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.     

Host species and habitat affect nodulation by specific Frankia genotypes in two species of Alnus in interior Alaska

Alders (Alnus spp.) are important components of northern ecosystems due to their ability to fix nitrogen (N) in symbiosis with Frankia bacteria. Availability of optimal Frankia may be a contributing factor in limiting the performance and ecological effects of Alnus, but the factors underlying distribution of Alnus-infective Frankia are not well understood. This study examined the genetic structure (nifD–K spacer RFLP haplotypes) of Frankia assemblages symbiotic with two species of Alnus (A. tenuifolia and A. viridis) in four successional habitats in interior Alaska. We used one habitat in which both hosts occurred to observe differences between host species independent of habitat, and we used replicate sites for each habitat and host to assess the consistency of symbiont structure related to both factors. We also measured leaf N content and specific N-fixation rate (SNF) of nodules (¹⁵N uptake) to determine whether either covaried with Frankia structure, and whether Frankia genotypes differed in SNF in situ. Frankia structure differed between sympatric hosts and among habitats, particularly for A. tenuifolia, and was largely consistent among replicate sites representing both factors. Leaf N differed between host species and among habitats for both hosts. SNF did not differ among habitats or host species, and little evidence for differences in SNF among Frankia genotypes was found, due largely to high variation in SNF. Consistency of Frankia structure among replicate sites suggests a consistent relationship between both host species and habitat among these sites. Correlations with specific environmental variables and possible underlying mechanisms are discussed. Nomenclature: Flora of North America ().     

Correlations between the ages of Alnus host species and the genetic diversity of associated endosymbiotic Frankia strains from nodules

Nodule samples were collected from four alder species: Alnus nepalensis, A. sibirica, A. tinctoria and A. mandshurica growing in different environments on Gaoligong Mountains, Yunnan Province of Southwest China and on Changbai Mountains, Jilin Province of Northeast China. PCR-RFLP analysis of the IGS between nifD and nifK genes was directly applied to uncultured Frankia strains in the nodules. A total of 21 restriction patterns were obtained. The Frankia population in the nodules of A. nepalensis had the highest genetic diversity among all four Frankia populations; by contrast, the population in the nodules of A. mandshurica had the lowest degree of divergence; the ones in the nodules of A. sibirica and A. tinctoria were intermediate. A dendrogram, which was constructed based on the genetic distance between the restriction patterns, indicated that Frankia strains from A. sibirica and A. tinctoria had a close genetic relationship. Frankia strains from A. nepalensis might be the ancestor of Frankia strains infecting other Alnus species. From these results and the inference of the ages of Alnus host species, it is deduced that there was a co-evolution between Alnus and its microsymbiont Frankia in China.     

Correlations between the ages ofAlnus host species and the genetic diversity of associated endosymbioticFrankia strains from nodules

Nodule samples were collected from four alder species:Alnus nepalensis, A. sibirica, A. tinctoria andA. mandshurica growing in different environments on Gaoligong Mountains, Yunnan Province of Southwest China and on Changbai Mountains, Jilin Province of Northeast China. PCR-RFLP analysis of the IGS betweennifD andnifK genes was directly applied to unculturedFrankia strains in the nodules. A total of 21 restriction patterns were obtained. TheFrankia population in the nodules ofA. nepalensis had the highest genetic diversity among all fourFrankia populations; by contrast, the population in the nodules ofA. mandshurica had the lowest degree of divergence; the ones in the nodules ofA. sibirica andA. tinctoria were intermediate. A dendrogram, which was constructed based on the genetic distance between the restriction patterns, indicated thatFrankia strains fromA. sibirica andA. tinctoria had a close genetic relationship.Frankia strains fromA. nepalensis might be the ancestor ofFrankia strains infecting otherAlnus species. From these results and the inference of the ages ofAlnus host species, it is deduced that there was a co-evolution betweenAlnus and its microsymbiontFrankia in China.

     

Lectin genes in the <Emphasis Type="Italic">Frankia alni</Emphasis> genome

Frankia alni strain ACN14a’s genome was scanned for the presence of determinants involved in interactions with its host plant, Alnus spp. One such determinant type is lectin, proteins that bind specifically to sugar motifs. The genome of F. alni was found to contain 7 such lectin-coding genes, five of which were of the ricinB-type. The proteins coded by these genes contain either only the lectin domain, or also a heat shock protein or a serine-threonine kinase domain upstream. These lectins were found to have several homologs in Streptomyces spp., and a few in other bacterial genomes among which none in Frankia EAN1pec and CcI3 and two in strain EUN1f. One of these F. alni genes, FRAAL0616, was cloned in E. coli, fused with a reporter gene yielding a fusion protein that was found to bind to both root hairs and to bacterial hyphae. This protein was also found to modify the dynamics of nodule formation in A. glutinosa, resulting in a higher number of nodules per root. Its role could thus be to permit binding of microbial cells to root hairs and help symbiosis to occur under conditions of low Frankia cell counts such as in pioneer situations.     

Phylogeny and assemblage composition of Frankia in Alnus tenuifolia nodules across a primary successional sere in interior Alaska

In nitrogen (N) fixing symbioses, host‐symbiont specificity, genetic variation in bacterial symbionts and environmental variation represent fundamental constraints on the ecology, evolution and practical uses of these interactions, but detailed information is lacking for many naturally occurring N‐fixers. This study examined phylogenetic host specificity of Frankia in field‐collected nodules of two Alnus species (A. tenuifolia and A. viridis) in interior Alaska and, for A. tenuifolia, distribution, diversity, spatial autocorrelation and correlation with specific soil factors of Frankia genotypes in nodules collected from replicated habitats representing endpoints of a primary sere. Frankia genotypes most commonly associated with each host belonged to different clades within the Alnus‐infective Frankia clade, and for A. tenuifolia, were divergent from previously described Frankia. A. tenuifolia nodules from early and late succession habitats harboured distinct Frankia assemblages. In early succession, a single genotype inhabited 71% of nodules with no discernable autocorrelation at any scale, while late succession Frankia were more diverse, differed widely among plants within a site and were significantly autocorrelated within and among plants. Early succession Frankia genotype occurrence was strongly correlated with carbon/nitrogen ratio in the mineral soil fraction, while in late succession, the most common genotypes were correlated with different soil variables. Our results suggest that phylogenetic specificity is a significant factor in the A. tenuifolia‐Frankia interaction and that significant habitat‐based differentiation may exist among A. tenuifolia‐infective genotypes. This is consistent with our hypothesis that A. tenuifolia selects specific Frankia genotypes from early succession soils and that this choice is attenuated in late succession.     

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.     

Frankia Populations in Soil and Root Nodules of Sympatrically Grown Alnus Taxa

The genetic diversity of Frankia populations in soil and in root nodules of sympatrically grown Alnus taxa was evaluated by rep-polymerase chain reaction (PCR) and nifH gene sequence analyses. Rep-PCR analyses of uncultured Frankia populations in root nodules of 12 Alnus taxa (n?=?10 nodules each) growing sympatrically in the Morton Arboretum near Chicago revealed identical patterns for nodules from each Alnus taxon, including replicate trees of the same host taxon, and low diversity overall with only three profiles retrieved. One profile was retrieved from all nodules of nine taxa (Alnus incana subsp. incana, Alnus japonica, Alnus glutinosa, Alnus incana subsp. tenuifolia, Alnus incana subsp. rugosa, Alnus rhombifolia, Alnus mandshurica, Alnus maritima, and Alnus serrulata), the second was found in all nodules of two plant taxa (A. incana subsp. hirsuta and A. glutinosa var. pyramidalis), and the third was unique for all Frankia populations in nodules of A. incana subsp. rugosa var. americana. Comparative sequence analyses of nifH gene fragments in nodules representing these three profiles assigned these frankiae to different subgroups within the Alnus host infection group. None of these sequences, however, represented frankiae detectable in soil as determined by sequence analysis of 73 clones from a Frankia-specific nifH gene clone library. Additional analyses of nodule populations from selected alders growing on different soils demonstrated the presence of different Frankia populations in nodules for each soil, with populations showing identical sequences in nodules from the same soil, but differences between plant taxa. These results suggest that soil environmental conditions and host plant genotype both have a role in the selection of Frankia strains by a host plant for root nodule formation, and that this selection is not merely a function of the abundance of a Frankia strain in soil.     

Molecular ecology ofFrankia: Advantages and disadvantages of the use of DNA probes

Ecological studies on the actinomyceteFrankia are often influenced by the difficulty to isolate and identify this microorganism. The application of molecular biological techniques offers possibilities to detect microbes without isolation and cultivation.Nif genes or whole plasmids can serve as targets for the design of specific probes. Alternatively, ribosomal RNA (rRNA), commonly used in modern phylogenetic studies, can be used as a target molecule in ecological studies. This paper gives an overview of new developments on the use of 16S rRNA as a target molecule for oligonucleotide probes. Group-specific sequences in the 16S rRNA ofFrankia can be used as targets for oligonucleotide probes that a) recognize ineffectiveFrankia strains onAlnus, b) recognize effective strains onAlnus, c) recognize allFrankia strains tested so far. The present paper summarizes the essential steps needed for the use of these probes for the detection ofFrankia strains in soil without isolation and cultivation.     

Frankia in the rhizosphere of nonhost plants: A comparison with root-associated N2-fixing Enterobacter,Klebsiella and Pseudomonas

Bacterial growth in the rhizosphere and resulting changes in plant growth parameters were studied in small aseptic seedlings of birch (Betula pendula and B. pubescens) and grasses (Poa pratensis and Festuca rubra). The seedlings were inoculated with three Frankia strains (Ai1a and Ag5b isolated from native Alnus root nodules and Ai17 from a root nodule induced by soil originating from a Betula pendula stand), and three associative N₂-fixing bacteria (Enterobacter agglomerans, Klebsiella pneumoniae and Pseudomonas sp., isolated from grass roots). Microscopic observations showed that all the Frankia strains were able to colonize and grow on the root surface of the plants tested without addition of an exogenous carbon source. No net growth of the associative N₂-fixers was observed in the rhizosphere, although inoculum viable counts were maintained over the experimental period. Changes in both the biomass and morphology of plant seedlings in response to bacterial inoculation were recorded, which were more dependent on the plant species than on the bacterial strain.     

Effects of some pure and mixedFrankia strains on seedling growth in differentAlnus species

In greenhouse experiments plants of eightAlnus species, from various parts of the world, and from different taxonomic sections, were inoculated with threeFrankia strains in order to show any possible interaction. Mixtures in equal parts of theseFrankia strains were also tried. The growth of inoculated plants was significantly higher than of the controls, with one of the three strains being superior. Mixtures of strains generally provided higher growth than the best individual strain. No interaction betweenFrankia strains andAlnus species was detected in the young plants 60 days after inoculation. Three clones ofAlnus glutinosa were inoculated with the same pure cultures ofFrankia, without producing any interaction. Inoculation time was studied in one clone and one progeny ofAlnus glutinosa. The best results were obtained with the earlier inoculation (at sowing for the progeny and at transfer to soil for thein vitro-propagated clone). The results are discussed in terms of nursery practice and field experiments for selection in breeding programmes.     

Acetylene, Not Ethylene, Inactivates the Uptake Hydrogenase of Actinorhizal Nodules during Acetylene Reduction Assays

Acetylene reduction assays were shown to inactivate uptake hydrogenase activity to different extents in one Casuarina and two Alnus symbioses. Inactivation was found to be caused by C₂H₂ and not by C₂H₄. Acetylene completely inactivated the hydrogenase activity of intact root systems of Alnus incana inoculated with Frankia strain Avcl1 in 90 minutes, as shown by a drop in the relative efficiency of nitrogenase from 1.0 to 0.73. The hydrogenase of Frankia preparations (containing vesicles) and of cell-free extracts (not containing vesicles) from the same symbiosis was much more susceptible to acetylene inactivation. Cell-free extracts lost all hydrogenase activity after 5 minutes of exposure to acetylene. The hydrogenase activity of intact root systems of Casuarina obesa was less sensitive to acetylene than that of root systems of A. incana, since the relative efficiency of nitrogenase changed only from 1.0 to 0.95 over 90 minutes. Frankia preparations and cell-free extracts of C. obesa still retained hydrogenase activity after a 10 minute-exposure to acetylene.     

Respiration of Malate and Glutamate in Symbiotic Frankia Prepared from Alnus incana

Frankia vesicle clusters were prepared from root nodules ofAlnus incana (L.) Moench inoculated either with a local sourceof Frankia or with Frankia Cpll. The capacity of vesicle clustersto respire was investigated by respirometric and enzymologicalstudies. Simultaneous addition of malate, glutamate, and NAD⁺supported respiration in both types of Frankia, though at asmaller rate compared to the substrates NADH or 6-phosphogluconate.The saturating concentrations of malate and glutamate were alsomuch higher than with the other substrates. No respiration wassupported by succinate. Activity of the enzymes malate dehydrogenase(EC 1.1.1.37 [EC] ) and glutamate oxaloacetate transaminase (EC 2.6.1.1 [EC] )was demonstrated in crude extracts from both types of symbioticFrankia. Their maximum rates were high enough to account forthe respiration of malate and glutamate. This respiration wasinhibited by mersalylic acid, an inhibitor of the dicarboxylateshuttle in mitochondria, but it was shown that inhibition ofrespiration could be due to a direct effect on the enzymes.We conclude that respiration of malate and glutamate is mostlikely mediated by malate dehydrogenase and glutamate oxaloacetatetransaminase, but no explicit evidence for or against the presenceof a dicarboxylate carrier was found. The utilization of respiratorysubstrates was largely similar in the two types of Frankia,except for some differences in maximum rates and cofactor dependency. Key words: Actinorhizal symbioses, Alnus, dicarboxylate shuttle, Frankia, reducing power, respiration     

Conservation of nif sequences in Frankia

Summary Southern blots of Frankia total DNAs were hybridized with nifHDK probes from Rhizobium meliloti, Klebsiella pneumoniae and Frankia strain Arl3. Differences between strains were noted in the size of the hybridizing restriction fragments. These differences were more pronounced among Elaeagnus-compatible strains than among Alnus- or Casuarina-compatible strains. Gene banks constructed for Frankia strains EUN1f, HRN18a, CeD and ACoN24d were used to isolate nif-hybridizing restriction fragments for subsequent mapping and comparisons. The nifH zone had the highest sequence conservation and the nifH and nifD genes were found to be contiguous. The complete nucleotide sequence of the nifH open reading frame (ORF) from Frankia strain Arl3 is 861 bp in length and encodes a polypeptide of 287 amino acids. Comparisons of these nucleic acid and amino acid sequences with other published nifH sequences suggest that Frankia is most similar to Anabaena and Azotobacter spp. and K. pneunoniae and least similar to the Gram-positive Clostridium pasteurianum and to the archaebacterium Methanococcus voltae.     

Diversity of frankiae in root nodules of Morella pensylvanica grown in soils from five continents

Bioassays with Morella pensylvanica as capture plant and comparative sequence analyses of nifH gene fragments of Frankia populations in nodules formed were used to investigate the diversity of Frankia in soils over a broad geographic range, i.e., from sites in five continents (Africa, Europe, Asia, North America, and South America). Phylogenetic analyses of 522-bp nifH gene fragments of 100 uncultured frankiae from root nodules of M. pensylvanica and of 58 Frankia strains resulted in a clear differentiation between frankiae of the Elaeagnus and the Alnus host infection groups, with sequences from each group found in all soils and the assignment of all sequences to four and five clusters within these groups, respectively. All clusters were formed or dominated by frankiae obtained from one or two soils with single sequences occasionally present from frankiae of other soils. Variation within a cluster was generally low for sequences representing frankiae in nodules induced by the same soil, but large between sequences of frankiae originating from different soils. Three clusters, one within the Elaeagnus and two within the Alnus host infection groups, were represented entirely by uncultured frankiae with no sequences from cultured relatives available. These results demonstrate large differences in nodule-forming frankiae in five soils from a broad geographic range, but low diversity of nodule-forming Frankia populations within any of these soils.     

Both the arbuscular mycorrhizal fungus Gigaspora rosea and Frankia increase root system branching and reduce root hair frequency in Alnus glutinosa

Alnus glutinosa is an important pioneer species that forms effective symbioses with Frankia and ecto and arbuscular mycorrhizal fungi (AMF). There is evidence that Frankia and AMF interact and the focus of this study was to investigate how interactions affected root system and root hair development. A. glutinosa seedlings were grown in pots in soil pre-inoculated with the AMF Gigaspora rosea. Seedlings were inoculated with Frankia either immediately on transfer to AMF-inoculated pots (day 0) on day 15 or on day 30 following AMF inoculation so the effect of timing of inoculation on interactions could be determined. Seedlings were harvested in batches at intervals of 10, 15, 20, 25 and 30 days after the commencement of each treatment. Both G. rosea and Frankia increased root branching and effects were greater when both were present. By contrast, both G. rosea and Frankia decreased root hair numbers markedly. Effects on root hair development were not a consequence of phosphorous, as P levels were not changed significantly in seedlings colonised by G. rosea or nodulated by Frankia. Effects are not due to differences in root system size but conceivably could offset some of the carbon costs incurred by the symbioses.     

Host plant growth response to inoculation withFrankia

Optimum growth conditions and inoculation regimes were determined for severalFrankia strains isolated from both Alnus and Casuarina host plants. Growth conditions were estabilished that allowed a reduction in generation time to less than 15 hours for certain Alnus derivedFrankia. Differences in plant growth response were observed with differing inoculum levels and soil mixtures. Elite strains of Alnus derivedFrankia were isolated that elicited similar growth reponses in allAlnus species tested; however, differences were observed betweenFrankia strains and plant growth response of variousCasuarina species tested.     

Sucrose Utilization via Invertase and Sucrose Synthase with Respect to Accumulation of Cellulose and Callose Synthesis in Wheat Roots under Oxygen Deficiency

The sucrose cleavage by sucrose synthase (SuSy) and neutral invertase was studied in wheat roots (Triticum aestivum L.) subjected to hypoxia or anoxia for 4 days. By in situ activity staining, increased SuSy activity was observed in the tip region and stele of root axes while the activity of invertase decreased. Cellulose content significantly increased in hypoxically treated roots. The cellulose deposition was correlated with regions of high SuSy activity, being mainly located in the pericycle and endodermis. Invertase activity was distributed along the root without clear difference between cortex and stele. Under root hypoxia, a significant increase in the structural carbohydrates, callose and especially cellulose, was shown. Increasing levels of soluble carbohydrates were partially used to synthesize cellulose for secondary wall thickening and callose to counteract the tissue injury following low-oxygen stress. Under strict anoxia, the roots were much more injured but sustained a high level of cellulose and callose while the soluble carbohydrates almost disappeared.     

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.