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.     

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.     

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.     

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.     

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.     

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.     

Mutants of Rhizobium capable of fixing N2 in the free-living condition

Six mutant strains of Rhizobium were isolated after UV treatment which could exhibit nitrogenase activity in Burk's N-free medium without any supplement. The activity ranged between 99.5 and 113 nmol/mg cell dry weight and hour. Two of the parent strains belonged to soybean, and one each to mungbean and Sesbania sp. Both the parent and mutant strains exhibited nitrogenase activity in CS 7 medium. One of the mutants retained its capacity to produce nodules on soybean roots.List of Abbreviations C.D. Critical difference - EMS ethylmethane sulphonate - NTG N-methyl-N-nitro, N-nitrosoguanidine     

Foliar nutrient concentrations of small Chamaecyparis in Taiwan

Summary Seedlings ofChamaecyparis lawsoniana, C. formosensis andC. taiwanensis were grown in a forest nursery in Taiwan. Native saplings of the latter two species were sampled in a clearcut and in the forest understory. Foliar nutrient concentrations were mostly in the mid-range of values for other conifers. However, Ca (.59–1.5%) and Cu (7–20 ppm) were almost always high. Concentrations of N, P, and K in field saplings in Taiwan were low enough (.7–1.5%, .07–.15%, .47–.60%, respectively) to indicate that these elements are probably limiting growth.Among the three species,C. lawsoniana usually had the lowest foliar nutrient values. When a difference among the species from Taiwan occurred,C. taiwanensis usually was lower. Foliar nutrient concentration varied among provenances, especially withinC. formosensis. Seasonal variability did not occur consistently; nutrients which did change increased their concentrations from the dormant to the growing season, except for K which decreased.     

Diversity of nitrogen-fixing cyanobacteria under various ecosystems of Thailand: I. Morphology,physiology and genetic diversity

The diversity among 853 isolates of nitrogen-fixing cyanobacteria obtained from soil samples collected from different ecosystems including mountainous, forest and cultivated areas in the central, northern and northeastern regions of Thailand was examined. Most isolates showed slow growth rate and had filamentous, heterocystous cells. The percentage of heterocysts in the filaments of different isolates varied from 8.3 to 9.6. Only a few strains showed high nitrogen-fixing potential, while most of the strains exhibited low capacity for nitrogen fixation. Anabaena and Nostoc were the dominant genera among these isolates. One hundred and two isolates were randomly selected from this diverse collection to determine the extent of genetic diversity on the basis of DNA fingerprinting using the PCR method. Based on the PCR products obtained by using a combination of three primers, all strains could be distinguished from one another. When a subset of 45 isolates of Nostoc and a subset of 44 isolates of Anabaena were further analysed by PCR, a wide range of diversity was observed within each of these genera.     

Inoculation of sugar mill by-products compost with N2-fixing bacteria

Inoculation of sugar mill by-products compost with N₂-fixing bacteria may improve its quality by increasing total N and available P. Compost was inoculated with Azotobacter vinelandii(ATCC 478), Beijerinckia derxii (ATCC 49361), and Azospirillumsp. TS8, each alone and all three together. Numbers of all N₂-fixing bacteria in compost declined from an initial population of 5×10⁵cellsg^–1 during incubation. The population of Azotobacter declined to approximately 2×10²cellsg^–1 and the population of Beijerinckia and Azospirillum declined to approximately 9×10³ and 3.5×10⁴cellsg^–1 respectively, at day 50. Inoculation with N₂-fixing bacteria increased acetylene reduction, total N by 6–16 and available P by 25–30% in comparison to the uninoculated control. Increasing the N content and P availability of compost increases its value and there may be additional benefit from providing N₂ fixing bacteria.     

Autumnal changes in tissue nitrogen of autumn olive,black alder and eastern cottonwood

Two experiments were conducted to determine patterns of N change in tissues of autumn olive (Elaeagnus umbellata Thunb.) and black alder (Alnus glutinosa [L.] Gaertn.) during autumn in central Illinois, U.S.A. In the first study leaf nitrogen concentrations of autumn olive decreased 40% at an infertile minespoil site and 39% at a fertile prairie site throughout autumn whereas nitrogen concentrations in respective bark samples increased by 39% and 37%. Salt-extractable protein concentrations increased in bark and decreased in leaves over the sampling period. Free amino acid concentrations of autumn olive leaves decreased over the course of the experiment from peak concentrations in August. Asparagine, glutamic acid and proline were major constituents of the free amino acid pools in leaves. Total phosphorus concentrations of autumn olive leaves declined by 40–46% during autumn while bark concentrations of P did not significantly change.In the second experiment non-nodulated seedlings of alder receiving a low level of N-fertilization did not exhibit net resorption of leaf N during autumn whereas foliar N concentration of contrasting nonactinorhizal cottonwood plants (Populus deltoides Bartr. ex. Marsh) under the same fertilization regime decreased by 27% after the first frost. A gradual but significant decrease of 38% in foliar N concentration of nodulated alder seedlings grown under a low N-fertilization regime was associated with the cessation of nitrogenase activity during autumn in nodules. Compared with the low N fertilization regime, the higher level of N-fertilization resulted in smaller autumnal decreases of foliar N concentration in nodulated alder (17%) and in cottonwood (20%); but there was no decrease in foliar N concentration in non-nodulated alder. The higher level of N-fertilization promoted a greater accumulation of N in the roots than in the bark of both tree species after the first frost.Our results suggest that black alder lackingFrankia symbionts does not exhibit net leaf N resorption and that autumnal decreases in leaf N ofFrankia-nodulated black alder result primarily from declining foliar N import relative to export due to low temperature inhibition of N₂ fixation. In contrast, autumn olive exhibited greater and more precipitous autumnal declines in foliar N concentration than those of alder, and the pattern of N decline was unaffected by site fertility.     

Nodulation and growth ofPhaseolus vulgaris in solution culture

Summary Conditions and techniques for achieving good nodulation ofPhaseolus vulgaris L. in continuously aerated solution were developed from greenhouse experiments.If nodules had been established, their growth and activity and the growth of the plant were at least as good in solution culture as in gravel culture. Nodule formation was observed within 10 days of inoculation in small volumes of solution culture (1 liter). In large volumes (19 liters), similarly prompt nodulation occurred only if the plants were inoculated before or immediately after the seedlings were transferred to the solution from gravel or vermiculite; and the nodules were restricted to the roots that had been present at the time of transfer. Delayed inoculation, 2 days after transfer to large volume solutions, led to sparse nodulation observed only after 3 weeks. Delay or inhibition of nodulation in large volumes of solution could not be explained by failute of bacteria to colonize roots or by sparsity of root hairs.Nodule initiation in solution culture was severely inhibited at pH below 5.4. An additional problem in growing N₂-dependent bean in solution culture was the buildup of Cl^– to toxic levels in the plant in nitrate-free media, even at solution concentrations as low as 0.4 mM Cl^–. Daily addition of 0.5 to 1.0 mg N per plant delayed nodule growth and activity slightly, but increased plant growth and alleviated the severe N-deficiency that otherwise developed before the onset of N₂-fixation.     

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.     

Nitrogen fixation by non-legumes in tropical agriculture with special reference to wetland rice

Summary Of the 143 million hectares of cultivated rice land in the world, 75% are planted to wetland rice. Wet or flooded conditions favour biological nitrogen fixation by providing (1) photic-oxic floodwater and surface soil for phototrophic, free-living or symbiotic blue-green algae (BGA), and (2) aphotic-anoxic soil for anaerobic or microaerobic, heterotrophic bacteria. TheAzolla-Anabaena symbiosis can accumulate as much as 200 kg N ha^–1 in biomass. In tropical flooded fields, biomass production from a singleAzolla crop is about 15 t fresh weight ha^–1 or 35 kg N ha^–1. Low tolerance for high temperature, insect damage, phosphorus requirement, and maintenance of inoculum, limit application in the tropics. Basic work on taxonomy, sporulation, and breeding ofAzolla is needed. Although there are many reports of the positive effect of BGA inoculation on rice yield, the mechanisms of yield increase are not known. Efficient ways to increase N₂-fixation by field-grown BGA are not well exploited. Studies on the ecology of floodwater communities are needed to understand the principles of manipulating BGA. Bacteria associated with rice roots and the basal portion of the shoot also fix nitrogen. The system is known as a rhizocoenosis. N₂-fixation in rhizocoenosis in wetland rice is lower than that ofAzolla or BGA. Ways of manipulating this process are not known. Screening rice varieties that greatly stimulate N₂-fixation may be the most efficient way of manipulating the rhizocoenosis. Stimulation of N₂-fixation by bacterial inoculation needs to be quantified.     

Carbon cost of nitrogenase activity in Frankia–Alnus incana root nodules

The carbon cost of nitrogenase activity was investigated to determine symbiotic efficiency of the actinorhizal root nodule symbiosis between the woody perennial Alnus incana and the soil bacterium Frankia. Respiration (CO₂ production) and nitrogenase activity (H₂ production) by intact nodulated root systems were continuously recorded in short-term assays in an open-flow gas exchange system. The assays were conducted in N₂:O₂, thus under N₂-fixing conditions, in all experiments except for one. This avoided the declines in nitrogenase activity and respiration due to N₂ deprivation that occur in acetylene reduction assays and during extended Ar:O₂ exposures in H₂ assays. Two approaches were used: (i) direct estimation of root and nodule respiration by removing nodules, and (ii) decreasing the partial pressure of O₂ from 21 to 15% to use the strong relationship between respiration and nitrogenase activity to calculate CO₂/H₂. The electron allocation of nitrogenase was determined to be 0.6 and used to convert the results into moles of CO₂ produced per 2e^– transferred by nitrogenase to reduction of N₂. The results ranged from 2.6 to 3.4mol CO₂ produced per 2e^–. Carbon cost expressed as gC produced per gN reduced ranged from 4.5 to 5.8. The result for this actinorhizal tree symbiosis is in the low range of estimates for N₂-fixing actinorhizal symbioses and crop legumes. Methodology and comparisons of root nodule physiology among actinorhizal and legume plants are discussed.     

Identification of nitrogen-fixing enterobacteria from living Sassafras (Atherosperma moschatum Labill.) trees

Nitrogen-fixing enterobacteria (Enterobacter agglomerans and Citrobacter freundii) were commonly found associated with the microflora of stained Sassafras (Atherosperma moschatum Labill.) in forests of SE Tasmania. However, their populations never exceeded 3× 10⁴ cells g⁻¹ dry wood and comprised at most 3% of the total bacterial flora. Bacterial colonization of the wood appeared to coincide with that by non-hymenocetous fungi: bacteria were never isolated in significant numbers from wood not infected with fungi. The contribution of the N₂-fixing flora to the N economy of the habitat is considered to be negligible.     

Effect of salinity and inoculation on growth,nitrogen fixation and nutrient uptake ofVigna radiata (L.) Wilczek

This study reports the effect of salinity and inoculation on growth, ion uptake and nitrogen fixation byVigna radiata. A soil EC_e level of 7.5 dS m⁻¹ was quite detrimental causing about 60% decline in dry matter and grain yield of mungbean plants whereas a soil EC_e level of 10.0 dS m⁻¹ was almost toxic. In contrast most of the studied strains of Rhizobium were salt tolerant. Nevertheless, nodulation, nitrogen fixation and total nitrogen concentration in the plant was drastically affected at high salt concentration. A noticeable decline in acetylene reduction activity occurred when salinity level increased to 7.5 dS m⁻¹.     

Amount of nitrogen fixed by forage,pasture and grain legumes in Cyprus,estimated by the A-value and a modified difference method

The increasing need for protein at low cost has created a need to evaluate the biological nitrogen fixing potential of legumes in Cyprus. In field studies which were conducted over the growing years of 1982–3 and 1983–4, legumes which are traditionally grown in the country were evaluated for dry matter and nitrogen yield and biological nitrogen fixation (BNF). The legumes studied were medic (Medicago truncatula Gearth), ochrus vetch (Lathyrus ochrus L.), bitter vetch (Vicia ervilia L.) and faba bean (Vicia faba L. var major) in the first year and in addition chickpea (Cicer arietinum L.), woollypod vetch (Vicia dasycarpa Ten.) and tickbean (Vicia faba L. var minor) in the second year. Using the A-value method with barley and oats as reference crops, nitrogen (N) fixed by the various legumes in the first year was 30–50% and from 55–67% of total N yield for the two reference crops, respectively. In the second year the estimates of N fixed ranged from 70 to 80% with similar results obtained for the two reference crops barley and ryegrass. However, in the second year chickpea, which had limited nodulation, fixed only 40% of its N yield. Estimates of nitrogen from the atmosphere (Ndfa) obtained by the difference method (DM) were 10 to 14% lower than those from the A-value method. These results were obtained after correcting for the amount of N derived from the applied fertilizer. The two methods were highly correlated (r=0.98) for estimates of amount of BNF. The rates of N₂ fixation of uninoculated legumes which are nodulated by the indigenous populations of Rhizobium in Cyprus are comparable to those of legumes inoculated with selected strains of Rhizobium in other countries. An exception was the amount of N fixed by chickpea. The appearance of the first nodules at late stages of growth may be the reason for the low BNF of this crop.     

Nitrogen fixation in Pseudomonas stutzeri

A recently developed oxygen gradient system and a complex medium were used to isolate a microaerobically N₂-fixing heterotrophic bacterium from the rhizosphere of a high fixing Sorghum nutans cultivar. The isolate was identified as nif(+) phenotype of Pseudomonas stutzeri on the basis of cultural, physiological and biochemical characteristics, including DNA/DNA hybridization. N₂ fixation was demonstrated by assimilation of ¹⁵N₂ into cellular protein; the physiology of nitrogen fixation was studied. The isolate contains one 30 MD plasmid and can be cured with associated loss of N₂ fixation capability.Dedicated to Prof. Dr. W. Nultsch on the occasion of his 60th birthday