Water-soluble carbon in roots of rape and barley: impacts on labile soil organic carbon, arylsulphatase activity and sulphur mineralization

Investigating the impact of plant species on sulphur (S) availability in the rhizosphere soil is agronomically important to optimize S fertilization. Bulk, rhizosphere soils and the roots of field-grown rape and barley were sampled 7 times (every fortnight), from March to June, at plant maturity. Root carbon (C) and nitrogen (N) in water extract, along with soil SO₄²⁻-S, labile soil organic-C (HWC) and -N (HWN) in hot water extract, as well as soil arylsulphatase activity were then monitored. The average concentrations of both HWC and HWN were observed in the following decreasing order: rape rhizosphere soil >barley rhizosphere soil >bulk soil. In parallel, the average contents of water extractable-C and -N in rape roots were higher than those in barley roots. These results suggest that soil C and N contents in hot water extract (including rhizodeposition) were correlated with C and N released by roots. Great ARS activities found in rape rhizosphere soil were accompanied by great SO₄²⁻-S mineralization over time. Finally, bulk and rhizosphere soils of rape and barley were pooled from the seven samplings and incubated with the corresponding pooled root water-soluble C of both plant species and glucose-C. After 1 and 9 weeks, a greater net S mineralization (gross mineralization - immobilization) was observed with rape root water-soluble C than with barley root water-soluble C and glucose-C. Conjointly, we found a higher average value of ARS activity in rape rhizosphere than in barley rhizosphere soil. Our findings suggest that plant species, via their rhizodeposition, determine the dynamic of S in soil.     

Inoculation of forage grasses with N2-fixing Enterobacteriaceae

Summary Previous investigations indicated some forage grass roots in Texas are heavily colonized with N₂-fixing bacteria. The most numerous N₂-fixing bacteria were in the genera Klebsiella and Enterobacter. In the present investigation inoculation experiments were conducted using 18 isolates of these bacteria to determine if a N₂-fixing association could be established between the bacteria and the grassesCynodon dactylon andPanicum coloratum. Plants were grown in soil for approximately 5 months in a greenhouse and were measured periodically for dry matter, nitrogen accumulation, and acetylene reduction activity. Results of the investigation indicated that 25% of the plant-soil systems were active in acetylene reduction and the activity was high enough to indicate agronomically significant quantities of N₂ were being fixed (>8kg N ha⁻¹). However, plant systems extrapolated to fix>8 kg N ha⁻¹ contained less nitrogen and accumulated less dry matter than plants less active in acetylene reduction. Inocula could not be re-isolated from healthy grass roots indicating that the N₂-fixing activity may have not have been closely assiciated with plant roots. Future research is needed to determine factors limiting colonization of grass roots.     

Response of nodulating and non-nodulatingPisum sativum L. to nitrate

This study examined whether ‘Risnod2’ and ‘Risnod27’ non-nodulating mutants of pea (Pisum sativum L.) provided with increasing concentrations of nitrate could achieve a growth and nitrogen accumulation comparable to their parental N₂-fixing cv. Finale. In the cv. Finale, nodule number, nodule dry mass accumulation, total C₂H₂-reducing activity of nodulated roots (TAR) and estimated N₂ fixation were considerably inhibited at 5.0 and 10.0 mM root medium NO₃ ⁻ concentrations. In contrast a 0.63 mM level stimulated both the nodule dry mass and TAR. The cv. Finale N₂-fixing plants grown on 0 to 2.5 mM NO₃ ⁻ levels had higher shoot N concentrations than the Nod⁻ mutants, but within the 5.0 to 10.0 mM levels the Nod⁻ mutants approached or even overtopped the N concentration of the cv. Finale plants. Compared with a high positive correlation found in the Nod⁻ mutants, shoot N concentration in the cv. Finale was negatively correlated with the root medium NO₃ ⁻ concentration. The pattern of nitrogen content in shoot dry mass was very similar to that seen in the shoot dry mass accumulation. The Nod⁻ mutants grown on the 5.0 and/or 10.0 mM NO₃ ⁻ level had plant dry mass, shoot nitrogen concentration, shoot nitrogen content, and root/shoot dry mass ratio comparable with those of the nodulating cv. Finale grown on the same nitrate levels.     

A new acid-tolerant nitrogen-fixing bacterium associated with sugarcane

During surveys of bacteria possibly responsible for N₂ fixation in sugarcane, root and stem samples were collected in four sugarcane-growing regions in Brazil. A new microaerobic N₂-fixing bacterium was isolated from most samples of washed roots and stems from all regions. Isolation procedures were based on semisolid diluted sugarcane juice medium followed by replication to N-free 10% sugar medium acidified with acetic acid to pH 4.5. The new bacterium is an aerobic rod, motile by 1 to 3 lateral flagella, fixes N₂ in semisolid media under air but not in liquid media except when a starter dose of N is added. It has no nitrate, reductase and N₂ fixation proceeds in the presence of 10mM NO ₃ ⁻ . Best growth occurs with high sucrose concentrations (10%). Growth occurs up to 30% sucrose but not at 35%. Acid is formed reaching a final pH of below 3.0. Growth and N₂ fixation proceed at this acidity. Ethanol is used for growth and is “overoxidised” (oxidized to CO₂ and H₂O). Acetic and lactic acids are also oxidized to CO₂ and H₂O. Acids produced from glucose are consumed with precipitation of CaCO₃. Dark brown colonies are formed on potato agar with 10% sugar and dark orange colonies on N poor agar (20 mg yeast extract per 1) containing bromothymol blue. In view of the distinct characteristics which do not allow identification within either Frateuria, Gluconobacter, Acetobacter or any known N₂-fixing bacterium a new genus and species are proposed and namedSaccharobacter nitrocaptans.     

Belowground responses of <Emphasis Type="Italic">Picea asperata</Emphasis> seedlings to warming and nitrogen fertilization in the eastern Tibetan Plateau

The impacts of global climatic change on belowground ecological processes of terrestrial ecosystems are still not clear. We therefore conducted an experiment in the subalpine coniferous forest ecosystem of the eastern edges of the Tibetan Plateau to study roots of Picea asperata seedlings and rhizosphere soil responses to soil warming and nitrogen availability from April 2007 to December 2008. The seedlings were subjected to two levels of temperature (ambient; infrared heater warming) and two nitrogen levels (0 or 25 g m⁻²year⁻¹ N). We used a free air temperature increase from an overhead infrared heater to raise both air and soil temperature by 2.1 and 2.6°C, respectively. The results showed that warming alone significantly increased total biomass, coarse root biomass and fine root biomass of P. asperata seedlings. Both total biomass and fine root biomass were increased, but coarse root biomass was significantly decreased by nitrogen fertilization and warming combined with nitrogen fertilization. Warming induced a prominent increase in soil organic carbon (SOC) and NO₃ ⁻-N of rhizosphere soil, while nitrogen fertilization significantly decreased SOC and NH₄ ⁺-N of rhizosphere soil. The warming, fertilization and warming × N fertilization interaction decreased soil microbial C significantly, but substantially increased soil microbial N. These results suggest that nitrogen deposition combined with warmer temperatures under future climatic change possibly will have no effect on fine root production of P. asperata seedlings, but could enhance the nitrification process of their rhizosphere soils in subalpine coniferous forests.     

Nitrogenase activity in the papyrus swamps of Lake Naivasha,Kenya

Nitrogenase activity (acetylene reduction activity) was found to occur universally in the Cyperus papyrus swamp in Lake Naivasha. Low rates of acetylene reduction activity (0.9–104.9 nmol C₂H₄ g d.wt. roots^-1 h^-1) were associated with excised roots of C. papyrus but higher rates of activity (89.0–280.4 nmol C₂H₄ g d.wt. roots^-1 h^-1) were associated with intact root systems of the plant. It was estimated that nitrogen fixation associated with young roots alone could supply about 26% of the nitrogen requirements of growing papyrus plants. Acetylene reduction activity in the lake bottom sediments was generally low and associated with adjacent papyrus stands. Plate counts of putative aerobic and facultatively anaerobic N₂-fixing bacteria associated with papyrus roots showed the presence of high numbers of diazotrophs (5.4 × 10⁶ CFU g d.wt. roots^-1). Fewer numbers of N₂-fixing bacteria were detected in the sediments (1.9 × 10³-3.2 × 10⁴ CFU g d.wt. sediment^-1).     

Critical evaluation of thein situ nitrate reductase assay

Anin situ method, derived from anin vivo method, was used to determine nitrate reductase activity (NRA) in:i) excised barley and corn shoots and excised soybean leaves during a N-depletion experiment and; ii) roots and shoots of N-depleted barley and corn seedlings during induction of nitrate, reductase (NR). Nitrate reduction, calculated from thesein situ RNA measurements, was compared with estimates of each organ's nitrate reduction in light aerobic conditions from NO ₃ ⁻ consumption and a¹⁵N model (Gojonet al., 1986b). Thein situ RNA of roots strongly underestimated their¹⁵NO ₃ ⁻ reduction. In contrast, in barley and corn shoots and in the first trifoliolate leaves from 26-day-old, soybean, thein situ NRA assay gave a fair approximation of the true NO ₃ ⁻ reduction rate (relative differences ranging from −14 to +32%). In young soybean leaves (from 20-day-old plants), however, thein situ NRA strongly underestimated the actual NO ₃ ⁻ reduction. The physiological significance of thein situ NRA assay in shoots and roots, and its value for field studies are discussed from these results.     

Ammonium versus Nitrate Nutrition of Plants Stimulates Microbial Activity in the Rhizosphere

Using an alkaline calcareous soil, pot experiments were conducted to elucidate the effects of NH ₄ ⁺ vs. NO ₃ ⁻ nutrition (50 or 100 mg kg⁻¹ soil) of wheat and maize on microbial activity in the rhizosphere and bulk soils. Dicyandiamide was used as nitrification inhibitor to maintain NH ₄ ⁺ as the predominant N source for plants grown in NH ₄ ⁺ -treated soil. While maize grew equally well on both N sources, root and shoot growth of wheat was higher under NH ₄ ⁺ than under NO ₃ ⁻ nutrition. Bacterial population density on roots, but not in the rhizosphere soil, was higher under NH ₄ ⁺ than under NO ₃ ^– supplied at 150 mg N kg⁻¹ soil; whereas at both N levels applied, NH ₄ ⁺ compared to NO ₃ ⁻ nutrition of wheat and maize significantly increased microbial biomass in the rhizosphere soil. Under both plant species, NH ₄ ⁺ vs. NO ₃ ⁻ nutrition also increased aerobic and anaerobic respiration, and dehydrogenase activity in the rhizosphere. As microbial activity in the planted bulk and unplanted soils was hardly affected by the N-source, we hypothesize that the stimulation by NH ₄ ⁺ of the rhizosphere microbial activity was probably due to higher availability of root exudates under NH ₄ ⁺ than under NO ₃ ⁻ nutrition.     

Most probable numbers of Azospirillum associated with the roots of inoculated pearl millet

Summary Inoculation of pearl millet (Pennisetum americanum (L.) Leeke) with Azospirillum significantly increased the numbers of this organism in the rhizosphere, rhizoplane, washed and crushed roots and surface sterilized and crushed roots. The maximum number of organisms plant^–1 were localized in the rhizosphere. The numbers of Azospirillum on the roots of inoculated plants grown under sterilized conditions were much higher than in the field grown plants. In both cases populations outside the roots were higher than in the surface sterilized roots. The highest numbers per unit root weight were recorded between 60–75 days of growth. N₂-ase activity throughout the growth cycle was very low and was not related to the populations of Azospirillum on the roots. Root exudates and extracts of pearl millet showed a stimulatory effect on the growth of Azospirillum suggesting their possible involvement in the colonization of this organism on the roots of inoculated plants.     

Responses of Wheat Roots to Exogenous Selenium Supply Under Enhanced Ultraviolet-B

Effects of selenium (Se) on growth and some physiological traits of roots in wheat (Triticum aestivum L. cv Han NO.7086) seedlings exposed to enhanced ultraviolet-B (UV-B) stress are reported. Responses of roots were different depending on the Se concentration. Compared with the control, root weight of wheat seedlings treated with 1.0 and 2.0 mg Se kg⁻¹ soil increased by 39.47% and 16.28%, respectively. The lower amount Se (0.5 mg kg⁻¹) and the higher amount Se treatments (3.0 mg kg⁻¹) did not significantly affect on root weight. Se treatments significantly increased root activity, flavonoids and proline content, and activities of peroxidase and superoxide dimutase in wheat roots exposed to enhanced UV-B. In addition, the treatments with 0.5, 1.0, and 2.0 mg Se kg⁻¹ significantly reduced malondialdehyde content and the rate of superoxide radical (O₂⁻) production of roots, whereas the higher amount Se treatment only induced a decrease in the rate of O₂⁻ production. The results of this study demonstrated that optimal Se supply promoted roots growth of wheat seedlings, and that optimal Se supply could reduce oxidative stress in wheat roots under enhanced UV-B radiation.     

Carbon balance and allocation of assimilated CO2 in Scots pine, Norway spruce, and Silver birch seedlings determined with gas exchange measurements and 14C pulse labelling

Carbon dioxide is released from the soil to the atmosphere in heterotrophic respiration when the dead organic matter is used for substrates for soil micro-organisms and soil animals. Respiration of roots and mycorrhiza is another major source of carbon dioxide in soil CO₂ efflux. The partitioning of these two fluxes is essential for understanding the carbon balance of forest ecosystems and for modelling the carbon cycle within these ecosystems. In this study, we determined the carbon balance of three common tree species in boreal forest zone, Scots pine, Norway spruce, and Silver birch with gas exchange measurements conducted in laboratory in controlled temperature and light conditions. We also studied the allocation pattern of assimilated carbon with ¹⁴C pulse labelling experiment. The photosynthetic light responses of the tree species were substantially different. The maximum photosynthetic capacity (P _max) was 2.21 μg CO₂ s⁻¹ g⁻¹ in Scots pine, 1.22 μg CO₂ s⁻¹ g⁻¹ in Norway spruce and 3.01 μg CO₂ s⁻¹ g⁻¹ in Silver birch seedlings. According to the pulse labelling experiments, 43–75% of the assimilated carbon remained in the aboveground parts of the seedlings. The amount of carbon allocated to root and rhizosphere respiration was about 9–26%, and the amount of carbon allocated to root and ectomycorrhizal biomass about 13–21% of the total assimilated CO₂. The ¹⁴CO₂ pulse reached the root system within few hours after the labelling and most of the pulse had passed the root system after 48 h. The transport rate of carbon from shoot to roots was fastest in Silver birch seedlings.     

The effect of the osmotic potential and specific ion concentration of the nutrient solution on the uptake and reduction of nitrate by barley seedlings

Summary Short-term absorption experiments were conducted with intact barley (Hordeum vulgare L.) seedlings to observe the effects of the osmotic potential (Ψ^π) and salt species on nitrate uptake andin vivo nitrate reduction. The experiments consisted of growing barley seedlings for 5 days in complete nutrient solutions salinized to (Ψ^π) levels of −0.6, −1.8, −3.0, −4.2, and −5.4 bars with NaCl, CaCl₂ or Na₂SO₄. After the absorption period, the seedlings were separated into shoots and roots, weighed, then analyzed for NO₃. The nutrient solutions were sampled for NO₃ analysis each day immediately before renewing the solutions. The accumulative loss of NO₃ from the solutions was considered to be uptake whereas NO₃ reduction was the difference between uptake and seedling content. Lowering the (Ψ^π) of the nutrient solutions resulted in decreased concentrations of NO₃ in the plant, little or no effect (except at the lowest (Ψ^π) level) on uptake, and increased nitrate reductase activity. Increased rates of NO₃ reduction were in particular associated with the Cl concentration of the nutrient solution.     

Comparative effects of salt-stress and alkali-stress on the growth,photosynthesis, solute accumulation,and ion balance of barley plants

We compared the effects of salt-stresses (SS, 1: 1 molar ratio of NaCl to Na₂SO₄) and alkali-stresses (AS, 1: 1 molar ratio of NaHCO₃ to Na₂CO₃) on the growth, photosynthesis, solute accumulation, and ion balance of barley seedlings, to elucidate the mechanism of AS (high-pH) damage to plants and the physiological adaptive mechanism of plants to AS. The effects of SS on the water content, root system activity, membrane permeability, and the content of photosynthetic pigments were much less than those of AS. However, AS damaged root function, photosynthetic pigments, and the membrane system, led to the severe reductions in water content, root system activity, content of photosynthetic pigments, and net photosynthetic rate, and a sharp increase in electrolyte leakage rate. Moreover, with salinity higher than 60 mM, Na⁺ content increased slowly under SS and sharply under AS. This indicates that high-pH caused by AS might interfere with control of Na⁺ uptake in roots and increase intracellular Na⁺ to a toxic level, which may be the main cause of some damage emerging under higher AS. Under SS, barley accumulated organic acids, Cl⁻, SO₄ ²⁻, and NO₃ ⁻ to balance the massive influx of cations, the contribution of inorganic ions to ion balance was greater than that of organic acids. However, AS might inhibit absorptions of NO₃ ⁻ and Cl⁻, enhance organic acid synthesis, and SO₄ ²⁻ absorption to maintain intracellular ion balance and stable pH.     

Seasonal accumulation and partitioning of nitrogen by lentil

Although wheat (Triticum aestivum L.) is the dominant crop of the semi-arid plains of Canada and the western United States, lentil (Lens culinaris Medik.) has become an important alternative crop. Sources and seasonal accumulation of N must be understood in order to identify parameters that can lead to increased N₂-fixing activity and yield. Inoculated lentil was grown in a sandy-loam soil at an irrigated site in Saskatchewan, Canada. Wheat was used as the reference crop to estimate N₂ fixation by the A-value approach. Lentil and wheat received 10 and 100 kg N ha⁻¹ of ammonium nitrate, respectively. Crops were harvested six times during the growing season and plant components analyzed. During the first 71 days after planting the wheat had a higher daily dry matter and N accumulation compared to lentil. However, during the latter part of the growing season, daily dry matter and N accumulation were greater for lentil. The maximum total N accumulation for lentil at maturity was 149 kg ha⁻¹. In contrast, wheat had a maximum N accumulation of 98 kg ha⁻¹ in the Feekes 11.1 stage, or 86 days after planting. The maximum daily rates of N accumulation were 3.82 kg N ha⁻¹ day⁻¹ for lentil and 2.21 kg N ha⁻¹ day⁻¹ for wheat. The percentage of N derived from N₂ fixation (% Ndfa) ranged from 0 at the first harvest to 92 % at final harvest. Generative plant components had higher values for % Ndfa than the vegetative components which indicates that N in the reproductive plant parts was derived largely from current N₂ fixation and lentil continued to fix N until the end of the pod fill stage. At final harvest, lentil had derived 129 kg N ha⁻¹ from N₂ fixation with maximum N₂-fixing activity (4.4 kg N ha⁻¹ day⁻¹) occurring during the early stages of pod fill. Higher maximum rates of N₂-fixing activity than net N accumulation (3.82 kg N ha⁻¹ day⁻¹) may have been caused by N losses like volatilization. In addition, lentil provided a net N contribution to the soil of 59 kg ha⁻¹ following the removal of the grain.     

盐分胁迫对啤酒大麦幼苗生长、离子平衡和根际pH变化的影响

盐分胁迫不仅影响植物的生长,而且会影响植物根际微域环境。根际pH的改变对土壤养分的有效性和微生物群落组成的变化有重要影响。为了探究啤酒大麦幼苗对不同类型盐分胁迫的生理生态响应机制和根际pH变化影响的生理机制,采用水培法,通过不同类型盐分(对照、混合Na盐、混合Cl盐和NaCl)胁迫处理啤酒大麦幼苗,对其生长、离子平衡和根际pH变化进行了研究。结果表明,1)在3种不同类型盐分胁迫下,啤酒大麦幼苗地上部干重、含水量均有所降低,而根冠比增加,尤其在NaCl胁迫下啤酒大麦幼苗地上部干重较对照显著降低了17.88%,而根干重和根冠比则分别增加了19.12%和43.86%。不同类型盐分胁迫抑制了啤酒大麦幼苗根长的生长,尤其在混合Na盐胁迫下根长降低明显(P0.05),但促进了根表面积和根体积的增加,尤其在混合Cl盐胁迫下,根表面积和根体积分别增加了41.76%和84.38%。2)不同类型盐分胁迫下啤酒大麦幼苗地上部离子平衡发生改变,在混合Na盐和NaCl胁迫下啤酒大麦幼苗主要吸收Na~+,地上部K~+/Na~+、Ca~(2+)/Na~+和Mg~(2+)/Na~+显著降低;混合Cl盐和NaCl胁迫下则过量吸收Cl~-,抑制了H_2PO_4~-、NO_3~-和SO_4~(2-)的吸收。3)在混合Na盐、混合Cl盐和NaCl盐分胁迫下,啤酒大麦幼苗对阴离子的吸收总量高于对阳离子的吸收总量,离子平衡计算结果表明根际呈碱化现象,与原位显色结果一致,且在混合Cl盐胁迫下根际碱化程度最大。     

Dinitrogen-fixation by three neotropical agroforestry tree species under semi-controlled field conditions

Cultivating dinitrogen-fixing legume trees with crops in agroforestry is a relatively common N management practice in the Neotropics. The objective of this study was to assess the N₂ fixation potential of three important Neotropical agroforestry tree species, Erythrina poeppigiana, Erythrina fusca, and Inga edulis, under semi-controlled field conditions. The study was conducted in the humid tropical climate of the Caribbean coastal plain of Costa Rica. In 2002, seedlings of I. edulis and Vochysia guatemalensis were planted in one-meter-deep open-ended plastic cylinders buried in soil within hedgerows of the same species. Overall tree spacing was 1 × 4 m to simulate a typical alley-cropping design. The ¹⁵N was applied as (NH₄)₂SO₄ at 10% ¹⁵N atom excess 15 days after planting at the rate of 20 kg [N] ha⁻¹. In 2003, seedlings of E. poeppigiana, E. fusca, and V. guatemalensis were planted in the same field using the existing cylinders. The ¹⁵N application was repeated at the rate of 20 kg [N] ha⁻¹ 15 days after planting and 10 kg [N] ha⁻¹ was added three months after planting. Trees were harvested 9 months after planting in both years. The ¹⁵N content of leaves, branches, stems, and roots was determined by mass spectrometry. The percentage of atmospheric N fixed out of total N (%N_f) was calculated based on ¹⁵N atom excess in leaves or total biomass. The difference between the two calculation methods was insignificant for all species. Sixty percent of I. edulis trees fixed N₂; %N_f was 57% for the N₂-fixing trees. Biomass production and N yield were similar in N₂-fixing and non-N₂-fixing I. edulis. No obvious cause was found for why not all I. edulis trees fixed N₂. All E. poeppigiana and E. fusca trees fixed N₂; %N_f was ca. 59% and 64%, respectively. These data were extrapolated to typical agroforestry systems using published data on N recycling by the studied species. Inga edulis may recycle ca. 100 kg ha⁻¹ a⁻¹ of N fixed from atmosphere to soil if only 60% of trees fix N₂, E. poeppigiana 60–160 kg ha⁻¹ a⁻¹, and E. fusca ca. 80 kg ha⁻¹ a⁻¹.     

Mercurial-sensitive water transport in barley roots

An isolated barley root was partitioned into the apical and basal part across the partition wall of the double-chamber osmometer. Transroot water movement was induced by subjecting the apical part to a sorbitol solution, while the basal part with the cut end was in artificial pond water. The rate of transroot osmosis was first low but enhanced by two means, infilitration of roots by pressurization and repetition of osmosis. Both effects acted additively. The radial hydraulic conductivity (Lp_r) was calculated by dividing the initial flow rate with the surface area of the apical part of the root, to which sorbitol was applied, and the osmotic gradient between the apical and basal part of the root. Lp_r which was first 0.02–0.04 pm s⁻¹ Pa⁻¹ increased up to 0.25–0.4 pm s⁻¹ Pa⁻¹ after enhancement. Enhancement is assumed to be caused by an increase of the area of the plasma membrane which is avallable to osmotic water movement. The increased Lp_r is in the same order of magnitude as the hydraulic conductivity (Lp) of epidermal and cortical cells of barley roots obtained by Steudie and Jeschke (1983). HgCl₂, a potent inhibitor of water channels, suppressed Lp_r of non-infiltrated and infiltrated roots down to 17% and 8% of control values, respectively. A high sensitivity of Lp_r to HgCl₂ suggests that water channels constitute the most conductive pathway for osmotic radial water movement in barley roots.     

High diversity of diazotrophic bacteria associated with the carnivorous plant Drosera villosa var. villosa growing in oligotrophic habitats in Brazil

Drosera villosa var. villosa A. St.-Hil is a carnivorous plant that grows in Brazilian flooded soils very poor on nutrients, including low levels of N. Under these conditions, the plant shows vigorous growth, low root number, low number of captured prey (less than 50%) and a great assemblage of bacteria associated with the roots and leaves that grow in N-free medium. These preliminary results have led us to investigate the number of colony forming units (log CFU) in the roots (rhizosphere and endorhizosphere) and leaves (phyllosphere and endophyllosphere) of D. villosa var. villosa by the tenfold serial dilution technique in two N-free culture media. The results showed that the phyllosphere had 6.65 log CFU g dry leaf⁻¹ and the rhizosphere 6.47 log CFU g dry soil⁻¹, with the lowest value detected in the endophyllosphere (4.39 log CFU g dry leaf⁻¹). Sixty-three different bacteria morphotypes were isolated from the surface and interior of the roots and leaves and the amplification of the DNA with specific primers detected the nifH gene in 34 of those strains. The DNAs of the 34 strains were compared by the BOX-PCR technique and a great diversity was observed, with the bacteria clustering at a final level of similarity of only 12%. The strains were also submitted to the partial sequencing of the 16S rRNA gene and several genera of N₂-fixing bacteria were detected, including Bacillus, Burkholderia, Methylobacterium, Paenibacillus, Pseudomonas and Sphingomonas.     

Sugar beet and barley yields in relation to inoculation with N2-fixing and phosphate solubilizing bacteria

Recently, there has been a resurgence of interest in bioorganic fertilizers as part of sustainable agricultural practices to alleviate drawbacks of intensive farming practices. N₂-fixing and P-solubilizing bacteria are important in plant nutrition increasing N and P uptake by the plants, and playing a significant role as plant growth-promoting rhizobacteria in the biofertilization of crops. A study was conducted in order to investigate the effects of two N₂-fixing (OSU-140 and OSU-142) and a strain of P-solubilizing bacteria (M-13) in single, dual and three strains combinations on sugar beet and barley yields under field conditions in 2001 and 2002. The treatments included: (1) Control (no inoculation and fertilizer), (2) Bacillus OSU-140, (3) Bacillus OSU-142, (4) Bacillus M-13, (5) OSU-140 + OSU-142, (6) OSU-140 + M-13, (7) OSU-142 + M-13, (8) OSU-140 + OSU-142 + M-13, (9) N, (10) NP. N and NP plots were fertilized with 120 kg N ha^–1 and 120 kg N ha^–1 + 90 kg P ha^- for sugar beet and 80 kg N ha^–1 and 80 kg N ha^–1 + 60 kg P ha^–1 for barley. The experiments were conducted in a randomized block design with five replicates. All inoculations and fertilizer applications significantly increased leaf, root and sugar yield of sugar beet and grain and biomass yields of barley over the control. Single inoculations with N₂-fixing bacteria increased sugar beet root and barley yields by 5.6–11.0% depending on the species while P-solubilizing bacteria alone gave yield increases by 5.5–7.5% compared to control. Dual inoculation and mixture of three bacteria gave increases by 7.7–12.7% over control as compared with 20.7–25.9% yield increases by NP application. Mixture of all three strains, dual inoculation of N₂-fixing OSU-142 and P-solubilizing M-13, and/or dual inoculation N₂-fixing bacteria significantly increased root and sugar yields of sugar beet, compared with single inoculations with OSU-140 or M-13. Dual inoculation of N₂-fixing Bacillus OSU-140 and OSU-142, and/or mixed inoculations with three bacteria significantly increased grain yield of barley compared with single inoculations of OSU-142 and M-13. In contrast with other combinations, dual inoculation of N₂-fixing OSU-140 and P-solubilizing M-13 did not always significantly increase leaf, root and sugar yield of sugar beet, grain and biomass yield of barley compared to single applications both with N₂-fixing bacteria. The beneficial effects of the bacteria on plant growth varied significantly depending on environmental conditions, bacterial strains, and plant and soil conditions.     

Short-term inhibition of legume N2 fixation by nitrate

The hypothesis of NO ₂ ⁻ toxicity as the causative factor of NO ₃ ⁻ inhibition of nitrogenase (N₂ase; EC 1.18.6.1) activity has been evaluated using a short-term exposure (3 d) of several legumes. Treatment of plants with 10 mM NO ₃ ⁻ induced nitrate reductase (NR) from bacteroids (EC 1.7.99.4) and nodule cytosol (EC 1.6.6.1) in most species. Regardless of the levels of both enzymes, significant accumulation of NO ₂ ⁻ did not occur in nodules. Dissection of nodules into cortical and infected regions, and subsequent NO ₂ ⁻ assays in conditions that suppressed enzyme activities, indicated that, in the short-term, bacteroid NR does not generate NO ₂ ⁻ in vivo. This is probably because NO ₃ ⁻ access is restricted to the nodule cortex. Accumulation of NO ₂ ⁻ at levels that are damaging for N₂ase and leghaemoglobin were only observed when a delay occurred between dissection and assaying of nodules. It is concluded that NO ₂ ⁻ is not responsible for the initial NO ₃ ⁻ -induced decline of N₂ase activity, and that toxic amounts of NO ₂ ⁻ only build up in nodules following longer exposures to NO ₃ ⁻ , when this anion is actively reduced by bacteroid and cytosol enzymes.