Nodule Lobes Research Articles

Localization of typical and atypical Frankia isolates from Casuarina sp. in nodules formed on Casuarina equisetifolia

Members of the nitrogen-fixing actinobacterial genus Frankia are typically isolated from root nodules and generally infective on the same plant species. Several Frankia strains originally isolated from Casuarina species, however, have been found to be non-infective on Casuarina species. The goal of this study was to investigate the potential role of infective isolates from Casuarina species on the potential establishment of these non-infective Frankia strains in root nodule formation on Casuarina equisetifolia. Soil microcosms were established with plants of C. equisetifolia and inoculated with Frankia casuarinae strain CcI3 or cluster 3 strain R43, or combinations of both at different densities. Basic plant growth characteristics, root nodule formation and localization of both Frankia strains in nodule periderm and cortex, as well as population development in soils were monitored. The presence of strain R43 did not affect plant growth performance nor root nodule formation, while inoculation with strain CcI3 enhanced plant growth and resulted in root nodule formation. qPCR analyses on selected nodule lobes revealed the presence of strain CcI3 in cortex samples in all treatments, while strain R43 was not detected in any cortex samples but in 40% of the periderm samples from lobes from treatments with highest inoculation values. In situ hybridization detected cells of strain R43 on the outside of the nodules, i.e. on the periderm only. These results demonstrate that the cluster 3 Frankia strain R43 is not co-infecting root nodules formed by the F. casuarinae strain CcI3 on C. equisetifolia, but has likely been isolated as a surface contaminant from Casuarina nodules.

Metastatic Papillary Thyroid Carcinoma in an Autonomous Hyperfunctioning Thyroid Nodule in an Adolescent: Case Report and Literature Review

Background: Although thyroid nodules are less common in children and adolescents, the malignancy rate for nodules is higher among children and adolescents than adults. A nodule that is discovered in a patient presenting with hyperthyroidism is usually a benign nodule or benign toxic adenoma and, thus, nodules are biopsied only infrequently. Objective: To report a case of papillary thyroid carcinoma presenting with a hyperfunctioning (hot) thyroid nodule. Case report: A 14-year-old female presented with symptoms of thyrotoxicosis and multi-nodular goitre. Laboratory findings were consistent with the diagnosis of thyrotoxicosis. Thyroid ultrasonography showed an enlarged thyroid gland with cystic and solitary nodules, diffuse heterogeneous echo, and microcalcifications. Doppler scans revealed increased vascularity within the left thyroid lobe and a solitary nodule. Thyroid scintigraphy revealed an autonomous nodule. Cytological examination of fine-needle aspirations from the solid nodule and left thyroid lobe revealed papillary thyroid carcinoma. The patient underwent total thyroidectomy. Histopathology of the thyroid gland and lymph nodes were also consistent with papillary thyroid carcinoma with nodal metastases. Conclusion: Contrary to the guidelines published by major endocrine societies, the detection of an autonomously functioning thyroid nodule in children and adolescents does not exclude the possibility of thyroid carcinoma. Suspicious hot nodules should be evaluated cytologically.

Inoculation of green alder ([Alnus crispa]) with Frankia-Ectomycorrhizal Fungal Inoculant Under Commercial Nursery Production Conditions

We examined the feasibility of producing container <i>Alnus crispa</i> (Ait.) Pursh (Betulaceae) seedlings (green alder) inoculated with a pure culture of <i>Frankia</i> sp., Brunchorst strain AvcI1 and an ectomycorrhizal fungus, <i>Hebeloma crustuliniforme</i> (Bull. ex st. Amans) Quél. in a commercial nursery setting. Alders are actinorhizal plants that fix atmospheric nitrogen in a symbiotic association with <i>Frankia</i> species and can also form mycorrhizal associations. <i>Frankia</i> inoculation significantly increased seedling biomass, number of nodule lobes, nodule weight, and plant nitrogen content of green alder at the end of nursery culture compared with control or “<i>Hebeloma</i> only” treatments. Improved seedling growth, root nodulation, and nitrogen nutrition achieved in this study was attributable to <i>Frankia</i> inoculation, suggesting <i>Frankia</i> inoculation in nursery may be beneficial for the production of superior alder seedlings to use in reclamation work. Actinorhizal plants have the potential to enhance plant establishment on disturbed sites and to improve soil fertility and stability. Seedlings inoculated with <i>Hebeloma</i> only or in combination with <i>Frankia</i> did not show any visible ectomycorrhizal colonization, suggesting <i>H. crustuliniforme</i> may not be compatible with green alder under these experimental conditions. This study demonstrated the suitability of producing large-scale inoculated alder seedlings in commercial nurseries without altering regular nursery operations.

Location and Anatomy of Nodules on Alnus maritima Subjected to Flooding

Although many species of Alnus Miller grow in wet soils, none is as closely associated with low-oxygen, waterlogged soils as Alnus maritima (Marsh.) Muhl. ex Nutt. (seaside alder). An actinorhizal species with promise for use in horticultural landscapes, land reclamation, and sustainable systems, A. maritima associates with Frankia Brunchorst, thereby forming root nodules in which gaseous nitrogen is fixed. Our objective was to determine how root-zone moisture conditions influence the occurrence, location, and anatomy of nodules on A. maritima. Plants of Alnus maritima subsp. maritima Schrader and Graves were established in root zones with compatible Frankia and subjected to four moisture regimens (daily watered/drained, partially flooded, totally flooded, and totally flooded with argon bubbled through the flood water) for 8 weeks. Oxygen content of the root zone, number and location of nodules on root systems, and dry weight and nitrogen content of shoots were determined. Root-zone oxygen content ranged from 17.3 kPa for daily watered/drained plants to 0.9 kPa for argon-treated plants. Across all treatments, 87% of the nodules were within the upper one-third (4 cm) of the root zone. Although shoot dry weights of daily watered/drained and partially flooded plants were not different, daily watered/drained plants had more nitrogen in their leaves (2.53 vs. 2.21 mg·g-1). Nodulation occurred in all treatments, but nodules on totally flooded roots (with or without argon) were limited to a single lobe; in contrast, multilobed nodules were prevalent on partially flooded and daily watered/drained plants. Frankia infection within submerged nodule lobes was limited to one or two layers of cortical cells. Submerged nodules developed large air spaces between cortical cells, and phenolic-containing cells appeared to inhibit Frankia expansion within the nodule. These data suggest that access to root-zone oxygen is critical to the Frankia-A. maritima subsp. maritima symbiosis, and that plants of this subspecies in the drained soils of managed landscapes may benefit more than plants in native wetland habitats from nodulation and nitrogen fixation.

Isolation of endophytic Frankia from root nodules of Casuarina equisetifolia and infectivity of the isolate to host plants

Attempts were made to isolate endophytic Frankia from the root nodules formed on Casuarina equisetifolia grown in a mixture of vermiculite and field soil obtained from the Okayama University Campus. The isolation of Frankia was performed by macerating a nodule lobe directly in a test tube containing modified Qmod medium with casamino acids and sodium pyruvate as nitrogen and carbon sources. One colony, which showed hyphal and sporangial characteristics similar to those of Frankia, appeared in a test tube after 1-month incubation. The root nodules were successfully formed on the roots of the host plants by inoculation of the isolate which was multiplied using the original Qmod medium containing yeast extract, peptone and propionate. The acetylene reduction activity (ARA) obtained was 6.29 ± 1.57 µmol C2H4 h−1 (g F.W. nod.)−1. Accordingly, we identified the isolate as an effective Frankia strain for C. equisetifolia and designated it as Ceq1. Since this strain also formed root nodules on Alnus sieboldiana and Myrica rubra, it was considered to have a wider host range than that of Frankia strains which had been previously isolated from Casuarina nodules. However, the values of the nodule fresh weight and ARA in A. sieboldiana and M. rubra were much lower than those in C. equisetifolia and only half of the plants tested were nodulated in M. rubra. This strain did not form root nodules on Elaeagnus macrophylla. Frankia vesicles, which are the specialized structures where nitrogenase is located, were not observed in the free-living culture grown in Qmod medium containing abundant nitrogen and in the nodules newly formed by inoculation of the Ceq1 strain.

Dg93, a nodule-abundant mRNA of Datisca glomerata with homology to a soybean early nodulin gene.

We have isolated a 590-bp full-length cDNA clone designated Dg93, an mRNA that is highly expressed in symbiotic root nodules of the actinorhizal host Datisca glomerata. Dg93 mRNA encodes a deduced polypeptide of 105 amino acids with significant identity (74%) to the soybean (Glycine max) early nodulin (ENOD) gene GmENOD93 (Kouchi and Hata, 1993). Dg93 mRNA is abundant in nodules at 4 weeks post inoculation, the earliest time assayed, and steady-state mRNA levels remain elevated 11 weeks after inoculation. Spatial patterns of Dg93 mRNA expression are complex, with transcript accumulation in the nodule lobe meristem, early infection zone, periderm, and cells of the vascular cylinder, but not in the surrounding uninfected cortical cells. Dg93 is encoded by a small gene family in D. glomerata. To our knowledge, this is the first report of a gene from an actinorhizal host that is expressed in the nodule meristem and that shares sequence homology with an early nodulin gene from a legume.

In situ analysis of introduced Frankia populations in root nodules of Alnus glutinosa grown under different water availability

The competitive ability for nodulation of Alnus glutinosa (L.) Gaertn. plants by Frankia strains inoculated into soil with indigenous Frankia populations was studied at two matric potentials representing "dry" (-0.016 MPa) and "wet" (-0.001 MPa) conditions. In pots kept at a matric potential of -0.001 MPa, nitrate concentrations decreased within 3 weeks more than 10-fold to an average of approx. 200 µmol·(g soil dry wt.)-1. After 4 months, nitrate concentrations in these pots were 16 ± 16 and 277 ± 328 µmol·(g soil dry wt.)-1 (mean ± SD) for non-inoculated and inoculated soils, respectively. At a matric potential of -0.016 MPa, nitrate concentrations for non-inoculated and inoculated soils were 687 ± 491 and 1796 ± 1746 µmol·(g soil dry wt.)-1, respectively. Inoculated plants always grew better than their non-inoculated counterparts. The largest plants were found on inoculated soil at a matric potential of -0.001 MPa, whereas the smallest plants were found on non-inoculated soil at the same matric potential. At a matric potential of -0.016 MPa, plants grown on non-inoculated soil were not as tall as those grown on inoculated soil and were slightly chlorotic, indicating that the high level of nitrate in the soil was not providing optimal plant growth conditions. The number of nodule lobes formed on plants was not significantly different among treatments, though size and weight of lobes differed. Nodules from plants grown on inoculated soils always harbored vesicle-producing Frankia populations, while nodules from plants grown on non-inoculated soils harbored only Frankia with distorted vesicles or no Frankia at all. All strains in nodules from plants grown on non-inoculated soil were of Alnus host infection group IIIa. Nodules from plants grown on soil inoculated with strains ArI3 (group IIIa), Ag45/Mut15 (group IV), and AgB1.9 (group I) were also infected by Frankia strain Ag45/Mut15. These results indicate that by inoculation, Frankia populations could be established under conditions that did not favour vesicle formation in root nodules formed by the indigenous Frankia population. Inoculation even in soils with high nitrogen content might therefore be an appropriate strategy to enhance plant growth.Key words: competition, fluorescent oligonucleotide probes, inoculation, in situ hybridization, matric potential, nitrate, rRNA.

Flavan-containing cells delimit Frankia-infected compartments in Casuarina glauca nodules.

We investigated the involvement of polyphenols in the Casuarina glauca-Frankia symbiosis. Histological analysis revealed a cell-specific accumulation of phenolics in C. glauca nodule lobes, creating a compartmentation in the cortex. Histochemical and biochemical analyses indicated that these phenolic compounds belong to the flavan class of flavonoids. We show that the same compounds were synthesized in nodules and uninfected roots. However, the amount of each flavan was dramatically increased in nodules compared with uninfected roots. The use of in situ hybridization established that chalcone synthase transcripts accumulate in flavan-containing cells at the apex of the nodule lobe. Our findings are discussed in view of the possible role of flavans in plant-microbe interactions.

Amplification of 16S rRNA genes from Frankia strains in root nodules of Ceanothus griseus, Coriaria arborea, Coriaria plumosa, Discaria toumatou, and Purshia tridentata.

To study the global diversity of plant-symbiotic nitrogen-fixing Frankia strains, a rapid method was used to isolate DNA from these actinomycetes in root nodules. The procedure used involved dissecting the symbiont from nodule lobes; ascorbic acid was used to maintain plant phenolic compounds in the reduced state. Genes for the small-subunit rRNA (16S ribosomal DNA) were amplified by the PCR, and the amplicons were cycle sequenced. Less than 1 mg (fresh weight) of nodule tissue and fewer than 10 vesicle clusters could serve as the starting material for template preparation. Partial sequences were obtained from symbionts residing in nodules from Ceanothus griseus, Coriaria arborea, Coriaria plumosa, Discaria toumatou, and Purshia tridentata. The sequences obtained from Ceonothus griseus and P. tridentata nodules were identical to the sequence previously reported for the endophyte of Dryas drummondii. The sequences from Frankia strains in Coriaria arborea and Coriaria plumosa nodules were identical to one another and indicate a separate lineage for these strains. The Frankia strains in Discaria toumatou nodules yielded a unique sequence that places them in a lineage close to bacteria that infect members of the Elaeagnaceae.

Le nodule actinorhizien

Summary Actinorhizal nodules or actinorhizae represent the most typical example of modified non pathogenic roots (nodule lobes), induced by signals synthetized by the microsymbiont. The formation of a nodule lobe occurs in four steps: infection of the root hair by Frankia, formation of the prenodule, initiation and nodule lobe infection. The nodule lobe comprises four distinct zones which are the result of the Frankia acropetal growth and of the differentiation of tissues originating from the apical meristem. In situ hybridization studies of the expression of nif genes of Frankia show that the zone of infected mature cells (zone III) is the zone where the nitrogen fixation is the greatest. Similitudes and differences with organogenesis of the nodules of legumes are displayed and the interest of this primitive symbiosis is emphasized.

Expression of Frankia nif genes in nodules of Alnus glutinosa

Expression of Frankia genes involved in nitrogen fixation was studied in Alnus glutinosa nodules using the in situ hybridization technique. The results show that high level expression of nif genes does not occur immediately upon infection of cortical cells by Frankia. Also, only in the infected cells near the tips of the nodule lobes, nif genes are expressed at high levels. In the majority of infected cells, nif gene expression is rather low.

The wall of the infected cell may be the major diffusion barrier in nodules of Myrica galE L

Nitrogenase activity (C 2H 2 reduction) by single- or two-lobed nodules of Myrica gale L. with long nodule roots was measured at 6 and 8 kPa O 2 in a flow-through system after various treatments. Neither excision of nodule roots nor covering the cut surface with silicone grease after nodule root removal had a significant effect on C 2H 2 reduction, while covering the entire nodule with grease eliminated C 2H 2 reduction. Likewise, covering the nodule lobe while leaving nodule roots uncovered had little effect on C 2H 2 reduction. These results suggest that the diffusion resistance to gases is low at the surface of the nodule lobe and the nodule roots. Nodule slices ( ca 100 μm thick) retained high C 2H 2 reduction rates which were O 2-limited up to 18 kPa, indicating that the diffusion barrier is within the slices. The O 2 gradient from the gas phase to the infected cells in the slices was calculated to be ca 0.65 kPa out of a total gradient of 20 kPa. The addition of 2 kPa CO had little effect on nodule respiration (CO 2 evolution), suggesting that the diffusion barrier is outside the cytoplasm of the infected cells. Thus the major diffusion barrier may be in the wall of the infected cells.

Diversity of Frankia nodule endophytes of the actinorhizal shrub Ceanothus as assessed by RFLP patterns from single nodule lobes

Root nodules of the actinorhizal shrub Ceanothus were collected from seven sites from its native range. DNA was extracted from individual nodule lobes using a cetyl trimethyl ammonium bromide (CTAB) extraction procedure. Three DNA probes were used in combination with two restriction endonucleases to evaluate the extent of restriction fragment length polymorphism (RFLP) diversity within and among populations of Ceanothus endophytes. We observed some diversity using a nif DH gene probe; however there was no correlation of RFLP pattern and geographic site. Using two random Frankia probes, we observed more diversity among the Ceanothus endophytes than with the nif probe. Differences in RFLP patterns were observed among plants at a single geographic site and between geographical sites. The results demonstrated that considerable diversity exists among Frankia strains symbiotic with Ceanothus, as has been shown for pure-cultured Frankia strains isolated from other actinorhizal genera. We have also demonstrated the usefulness of this method for the study of Frankia ecology in planta.

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

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.

Root nodule initiation in Gymnostoma (Casuarinaceae) and Shepherdia (Elaeagnaceae) induced by Frankia strain HFPGpI1

Seedlings of Gymnostoma papuanum (Casuarinaceae) and Shepherdia argentea (Elaeagnaceae) were grown in water culture and inoculated with Frankia strain HFPGpI1. Root nodule initiation and early nodule development were examined using light microscopy. Gymnostoma papuanum was infected by penetration of Frankia into deformed root hairs, followed by development of a prenodule region and one to several nodule lobe primordia in the root cortex. Frankia hyphae grew directly through cell walls from cell to cell, colonizing cells of the prenodule prior to invading nodule lobe cells. Shepherdia argentea roots were infected by Frankia via intercellular penetration of the root epidermis and cortex with direct infection of cells of the nodule lobe primordia. No prenodule region was formed. Thus far, the mode of infection appears to be characteristic for each of the plant families. Subsequent to nodule initiation, plants were assayed at 4-week intervals (up to 12 or 16 weeks) for acetylene reduction activity. Low and variable activity was observed. The presence of symbiotic vesicles in G. papuanum nodules is reported as the first instance of vesicles seen in nodules taken from a member of the family Casuarinaceae.

Development of Alnus japonica root nodules after inoculation with Frankia strain HFPArI3

The ontogeny and structure of nodules on an exotic tree species, Alnus japonica (Thunb.) Steud. were studied for up to 3 months after inoculation of seedlings with a North American Frankia strain, HFPArI3. Nodules developed with Frankia growth first restricted to one side of the developing vascular cylinder. Nodules became multilobed, developed a complex network of vascular cylinders, and infected cortical cells organized in a concentric ring around each vascular cylinder. Each nodule lobe showed a zonation from the meristem basipetally. Cortical cells adjacent to the meristem contained hyphae, while those distal to this zone developed hyphae and endophytic vesicles. Staining for suberin induced fluorescence of root hairs, the endodermal Casparian strip, endophytic vesicle clusters, and periderm. Staining for callose resulted in fluorescence of the encapsulation material at one stage of Frankia development. Infected cells had few, small starch grains, while uninfected cells had several, large starch grains. The adjacent pericycle was multilayered. Its cells, viewed with the transmission electron microscope, had lobed nuclei, small vacuoles, many mitochondria, and dense plastids containing small starch grains.

Early development of Rhizobium-induced root nodules of Parasponia rigida. II. Nodule morphogenesis and symbiotic development

The Rhizobium-induced root nodules of Parasponia rigida (Ulmaceae) outwardly resemble those formed on actinorhizal plants, being coralloid in shape and consisting of multiple, branched lobes. The details of nodule morphogenesis also resemble more closely those which occur in an actinorhizal association than a typical Rhizobium–legume association and include prenodule formation, initiation of modified lateral roots which are termed nodule lobe primordia, and rhizobial colonization of tissues derived from the nodule lobe primordia to form the primary nodule lobes. Mature nodule lobe structure is actinorhizallike. Each lobe has an apical meristem and a central vascular cylinder which is surrounded by an uninfected inner cortex and then a zone of infected tissue. Peripheral to the infected tissue is an uninfected outer cortex. Infection threads and intercellular rhizobia progress continuously toward the apical meristem but do not infect the meristem itself. The establishment of the symbiosis in the host cells involves continuous thread formation after the initial infection until the host cells are nearly filled with rhizobia enclosed in threads. The rhizobia remain in threads throughout the symbiotic relationship and are not released from the threads as occurs in bacteroid formation in legumes.

Preliminary evidence for the involvement of suberization in infection of Casuarina

Histochemistry of infected cells in mature nodule lobes of Casuarina showed that walls of infected host cells had a ligninlike component (ultraviolet-stimulated autofluorescence and staining with auramine O, phloroglucinol staining, and resistance to degradation by hydrolytic enzymes). Cytoplasm of infected cells had a pronounced affinity for lipid stains (Sudan black B, Rose Bengal fluorescence), though walls of infected cells were less clearly stained. When nodules were digested several days in cold 50% chromic acid, the walls of infected cells and suberized host tissue (epidermis, endodermis) were not degraded. Endophyte cell wall components were also found to be resistant to chromic acid digestion. The digested tissue retained the capacity to adsorb lipid dyes. These observations suggested that walls of infected host cells had become impregnated with a suberinlike compound. The hydrophobic quality of this wall was evident when its ultrastructure was examined after en bloc staining with the polar stain KMnO4. This stain did not penetrate the walls of mature infected cells, perhaps because of the presence of aliphatic compounds similar to those found in suberin. As is known for suberizing tissue, peroxidase activity (via diaminobenzidine oxidation) was high in nodule cortical tissue cell walls. The peroxidase stain was also localized on endophyte hyphae. This report is the first instance associating a suberizationlike host reaction with infection of an actinorhizal plant.

DEVELOPMENTAL ANATOMY OF LIGHT‐INDUCED ROOT NODULATION BY ZAMIA PUMILA L. SEEDLINGS IN STERILE CULTURE

The developmental anatomy of Zamia pumila L. root apices was studied during light‐induced nodulation. Dark‐grown roots had an apical organization identical to that of other cycads and similar to that of other gymnosperms. A distinct protoderm was not observed in these roots, which had a large open meristem and a root cap with a well‐defined columella. During nodulation, the meristem became reduced in size, and its constituent cells became vacuolate until all but a few resembled ground tissue. The root cap senesced during nodulation, and a recognizable root cap was absent from mature nodules. A file of densely cytoplasmic cells with centrally positioned nuclei developed in the nodule cortex. This layer was continuous across the nodule apex, and was identical to the presumptive algal‐zone described previously by other authors. Light‐induced nodules branched dichotomously and were identical to algal‐free nodules described by other authors. In dichotomously branched nodules, each lobe was covered by a parenchymatous mantle analogous to a root cap. A unicellular layer similar to the presumptive algal zone spanned the gap between opposite nodule lobes, and extended beneath each lobe before terminating in the cortex. Typical meristematic regions were not observed in these nodules. Based on cell sizes and patterns, a meristematic zone was thought to exist between the mantle and the inner cortex.

Developmental Anatomy of Light-Induced Root Nodulation by Zamia pumila L. Seedlings in Sterile Culture

The developmental anatomy of Zamia pumila L. root apices was studied during light-induced nodulation. Dark-grown roots had an apical organization identical to that of other cycads and similar to that of other gymnosperms. A distinct protoderm was not observed in these roots, which had a large open meristem and a root cap with a well-defined columella. During nodulation, the meristem became reduced in size, and its constituent cells became vacuolate until all but a few resembled ground tissue. The root cap senesced during nodulation, and a recognizable root cap was absent from mature nodules. A file of densely cytoplasmic cells with centrally positioned nuclei developed in the nodule cortex. This layer was continuous across the nodule apex, and was identical to the presumptive algal-zone described previously by other authors. Light-induced nodules branched dichotomously and were identical to algal-free nodules described by other authors. In dichotomously branched nodules, each lobe was covered by a parenchymatous mantle analogous to a root cap. A unicellular layer similar to the presumptive algal zone spanned the gap between opposite nodule lobes, and extended beneath each lobe before terminating in the cortex. Typical meristematic regions were not observed in these nodules. Based on cell sizes and patterns, a meristematic zone was thought to exist between the mantle and the inner cortex.