Persistence and recovery of introduced Rhizobium ten years after inoculation on Leucaena leucocephala grown on an Alfisol in southwestern Nigeria

Establishment of Leucaena leucocephala was poor at Ibadan (Transition forest-savanna zone) and Fashola (savanna zone, 70 km north of Ibadan) in southwestern Nigeria as a result of low soil fertility and the presence of only a few native rhizobia capable of nodulating it. Inoculation with L. leucocephala at these two locations in 1982 resulted in striking responses with Rhizobium strains IRc 1045 and IRc 1050 isolated from L. leucocephala grown in Nigeria. The persistence of inoculated effective Rhizobium strains after inoculation is desirable since it removes the need for reinoculation. Because of the perennial nature of L. leucocephala and its use in long-term alley farming experiments, we examined the persistence of inoculated rhizobial strains after inoculation, and their ability to sustain N₂-fixation and biomass production at Ibadan. In 1992, ten years after Rhizobium introduction, uninoculated, L. leucocephala fixed about 150 kg N ha^-1 yr^-1 or about 41% of total plant N compared to 180 kg N ha^-1 yr^-1 or 43% measured in 1982. Serological typing of the nodules using the Enzyme-Linked-Immunosorbent Assay (ELISA) and intrinsic resistance to the streptomycin test revealed that most of the nodules (96%) formed on L. leucocephala in 1992 were by Rhizobium strains IRc 1045 and IRc 1050, which were inoculated in 1982. Nodules were absent on uninoculated L. leucocephala grown on the adjacent field with no history of L. leucocephala cultivation. We conclude that the N₂ fixed by Rhizobium strains IRc 1045 and IRc 1050 persisted for many years in the absence of L. leucocephala and sustained effectively fixed N₂ which growth and yield of L. leucocephala after several years, thus encouraging a possible low-input alley farming system by smallholder farmers in Nigeria.     

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.     

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.     

Residual toxicity of soil-applied chlorothalonil on mycorrhizal symbiosis in Leucaena leucocephala

The residual effect of the fungicide chlorothalonil on the vesicular-arbuscular mycorrhizal (VAM) symbiosis was evaluated in a greenhouse experiment. The soil used was an oxisol (Tropeptic Eutrustox) treated with P to obtain target levels near-optimal for VAM activity or sufficient for nonmycorrhizal host growth. In the uninoculated soil treated with the former P level, the fungicide reduced VAM colonization of roots and completely suppressed symbiotic effectiveness measured in terms of pinnule P content. When this soil was inoculated with Glomus aggregatum, symbiotic effectiveness was significantly reduced but not eliminated by 50 mg of the fungicide kg⁻¹. At higher chlorothalonil levels, VAM effectiveness but not VAM colonization was completely suppressed in the inoculated soil. The pattern with which chlorothalonil influenced tissue P content and dry matter yield at the time of harvest closely paralleled its effect on VAM effectiveness. In the soil treated with P level sufficient for nonmycorrhizal host growth, the adverse effect of the fungicide on the above variables was appreciably milder than when the host relied on VAM fungi for its P supply. The toxic effect of the fungicide, therefore, was partly offset by P fertilization, suggesting that VAM fungi were more sensitive to chlorothalonil than the host. Our results demonstrate that although the toxic effect of chlorothalonil declined as a function of time, a significant level of toxicity persisted 12.5 weeks after the chemical was applied to soil. Contribution from Hawaii Institute of Tropical Agriculture and Human Resources Journal Series No. 3625. Contribution from Hawaii Institute of Tropical Agriculture and Human Resources Journal Series No. 3625.     

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.     

Inhibition of mycorrhizal symbiosis in Leucaena leucocephala by chlorothalonil

The effect of the fungicide, chlorothalonil, on vesicular-arbuscular mycorrhizal (VAM) symbiosis was studied in a greenhouse using Leucaena leucocephala as test plant. Chlorothalonil was applied to soil at 0, 50, 100 and 200 μg g⁻¹. The initial soil solution P levels were 0.003 μg mL⁻¹ (sub-optimal) and 0.026 μg mL⁻¹ (optimal). After 4 weeks, the sub-optimal P level was raised to 0.6 μg mL⁻¹ (high). The soil was either uninoculated or inoculated with the VAM fungus, Glomus aggregatum. The fungicide reduced mycorrhizal colonization of roots, development of mycorrhizal effectiveness, shoot P concentration and uptake and dry matter yields at all concentrations tested, although the highest inhibitory effect was noted as the concentration of the fungicide was increased from 50 to 100 μg g⁻¹. Phosphorus applied after four weeks tended to partially offset the deleterious effects of chlorothalonil in plants grown in the inoculated and uninoculated soil which suggests that the fungicide was interfering with plant P uptake. The results suggest that the use of chlorothalonil should be restricted to levels below 50 μg g⁻¹ if the benefits of mycorrhizal symbiosis are to be expected. Contribution from Hawaii Institute of Tropical Agriculture and Human Resources Journal Series No. 3464. Contribution from Hawaii Institute of Tropical Agriculture and Human Resources Journal Series No. 3464.     

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.     

Root exudation from Leucaena leucocephala in relation to mycorrhizal colonization

Lower amounts of root eduxates (13 mg/g dry root) emerged from leucaena plants inoculated with the mycorrhizal fungus, Glomus fasciculatum, than uninoculated plants (21 mg/g dry root). Mycorrhizal plants exuded less K⁺, P_i and sugars (mainly glucose) but more protein, nitrogen, phenolics and gibberellins than uninoculated plants. Glycine, alanine, cysteine, arginine, tryptophan and valine occurred only in the root exudates of the former. Uninoculated plants exuded more of a root-elongation inhibitory substance than the uninoculated ones.R.J. Mada and D.J. Bagyaraj are with the Department of Agricultural Microbiology, University of Agricultural Sciences, GKVK Campus, Bangalore 560065, India.     

Growth characteristics and total N content of a Leucaena/Sorghum agroforestry system

The growth characteristics and total N content of the woody legume Leucaena leucocephala and the companion crop Sorghum bicolor grown in the greenhouse with N-limiting (NH₄ ⁺ concentration = 447 mM) nutrient medium in sole and mixed cropping were examined. Plant height, dry weight, and total N content increased significantly in sorghum grown intercropped with nodulated Leucaena over the control, sole sorghum. Evidence is presented to indicate that in mixed culture, sorghum gains an average of 0.03 mg N day^–1 plant^–1 relative to sole-cropped sorghum in N-limiting sand culture. The gain in N content of intercropped sorghum, however, represented less than 1% of the N budget of N-fixing Leucaena and was inadequate to sustain normal physiological development of sorghum.     

Assessment of nodulation of Mucuna pruriens by promiscuous indigenous rhizobia in the moist savanna zone of Nigeria

Leucaena leucocephala failed to nodulate effectively with promiscuous indigenous rhizobia with which Mucuna pruriens nodulated effectively. Mucuna pruriens was adequately established and well nodulated due to the presence of favourable climatic and edaphic factors which enhanced its establishment in the humid/moist savanna zone of Nigeria. The microsymbiont for M. pruriens seems to thrive more in an alkaline rhizosphere. Introduction of M. pruriens into the farming system in Nigeria may serve as a low input agricultural system, which is not only sustainable but also economically viable.     

The cloned 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene from Sinorhizobium sp. strain BL3 in Rhizobium sp. strain TAL1145 promotes nodulation and growth of Leucaena leucocephala

The objective of this study was to determine the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase of symbionts in nodulation and growth of Leucaena leucocephala. The acdS genes encoding ACC deaminase were cloned from Rhizobium sp. strain TAL1145 and Sinorhizobium sp. BL3 in multicopy plasmids, and transferred to TAL1145. The BL3-acdS gene greatly enhanced ACC deaminase activity in TAL1145 compared to the native acdS gene. The transconjugants of TAL1145 containing the native or BL3 acdS gene could grow in minimal media containing 1.5mM ACC, whereas BL3 could tolerate up to 3mM ACC. The TAL1145 acdS gene was inducible by mimosine and not by ACC, while the BL3 acdS gene was highly inducible by ACC and not by mimosine. The transconjugants of TAL1145 containing the native- and BL3-acdS genes formed nodules with greater number and sizes, and produced higher root mass on L. leucocephala than by TAL1145. This study shows that the introduction of multiple copies of the acdS gene increased ACC deaminase activities of TAL1145 and enhanced its symbiotic efficiency on L. leucocephala.     

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.     

矿质养分输入对森林生物固氮的影响

生物固氮是森林生态系统重要的氮素来源,并且在全球氮循环中占有重要的地位。近代以来,因人类活动加剧而导致氮沉降的增加以及其它矿质养分元素(如磷、钼、铁等)输入的改变已成为影响森林生态系统生物固氮的重要因素之一,并引起了学术界的普遍关注。综述了国内外关于森林生物固氮对矿质养分输入的响应及机理。主要内容包括:(1)森林生物固氮的概念及主要的测定方法;(2)矿质养分输入对森林生物固氮的影响。整体上讲,氮素输入抑制了森林生物固氮,磷和其他营养元素输入则表现为促进作用。氮和磷、磷和微量元素同时添加均提高了森林的固氮量;(3)矿质养分改变森林生物固氮的机理。包括生物作用机制(如改变地表层固氮菌的数量或群落丰度、改变结瘤植物的根瘤生物量和附生植物的丰度或盖度)和环境作用机制(如引起土壤酸化、改变碳源物质的含量);(4)探讨了矿质养分输入对森林生物固氮影响研究中所存在的问题,并对未来该领域的研究提出建议。     

Changes in nodule and root biomass of Sesbania sesban and Leucaena leucocephala following coppicing

A pot study was conducted to measure the extent of and determine the factors controlling fine root and nodule shedding following coppicing of Sesbania sesban and Leucaena leucocephala. Fine (<2 mm) root biomass decreased below pre-cutting values, but the decreases were not statistically significant in either species. Living (white) nodule biomass decreased and dead (brown) nodule biomass increased significantly two weeks after cutting in both species. These changes were relatively greater in Sesbania than in Leucaena. In the uncut treatments of both species, fine root and nodule biomass were correlated with leaf biomass, and in the cut treatments, root and nodule biomass returned to near this apparent equilibrium by two weeks after cutting. Stem growth rate per unit leaf area was not different between cut and uncut treatments, nor was it correlated with root:leaf ratios in either species. Leucaena allocated a greater fraction of its total biomass below-ground, and a greater fraction of its below-ground biomass to coarse (>2 mm) roots than Sesbania. These results are consistent with the hypothesis that relatively lower allocation to below-ground storage tissue is the cause for Sesbania's relatively greater sensitivity to cutting.     

The effect of VA mycorrhizal fungi,phosphorus and drought stress on the growth of Acacia nilotica and Leucaena leucocephala seedlings

The effect of vesicular-arbuscular mycorrhizal (VAM) fungi on growth and drought resistance of Acacia nilotica and Leucaena leucocephala seedlings was studied in a glasshouse experiment. The experimental design was a 2·2·2 factorial: ± mycorrhizal inoculation, ± application of phosphorus fertilizer and ± repeated drought treatment. The growth promoting effect of VAM fungi equalled the effect of phosphorus fertilization after 12 weeks. The drought treatment reduced seedling biomass and nodulation. Differences between the plant species were found with respect to growth improvements due to VAM inoculation and/or phosphorus fertilization under drought stress conditions. The results are discussed in relation to plant drought resistance and reforestation in the subhumid to arid tropics.     

The role of plant growth regulators on direct and indirect plant regeneration from various organs of <Emphasis Type="Italic">Leucaena leucocephala</Emphasis>

Prolific differentiation of shoot buds of Leucaena leucocephala was induced from the different plant parts viz. cotyledon, hypocotyl and leaf. Adventitious shoot bud formation was recorded with prudent application of N⁶-2- (isopentenyl) adenosine and 15% (v/v) coconut water. Coconut water alone was unable to produce any beneficial effect with regard to the shoot bud proliferation but the response was augmented with the increase in concentration of N⁶-2- (isopentenyl) adenosine. However supra-optimal level of N⁶-2-(isopentenyl) adenosine was inhibitory. Best response was recorded from the cotyledon explant at 2 mg dm⁻³ N⁶-2-(isopentenyl) adenosine compared to the other two explants. Comparative assessment was undertaken following the same experimental protocol in liquid shake culture. The regenerated shoot buds were subcultured in plant growth regulator-free medium where leafy shoot emergence was recorded. Optimum regeneration of roots was observed in these shoots in presence of 1 mg dm⁻³ α-naphthalene acetic acid. Plantlets were finally hardened following standard procedures before transplantation to the field. In another experimental set up, the de-embryonated cotyledons regenerated shoot buds via callus formation. The regenerated shoots and plantlets obtained through callus mediated organogenesis could be used for rapid multiplication and also for the genetic improvement of individual clones of Leucaena leucocephala.     

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.     

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%.     

Applications of mineral nutrients to heavily N-fertilized Scots pine trees: Effects on arginine and mineral nutrient concentrations

The aim of the investigation was to study if improved nutrient status in Scots pine (Pinus sylvestris L) trees would be reflected in decreased concentrations of arginine in the needles. The studies trees had imbalanced mineral nutrient composition and elevated needle arginine concentrations caused by long-term fertilization with N. Concentrations of arginine and mineral nutrients in needles were followed over three consecutive years of additional fertilization with N alone or with P, K, Mg and micronutrients in combination with and without N.Analysis of needle mineral concentrations suggested that there were deficiencies only in K and Mg. The N concentration increased both in trees fertilized with N alone and in trees fertilized with N in combination with mineral nutrients. In the control treatment and in trees fertilized with mineral nutrients other than N the N concentration remained fairly constant. The highest Ca/N, K/N and P/N ratios were found in trees fertilized with mineral nutrients other than N while the lowest ratios were found in trees fertilized with N alone. Arginine concentrations in needles from trees fertilized with N alone remained at a high level throughout the experiment while arginine concentrations in trees given the other treatments decreased.The results show that the mineral nutrient balance can be improved with appropriate fertilization and that this improvement is reflected in decreasing arginine levels. Furthermore the study demonstrates that when N supply is reduced the arginine concentration also decreases also as an effect of reduced N supply per se. The study also indicates that arginine may be a better measure of the N status in pine trees than total N.     

Propagation of the tropical tree,Leucaena leucocephala K67, by in vitro bud culture

A tissue culture method is described for clonal multiplication of Leucaena leucocephala K67 using single lateral bud explants from 2–3 m tall greenhouse grown trees. N-6 benzyladenine (BA: 3.0 mg.1^-1) and napthaleneacetic acid (NAA: 0.05 mg.1^-1) in Murashige & Skoog's (MS) medium were found to be best suited for multiple shoot differentiation in 4–5 week old cultures. Analysis of variance of the main treatment effects of BA and NAA on shoot parameters showed that BA significantly (P=0.001) affected shoot development while NAA did not. A shoot multiplication rate of 22±3.63 shoots per bud explant was obtained in 150 days on 1/2 strength MS medium with 3.0 mg.1^-1 BA and 0.05 mg.1^-1 NAA. Shoots developed adventitious roots within 15 days in 1/2 strength MS medium containing indole-3-butyric acid (IBA: 3.0 mg.1^-1) and Kinetin (0.05 mg.1^-1). Eighty percent of the transplanted plantlets are being grown in greenhouse conditions.