Nodulation and growth ofLeucaena leucocephala (Lam.) de Wit as affected by inoculation and N fertilizer

Leonard jar, pot and field experiments examined the effects of inoculation and the influence of nitrogen fertilizer on nodulation, nitrogen fixation and growth ofLeucaena leucocephala (Lam.) de Wit at IITA, Ibadan, Nigeria. Leucaena responded to both inoculation and/or nitrogen application. Shoot growth and total N and P of inoculated plants were comparable to those of the highest N treatment, and the values were about 55% greater than those of uninoculated ones. Field data indicated that toal N yields of inoculated leucaena were increased by 50% with 40 or 80 kg ha^–1 of N fertilizer. However, N fertilizer depressed N fixation by 56% as was expected from nodule mass data. N-fixation was delayed for about 8 weeks in the plots without N. Application of small amounts of N starter (20 ppm) proved to be beneficial to satisfy the plant need during the early stage of leucaena growth. The rhizobial strains IRc 1045 and IRc 1050 were effective, competitive and survived well in the field one year after their establishment.     

Nitrogen fixation of field-inoculatedLeucaena leucocephala (Lam.) de Wit estimated by the15N and the difference methods

The amount of nitrogen fixed byLeucaena leucocephala (Lam.) de Wit was assessed on an Alfisol at the International Institute of Tropical Agriculture located in southwestern Nigeria. Estimated by the difference method, nitrogen fixation of leucaena inoculated with Rhizobium strain IRc 1045 was 133 kg ha^–1 in six months. Inoculation with Rhizobium strain IRc 1050 gave a lower nitrogen fixation of 76 kg ha^–1. Fertilization with 40 and 80 kg N ha^–1 inhibited nitrogen fixation by 43–76% and 49–71%, respectively. Estimates with the¹⁵N dilution method gave nitrogen fixation of 134 kg ha^–1 in six months when leucaena was inoculated with Rhizobium strain IRc 1045 and 98 kg ha^–1 for leucaena inoculated with Rhizobium strain IRc 1050. This nitrogen fixation represented 34–39% of the plant nitrogen. Inoculated leucaena derived 5–6% of its nitrogen from applied fertilizer and 56–54% from soil.     

Persistence and competitiveness of threeBradyrhizobium japonicum inoculant strains in clay loam Nile valley soil

The nitrogen contribution from the shoot and root system of symbiotically grown leucaena was evaluated in a field experiment on an Alfisol at IITA in Southern Nigeria. Maize in plots that received prunings from inoculated leucaena contained more N and grain yield was increased by 1.9 t.ha.^–1. Large quantities of nitrogen were harvested with leucaena prunings (300 kg N ha^–1 in six months) but the efficiency of utilization of this nitrogen by maize was low compared to inorganic N fertilizer (ammonium sulphate) at 80 kg N ha^–1. Maize yield data indicated that nitrogen in leucaena prunigs was 34 and 45% as efficient as 80 kg N ha^–1 of (NH₄)₂SO₄ for uninoculated and inoculated plants with Rhizobium IRc 1045, respectively. In plots where the prunings were removed, the leaf litter and decaying roots and nodules contributed N equivalent of 32 kg ha^–1. Twenty-five kg ha^–1 was the inorganic N equivalent from nitrogen fixed symbiotically by leucaena when inoculated with Rhizobium strain IRc 1045. Application of prunings from inoculated leucaena resulted in higher soil ogranic C, total N, pH and available NO₃.     

Response of Leucaena/Rhizobium symbiosis to mineral nutrients in Southwestern Nigeria

Pot and field experiments carried out at the International Institute of Tropical Agriculture (IITA) and at Fashola, Southwestern Nigeria, examined the effect of inoculation and N, P and micronutrients on nodulation and growth ofLeucaena leucocephala (Lam.) de Wit. In pot studies all parameters measured, except the percentage of nitrogen in shoots, were improved by inoculation, nitrogen and phosphorus. Micronutrients increased only nitrogen and allantoin contents. Interactions between inoculation and P, N and micronutrients on nodulation and growth of leucaena were observed. The effect of inoculation and fertilization with phosphorus or micronutrients was further investigated in field experiments. Establishment of uninoculated and unfertilized leucaena was poor at both locations due to low soil fertility and the presence of only a few native leucaena rhizobia. At one site, only inoculated plants were nodulated, while at the other, all plants produced nodules. Shoot dry weight, total nitrogen and phosphorus of inoculated plants were statistically equal to nitrogen-fertilized plants. Uninoculated plants were stunted. Generally, micronutrients did not influence nodulation, total nitrogen or growth of leucaena. They had only a positive effect on nitrogenase activity. Phosphorus increased total nitrogen and phosphorus uptake and plant growth. A 75% increase in shoot dry weight was obtained when 80 kg P ha^–1 was applied to inoculated leucaena with Rhizobium strain IRc 1045. Inoculated plants contained more allantoins than uninoculated ones but no significant correlation was found between these compounds and other parameters of N fixation.     

Long term field study shows increased biomass production in tree legumes inoculated with Rhizobium

Nodulation potential, nitrogen fixation efficiency (nitrogenase activity) and biomass yield response of Leucaena leucocephala and Acacia nilotica to inoculation with 6 selected fast growing Rhizobium strains was explored in long-term (5 year) field trials. All the strains formed nodules and fixed nitrogen in L. leucocephala and A. nilotica. Seasonal effects on nitrogenase activity was observed and in winter (ambient temperature about 20 °C), nitrogenase activity could not be detected. However, with the onset of spring and a rise in temperature, fresh nodulation (renodulation) by all the inoculant rhizobial strains was observed in both the tree legumes. In L. leucocephala, maximum renodulation was exhibited by strain A1 while in A. nilotica, strain AB3 formed the maximum renodulation 24 months after transplantation. Dry matter yield of all the inoculated plants demonstrated a significant increase over that of the uninoculated plants at the end of five years after transplanting. In L. leucocephala, strain NGR8 gave the maximum response (45% more dry matter yield) in dry matter production while in A. nilotica, strain USDA 3325 showed a 25% increase in total dry matter yield five years after transplantation.     

Assessment of genetic variability for N2 fixation between and within provenances of Leucaena leucocephala and Acacia albida estimated by 15N labelling techniques

Nitrogen fixed in 13 provenances of Acacia albida and 11 isolines of Leucaena leucocephala inoculated with effective Rhizobium strains was measured by ¹⁵N techniques and the total N difference method. In the test soil, on the average, L. leucocephala derived about 65% of its total N from atmospheric N₂ fixation compared to about 20% by A. albida. Significant differences in the percentage of N derived from atmospheric N₂ (% Ndfa) occurred, between provenances or isolines within species. The % Ndfa ranged from 37 to 74% within L. leucocephala and from 6 to 37 within A. albida; (equivalent to 20–50 mg N plant^–1 and 4–37 mg N plant^–1 for the two species over three months, respectively) and was correlated with the nodule mass (r=0.91). The time course of N₂ fixation of three selected provenances (low, intermediate and good fixers) was followed at 12 weekly intervals over a 36 week period. The % Ndfa of all provenances and isolines increased with time; and except for one of the L. leucocephala provenances, % Ndfa was similar within species at the 36 weeks harvest. There was a significant correlation between % Ndfa and the amount of N₂ fixed (r=0.96). Significant interactions occurred between provenances and N treatments and often growth of uninoculated but N fertilized plants was less variable than for inoculated unfertilized plants.     

Effect of Rhizobium inoculation methodologies on nodulation and growth of Leucaena leucocephala

The aim of this study was to evaluate the effect of five methods of Rhizobium inoculum application on nodulation and nitrogen fixation in Leucaena leucocephala seedlings cultivated for 6 months in the greenhouse. Plants inoculated with alginate beads were significantly more developed and more nodulated than plants inoculated with the other methodologies used.     

Effect ofAzospirillum inoculation on nitrogen fixation and growth of several winter legumes

Summary Inoculation of naturally nodulatedPisum sativum L. (garden pea) withAzospirillum in the greenhouse caused a significant increase in nodule numbers above controls. Field inoculation of garden peas in the winter 1981–1982 andCicer arietinum L. (chick pea), in winter 1982–1983, withAzospirillum one week after plant emergence, produced a significant increase in seed yield, but did not affect plant dry matter yield. ForVicia sativa L. (vetch) grown in soil in the greenhouse and in the field for forage, winter 1980–1981, inoculation significantly increased dry matter yield, %N, N-content, and acetylene reduction (nitrogen fixation) activity. InHedysarum coronarium L. (sulla clover), winter 1981–1982, inoculated with both its specificRhizobium (by the slurry method) andAzospirillum, 7 days after emergence, there was an increase in acetylene reduction above controls inoculated withRhizobium alone. These results suggest that it is possible, under conditions tested in this work, to increase nodulation, nitrogen fixation, and crop yields of winter legumes by inoculation withAzospirillum.     

Interaction between alfalfa cultivars and Rhizobium strains for nitrogen Fixation

Summary Two experiments were conducted in the greenhouse to study the interaction between alfalfa cultivars (Medicago sativa L. and M. falcata L.) and strains of Rhizobium meliloti Dang. for acetylene reduction rate, plant height and dry weights of shoot, root and whole plant. Fifteen alfalfa cultivars were inoculated with 10 strains of Rhizobium in Experiment I. Variance component analysis revealed that more than 30% of the total variance was due to alfalfa cultivars for acetylene reduction rate and 26% was accounted for by Rhizobium strains. More than 36% of the total variation was attributed to the interaction between alfalfa cultivars and Rhizobium strains for this character. Twenty-five host cultivars and 11 Rhizobium strains were included in Experiment II. The results also showed that the interaction of alfalfa cultivars and Rhizobium strains contributed the largest portion of the total variation for dry weights of shoot, root and whole plant and acetylene reduction rate. The results clearly demonstrated that the non-additive effects were the major component of variation for these characters associated with nitrogen fixation in alfalfa. Therefore, an effective way of improving nitrogen fixation in alfalfa is to select for a favourable combination of specific Rhizobium strains and alfalfa cultivars.     

Rhizobium strains expressing uptake hydrogenase in different host species of cowpea miscellany

Uptake hydrogenase activity in nodules of green gram (Vigna radiata (L.) (Wilczek)), black gram (Vigna mungo (L.) (Hepper)), cowpea (Vigna unguiculata (L.) and cluster bean (Cyamopsis tetragonoloba (L.) (Taub.)), formed with two Hup⁺ (S24 and CT2014) and one Hup⁻ (M11)Rhizobium strains, was determined at different levels of external H2 in air atmosphere. Nodules of all the 4 host species formed by inoculation with strains S24 and CT2014, showed H2 uptake but not those formed with strain M11. H₂ uptake rates were higher in 1 and 2% H₂ in air atmosphere (v/v) than at 5 or 10% levels in all the host species. Variations in the relative rates of H₂ uptake were observed both, due to host species as well as due toRhizobium strains. However, no host dependent complete repression of the expression of H₂ uptake activity was observed in nodules of any of the host species formed with Hup⁺ strains.     

Production of gibberellins and indole-3-acetic acid by Rhizobium phaseoli in relation to nodulation of Phaseolus vulgaris roots

Similar ranges of gibberellins (GAs) were detected by high-performance liquid chromatography (HPLC)-immunoassay procedures in ten cultures of wild-type and mutant strains of Rhizobium phaseoli. The major GAs excreted into the culture medium were GA₁ and GA₄. These identifications were confirmed by combined gas chromatographymass spectrometry. The HPLC-immunoassays also detected smaller amounts of GA₉- as well as GA₂₀-like compounds, the latter being present in some but not all cultures. In addition to GAs, all strains excreted indole-3-acetic acid (IAA) but there was no obvious relationship between the amounts of GA and IAA that accumulated. The Rhizobium strains studied included nod ^– and fix ^– mutants, making it unlikely that the IAA- and GA-biosynthesis genes are closely linked to the genes for nodulation and nitrogen fixation.The HPLC-immunoassay analyses showed also that nodules and non-nodulated roots of Phaseolus vulgaris L. contained similar spectra of GAs to R. phaseoli culture media. The GA pools in roots and nodules were of similar size, indicating that Rhizobium does not make a major contribution to the GA content of the infected tissue.Abbreviations EIA enzyme immunoassay - GA_n gibberellin A_n - GC-MS gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - Me methyl ester - RIA radioimmunoassay - TLC thin-layer chromatography     

The effect of inoculation with Azospirillum brasilense on growth and nitrogen utilization by wheat plants

Azospirillium brasilense is a rhizosphere bacteria that has been reported to improve yield when inoculated on wheat plants. However, the mechanisms through which this effect is induced is still unclear. In the present work, we have studied the effects of inoculating a highly efficient A. brasilense strain on wheat plant grown in 5 kg pots with soil in a greenhouse, under three N regimes (0, 3 or 16 mM NO₃ ^–, 50 ml/pot once or twice-a -week), and in disinfected or non-disinfected soil. At the booting stage, the inoculated roots in both soils showed a similar colonization by Azospirillum sp. that was not affected by N addition. The plants grown in the disinfected soil showed a higher biomass, N content and N concentration than those in the non-disinfected soil, and in both soils the inoculation stimulated plant growth, N accumulation, and N and NO₃ ^– concentration in the tissues.At maturity, the inoculated plants showed a higher biomass, grain yield and N content than the uninoculated ones in both soils, and a higher grain protein concentration than the uninoculated. It is concluded that in the present experiments, A. brasilenseincreased plant growth by stimulating nitrogen uptake by the roots.     

Effect of amaranth on the growth of Rhizobium and Bradyrhizobium strains

The growth rate of different strains of Bradyrhizobium and Rhizobium was studied in media containing amaranth seed meal instead of yeast extract. Results obtained in erlenmeyer flasks and stirred fermenters show that both Bradyrhizobium japonicum strains E109, E110, 5019, 587 and Rhizobium melilotistrains B36, B323, B399, Lq₂₂, Lq₄₂, Lq₅₁ and U₃₂₂, grow satisfactorily in amaranth seed meal medium. Cell count obtained for the strains tested was greater than 4 × 10¹⁰ viable cells.ml^–1. Amaranth seed meal (4 g.l^–1) is a suitable component for culture media that can be used instead of yeast extract.     

The potential of <Emphasis Type="Italic">Rhizobium</Emphasis> strains for biological control of <Emphasis Type="Italic">Orobanche crenata</Emphasis>

Orobanche crenata Forsk is a chlorophyll lacking holoparasite that subsists on the roots of plants and causes significant damage to the culture of leguminous plants and, in particular, to peas (Pisum sativum L.). Here, we investigated the potential of Rhizobium strains for biological control of Orobanche crenata using a commercial pea cultivar (Douce de province) and different Rhizobium strains. Firstly, benefit of bacterial inoculation on plant growth and efficiency in N-incorporation were demonstrated with four isolates, P.SOM, P.1001, P.Mat.95 and P.1236. After five Rhizobium strains (three efficient: P.SOM, P.1236, P.Mat.95 and two not efficient: P.OM1.92, P.MleTem.92) were investigated for their ability to control Orobanche crenata using pot and Petri dish experiments. Inoculation of peas with two (P.SOM and P.1236) of the five strains induced a significant decrease in O. crenata seed germination and in the number of tubercles on pea roots. Furthermore, other symptoms, including the non-penetration of the germinated seeds into pea roots followed by radicle browning and death of the parasites, were observed in the presence of these inoculated pea plants. The hypothesis that roots secrete toxic compounds related to Rhizobium inoculation is discussed.     

Management of biological N2 fixation in alley cropping systems: Estimation and contribution to N balance

Alley cropping is being widely tested in the tropics for its potential to sustain adequate food production with low agricultural inputs, while conserving the resource base. Fast growth and N yield of most trees used as hedgerows in alley cropping is due greatly to their ability to fix N₂ symbiotically with Rhizobium. Measurements of biological N₂ fixation (BNF) in alley cropping systems show that some tree species such as Leucaena leucocephala, Gliricidia sepium and Acacia mangium can derive between 100 and 300 kg N ha^-1 yr^–1 from atmospheric N₂, while species such as Faidherbia albida and Acacia senegal might fix less than 20 kg N ha^-1 yr^-1. Other tree species such as Senna siamea and S. spectabilis are also used in alley cropping, although they do not nodulate and therefore do not fix N₂. The long-term evaluation of the potential or actual amounts of N₂ fixed in trees however, poses problems that are associated with their perennial nature and massive size, the great difficulty in obtaining representative samples and applying reliable methodologies for measuring N₂ fixed. Strategies for obtaining representative samples (as against the whole tree or destructive plant sampling), the application of ¹⁵N procedures and the selection criteria for appropriate reference plants have been discussed.Little is known about the effect of environmental factors and management practices such as tree cutting or pruning and residue management on BNF and eventually their N contribution in alley cropping. Data using the ¹⁵N labelling techniques have indicated that up to 50% or more of the tree's N may be below ground after pruning. In this case, quantification of N₂ fixed that disregards roots, nodules and crowns would result in serious errors and the amount of N₂ fixed may be largely underestimated. Large quantities of N are harvested with hedgerow prunings (>300 kg N ha^-1 yr^-1) but N contribution to crops is commonly in the range of 40–70 kg N ha^-1 season. This represents about 30% of N applied as prunings; however, N recoveries as low as 5–10% have been reported. The low N recovery in maize (Zea mays) is partly caused by lack of synchronization between the hedgerow trees N release and the associated food crop N demand. The N not taken up by the associated crop can be immobilized in soil organic matter or assimilated by the hedgerow trees and thus remain in the system. This N can also be lost from the system through denitrification, volatilization or is leached beyond the rooting zone. Below ground contribution (from root turnover and nodule decay) to an associated food crop in alley cropping is estimated at about 25–102 kg N ha^-1 season^-1. Timing and severity of pruning may allow for some management of underground transfer of fixed N₂ to associated crops. However many aspects of root dynamics in alley cropping systems are poorly understood. Current research projects based on ¹⁵N labelling techniques or ¹⁵N natural abundance measurements are outlined. These would lead to estimates of N₂ fixation and N saving resulting from the management of N₂ fixation in alley cropping systems.     

Inoculation of Rhizobium (VR-1 and VA-1) induces an increasing growth and metal accumulation potential in Vigna radiata and Vigna angularis L. growing under fly-ash

Fly-ash-tolerant Rhizobium strains were isolated from plants grown in fly-ash-contaminated soil, axenically under laboratory conditions. Saplings of both plants were raised in N₂-free Jenson medium and inoculated with 2.6 × 10⁸ cell ml⁻¹ and 5.2 × 10⁸ cell ml⁻¹ of culture after 10 d of growth. Plants were transferred into 100% fly-ash under natural condition. Rhizobium-inoculated plants grown on 100% fly-ash showed marked increase in relation to root-shoot length, biomass yield, photosynthetic pigment, protein content and nodulation frequency compared to uninoculated plant grown in control (100% fly-ash). Inoculation of fly-ash-tolerant Rhizobium increased the accumulation of Fe, Zn, Cu Cd and Cr in different tissues vis-à-vis enhanced translocation of metals to the aboveground part of plant. Although inoculation of fly-ash-tolerant Rhizobium strains (VR-1 and VA-1) enhanced the translocation of more Fe to shoot parts, nevertheless, the amount of Rhizobium inoculants supplied to the plant was found to be very important since it has a positive role in increasing plant growth through increased N₂ supply via nitrogenase activity. Results suggest that an integrated approach employing biotechnological means and inoculation of plants with host-specific fly-ash-tolerant Rhizobium strain may prove a stimulus to a fly-ash management programme.     

Short communication: Screening of native and foreign Bradyrhizobium japonicum strains for high N2 fixation in soybean

Four local rhizobia isolates selected after two screening experiments and five USDA Bradyrhizobium japonicum strains were estimated for N₂ fixation in soybean using the ¹⁵N isotope dilution technique. Strain USDA 110 was superior to the local isolates in nodulation and N₂ fixation when inoculated onto soybean cv TGX 1497-ID in a Nigerian soil and could therefore be used as an inoculant for enhanced N₂ fixation in soybean in Nigeria.     

Mimosine produced by the tree-legume Leucaena provides growth advantages to some Rhizobium strains that utilize it as a source of carbon and nitrogen

Growth of most Rhizobium strains is inhibited by mimosine, a toxin found in large quantities in the seeds, foliage and roots of plants of the genera Leucaena and Mimosa. Some Leucaena-nodulating strains of Rhizobium can degrade mimosine (Mid⁺) and are less inhibited by mimosine in the growth medium than the mimosine-nondegrading (Mid^-) strains. Ten Mid⁺ strains were identified that did not degrade 3-hydroxy-4-pyridone (HP), a toxic intermediate of mimosine degradation. However, mimosine was completely degraded by these strains and HP was not accumulated in the cells when these strains were grown in a medium containing mimosine as the sole source of carbon and nitrogen. The mimosine-degrading ability of rhizobia is not essential for nodulation of Leucaena species, but it provides growth advantages to Rhizobium strains that can utilize mimosine, and it suppresses the growth of other strains that are sensitive to this toxin.     

Long-term integrated soil fertility management in South-western Nigeria: Crop performance and impact on the soil fertility status

Crop response, tree biomass production and changes in soil fertility characteristics were monitored in a long-term (1986–2002) alley-cropping trial in Ibadan, Nigeria. The systems included two alley cropping systems with Leucaena leucocephala and Senna siamea on the one hand and a control (no-trees) system on the other hand, all cropped annually with a maize–cowpea rotation. All systems had a plus and minus fertilizer treatment. Over the years, the annual biomass return through tree prunings declined steadily, but more drastically for Leucaena than for Senna. In 2002, the nitrogen contribution from Leucaena residues stabilized at about 200 kg N/ha/year, while the corresponding value for Senna was about 160 kg N/ha/year. On average, the four Leucaena prunings were more equal in biomass as well as in amounts of N, P and cations, while the first Sennapruning was always contributing up to 60% of the annual biomass or nutrient return. Maize crop yields declined steadily in all treatments, but the least so in the Senna + fertilizer treatment where in 2002 still 2.2 tonnes/ha of maize were obtained. Nitrogen fertilizer use efficiency was usually higher in the Senna treatment compared to the control or the Leucaena treatment. Added benefits due to the combined use of fertilizer N and organic matter additions were observed only for the Sennatreatment and only in the last 6 years. At all other times, they remained absent or were even negative in the Leucaenatreatments for the first 3 years. Most chemical soil fertility parameters decreased in all the treatments, but less so in the alley cropping systems. The presence of trees had a positive effect on remaining carbon stocks, while they were reduced compared to the 1986 data. Trees had a positive effect on the maintenance of exchangeable cations in the top soil. Exchangeable Ca, Mg and K – and hence ECEC – were only slightly reduced after 16 years of cropping in the tree-based systems, and even increased in the Senna treatments. In the control treatments, values for all these parameters reduced to 50% or less of the original values after 16 years. All the above points to the Senna-based alley system with fertilizers as the more resilient one. This is reflected in all soil fertility parameters, in added benefits due to the combined use of fertilizer nitrogen and organic residue application and in a more stable maize yield over the years, averaging 2.8 tonnes/ha with maximal deviations from the average not exceeding 21%.     

Ecology of Hup+ Rhizobium strains of cow pea miscellany: native frequency and competence

Rhizobium strains nodulating summer legumes cow pea [Vigna unguiculata (L.)], green gram [V. radiata (L.) (Wilczek)], black gram [V. mungo (L.) (Hepper)] and cluster bean [Cyamopsis tetragonoloba (L.) (Taub)] and a winter legume chick pea [Cicer arietinum (L.)] were surveyed in the Northern Plains of India and screened for hydrogenase activity to determine distribution of Hup character in the native ecosystem. It was observed that 56% of the Rhizobium strains of summer legumes were Hup⁺ whereas that of the winter legume, chick pea, were all Hup^-. Ex planta acetylene reduction activity was observed in most of the Hup⁺ but not in the Hup^- strains of any of the host species. In summer legume, mixed inoculation of Hup⁺ and Hup^- strains, under sterilized as well as unsterilized soil conditions, showed that the host species were predominantly nodulated with Hup⁺ strain.