Showing papers on "Frankia published in 1985"

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

Abstract: 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.

Restriction enzyme digestion patterns of Frankia plasmids

Abstract: After the initial screening of more than 200 Frankia strains, the plasmid DNA observed in eight Frankiae was analyzed. In situ lysis was performed to obtain an estimate of their copy number and molecular weight. Four plasmid classes were distinguished, 7–9, 18–20, 30–35 and 50–55 kb. Twelve plasmids were thus analysed with restriction enzymes to determine their plasmid restriction patterns.

Growth, acetylene reduction activity and localization of nitrogenase in relation to vesicle formation in Frankia strains Cc1.17 and Cp1.2

Abstract: A comparative study was conducted on the effect of NH4Cl on growth, vesicle formation and formation of nitrogenase of Frankia strains Cc1.17 and Cp1.2, derived from root nodules of Colletia cruciata and Comptonia peregrina, respectively. On a medium without combined nitrogen (“P-N”), both strains formed spherical cells, called vesicles, like many other Frankia strains. Data are presented on the number of vesicles per mg protein, after cultivation in media with sodium propionate as C-source without combined nitrogen (“P-N”) or with 0.2 g NH4Cl/l (“P+N”). Strain Cp1.2 as may other Frankia strains, showed on “P+N” medium a very strong reduction of vesicle formation of 99% relative to the number of vesicles formed on “P-N” medium, after 11 days growth. However, in strain Cc11.17 this reduction was only 70%. The occurence of relatively large numbers of vesicles in “P+N” media has not yet been reported for other Frankia strains. No acetylene reduction activity was found in NH4+-grown cells. The regulation of induction of nitrogenase in Frankia by NH4Cl was tested by immuno-gelectrophoresis using antisera against nitrogenase of Rhizobium leguminosarum PRE. The component I of the enzyme showed crossreactivity while the component II had only a weak crossreaction. The experiments indicated that no nitrogenase was detectable in the NH4+-grown cells. For the localization of nitrogenase, relative amounts of the enzyme were compared in whole cells and vesicle-enriched fractions. Western blots showed a significant enrichment of nitrogenase in the vesicle fractions, which indicated that most of the nitrogenase was localized in the vesicle.

Effect of O2 on vesicle formation, acetylene reduction, and O2-uptake kinetics in Frankia sp. HFPCcI3 isolated from Casuarina cunninghamiana

Abstract: The effect of the partial pressure of oxygen (PO2) on the formation of vesicles, which are thought to be the site of N2 fixation in Frankia, was studied in HFPCcI3, an effective isolate from Casuar...

Relationships of Frankia Isolates Based on Deoxyribonucleic Acid Homology Studies

Abstract: A molecular approach was used to examine the genetic relatedness of 19 Frankia isolates by measuring the extent of deoxyribonucleic acid-deoxyribonucleic acid homology and the fidelity of hybrid duplex molecules. The Frankia isolates examined were divided into two groups based on the results of hybridization tests. The members of genogroup 1, consisting of isolates from Alnus, Myrica, and Comptonia host plants, exhibited high levels of homology (67.4 to 94.1%) with strain ArI4, an isolate from Alnus rubra. Isolates from Elaeagnus, Ceanothus, Purshia, and Casuarina and isolate AirI2 from Alnus did not exhibit significant homology (less than 39%) with strain ArI4. None of the strains showed a high degree of homology with strain EuI1a (less than 33%), an isolate from Elaeagnus umbellata. Among the isolates not belonging to genogroup 1, subgroupings seemed to exist, as evidenced by a very high level of homology (97%) between two isolates from Casuarina, strains D11 and the G2, but a low level of homology (27%) between other strains and strain G2. Thermal stability studies of the hybrid deoxyribonucleic acids which showed high levels of homology revealed an average mismatch of 3%, whereas the low-homology duplexes exhibited about 5% mismatch. The genome molecular weights of two probe Frankia strains, strains ArI4 and EuI1a, were 8.3 × 109and 6.0 × 109, respectively.

Frankia source affects growth, nodulation and nitrogen fixation in casuarina species

Abstract: Summary Two species of Casuarina [C. equisetifolia ssp. incana (Benth.) L.J. and C. cunninghamiana Miq.] differed in growth responses to inoculation with Frankia from five sources. Casuarina equisetifolia ssp. incana formed nodules with all five Frankia inocula whereas C. cunninghamiana formed nodules with only two. A range of response from ineffectiveness in N2 fixation to high effectiveness was observed between Casuarina species - Frankia source combinations. Strains of Frankia highly effective on one species were frequently ineffective on the other. Effectiveness was related to nodule development after infection and to the ability of developed nodules to fix N2.

Assessment of N2 fixation by Casuarina equisetifolia inoculated with Frankia ORS021001 using 15N methods

Abstract: Casuarina equisetifolia seedlings, uninoculated or inoculated with Frankia strain ORS021001 were grown for 4.5 months in pouches, then transplanted into 1 m3 concrete containers forming 1 m2 microplots. Trees were harvested 6.5 months later when they were 11 months old. N2 fixation was measured using three methods of assessment: the direct isotopic method, the A-value method and the difference method. Estimations of N2 fixation during the 6.5 months following transplantation were respectively 3.27, 2.31 and 3.07g N2 per tree. From these values it was calculated that about 40–60 kg N2 would be fixed per hectare in a year at normal densities of 10,000 trees ha−1. The results of this experiment confirm that Frankia strain ORS021001 can be confidently recommended to inoculate casuarinas in the field. Means to improve nodulation and subsequently N2 fixation by casuarinas are discussed.

Large scale inoculation of actinorhizal plants with Frankia

Abstract: From 1979 to 1984 more than seven million seedlings of actinorhizal plants were successfully inoculated on an industrial scale with Frankia inoculants. Nodulated seedlings were produced in greenhouses to be used for land reclamation in northern Quebec by the Societe d’Energie de la Baie James (SEBJ) and also by the City of Montreal for a revegetation program. Crushed-nodule homogenates and Frankia pure culture formulations were compared for large scale inoculation of green alder. Pure culture inoculant was found to be superior than crushed-nodule homogenates yielding reproducible nodulation of seedlings. Two inoculation methods of the Frankia pure culture inocula were compared: soil injection and spraying with greenhouse watering devices. Both methods resulted in efficient nodulation of Alnus crispa, A. glutinosa, A. rugosa, Elaeagnus angustifolia, E. commutata, Hippophae rhamnoides, Myrica gale and Shepherdia argentea.

Studies of an effective strain of Frankia from Allocasuarina lehmanniana of the Casuarinaceae

Abstract: 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.

Development of endophytic frankia sporangia in field- and laboratory-grown nodules of comptonia peregrina and myrica gale

Abstract: Field-collected nodules of Comptonia peregrina (L.) Coult. and Myrica gale L. (Myricaceae), infected by the nitrogen-fixing actinomycete Frankia sp., were of two types: those that lacked sporangia entirely, designated sporef-), and those that showed extensive sporangial development, designated spore(+). In spore(+) nodules of C. peregrina, sporangia began to develop after the differentiation of endophytic vesicles and the concomitant onset of nitrogenase activity. At the onset of sporangial differentiation, infected host cells appeared healthy. However, endophytic vesicles and host cell cytoplasm and nuclei began to senesce rapidly as sporangia developed. Staining of sectioned material with the fluorescent stain Calcofluor White suggested that vesicles, hyphae and young sporangia were enclosed within a host-derived encapsulation layer, but mature sporangia were no longer encapsulated. In both C. peregrina and M. gale, vesicles were more short-lived in spore(+) than in sporef-) nodules. Field-collected spore(+) M. gale nodules exhibited a pronounced seasonality of sporangial formation. Sporangia began to differentiate in June, after the formation of vesicles and became more prominent in late summer. Inter- and intraspecific cross-inoculations suggest that the ability to form sporangia in the symbiotic state is controlled by endophytic strain type rather than host genotype or host/ endophyte combination. The host may, however, influence the number and seasonal appearance of sporangia formed. MEMBERS of the genus Frankia, soil-inhabiting actinomycetes, are capable of infecting the roots of woody plants belonging to eight dicotyledonous families, and of inducing the formation of nodules (Bond, 1976; Torrey, 1978). These nodules are specialized symbiotic organs in which the endophyte, Frankia, fixes atmospheric nitrogen which is then available to the host in reduced form. That Frankia is a pleomorphic organism capable of diffierentiating both vesicles and sporangia from its hyphae, was known long before it was first successfully isolated from nodules (Callaham, Del Tredici and Torrey 1978) or characterized as an actinomycete. Schaede (1933) first noticed that nodules ob

The effects of soil temperature on

Abstract: The effects of soil temperatures between 15 and 30 ?C on plant growth, nodulation and nitrogen fixation in seedlings of Casuarina cunninghamiana Miq. inoculated with Frankia from two different sources were examined. The optimum soil temperature for the growth of plants dependent on symbiotic nitrogen fixation was 25 'C. Decreasing the soil temperature below 25 'C markedly decreased plant growth that was reliant on symbiotically fixed nitrogen; effects on the growth of plants supplied with mineral nitrogen were much smaller. At 15 'C there was no response in plant growth to inoculation after 148 d, whereas plants supplied with nitrogenous fertilizer were 10 times the weight of uninoculated plants. Nodulation was delayed at 15 and 20 'C with nodules formed at 15 'C fixing no nitrogen in these studies. The production of fewer nodules at 20 'C than at 25 'C was partly compensated by the production of larger nodules. Nodule growth at 20 to 30 'C was a prime determinant of nitrogen fixed, with the exception of one Frankia at 20 'C. The amount of nitrogen-fixed g-' nodule was the same for the two Frankia sources at 25 and 30 'C, differences in effectiveness being due to nodule development. However, differences in the effectiveness of the two Frankia sources at 20 'C were related to differences both in nodule development and in nitrogen-fixing ability. The absence of nitrogen fixation at 15 'C would be expected to limit the natural distribution of Casuarina species reliant on symbiotically fixed nitrogen to areas where soil temperatures exceed 15 'C for a major part of the potential growing season.

The effects of soil temperature on plant growth, nodulation and nitrogen fixation in Casuarina cunninghamiana Miq.

Abstract: Summary The effects of soil temperatures between 15 and 30°C on plant growth, nodulation and nitrogen fixation in seedlings of Casuarina cunninghamiana Miq. inoculated with Frankia from two different sources were examined. The optimum soil temperature for the growth of plants dependent on symbiotic nitrogen fixation was 25°C. Decreasing the soil temperature below 25°C markedly decreased plant growth that was reliant on symbiotically fixed nitrogen; effects on the growth of plants supplied with mineral nitrogen were much smaller. At 15°C there was no response in plant growth to inoculation after 148 d, whereas plants supplied with nitrogenous fertilizer were 10 times the weight of uninoculated plants. Nodulation was delayed at 15 and 20°C with nodules formed at 15°C fixing no nitrogen in these studies. The production of fewer nodules at 20°C than at 25°C was partly compensated by the production of larger nodules. Nodule growth at 20 to 30°C was a prime determinant of nitrogen fixed, with the exception of one Frankia at 20°C. The amount of nitrogen-fixed g−1 nodule was the same for the two Frankia sources at 25 and 30°C, differences in effectiveness being due to nodule development. However, differences in the effectiveness of the two Frankia sources at 20°C were related to differences both in nodule development and in nitrogen-fixing ability. The absence of nitrogen fixation at 15°C would be expected to limit the natural distribution of Casuarina species reliant on symbiotically fixed nitrogen to areas where soil temperatures exceed 15°C for a major part of the potential growing season.

Do single nodules of Casuarinaceae contain more than oneFrankia strain

Abstract: Studies using inoculum suspensions prepared from single nodules of Casuarinaceae suggest that more than oneFrankia strain may be found in some nodules. Evidence is also presented to support the concept of intergeneric specificity in host-Frankia relations in the family Casuarinaceae.

Restriction analysis of the Frankia spp. genome

Abstract: Total cellular DNAs of 10 Frankia isolates from Alnus, Elaeagnus and Colletia spp. root nodules were cleaved with ten site-specific restriction endonucleases and analysed by agarose gel electrophoresis. Among the endonucleases tested, Bam HI, Bgl II, Sal I and Sma I proved to be the most suitable for the genome analysis in Frankia spp. DNA restriction banding patterns were reproducible and characteristic of each Frankia strain. The patterns of different strains differed marked indicating considerable genotypic heterogeneity among the isolates. The approach can be used for strain identification in Frankia spp. as well as for differentiation between phenotypically similar strains.

Performance of in vitro propagated Alnus glutinosa (L.) Gaertn. clones inoculated with Frankiae

Abstract: Three Alnus glutinosa (L.) Gaertn. clones, obtained by in vitro propagation techniques, were inoculated with four strains of Frankia. The ability of these clones to nodulate and fix nitrogen was previously reported; this study deals with the performance of 12 different combinations of pairs of symbionts.

Frankia symbiosis as a source of nitrogen in forestry: a case study of symbiotic nitrogen-fixation in a mixed Alnus-Picea plantation in Scotland

Abstract: Alnus rubra growing in the field in Scotland is nodulated effectively by local strains of Frankia. Strains which have been isolated from Alnus rubra at Lennox Forest show different growth medium requirements and colony morphology compared with isolates from Alnus glutinosa and several different strains have been isolated from the same locality. Preliminary evidence suggests that some spore negative, northwest American Frankia strains may be more effective than local spore positive crushed nodule inoculum for nitrogen-fixation in Alnus rubra.On the moderately fertile clay soil of Lennox Forest, a mixed plantation of alternate Picea silchensis and Alnus rubra showed no improvement in growth of spruce compared with pure spruce plots. However, the presence of alder increased upper soil nitrogen status by 585 kg ha ' which approximates the standard 150 kg N ha 'of fertiliser nitrogen applied in practice to nitrogen deficient stands at about 5-year intervals. Although the alder had penetrated the subsoil, there was no apparent effect on spruce rooting depth in the mixed stands in this high clay soil. In addition to nitrogen content, the total phosphorus of the upper soil horizons was improved in the mixed plots by an estimated 3–6 kg ha -1 y 1 and it is suggested that this phosphorus may be brought from the subsoil by the deeper rooting alder and deposited on the surface in its litter.Although improved growth of spruce in mixture with Alnus rubra is only likely where pure spruce stands are stressed for nutrients, the potential benefits of symbiotic nitrogen-fixation in silvicultural practice make it desirable to investigate other species and provenances of Alnus suited to British conditions, to achieve maximum symbiotic fixation of nitrogen by selection and inoculation with superior strains of Frankia and to include such plants in trials of mixtures on sites where nitrogen-availability may be critical.

Host range of Frankia endophytes

Abstract: 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 from Alnus 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 of Myrica californica, but not the seedlings of Elaeagnus, Casuarina and Myrica rubra. The pure cultured strains from Elaeagnus can infect and nodulate the host species in the same genus and family with an exception of E. viridis var. delavayi, which can be only poorly nodulated by a few strains from Elaeagnus. The strains from Elaeagnus cannot infect the seedlings of Alnus and Myrica rubra. The results presented here suggest that Frankia endophytes can be divided into two groups: Alnus group and Elaeagnus group.

In vitro propagation and nodulation by Frankia of actinorhizal Russian Olive (Elaeagnus angustifolia L.)

Abstract: Following the evaluation of the nutritional requirements for thein vitro propagation ofElaeagnus angustifolia, this actinorhizal species was routinely multiplied on MS, supplemented with 100 mM sucrose and 5 μM kinetin. On this medium, at a 3 week-interval, a multiplication rate of 5–10 was observed. A morphological variant occurred in culture (wet type) but it was converted into the normal type (pubescent type) by a passage on 1/2 macro MS and 1.5% agar. One hundred percent rooting was achieved in liquid medium containing 1/2 MS without growth regulators. The plantlets were transferred aseptically to a nitrogen-free artificial soil substrate and inoculated with pure cultures of differentFrankia strains which had been isolated from Elaeagnus, Shepherdia and Hippophae host plants. We thus ascertained that afterin vitro propagation, the plants retained their capacity to nodulate and sustain nitrogen fixation.

The Site of Nitrogenase in Frankia in Free-Living Culture and in Symbiosis

Abstract: Actinorhizal plants comprise a diverse array of woody dicotyledonous species whose roots are susceptible to infection by the filamentous soil bacterium Frankia of the Actinomycetales. The present tally of host plants includes in excess of 200 species distributed among twenty-three genera in eight families (Moiroud and Gianinazzi-Pearson 1984). The filamentous bacterium infects susceptible plants either via root hair deformation and invasion (Callaham et al. 1979, Berry et al. 1985) or through root epidermal penetration via intercellular spaces and cortical cell penetration (Miller, Baker 1985). Within the host cortical cells produced from proliferating modified lateral roots which form the multilobed nodule (Bowes et al. 1977), the invading bacterium differentiates branched filaments which typically end in terminal swellings called vesicles. Within the vesicles the prokaryotic enzyme nitrogenase is formed, catalyzing the reduction of dinitrogen to ammonia which is assimilated into the metabolism of both the endophyte and its symbiotic host.

Preinfection events in the establishment of Alnus-Frankia symbiosis: Development of a spot inoculation technique

Abstract: The early biochemical and structural events associated with the infection of Alnus byFrankia are still largely unknown. These studies have been hampered by the difficulty of localizing precisely the site of inoculation on the root surface and of standardizing the inoculum dose. To facilitate these investigations, we describe a new spot inoculation method wellsuited to study the Alnus-Frankia system. This method involves the growth ofFrankia in the presence of microcarrier and their subsequent deposition on the alder root surface as an inoculum dose. The ability of this new procedure to induce nodulation close to the point of inoculation has been observed.

In vitro production of specialized reproductive torulose hyphae by Frankia strain ORS 021001 isolated from Casuarina junghuhniana root nodules

Abstract: AFrankia strain (ORS 021001) isolated fromCasuarina junghuhniana root nodules was shown to produce four type of structuresin vitro: vegetative hyphae, sporangiospores within sporangia, N2-fixing vesicles, and a fourth type of structure which is described in detail in this report. Structures of this latter type which we propose to call ‘reproductive torulose hyphae: (RTH) result from enlargement and multiple segmentation of vegetative hyphae into torulose chaions of spore-like cells. RTH differ from sporangia in three major aspects: morphology, morphogenesis and outgrowth. RTH play an important role in survival and reproduction ofFrankia strain ORS 021001. Adding activated charcoal to the nutrient medium promotes the formation ofFrankia colonies orginating from RTH.

Homogeneity of superoxide dismutase patterns in Frankia strains from Casuarinaceae

Abstract: Seven Frankia strains from Casuarina and Allocasuarina were analyzed by polyacrylamide gel electrophoresis to determine the patterns of several enzymes. No relatedness could be established between the strains as far as polyphenol oxidase, esterase and diaphorase were concerned. A first attempt at grouping the Frankia isolates could be achieved with catalase. It was confirmed by the study of superoxide dismutase: only one activity band, with the same mobility, was found in all cases. We propose to use this enzyme as a marker for identification of Frankia strains issued from Casuarinaceae.

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

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

Purification and properties of trehalase in Frankia ArI3

Abstract: Trehalase was purified from cultures of Frankia strain ArI3 grown on media with or without NH4Cl. The purified enzyme was specific for trehalose, exhibited a broad pH optimum of pH 4.5 to 5.3 and had a Km for trehalose of 4.2 mM. The trehalase was inhibited in vitro completely by sucrose, glucose and mannose and partially by mannitol and sorbitol. In addition to the specific trehalase, a mixture of non-specific α- and β-glucosidases which exhibited some activity with α,α-trehalose as a substrate were also partially purified in Frankia extracts made from nitrogen-fixing cells. These enzymes were not detected in the purifications of crude extracts made from non-nitrogen-fixing cells (grown on media supplemented with NH4Cl). Trehalase activity in crude extracts increased over time when cells were induced to fix nitrogen, and the maximum specific activity of trehalase from nitrogen-fixing cultures was 4 times the maximum activity from non-fixing cultures. Trehalase activity was also examined in crude extracts made from Frankia vesicle clusters isolated from Alnus rubra nitrogen-fixing nodules infected with ArI3. The maximum activity of trehalase in these clusters was 6–7 times greater than in the nitrogenfixing pure cultures of ArI3 and 26–33 times greater than the non-fixing pure cultures.

Nodulation speed of Frankia sp. on Alnus glutinosa, Alnus crispa, and Myrica gale

Abstract: The question of compatibility between actinorhizal host plants and Frankia sp. was addressed using nodulation speed on Alnus spp. seedlings and Myrica gale seedlings. It was found that the speed of nodulation, defined as the mean time taken for the formation of the first prenodule, was a stable phenotype of both the Frankia strains and the host plants and that a distinction between slow-, medium-, and fast-nodulating Frankia strains could be made. The speed of nodulation of a given Frankia strain did not appear to be positively correlated to the original host plant from which isolation was first performed. It was, however, positively correlated with the Frankia strain and with the host plant species used for inoculation. Some optimal host plant – endophyte combinations were thus defined. Pure spore inocula of Frankia and in vitro propagated Alnus glutinosa plantlets were used to confirm that both the host plant and the microbial partners genetically influenced the nodulation process.

The detection of leghemoglobin-line sequences in legumes and non-legumes.

Abstract: Leghemoglobin is a major component of the nitrogen-fixing nodules formed by legumes in association with bacterial symbionts of the genusRhizobium. It is thought to be involved in regulating the oxygen tension within nodules. In a series of Southern blot experiments using cloned soybean leghemoglobin cDNAs as hybridization probes, cross-hybridizing sequences have been detected in legumes closely related to soybean (members of the Leguminosae subfamily Papilionoideae), as well as in a distantly related legume not reported to be nodulated (subfamily Caesalpinioideae). With the same probes, the presence of cross-hybridizing sequences has also been detected in plants outside the Leguminosae, including two nitrogen-fixing non-legumes and one species which is not nodulated. These results suggest that the genes for oxygen-binding proteins may be more widely dispersed than previously thought.

Inoculation and production of container-grown red alder seedlings

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

Characterization and infectivity of a spontaneous variant isolated from Frankia sp. WEY 0131391

Abstract: A spontaneous variant, obtained from a Frankia isolate from Alnus rubra nodules, was compared with the parent strain with regard to infeetivity, nitrogenase activity, and electro- phoretic and immunological profiles. Both the parent and the variant strain were equally effective in inducing nodulation in seedlings of A. rubra. All inoculated plants had an active nitrogenase system as measured by the acetylene reduction assay. Electrophoresis of whole cell homogenates on SDS-polyacrylamide slab gels showed similar electrophoretic profiles; however, the variant strain also exhibited striking differences in protein patterns that distinguish it from the parent strain. Immunological analysis of the original Frankia strain and its variant revealed shared antigens as well as immunologically distinct antigenic determinants in the two strains. The variant strain exhibits a distinct morphology and growth patterns which remain stable after many passages through culture.