Effects of symbiosis with Rhizobium fredii on transport of fixed nitrogen in the xylem of soybean plants

An experiment was conducted to identify the main nitrogenous compound transported in the xylem sap of soybean plants nodulated with Rhizobium fredii. Soybean (Glycine max L. Merr.) cultivars, wild type Bragg (nod⁺, fix⁺) and its nitrate tolerant, hypernodulating mutant ntsll16 (nod⁺⁺, fix⁺) were used for this experiment. These soybean plants were inoculated with a slowgrowing rhizobium, Bradyrhizobium japonicum USDAllO or fast-growing rhizobia consisting of a mixture of R. fredii USDA191, USDA193, and USDA-194 and grown in a phytotron under natural light and controlled temperature conditions. Xylem sap was collected from Bragg and ntsll16 plants at the flowering and pod elongation stages. Acetylene reduction activity per plant or per nodule weight was not different between soybean lines and inoculums. The composition of the nitrogenous compounds in the xylem sap was compared between the symbionts, with B. japonicum and R. fredii. At the flowering stage, ureide-N and amide-N accounted for 53 to 70% and 20 to 27% respectively of the total N in the sap collected from the plants inoculated either with B. japonicum or R. fredii. At the pod elongation stage, ureide-N and amide-N accounted for 74 to 85%, and 7 to 19% of total sap N. With the growth of the soybean plants, the ratio of ureide-N in the xylem sap increased. These results suggest that in the case of wild soybean and the hypernodulating mutant line nodulated by R. fredii, ureide is transported as the main nitrogenous compound of fixed nitrogen in the xylem sap in the same way as in plants nodulated with B. japonicum.     

N natural abundance of biologically fixed N2 in soybean is controlled more by the Bradyrhizobium strain than by the variety of the host plant

The ¹⁵N natural abundance technique is one of those most easily applied ‘on farm’ to evaluate the contribution of biological N₂ fixation (BNF) to legume crops. When proportional BNF inputs are high, the accuracy of this technique is highly dependent on an accurate estimate of the ¹⁵N abundance of the N derived from N₂ fixation (the ‘B’ value). The objective of this study was to determine the influence of soybean variety on ‘B’ value. Plants of five soybean varieties were inoculated separately with two Bradyrhizobium strains (one Bradyrhizobium japonicum and one Bradyrhizobium elkanii) grown in pots of soil virtually free of bradyrhizobia capable of nodulating soybean. The proportion of N derived from BNF (%Ndfa) was estimated in separate pots where a small quantity of enriched ¹⁵N ammonium sulphate was added. The %Ndfa was then used with the ¹⁵N natural abundance data of the nodulated soybean and non-N₂-fixing reference plants, to determine the ‘B’ value for each soybean variety/Bradyrhizobium association. The varieties nodulated by the B. japonicum strain showed significantly greater N content and %Ndfa than those nodulated by the B. elkanii strain, and in all cases the ‘B’ value of the shoot tissue (‘B_s’) was higher. The differences in ‘B_s’ values between varieties nodulated by the same Bradyrhizobium strain were insignificant, indicating that this parameter is influenced much more by the Bradyrhizobium strain than by the variety of the host plant.     

Effects of deep placement of controlled-release nitrogen fertilizer on soybean growth and yield under sulfur deficiency

Sulfur (S) and Nitrogen (N) metabolisms in plants are interacted and it is known that S deficiency decrease N absorption and metabolism. In leguminous plants S deficiency also decreases N₂ fixation by rhizobia in the nodules. Deep placement of a controlled-release N fertilizer is a good method to provide nitrogen to soybean without inhibiting N₂ fixation; thus, it was hypothesized that this method is able to provide nitrogen effectively to sulfur-deficient soybean plants. In this study effects of deep placement of coated urea on S-N physicological interaction, growth and productivity in soybean plants were examined using pot experiments. Soybean plants were grown with sulfate concentrations of 30, 100, or 1000 μM, with or without deep placement of coated urea. Shoot weights at the developing stage were not affected by S deficiency. SPAD values of leaves during the flowering stage decreased with S deficiency and increased with the deep placement of coated urea. S deficiency decreased seed weight per plant at the harvesting stage, but this decrease was attenuated by the deep placement of coated urea. N and S content in shoots at the developing stage increased with the deep placement of coated urea, whereas in seeds, only the N content increased. N₂ fixation activity based on the relative ureide-N content in xylem sap indicated that the deep placement of coated urea did not inhibit N₂ fixation activity at the early flowering stage. Without deep placement of coated urea, the relative ureide-N content decreased under S deficiency at the seed filling stage. These results suggest that the deep placement of coated urea is an efficient method to supply N to support soybean yield under S deficiency.

Abbreviations: Deep+: with deep placement of coated urea; Deep–: without deep placement of coated urea     

Isotopic fractionation during N2 fixation by four tropical legumes

To quantify the contribution of biological nitrogen fixation (BNF) to legume crops using the ¹⁵N natural abundance technique, it is necessary to determine the ¹⁵N abundance of the N derived from BNF—the B value. In this study, we used a technique to determine B whereby both legume and non-N₂-fixing reference plants were grown under the same conditions in two similar soils, one artificially labelled with ¹⁵N, and the other not. The proportion of N derived from BNF (%Ndfa) was determined from the plants grown in the ¹⁵N-labelled soil and it was assumed that the %Ndfa values of the legumes grown in the two soils were the same, hence the B value of the legumes could be calculated. The legumes used were velvet bean (Mucuna pruriens), sunnhemp (Crotalaria juncea), groundnut (Arachis hypogaea) and soybean (Glycine max) inoculated, or not, with different strains of rhizobium. The values of %Ndfa were all over 89%, and all the legumes grown in unlabelled soil showed negative δ¹⁵N values even though the plant-available N in this soil was found to be approximately +6.0‰. The B values for the shoot tissue (B_s) were calculated and ranged from approximately −1.4‰ for inoculated sunnhemp and groundnut to −2.4 and −4.5‰ for soybean inoculated with Bradyrhizobium japonicum strain CPAC 7 and Bradyrhizobium elkanii strain 29W, respectively. The B (B_wp) values for the whole plants including roots, nodules and the original seed N were still significantly different between the soybean plants inoculated with CPAC 7 (−1.33‰) and 29W (−2.25‰). In a parallel experiment conducted in monoxenic culture using the same soybean variety and Bradyrhizobium strains, the plants accumulated less N from BNF and the values were less negative, but still significantly different for soybean inoculated with the two different Bradyrhizobium strains. The results suggest that the technique utilized in this study to determine B with legume plants grown in soil in the open air, yields B values that are more appropriate for use under field conditions.     

Response of Determinate and Indeterminate Common Bean Genotypes to Rhizobium Inoculant in a Short Season Rainfed Production System in the Canadian Prairie

ABSTRACT

Common bean (Phaseolus vulgaris L.) has been shown to be a poor di-nitrogen (N₂) fixer and nitrogen (N) fertilizers are usually recommended in bean production. Recent research results suggest that the success of the bean/Rhizobium symbiosis may depend, in part, on the specific bean genotype. Twelve dry bean genotypes differing in growth habit, commercial class, and maturity were evaluated for N₂ fixation in field experiments. Response to inoculant application was highly influenced by environmental conditions. Genotypes differed in nodule dry mass, seed yield, seed N yield, and in amount of N₂ fixed. Growth habit alone was not adequate in classifying bean genotypes for N₂ fixation. The actual amount of N₂ fixed was low ranging from 16 kg ha^?1 to 27 kg ha^?1, suggesting that the symbiotic process alone may not provide adequate N for optimum seed yield in dry environments.     

Determining Symbiotic Nitrogen Fixation in Dry Bean Cultivars Using Ureide Method and Isotope Dilution Techniques

Symbiotic nitrogen fixation (SNF) is an environmentally safe source of nitrogen (N) to the crop plants. In total, 12 dry bean (Phaseolus vulgaris L.) cultivars from pinto, navy, black, and kidney market classes were inoculated with rhizobia and grown in a greenhouse. SNF was estimated using isotope dilution technique and ‘ureide’ method. The amount of SNF ranged between 33 and 68 mg N plant^–1 when determined using ¹⁵N isotope dilution and followed the order: pinto > navy > black > kidney. Percent N derived from atmosphere (%Ndfa) significantly varied between 49% and 90% at V₃ and between 71% and 98% at R₂ stages. The outcomes of the experiment suggested that dry bean cultivars from different market classes have variable N₂ fixation ability, and fertilizer N required should be calculated according to their SNF potentials and N need of a specific market class or cultivar. Stable isotope dilution should be used as the standard procedure to estimate the SNF in dry bean.     

Ureide production by N2-fixing and non-N2-fixing leguminous trees

Xylem-sap and stem and leaf extracts from 35 species, comprising 14 genera, of leguminous trees were analyzed for ureides, nitrate and -amino acids. Trees were either inoculated with Rhizobium or fertilized with NH₄NO₃. The dominant form of soluble N in stem and leaf extracts and xylem sap was 2-amino acids. Certain non-N₂-fixing species, i.e. Tamarindus indica and Adenantherapavonina, produced significant amounts of ureides. Several N₂-fixing species. Mimosa scabrella, Sesbania grandiflora. Acacia mearnsii and Gliricida sepium, grown on mineral-N had higher absolute amounts of ureides in both extracts and exudates than did most nodulated species. Nodulated A. meamsii and S. grandiflora, had the highest amounts of ureides in xylem sap. The relative abundance of ureides in stem and leaf extracts was lower than in xylem sap, but was correlated. Results indicated that the presence of ureides, per se, was not a reliable indicator of N₂-fixing activity. Moreover, the relative abundance of ureides in most of the species tested was too low to use as a presumptive test for, or as a means of, estimating N₂ fixation.     

Size, symbiotic effectiveness and genetic diversity of field pea rhizobia (Rhizobium leguminosarum bv. viciae) populations in South Australian soils

Field pea (Pisum sativum L.) is widely grown in South Australia (SA), often without inoculation with commercial rhizobia. To establish if symbiotic factors are limiting the growth of field pea we examined the size, symbiotic effectiveness and diversity of populations of field pea rhizobia (Rhizobium leguminosarum bv. viciae) that have become naturalised in South Australian soils and nodulate many pea crops. Most probable number plant infection tests on 33 soils showed that R. l. bv. viciae populations ranged from undetectable (six soils) to 32×10³ rhizobia g⁻¹ of dry soil. Twenty-four of the 33 soils contained more than 100 rhizobia g⁻¹ soil. Three of the six soils in which no R. l. bv. viciae were detected had not grown a host legume (field pea, faba bean, vetch or lentil). For soils that had grown a host legume, there was no correlation between the size of R. l. bv. viciae populations and either the time since a host legume had been grown or any measured soil factor (pH, inorganic N and organic C). In glasshouse experiments, inoculation of the field pea cultivar Parafield with the commercial Rhizobium strain SU303 resulted in a highly effective symbiosis. The SU303 treatment produced as much shoot dry weight as the mineral N treatment and more than 2.9 times the shoot dry weight of the uninoculated treatment. Twenty-two of the 33 naturalised populations of rhizobia (applied to pea plants as soil suspensions) produced prompt and abundant nodulation. These symbioses were generally effective at N₂ fixation, with shoot dry weight ranging from 98% (soil 21) down to 61% (soil 30) of the SU303 treatment, the least effective population of rhizobia still producing nearly double the growth of the uninoculated treatment. Low shoot dry weights resulting from most of the remaining soil treatments were associated with delayed or erratic nodulation caused by low numbers of rhizobia. Random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) fingerprinting of 70 rhizobial isolates recovered from five of the 33 soils (14 isolates from each soil) showed that naturalised populations were composed of multiple (5-9) strain types. There was little evidence of strain dominance, with a single strain type occupying more than 30% of trap host nodules in only two of the five populations. Cluster analysis of RAPD PCR banding patterns showed that strain types in naturalised populations were not closely related to the current commercial inoculant strain for field pea (SU303, ≥75% dissimilarity), six previous field pea inoculant strains (≥55% dissimilarity) or a former commercial inoculant strain for faba bean (WSM1274, ≥66% dissimilarity). Two of the most closely related strain types (≤15% dissimilarity) were found at widely separate locations in SA and may have potential as commercial inoculant strains. Given the size and diversity of the naturalised pea rhizobia populations in SA soils and their relative effectiveness, it is unlikely that inoculation with a commercial strain of rhizobia will improve N₂ fixation in field pea crops, unless the number of rhizobia in the soil is very low or absent (e.g. where a legume host has not been previously grown and for three soils from western Eyre Peninsula). The general effectiveness of the pea rhizobia populations also indicates that reduced N₂ fixation is unlikely to be the major cause of the declining field pea yields observed in recent times.     

Use of 15N isotope dilution for quantification of N2 fixation associated with roots of kallar grass (Leptochloa fusca (L.))

Summary Leptochloa fusca (L.) Kunth (kallar grass) has previously been found to exhibit high rates of nitrogen fixation. A series of experiments to determine the level of biological nitrogen fixation using ¹⁵N isotopic dilution were carried out in nutrient solution and saline soil. In the nutrient solution, E. coli inoculated plants were taken as non-nitrogen-fixing control. It was observed that nearly 60%–80% of the plant N was derived from atmospheric fixation. Estimations based on the N difference method gave much lower values (18%–35%). In experiments with saline soil which was initially sterilized with chloroform fumigation, a mixed culture of N₂-fixing rhizospheric isolates from kallar grass roots was inoculated and planted to kallar grass. Uninoculated treatments were regarded as controls. The soil was previously labelled with ¹⁵N by adding cellulose and (¹⁵NH₄)₂SO₄. The results of these studies showed fixation values of 6%–32% when estimated by ¹⁵N dilution, whereas by the N difference method 54% of the plant N was estimated to be derived from fixation. This discrepancy is due to the increase in root proliferation due to inoculation, which results in greater uptake of soil N. The distribution of ¹⁵N in different fractions of the soil-N indicted isotopic dilution due to bacterial fixation of atmospheric N₂.     

Comparison of 15N natural abundance and difference methods for estimating N2 fixation of soybeans grown in a tropical soil

Abstract

A study was carried out to compare the difference or N-yield method with the ¹⁵N natural abundance method for the estimation of the fractional contribution of biological N₂ fixation in the different plant parts of nodulating and non-nodulating isolines of soybeans. The results indicated that the δ¹⁵N values of most plant parts of soybeans were significantly lower (p<0.05) in the nodulating than in the non-nodulating isoline. However, in the case of the root+nodule component, the δ¹⁵N value was higher in the nodulating than in the non-nodulating isoline possibly due to isotopic discrimination of ¹⁵N over ¹⁴N which may have occurred in the nodules. Inoculation of soybeans with the Bradyrhizobium japonicum strain CB 1809 increased significantly (p<0.05) the δ¹⁵N value of the root+nodule component implying that the effectiveness of the soybean-rhizobium symbiosis had increased by inoculation.

Percentage of plant N derived from atmospheric N₂ fixation (%Ndfa) estimated by the ¹⁵N natural abundance method was highly correlated (r=0.762, p<0.01) with that by the difference or N-yield method and the differences between the two methods were not statistically significant. The agreement between the two methods was closer at maturity than at the early reproductive stage.

The %Ndfa obtained by the difference method ranged from 48.4 to 92.6% whereas the %Ndfa obtained by the ¹⁵N natural abundance method ranged from 43.2 to 92.4% in the different plant parts. Based on the ¹⁵N natural abundance method, approximately 15% of the N in pod, shoot, grain, and shell was derived from the soil but in the case of stover, this fraction was about 55%.     

Nodulation and N2 fixation of faba bean (Vicia faba L.) after soil amendment with crop residues

The effect of prior soil amendment with different N sources at 50 mg N (kg soil)^—1 on nodulation and N₂ fixation of faba bean (Vicia faba L. cv. Troy) using wheat (Triticum aestivum L. cv. Star) as reference crop was assessed in a pot experiment. Four treatments viz legume manure (LEGM) as clover shoots, cereal manure (CEREM) as barley straw, N fertilizer (FERT‐N) as Ca(NO₃)₂, and no‐manure control (NOMAN) were investigated consecutively at 45, 70, and 90 days after sowing (DAS). Faba bean nodulated profusely, with an increase on average from 629 nodules per pot at 45 DAS to nearly 2.3‐ and 3.3‐fold at 70 and 90 DAS, respectively. Low nodule numbers and nodule dry matter occurred under FERT‐N and CEREM, whereas high values were found for NOMAN and LEGM. Soil amendment affected percent N₂ fixation in relation to N source and plant age. Highest percent N₂ fixation (≥ 90 %) was found under the lowest N‐supplying amendments, no‐manure, and cereal manure, respectively. FERT‐N depressed N₂ fixation particularly at 45 DAS when N₂ fixation was reduced to as low as 23 %. The rise in N₂ fixation thereafter suggests that faba bean adjusted after depletion of mineral N in the soil. N₂ fixation was also decreased after cereal straw application, even though N concentration in faba bean plants was high. The results indicate that plant residues, both with high and low N concentration, applied to soil to raise its fertility may interfere with N₂ fixation of faba bean.     

Yield,nodulation, and N2 fixation by cowpea cultivars at different phosphorus levels

A greenhouse experiment was conducted to investigate the effect of a P application (0 vs. 50 mg P kg^-1) on yield, nodulation, and N₂ fixation by three cowpea cultivars (Soronko, Amantin, and IT81D-1137) using the ¹⁵N isotope-dilution method. When P was not applied the inoculated cowpea genotypes showed significant differences (Soronko>Amantin> IT81D-1137) in N accumulation, in contrast to the uninoculated cowpea cultivars, which accumulated similar amounts of N. The differences in shoot N in inoculated plants were thus caused by differences in N₂ fixation. The average values of N fixed (for both P levels) were 74% in Soronko, 59% in Amantin, and 42% in IT81D-1137, corresponding to 80, 51, and 24 mg N plant^-1, respectively. Inoculation increased the total shoot-N accumulation in cv. Soronko by 270% without P and by 204% with P, cv. Amantin by 152 and 104%, and cv. IT81D-1137 by 74 and 58%, respectively. With P, the % N derived from atmosphere (%Ndfa) was 42% for IT81D-1137, 62% for Amantin, and 76% for Soronko. The high value for Soronko indicates that in a soil of medium fertility, certain cowpea cultivars are capable of satisfying their total N requirement through N₂ fixation. The P effect on N₂ fixation was mainly in the total amount of N fixed rather than on the percentage derived from the atmosphere.     

Effect of soil physical properties on soybean nodulation and N2 fixation at the early growth stage in heavy soil field in Hachirougata Polder,Japan

We studied the effect of the soil physical properties on soybean nodulation and N₂ fixation in the heavy soil of an upland field (UF) and an upland field converted from a paddy field (UCPF) in the Hachirougata polder, Japan. Seeds of the soybean cultivar Ryuho were sown in each field with or without inoculation of Bradyrhizobium japonicum A1017. The soybean plants were sampled at 35 (V3) and 65 (Rl) d after sowing (DAS), and then nodulation and the percentage of N derived from N₂ fixation in the xylem sap were determined. The soil physical properties were different between UF and UCPF, especially the air permeability and soil water regime. Nodule growth was restricted in UCPF irrespective of rhizobial inoculation, though rhizobial infection was not inhibited by the unfavorable soil physical conditions. Soybean plant growth was closely related to the nodule mass and N₂ fixation activity, and the inoculation of a superior rhizobium strain was effective only at 35 DAS. These results indicate that soybean nodulation and N₂ fixation was considerably affected by the physical properties of heavy soil, and that it is important to maintain the N₂ fixation activity and inoculate the soybean plants with a superior rhizobium strain at a later growth stage in order to increase soybean production in heavy soil fields.     

Nitrogen fixation and soil N level during maturation affect the contents of storage compounds of soybean seeds

We compared the concentrations and contents of protein and oil in mature seeds from nodulated and non-nodulated soybean plants grown on soils with four different N levels during maturation. We observed a positive correlation between the contents of protein and oil in seeds from nodulated plants. Seeds from nodulated plants grown on urea-treated soil showed higher protein and lower oil contents than those from plants grown on soil treated with coated slow release N fertilizer (LP-100). Contents of these compounds in seeds from nodulated plants grown on LP-100 soil were almost the same as those from non-nodulated plants on the same soil. These observations indicated that N economy in roots during seed maturation affects the contents of storage compounds. We suggested that the control of the N₂ fixation activity of soybean plants and management of soil N level during seed maturation are important to determine the contents of protein and oil in seeds.     

Relative efficiency,ureide transport and harvest index in soybeans inoculated with isogenic HUP mutants of Bradyrhizobium japonicum

Summary Differences between isogenic uptake hydrogenase (HUP) mutants of Bradyrhizobium japonicum in terms of nodule efficiency, N₂ fixation and N incorporation into various plant parts were studied in a monoxenic greenhouse experiment in order to confirm previous results with soybeans and beans inoculated with various HUP⁺ and HUP^– strains. The HUP+ revertant PJ17-1 of a HUP^– mutant (PJ17) of strain USDA DES 122 showed a completely restored relative efficiency (100% versus 78±2% for the HUP^– mutant), higher nodule efficiency (N₂ fixed per g nodules), higher ureide-N transport rates, higher N contents in pods and higher N harvest indices. All these observations confirm previous experiments with HUP⁺ and HUP^– strains.     

Nitrogen fixation by nodulated soybean under tropical field conditions estimated by the 15N isotope dilution technique

The contribution of biological N₂ fixation to the N nutrition of nodulated soybean was estimated using the ¹⁵N isotope dilution technique and a non-nodulating soybean isoline as a non-fixing control plant. The plants were grown in the field in concrete cylinders (60 cm dia) and harvested at seven stages of plant growth. Labelled N was added to the soil either as labelled organic matter before planting or in seven small additions (2kg N ha^?1) of (NH₄)₂SO₄ during the growing period.There was good agreement between isotope dilution estimates of nitrogen fixation for the two labelling methods. Acetylene reduction assays on intact root systems greatly underestimated N₂ fixing activity. The difference in total N between nodulated and non-nodulated plants generally gave higher estimates compared with the isotope technique. The data indicate that this was because nodulated plants recovered more N from the soil than the non-nodulated plants. After 92 days of growth, the soybean derived approximately 250kg N ha^?1 from biological N₂ fixation.     

Short-term measurements of uptake of nitrogen fixed in the rhizospheres of sorghum (Sorghum bicolor) and millet (Pennisetum americanum)

Summary In a series of short-term experiments root systems of young sorghum and millet plants inoculated with N₂-fixing bacteria were exposed to ¹⁵N₂-enriched atmospheres for 72 h. The plants were grown in a normal atmosphere for up to 22 days after the end of the exposure to allow them to take up the fixed N₂. Environmental conditions and genotypes of sorghum and millet were selected to maximise N₂-fixation in the rhizosphere. Detectable amounts of fixed N (> 16 g/plant) were rapidly incorporated into sorghum plants grown in a sand/farmyard manure medium, but measurable fixation was found on only one occasion in plants grown in soil. N₂ fixation was detectable in some experiments with soil-grown millet plants but the amounts were small (2–4 g/plant) and represented less than 1 % of plant N accumulated over the same period. In many cases there was no detectable ¹⁵N₂ incorporation despite measurable increases in ethylene concentration found during an acetylene reduction assay.Published as ICRISAT Journal Article No. JA 740     

Salinity effects on nitrogen metabolism in plants – focusing on the activities of nitrogen metabolizing enzymes: A review

Nitrogen (N) metabolism is of great economic importance because it provides proteins and nucleic acids which in turn control many cellular activities in plants. Salinity affects different steps of N metabolism including N uptake, NO₃^? reduction, and NH₄⁺ assimilation, leading to a severe decline in crop yield. Major mechanisms of salinity effects on N metabolism are salinity-induced reductions in water availability and absorption, disruption of root membrane integrity, an inhibition of NO₃^? uptake by Cl^?, low NO₃^? loading into root xylem, alteration in the activities of N assimilating enzymes, decrease in transpiration, and reduction in relative growth rate which results in a lower N demand. However, the effects of salinity on N metabolism are multifaceted and may vary depending on many plant and soil factors. The present review deals with salinity effects on N metabolism in plants, emphasizing on the activities of N metabolizing enzymes in a saline environment.     

Competition and persistence of Rhizobium tropici and Rhizobium etli in tropical soil during successive bean (Phaseolus vulgaris L.) cultures

Strains of Rhizobium tropici IIB, CIAT899 and F98.5, both showing good N₂ fixation, and a R. etli strain W16.3SB were introduced into a field which had no history of bean culture. Plant dilution estimates showed that in the presence of its host (Phaseolus vulgaris cv. Carioca) during the cropping seasons and the subsequent fallow summer periods, the bean rhizobial populations increased from less than 30 to 10³ g^–1 dry soil after 1 year and to 10⁴ g^–1 dry soil after 2 years. In the 1st year crop, the inoculated strains occupied most of the nodules, which resulted in a higher nodulation and C₂H₂ reduction activity. Without reinoculation for the second and third crops, however, little R. tropici IIB was recovered from the nodules and the bean population consisted mainly of R. etli, R. leguminosarum bv. phaseoli, and R. tropici IIA. Reinoculation with our superior R. tropici IIB strains before the second crop resulted in R. tropici IIB occupying the main part of the nodules and a positive effect on nodulation and C₂H₂ reduction activity, but reintroduction of the inoculant strain in the third season did not have any effect.     

Effect of successive cuttings on uptake and partitioning of 15N among plant parts of Leucaena leucocephala

Summary We studied the effect of three successive cuttings on N uptake and fixation and N distribution in Leucaena leucocephala. Two isolines, uninoculated or inoculated with three different Rhizobium strains, were grown for 36 weeks and cut every 12 weeks. The soil was labelled with 50 ppm KNO₃ enriched with 10 atom % ¹⁵N excess soon after the first cutting. Except for the atom % ¹⁵N excess in branches of K28 at the second cutting, both the L. leucocephala isolines showed similar patterns of total N, fixed N₂, and N from fertilizer distribution in different parts of the plant at each cutting. The Rhizobium strain did not influence the partitioning of ¹⁵N among the different plant parts. Significant differences in ¹⁵N enrichment occurred in different parts. Live nodules of both isolines showed the lowest atom % ¹⁵N excess values (0.087), followed by leaves (0.492), branches (0.552), stems (0.591), and roots (0.857). The roots contained about 60% of the total plant N and about 70% of the total N derived from fertilizer over the successive cuttings. The total N₂ fixed in the roots was about 60% of that fixed in the whole plant, while the shoots contained only 20% of the fixed N₂. We conclude that N reserves in roots and nodules constitute another N source that must be taken into account when estimating fixed N₂ or the N balance after pruning or cutting plants. ¹⁵N enrichment declined up to about fivefold in the reference and the N₂-fixing plants over 24 weeks following the ¹⁵N application. The proportion and the amounts of N derived from fertilizer decreased, while the amount derived from N₂ fixation increased with time although its proportion remained constant.