Water-facilitated dispersal of inoculant Bradyrhizobium japonicum in soils

Summary Studies were performed to assess the influence of percolating water and an advancing wetting front on the transport of Bradyrhizobium japonicum in sand and silt loam soils, and to assess the influence of clay content on water-facilitated dispersal of these bacteria in a sand amended with various amounts of kaolin. The data obtained showed that movement of B. japonicum in soil was dependent upon water movement and that both percolating water and an advancing wetting front readily transported bacteria in coarse-textured soils. Percolation with the equivalent of 10 cm of rainfall dispersed B. japonicum throughout 40-cm columns containing sand and silt loam soils. Percolation with 5 cm of water was sufficient to disperse B. japonicum throughout 20-cm columns of these soils but did not transport these bacteria below the surface 4 cm of a sand amended with 12% kaolin. Our finding that cells of B. japonicum are readily transported by an advancing wetting front indicates that non-saturated flow of soil water contributes to dispersal of inoculum in soils.     

Dinitrogen (C2H2) fixation in relation to nitrogen fertilization of grey alder [Alnus incana (L.) Moench.] plantations in a peat bog

Summary Nitrogenase activity was measured in young grey alder plantations in a peat bog in central Sweden. The stands were treated in three ways: (1) daily irrigation during the growing season with a complete nutrient solution, including N; (2) application of bark ash or wood ash before planting; and (3) fertilization every second year with solid PK fertilizers. Acetylene reduction assays were performed on (1) detached nodules and attached nodules, either on (2) whole enclosed plants or (3) enclosed nodules. The acetylene reduction rate for the enclosed plants showed a maximum in July when mean values of nearly 80 mol C₂H₄ (g nodule dry matter)^-1 h^-1 were reached. No diurnal patterns were observed. The irrigated stands, with an N supply, showed overall nitrogenase activities that corresponded well with those of the other treatments. Only in the case of temporarily increased soil nutrient concentrations in the irrigated stands did the nitrogenase activity fall considerably. In 6-to 7-year-old intensively managed irrigated stands N₂-fixation was estimated as 85–115 kg N ha^-1 year^-1 which was about 55% of the total N uptake of the trees.     

Diversity of diazotroph populations in the rhizosphere of maize (Zea mays L.) growing on different French soils

Summary We studied the dominant diazotrophs associated with maize roots and rhizosphere soil originating from three different locations in France. An aseptically grown maize plantlet, the spermosphere model, was used to isolate N₂-fixing (acetylene-reducing) bacteria. Bacillus circulans was the dominant N₂-fixing bacterium in the rhizosphere of maize-growing soils from Ramonville and Trogny, but was not found in maize-growing sandy soil from Pissos. In the latter soil, Enterobacter cloacae, Klebsiella terrigena, and Pseudomonas sp. were the most abundant diazotrophs. Azospirillum sp., which has been frequently reported as an important diazotroph accociated with the maize rhizosphere, was not isolated from any of these soils. The strains were compared for their acetylene-reducing activity in the spermosphere model. The Bacillus circulans strains, which were more frequently isolated, also exhibited significantly greater acetylene-reducing activity (3100 nmol ethylene day^-1 plant^-1) than the Enterobacteriaceae strains (180 nmol ethylene day^-1 plant^-1). This work indicates for the first time that Bacillus circulans is an important maizerhizosphere-associated bacterium and a potential plant growth-promoting rhizobacterium.     

Risk of erosion in peat soils – an investigation using Bayesian belief networks

Peat is an important carbon sink in the context of climate change. However, well‐documented examples suggest that risk of peat erosion is widespread and significant. Our understanding of peat vulnerability to erosion is commonly constrained by the complexity of drivers, and their interactions, in this process. However, the key constraints are: limited, consistent and comprehensive quantitative data relating to this process and, more significantly, the explicit relationships between the occurrence of peat erosion and its causes and drivers. Bayesian belief networks (BBNs) provide a methodology for integrating qualitative and quantitative knowledge. BBNs can capture and structure available knowledge and rationalize complex interactions, where empirical data are limited or poorly compatible and processes are complex or uncertain. In this article we explore the BBN potential to advance our understanding and to identify gaps in current knowledge. BBN has been demonstrated to be a useful tool in structuring and utilizing currently available knowledge, often with limited evidence, of peat’s actual exposure to erosive forces. Despite considerable research into peat erosion processes and understanding the inherent vulnerability of peat, results presented indicate clear gaps in knowledge regarding the role of land management, spatially explicit data related to land management as well as limited evidence of the relevant relationships between many of the variables. The attention of further research will focus on these gaps. The BBN approach provides a framework in which different scenarios of biophysical, climatic and land management (social and economic) conditions can examine and assess the probability of erosion.     

Population size and N2-fixing activity of native peanut rhizobia in soils of Thailand

Summary The objective of this study was to assess the number and effectiveness of peanut rhizobia in soils of the major peanut-growing areas of Thailand. Three cropping areas, (1) continuously cropped with peanuts, (2) continuously cropped with non-legumes, and (3) non-cultivated fields, were chosen in each region. Peanut rhizobia were found in the soil at 38 to 55 sites sampled. Cultivated fields with a peanut cultivation history contained (as estimated by most probable numbers) an average of 1.6×10³ cells g^-1 of soil. The numbers of peanut rhizobia in most of the fallow fields and some of the noncultivated shrub or forest locations were much the same as at the sites where Arachis hypogaea was cultivated. In contrast, there were no or few (28–46 cells g^-1 soil) peanut rhizobia in the majority of fields continuously cultivated with sugarcane, cassava, corn, and pineapple. It appears that in these areas the indigenous peanut rhizobial populations are not adequate in number for a maximal nodulation of peanuts. A total of 343 Bradyrhizobium isolates were tested for effectiveness and were found to vary widely in their ability to fix N₂. In some areas the majority of rhizobia were quite effective while in others they were less effective than the inoculum strain THA 205 recommended in Thailand.     

Two- and three-domain bacterial laccase-like genes are present in drained peat soils

Laccases of fungal origin have been intensively studied due to their importance in various biotechnological applications. There is a constant demand for new laccases with improved properties such as stability at higher temperatures or at an alkaline pH. Growing molecular evidence suggests that laccases may also be widespread in bacteria. While only a handful of bacterial laccases have been purified and characterized, several novel traits have already been discovered (e.g. pH-stability and 2-domain organization of the enzyme as opposed to the usual 3-domain structure of fungal laccases). The aim of this study was to examine the diversity of bacterial laccase-like genes in two types of high-organic peat soil using a cloning and sequencing approach. Gene libraries prepared of small fragments (150 base pairs) revealed an amazing diversity of bacterial laccases. The fragments clustered in 11 major lineages, and one third of the 241 sequences resembled laccase-like genes of Acidobacteria. Additionally, a new primer was used to retrieve several larger fragments of the putative bacterial laccase genes that spanned all four copper-binding sites. Both “conventional” 3-domain laccases and the recently described 2-domain small laccases have been obtained using this approach, demonstrating the potential of the primer. The present study thus contributes to the understanding of the diversity of bacterial laccases and provides a new tool for finding laccase-like sequences in bacterial strains and soil samples.     

Biological control of Rhizoctonia sp. root rot of Casuarina equisetifolia seedlings by Frankia spp. strains

Seventy Frankia spp. strains (nodulating N₂-fixing actinomycetes) were isolated from root nodules of Casuarina equisetifolia from different localities of Tamil Nadu state, India. From these, four strains (UMCe12, UMCe23, UMCe35, and UMce55) were selected. Their potential use as biological control agents for Rhizoctonia solani root rot disease of C. equisetifolia seedlings and their relative efficiency in nodule production were investigated. Between the two inoculum broadcast systems tested, seed-coating with Frankia spp. cell suspension was superior to the soil application of cells as sand-vermiculite-basal ammonium propionate inoculum. UMCe12 was the promising strain, offering the highest level of disease protection (81.1%) and nodule production (88.1%) in the R. solani-infested soil, followed by UMCe23 (60.3 and 65.5% of disease protection and nodule production, respectively), UMCe55 (53.5 and 58.2%), and UMCe35 (45.4 and 44.5%). Further, a significant positive correlation was observed between the dose of Frankia spp. and efficiency in both disease control and nodule production.     

Nodulation status of native woody legumes and phenotypic characteristics of associated rhizobia in soils of southern Ethiopia

The nodulation of provenances of Acacia seyal, Acacia tortilis and Faidherbia albida, and other indigenous multipurpose tree species were tested in 14 different soil samples collected from diverse agro-ecological zones in southern Ethiopia. Associated rhizobia were isolated from these and from excavated nodules of field standing mature trees, and phenotypically characterized. Indigenous rhizobia capable of eliciting nodules on at least one or more of the woody legume species tested were present in most of the soils. Tree species were markedly different in nodulation in the different site soils. Sesbania sesban and Acacia abyssinica showed higher nodulation ability across the different sites indicating widespread occurrence of compatible rhizobia in the soils. The nodulation patterns of the different provenances of Acacia spp. suggested the existence of intraspecific provenance variations in rhizobial affinity which can be exploited to improve N fixation through tree selection. Altogether, 241 isolates were recovered from the root nodules of trap host species and from excavated nodules. Isolates were differentiated by growth rate and colony morphology and there were very fast-, fast-, slow-, and very slow-growing rhizobia. The bulk of them (68.5%) were fast-growing acid-producing rhizobia while 25.3% were slow-growing alkali-producing types. Fast-growing alkali-producing (2.9%) and slow-growing acid-producing strains (3.3%) were isolated from trap host species and excavated nodules, respectively. All isolates fell into four colony types: watery translucent, white translucent, dull glistering and milky (curdled) type. The diversity of indigenous rhizobia in growth rate and colony morphology suggested that the collection probably includes several rhizobial genera.     

Dynamics of nodulation,nitrogen fixation,nitrogen use and biomass yield over time in pot-grown Leucaena leucocephala (Lam.) de Wit

The dynamics of nodulation, N₂-fixation and N use in Leucaena leucocephala cv. K28 over time was investigated in a screenhouse at 4, 8, 12 and 16 months after planting (MAP) using the ¹⁵N-labelling method. Leucaena had a consistently increasing pattern of nodulation, dry biomass and nitrogen yield. A sharp rise in nodulation was observed between 12 and 16 MAP, whereas for biomass, N accumulation and N₂-fixation, and N₂-fixation, an upward surge occurred between 4 and 12 months. Nodulation, N accumulation, N₂-fixation and biomass yield all peaked at 16 MAP. Along with the steady increase in N₂-fixation throughout the 16-month growth period, the % N derived from the atmosphere rose from 17.9% to 61.5%, 70.1% and 74%, equivalent to 191, 1623, 2395 and 3385 mg N₂ fixed plant^-1 at 4, 8, 12 and 16 MAP, respectively. Nitrogen assimilation from soil and fertilizer decreased inversely to the increase in symbiotic nitrogen fixation with time.     

Differences in the activity and bacterial community structure of drained grassland and forest peat soils

The microbial activity and bacterial community structure were investigated in two types of peat soil in a temperate marsh. The first, a drained grassland fen soil, has a neutral pH with partially degraded peat in the upper oxic soil horizons (16% soil organic carbon). The second, a bog soil, was sampled in a swampy forest and has a very high soil organic carbon content (45%), a low pH (4.5), and has occasional anoxic conditions in the upper soil horizons due to the high water table level. The microbial activity in the two soils was measured as the basal and substrate-induced respiration (SIR). Unexpectedly, the SIR (μl CO₂ g⁻¹ dry soil) was higher in the bog than in the fen soil, but lower when CO₂ production was expressed per volume of soil. This may be explained by the notable difference in the bulk densities of the two soils. The bacterial communities were assessed by terminal restriction fragment length polymorphism (T-RFLP) profiling of 16S rRNA genes and indicated differences between the two soils. The differences were determined by the soil characteristics rather than the season in which the soil was sampled. The 16S rRNA gene libraries, constructed from the two soils, revealed high proportions of sequences assigned to the Acidobacteria phylum. Each library contained a distinct set of phylogenetic subgroups of this important group of bacteria.     

Time-course of nitrogen fixation in two bambara groundnut (Vigna subterranea L. Verdc.) cultivars

TheA-value method, involving the application of a higher¹⁵N rate to a reference non-N₂-fixing plant, was used to assess the magnitude of N₂ fixation in two bambara groundnut cultivars at four growth stages [vegetative, 0–47 days after planting (DAP); early pod-filling, 47–99 DAP; mid-pod-filling, 99–120 DAP; physiological maturity, 120–148 DAP). The cultivars were Ex-Ada, a bunchy type, and CS-88-11, a slightly spreading type. They were grown on a loamy sand. Uninoculated Ex-Ada and CS-88-11 were used as reference plants to measure the N₂ fixed in the inoculated bambara groundnuts. In this greenhouse study, soil was the major source of N in bambara groundnuts during vegetative growth, and during this period it accounted for over 80% of the N accumulaed in the plants. However, N₂ fixation became the major source of plant N during reproductive growth. There were significant differences between the two cultivars in the ability to fix N₂, and at physiological maturity, almost 75% of the N in CS-88-11 was derived from the atmosphere compared to 55% in Ex-Ada. Also, the total N fixed in CS-88-11 at physiological maturity was almost double that in Ex-Ada. Our data indicate that the higher N₂ fixation in CS-88-11 was due to two factors, a higher intensity of N₂ fixation and a longer active period of N₂ fixation. The results also suggest that bambara groundnut genotypes could be selected for higher N₂ fixation in farining systems.     

Denitrification losses from puddled rice soils in the tropics

Summary Although denitrification has long been considered a major loss mechanism for N fertilizer applied to lowland rice (Oryza sativa L.) soils, direct field measurements of denitrification losses from puddled rice soils in the tropics have only been made recently. This paper summarizes the results of direct measurement and indirect estimation of denitrification losses from puddled rice fields and reviews the status of research methodology for measurement of denitrification in rice fields. The direct recovery of (N₂+N₂O)-¹⁵N from ¹⁵N-enriched urea has recently been measured at sites in the Philippines, Thailand, and Indonesia. In all 12 studies, recoveries of (N₂+N₂O)-¹⁵N ranged from less than 0.1 to 2.2% of the applied N. Total gaseous N losses, estimated by the ¹⁵N-balance technique, were much greater, ranging from 10 to 56% of the applied urea-N. Denitrification was limited by the nitrate supply rather than by available C, as indicated by the values for water-soluble soil organic C, floodwater (nitrate+nitrite)-N, and evolved (N₂+N₂O)-¹⁵N from added nitrate. In the absence of runoff and leaching losses, the amount of (N₂+N₂O)-¹⁵N evolved from ¹⁵N-labeled nitrate was consistently less than the unrecovered ¹⁵N in ¹⁵N balances with labeled nitrate, which presumably represented total denitrification losses. This finding indicates that the measured recoveries of (N₂+N₂O)-¹⁵N had underestimated the denitrification losses from urea. Even with a probable two-or threefold underestimation, direct measurements of (N₂+N₂O)-¹⁵N failed to confirm the appreciable denitrification losses often estimated by the indirect difference method. This method, which determines denitrification losses by the difference between total ¹⁵N loss and determined ammonia loss, is prone to high variability. Measurements of nitrate disappearance and ¹⁵N-balance studies suggest that nitrification-denitrification occurs under alternate soil drying and wetting conditions both during the rice cropping period and between rice crops. Research is needed to determine the magnitude of denitrification losses when soils are flooded and puddled for production of rice.     

Nitrogenase activity and nitrogen-fixing bacteria associated with the roots ofAtriplex spp. growing in saline sodic soils of Pakistan

Summary TwoAtriplex spp. growing in low-fertility saline sodic soils were assayed for root-associated nitrogenase activity. The excised washed and unwashed root of the two species.A. lentiformis andA. amnicola, showed high root-associated nitrogenase activity. Acetylene-reducing activity seemed to be directly influenced by moisture. The highest number of diazotrophs, enumerated using a most probable number technique was observed on the root surface. Most of the isolated diazotrophs were identified asEnterobacter agglomerans. Root-associated nitrogenase activity inAtriplex spp. may explain the high protein and biomass content of these plants growing in low-fertility saline sodic soils.     

Nitrogen fixation,biomass production,and nutrient uptake by annual Sesbania species in an alkaline soil

Summary In three field trials conducted during the summer season of 1986, 1987 and 1989 in an alkaline soil, 17 accessions of annual Sesbania spp. were evaluated for nodulation, N₂ fixation (acetylene reduction assay), dry weight of roots and shoots, woody biomass production, and nutrient uptake. At 50 days after sowing all the accessions were effectively nodulated (average 36.4 root nodules plant^-1) with a high nodule score (3.4). There was a lot of variation in nodule volume and mass and in acetylene reduction activity but not in N content (5.2%). N uptake in shoots, roots and nodules averaged 639, 31, and 13 mg plant^-1, respectively, and much of the fixed N remained in shoots. Accessions of S. cannabina complex performed better than others. S. rostrata had poor root nodulation but exhibited excellent stem nodulation (300 nodules plant^-1) even though not inoculated with Azorhizobium sp. Average concentrations of N, P, K, S, Ca, and Mg in the shoots were high, at 3.2, 0.28, 1.5, 0.28, 1.5, and 0.4% respectively, and Na was low (0.15%), reflecting the usefulness of Sesbania spp. as an integrated biofertilizer source. Green matter production was 26.0 Mg ha^-1 (5.9 Mg dry matter) and N uptake was 158 kg ha^-1, 54 days after sowing. Average woody biomass of six accessions at maturity, 200 days after sowing, was high (19.9 Mg ha^-1), showing its potential for shortterm firewood production. Total nutrient uptake for production of woody biomass (200 days of growth) was no more demanding than growing the plant to the green-manuring stage of 50–60 days' growth.     

Survival and symbiotic properties of Bradyrhizobium japonicum in the presence of thiram: isolation of fungicide resistant strains

The fungicide thiram, widely used as a chemical seed protectant, induces a strong inhibition of primary nodulation in the crown zone of soybean roots. The present work reports on the isolation of Bradyrhizobium japonicum strains resistant to thiram, some of which (T3B, A86 and A2) maintained their capacity for nodulation and were still efficient symbionts, but some (A1, C1 and C6) lost the ability to stimulate nodulation. Characterization tests such as growth at different pH, denitrifying ability, salt tolerance, production of siderophores and phosphate solubilization were performed on the resistant strains. Inoculants produced from these strains could be appropriate for use with thiram-treated seeds, without causing a loss of bacteria viability. Received: 16 September 1996     

Nitrogen-supplying capacity of leaves of Dactyladenia barteri (Hook ex olw) and Leucaena leucocephala (Lam.) de Wit in two soils of different acidity from southern nigeria

Summary The evolution of mineral and hydrosoluble organic N released from two soils differing in pH and treated with leaves of Leucaena leucocephala (0, 8.3, 16.7, and 33. g kg^-1 soil), Dactyladenia barteri (syn. Acioa barteri; 0 and 16.7 g), and their mixtures was studied in the laboratory using the aerobic incubation-leaching method. N mineralization in untreated soils and in soils supplemented with 8.3 g leucaena leaves was 41–53% higher in the soil from Onne (pH 4.7) than in the soil from Ibadan (pH 6.2), but the organic N content was similar with these treatments in the leachates of the soils from both locations. The application of 16.7 or 33.3 g of either or both type of leaves reduced the rate of mineral N production during the first 4 weeks, particularly in soils treated with dactyladenia leaves (C:N=36). After this lag period, N mineralization proceeded at a faster rate in the soil from Ibadan treated with 16.7 or 33.3 g of leucaena leaves (C:N=12), even in the presence of dactyladenia leaves. In Ibadan soil, after 12 weeks, mineral N apprently derived from leaves of both dactyladenia and leucaena averaged 6.3% of the N applied, and organic N from leaves averaged 9.5%. The addition of dactyladenia and leucaena leaves did not increases the mineral N content in the acid soil from Onne but leaching of soluble organic N with addition of 16.7 or 33.3 g of leaves contributed an N-mineralizable pool of 5.9% of the N applied.     

Nitrogen use in maize-grain legume cropping systems in semi-arid Kenya

Locally suitable cultivars of maize, beans, and cowpeas were grown in field experiments for four seasons in semi-arid Kenya. For three seasons, the dry matter production and grain yield of maize and beans were not increased by N fertilizer additions up to 120 kg N ha^-1. Fertilizer recoveries measured by ¹⁵N isotope dilution techniques were low, less than 20%. Inoculated and uninoculated beans failed to fix N₂. By contrast the cowpea derived 50% of its N from fixation, equivalent to 197 kg N ha^-1. The N content of the grain generally exceeded 40 kg N ha^-1, and the N content of the seeds from the grain legumes were greater than those from the cereals. Large inputs of N fertilizer or N by fixation are required if maize-grain legume cropping system in semiarid Kenya are to be sustained in the long term.     

Phosphorus requirements and nitrogen accumulation by N2-fixing and non-N2-fixing leguminous trees growing in low P soils

The variation in P uptake and use efficiency and N accumulation by Gliricidia sepium (N₂-fixing tree), Senna siamea and S. spectabilis (leguminous non-N₂-fixing trees) were examined in the field at Fashola (savanna zone), southwestern Nigeria, using four P rates, 0, 20, 40 and 80 kg P ha^-1. Growth of G. sepium and S. spectabilis responded to P application at 24 weeks after planting (WAP) and average yield increases of 58% and 145% were observed by the application of 40 kg P ha^-1 for the two species, respectively. Such a P response was not found in S. siamea at 24 WAP and for any of the species at 48 WAP. G. sepium accumulated more P (on average 162%) than S. siamea and S. spectabilis at 24 WAP and had greater root length and a higher percentage of mycorrhizal infection. However, at 48 WAP S. siamea had 2.5 times more P than G. sepium. Differences in the physiological P use efficiency (PPUE) between G. sepium and the non-N₂-fixing trees were significant at the 0 P level, being higher for S. siamea (average, 0.61 g shoot mg^-1 P) than for G. sepium (0.27 g shoot mg^-1 P). G. sepium had a consistently lower atom % ¹⁵N than S. spectabilis, while that of S. siamea for most of the time did not differ from that of G. sepium. The reference plant affected N₂ fixation extimates, with negative values and a higher variability (CV 60%) associated with S. siamea than with S. spectabilis (CV<20%). Consequently, S. spectabilis was selected as a better reference plant for measuring N₂ fixation in G. sepium. G. sepium fixed on average 35% and 54% of its N at 24 and 48 WAP, respectively. Except at the lowest P rate, percentage and amount of N fixed were not generally enhanced by P application.     

Benefits of inoculation of the common bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>) crop with efficient and competitive<Emphasis Type="Italic"> Rhizobium tropici</Emphasis> strains

Cropping in low fertility soils, especially those poor in N, contributes greatly to the low common bean (Phaseolus vulgaris L.) yield, and therefore the benefits of biological nitrogen fixation must be intensively explored to increase yields at a low cost. Six field experiments were performed in oxisols of Paraná State, southern Brazil, with a high population of indigenous common bean rhizobia, estimated at a minimum of 10³ cells g^–1 soil. Despite the high population, inoculation allowed an increase in rhizobial population and in nodule occupancy, and further increases were obtained with reinoculation in the following seasons. Thus, considering the treatments inoculated with the most effective strains (H 12, H 20, PRF 81 and CIAT 899), nodule occupancy increased from an average of 28% in the first experiment to 56% after four inoculation procedures. The establishment of the selected strains increased nodulation, N₂ fixation rates (evaluated by total N and N-ureide) and on average for the six experiments the strains H 12 and H 20 showed increases of 437 and 465 kg ha^–1, respectively,in relation to the indigenous rhizobial population. A synergistic effect between low levels of N fertilizer and inoculation with superior strains was also observed, resulting in yield increases in two other experiments. The soil rhizobial population decreased 1 year after the last cropping, but remained high in the plots that had been inoculated. DGGE analysis of soil extracts showed that the massive inoculation apparently did not affect the composition of the bacterial community.