Nitrogen and Carbon Utilization in Alnus incana Fixing N2 or Supplied with NO3- at the Same Rate

The purpose of this study was (i) to evaluate biomass productionand nitrogen utilization in grey alder and (ii) to investigatecarbon utilization in alders fixing nitrogen or supplied withnitrate. Two experiments were made, each using two groups ofalders (Alnus incana (L.) Moench) growing in a climate chamber.In each experiment one group was inoculated with a local sourceof Frankia. The other group was not inoculated but receivednitrate in the same amount as the first group fixed nitrogen.Therefore, the rate of nitrate application increased duringthe experimental period. Biomass production, growth, nitrogencontent and nitrogen utilization were determined in one experiment.Growth, nitrate reductase activity, net photosynthesis and rootrespiration were measured in the second experiment. In experimentone there was no significant difference in biomass productionbetween the two groups of plants. Of the nitrate given 99% wastaken up and 1% of this uptake was excreted in an organic form.In the second experiment the plant development was similar innodulated and non-nodulated alders during the period studied.Nitrate reductase activity was found in both root- and shoot-tipsof the non-nodulated alders. At both days 24 and 30, the amountof carbon respired compared to the amount of carbon assimilatedwas similar for the nodulated alders as for the non-nodulatedalders. As nitrogen fixation increased, photosynthesis alsoincreased. Thus, there was an inter-relationship between netphotosynthesis and nitrogen fixation. Key words: Alnus-Frankia symbioses, carbon utilization, nitrogen (N₂ ) utilization     

Seasonal flooding regimes influence survival,nitrogen fixation,and the partitioning of nitrogen and biomass in Alnus incana ssp. rugosa

Alteration of natural flooding regimes can expose lowlands to waterlogged soil conditions during any month of the year. The seasonality of flooding may have profound effects on the carbon and nitrogen budgets of N-fixing alders (Alnus spp.), and in turn, may impact the C and N economy of extensive alder-dominated, wetland ecosystems, including those dominated by speckled alder (Alnus incana ssp. rugosa). To better understand this process, two-year-old, nodulated seedlings of speckled alder were subjected to late spring (May 10 – July 10), summer (July 10 – September 8), and fall (September 8 – November 8) flooding treatments. Alders were root-flooded outdoors in tanks containing an N-free nutrient solution and compared with unflooded alders at the experimental site. Flooding arrested N fixation, photosynthesis, and growth of alders without recovery in all flooding treatments for the remainder of the growing season. Late spring and summer flooding resulted in complete mortality of alders while all seedlings survived flooding in the fall. Fall flooding increased foliar N resorption by 140% over unflooded seedlings. Eighty-seven percent of the total N fixed and 89% of biomass accumulation for the entire growing season occurred in unflooded alders after July 10. In unflooded alders, nitrogen fixation rates per unit mass declined by 63% for nodules, 28% for leaves, and 48% for whole seedlings during the fall, while total N fixed per plant in the fall was similar to that fixed in the summer. The majority of newly fixed N in unflooded alders was allocated to leaves before September 8 and to roots/nodules combined after September 8. In unflooded plants, the greatest proportion of new biomass was partitioned to leaves before July 10, to stems between July 10 and September 8, and equally to stems and roots/nodules after September 8. Fall-flooded alders did not increase root or nodule biomass. Proportional allocation of plant resources were such that the ratio of N fixed to seedling growth of unflooded alders decreased by 19% during summer before rebounding by 6% in fall. Seasonality of flooding alters seedling survival, growth, and resource allocation, and may be a critical determinant of speckled alder recruitment and occurrence in wetlands.     

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.     

Growth and acetylene reduction activity by intact plants ofAlnus incana under field conditions

The nitrogen-fixing grey alder,Alnus incana (L.) Moench, has a potential use in forest soil restoration and as part of energy forestry plantations. As a first step to estimate nitrogen fixation byA. incana under field conditions we performed studies on nitrogenase activity and its possible relation to abiotic factors and growth of the alders. Nitrogenase activity was measured as acetylene reduction activity (ARA) on eleven 1-year-old seedlings ofA. incana inoculated with a local source ofFrankia and planted in an experimental plot located in Umeå, northern Sweden. Each alder was planted into an open-ended cylinder which was closed with a gas tight lid around the stem base to serve as cuvette during ARA measurements. Propane served as tracer gas. ARA was measured in the middle of the day at 15 occasions during 26 June to 29 September 1987. Growth was recorded as leaf area and top shoot length at each ARA measurement until the end of August. Weather conditions were recorded for the whole growing season.Maximal ARA was recorded in late July or early August and ranged from 1.86 to 106mol C₂H₄plant^–1h^–1. Final leaf area ranged from 0.022 to 0.124 m². A relationship between ARA and the number of hours of sunshine during the same day was observed. ARA in relation to soil temperature increased during the study period, except for the last measurements. ARA in relation to leaf area was initially high but decreased later on. It is suggested that as leaves got older their contribution to photosynthesis per unit leaf area decreased and their potential to deliver nitrogen for retranslocation within plant increased. Both of these events would cause reduced ARA per unit leaf area. The data on ARA, growth, and abiotic factors taken together supported the view that sunshine and weather conditions affected photosynthesis and thereby delivery of assimilates to the nodules.     

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.     

Water use and yield of tomatoes under limited water and excess boron

The role of tripartite associations among Frankia, Alpova diplophloeus (an ectomycorrhizal fungus) and Alnus tenuifolia in growth, nitrogen fixation, ectomycorrhizal formation, and mineral acquisition of A. tenuifolia was investigated. Seedlings of A. tenuifolia were planted in pots containing a mixture of ground basalt–perlite, or perlite alone, which served as the control. The seedlings were inoculated with Frankia isolated from root nodules of alder, followed by spores of A. diplophloeus and grown for 5 months in a greenhouse. The seedlings grown in the pots with a mixture of ground basalt–perlite after dual inoculation with Frankia and A. diplophloeus had the heaviest shoots and root nodules in dry weight, and showed the greatest nitrogen-fixing ability measured by acetylene reduction. Ectomycorrhizae formed with A. diplophloeus increased when this fungus was inoculated together with Frankia. The mineral composition (P, K, Ca, Fe, Mg, Mn, Na, Si and Al) in the seedlings was also determined. The results of these experiments showed that the tripartite associations could improve the growth, nitrogen fixation and mineral acquisition (rock solubilization) of A. tenuifolia.     

Nitrogen in shoot litter,root litter and root exudates from nitrogen-fixingAlnus incana

Summary A pot experiment withAlnus incana (L.) Moench growing in sand was set up to compare the amounts of nitrogen released from plants shoot litter with that released below ground as root litter and/or root exudation. No nitrogen fixation by free-living microorganisms was found in the sand and the increased nitrogen content of the plant + soil system was therefore due to nitrogen fixation byFrankia in the alder root-nodules. Most of the nitrogen released from the plants was in the nitrogen-rich leaf and other shoot litter. Only small amounts of nitrogen were found in the drainage water from the pots and were recorded as increased nitrogen content of the sand.     

Growth,nitrogen fixation and relative efficiency of nitrogenase inAlnus incana grown in different cultivation systems

Summary Three cultivation systems were compared. In one system the alders were grown hydroponically. In the two other systems the alders were planted in gravel and either given water and nutrients at intervals or the nutrient solution was continuously supplied. Alders continuously supplied with nutrients and water showed a significantly more rapid growth, higher biomass production and higher nitrogen content than did alders given nutrients and water at intervals or alders hydroponically grown. Alders continuously supplied with water and nutrients had a constant RE (relative efficiency of nitrogenase) of about 0.80 throughout the experimental period while alders supplied with water and nutrients at intervals showed a slight decrease in RE at the end of the experimental period. No strict relationship was found between RE and nitrogen content or between RE and plant productivity.     

Effects of Frankia on field performance of Alnus clones and seedlings

Field performance of tissue cultured clones and seedlings of Alnus viridis ssp. crispa, A. glutinosa, A. incana, and A. japonica was assessed five years after outplanting in central Ontario. Half the individuals were inoculated with a mixture of four Frankia isolates prior to planting. Inoculation produced significant increases (25% to 33%) in biomass production of two clones of A. glutinosa and one of A. incana. Woody biomass increments for the first five years, averaged across all clones and seedlings, were highest in A. japonica and A. incana (4.3 and 3.7 Mg ha^–1 yr^–1, respectively). Individual tree growth improved markedly in lower slope positions, but total plot biomass did not show similar gains in downslope positions owing to higher mortality and aphid (Paraprociphilus tessellatus) infestation. Aphids occurred in 22% of Frankia-inoculated individuals, and 15% of non-inoculated individuals. The fastest growing species, A. incana and A. japonica, were most susceptible to aphid attack. Growth of the best clones of A. glutinosa and A. incana exceeded seedling growth by 51% and 76%, respectively. The high growth variation in clones of the same species with similar geographic origins and the excellent performance of tissue cultured stock suggest that rapid genetic gains in an Alnus breeding program might be obtained by clonal propagation.     

Lipid composition and nitrogenase activity of symbioticFrankia (Alnus incana) in response to different oxygen concentrations

Summary The role ofFrankia vesicle envelope lipids in regulating oxygen diffusion of symbiotic nitrogen fixation inAlnus incana was examined. Total lipids of symbioticFrankia (vesicle clusters) that had been adapted to oxygen tensions of 5,21, or 40 kPa were analyzed with a normal phase HPLC system. During the oxygen treatment, nitrogenase activity was measured as hydrogen evolution in an open flow-through system. When plants were transferred to low oxygen (5 kPa) or high oxygen (40 kPa), nitrogenase activity dropped initially. Activity recovered in both treatments with a rate comparable to the controls (21 kPa O₂). Both lipid content and lipid composition of vesicle clusters were affected by the oxygen treatments. With increasing oxygen tension, the vesicle cluster lipid content increased. This correlated with structural data (fluorescence microscopy and TEM) which showed a thicker vesicle envelope at higher oxygen tension. Three hopanoid lipids, bacteriohopanetetrol (bht) and two isomers of phenylacetyl monoester of bht, made up approximately 80% of the vesicle cluster lipids. With changing oxygen concentrations, the ratio of the two bht esters changed whereas the relative proportion of bht remained fairly constant. Therefore, in theFrankia-Alnus incana symbiosis, adaptation to different ambient oxygen tensions occurs at least partly by increasing the thickness of theFrankia vesicle envelope and by changing its lipid composition.Abbreviations dw dry weight - bht bacteriohopanetetrol - SE standard error - TEM transmission electron microscopy Dedicated to the memory of Professor John G. Torrey     

Nodulation and growth response ofAllocasuarina andCasuarina species to phosphorus fertilization

To examine how soil phosphorus status affects nitrogen fixation by the Casuarinaceae —Frankia symbiosis,Casuarina equisetifolia and two species ofAllocasuarina (A. torulosa andA. littoralis) inoculated or fertilized with KNO₃ were grown in pots in an acid soil at 4 soil phosphate levels. InoculatedC. equisetifolia nodulated well by 12 weeks after planting and the numbers and weight of nodules increased markedly with phosphorus addition. Growth ofC. equisetifolia dependent on symbiotically fixed nitrogen was more sensitive to low levels of phosphorus (30 mg kg^–1 soil) than was growth of seedings supplied with combined nitrogen; at higher levels of phosphorus, the growth response curves were similar for both nitrogen fertilized and inoculated plants. The interaction between phosphorus and nitrogen treatments (inoculated and nitrogen fertilized) demonstrated that there was a greater requirement of phosphorus for symbiotic nitrogen fixation than for plant growth when soil phosphorus was low.WithAllocasuarina species, large plant to plant variation in nodulation occurred both within pots and between replicates. This result suggests genetic variation in nodulation withinAllocasuarina species. Nodulation ofAllocasuarina species did not start until 16 weeks after planting and no growth response due toFrankia inoculation was obtained at the time of harvest. Addition of nitrogen starter is suggested to boost plant growth before the establishment of the symbiosis. Growth ofAllocasuarina species fertilized with nitrogen responded to increasing levels of phosphorus up to 90 mg P/kg soil after which it declined by 69% forA. littoralis. The decrease in shoot weight ofA. littoralis, A. torulosa, C. equisetifolia andC. cunninghamiana at high phosphorus was confirmed in a sand culture experiment, and may be atributable to phosphorus toxicity.     

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.     

Inoculation and production of container-grown red alder seedlings

Summary The inoculation ofAlnus rubra (red alder) withFrankia sp. can lead to a highly efficient symbiosis. Several factors contribute to the successful establishment of nitrogenfixing nodules: (1) quantity and quality ofFrankia 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.     

Control of dinitrogen fixation in ammonium-assimilating cultures of Azotobacter vinelandii

Azotobacter vinelandii was grown at constant growth rate in a chemostat with different molar ratios of sucrose to ammonium (C/N) in the influent media. Both compounds were consumed at essentially the same ratios as were present in the influent media. At low (C/N)-ratios, the cultures were ammonium-limited. At increased (C/N)-ratio ammonium-assimilating cultures additionally began to fix dinitrogen. The (C/N)-ratio at which nitrogenase activity became measurable, increased when the ambient oxygen concentration was increased. Immunoblotting revealed the appearance of nitrogenase proteins when the activity became detectable. Nitrogenase activity as determined either by acetylene reduction or by total nitrogen fixation gave constant relative activities of 1:3.8 (mol of N₂ fixed per mol of acetylene reduced) under all sets of conditions used in this investigation. In spite of the oxygen dependent variation of the (C/N)-ratio, nitrogenase became active when the ammonium supply was less than about 14 nmol of ammonium per g of protein. This suggests that oxygen was not directly involved in the onset of dinitrogen fixation.     

Creation of novel nitrogen-fixing actinomycetes by protoplast fusion of Frankia with streptomyces

Protoplast fusion was used for the creation of a novel actinomycete capable of fixing atmospheric nitrogen. Protoplasts of Streptomyces griseofuscus, a fast-growing actinomycete, and Frankia, a slow-growing actinomycete which fixes atmospheric nitrogen in culture and in symbiotic association with alders, were allowed to fuse and regenerate on media without supplied nitrogen. Colonies which regenerated acquired the fast-growing characteristic of Streptomyces and the ability to grow on nitrogen-deficient media from Frankia. These colonies resembled Streptomyces in their morphology and fixed atmospheric nitrogen in culture. They contained both the parent Streptomyces DNA sequences and the Frankia DNA sequences homologous to nif structural genes HDK of K. pneumoniae. In addition to in vitro nitrogen-fixing capacity, one out of 20 colonies also formed nitrogen-fixing root nodules on Alnus rubra, the host plant for the Frankia strain. Examination of the root nodules induced by the hybrids showed only the presence of hyphae-like structures. The typical vesicle-like structures present in Frankia were absent.     

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.     

Studies of an effective strain ofFrankia fromAllocasuarina lehmanniana of the Casuarinaceae

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

Studies in tetrapartite symbioses

Alnus incana seedlings were successfully inoculated with an endomycorrhizal fungus (Glomus fasciculatus), an ectomycorrhizal fungus (Paxillus involutus) and an isolate ofFrankia (ACN1) simultaneously. The effects of the inoculation treatments on the growth performance of the seedlings were evaluated under controlled conditions.The overall growth performance of the seedlings inoculated with the three organisms was better than those inoculated withFrankia, G. fasciculatus andP. involutus individually or withFrankia+G. fasciculatus andFrankia+P. involutus combinations. The highest growth performance and mycorrhizal infection occurred when the seedlings were inoculated simultaneously withFrankia+G. fasciculatus+P. involutus.     

Nitrogenase activity in root nodule homogenates ofAlnus incana

Summary Root nodule homogenates of actinorhizal plants may representFrankia in a symbiotic stage but released from environmental influence of the host plant. Anaerobic homogenization with a blender in buffer supplied with sucrose, polyvinylpyrrolidone and reducing substances gave three times higher yields of nitrogenase activity (C₂H₂-reduction) than crushing the nodules in liquid nitrogen. The activity in the homogenates was very reproducible and was, on average, nearly twice as high as the activity in excised nodules and c. 10% of the activity in intact plants. The difference in activity between excised nodules and intact plants was, roughly by halves, due to removal of the root system from the pot and to excision of the nodules. The nitrogenase activity in the homogenates was slightly higher when nodule excision was done in Ar or under water as well as after treatment of the homogenate with toluene or Triton X-100 or osmotic shock. These gains in activity were considered too small to outweigh the increased complications of preparing homogenates for routine use. Due to the reproducible recovery of nitrogenase in the homogenates the technique seems useful for physiological studies on nitrogen fixation inAlnus incana.     

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.