Nutrient composition and in vitro ruminal fermentation of tropical legume mixtures with contrasting tannin contents

Various combinations of a low-tannin herbaceous legume (Vigna unguiculata) and foliage of tanniniferous shrub legumes (Calliandra calothyrsus, Flemingia macrophylla and Leucaena leucocephala) or a low-tannin shrub legume (Cratylia argentea), all mixed together with a low-quality tropical grass (Brachiaria humidicola), were tested in vitro for differences in the effects on ruminal fermentation. Two experiments with the gas transducer technique were carried out, where each forage mixture was tested either with or without polyethylene glycol in order to be able to identify tannin-related effects (n = 3). In Experiment 1, a stepwise replacement of V. unguiculata by C. calothyrsus (5:0, 4:1, 3:2, 2:3, 1:4, 0:5) at a legume proportion of 1/3 or 2/3 in the mixture was evaluated. Together with two grass-alone and four pure legume treatments this added up to 30 treatments. In Experiment 2, V. unguiculata was gradually replaced by each of the four shrub legumes (3:0, 2:1, 1:2, 0:3) in grass–legume ratios of 2:1, adding up, together with two grass-alone treatments, to 28 treatments. When added alone, V. unguiculata resulted in high fermentative activity as measured by gas production and kinetics as well as low proportion of undegraded crude protein. When V. unguiculata was replaced by the low-tannin C. argentea in Experiment 2, there was no noticeable difference (P>0.05) in fermentative activity. In both experiments, the effect of the substitution of V. unguiculata by tanniniferous shrub legumes resulted in a declining gas production and an increasing proportion of undegraded crude protein (P<0.001). However, the extent of these changes depended on the level of replacement and the shrub legume species (P<0.001). The results of Experiment 2 illustrate that this was the consequence not only of different tannin contents (less adverse effects with L. leucocephala than with C. calothyrsus) but also differences in the chemical properties of the tannins present in these shrub legume species (much less adverse effects with L. leucocephala than with F. macrophylla despite similar tannin contents). Furthermore these results indicate that, once the extent of the effects of a tanniniferous legume is known, one may calculate the maximal level of replacement of a low-tannin legume in a grass diet possible without negative effects on ruminal fermentation. This allows to improve dry season grass-based diets with as few as possible of the expensive and less well growing low-tannin legume.     

Application of 15N and xylem ureide methods for assessing N2 fixation of three shrub legumes periodically pruned for forage

The apparently diminished capacity for N₂ fixation by the shrub legume Calliandra calothyrsus (Calliandra) relative to other woody perennial legumes was investigated in a field experiment in northern Queensland, Australia. In this trial, (i) the proportion of plant nitrogen (N) derived from symbiotic N₂ fixation (%Pfix) and the amounts of N₂ fixed were compared in Calliandra, Gliricidia sepium (Gliricidia) and Codariocalyx gyroides (Codariocalyx), (ii) variations in N₂ fixation due to season or tree age were determined, (iii) estimates of Pfix derived with the ¹⁵N natural abundance technique were compared with values obtained from ¹⁵N enrichment or xylem sap ureide procedures to determine whether the previous conclusions about Calliandra's ability to fix N had resulted from specific problems with the natural abundance methodology used in the earlier studies.Inoculated seedlings of each of the three shrub legume species were planted in dense stands (1.5 m rows, 0.5 m between trees) in two randomised blocks. The northern block was used solely for natural abundance measurements, while ¹⁵N-enriched KNO₃ (10 atom % ¹⁵N excess) was applied four times over a 52 week period to plots in the southern block. The non-nodulating tree legume Senna spectabilis (formally Cassia spectabilis) was used as a non-N₂-fixing reference for the ¹⁵N-based procedures, with Guinea grass (Panicum maximum) included as an additional non-fixing check. Growth by the trees above 75 cm was first cut and removed after 22 weeks and regrowth was subsequently pruned periodically for another 95 weeks. Sampling for dry matter production, N yield and estimates of Pfix were restricted to the central four of the 32 plants which constituted each replicate plot. Information generated during the 117 week study indicated that estimates of Pfix by ¹⁵N natural abundance were closely similar to values derived with ¹⁵N-enrichment or sap ureides. The data indicated that Calliandra had a reduced reliance upon N₂ fixation relative to Gliricidia and Codariocalyx for the first 65 weeks after establishment. This appeared to be due to more prolifc root growth by Calliandra than either of the other N₂-fixing species and an ability to extract a greater proportion of its N requirements from soil mineral N. However, after week 65 and for the remainder of the experiment, estimates of Pfix for Calliandra were similar to the other shrub legumes. Over 117 weeks, prunings from Calliandra and Gliricidia had removed 52–58 t dry matter ha^-1, and between 1471 and 1678 kg N ha^-1, of which 1026–1063 kg N ha^-1 was estimated to have been derived from N₂ fixation. At the time of final harvest, 65–73% of the fixed N was present in shoot regrowth of the N₂ fixing shrubs, 9–18% in the roots, 15% in the trunk, and 2–6% in fallen leaves.     

The Effect of Mixing Prunings of Two Tropical Shrub Legumes (Calliandra Houstoniana and Indigofera zollingeriana) with Contrasting Quality on N Release in the Soil and Apparent N Degradation in the Rumen

Lack of synchronization between N released from prunings applied to the soil as green manures and crop uptake as well as optimization of protein digestibility for ruminants, remain major research objectives for the selection of multipurpose tree and shrub legumes (MPT) for mixed smallholder systems in the tropics. Prunings of the high tannin, low quality MPT Calliandra houstoniana CIAT 20400 (Calliandra) and the tannin free, high quality MPT Indigofera zollingeriana (Indigofera) were mixed in the proportions 100:0, 75:25, 50:50, 25:75, and 0:100 (w/w) in order to measure the aerobic rate and extent of N release in a leaching tube experiment, and the anaerobic extent of N degradation in an in vitro gas production experiment. Parameters measured in Calliandra:Indigofera mixtures were compared to theoretical values derived from single species plant material (i.e. 100:0 and 0:100). Aerobic N release and apparent anaerobic N degradation increased with increasing proportion of the high quality legume (Indigofera) in the mixture. While N release in the soil was lower than theoretical values in the mixture 50% Calliandra/50% Indigofera, this was not the case with apparent anaerobic N degradation with the same mixture. Aerobic N immobilization was more pronounced for the mixture 75% Calliandra/25% Indigofera than for 100% Calliandra and negative interaction was observed with apparent anaerobic N degradation in the mixture 75% Calliandra/25% Indigofera. Plant quality parameters that best correlated with aerobic N release and apparent anaerobic N degradation in the rumen were lignin + bound condensed tannins (r=−0.95 and −0.95 respectively, P<0.001). In addition, a positive correlation (r=0.89, P<0.001) was found between aerobic N release in the leaching tube experiment and apparent N degradation in the in vitro anaerobic gas production experiment. Results show that mixing prunings of MPT materials with contrasting quality is an effective way to modify aerobic N release pattern as well as apparent anaerobic N degradation and could possibly be applied to minimize N losses in the rumen and in the soil. In addition, apparent anaerobic N degradation was identified as good predictor of aerobic N release in the soil, which has resource saving implications when screening MTP to be used as green manures.     

Effects of long-term supplementation of chestnut and valonea extracts on methane release,digestibility and nitrogen excretion in sheep

The long-term effects of adding chestnut (CHE; Castanea sativa) and valonea (VAL; Quercus valonea) tannin-rich extracts to sheep feed were investigated. In Experiment 1, sheep (65 kg BW) were fed 842 g/day of a ryegrass-based hay. The control-treated animals (CON) received 464 g/day of concentrate, and tannin-treated animals received the same amount of concentrate additionally containing 20 g of the respective tannin-rich extract. Hay and concentrates were offered together in one meal. After the onset of treatment, methane release was measured in respiration chambers for 23.5-h intervals (nine times) in a 190-days period. Faeces and urine were collected three times (including once before the onset of the tannin treatment) to assess digestibility and urinary excretion of purine derivatives. Based on the results obtained from Experiment 1, a second experiment (Experiment 2) was initiated, in which the daily tannin dosage was almost doubled (from 0.9 (Experiment 1) to 1.7 g/kg BW^0.75). With the exception of the dosage and duration of the treatment (85 days), Experiment 2 followed the same design as Experiment 1, with the same measurements. In an attempt to compare in vitro and in vivo effects of tannin supplementation, the same substrates and tannin treatments were examined in the Hohenheim gas test. In vitro methane production was not significantly different between treatments. None of the tannin-rich extract doses induced a reduction in methane in the sheep experiments. On the 1st day of tannin feeding in both experiments, tannin inclusion tended to decrease methane release, but this trend disappeared by day 14 in both experiments. In balance period 3 of Experiment 1, lower dry matter and organic matter digestibility was noted for tannin treatments. The digestibility of CP, but not NDF or ADF, was reduced in both experiments. A significant shift in N excretion from urine to faeces was observed for both tannin-rich extracts in both experiments, particularly in Experiment 2. In balance period 2 of Experiment 2, an increased intake of metabolisable energy for VAL was observed. The urinary excretion of purine derivatives was not significantly different between treatments, indicating that microbial protein synthesis was equal for all treatments. Thus, we concluded that both tannin-rich extracts temporary affect processes in the rumen but did not alter methane release over a longer period.     

Assessing the symbiotic dependency of grain and tree legumes on N2 fixation for their N nutrition in five agro-ecological zones of Botswana

To assess the symbiotic dependency of grain and shrub/tree legumes within five agro-ecological zones of Botswana, fully expanded leaves of the test species were sampled from about 26 study sites within Ngwaketse, Gaborone, Central, Ghanzi and Kalahari agro-ecological zones. Isotopic analysis revealed significant differences in δ¹⁵N values of the grain legumes [cowpea (Vigna unguiculata L. Walp), Bambara groundnut (Vigna subterranea L. Verde.), and groundnut (Arachis hypogaea L.)] from the 26 farming areas in both 2005 and 2006. Estimates of %Ndfa of leaves also showed significant differences between farming areas, with cowpea deriving more than 50% of its N nutrition from symbiotic fixation. In terms of distribution, many more symbiotic shrub/tree species were found in the wetter Ngwaketse agro-zone compared to the fewer numbers in the drier Kalahari region. Acacias were the more dominant species at all sites. Leaf δ¹⁵N values of shrub/tree species also varied strongly across Botswana, with 11 out of 18 of these legumes deriving about 50%, or more, of their N from symbiotic N₂ fixation.Acacia caffra, in particular, obtained as much as 93.6% of its N nutrition from symbiotic fixation in the wetter Ngwaketse agro-zone. This study has shown that grain legumes sampled from farmer’s fields in Botswana obtained considerable amounts of their N from symbiotic fixation. We have also shown that shrub and tree legumes probably play an important role in the N economy of the savanna ecosystems in Botswana. However, the decline in the number of functional N₂-fixing shrub/tree legumes along an aridity gradient suggests that soil moisture is a major constraint to N₂ fixation in the tree legumes of Botswana.     

Bi-directional transfer of nitrogen between alfalfa and bromegrass: Short and long term evidence

Transfer of N from legumes to associated non-legumes has been demonstrated under a wide range of conditions. Because legumes are able to derive their N requirements from N₂ fixation, legumes can serve, through the transfer of N, as a source of N for accompanying non-legumes. Studies, therefore, are often limited to the transfer of N from the legume to the non-legume. However, legumes preferentially rely on available soil N as their source of N. To determine whether N can be transferred from a non-legume to a legume, two greenhouse experiments were conducted. In the short-term N-transfer experiment, a portion of the foliage of meadow bromegrass (Bromus riparius Rhem.) or alfalfa (Medicago sativa L.) was immersed in a highly labelled ¹⁵N-solution and following a 64 h incubation, the roots and leaves of the associated alfalfa and bromegrass were analyzed for ¹⁵N. In the long-term N transfer experiment, alfalfa and bromegrass were grown in an ¹⁵N-labelled nutrient solution and transplanted in pots with unlabelled bromegrass and alfalfa plants. Plants were harvested at 50 and 79 d after transplanting and analyzed for ¹⁵N content. Whether alfalfa or bromegrass were the donor plants in the short-term experiment, roots and leaves of all neighbouring alfalfa and bromegrass plants were enriched with ¹⁵N. Similarly, when alfalfa or bromegrass was labelled in the long-term experiment, the roots and shoots of neighbouring alfalfa and bromegrass plants became enriched with ¹⁵N. These two studies conclusively show that within a short period of time, N is transferred from both the N₂-fixing legume to the associated non-legume and also from the non-legume to the N₂-fixing legume. The occurrence of a bi-directional N transfer between N₂-fixing and non-N₂-fixing plants should be taken into consideration when the intensity of N cycling and the directional flow of N in pastures and natural ecosystems are investigated.     

Effects of different tannin-rich extracts and rapeseed tannin monomers on methane formation and microbial protein synthesis in vitro

Tannins, polyphenolic compounds found in plants, are known to complex with proteins of feed and rumen bacteria. This group of substances has the potential to reduce methane production either with or without negative effects on digestibility and microbial yield. In the first step of this study, 10 tannin-rich extracts from chestnut, mimosa, myrabolan, quebracho, sumach, tara, valonea, oak, cocoa and grape seed, and four rapeseed tannin monomers (pelargonidin, catechin, cyanidin and sinapinic acid) were used in a series of in vitro trials using the Hohenheim gas test, with grass silage as substrate. The objective was to screen the potential of various tannin-rich extracts to reduce methane production without a significant effect on total gas production (GP). Supplementation with pelargonidin and cyanidin did not reduce methane production; however, catechin and sinapinic acid reduced methane production without altering GP. All tannin-rich extracts, except for tara extract, significantly reduced methane production by 8% to 28% without altering GP. On the basis of these results, five tannin-rich extracts were selected and further investigated in a second step using a Rusitec system. Each tannin-rich extract (1.5 g) was supplemented to grass silage (15 g). In this experiment, nutrient degradation, microbial protein synthesis and volatile fatty acid production were used as additional response criteria. Chestnut extract caused the greatest reduction in methane production followed by valonea, grape seed and sumach, whereas myrabolan extract did not reduce methane production. Whereas chestnut extract reduced acetate production by 19%, supplementation with grape seed or myrabolan extract increased acetate production. However, degradation of fibre fractions was reduced in all tannin treatments. Degradation of dry matter and organic matter was also reduced by tannin supplementation, and no differences were found between the tannin-rich extracts. CP degradation and ammonia-N accumulation in the Rusitec were reduced by tannin treatment. The amount and efficiency of microbial protein synthesis were not significantly affected by tannin supplementation. The results of this study indicated that some tannin-rich extracts are able to reduce methane production without altering microbial protein synthesis. We hypothesized that chestnut and valonea extract have the greatest potential to reduce methane production without negative side effects.     

Microbial colonisation of tannin-rich tropical plants: Interplay between degradability,methane production and tannin disappearance in the rumen

Condensed tannins in plants are found free and attached to protein and fibre but it is not known whether these fractions influence rumen degradation and microbial colonisation. This study explored the rumen degradation of tropical tannin-rich plants and the relationship between the disappearance of free and bound condensed tannin fractions and microbial communities colonising plant particles using in situ and in vitro experiments. Leaves from Calliandra calothyrsus, Gliricidia sepium, and Leucaena leucocephala, pods from Acacia nilotica and the leaves of two agricultural by-products: Manihot esculenta and Musa spp. were incubated in situ in the rumen of three dairy cows to determine their degradability for up to 96 h. Tannin disappearance was determined at 24 h of incubation, and adherent microbial communities were examined at 3 and 12 h of incubation using a metataxonomic approach. An in vitro approach was also used to assess the effects of these plants on rumen fermentation parameters. All plants contained more than 100 g/kg of condensed tannins with a large proportion (32–61%) bound to proteins. Calliandra calothyrsus had the highest concentration of condensed tannins at 361 g/kg, whereas Acacia nilotica was particularly rich in hydrolysable tannins (350 g/kg). Free condensed tannins from all plants completely disappeared after 24-h incubation in the rumen. Disappearance of protein-bound condensed tannins was variable with values ranging from 93% for Gliricidia sepium to 21% for Acacia nilotica. In contrast, fibre-bound condensed tannin disappearance averaged ～ 82% and did not vary between plants. Disappearance of bound fractions of condensed tannins was not associated with the degradability of plant fractions. The presence of tannins interfered with the microbial colonisation of plants. Each plant had distinct bacterial and archaeal communities after 3 and 12 h of incubation in the rumen and distinct protozoal communities at 3 h. Adherent communities in tannin-rich plants had a lower relative abundance of fibrolytic microbes, notably Fibrobacter spp. whereas, archaea diversity was reduced in high-tannin-containing Calliandra calothyrsus and Acacia nilotica at 12 h of incubation. Concurrently, in vitro methane production was lower for Calliandra calothyrsus, Acacia nilotica and Leucaena leucocephala although for the latter total volatile fatty acids production was not affected and was similar to control. Here, we show that the total amount of hydrolysable and condensed tannins contained in a plant govern the interaction with rumen microbes affecting degradability and fermentation. The effect of protein- and fibre-bound condensed tannins on degradability is less important.     

Does nitrogen transfer between plants confound 15N-based quantifications of N2 fixation?

Background and aims

Transfer of fixed N from legumes to non-legume reference plants may alter the ¹⁵N signature of the reference plant as compared to the soil N available to the legume. This study investigates how N transfer influences the result of ¹⁵N-based N₂ fixation measurements.

Methods

We labelled either legumes or non-legumes with ¹⁵N and performed detailed analyses of ¹⁵N enrichment in mixed plant communities in the field. The results were used in a conceptual model comparing how different N transfer scenarios influenced the ¹⁵N signatures of legumes and reference plants, and how the resulting N₂ fixation estimate was influenced by using reference plants in pure stand or in mixture with the legume.

Results

Based on isotopic signatures, N transfer was detected in all directions: from legume to legume, from legume to non-legume, from non-legume to legume, from non-legume to non-legume. In the scenario of multidirectional N transfer, N₂ fixation was overestimated by using a reference plant in pure stand.

Conclusions

Fixed N transferred to neighbouring reference plants modifies the ¹⁵N signature of the soil N available both to the reference plant and the N₂-fixing legume. This provides strong support for using reference plants growing in mixture with the legumes for reliable quantifications of N₂ fixation.     

Legume agglutinins that bind to Rhizobium meliloti.

A protein found in seeds and roots of alfalfa (Medicago sativa) was implicated in the specificity of the infection process, based on its binding to the symbiont Rhizobium meliloti. We found an agglutinin with similar properties in seeds and roots of sweet clover (Melilotis alba). The sweet clover differed from alfalfa in nodulation by a mutant strain of R. meliloti, but the agglutinins were indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Rhizobium agglutination, and cross-reactivity to antibodies. Similar agglutinins binding R. meliloti were found in seeds of legumes from different cross-inoculation groups, including soybean (Glycine max), cowpea (Vigna unguiculata), pea (Pisum sativum L), and mung bean (Vigna mungo). The agglutinins from these legumes were recognized by antibodies raised against the agglutinins of alfalfa and sweet clover. Seeds of corn (Zea mays) and tomato (Lycopersicon esculentum) contained a protein similar to the legume agglutinin, but it did not react with the antibodies. We conclude that the alfalfa agglutinin is representative of a common legume protein and that there is no evidence for its role in specificity or nodule initiation.     

Nitrogen fixation in legumes and actinorhizal plants in natural ecosystems: values obtained using 15N natural abundance

Background: Nitrogen fixation has been quantified for a range of crop legumes and actinorhizal plants under different agricultural/agroforestry conditions, but much less is known of legume and actinorhizal plant N₂ fixation in natural ecosystems.

Aims: To assess the proportion of total plant N derived from the atmosphere via the process of N₂ fixation (%Ndfa) by actinorhizal and legume plants in natural ecosystems and their N input into these ecosystems as indicated by their ¹⁵N natural abundance.

Methods: A comprehensive collation of published values of %Ndfa for legumes and actinorhizal plants in natural ecosystems and their N input into these ecosystems as estimated by their ¹⁵N natural abundance was carried out by searching the ISI Web of Science database using relevant key words.

Results: The %Ndfa was consistently large for actinorhizal plants but very variable for legumes in natural ecosystems, and the average value for %Ndfa was substantially greater for actinorhizal plants. High soil N, in particular, but also low soil P and water content were correlated with low legume N₂ fixation. N input into ecosystems from N₂ fixation was very variable for actinorhizal and legume plants and greatly dependent on their biomass within the system.

Conclusions: Measurement of ¹⁵N natural abundance has given greater understanding of where legume and actinorhizal plant N₂ fixation is important in natural ecosystems. Across studies, the average value for %Ndfa was substantially greater for actinorhizal plants than for legumes, and the relative abilities of the two groups of plants to utilise mineral N requires further study.     

In vitro ruminal fermentation characteristics and utilisable CP supply of sainfoin and birdsfoot trefoil silages and their mixtures with other legumes

The extensive protein degradation occurring during ensiling decreases the nutritive value of silages, but this might be counteracted by tannins. Therefore, silages from two legume species containing condensed tannins (CT) – sainfoin (SF) and birdsfoot trefoil (two cultivars: birdsfoot trefoil, cv. Bull (BTB) and birdsfoot trefoil, cv. Polom) – were compared for their in vitro ruminal fermentation characteristics. The effect of combining them with two CT-free legume silages (lucerne (LU) and red clover (RC)) was also determined. The supply of duodenally utilisable CP (uCP) in the forages was emphasised. The legumes were each harvested from three field sites. After 24 h of wilting on the field, the legumes were ensiled in laboratory silos for 86 days. Proximate constituents, silage fermentation characteristics, CT content and CP fractions were determined. Subsequently, silage samples and 1 : 1 mixtures of the CT-containing and CT-free silages were incubated for 24 h in batch cultures using ruminal fluid and buffer (1 : 2, v/v). Each treatment was replicated six times in six runs. The effects on pH, ammonia and volatile fatty acid concentrations, protozoal counts, and total gas and methane production were determined. uCP content was calculated by considering the CP in the silage and the ammonia in the incubation fluid from treatments and blanks. Statistical evaluation compared data from single plants alone and together with that from the mixtures. Among treatments, SF silage contained the least CP and the most CT. The non-protein nitrogen content was lower, favouring neutral detergent soluble and insoluble protein fractions, in the SF and RC silages. Absolute uCP content was lowest in SF and SF mixtures, although the ratio to total CP was the highest. In comparison with LU, the ammonia concentration of the incubation fluid was lower for SF, RC and BTB and for the mixture of SF with LU. The total gas and methane production was similar among the treatments, and the total volatile fatty acid production was decreased with the CT-containing legumes. Protozoal count was increased with the mixtures containing LU and either SF or BTB compared with single LU. In conclusion, compared with the other legumes, SF and RC have similar advantages as they show limited proteolysis during ensiling. In addition, SF supplies more uCP relative to total CP. The CT-containing legumes also differed in their effect on ruminal fermentation and ammonia formation, probably because of their different CT contents. Thus, SF and its mixtures appear promising for improving the protein utilisation of ruminants.     

Natural 15N abundance in shrub and tree legumes,Casuarina, and non N2 fixing plants in Thailand

The leaves and nodules from the shrub and tree legumes, particularly, Aeschynomene spp., Sesbania spp., Mimosa spp. and Leucaena spp., and Casuarina spp. and the leaves from neighbouring non-fixing plants were analyzed for their natural abundances of ¹⁵N ( ¹⁵N).The ¹⁵N in the leaves of non-fixing plants was +5.9% on average, whereas those from shrub legumes and Casuarina spp. were lower and close to the values of atmospheric N₂, suggesting the large contribution of N₂ fixation as the N source in these plants. The ¹⁵N values of the leaves from tree legumes except for Leucaena spp. were between the shrub legumes and non-fixing plants, which suggests that the fractional contribution of fixed N₂ in tree legumes may be smaller than that in the shrub legumes. Casuarina spp. was highly dependent on N₂ fixation. The ¹⁵N values of the nodules from most of the shrub legumes investigated were higher than those of the leaves.     

Diversity in symbiotic specificity of cowpea rhizobia indigenous to Zimbabwean soils

Tropical cowpea rhizobia are often presumed to be generally promiscuous but poor N fixers. This study was conducted to evaluate symbiotic interactions of 59 indigenous rhizobia isolates (49 of them from cowpea (Vigna unguiculata)), with up to 13 other (mostly tropical) legume species. Host ranges averaged 2.4 and 2.3 legume species each for fast- and slow-growing isolates respectively compared to 4.3 for slow-growing reference cowpea strains. An average of 22% and 19% of fast- and slow-growing cowpea isolates respectively were effective on each of 12 legume species tested. We conclude that the indigenous cowpea rhizobia studied have relatively narrow host ranges. The ready nodulation of different legumes in tropical soils appears due to the diversity of indigenous symbiotic genotypes, each consisting of subgroups compatible with a limited number of legume species.     

Isolation of insecticidal lectin-enriched extracts from African yam bean (Sphenostylis stenocarpa) and other legume species

Insecticidal lectins were isolated from 20 resistant Vigna and non-Vigna legumes and tested againstn 3 pests of cowpea namely: Maruca vitrata, Callosobruchus maculatus and Clavigralla tomentosicollis. Crude lectins were separated from seeds using sodium chloride extraction, ammonium sulfate fractionation, and dialysis. SDS-PAGE indicated the molecular size of ca. 30 kDa for the most intense (and presumably active) band. Haemagglutination assays using trypsin-treated rabbit erythrocytes suggested that lectins were among the extracted proteins. Extracts from Lablab purpureus and Sphenostylis stenocarpa both non-Vigna spp., caused greater agglutination than those from the wild Vigna species. Bioassays on all three insect species using the lectin extracts incorporated in either artificial cowpea seeds (5% w/w) or in modified Vanderzant legume pod borer diet (1% w/v) indicated that the non-Vigna extracts were highly toxic to the insects. Mortality after 10 days was >80% in the most toxic extracts. The extract from one of the accessions of Sphenostylis stenocarpa, an edible legume, was singled out for lectin purification and future gene cloning with the view of using it for engineering resistance to cowpea pests.     

Root-hair infection and nodulation of four grain legumes as affected by the form and the application time of nitrogen fertilizer

The effects of application of combined nitrogen fertilizer (ammonium nitrate or urea) on root-hair infection and nodulation of four grain legumes were studied. Young roots of each legume were inoculated with their compatible rhizobia. The application of the two forms of combined N either at the early stages of plant growth and/or at the time of nodule formation depressed root-hair curling, infection and nodulation. Infection of hairs on the primary roots was more sensitive to the N fertilizer than hair infection of secondary roots in bothVicia faba andPisum sativum. The nodule number and total fresh mass of the four legumes were drastically affected by fertilizer application. The combined N added both at early and at later stages significantly reduced the nodulation ofV. faba, Phaseolus vulgaris andVigna sinensis. The inhibitory effect of urea on nodulation ofP. sativum was only observed when the fertilizer was applied at the late stages of plant growth. It is concluded that, although the nodulation of the four legumes was suppressed by combined N, the initial events ofRhizobium-legume symbiosis (infection of roots and nodule initiation) are more sensitive to combined N than the stages after nodule formation.     

Herbage intake,methane emissions and animal performance of steers grazing dwarf elephant grass v. dwarf elephant grass and peanut pastures

Management strategies for increasing ruminant legume consumption and mitigating methane emissions from tropical livestock production systems require further study. The aim of this work was to evaluate the herbage intake, animal performance and enteric methane emissions of cattle grazing dwarf elephant grass (DEG) (Pennisetum purpureum cv. BRS Kurumi) alone or DEG with peanut (Arachis pintoi cv. Amarillo). The experimental treatments were the following: DEG pastures receiving nitrogen fertilization (150 kg N/ha as ammonium nitrate) and DEG intercropped with peanut plus an adjacent area of peanut that was accessible to grazing animals for 5 h/day (from 0700 to 1200 h). The animals grazing legume pastures showed greater average daily gain and herbage intake, and shorter morning and total grazing times. Daily methane emissions were greater from the animals grazing legume pastures, whereas methane emissions per unit of herbage intake did not differ between treatments. Allowing animals access to an exclusive area of legumes in a tropical grass-pasture-based system can improve animal performance without increasing methane production per kg of dry matter intake.     

Legumes regulate grassland soil N cycling and its response to variation in species diversity and N supply but not CO2

Legumes are an important component of plant diversity that modulate nitrogen (N) cycling in many terrestrial ecosystems. Limited knowledge of legume effects on soil N cycling and its response to global change factors and plant diversity hinders a general understanding of whether and how legumes broadly regulate the response of soil N availability to those factors. In a 17‐year study of perennial grassland species grown under ambient and elevated (+180 ppm) CO₂ and ambient and enriched (+4 g N m^?2 year^?1) N environments, we compared pure legume plots with plots dominated by or including other herbaceous functional groups (and containing one or four species) to assess the effect of legumes on N cycling (net N mineralization rate and inorganic N pools). We also examined the effects of numbers of legume species (from zero to four) in four‐species mixed plots on soil N cycling. We hypothesized that legumes would increase N mineralization rates most in those treatments with the greatest diversity and the greatest relative limitation by and competition for N. Results partially supported these hypotheses. Plots with greater dominance by legumes had greater soil nitrate concentrations and mineralization rates. Higher species richness significantly increased the impact of legumes on soil N metrics, with 349% and 505% higher mineralization rates and nitrate concentrations in four‐species plots containing legumes compared to legume‐free four‐species plots, in contrast to 185% and 129% greater values, respectively, in pure legume than nonlegume monoculture plots. N‐fertilized plots had greater legume effects on soil nitrate, but lower legume effects on net N mineralization. In contrast, neither elevated CO₂ nor its interaction with legumes affected net N mineralization. These results indicate that legumes markedly influence the response of soil N cycling to some, but not all, global change drivers.     

Leguminous plants significantly increase soil nitrogen cycling across global climates and ecosystem types

Leguminous plants are an important component of terrestrial ecosystems and significantly increase soil nitrogen (N) cycling and availability, which affects productivity in most ecosystems. Clarifying whether the effects of legumes on N cycling vary with contrasting ecosystem types and climatic regions is crucial for understanding and predicting ecosystem processes, but these effects are currently unknown. By conducting a global meta-analysis, we revealed that legumes increased the soil net N mineralization rate (R_min) by 67%, which was greater than the recently reported increase associated with N deposition (25%). This effect was similar for tropical (53%) and temperate regions (81%) but was significantly greater in grasslands (151%) and forests (74%) than in croplands (−3%) and was greater in in situ incubation (101%) or short-term experiments (112%) than in laboratory incubation (55%) or long-term experiments (37%). Legumes significantly influenced the dependence of R_min on N fertilization and experimental factors. The R_min was significantly increased by N fertilization in the nonlegume soils, but not in the legume soils. In addition, the effects of mean annual temperature, soil nutrients and experimental duration on R_min were smaller in the legume soils than in the nonlegume soils. Collectively, our results highlighted the significant positive effects of legumes on soil N cycling, and indicated that the effects of legumes should be elucidated when addressing the response of soils to plants.     

Monovalent cation transporters; establishing a link between bioinformatics and physiology

Toxic aluminum (Al) ion is a major constraint to plant growth in acid soils. Aluminum tolerance in wheat (Triticum aestivum L.) is strongly related to the Al-triggered efflux of malate from root apices. A role of the secreted malate has been postulated to be in chelating Al and thus excluding it from root apices (malate hypothesis), but the actual process has yet to be fully elucidated. We measured Al content and root growth during and after Al exposure using seedlings of near-isogenic lines [ET8 (Al tolerant) and ES8 (Al sensitive)] differing in the capacity to induce Al-triggered malate efflux. Aluminum doses that caused 50% root growth inhibition during 24-h exposure to Al in calcium (Ca) solution (0.5 mM CaCl₂, pH 4.5) were 50 μM in ET8 and 5 μM in ES8. Under such conditions, the amount of Al accumulated in root apices was approximately 2-fold higher in ET8 than ES8. Al-treated seedlings were then transferred to the Al-free Ca solution for 24 h. Compared to control roots (no Al pretreatment), root regrowth of Al-treated roots was about 100% in ET8 and about 25% in ES8. The impaired regrowth in ES8 was observed even after 24-h exposure to 2.5 μM Al which had caused only 20% root growth inhibition. The addition of malate (100 μM) during exposure to 50 μM Al in ES8 enhanced root growth 1.6 times and regrowth in Al-free solution 7 times, resulting in similar root growth and regrowth as in ET8. Short-term Al treatments of ES8 for up to 5 h indicated that the Al-caused inhibition of root regrowth started after 1-h exposure to Al. The stimulating effect of malate on root regrowth was observed when malate was present during Al exposure, but not when roots previously exposed to Al were rinsed with malate, although Al accumulation in root apices was similar under these malate treatments. We conclude that the malate secreted from root apices under Al exposure is essential for the apices to commence regrowth in Al-free medium, the trait that is not related to the exclusion of Al from the apices.