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.     

Host range ofFrankia endophytes

Summary Cross-inoculation experiments with 10 pure cultured strains and 17 host species were carried out. The 10 strains were isolated from the root nodules on actinorhizal trees ranging in 9 species, 5 genera and 4 families. The host species belong to 5 genera. The pure cultured strains fromAlnus are of strong ability to infect different species of the same genus. The seedlings inoculated with these strains are able to nodulate normally. These strains can also infect and nodulate the seedlings ofMyrica californica, but not the seedlings of Elaeagnus, Casuarina andMyrica rubra. The pure cultured strains from Elaeagnus can infect and nodulate the host species in the same genus and family with an exception ofE. viridis vardelavayi, which can be only poorly nodulated by a few strains from Elaeagnus. The strains from Elaeagnus cannot infect the seedlings of Alnus andMyrica rubra. The results presented here suggest thatFrankia endophytes can be divided into two groups: Alnus group and Elaeagnus group.     

The occurrence of nitrogen-fixing root nodules on non-leguminous plants

At present it is known that some 13 genera of non-leguminous Angiosperms include at least some species which bear root nodules with the property of nitrogen fixation. Alnus is the best known example. To assist in the assessment of the ecological importance of these plants, the author has surveyed the relevant literature to discover how many species in each of the genera have been recorded to bear noduleS. He also provides evidence of this feature in several species not hitherto reported. Of an estimated total complement of 342 species in the 13 genera, 118 species haye so far been recorded to bear nodules. The remaining species do not appear to have been examined for the presence of nodules.     

Actinomycetous root nodules in angiosperms of Pakistan

Summary In a survey, out of the nineteen genera of non-leguminous angiosperms that are already reported to bear actinomycete-induced nitrogen-fixing root nodules from various parts of the world, the species of seven genera (Alnus, Elaeagnus, Hippophaë, Coriaria, Datisca, Rubus and Casuarina) have been located in Pakistan and checked for nodulation. All the investigated species of the these genera, except those of Rubus and some of the introduced species of Casuarina, were found to bear nodules. In all 12 species belonging to six genera of five families have been recorded to bear actinorhizal nodules in Pakistan.     

Endophyte sporulation in root nodules of actinorhizal plants

All strains of isolated Frankia possess the genetic capacity to form sporangia since, when grown in vitro, they usually sporulate freely, depending on the physical and chemical environment in which they are cultured. Endophytic sporulation involving Frankia differentiation of sporangia within root nodules has been described in only 16 host species in 9 genera within six families of actinorhizal plants. From studies published to date, endophytic sporulation cannot be correlated with specific environmental conditions surrounding the host plants. Based on the literature and on previously unpublished observations from field and greenhouse studies, an account is given of the occurrence of sporulation in actinorhizal plants with emphasis on Alnus, Casuarina, Comptonia, Elaeagnus and Myrica . The possible role of the host plant in controlling Frankia sporulation as contrasted to the control exerted by the genetic constitution of the microbial symbiont is explored.     

Total and CO-reactive heme content of actinorhizal nodules and the roots of some non-nodulated plants

Summary The concentration of total and CO-reactive heme was measured in actinorhizal nodules from six different genera. This gave the upper limit to hemoglobin concentration in these nodules. Quantitative extraction of CO-reactive heme was achieved under anaerobic conditions in a buffer equilibrated with CO and containing Triton X-100. The concentration of CO-reactive heme in nodules of Casuarina and Myrica was approximately half of that found in legume nodules, whereas in Comptonia, Alnus and Ceanothus the concentrations of heme were about 10 times lower than in legume nodules. There was no detectable CO-reactive heme in Datisca nodules, but low concentrations were detected in roots of all non-nodulating plants examined, includingZea mays. Difference spectra of CO treated minus dithionite-reduced extracts displayed similar wavelengths of maximal and minimal light absorption for all extracts, and were consistent with those of a hemoglobin. The concentration of CO-reactive heme was not correlated to the degree to which CO inhibited nitrogenase activity nor was it affected by reducing the oxygen concentration in the rooting zone. However, there was a positive correlation between heme concentration and suberization or lignification of the walls of infected host cells. These observations demonstrate that, unlike legume nodules, high concentrations of heme or hemoglobin are not needed for active nitrogen fixation in most actinorhizal nodules. Nonetheless, a significant amount of CO-reactive heme is found in the nodules of Alnus, Comptonia, and Ceanothus, and in the roots ofZea mays. The identity and function of this heme is unknown.     

Methods of isolation and cultivation of Frankia species from actinorhizas

A critical review is given about the isolation and cultivation methods of Frankia species fromAlnus glutinosa root-nodules. The best results so far are obtained with a combination of sucrose (60% w/v)-sedimentation of root-nodule homogenate and subsequent suspension in the top-layer of a doubleagar layer system. The top-layer needs to contain a suitable C-source, in this study often a lipid factor from an alcoholic root-extract and an organic N-source.The isolation and cultivation of Sp(–) and Sp(+) strains fromAlnus glutinosa root nodules and a Frankia from the root-nodules ofMyrica gale is reported. The regular observation of growing colonies appears to be very important for the interpretation of results. The latter was illustrated by the remarkable diauxic growth of the strains isolated fromAlnus glutinosa Sp(+) root nodules.     

Phylogenetic characterization of ineffective Frankia in Alnus glutinosa (L.) Gaertn. nodules from wetland soil inoculants

Ineffective Frankia endophytes were retrieved from various wet soils by using Alnus glutinosa clones as trapping plants. No pure cultures could be isolated from these ineffective nodules. Therefore, the phylogenetic position of these endophytes was determined by sequence analysis of cloned PCR products of bacterial 16S rDNA, derived from nodules. The results showed that all nodule endophytes belong to a hitherto undescribed cluster of the Frankia phylogenetic tree. The position of these uncultured ineffective Frankia nodule endophytes is different from that of the ineffective Frankia isolates derived from A. glutinosa nodules, even when originating from the same geographical location. This suggests a bias in current isolation techniques.     

The effect of ammonium nitrogen on fixation of elemental nitrogen inAlnus andMyrica

Summary 1. A substantial formation of nodules occurred on plants ofAlnus glutinosa andMyrica gale grown in water culture with different levels of ammonium nitrogen labelled with N¹⁵ present in the culture solution. The nodules tended to be fewer but larger than on plants in solution free of combined nitrogen.2. The nodules continued to fix atmospheric nitrogen despite the presence of ammonium nitrogen in the rooting medium, though fixation per unit weight of nodule tissues formed was somewhat lower than in nitrogen-free solution. Among other possible reasons this could have been due to a substitution of ammonium nitrogen for elemental nitrogen at the nitrogen-fixing centres of the nodule, but evidently this does not occur to any great extent.3. In Alnus but not in Myrica fixation per plant was considerably enhanced in the presence of a low level of ammonium nitrogen, owing to greater nodule development. At higher ammonium levels, in excess of the plants' requirements, fixation per plant was still of comparable order to that in nitrogen-free solution, but now only represented some 24 to 45 per cent of the total nitrogen accumulated by the plants.4. The results suggest that under field conditions some fixation of atmospheric nitrogen will always be associated with nodules present.     

The Cobalt Requirement of Non-legume Root Nodule Plants

As already shown for Alnus glutinosa, cobalt is found to beessential for the proper growth of nodulated plants of Casuarinacunninghamiana and Myrica gale in a nitrogen-free rooting medium.If cobalt is not supplied, the plants develop symptoms of nitrogendeficiency; under the conditions of the experiments such symptomsbecame pronounced during the second season of growth of theseperennial plants. No cobalt requirement could be detected innon-nodulated plants of Alnus and Myrica supplied with nitrateor ammonium-nitrogen, and this suggests that in nodulated plantsthe need for cobalt is confined to the nodules. Vitamin B₁₂analogues are shown to be present in the nodules in relativelylarge amounts when cobalt is supplied, their formation beingattributed to the endophytes, which may therefore require cobaltfor their growth. The great reduction in fixation of atmosphericnitrogen in cobalt-deficient nodules may be due to a retardedgrowth of the endophyte, though this is not the only possibility.The cobalt relation of these non-legumes appears to be basicallysimilar to that of legumes.     

Hydrogenase in actinorhizal root nodules and root nodule homogenates.

Hydrogenases were measured in intact actinorhizal root nodules and from disrupted nodules of Alnus glutinosa, Alnus rhombifolia, Alnus rubra, and Myrica pensylvanica. Whole nodules took up H2 in an O2-dependent reaction. Endophyte preparations oxidized H2 through the oxyhydrogen reaction, but rates were enhanced when hydrogen uptake was coupled to artificial electron acceptors. Oxygen inhibited artifical acceptor-dependent H2 uptake. The hydrogenase system from M. pensylvanica had a different pattern of coupling to various electron acceptors than the hydrogenase systems from the alders; only the bayberry system evolved H2 from reduced viologen dyes.     

Micromonospora is a normal occupant of actinorhizal nodules

Actinorhizal plants have been found in eight genera belonging to three orders (Fagales, Rosales and Cucurbitales). These all bear root nodules inhabited by bacteria identified as the nitrogen-fixing actinobacterium Frankia. These nodules all have a peripheral cortex with enlarged cells filled with Frankia hyphae and vesicles. Isolation in pure culture has been notoriously difficult, due in a large part to the growth of fast-growing contaminants where, it was later found, Frankia was slow-growing. Many of these contaminants, which were later found to be Micromonospora, were obtained from Casuarina and Coriaria. Our study was aimed at determining if Micromonospora were also present in other actinorhizal plants. Nodules from Alnus glutinosa, Alnus viridis, Coriaria myrtifolia, Elaeagnus x ebbingei, Hippophae rhamnoides, Myrica gale and Morella pensylvanica were tested and were all found to contain Micromonospora isolates. These were found to belong to mainly three species: Micromonospora lupini, Micromonospora coriariae and Micromonospora saelicesensis. Micromonospora isolates were found to inhibit some Frankia strains and to be innocuous to other strains.     

Diversity and specificity of Frankia strains in nodules of sympatric Myrica gale, Alnus incana, and Shepherdia canadensis determined by rrs gene polymorphism

The identity of Frankia strains from nodules of Myrica gale, Alnus incana subsp. rugosa, and Shepherdia canadensis was determined for a natural stand on a lake shore sand dune in Wisconsin, where the three actinorhizal plant species were growing in close proximity, and from two additional stands with M. gale as the sole actinorhizal component. Unisolated strains were compared by their 16S ribosomal DNA (rDNA) restriction patterns using a direct PCR amplification protocol on nodules. Phylogenetic relationships among nodular Frankia strains were analyzed by comparing complete 16S rDNA sequences of study and reference strains. Where the three actinorhizal species occurred together, each host species was nodulated by a different phylogenetic group of Frankia strains. M. gale strains from all three sites belonged to an Alnus-Casuarina group, closely related to Frankia alni representative strains, and were low in diversity for a host genus considered promiscuous with respect to Frankia microsymbiont genotype. Frankia strains from A. incana nodules were also within the Alnus-Casuarina cluster, distinct from Frankia strains of M. gale nodules at the mixed actinorhizal site but not from Frankia strains from two M. gale nodules at a second site in Wisconsin. Frankia strains from nodules of S. canadensis belonged to a divergent subset of a cluster of Elaeagnaceae-infective strains and exhibited a high degree of diversity. The three closely related local Frankia populations in Myrica nodules could be distinguished from one another using our approach. In addition to geographic separation and host selectivity for Frankia microsymbionts, edaphic factors such as soil moisture and organic matter content, which varied among locales, may account for differences in Frankia populations found in Myrica nodules.     

Rapid assimilation of recently fixed N2 in root nodules of Myrica gale

In Myrica gale L. plants the assimilation of ammonia released by symbiotic Frankia was observed by ¹⁵N₂ labelling and subsequent analysis of the isotopic enrichment of nodule amino acids over time by single ion monitoring gas chromatography-mass spectrometry. In detached nodules of Myrica , glutamine was the first amino acid labelled at 30 s and subsequently the amino acids glutamate, aspartate, alanine and γ-amino butyric acid (GABA) became labelled. This pattern of labelling is consistent with the incorporation of ammonium via glutamine synthetase [GS; EC 6.3.1.2]. No evidence for the ammonium assimilation via glutamate dehydrogenase [GDH; EC 1.4.1.2] was observed as glutamate became labelled only after glutamine. Using attached nodules and pulse-chase labelling, we observed synthesis of glutamine, glutamate, aspartate, alanine, GABA and asparagine, and followed the transport of fixed nitrogen in the xylem largely as glutamine and asparagine. Estimation of the cost of nitrogen fixation and asparagine synthesis in Myrica nodules suggests a minimum of one sucrose required per asparagine produced. Rapid translocation of recently fixed nitrogen was observed in Myrica gale nodules as 80% of the nitrogen fixed during a 1-h period was translocated out of the nodules within 9 h. The large pool of asparagine that is present in nodules may buffer the transport of nitrogen and thus act to regulate nitrogen fixation via a feedback mechanism.     

Molecular phylogeny of Myricaceae: a reexamination of host-symbiont specificity

The phylogeny of 13 species of Myricaceae, the most ancient actinorhizal family involved in a nitrogen-fixing symbiosis with the actinomycete Frankia, was established by the analysis of their rbcL gene and 18S-26S ITS. The phylogenetic position of those species was then compared to their specificity of association with Frankia in their natural habitat and to their nodulation potential determined on greenhouse-grown seedlings. The results showed that Genus Myrica, including M. gale and M. hartwegii, and Genus Comptonia, including C. peregrina, belong to a phylogenetic cluster distinct from the other Myrica species transferred in a new genus, Morella. This grouping parallels the natural specificity of each cluster with Comptonia-Myrica and Morella being nodulated by two phylogenetically divergent clusters of Frankia strains, the Alnus and Elaeagnaceae-infective strains clusters, respectively. Under laboratory conditions, Comptonia and Morella had a nodulation potential larger than under natural conditions. From this study it appears that the Myricaceae are split into two different specificity groups. It can be hypothesized that the early divergence of the genera led to the selection of genetically diverse Frankia strains which is contradictory to the earlier proposal that evolution has proceeded toward narrower promiscuity within the family.     

An Isotopic Study of the Fixation of Nitrogen associated with Nodulated Plants of Alnus, Myrica, and Hippophae

Nodulated plants of Alnus glutinosa, Myrica gale, and Hippophaërhamnoides, the root systems of which had been exposed to excessfree ¹⁵N, showed substantial enrichment in fixed ¹⁵N contentin all parts of the plant, but particularly in the root nodules.The data resemble closely those obtained by the present andprevious authors with legumes under comparable conditions, andsupport the conclusion already drawn from experiments of traditionaltype that the nodules of these non-legumes are similar to thoseof legumes in their function and relation to the rest of theplant. Nodulated plants of Alnus and Myrica continued to fixfree nitrogen concurrently with the uptake of combined nitrogenwhen the latter was supplied in the rooting medium in amountsunlikely to be exceeded in the field. Isotopic tests on detachednodules of one of the species (Alnus glutinosa) showed thatfixation continued, and though much reduced as compared withthat shown by attached nodules it considerably exceeded thatexperienced by the present and previous authors with detachedlegume nodules, on the basis of fixation per unit of total nodulenitrogen. It is probable that detached Alnus nodules presentconvenient material for the further study of various aspectsof the fixation process.     

Root Nodule Symbiosis II

Nodule-roots of Myrica cerifera (Southern Wax Myrtle) and Casuarina cunning hamiana (Australian Pine) have a negative geotropic curvature. Studies of their endogenotts auxin content revealed a pattern of correlation: the absence of detectable auxin when the geotropisni was negative. Non-nodulated roots of Myrica exhibited a normal positive geotropic curvature and possessed an auxin content within an anticipated range (10 mg/kg). Root nodules of Alnus species, whose roots exhibit a positive geotropic curvature, contained measurable endogenous auxin (20 mg IAA/kg). The presence of an indoleaectic acid oxidase system in Myrica and Casuarina root nodules has heen described and correlations are drawn between non-detectable endogenous auxin concentrations and high enzymatic activities. It is suggested that the negative geotropic curvature of the nodule-roots of Myrica and Casuarina is due to the presence of a sub-optimal concentration of auxin which in turn results from the activity of an indoleacetic aeid destroying system.     

Isolation and characterization of non-Frankia actinobacteria from root nodules of Alnus glutinosa, Casuarina glauca and Elaeagnus angustifolia

Actinobacterial isolates randomly obtained on nitrogen-free BAP medium from surface sterilized root nodules of Alnus glutinosa, Casuarina glauca and Elaeagnus angustifolia sampled from fields were reported. They were assigned on the basis of partial 16S rRNA sequences to Micromonospora, Nocardia and Streptomyces genera. The isolates have been screened for hydrolytic activities, indole acetic acid (IAA) and siderophores production, phosphate solubilization and antagonistic activities. Results suggest putative traits as plant growth promoting bacteria proprieties of the isolates that occur in unique association in root nodules of the three analysed actinorhizal host species.     

Effects of substrate nitrate concentration on symbiotic nodule formation in actinorhizal plants

The response ofAlnus glutinosa, Casuarina cunninghamiana, Elaeagnus angustifolia andMyrica cerifera to a range of substrate nitrogen levels in solution, in relation to plant growth, infection, nodulation and root fine structure was studied. Nine concentrations of potassium nitrate ranging from 0.05 to 3.0 mM, were tested on each of the species. Plants were inoculated withFrankia pure cultures after a two week exposure to one of the nine levels of added nitrate. After six more weeks with constant exposure to nitrate, plants were harvested and assayed. With the exception of Myrica, regression analyses of whole plant dry weights as a function of added nitrate were highly significant. There was a tendency for nodulated plants grown at intermediate levels of added nitrate to exhibit higher relative growth rates, probably due to the additive effect of substrate nitrogen and fixation of atmospheric nitrogen. The mean numbers of nodules per plant were, with the exception of Alnus, significantly higher at intermediate levels of added nitrate, as were mean nodule dry weights. A highly significant inverse relationship between nodule weight as a percentage of whole plant weight was found in Elaeagnus and Myrica. The observed response of Elaeagnus to added nitrate compared to other actinorhizal plants appears to demonstrate that root hair infected plants are much more sensitive to the inhibitory effects of added nitrate than plants infected by intercellular penetration. A sharp reduction in the presence of root hairs at high concentrations of nitrate was observed. This indicates that the inhibition of nodulation in some actinorhizal plant species results from nitrate induced root hair suppression.     

Symbionts of alder nodules in New Zealand

Summary Micro-organisms capable of forming effective nodules on roots ofAlnus viridis have been found to be present in New Zealand soils. It is concluded that endemic soil organisms suitable for nodulation ofAlnus viridis occur, and cross inoculation trials have indicated that there are probably differences between organisms forming nodules with various species of alders.