Nodulation potential of soils from red alder stands covering a wide age range

Red alder (Alnus rubra Bong.) stands in the Pacific Northwest are the common first stage in succession following disturbance. These stands are highly productive and contribute a large amount of N to the soils as a result of their N₂-fixing symbiosis with Frankia. As these alder stands age, the soils not only increase in total N, but concentrations of NO^– ₃ increase and pH decreases as a result of nitrification. The objective of this study was to determine how the nodulation capacity of Frankia varies as red alder stands age and if differences in nodulation capacity are related to changes in soil properties. Nodulation capacity was determined by a red alder seedling bioassay for soils from red alder stands in the Oregon coast range covering a wide range of ages. Six chronosequences were sampled, each containing a young, an intermediate, and an older alder stand. Soil total N, total C, NO^– ₃, NH⁺ ₄, and pH were measured on the same soil samples. These factors as well as alder stand characteristics were compared with nodulation capacity in an attempt to identify soil characteristics typical in developing alder stands that most strongly affect nodulation capacity. Soil pH and NO^– ₃ concentration were highly correlated with nodulation capacity and with each other. Cluster analysis of the sites using these two variables identified two groups with distinctly different nodulation capacities. The cluster with the higher nodulation capacity was lower in NO^– ₃ and higher in pH than the other cluster, which included the majority of sites. There was substantial overlap in the age ranges for the two clusters and there was no significant correlation between age and nodulation capacity. Thus nodulation capacity appears to be most closely related to soil properties than to stand age.     

Nutrient relations in coppiced black cottonwood and red alder

Two-year-old coppice of black cottonwood and red alder, grown in pure culture and in mixture, were compared using terminal twigs and leafless shoots harvested in the winter. Terminal twigs were taken with buds intact; they were about 15 cm long. Leafless shoot samples included all above-ground components. In pure culture, dry weights of the leafless shoots per plant were similar for the two species. In mixture with alder, however, weight of the cottonwood plants was enhanced and that of alder was reduced, but neither response was statistically significant. Nutrient concentration, content per plant, and utilization varied by the plant tissues analyzed, cultural treatment (purevs. mixed), and species. In general, nutrient concentrations were higher in the terminal twigs than in the leafless shoots of both species. Cultural treatment did not significantly affect nutrient concentration in cottonwood twigs or in the leafless shoots of either species. Concentrations of N and Fe were significantly higher and those of Mn were lower in twigs of mixed alder than in twigs of pure alder. Twigs of cottonwood were significantly higher than those of alder in concentration of P and Zn, and lower in N, Mn, and Cu. Compared with alder, cottonwood leafless shoots were significantly higher in concentration of Ca, but lower in N, S, Cu, and Mn. With few exceptions, nutrient content was highest in the shoots of the large plants of mixed cottonwood, intermediate in medium-sized pure cottonwood and pure alder, and lowest in the small mixed alder. Cottonwood was significantly more efficient than alder in use of N, S, and Cu, and less efficient in use of Ca. Some of the differences between cultural treatments and species may be associated directly or indirectly with the N₂-fixing ability of red alder. Mixed culture of the two species appears promising because of the increased growth of cottonwood. Planted separately in pure culture, the choice between cottonwood and alder may be determined, in part, by the nutritional status of the soil where plantations are established.     

Preinfection cell wall formation in roots and developing nodules ofAlnus rubra Bong

Summary The establishment of actinorhizal root nodules involves penetration of host cell walls and intracellular colonization by the nitrogen-fixing endosymbiont,Frankia (Actinomycetales). In the early stages of the infection process inAlnus, unusual cell walls with undulate profiles were observed in root tip meristematic derivatives, and in early (preinfection) derivatives of the nodule lobe meristem, inFrankia-inoculated plants. The irregular cell walls attached obliquely to preexisting walls, but were not discontinuous. Serial sections revealed that the unusual walls divided two daughter cells. Microtubules in bundled arrays were abundant near the undulate walls, and radiated in several planes. In the root tips, the anomalous cell walls were observed within one day of inoculation withFrankia.     

A comparison of cultural characteristics and infectivity ofFrankia isolates from root nodules ofCasuarina species

Summary The isolations of three new strains ofFrankia were made from root nodules ofCasuarina cunninghamiana growing aeroponically. Two strains, HFPCCI1 and HFPCcI2 isolated by Lopez are typicalFrankia strains, producing sporangia among filamentous mats in culture and, in the absence of combined nitrogen, forming vesicles and showing acetylene reduction. They are red-pigmented and, although failing to nodulateCasuarina hosts, effectively nodulatedElaeagnus andHippophae. A third strain HFPCcI3 isolated by Zhang from the same source, also a typicalFrankia, can form sporangia and vesicles in culture and reduce acetylene, is unpigmented, fails to nodulateElaeagnus but effectively nodulatesC. cunninghamiana andC. equisetifolia. Comparisons are made among all of theCasuarina isolates in our collection from around the world (twelve in all) with regard to their cultural characteristics and capacity to infect host plant species. Questions are raised about the specificity of the various isolates and their possible affinities. Opportunities are suggested for inoculation of seedlings for forestry and field application using the infective, effective strains now available.     

Foliar chemical concentrations in red alder stands of various ages

Summary Six stands of red alder (Alnus rubra Bong.) were sampled to assess the relationship between stand age and foliar chemical concentrations. The stands ranged in age from 9 to 45 years, and were located near Olympia, Washington, on sites of similar soil, aspect, and slope. Foliage was collected in July, and concentrations of most essential nutrients and aluminum were determined. Foliar concentrations of these elements varied among the different stands. Highest concentrations of N, P, Ca, S, Fe, and Al were found in trees of the youngest stand. Concentrations of N, P, S, and Fe were negatively correlated with stand age. The strongest correlation was obtained with P (r=–0.96,P<0.01), followed by N (r=–0.85,P=0.03). Results suggest that declining growth and deterioration of red alder stands with age may be related, at least in part, to limiting supplies of essential nutrients, especially P, N, and S.     

Flavonoid-like compounds from seeds of red alder (Alnus rubra) influence host nodulation by Frankia (Actinomycetales)

Nitrogen-fixing root nodules are formed by Frankia spp. (Actinomycetales) on dicotyledonous hosts such as alders ( Alnus spp.). Flavonoid-containing preparations from seed washes of red alder ( Alnus rubra Bong.), and individual compounds isolated from such preparations, influenced nodulation of A. rubra by Frankia. Nodulation was enhanced by one flavonoid-like compound, and apparently inhibited by two other such compounds. Four flavonoid-like compounds had no significant effect on nodulation. The seven individual compounds purified from the seed washes were characterized spectrally as possible flavanones and isoflavones. Both the enhancer and the inhibitors appeared to be possible flavanones.     

Litterfall and nutrient returns in red alder stands in western Washington

Summary Litterfall was collected over 1 year from eight natural stands of red alder growing on different sites in western Washington. The stands occurred at various elevations and on different soils, and differed in age, basal area, and site index. Most litterfall was leaf litter (average 86 percent). Amounts of litterfall and leaf litter varied significantly (P<0.05) among the sites. Average weights of litterfall and leaf litter in kg ha^–1 yr^–1, were 5150 and 4440, respectively. Weight of leaf litter was not significantly (P<0.05) related to site index, stand age, or basal area. The sites varied significantly (P<0.05) in concentrations of all elements determined in the leaf litter, except Zn. Average chemical concentrations were: N, 1.98 percent; P, 0.09 percent; K, 0.44 percent; Ca, 1.01 percent; Mg, 0.21 percent; S, 0.17 percent; SO₄–S, nil; Fe, 324 ppm; Mn, 311 ppm; Zn, 53 ppm; Cu, 13 ppm; and Al, 281 ppm. There were significant correlations between some stand characteristics and concentrations of some elements, and among the different chemical components of the leaf litter. Important correlations were found between stand age and P concentration (r=–0.84,P<0.01); weight of leaf litter and P concentration (r=0.74,P<0.05); weight of leaf litter and K concentration (r=0.71,P<0.05); concentrations of N and S (r=0.81,P<0.05); and concentrations of Fe and Al (r=0.98,P<0.01). Returns of the different elements to the soil by leaf litter varied among the different sites. Average nutrient and Al returns, in kg ha^–1 yr^–1, were: N, 82; Ca, 41; K, 19; Mg, 8; S, 7; P, 4; Fe, 1; Mn, 1; Al, 1; Zn, 0.2, and Cu, <0.1.     

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.     

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.     

Distribution of spore-positive and spore-negative nodules in stands ofAlnus glutinosa andAlnus incana in Finland

Summary The distribution of spore positive (Sp⁺) and spore negative (Sp⁻) nodules on the two native alder species (A. incana andA. glutinosa) in Finland was investigated. Nodules were collected throughout the country from different ecosystems (forests, swamps, lake- sea- and riversides, old pastures and fields as well as from alder plantations). OnA. incana Sp⁺ nodules predominated, whereas onA. glutinosa the vast majority of the nodules were of the Sp⁻ type. Sp⁺ nodules onA. glutinosa were found only at sites where the two alder species grew close together. This distribution pattern indicates an association of nodule type with alder species, the reasons for which are discussed. Indications of saprophytic growth in the Sp⁻ strain were also found.     

Oxygen protection of nitrogenase in Frankia sp. HFPArI3

O₂ protection of nitrogenase in a cultured Frankia isolate from Alnus rubra (HFPArI3) was studied in vivo. Evidence for a passive gas diffusion barrier in the vesicles was obtained by kinetic analysis of in vivo O₂ uptake and acetylene reduction rates in response to substrate concentration. O₂ of NH ₄ ⁺ -grown cells showed an apparent K _m O₂ of approximately 1M O₂. In N₂-fixing cultures a second K _m O₂ of about 215 M O₂ was observed. Thus, respiration remained unsaturated by O₂ at air-saturation levels. In vivo, the apparent K _m for acetylene was more than 10-fold greater than reported in vitro values. These data were inter oreted as evidence for a gas diffusion barrier in the vesicles but not vegetative filaments of Frankia sp. HFPArI3.     

Production of growth hormones inCasuarina cunninghamiana root nodules induced byFrankia strain HFPCgI4

Summary Measurements of auxin and cytokinin activities in extracts ofCasuarina root nodules were made. The nodules were induced either by pure culture ofFrankia strain CgI4 or by crushed nodule inoculum. Levels of cytokinin activity were significantly higher in root nodules induced by pureFrankia culture than in those induced by crushed nodule inoculum. However, the reasons for this are unknown. Seasonal variation in levels of cytokinin activity inCasuarina nodules has also been detected.Dedicated to the memory of Professor John G. Torrey     

N2 fixation by red alder (Alnus rubra) and scotch broom (Cytisus scoparius) planted under precommerically thinned Douglas-fir (Pseudotsuga menziesii)

Summary From acetylene reduction assays over a 10-month period starting in April 1979, nodule activities averaged 18.78 (se 4.67) moles C₂H₄ g nodule dw^–1 h^–1 forAlnus rubra and 59.95 (se 12.14) moles C₂H₄ g nodule dw^–1 h^–1 forCytisus scorparius. Plant rates were 1.91 (se. 47) moles C₂H₄ plant^–1 h^–1 forA. rubra and 0.55 (se. 17) moles C₂H₄ plant^–1 h^–1 forC. Scoparius. Plant activity and total leaf N were strongly correlated with the dw of other plant parts, but nodule activity and percent leaf N were not. Plant and nodule activities were not associated with temperature, moisture stress, precipitation events or percent light for either species over the growing season nor for 54A. rubra sampled in mid-season 1979 on one replication. After 5 to 6 growing seasons, 14A. rubra on the same site ranged from 30 to 332 cm in height and showed strong correlation between nodule dw, leaf dw, plant size and total leaf N. Results from this study and others indicate logistic equations may be modified to predict the effect of adding a N₂ fixing plant to a population of non N₂ fixing trees.     

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.     

Seasonal fluctuations of the mineral concentration of alder (Alnus glutinosa (L.) Gaertn.) from the field

Summary The seasonal fluctuation of N, P, K, Ca, Mg, Fe, Mn, Mo, and Co, in leaves, roots and nodules of 40–50 year oldAlnus glutinosa trees growing at four different locations along the banks of the Tormes river, in the province of Salamanca, was studied. Also, the evolution of the soil organic matter under the trees sampled was evaluated. The data obtained for the various nutrient elements in the three plant parts are statistically treated at the significance levels of 99–95 per cent, and some remarks as to the nutritional status of the European alder in respect to the nutrients and its contribution to soil nutrient-cycling are provided. A positive correlation was found between N–P, N–K, N–Mg, and N–Mo, in leaves, and between N–P, N–K, N–Fe, N–Mn, and N–Mo in root nodules. In roots only, no significance at any level was obtained between N and any of the elements analyzed.     

Early colonization of red alder and Douglas fir by ectomycorrhizal fungi and Frankia in soils from the Oregon coast range

The potential for mycorrhizal formation and Frankia nodulation were studied in soils from six sites in the Pacific Northwest. The sites included young and old alder stands, a 1-year-old conifer clear-cut, a young conifer plantation, and rotation-aged and old-growth conifer stands. A bioassay procedure was used with both red alder and Douglas fir seedlings as hosts. After 6 weeks growth, seedlings of both hosts were harvested every 3 weeks for 21 weeks and numbers of nodules and ectomycorrhizal types estimated. Nodules formed on red alder and ectomycorrhizae formed on both alder and Douglas fir in soil from all sites. Nodulation potential was highest in soil from the alder stands and the conifer plantation. Seven morphologically distinct ectomycorrhizal types were recovered on Douglas fir and five on alder. Only Thelephora terrestris, a broad-host-range mycobiont, formed mycorrhizae on both hosts. New ectomycorrhizal types formed on both hosts throughout the bioassay. Ectomycorrhizal colonization of alder was greatest in the alder and clear-cut soils. Low ectomycorrhizal colonization on alder was found in soils from sites where conifers were actively growing. Ectomycorrhizal colonization of Douglas fir was highest in the young alder and conifer plantation soils and was low in the rotation-aged conifer soil. The highest diversity of ectomycorrhizal types was found on alder in the conifer clear-cut soil and on Douglas fir in the rotation-aged conifer soil. Effects of host specificity, nodulation and mycorrhiza-forming potential and nodule-mycorrhiza interactions on seedling establishment are discussed in relation to seral stage dynamics and attributes of pioneer ectomycorrhizal fungal species.     

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

Summary A spontaneous variant, obtained from aFrankia isolate fromAlnus rubra nodules, was compared with the parent strain with regard to infectivity, nitrogenase activity, and electrophoretic and immunological profiles. Both the parent and the variant strain were equally effective in inducing nodulation in seedlings ofA. 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 originalFrankia 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.     

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.     

Responses of red alder and black cottonwood seedlings to flooding

Red alder ( Alnus rubra Bong.) and black cottonwood ( Populus trichocarpa Torr. & Gray) seedlings were monitored to evaluate response during a 20-day period of artificial flooding and a 20-day recovery period following flooding. During the flooding period, both species showed changes in nutrient uptake and transport, initiated stemderived adventitious roots that became aerenchymatous, and exhibited hypertrophied lenticels. Flooded red alder seedlings also showed reduced height and leaf area growth and developed lower-stem hypertrophy. Flooded black cottonwood seedlings exhibited root dieback, aerenchyma in below ground root tips, and changes in root hydraulic conductance and xylem pressure potential. Contrary to expectations, however, stomatal closure following flooding was not observed in either species. Flooded red alder seedlings increased growth rapidly when drained, and by the end of the recovery period, formerly flooded and non-flooded red alder seedlings differed only minimally in this respect. In contrast, several characteristics of black cottonwood – including growth rate and nutrient content – still differed between formerly flooded and non-flooded seedlings at the end of the recovery period. Based on observed treatment differences at the end of the experiment, red alder seedlings were judged to be more tolerant of flooding than black cottonwood.