Polyphasic characterization of Brazilian Rhizobium tropici strains effective in fixing N2 with common bean (Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris) is native to the Americas, and Rhizobium etli is the dominant microsymbiont in both the Mesoamerican and the Andean centers of genetic diversification. Wild common beans are not found in Brazil, although the legume has been cropped in the country throughout time and all but one of the rhizobial species that nodulate it (Rhizobium gallicum) have been broadly detected in Brazilian soils. However, the majority of the effective rhizobial strains isolated so far from field-grown plants belong to R. tropici. This study describes the analysis of symbiotic and non-symbiotic genes of 15 effective R. tropici strains, isolated from four geographically distant regions in Brazil. With RFLP-PCR of the 16S and 23S rRNA genes and sequence analysis of 16S rRNA, two clusters were observed, one related to R. tropici type A and another to type B strains. Diversity in ribosomal genes was high, indicating that type A strains might represent a new species. High intraspecies diversity was also observed in the rep-PCR analysis with BOX, ERIC and REP primers. However, in the RFLP-PCR analysis of nifH and nodC genes, all R. tropici showed unique combinations of profiles, which might reflect an evolutionary strategy to maximize N₂ fixation.     

Isotopic fractionation during N2 fixation by four tropical legumes

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

Effect of soil treatment with cereal straw and method of crop establishment on field pea (Pisum sativum L.) N2 fixation

The effects of soil incorporation with cereal straw (nil, 2.5, 5 and 10 t straw ha^–1) and direct drilling on the proportion and amount of pea N derived from biological N fixation were investigated in three field experiments. Fixed N was determined by¹⁵N dilution using barley as a reference plant. The three sites were on acidic, red clay-loams in the cropping zone of southeastern Australia. Seasonal plant available soil N, as determined by the N accumulated in barley, was 31, 56 and 158 kg N ha^–1, for the three sites. Incorporated straw reduced soil nitrate at sowing by 10–50 kg N ha^–1 (0–30 cm), and 5 or 10 t straw ha^–1 reduced barley uptake of N by 10–38 kg N ha^–1. However, reducing plant available soil N was generally ineffective for increasing the N fixed by pea. Fixed N increased only at the site with the least plant-available N, and only one-third of the increase could be attributed to lower soil N uptake by pea. There was no evidence that direct drilling pea increased fixed N by decreasing crop uptake of soil N. It is proposed that a lower requirement for soil N by pea as compared to barley, and availability of mineral N beneath the soil layer treated with straw, minimise the effectiveness of straw incorporation for increasing the N fixed by pea.     

Distribution and genetic diversity of rhizobia nodulating natural populations of Medicago truncatula in tunisian soils

A collection of 299 isolates of rhizobia nodulating Medicago truncatula was isolated from 10 Tunisian soils and was characterized by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR/RFLP) of 16S rRNA gene. Results showed that 227 and 72 isolates were assigned, respectively, to Sinorhizobium meliloti and Sinorhizobium medicae. In 9 out of 10 soils S. meliloti was detected, whereas S. medicae was recovered from only 5 out of 10 soils. The cross-nodulation of three populations of M. truncatula grown on Bulla Regia soil, which contained naturally the two Sinorhizobium species, showed that M. truncatula population collected from Amra site was selective to S. meliloti at least in soil conditions. Forty-eight isolates of each Sinorhizobium species trapped by M. truncatula populations collected from Bulla Regia, Soliman and Rhayet sites on Bulla Regia soil were characterized by repetitive extragenic palindromic-PCR (REP-PCR) and showed a clear distinction between the two Sinorhizobium species and a higher diversity for S. meliloti.     

Can a mixed stand of N2-fixing and non-fixing plants restrict N2O emissions with increasing CO2 concentration?

Initial effects of elevated atmospheric CO₂ concentration on N₂O fluxes and biomass production of timothy/red clover were studied in the laboratory. The experimental design consisted of two levels of atmospheric CO₂ (ca. 360 and 720 μmol CO₂ mol⁻¹) and two N fertilisation levels (5 and 10 g N m⁻²). There was a total of 36 mesocosms comprising sandy loam soil, which were equally distributed in four thermo-controlled greenhouses. In two of the greenhouses, the CO₂ concentration was kept at ambient concentration and in the other two at doubled concentration. Forage was harvested and the plants fertilised three times during the basic experiment, followed by harvest, a fertilisation with the double amount of nitrogen and rise of water level. Under elevated CO₂, harvestable and total aboveground dry biomass production of a mixed Trifolium/Phleum stand was increased at both N treatments compared to ambient CO₂. The N₂O flux rates under ambient CO₂ were significantly higher at both N treatments during the early growth of mixed Phleum/Trifolium mesocosms compared to the N₂O flux rate under elevated CO₂. However, when the conditions were favourable for denitrification at the end of the experiment, i.e. N availability and soil moisture were high enough, the elevated CO₂ concentration enhanced the N₂O efflux.     

First indications for the involvement of strigolactones on nodule formation in alfalfa (Medicago sativa)

Strigolactones have recently been suggested to be phytohormones that are present in all plants. Strigolactones are released by roots into the rhizosphere, stimulating the seed germination of parasitic plants such as Striga spp. and Orobanche spp. and play a crucial role in the interaction between plants and symbiotic arbuscular mycorrhizal fungi.By applying different concentrations of the synthetic strigolactone analogue GR24 to alfalfa (Medicago sativa) inoculated with Sinorhizobium meliloti we could show that in alfalfa nodulation is positively affected by the presence of the strigolactone analogue GR24. Moreover, we could show that this increased nodulation cannot be linked with a stimulatory effect of GR24 on the growth or the expression of nod genes of S. meliloti.Putative mechanisms operating in the plant in response to the addition of GR24 and leading to increased nodule formation by rhizobia are discussed.     

Displacement of native rhizobia nodulating chickpea (Cicer arietinum L.) by an inoculant strain through soil solarization

Summary Soil solarization greatly reduced the native chickpea Rhizobium population. With inoculation, it was possible to increase the population of the Rhizobium in solarized plots. In the 1st year, 47% nodulation was obtained with chickpea inoculant strain IC 59 when introduced with a cereal crop 2 weeks after the soil solarization and having a native Rhizobium count of <10 g^-1 soil, and only 13% when introduced 16 weeks after solarization at the time the chickpeas were sown, with 2.0×10² native rhizobia g^-1 soil. In the non-solarized plots inoculated with 5.6×10³ native rhizobia g^-1 soil, only 6% nodulation was obtained with the inoculant. In the succeeding year, non-inoculated chickpea was grown on the same plots without any solarization or Rhizobium inoculation. The treatment that showed good establishment of the inoculant strain in year 1 formed 68% inoculant nodules. Other treatments indicated a further reduction in inoculant success, from 1%–13% to 1%–9%. Soil solarization thus allowed an inoculant strain to successfully displace the high native population in the field and can serve as a research tool to compare strains in the field, irrespective of competitive ability. In year 1, Rhizobium inoculation of chickpea gave increased nodulation and increased plant growth 20 and 51 days after sowing, and increased dry matter, grain yield, and grain protein yield at maturity. These beneficial effects of inoculation on plant growth and yield were not measured in the 2nd year.Submitted as Journal Article No. JA 945 by the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, Andhra Pradesh 502 324, India     

Competitive ability of selected Cyclopia Vent. rhizobia under glasshouse and field conditions

This study tested the competitive ability of three locally isolated Cyclopia rhizobia and strain PPRICI3, the strain currently recommended for the cultivation of Cyclopia, a tea-producing legume. Under sterile glasshouse conditions, the three locally isolated strains were equally competitive with strain PPRICI3. In field soils, the inoculant strains were largely outcompeted by native rhizobia present in the soil, although nodule occupancy was higher in nodules growing close to the root crown (the original inoculation area). In glasshouse experiments using field soil, the test strains again performed poorly, gaining less than 6% nodule occupancy in the one soil type. The presence of Cyclopia-compatible rhizobia in field soils, together with the poor competitive ability of inoculant strains, resulted in inoculation having no effect on Cyclopia yield, nodule number or nodule mass. The native rhizobial population did not only effectively nodulate uninoculated control plants, they also out-competed introduced strains for nodule occupancy in inoculated plants. Nonetheless, the Cyclopia produced high crop yields, possibly due to an adequate supply of soil N.     

Changes in soil aggregation and glomalin-related soil protein content as affected by the arbuscular mycorrhizal fungal species Glomus mosseae and Glomus intraradices

Arbuscular mycorrhizal (AM) fungi are key organisms of the soil/plant system, influencing soil fertility and plant nutrition, and contributing to soil aggregation and soil structure stability by the combined action of extraradical hyphae and of an insoluble, hydrophobic proteinaceous substance named glomalin-related soil protein (GRSP). Since the GRSP extraction procedures have recently revealed problems related to co-extracting substances, the relationship between GRSP and AM fungi still remains to be verified. In this work the hypothesis that GRSP concentration is positively correlated with the occurrence of AM fungi was tested by using Medicago sativa plants inoculated with different isolates of Glomus mosseae and Glomus intraradices in a microcosm experiment. Our results show that (i) mycorrhizal establishment produced an increase in GRSP concentration - compared to initial values - in contrast with non-mycorrhizal plants, which did not produce any change; (ii) aggregate stability, evaluated as mean weight diameter (MWD) of macroaggregates of 1-2 mm diameter, was significantly higher in mycorrhizal soils compared to non-mycorrhizal soil; (iii) GRSP concentration and soil aggregate stability were positively correlated with mycorrhizal root volume and weakly correlated with total root volume; (iv) MWD values of soil aggregates were positively correlated with values of total hyphal length and hyphal density of the AM fungi utilized.The different ability of AM fungal isolates to affect GRSP concentration and to form extensive and dense mycelial networks, which may directly affect soil aggregates stability by hyphal enmeshment of soil particles, suggests the possibility of selecting the most efficient isolates to be utilized for soil quality improvement and land restoration programs.     

Temporal and spatial dynamics of soil solution C and N concentrations during Lolium perenne L. sward establishment and the effects of elevated CO2 and N additions

There is now clear evidence for a prolonged increase in atmospheric CO₂ concentrations and enrichment of the biosphere with N. Understanding the fate of C in the plant-soil system under different CO₂ and N regimes is therefore of considerable importance in predicting the environmental effects of climate change and in predicting the sustainability of ecosystems. Swards of Lolium perenne were grown from seed in a Eutric Cambisol at either ambient (ca. 350 μmol mol⁻¹) or elevated (700 μmol mol⁻¹) atmospheric pCO₂ and subjected to two inorganic N fertilizer regimes (no added N and 70 kg N ha⁻¹ month⁻¹). After germination, soil solution concentrations of dissolved organic C (DOC), dissolved inorganic N (DIN), dissolved organic N (DON), phenolics and H⁺ were measured at five depths down the soil profile over 3 months. The exploration of soil layers down the soil profile by roots caused transient increases in soil solution DOC, DON and phenolic concentrations, which then subsequently returned to lower quasi-stable concentrations. In general, the addition of N tended to increase DOC and DON concentrations while exposure to elevated pCO₂ had the opposite effect. These treatment effects, however, gradually diminished over the duration of the experiment from the top of the soil profile downwards. The ambient pCO₂ plus added N regime was the only treatment to maintain a notable difference in soil solution solute concentration, relative to other treatments. This effect on soil solution chemistry appeared to be largely indirect resulting from increased plant growth and a decrease in soil moisture content. Our results show that although plant growth responses to elevated pCO₂ are critically dependent upon N availability, the organic chemistry of the soil solution is relatively insensitive to changes in plant growth once the plants have become established.     

Long-term soil organic carbon dynamics in a subhumid tropical climate: C data in mixed C3/C4 cropping and modeling with ROTHC

Scanty information on long-term soil organic carbon (SOC) dynamics hampers validation of SOC models in the tropics. We observed SOC content changes in a 16-year continuously cropped agroforestry experiment in Ibadan, south-western Nigeria. SOC levels declined in all treatments. The decline was most pronounced in the no-tree control treatments with continuous maize and cowpea cropping, where SOC levels dropped from the initial 15.4 to 7.3-8.0 Mg C ha⁻¹ in the 0-12 cm topsoil in 16 years. In the two continuously cropped alley cropping (AC) systems, one with Leucaena leucocephala and one with Senna siamea trees, SOC levels dropped to 10.7-13.2 Mg C ha⁻¹. Compared to the no-tree control treatments, an annual application of an additional 8.5 Mg ha⁻¹ (dry matter) of plant residues, mainly tree prunings, led to an extra 3.5 Mg C ha⁻¹ (∼0.2% C) in the 0-12 cm top soil after 11 years, and 4.1 Mg C ha⁻¹ after 16 years. The addition of NPK fertilizer had little effect on the quantities of above-ground plant residues returned to the soil, and there was no evidence that the fertilizer affected the rate of SOC decomposition. The fact that both C₃ and C₄ plants returned organic matter to the soil in all cropping systems, but in contrasting proportions, led to clear contrasts in the ¹³C abundance in the SOC. This ¹³C information, together with the measured SOC contents, was used to test the ROTHC model. Decomposition was very fast, illustrated by the fact that we had to double all decomposition rate constants in the model in order to simulate the measured contrasts in SOC contents and δ¹³C between the AC treatments and the no-tree controls. We hypothesized (1) that the pruning materials from the legume trees and/or the extra rhizodeposition from the tree roots in the AC treatments accelerated the decomposition of the SOC present at the start of the experiment (true C-priming), and/or (2) that the physical protection of microbial biomass and metabolites by the clay fraction on this site, having a sandy top soil in which clay minerals are mainly of the 1:1 type, is lower than assumed by the model.     

Short-term effects of clearfelling on soil CO2, CH4, and N2O fluxes in a Sitka spruce plantation

We examined the effects of forest clearfelling on the fluxes of soil CO₂, CH₄, and N₂O in a Sitka spruce (Picea sitchensis (Bong.) Carr.) plantation on an organic-rich peaty gley soil, in Northern England. Soil CO₂, CH₄, N₂O as well as environmental factors such as soil temperature, soil water content, and depth to the water table were recorded in two mature stands for one growing season, at the end of which one of the two stands was felled and one was left as control. Monitoring of the same parameters continued thereafter for a second growing season. For the first 10 months after clearfelling, there was a significant decrease in soil CO₂ efflux, with an average efflux rate of 4.0 g m⁻² d⁻¹ in the mature stand (40-year) and 2.7 g m⁻² d⁻¹ in clearfelled site (CF). Clearfelling turned the soil from a sink (−0.37 mg m⁻² d⁻¹) for CH₄ to a net source (2.01 mg m⁻² d⁻¹). For the same period, soil N₂O fluxes averaged 0.57 mg m⁻² d⁻¹ in the CF and 0.23 mg m⁻² d⁻¹ in the 40-year stand. Clearfelling affected environmental factors and lead to higher daily soil temperatures during the summer period, while it caused an increase in the soil water content and a rise in the water table depth. Despite clearfelling, CO₂ remained the dominant greenhouse gas in terms of its greenhouse warming potential.     

The tripartite symbiosis between legumes, rhizobia and indigenous mycorrhizal fungi is more efficient in undisturbed soil

We investigated how the rate of colonization by indigenous arbuscular mycorrhizal fungi (AMF) affects the interaction between AMF, Sinorrhizobium meliloti and Medicago truncatula Gaertn. To generate a differential inoculum potential of indigenous AMF, five cycles of wheat, each of 1 month, were grown in sieved or undisturbed soil before M. truncatula was sown. The early colonization of M. truncatula roots by indigenous AMF was faster in undisturbed soil compared with sieved soil, but by pod-fill the frequency of hyphae, arbuscules and vesicles was similar in both treatments. At this latter stage, M. truncatula grown in undisturbed soil had accumulated a greater biomass in aboveground tissues, had a greater P concentration and derived more N from the atmosphere than plants grown in disturbed soil, although soil compaction resulted in plants having a smaller root system than those from disturbed soil. The difference in plant P content could not be explained by modifications in hydrolytic soil enzymes related to the P cycle as the activity of acid phosphatase was greater in sieved than in undisturbed soil, and the activity of alkaline phosphatase was unaffected by the treatment. Thus, the results observed were a consequence of the different rates of AMF colonization caused by soil disturbance. Together with earlier results for soybean, this study confirms that soil disturbance modifies the interaction between indigenous AMF, rhizobia and legumes leading to a reduced efficacy of the bacterial symbiont.     

Soil CO2 efflux vs. soil respiration: Implications for flux models

In the long term, all CO₂ produced in the soil must be emitted by the surface and soil CO₂ efflux (FCO₂) must correspond to soil respiration (R_soil). In the short term, however, the efflux can deviate from the instantaneous soil respiration, if the amount of CO₂ stored in the soil pore-space (SCO₂) is changing. We measured FCO₂ continuously for one year using an automated chamber system. Simultaneously, vertical soil profiles of CO₂ concentration, moisture, and temperature were measured in order to assess the changes in the amount of CO₂ stored in the soil. R_soil was calculated as the sum of the rate of change of the CO₂ storage over time and FCO₂. The experiment was split into a warm and a cold season. The dependency of soil respiration and soil efflux on soil temperature and on soil moisture was analyzed separately. Only the moisture-driven model of the warm season was significantly different for FCO₂ and R_soil. At our site, a moisture-driven soil-respiration model derived from CO₂ efflux data would underestimate the importance of soil moisture. This effect can be attributed to a temporary storage of CO₂ in the soil pore-space after rainfalls where up to 40% of the respired CO₂ were stored.     

New insights into the origins and evolution of rhizobia that nodulate common bean (Phaseolus vulgaris) in Brazil

It is generally accepted that there are two major centers of genetic diversification of common beans (Phaseolus vulgaris L.): the Mesoamerican (Mexico, Colombia, Ecuador and north of Peru, probably the primary center), and the Andean (southern Peru to north of Argentina) centers. Wild common bean is not found in Brazil, but it has been grown in the country throughout recorded history. Common bean establishes symbiotic associations with a wide range of rhizobial strains and Rhizobium etli is the dominant microsymbiont at both centers of genetic diversification. In contrast, R. tropici, originally recovered from common bean in Colombia, has been found to be the dominant species nodulating field-grown common-bean plants in Brazil. However, a recent study using soil dilutions as inocula has shown surprisingly high counts of R. etli in two Brazilian ecosystems. In the present study, RFLP-PCR analyses of nodABC and nifH genes of 43 of those Brazilian R. etli strains revealed unexpected homogeneity in their banding patterns. The Brazilian R. etli strains were closely similar in 16S rRNA sequences and in nodABC and nifH RFLP-PCR profiles to the Mexican strain CFN 42^T, and were quite distinct from R. etli and R. leguminosarum strains of European origin, supporting the hypothesis that Brazilian common bean and their rhizobia are of Mesoamerican origin, and could have arrived in Brazil in pre-colonial times. R. tropici may have been introduced to Brazilian soils later, or it may be a symbiont of other indigenous legume species and, due to its tolerance to acidic soils and high temperature conditions became the predominant microsymbiont of common bean.     

CO2 and inorganic N supply modify competition for N between co-occurring grass plants, tree seedlings and soil microorganisms

Plant-plant and plant-soil interactions play a key role in determining plant community structure and ecosystem function. However, the effects of global change on the interplay between co-occurring plants and soil microbes in successional communities are poorly understood. In this study, we investigated competition for nitrogen (N) between soil microorganisms, grass plants and establishing tree seedlings under factorial carbon dioxide (CO₂) and N treatments. Fraxinus excelsior seedlings were germinated in the presence or absence of grass competition (Dactylis glomerata) at low (380 μmol mol⁻¹) or high (645 μmol mol⁻¹) CO₂ and at two levels of N nutrition in a mesocosm experiment. Pulse ¹⁵N labelling was used to examine N partitioning among plant and soil compartments. Dactylis exerted a strong negative effect on Fraxinus biomass, N capture and ¹⁵N recovery irrespective of N and CO₂ treatment. In contrast, the presence of Dactylis had a positive effect on the microbial N pool. Plant and soil responses to N treatment were of a greater magnitude compared with responses to elevated CO₂, but the pattern of Fraxinus- and microbial-N pool response to N and CO₂ varied depending on grass competition treatment. Within the Dactylis competition treatment, decreases in Fraxinus biomass in response to N were not mirrored by decreases in tree seedling N content, suggesting a shift from below- to above-ground competition. In the Dactylis-sown pots, ¹⁵N recovery could be ranked Dactylis > microbial pool > Fraxinus in all N and CO₂ treatment combinations. Inequalities between Fraxinus and soil microorganisms in terms of ¹⁵N recovery were exacerbated by N addition. Contrary to expectations, elevated CO₂ did not increase plant-microbe competition. Nevertheless, microbial ¹⁵N recovery showed a small positive increase in the high CO₂ treatment. Overall, elevated CO₂ and N supply did not interact on plant/soil N partitioning. Our data suggest that the competitive balance between establishing tree seedlings and grass plants in an undisturbed sward is relatively insensitive to CO₂ or N-induced modifications in N competition between plant and soil compartments.     

Elevated CO2 stimulates N2O emissions in permanent grassland

To evaluate climate forcing under increasing atmospheric CO₂ concentrations, feedback effects on greenhouse gases such as nitrous oxide (N₂O) with a high global warming potential should be taken into account. This requires long-term N₂O flux measurements because responses to elevated CO₂ may vary throughout annual courses. Here, we present an almost 9 year long continuous N₂O flux data set from a free air carbon dioxide enrichment (FACE) study on an old, N-limited temperate grassland. Prior to the FACE start, N₂O emissions were not different between plots that were later under ambient (A) and elevated (E) CO₂ treatments, respectively. However, over the entire experimental period (May 1998–December 2006), N₂O emissions more than doubled under elevated CO₂ (0.90 vs. 2.07 kg N₂O-N ha⁻¹ y⁻¹ under A and E, respectively). The strongest stimulation occurred during vegetative growth periods in the summer when soil mineral N concentrations were low. This was surprising because based on literature we had expected the highest stimulation of N₂O emissions due to elevated CO₂ when mineral N concentrations were above background values (e.g. shortly after N application in spring). N₂O emissions under elevated CO₂ were moderately stimulated during late autumn–winter, including freeze–thaw cycles which occurred in the 8th winter of the experiment. Averaged over the entire experiment, the additional N₂O emissions caused by elevated CO₂ equaled 4738 kg CO₂-equivalents ha⁻¹, corresponding to more than half a ton (546 kg) of CO₂ ha⁻¹ which has to be sequestered annually to balance the CO₂-induced N₂O emissions. Without a concomitant increase in C sequestration under rising atmospheric CO₂ concentrations, temperate grasslands may be converted into greenhouse gas sources by a positive feedback on N₂O emissions. Our results underline the need to include continuous N₂O flux measurements in ecosystem-scale CO₂ enrichment experiments.     

A new technique to measure soil CO2 efflux at constant CO2 concentration

A new principle for measuring soil CO₂ efflux at constant ambient concentration is introduced. The measuring principle relies on the continuous absorption of CO₂ within the system to achieve a constant CO₂ concentration inside the soil chamber at ambient level, thus balancing the amount of CO₂ entering the soil chamber by diffusion from the soil. We report results that show reliable soil CO₂ efflux measurements with the new system. The novel measuring principle does not disturb the natural gradient of CO₂ within the soil, while allowing for continuous capture of the CO₂ released from the soil. It therefore holds great potential for application in simultaneous measurements of soil CO₂ efflux and its δ¹³C, since both variables show sensitivity to a distortion of the soil CO₂ profile commonly found in conventional chamber techniques.     

Influence of arbuscular mycorrhizae and phosphorus fertilization on growth, nodulation and N2 fixation (15N) inMedicago sativa at four salinity levels

The rose of an isolate of the arbuscular mycorrhizal (AM) fungusGlomus mosseae in the protection ofMedicago sativa (+Rhizobium meliloti) against salt stress induced by the addition of increasing levels of soluble salts was studied. The interactions between soluble P in soil (four levels), mycorrhizal inoculum and degree of salinity in relation to plant growth, nutrition and infective parameters were evaluated. Salt stress was induced by sequential irrigation with saline water having four concentrations of three salts (NaCl, CaCl₂, and MgCl₂).¹⁵N-labelled ammonium sulphate was added to provide a quantitative estimate of N₂ fixation under moderate to high salinity levels. N and P concentration and nodule formation increased with the amount of plant-available P or mycorrhizal inoculum in the soil and generally declined as the salinity in the solution culture increased from a moderate to a high level. The mycorrhizal inoculation protected the plants from salt stress more efficiently than any amount of plant-available P in soil, particularly at the highest salinity level applied (43.5 dS m^–1). Mycorrhizal inoculation matched the effect on dry matter and nutrition of the addition in the soil of 150 mg P kg^–1. Nevertheless the highest saline solution assayed (43.5 dS m^–1) affected more severely plants supplemented with phosphorus than those with the addition of mycorrhizal inoculum. Such a saline-depressing effect was 1.5 (biomass), 1.4 (N) and 1.5 (P) times higher in plants supplied with soluble phosphate than with AM inoculum. Mechanisms beyond those mediated by P must be involved in the AM-protectioe effect against salinity. The¹⁵N methodology used allowed the determination of N₂ fixation as influenced by different P applications compared to mycorrhizal inoculation. A lack of correlation between nodule formation and function (N₂ fixation) was evidenced in mycorrhizal-inoculated plants. In spite of the reduced activity per nodule in mycorrhizal-inoculated In spite of the reduced activity per nodule in mycorrhizal-inoculated plants, the N contents determined indicated the highest acquisition of N occurred in plants with the symbiotic status. Moreover, N and P uptake increased while Ca and Mg decreased in AM-inoculated plants. Thus P/Ca ratios and cation/anion balance in general were altered in mycorrhizal treatments. This study therefore confirms previous findings that AM-colonized plants have optional and alternative mechanisms available to satisfy their nutritive requirements and to maintain their physiological status in stress situations and in disturbed ecosystems.     

Seasonal changes in the spatial structures of N2O, CO2, and CH4 fluxes from Acacia mangium plantation soils in Indonesia

We evaluated the spatial structures of nitrous oxide (N₂O), carbon dioxide (CO₂), and methane (CH₄) fluxes in an Acacia mangium plantation stand in Sumatra, Indonesia, in drier (August) and wetter (March) seasons. A 60 × 100-m plot was established in an A. mangium plantation that included different topographical elements of the upper plateau, lower plateau, upper slope and foot slope. The plot was divided into 10 × 10-m grids and gas fluxes and soil properties were measured at 77 grid points at 10-m intervals within the plot. Spatial structures of the gas fluxes and soil properties were identified using geostatistical analyses. Averaged N₂O and CO₂ fluxes in the wetter season (1.85 mg N m⁻² d⁻¹ and 4.29 g C m⁻² d⁻¹, respectively) were significantly higher than those in the drier season (0.55 mg N m⁻² d⁻¹ and 2.73 g C m⁻² d⁻¹, respectively) and averaged CH₄ uptake rates in the drier season (−0.62 mg C m⁻² d⁻¹) were higher than those in the wetter season (−0.24 mg C m⁻² d⁻¹). These values of N₂O fluxes in A. mangium soils were higher than those reported for natural forest soils in Sumatra, while CO₂ and CH₄ fluxes were in the range of fluxes reported for natural forest soils. Seasonal differences in these gas fluxes appears to be controlled by soil water content and substrate availability due to differing precipitation and mineralization of litter between seasons. N₂O fluxes had strong spatial dependence with a range of about 18 m in both the drier and wetter seasons. Topography was associated with the N₂O fluxes in the wetter season with higher and lower fluxes on the foot slope and on the upper plateau, respectively, via controlling the anaerobic-aerobic conditions in the soils. In the drier season, however, we could not find obvious topographic influences on the spatial patterns of N₂O fluxes and they may have depended on litter amount distribution. CO₂ fluxes had no spatial dependence in both seasons, but the topographic influence was significant in the drier season with lowest fluxes on the foot slope, while there was no significant difference between topographic positions in the wetter season. The distributions of litter amount and soil organic matter were possibly associated with CO₂ fluxes through their effects on microbial activities and fine root distribution in this A. mangium plantation.