A comparison of symbiotic nitrogen fixation in Scotland inAlnus glutinosa andAlnus rubra

Summary Alnus glutinosa andAlnus rubra growing in the field in Scotland show specific nitrogenase activities of the same order of magnitude. The period of maximum potential nitrogenase activity coincides with that of maximum growth in late Spring and Summer. It is suggested that the retention of nitrogenase activity into the Autumn when growth has virtually ceased may be important as a contribution to the nitrogenous reserves of the tree.Bioassay of different Scottish soils, all collected from the locality of natural stands ofAlnus glutinosa, showed wide variation in the nodulation of seedlings, although generally a soil poor for nodulation ofAlnus glutinosa generally gave poor nodulation ofAlnus rubra. Soils of pH 4.5 to 6.5, best suited for growth and nitrogen fixation of the two species, often gave nodules showing highest specific nitrogen fixing activity. Young (2 to 3 year old) plants in glasshouse or controlled environment cabinet, inoculated withAlnus glutinosa endophyte, differed from mature field grown plants, however, sinceAlnus rubra required a much larger (up to 2.5 times) mass of root nodules to fix a unit quantity of N. Microscopic comparison of the nodules of glasshouse plants showed that the proportion of cells containing the vesicular (nitrogen fixing) form of the endophyte was only slightly lower inAlnus rubra than inAlnus glutinosa and it is suggested that the differences in specific nitrogen fixing activity between the two species may reflect some incompatibility of function of theAlnus glutinosa endophyte when in symbiosis withAlnus rubra.     

Distribution ofFrankia in soils from forest and afforestation sites in northern Sweden

Summary The presence in soil ofFrankia, capable of forming nitrogen-fixing root nodules onAlnus incana (L.) Moench, was investigated. Intact soil cores from forested as well as disturbed sites were sampled and both alder-rich and alder-free sites were included in the study. Surface-sterilized alder seeds were sown in the soil cores which were kept in sterile culture tubes in a growth chamber. Root nodules with nitrogenase activity developed in soil cores from all sites studied. Thus, infective and effectiveFrankia was present in all of the soils sampled, even from sites free from actinorhizal plants and irrespective of pH and nitrogen content of the soils.     

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.     

Photosynthetic responses of birch and alder saplings grown in a free air CO2 enrichment system in northern Japan

Though birch and alder are the common pioneer tree species which dominate in northeast Asia, little is known about the effects of the predicted increase in atmospheric CO₂ concentrations ([CO₂]) upon their photosynthesis in field conditions. To investigate this, we grew 2-year-old saplings of three Betulaceae species (Betula platyphylla var. japonica Hara, Betula maximowicziana Regel, and Alnus hirsuta Turcz) for 2 years in a free air CO₂ enrichment system in northern Japan. Since the effect of high [CO₂] is known to depend on soil conditions, we evaluated the responses in two soils which are widely distributed in northern Japan: infertile and immature volcanic ash (VA) soil, and fertile brown forest (BF) soil. For B. platyphylla, photosynthetic down-regulation occurred in both soils, but for B. maximowicziana, down-regulation occurred only in VA soil. The explanation is reduced nitrogen and Rubisco content in the leaf. For A. hirsuta, down-regulation occurred only in BF soil because of the accumulation of starch in foliage, which restricts CO₂ diffusion inside the chloroplast. The higher photosynthetic rate of A. hirsuta in infertile VA soil could be due to the sink for photosynthates in the N₂-fixing symbiont. These three species are all able to down-regulate at high [CO₂]. However, it is possible that A. hirsuta would dominate in VA soil and B. maximowicziana in BF soil in the early stages of forest succession in a CO₂-enhanced world.     

The Effect of Pressurized Gas Transport on Nutrient Uptake During Hypoxia of Alder Roots*

The uptake of nitrate, sulphate, phosphate, and potassium from a nutrient solution by young alder trees (Alnus glutinosa) was reduced during hypoxia. Such a decrease of ion uptake was not observed when the internal oxygen supply of the tree roots was improved by pressurized gas transport. These results demonstrate the beneficial effect of this gas transport phenomenon on the nutrition of trees growing on wet sites characterized by anaerobic soil.     

Alnus rubra nodulation capacity of soil under five species from harvested forest sites in coastal British Columbia

Nodulation of Alnus rubra seedlings after inoculation with soil from under A. rubra, Betula papyrifera. Rubus lacianutus, R. spectabilis, and R.ursinus on 2 recently harvested sites was compared. Nodulation capacity was low compared to other published reports, ranging from 0 to 18.9 infective units cm^-3 of soil and was significantly affected by the site and plant species. Nodulation capacity of soil under alder was significantly higher than under all other species except R. spectabilis, regardless of site. The lowest nodulation capacity was found in soil under B. papyrifera.Joint appointment with Dept. of Soil Science, Faculty of Agricultural Sciences     

Effects of elevated CO<Subscript>2</Subscript> and nitrogen on wood structure related to water transport in seedlings of two deciduous broad-leaved tree species

Wood structure might be altered through the physiological responses to atmospheric carbon dioxide concentration ([CO₂]) and nitrogen (N) deposition. We investigated growth, water relations and wood structure of 1-year-old seedlings of two deciduous broad-leaved tree species, Quercus mongolica (oak, a ring-porous species) and Alnus hirsuta (alder, a diffuse-porous species and N₂–fixer), grown under a factorial combination of two levels of [CO₂] (36 and 72 Pa) and nitrogen supply (N; low and high) for 141 days in phytotron chambers. In oak, there was no significant effect of [CO₂] on wood structure, although elevated [CO₂] tended to decrease stomatal conductance (g _s) and increased water use efficiency regardless of the N treatment. However, high N supply increased root biomass and induced wider earlywood and larger vessels in the secondary xylem in stems, leading to increased hydraulic conductance. In alder, there was significant interactive effect of [CO₂] and N on vessel density, and high N supply increased the mean vessel area. Our results suggest that wood structures related to water transport were not markedly altered, although elevated [CO₂] induced changes in physiological parameters such as g _s and biomass allocation, and that N fertilization had more pronounced effects on non-N₂-fixing oak than on N₂-fixing alder.     

Survival ofFrankia strains introduced into soil

Factors affecting the establishment of Alnus/Frankia symbioses were studied partly by following the survival ofFrankia strains exposed to different soil conditions, and partly by investigating the effect of pH on nodulation. TwoFrankia strains were used, both of the Sp⁻ type (sporangia not formed in nodules). One of the strains sporulated heavily, while the other formed mainly hyphae. The strains originated fromAlnus incana root nodules growing in soils of pH 3.5 and 5.0. The optimum pH for their growth in pure culture was found to be 6.7 and 6.2, respectively. The strains were introduced into twoFrankia-free soils, peat and fine sand. Their survival, measured as the persistance of nodulation capacity using the plant infection technique, was followed for 14 months. The survival curves of the strains were similar despite the morphological differences between the strains in pure culture. The nodulation capacities declined over time both at 14 and 22°C. Survival was better in soils limed to a pH above 6 than in soils at their original pH (peat 2.9, fine sand 4.2). The effect of pH on nodule formation in Alnus seedlings by theFrankia strains was studied in liquid culture. The number of nodules increased linearly within the pH range studied (3.5–5.8). No nodules were formed at pH 3.5.     

Seed germination,seedling inoculation and establishment ofAlnus spp. in containers in greenhouse trials

Methods for production of containerized seedlings ofAlnus species were developed which permit nitrogen-fixing nodules to form on the root systems prior to outplanting, in order to provide an early nitrogen input during seedling establishment. The methods are based on procedures for inoculating root systems with suspensions ofFrankia (Actinomycetales), applied either directly in the container cell as a soil drench at the time of seeding, or as a root dip for seedlings transplanted into the containers. Germination of dried, stored seed was enhanced by light and by presoaking for 16 h in water. Pretreatments to overcome seed dormancy or to eliminate fungal pathogens did not further enhance germination. Some loss of seedlings was recorded in the early stages of growth shortly after germination, which is a factor in calculating projected seedling yield. Nodulation and seedling growth were evaluated in terms of growth media characteristics. Seedlings performed well in peat-vermiculite, at soil pH between 5.5 and 8.0.     

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.     

Characterization of a thermo-osmotic gas transport mechanism in Alnus glutinosa (L.) Gaertn.

Summary A gas transport system based upon the physico-chemical effect of thermo-osmosis of gases in described for the black alder, Alnus glutinosa (L.) Gaertn. Air is transported through the alder's stem to the roots, thus improving O₂ supply to respiring tissues of the root system. The gas transport system is investigated by means of a tracer gas technique (11% ethane in air, v/v). Gas transport depends on any source of radiant heat generating a temperature difference between the tree's stems and the atmosphere. The amount of gas transported in leafless trees is four times higher than the amount of gas reaching the roots by gas diffusion. Two-thirds of the gas is transported in the wood, only one-third in the bark. Intercellular spaces inside the porous lenticels of the bark are responsible for this kind of gas transport. Their diameters are estimated by the effusion rates of different tracer gases to be in the range of 1 m.     

Methods of isolation and cultivation of Frankia species from actinorhizas

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

Distribution of spore-positive and spore-negative nodules ofAlnus incana ssp.rugosa in Maine,USA

The distribution of spore-positive (sp+) and spore-negative (sp−) root nodules ofAlnus incana ssp.rugosa (DuRoi) Clausen (speckled alder) was examined at 29 sites with a wide range of environmental conditions in Maine, USA. These included: pH 3.4 to 7.0, soil texture ranging from coarse gravel to clay to organic soils, elevation from 3 to 591 m and latitude 43 to 47°N. Habitat types included disturbed areas, streamsides, swamps and old fields. Sp (−) nodules were substantially more common, making up 76% of all nodules, whereas only 24% were sp (+). Sp (−) nodules often occurred in pure stands and predominated at disturbed sites with mineral soils at the surface and in old fields and swamps with pH>4.0 Sp (+) nodules were nearly always found in mixture with sp (−) nodules. They occurred primarily at streamside and lakeshore sites where they made up 40% of the nodules and at sites with pH<4.0 regardless of habitat type. It is suggested that sp (−) strains ofFrankia may be maintained at a site by saprophytic growth in soil and thus nodulate newly established hosts, whereas sp (+) strains may be maintained primarily by spore production within nodules and thus depend on extended presence of the host.     

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.     

Intraspecific variability of isozymes in Penicillium nodositatum Valla,a fungus inducing ‘myconodules’ on the root-system of Alnus sp.

A comparison of mycelial extracts of 17 isolates of Penicillium nodositatum collected from the root system of grey alder (Alnus incana (L.) Moench.) and Italian alder (A. cordata Desf.) grown at different sites was made using isoenzyme analysis of esterases, acid phosphatase, malate dehydrogenase and phosphoglucose isomerase. Jaccard similarity coefficient analysis demonstrated that interspecific variability existed both among and within sites. In most cases, the enzyme patterns of isolates were distinct. Comparison of these isolates and three other Penicillium species closely related to P. nodositatum showed no similarity for the enzymes tested.     

Large scale inoculation of actinorhizal plants withFrankia

Summary From 1979 to 1984 more than seven million seedlings of actinorhizal plants were successfully inoculated on an industrial scale withFrankia inoculants. Nodulated seedlings were produced in greenhouses to be used for land reclamation in northern Québec by the Societe d'Energie de la Baie James (SEBJ) and also by the City of Montréal for a revegetation program. Crushed-nodule homogenates andFrankia 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 theFrankia pure culture inocula were compared: soil injection and spraying with greenhouse watering devices. Both methods resulted in efficient nodulation ofAlnus crispa, A. glutinosa, A. rugosa, Elaeagnus angustifolia, E. commutata, Hippophaë rhamnoides, Myrica gale andShepherdia argentea.     

Glasshouse evaluation of the growth ofAlnus rubra andAlnus glutinosa on peat and acid brown earth soils when inoculated with four sources ofFrankia

The effects of soil type (an acid peat and 2 acid brown earths) andFrankia source (3 spore-positive crushed nodule inocula and spore-negative crushed nodules containing the singleFrankia ArI5) on nodulation, N content and growth ofAlnus glutinosa andA. rubra were determined in a glasshouse pot experiment of two years duration. Plants on all soils required additional P for growth. Growth of both species was very poor on peat withA. glutinosa superior toA. rubra. The former species was also superior toA. rubra on an acid brown earth with low pH and low P content. Some plant-inoculum combinations were of notable effectivity on particular soils but soil type was the major source of variation in plant weight. Inoculation with crushed nodules containingFrankia ArI5 only gave poor infection of the host plant, suggesting that inoculation with locally-collected crushed nodules can be a preferred alternative to inoculation withFrankia isolates of untested effectivity. Evidence of adaptation ofFrankia to particular soils was obtained. Thus, while the growth of all strains was stimulated by mineral soil extracts, inhibitory effects of peat extracts were more apparent with isolates from nodules from mineral soils than from peat, suggesting that survival ofFrankia on peat may be improved by strain selection.     

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.     

Functional Diversity of Culturable Bacterial Communities in the Rhizosphere in Relation to Fine-root and Soil Parameters in Alder Stands on Forest, Abandoned Agricultural, and Oil-shale Mining Areas

Grey alder (Alnus incana) and black alder (Alnus glutinosa) stands on forest land, abandoned agricultural, and reclaimed oil-shale mining areas were investigated with the aim of analysing the functional diversity and activity of microbial communities in the soil–root interface and in the bulk soil in relation to fine-root parameters, alder species, and soil type. Biolog Ecoplates were used to determine community-level physiological profiles (CLPP) of culturable bacteria in soil–root interface and bulk soil samples. CLPP were summarized as AWCD (average well color development, OD 48 h⁻¹) and by Shannon diversity index, which varied between 4.3 and 4.6 for soil–root interface. The soil–root interface/bulk soil ratio of AWCD was estimated. Substrate-induced respiration (SIR) and basal respiration (BAS) of bulk soil samples were measured and metabolic quotient (Q = BAS/SIR) was calculated. SIR and Q varied from 0.24 to 2.89 mg C g⁻¹ and from 0.12 to 0.51, respectively. Short-root morphological studies were carried out by WinRHIZO^TM Pro 2003b; mean specific root area (SRA) varied for grey alder and black alder from 69 to 103 and from 54 to 155 m² kg⁻¹, respectively. The greatest differences between AWCD values of culturable bacterial communities in soil–root interface and bulk soil were found for the young alder stands on oil-shale mining spoil and on abandoned agricultural land. Soil–root interface/bulk soil AWCD ratio, ratio for Shannon diversity indices, and SRA were positively correlated. Foliar assimilation efficiency (FOE) was negatively correlated with soil–root interface/bulk soil AWCD ratio. The impact of soil and alder species on short-root morphology was significant; short-root tip volume and mass were greater for black alder than grey alder. For the investigated microbiological characteristics, no alder-species-related differences were revealed.     

Host range ofFrankia endophytes

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