SYMBIOTIC NITROGEN FIXATION IN CEANOTHUS ROOTS

Ceanothus greggii var. perplexans is a common shrub in the southern California chaparral. Clusters of nodules found under the canopy of this species are modified roots which contain a nitrogen-fixing endophyte, Frankia ceanothi (Actinomycetales), within the cortex. The nodule density per m² obtained from root system excavations is much lower than that reported for different Ceanothus species in northern California. Field observations indicate that soil moisture is an important factor in nodule formation. Anatomical studies with the scanning electron microscope and acetylene reduction assays support the hypothesis that the vesicles, spherical swellings of hyphal endings (1.2–3.0 μm in diam), are indeed the sites of N₂ fixation. No bacteria-like bodies were found. The acetylene reduction rates of C. greggii endophytes were of the same order of magnitude as those reported for other members of the genus Frankia. It is estimated that 100 grams of nitrogen are fixed per year per hectare for a specific area in the southern Californian chaparral where C. greggii comprises 1/3 of the ground cover. This amount appears to be large enough to replace the nitrogen that is lost annually by drainage and runoff from winter rain storms.     

Plant-endophyte symbiosis in non-leguminous plants

Summary A wide taxonomic range of non-leguminous dicotyledonous plants bear root nodules and are able to fix atmospheric nitrogen. These plants belong to the orders Casuarinales, Myricales, Fagales, Rhamnales, Coriariales, and Rosales. Actinomycetes are involved in the root-nodule symbiosis. Nitrogen fixation is inhibited by hydrogen and carbon monoxide. Combined nitrogen depress nodule formation, but nitrogen fixation still occurs in the presence of combined nitrogen in the medium. In nitrogen-free medium Alnus plants fix in one season of 48 weeks 500 mg N per plant and Ceanothus plants 760 mg N per plant. Fixation by the other plant species is about of the same order. Field estimates showed that the nitrogen increase of the soil was about 61.5–157 kg N per ha per annum, depending on the age of the trees, under Alnus, 58.5 kg N per ha per annum under Casuarina, about 60 kg N per ha per annum under Ceanothus, 27–179 kg N per ha per annum underHippopha? rhamnoides, and about 61.5 kg N per ha per annum underDryas drummondii with someShepherdia spp. Non-leguminous root nodules belong to two types: coralloid root nodules and root nodules where the apex of each nodule lobe produces a negatively geotropic root. The primary infection occurs through the root hairs where a curling effect is observed. In the host cells the endophyte presents itself in three forms: hyphae, vesicles and bacteria-like cells. Vesicles are probably associated with nitrogen fixation, whereas the bacteria-like cells function in the endophyte's survival and dispersal. The endophyte is an obligate symbiont. TheAlnus glutinosa endophyte has been isolated and grownin vitro in root-nodule callus tissue. However, the isolated endophyte produces only ineffective root nodules in re-inoculation tests.     

Fine structure of actinorhiza of theRhamnaceae growing in Chile I.Talguenea quinquenervia (Gill et Hook)

Summary Root nodulesTalguenea quinquenervia Gill et Hook (Rhamnaceae) are restricted to the middle region of the root cortex. The root endophyte possesses hyphae which are septate and vesicles. The vesicles are spherical and are continuous with that of the hyphae. The endophyte fine structure is similar to otherFrankia-induced root nodules.     

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.     

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.     

Cell and endophyte structure of the nitrogen-fixing root nodules ofCeanothus integerrimus H.and A.

Summary The nitrogen fixing root nodules ofCeanothus integerrimus were very similar in appearance to other non-legume nodules. Each nodule was a cluster of small lobes. Each lobe in cross section had a central vascular cylinder and a hypertrophied cortex. The cortex contained very large infected cells, with large nuclei; among these infected cells were scattered small, normal-appearing cortex cells. The actinomycete endophyte consisted of wavy hyphae 0.4 m in diameter which terminated in pear-shaped vesicles 1.6 m×2 m. The vesicles were not septate. The function of the vesicles was unknown. The infected cells had apparently normal nuclei, chloroplasts and mitochondria and were probably alive, except at the base of the nodule where both infected cells and the endophyte they contained were dead.     

Studies of an effective strain ofFrankia fromAllocasuarina lehmanniana of the Casuarinaceae

Summary Seedlings ofCasuarina spp. andAllocasuarina spp. were grown from seed in the greenhouse and inoculated with a nodule suspension fromC. equisetifolia. Plants ofCasuarina spp. nodulated regularly and were effective in nitrogen-fixation. Only one species ofAllocasuariona, A. lehmanniana formed root nodules. Using these plants as source of inoculum, the isolation of a newFrankia sp. HFPA11I1 (HFP022 801) was made and the strain was grown in pure culture.Frankia sp. HFPA11I1 grows well in a defined medium and shows typical morphological characteristics. In media lacking combined nitrogen, the filamentours bacterium forms terminal vesicles in abundance and differentiaties large intrahyphal or terminal sporangia containing numerous spores. This strain, used as inoculum, nodulates effectively seedlings ofC. equisietifolia andC. cunninghamiana, forming nodules with verically-growing nodule roots. Although effective in acetylene reduction, the endophyte within the nodules is filamentous and lacks veiscles. When used to inoculated seedlings ofA llocasuarina lehmanniana, Frankia sp. HFPA11I1 induces root nodules which are coralloid and lacking nodule roots. The nodules are effective in acetylene reduction and the filamentous hyphae ofFrankia within the nodule lobes lack vesicles. Effective nodulation inA. Lehmanniana depends upon environmental conditions of the seedlings and proceeds much more slowly than in Casuariana.     

Scanning electron microscopy of the Alnus crispa var. mollis Fern. root nodule endophyte

Nitrogen-fixing root nodules of the Alnus crispa var. mollis Fern. were studied by scanning electron microscopy (SEM). The critical point drying of glutaraldehyde-osmium fixed nodular tissue permitted an excellent morphological preservation of the three-dimensional structures of the host and endophyte cells. The nodule endophyte was observed as two forms: the hypha which can be branched, and the vesicle which developed at the parental hypha tip. The actinomycetal endophyte penetrated through the host cortical cell wall and became enveloped by a membrane. This enclosing membrane is suggested to be the invaginated host plasmalemma. Perforations of the cell wall of the host infected cell were observed. These perforations are suggested to be the result of an enzymatic degradation process, probably regulated by the penetrating endophyte hyphae. In addition to the polymorphic endophyte, endogenous bacterial contaminants were observed in the nodular tissue. The present SEM study confirms previous light microscopy and transmission electron microscopy studies of the same species of root nodule symbiosis.     

<Emphasis Type="Italic">Rhizobium etli</Emphasis> maize populations and their competitiveness for root colonization

Rhizobium etli, which normally forms nitrogen-fixing nodules on Phaseolus vulgaris (common bean), is a natural maize endophyte. The genetic diversity of R. etli strains from bulk soil, bean nodules, the maize rhizosphere, the maize root, and inside stem tissue in traditional fields where maize is intercropped with P. vulgaris-beans was analyzed. Based on plasmid profiles and alloenzymes, it was determined that several R. etli types were preferentially encountered as putative maize endophytes. Some of these strains from maize were more competitive maize-root colonizers than other R. etli strains from the rhizosphere or from bean nodules. The dominant and highly competitive strain Ch24-10 was the most tolerant to 6-methoxy-2-benzoxazolinone (MBOA), a maize antimicrobial compound that is inhibitory to some bacteria and fungi. The R. tropici strain CIAT899, successfully used as inoculant of P. vulgaris, was also found to be a competitive maize endophyte in inoculation experiments.     

N2(C2H2−C2H4) fixation in two species of Ceanothus seedlings in second year postfire chaparral

Nitrogen fixation in excised root nodules of 2-year-old, postfireCeanothus tomentosus andC. leucodermis seedlings was measured over an 8-month period using the acetylene reduction method. High levels of NO₃–N and NH₄–N present in postfire soils were limited to the upper 10 cm and did not inhibit nodulation in these deeper-rooting seedlings. Decreases in acetylene reduction activity occurred with decreased soil moisture and increased soil temperature. Nitrogen gains from these two Ceanothus shrub seedlings totalled 1.6 kg N ha^–1 yr^–1.     

Non-leguminous root nodules in Japan

Summary Among plants native to Japan, nodule formation is confirmed in 14 species and varieties of Alnus, 10 of Elacagnus, 2 of Myrica and 1 of Coriaria, in a number of instances for the first time. Plants of 20 foreign species, in 8 genera, which bear nodules in their native countries, were raised in the nursery in Tokyo; only species of Alnus, Myrica and Ceanothus formed nodules. No nodules were found on native plants ofDryas octopetala var.asiatica. In a trial extending over 12 years evidence was obtained that the growth ofPinus thunbergii was benefited by underplanting withMyrica rubra, a result attributed to nitrogen fixation in the root nodules of the latter species. In the attempted isolation of the endophytes from the nodules of Alnus and other non-legume Angiosperms, although actinomycetes peculiar to the host species were usually obtained from the nodules, none of the isolates induced nodules in re-inoculation tests. Also from Podocarpus nodules actinomycetal and bacterial strains were commonly isolated; re-inoculation tests with these are in progress.     

TIME-LAPSE PHOTOGRAPHIC OBSERVATIONS OF MORPHOGENESIS IN ROOT NODULES OF COMPTONIA PEREGRINA (MYRICACEAE)

Seedlings of the sweet fern Comptonia peregrina (L.) Coult. were grown aeroponically with their roots bathed in a nutrient mist lacking nitrogen except for 10 ppm N at the outset. The initiation and early development of root nodules capable of fixing atmospheric nitrogen were recorded with time-lapse photography through early development to the establishment of highly branched, roughly spherical nodules. In Comptonia multiple primary nodule lobes are formed at or near the site of infection with as many as 10 primary lobes occurring together. On the shoulders of the swollen primary lobes new primordia develop, forming secondary nodule lobes, which may persist without nodule root elongation, giving a coralloid appearance. The tips of the lobes may elongate, forming nodule roots which grow vertically upward, or, if disturbed, in random orientation. Nodule roots occasionally form lateral roots. The root axis upon which the nodule forms undergoes secondary thickening on the proximal side of the nodule attachment; the distal portion of the root shows no secondary thickening and later atrophies. Thus, nodules are perennial structures on a woody root system. The endophyte infects and occupies the basal cortical tissues of the primary nodule lobes and successive nodule lobes as they are formed, being restricted to the swollen bases and not infecting the elongate nodule roots. Development of the nodule is interpreted in terms of complex host-endophyte interactions involving the initiation of multiple primordia forming nodule lobes, the active inhibition of nodule lobes and finally nodule root elongation. Anatomical evidence for the endogenous origin of nodule primordium formation substantiates the view obtained from time-lapse photomacrography.     

NITROGEN FIXATION IN THE BAYBERRY (MYRICA PENSYLVANICA) AND ITS ROLE IN COASTAL SUCCESSION

The nodules on roots of Myrica pensylvanica (bayberry) contain a bacterial endophyte. By using the acetylene reduction technique these plant endophyte associations were shown to be capable of fixing nitrogen. As nodulation was plentiful and fixation vigorous, it is proposed that the success of M. pensylvanica as an early successional plant of dunes and impoverished coastal soils is due in part to the nitrogen-fixing capacity of its nodular association.     

The Development and Structure of the Root-nodules of Myrica gale L. with Special Reference to the Nature of the Endophyte

The development and structure of the root nodules of Myricagale are described, and evidence advanced showing that theyrepresent modified lateral roots. Attempts at the isolationof the endophyte were unsuccessful, but on the basis of a cytologicalstudy of the nodules the author adheres to the view of someprevious investigators that the endophyte is actinomycetal.     

Ammonium effects on function and structure of nitrogen-fixing root nodules of Alnus incana (L.) Moench

Cloned plants of Alnus incana (L.) Moench were inoculated and grown without combined nitrogen for seven weeks. The effects of ammonium on the function and structure of the root nodules were studied by adding 20 mM NH₄Cl (20 mM KCl=control) for four days. Nitrogenase activity decreased to ca. 50% after one day and to less than 10% after two days in ammonium treated plants, but was unaffected in control plants. The results were similar at photon flux densities of 200 and 50 mol m^-2 s^-1. At the higher light level the effect was concentration dependent between 2 and 20 mM NH₄Cl. The recovery was slow, and more than 11 d were needed for plants treated with 20 mM ammonium to reach initial activity. The distribution of ¹⁴C to the root nodules after assimilation of ¹⁴CO₂ by the plants was not changed by the ammonium treatment. Microscopical studies of root nodules showed high frequencies of endophyte vesicles being visually damaged in nodules from ammonium-treated plants, but not in nodules from control plants. When nitrogenase activity was restored, visually damaged vesicles were again few, whereas young developing vesicles were numerous. The slow recovery, the ¹⁴C-translocation pattern, and the structural changes of the endophyte indicate a more complex mechanism of ammonium influence than simply a short-term reduction in supply of carbon compounds to the nodules.     

Observations on the fine structure of the root nodule endophyte of Hippophaë rhamnoides L.

Summary Electron microscopy of ultra-thin sections of Hippophaë rhamnoides root nodules has been carried out in order to elucidate the nature of the endophyte. The organism is seen as a branching, septate filament approximately 0.6 microns in diameter bearing on its terminal ends spherical sub-divided vesicles 3–4 microns in diameter. In the mature nodule the vesicles are the most prominent endophyte form and appear to be formed by swelling of the hyphal tips. It is concluded that the endophyte is an actinomycete closely related to but not identical with that of Alnus glutinosa.     

Demonstration of the isolation of non-infective Alnus crispa var. mollis Fern, nodule endophyte by morphological immunolabelling and whole cell composition studies.

Two filamentous, branched, and septate actinomycetes were isolated from field-collected and from axenic in vitro produced root nodules of Alnus crispa var. mollis Fern. host plant. After their transfer to a chemically defined medium, these nodule isolates could not be distinguished from each other on the basis of morphology, cultural reactions, and whole cell composition and were considered to be the same species. They were morphologically similar to the root nodule endophyte, but were incapable of nodulating aseptic host plants growing in a nitrogen-deficient substrate. Whole cells of the nodule isolates were used for the production of rabbit antibodies. The resulting specific antiisolate antibodies were conjugated with fluorescein isothiocyanate and used in staining tests of the nodule endophyte. The immunofluorescence reactions demonstrated the homology of the nodule isolates with the nodule endophyte. After pectinase degradation of the endophyte capsule, the indirect immunoferritin method corroborated the fluorescent anti-body (FA) staining reactions. There was no antigenic relationship between the nodule isolates and 13 known strains of actinomycetes as determined by the FA techique. Fluorescent antibody reactions of adsorbed conjugates suggested that endophytes of both Alnus crispa var. mollis Fern. and Alnus rugosa (DuRoi) Spreng. root nodules belong to a common serotype. The LL and mesoisomers of diaminopimelic acid were present in similar proportions in the nodule endophyte and in the nodule isolates. Glucose, mannose, and an unknown sugar were the predominant whole cell sugars in the nodule isolates, although trace amounts of arabinose and rhamnose were also displayed. The unknown sugar found in the nodule isolates was also present in trace amounts in the endophyte-suspension hydrolysate.     

Identification of the endophypte ofDryas andRubus (Rosaceae)

Summary Root nodules ofDryas drummondii are of the coralloid type (Alnus type). The endophyte is present in the middle cortical cells of the root-nodule tissue. Transmission electron micrographs revealed an actinorhizal endophyte with septate hyphae and non-septate spherical or ovoid vesicles. Vesicles always possess at the base a septum; septa formation in the endophyte is always associated with the presence of mesosomes. Branching of the endophyte is not necessarily correlated with septum formation. Hyphal structures are more prominent in the apical part of the root nodule and vesicles are more numerous in a broad zone below this. In the middle and towards the base of the root nodule the endophytic structures appear in a stage of disintegration. Vesicles appear in a broad region near the periphery of the host cell and regularly show no strict orientation towards the host-cell wall. In the center of the host cells only hyphae occur. In the intercellular spaces between the host cells theFrankia endophyte produces spore-like structures although the outline of the sporangia is often faint.The coralloid root ofRubus ellipticus shows characteristically a basal rootlet initiated below the dichotomous branching of the nodular lobes, but extending beyond the root nodule. The endophyte is only present in the outer cortex of the root nodule in a 1–2 cell wide layer. This endophytic layer is bounded, internally as well as externally, with a 4–5 cell wide layer of tannin-filled host cells. The implications of this situation are discussed. Tannin-filled cells occur regularly inRubus species and their arrangement has been used for taxonomic purposes within the genus. TheRubus endophyte is aFrankia species with septate hyphae and distinctly septate spherical vesicles. The ultrastructure of the vesicles of theRubus endophyte is very similar to that of theAlnus endophyte.     

Immuno-Gold Localization of Nitrogenase in Root Nodules of Elaeagnus pungens Thunb

The molybdenum-iron component of nitrogenase (Mo-Fe component)was purified from soybean nodule bacteroids and antibody wasraised against it in rabbits. Antibody raised against the 53kDa polypeptide which was the major protein in the Mo-Fe componentfraction of soybean nitrogenase was confirmed to be specificto the nitrogenase by immunodiffusion and immunotitration. Thenitrogenase from root nodules of Elaeagnus pungens cross-reactedwith the antibody and appeared from the results of the immunodiffusionto be partially identical to soybean nitrogenase. Using the antibody, we examined intracellular localization ofnitrogenase in root nodules of Elaeagnus pungens, in which Frankiais present as a symbiont, by immuno-gold labelling. Thin sectionsof nodules of Elaeagnus pungens were first treated with anti-nitrogenasespecific antibody and then with colloidal gold-protein A asa marker. The gold particles were observed to be concentratedin the vesicles of the endophyte Frankia. This provides strongsupport for the existence E of nitrogenase in the vesicles.Furthermore, our results suggested that nitrogenase localizesin the hyphae of the endophyte Frankia in Elaeagnus pungensnodules. ¹Present address: Iwata Experiment Station, Japan Tobacco Inc.,Iwata-gun, Shizuoka 438, Japan. (Received March 9, 1988; Accepted July 28, 1988)     

Some effects of combined nitrogen on the nodule symbioses of Casuarina and Ceanothus

Summary 1. The effect of ammonium-nitrogen on the further growth and activity in fixation of nodules already present at the commencement has been studied inCasuarina cunninghamiana growing in water culture and inCeanothus velutinus var.laevigatus in Peralite culture. 2. In Casuarina, at a low level of ammonium-nitrogen nodule growth remained similar to that in plants in nitrogen-free solution, but was stimulated in Ceanothus. In both genera nodule growth was strongly retarded at higher levels. 3. Fixation of nitrogen fell continuously in Casuarina as the level of ammonium-nitrogen was increased, while that in Ceanothus was unaffected at a low level but markedly decreased at a higher one. These effects were compounded from the changes noted in 2 and a tendency for the efficiency of nodule tissues in fixation to fall in the presence of ammonium-nitrogen, though this was not always shown. 4. In both genera but especially in Casuarina the growth of plants entirely dependent on nodule nitrogen was inferior to that of plants additionally supplied with ammonium-nitrogen. The reasons are discussed. 5. The results indicate the effects likely to be produced in the field as the soil nitrogen level rises through the action of the nodules of these species.