogen in the medium strain An 1 formed vesicles and fixed N₂ (up to 200 nmol C₂H₄. mg protein^–1.h^–1), while strain An 2 under the experimental conditions formed only few vesicles and fixed N₂ at a very low rate (ca 10 nmol C₂H₄. mg protein^–1 .h^–1). The nitrogenase activity of strain An 1 was strongly affected by the O₂ concentration.Frankia An 1 and An 2 were infective and effective onA. nitida andA. glutinosa but not onDatisca cannabina andElaeagnus umbellata. Both An 1 and An 2 strains were more infective and effective onA. glutinosa thanFrankia strains AvcIl and CpI1.     

Observations on the ultrastructure ofFrankia sp. in root nodules ofDatisca cannabina L.

Summary The fine structures of the microsymbiont inside the root nodules ofDatisca cannabina have been studied by light, by transmission- and by scanning-electron microscopy. The endophyte is prokaryotic and actinomycetal in nature. The hyphae are septate and branched, diameter 0.3–0.5 m. The tips of hyphae are swollen to form electron-dense, clubshaped to filamentous vesicles, ranging in diameter: 0.4–1.4 m. The endophyte penetrates through walls of the cortial cells. The infected zone is kidney shaped and confined to one side of the acentric stele. The orientation of infection is reversed from other actinorhizae exceptCoriaria. The hyphae are near the host cell wall and vesicles are directed towards the central vacuole. Vesicles are aseptate and no collapsing of the vesicle cell wall (void area) has been observed. Vesicle clusters structures are globular with an opening at one side of the cluster. The host cell is multinucleate or contains a lobed nucleus. Groups of mitochondria are located in between the hyphae, suggesting a strong association between the host and the endophyte for energy supply and amino acid production. The consequences of the inability to separate the mitochondria from the vesicle clusters in nodule homogenates in physiological studies have been discussed.Isolated vesicles clusters showed dehydrogenase activity, indicated by the presence of formazan crystals, after incubation with NADH and NBT. Strongest reducing activity was found within the vesicles. The possible role of filamentous vesicles in nitrogen fixation has been discussed.     

Preinfection cell wall formation in roots and developing nodules ofAlnus rubra Bong

Summary The establishment of actinorhizal root nodules involves penetration of host cell walls and intracellular colonization by the nitrogen-fixing endosymbiont,Frankia (Actinomycetales). In the early stages of the infection process inAlnus, unusual cell walls with undulate profiles were observed in root tip meristematic derivatives, and in early (preinfection) derivatives of the nodule lobe meristem, inFrankia-inoculated plants. The irregular cell walls attached obliquely to preexisting walls, but were not discontinuous. Serial sections revealed that the unusual walls divided two daughter cells. Microtubules in bundled arrays were abundant near the undulate walls, and radiated in several planes. In the root tips, the anomalous cell walls were observed within one day of inoculation withFrankia.     

Ultrastructure of freeze-substitutedFrankia strain HFPCcI 3, the actinomycete isolated from root nodules ofCasuarina cunninghamiana

Summary Frankia strain HFPCcI 3 is an actinomycete isolated from root nodules ofCasuarina cunninghamiana. In culture it exhibits typicalFrankia morphology and may produce three distinct morphological forms: branching septate hyphae, terminal or intercalary sporangia, and specialized structures termed vesicles which are the purported site of nitrogenase activity. An examination of the ultrastructure of all three morphological forms using both conventional chemical fixation (CF) and quick-freezing followed by freeze-substitution (FS) reveals some interesting differences between the two fixation methods. Unique to FS material are: 1. smooth membrane profiles; 2. lack of mesosomes; 3. lack of discernible nucleoid regions with condensed chromatin; 4. clarity of cytoplasmic elements such as ribosomes and granular bodies; 5. large cytoplasmic tubules in hyphae and young sporangia; 6. outer wall layer not widely separated from the spherical portion of the vesicle, and 7. bundles of microfilaments in vesicles. The quality of preservation after FS appears to be far superior to that obtained with CF. Accordingly the structures observed after FS are thought to represent more faithfully the structure of the living cell.     

The occurrence of calyx nodules inPsychotria spp. (Rubiaceae)

Summary The occurrence and structure of calyx nodules in the flowers of two leaf nodulated rubiaceous speciesPsychotria punctata Vatke andPsychotria kirkii Hiern. has been described for the first time at the ultrastructural level. Bacteria, resident in colleter-secreted mucilage in the space between calyx and corolla, invade stomatal pores which develop on the calyx protoderm. The bacteria proliferate in the substomatal cavity and then invade the calyx mesophyll. This invasion is most pronounced inP. punctata where the bacteria even penetrate and enter the cells of the vascular tissue. Although no sheath forms around the calyx nodules, the calyx mesophyll cells surrounded by the bacteria become identical in shape, size and secretory function to the invasive mesophyll cells of leaf nodules. The functional and evolutionary significance of calyx nodulation is discussed.     

Frankia inoculation,soil biota,and host tissue amendment influence Casuarina nodulation capacity of a tropical soil

The effects of soil biota, Frankia inoculation and tissue amendment on nodulation capacity of a soil was investigated in a factorial study using bulked soil from beneath a Casuarina cunninghamiana tree and bioassays with C. cunninghamiana seedlings as capture plants. Nodulation capacities were determined from soils incubated in sterile jars at 21 °C for 1, 7, and 28 days, after receiving all combinations of the following treatments: ± steam pasteurization, ± inoculation with Frankia isolate CjI82001, and ± amendment with different concentrations of Casuarina cladode extracts. Soil respiration within sealed containers was determined periodically during the incubation period as a measure of overall microbial activity. Soil respiration, and thus overall microbial activity, was positively correlated with increasing concentrations of Casuarina cladode extracts. The nodulation capacity of soils inoculated with Frankia strain Cj82001 decreased over time, while those of unpasteurized soils without inoculation either increased or remained unaffected. The mean nodulation capacity of unpasteurized soil inoculated with Frankia CjI82001 was two to three times greater than the sum of values for unpasteurized and inoculated pasteurized soils. Our results suggest a positive synergism between soil biota as a whole and Frankia inoculum with respect to host infection.     

A method for the quantification ofFrankia by estimation of hyphal length

Summary The cellular mass ofFrankia, a filamentous actinomycete, was readily quantified by estimating hyphal length, using a modification of Tennant's method for the estimation of root length. Each sample ofFrankia was stained with Coomassie Brilliant blue G 250, dispersed well, and suspended in a 0.5% agar solution. One drop of the suspension was placed in a Petroff-Hausser counting chamber with 0.05 by 0.05mm grid squares. The number of intersections betweenFrankia hyphae and the grid lines in a standard area were counted under a microscope and converted to hyphal length. Using the formula: hyphal length (HL) in mm equals (11/14) times the number of intersections (n) times the grid dimension (0.05 mm). The validity of the line intersection method was tested by comparison with total protein estimates of replicate aliquots ofFrankia culture. Correlations between total protein and hyphal length estimates were strong (r² from 0.76 to 0.95; standard errors of 3 to 9% of estimated length). These results show that line intersection counts may be a satisfactory routine method for quantifyingFrankia in culture and may be especially suitable for detecting small amounts of livingFrankia in less time than with other methods.Dedicated to the memory of Professor John G. Torrey