Effective nodulation ofCoriaria myrtifolia L. induced byFrankia strains fromAlnus glutinosa L. (Gaertn.)

The present contribution covers the cross-inoculation between two actinorhizae belonging to different genera and families, mainlyAlnus glutinosa andCoriaria myrtifolia. Frankia strains isolated fromA. glutinosa received from the Netherlands (LDAgp₁r₁, LDAgn₁) and from Scotland (UGL010708), induced a fully effective nodulation onC. myrtifolia. The same effect was caused by a nodule extract fromA. glutinosa. The reverse, a crushed-nodule inoculum fromC. myrtifolia nodulated all theA. glutinosa seedlings, though nodules formed were less effective than those induced by the other inocula. Re-isolation of thoseFrankia strains from the nodules formed onA. glutinosa was readily obtained, whereas attempts to re-isolate them from the nodules formed onC. myrtifolia failed, suggesting that isolation procedures different to those employed should be tried.     

Evolutionary implications of nucleotide sequence relatedness between Alnus nepalensis and Alnus glutinosa and also between corresponding Frankia microsymbionts

Frankia DNAs were isolated directly from root nodules of Alnus nepalensis and Alnus nitida collected from various natural sites in India. For comparison, a nodule sample from Alnus glutinosa was also collected from Tuebingen, Germany. Nucleotide sequence analyses of amplified 16S–23S ITS region revealed that one of the microsymbionts from Alnus nepalensis was closely related to the microsymbiont from Alnus glutinosa. A similar exercise on the host was also carried out. It was found that one sample of Alnus nepalensis was closely related to Alnus glutinosa sequence from Europe. Since both Frankia and the host sequences studied revealed proximity between Alnus glutinosa and Alnus nepalensis, it is hypothesised that the common progenitor of all the alders first entered into an association with Frankia, and the symbiotic association has evolved since.     

The improvement and utilization in forestry of nitrogen fixation by actinorhizal plants with special reference toAlnus in Scotland

Summary Alnus species are used widely in Britain for land reclamation, forestry and other purposes. Rapid juvenile growth of the AmericanAlnus rubra makes it an attractive species for planting on N-deficient soils, particularly those of low organic content. In small plot trials, this species is nodulated by indigenous soil frankiae as effectively asAlnus glutinosa. Over a three year period both species return similar amounts of N to the ecosystem, estimated at up to 10–12 kg N ha^–1. Several strains ofFrankia have been isolated from local (Lennox Forest)A. rubra nodules. These differ morphologically and in their growth on different culture media, both from each other and fromA. glutinosa nodule isolates. AllAlnus isolates, however, have a total cellular fatty acid composition qualitatively similar to some other Group B frankiae. Glasshouse tests in N free culture suggest thatA. rubra nodules formed after inoculation of seedlings with American spore (–) isolates are three times more effective in N fixation than those inoculated with LennoxA. rubra spore (+) nodule homogenates. By contrast, the early growth of seedlings inoculated with spore (–)Frankia strains suggests at best a 35% improvement in N fixing activity over seedlings inoculated with LennoxA. rubra nodule isolates. Nevertheless, this improvement in activity, together with the better performance of seedlings inoculated with isolates compared with those treated with crushed nodule preparations, suggest that it would be worthwhile commercially to inoculate nursery stock with a spore (–)Frankia strain.     

Selection and micropropagation of nodulating and non-nodulating clones ofAlnus crispa (Ait.) Pursh

Summary 600,000 seedlings ofAlnus crispa were inoculated with a 111 mixture of theFrankia strains ACN1 ^AG , AGN1 _exo ^AG and MGP10i. After 3 successive inoculations and screenings, one individual, AC-4, was selected as non-nodulating (Nod^–) with Frankiae. This selected individual AC-4 (Nod^–) and two other clones ofA. crispa, AC-2 and AC-5, known for their ability to nodulate (Nod⁺) and two other clones ofA. crispa, AC-2 and AC-5, known for their ability to nodulate (Nod⁺) withFrankia werein vitro propagated. The different clones ofA. crispa in culture required different kinds and concentrations of sugar during the in vitro multiplication and rooting stages. Nodulation tests using 7Frankia strains indicated that the clone AC-4 (Nod^–) was non-nodulating with 6 of the 7Frankia strains tested. One strain,Frankia ANNI, isolated from one unique nodule produced on the mother-plant AC-4, induced 38% of the AC-4 plantlets to nodulate but with a number of nodules 10 to 20 times less than the clones AC-2 (Nod⁺) and AC-5 (Nod⁺). Morphological observations of the roots of AC-4 (Nod^–) indicated that this clone had few and abnormally short root hairs.     

Variable compatibility of clonedAlnus glutinosa ecotypes against ineffectiveFrankia strains

Nodulation tests onin-vitro propagated clones ofAlnus glutinosa ecotypes (forest ecotype, pioneer ecotype) withFrankia strains originating from both ecotypes indicated differences in host-plant compatibility. Inoculated plants of the pioneer ecotype clone were not infected by strains, that were unable to fix nitrogen in pure culture. Nodulation could only be induced on the clone of the forest ecotype, but no nitrogen-fixing activity could be detected. Ultra-structural observations of the nodules by SEM and TEM indicated that ineffectivity of these strains was correlated with the lack of vesicles in the infected cells. Cells were only filled with hyphae: neither sporangia nor vesicles could be detected. In contrast, effective nodules could be obtained on both alder clones after inoculation with an effective strain, showing normal development of vesicle clusters in infected cells. In pure culture the ineffective strains produced no vesicles; sporangia were found only during early stage of growth. The results demonstrate the existence ofFrankia strains which were either non-infective or ineffective on different clones ofAlnus glutinosa.     

Alder-Frankia interaction and alder-poplar association for biomass production

Summary Alders have an important role to play in biomass producing stands because of their N₂-fixing ability and their capacity to withstand soils having an excess of moisture. The objectives of preliminary trials were (1) to find if there is any alder-genotype xFrankia-strain interaction when the effect of inoculating the bacteria was compared to no inoculation in seed beds of different species and provenances of alder, (2) to measure the possible effect of black alders interplanted in poplars compared to pure poplar plots. Two trials were laid out to study the alder-Frankia interaction. Both produced interaction. In the first one the inoculation had a favorable effect onAlnus glutinosa at age 2 years andA. cordata at age 1 and 2 and no effect onA. rubra. In the second one the inoculation had a depressive effect at age 1 on 2 of 3 provenances ofA. rubra and no effect on 1A. rubra, 3A.glutinosa and 3A. cordata provenances.A closely spaced field trial associating one black alder provenance and the poplar clone UNAL gives no superiority of mixed plots compared to pure plots. The results suggest that the N₂-fixation of alders is not profitable to poplars at age 3 with a 1.5×2 m spacing.     

Growth increment ofAlnus glutinosa upon dual inoculation with effective and ineffectiveFrankia strains

Investigations on the ecological function of ineffectiveFrankia strains and their behaviour in competition with effectiveFrankia strains indicated an enhanced plant growth upon dual inoculation with increasing amounts of effective (i.e. N₂-fixing)Frankia strains and simultaneous inoculation with a constant amount of an ineffectiveFrankia strain. Enhanced plant growth was measured as increase in plant height and total dry weight at constant shoot/root ratio. The stimulating effect of the ineffective strain was independent of the plant clone and was obtained with bothAlnus glutinosa clones W I and B II, which were resistant and susceptable, respectively, to the ineffective strain. Stimulation was also independent of the nodulation conditions. Short-term studies (7 weeks) under axenic conditions and greenhouse experiments during 3 months showed comparable results, not only in plant growth but also in nodule formation. Increment in plant growth was not necessarily correlated to higher nodule formation with the effectiveFrankia strains.     

Infectivity and effectivity of Frankia strains from the Rhamnaceae family on different actinorhizal plants

Ten strains of Frankia isolated from root nodules of plant species from five genera of the host family Rhamnaceae were assayed in cross inoculation assays. They were tested on host plants belonging to four actinorhizal families: Trevoa trinervis (Rhamnaceae), Elaeagnus angustifolia (Elaeagnaceae), Alnus glutinosa (Betulaceae) and Casuarina cunninghamiana (Casuarinaceae). All Frankia strains from the Rhamnaceae were able to infect and nodulate both T. trinervis and E. angustifolia. Strain ChI4 isolated from Colletia hystrix was also infective on Alnus glutinosa. All nodules showed a positive acetylene reduction indicating that the microsymbionts used as inoculants were effective in nitrogen fixation. The results suggest that Frankia strains from Rhamnaceae belong to the Elaeagnus-infective subdivision of the genus Frankia.     

Frankia-Alnus incana symbiosis: Effect of endophyte on nitrogen fixation and biomass production

The efficiency of different FinnishFrankia strains as symbionts onAlnus incana (L.) Moench was evaluated in inoculation experiments by measuring nitrogen fixation and biomass production. Since all available pure cultures ofFrankia are of the Sp⁻ type (sporangia not formed in nodules), but the dominant nodule endophyte ofA. incana in Finland is of the Sp⁺ type (sporangia formed in nodules), crushed nodules of thisFrankia type were included. The Sp⁻ pure cultures, whether originating fromA. incana orA. glutinosa, produced with one exception, similar biomass withA. incana. The highest biomass was produced with an American reference strain fromA. viridis crispa. Using Sp⁺ nodule homogenates fromA. incana as inoculum, the biomass production was only one third of that produced by Sp⁻ pure cultures from the same host. Hence, through selection of the endophyte it is possible to exert a considerable influence on the productivity ofAlnus incana.     

Effect of Inoculation and Leaf Litter Amendment on Establishment of Nodule-Forming Frankia Populations in Soil

High-N₂-fixing activities of Frankia populations in root nodules on Alnus glutinosa improve growth performance of the host plant. Therefore, the establishment of active, nodule-forming populations of Frankia in soil is desirable. In this study, we inoculated Frankia strains of Alnus host infection groups I, IIIa, and IV into soil already harboring indigenous populations of infection groups (IIIa, IIIb, and IV). Then we amended parts of the inoculated soil with leaf litter of A. glutinosa and kept these parts of soil without host plants for several weeks until they were spiked with [¹⁵N]NO₃ and planted with seedlings of A. glutinosa. After 4 months of growth, we analyzed plants for growth performance, nodule formation, specific Frankia populations in root nodules, and N₂ fixation rates. The results revealed that introduced Frankia strains incubated in soil for several weeks in the absence of plants remained infective and competitive for nodulation with the indigenous Frankia populations of the soil. Inoculation into and incubation in soil without host plants generally supported subsequent plant growth performance and increased the percentage of nitrogen acquired by the host plants through N₂ fixation from 33% on noninoculated, nonamended soils to 78% on inoculated, amended soils. Introduced Frankia strains representing Alnus host infection groups IIIa and IV competed with indigenous Frankia populations, whereas frankiae of group I were not found in any nodules. When grown in noninoculated, nonamended soil, A. glutinosa plants harbored Frankia populations of only group IIIa in root nodules. This group was reduced to 32% ± 23% (standard deviation) of the Frankia nodule populations when plants were grown in inoculated, nonamended soil. Under these conditions, the introduced Frankia strain of group IV was established in 51% ± 20% of the nodules. Leaf litter amendment during the initial incubation in soil without plants promoted nodulation by frankiae of group IV in both inoculated and noninoculated treatments. Grown in inoculated, amended soils, plants had significantly lower numbers of nodules infected by group IIIa (8% ± 6%) than by group IV (81% ± 11%). On plants grown in noninoculated, amended soil, the original Frankia root nodule population represented by group IIIa of the noninoculated, nonamended soil was entirely exchanged by a Frankia population belonging to group IV. The quantification of N₂ fixation rates by ¹⁵N dilution revealed that both the indigenous and the inoculated Frankia populations of group IV had a higher specific N₂-fixing capacity than populations belonging to group IIIa under the conditions applied. These results show that through inoculation or leaf litter amendment, Frankia populations with high specific N₂-fixing capacities can be established in soils. These populations remain infective on their host plants, successfully compete for nodule formation with other indigenous or inoculated Frankia populations, and thereby increase plant growth performance.     

Physiological studies on N2-fixing root nodules ofDatisca cannabina L. andAlnus nitida Endl. from Himalaya region in Pakistan

Summary The nodulation and the morphology and physiology of the nodules were studied onDatisca cannabina, a perennial herb from northern Pakistan andAlnus nitida, a nodulated tree in the same locality. Both species bear coralloid clusters of actinorhizal nodules. The main free amino acid inD. cannabina nodules was arginine while the predominant free amino acid inA. nitida nodules was citrulline. The infectivity of crushed nodules of both types of plants on their respective host was about 10⁶ infective particles per gram of nodule fresh wt. In cross-inoculation experiments crushed nodule inoculum fromA. nitida failed to induce nodulation onD. cannabina seedlings but the crushed nodule inoculum fromD. cannabina caused low nodulation on seedlings ofA. nitida (10³ infective particles. g. nodule fresh wt.).The activity of nitrogenase, hydrogenase and respiration (O₂ uptake) were measured in detached nodules, nodule homogenates and the 20 m residue and 20 m filtrate preparations from the nodules of both species. Both species showed similar patterns of activities except that only the nodule homogenate and 20 m residue preparations fromD. cannabina showed pronounced enhancement of the O₂ uptake by succinate which was further stimulated by ADP. This has in part been explained by the presence of mitochondria in close connection with the endophyte.     

Utilization of the tricarboxylic acid cycle intermediates and symbiotic effectiveness inRhizobium meliloti

Summary The utilization of the tricarboxylic acid cycle intermediates and related compounds was studied in strains ofRhizobium meliloti having different symbiotic effectiveness. In general, the very effective (VE) strains used these compounds as sole carbon source better than the ineffective (I) strains. However, a significant different was observed between VE and I strains in their ability to use acetate or oxaloacetate for growth. In fact, at a concentration of 2 mM, 80% of the VE strains used acetate or oxaloacetate white 50% of the I strains used acetate and none was able to grow on oxaloacetate. No correlation was found between the symbiotic effectiveness of the strains and their ATP content, when grown on mannitol. The highest ATP content (9.21 nM×g protein^–1) was found in the I strain S₂₀ and the lowest (0.69 nM×g protein^–1 was found in the effective strain S₈. Numerical analysis of the patterns of utilization of the TCA cycle intermediates and related compounds indicated that the 49 strains tested formed 11 distinct groups at 86% similarity, according to Jaccard's coefficient. Several strains showed unique patterns of utilization and can be clearly identified under laboratory conditions.Contribution no.225 Station de Recherches, Agriculture Canada.     

The isolation,culture and infectivity of aFrankia strain fromGymnostoma papuanum (Casuarinaceae)

A strain ofFrankia was isolated fromGymnostoma papuanum(Casuarinaceae) nodules harvested from rooted cuttings which had been inoculated with a suspension of crushedCasuarina equisetifolia nodules. Designated HFPGpI1 (catalogue #HFP021801), this strain is pigmented and similar to other pigmentedFrankia strains in cultural characteristics. A previously unknown spiraled hyphal morphology was observed at very low frequency in some cultures of this strain. HFPGpI1 is infective and effective onG. papuanum but not on anyCasuarina species tested. It also infects members of the family Elaeagnaceae andMyrica gale. The host plantG. papuanum can be infected with a wide range ofFrankia isolates and thus can be considered a promiscuous host, unlike its close relatives in the genera Casuarina and Allocasuarina which are very restrictive as to which strains may nodulate them.     

Host range differentiation of spore-positive and spore-negative strain types of Frankia in stands of Alnus glutinosa and Alnus incana in Finland

Host compatibility of different spore-positive (Sp⁺)and spore-negative (Sp^?) strain types of Frankia from alder stands in Finland was studied in Modulation tests with hydrocultures of Alnus glutinosa (L.) Gaertner, A. incana (L.) Moench and A. nitida Endl. Root nodules and soil samples from stands of A. incana (Lammi forest and Hämeenlinna forest) were dominated by Sp ⁺ types of Frankia (coded AiSp⁺ and AiSp⁺ H. respectively), which caused effective root nodules in test plants of A. incana, but failed to induce nodules in A. nitida. In A. glutinosa Frankia strain types AiSp ⁺ and AiSp ⁺ H caused small, ineffective root nodules with sporangia (coded Ineff ^?), which were recognized by the absence or near absence of vesicles in the nodule tissue. Ineffective nodules without sporangia (coded Ineff ^?) were induced on A. glutinosa with soil samples collected at Lammi swamp. The spore-negative strain type of Frankia was common in root nodules of A. glutinosa in Finland (Lammi swamp) and caused effective Sp^? type root nodules (coded AgSp ^?) in hydrocultures of A. incana, A. glutinosa and A. nitida. A different Sp ⁺ strain type of Frankia. coded AgSp⁺ Finland, was occasionally found in stands of A. glutinosa. It was clearly distinguished from strain type AiSp ⁺ by the ability to produce effective nodules on both A. glutinosa and A. incana. The nodulation capacities of soil and nodule samples were calculated from the nodulation response in hydrocutlure and served as a measure for the population density of infective Frankia particles. Sp ⁺ nodules from both strain types had equal and high nodulation capacities with compatible host species. The nodulation capacities of Sp type root nodules from A. glutinosa were consistently low. High frequencies of Frankia AiSp⁺ and AiSp⁺ H were found in the soil environment of dominant AiSp ⁺ nodule populations on A. incana. The numbers of infective particles of this strain type were insignificant in the soil environment of nearby Sp ^? nodule populations on A. glutinosa and in the former field at Hämeen-linna near the Sp⁺ nodule area in Hämeenlinna forest. Strain type AgSp^? had low undulation capacity in the soil environment of both A. incana and A. glutinosa stands, Explanations for the strong associations between Frankia strain types AiSp⁺ and AiSp ^? H and A. incana and between strain type AgSp^? and A. glutinosa are discussed in the light of host specificity and of some characteristics of population dynamics of both strain types. The possible need to adapt the concept of Frankia strain types Sp ⁺ and Sp ^? to strains with some variation in spore development was stressed by the low potentials of strain type AiSp ⁺ H to develop spores in symbioses with hydrocultures of A. incnna.     

Study of the contribution of the shoot and/or root ofAlnus sp. in the compatibility between the host and a Sp+ Frankia strain using a grafting technique

Two alder species,Alnus glutinosa (L.) Gaertn. andAlnus incana (L) Moench, were inoculated with a Sp⁺ Frankia homogenate obtained fromA. incana root nodules. This inoculum formed effective nodules on the original host plant and ineffective nodules onA. glutinosa. Grafts between the two alder species were made to determine which part of the plant is involved in this phenomenon. The results obtained indicate that the compatibility between Alnus andFrankia is restricted to the root system.     

N2-fixation in Erwinia herbicola

Four from 18 strains of Erwinia herbicola tested had nitrogenase activity and grew with N₂ as sole source of nitrogen under strict anaerobic conditions with a doubling time of 20–24 h. Nitrogenase activity started only 96–120 h after transfer to a special medium maintained under anaerobic conditions. A ten fold increase in protein per culture found after the maximum nitrogenase activity of 80–130 nmol C₂H₄. mg protein^-1·min^-1 was accompanied by a fall in pH of the medium (20 mM phosphate buffer and in 125 mM Tris-buffer) from pH 7.2 to 5.4 or less, but only to 6.8 in 100 mM phosphate buffer. In all cases we found a sharp curtailing of nitrogenase activity 48 h after the maximum. The bacteria utilized only 35–50% of the nitrogen fixed for growth. Erwinia herbicola strains differed from two strains of Enterobacter agglomerans in being unable to fix nitrogen on agar surfaces exposed to air. Specific nitrogenase activity in Erwinia herbicola is compared with data reported for other Enterobacteriaceae and is found to be higher than that reported for Klebsiella pneumoniae, Enterobacter cloacae or Citrobacter freundii.     

Diversity of Frankia strains isolated from single nodules of Alnus glutinosa

Diversity of Frankia isolates originating from lobes of single nodules collected on Alnus glutinosa root systems has been analyzed using isozyme electrophoresis method. Analysis of isozyme patterns showed no divergence among strains isolated from the same nodule. Each nodule (among 10 assayed) was inhabited by a single Frankia strain.     

Effect of juglone on growthin vitro ofFrankia isolates and nodulation ofAlnus glutinosa in soil

Summary In vitro growth (total protein content) of 5Frankia isolates was significantly inhibited at 10^–4 M juglone (5-hydroxy-1, 4-napthoquinone) concentration, but the degree of inhibition varied with theFrankia isolate. Isolates fromAlnus crispa [Alnus viridis ssp.crispa (Ait.) Turril] were most tolerant of 10^–4 M juglone relative to controls, while an isolate fromPurshia tridentata (Pursh.) D.C. was most inhibited, displaying a dramatic decrease in growth and greatly altered morphology.Nodulation of black alder [Alnus glutinosa L. (Gaertn.)] in an amended prairie soil inoculated with aFrankia isolate from red alder (Alnus rubra Bong.) was significantly decreased by the addition of aqueous suspensions of 10^–3 M and 10^–4 M juglone. This decrease was partially independent of decreased plant growth. The addition of an equal volume of sand to the soil mixture further decreased nodulation of black alder.Frankia inoculation of the soil mixtures significantly increased the total number of nodules formed per seedling, and the degree of differences in seedling nodulation owing to juglone and soil treatments.     

Isolation of infective and effective Frankia strains from root nodules of Alnus acuminata (Betulaceae)

Two Frankia strains were isolated from root nodules of Alnus acuminata collected in the Tucumano-oranense forest, Argentina. Monosporal cultures were obtained by plating a spore suspension of each strain and isolating a single colony. The strains (named AacI and AacIII) showed branched mycelia with polymorphic sporangia and NIR-vesicles. They differed in their ability to use carbon sources: the AacI strain grew well on pyruvate, while the AacIII strain grew on mineral medium supplemented with glucose or, alternatively, with sucrose. The two strains were sensitive to oleandomycin, erythromycin, kanamycin, penicillin G, streptomycin and chloramphenicol at 5 μg/ml. The AcIII strain exhibited a moderate resistance to rifampicin, ampicillin and vancomycin. The nitrogenase activity in vitro of the strains was significantly higher in basal medium without nitrogen than that determined in the presence of ammonium chloride. Both strains were infective on seedlings of Alnus glutinosa, inducing an approximately similar percentage of nodulated plants (80%), although strain AacIII produced a higher number of nodules per plant (≤15) than strain AacI (≤6). They were also effective for nitrogen fixation in planta, determined by the acetylene reduction assay. This revised version was published online in July 2006 with corrections to the Cover Date.     

Preliminary characterization of an ineffective Frankia derived from a spontaneously neomycin-resistant strain

Five strains of Frankia isolated from actinorhizae of 3 alder species were cultivated with various concentrations of neomycin in the medium. For one strain, resistance to neomycin was associated with lost of effectiveness. The ineffective strain was as infective as the parent strain. Protein and isoenzymes patterns showed that the ineffective and the parent strains were quite similar. The ineffective strain differentiated vesicles inside the infected host-cells.