Conservation in Soil of H2 Liberated from N2 Fixation by Hup- Nodules

Pigeon peas (Cajanus cajan) were grown in large soil columns (90-cm length by 30-cm diameter) and inoculated with four different strains of cowpea rhizobia, which varied with respect to hydrogen uptake activity (Hup). Despite the profuse liberation of H₂ from Hup^- nodules in vitro, H₂ gas was not detected in any of the soil columns. When H₂ was injected into the columns, the rates of consumption were highest in the treatments (including control) containing Hup^- nodules (218 and 177 nmol · h⁻¹ · cm⁻²) and lowest in the Hup⁺ treatments (158, 92, and 64 nmoles · h⁻¹ · cm⁻²). In situ H₂ uptake rates in small soil cores at fixed distances from the nodules decreased exponentially with distance from the nodule (R² = 0.99). This decrease in H₂ consumption was associated with a similar decrease in numbers of H₂-oxidizing chemolithotrophic bacteria as determined by the most-probable-number method. On the basis of two equations derived separately upon diffusive theory (Fix's Law) and kinetic theory (Michaelis-Menten), the empirically derived rate constants and coefficients indicated that all of the H₂ emitted from Hup^- nodules would be consumed by H₂-oxidizing bacteria within a 3- to 4.5-cm radius of the nodule surface. It is concluded that H₂ is not lost from the soil-plant ecosystem during N₂ fixation in C. cajan but is conserved by H₂-oxidizing bacteria.     

Transformation of inorganic phosphorus in flooded soils under rice cropping

Summary The concentrations of water soluble and ammonium acetate extractable phosphorus in all the soils under investigation first increased and then decreased with time of submergence under rice cropping. The increase in soluble phosphorus in the three acid soils of Luisiana, Casiguran and Guadalupe was related to decrease in the concentration of iron, calcium and reductant soluble phosphates, while in slightly acidic Maahas clay, the increase was associated with decrease in iron and aluminium phosphates. But in the calcareous soil, the increase was due to decrease in the concentration of aluminium and reductant soluble phosphates. The decrease was due to the re-formation of insoluble aluminium, iron and calcium phosphates in Luisiana and Guadalupe clays, to the formation of aluminium and calcium phosphates in Maahas and to the formation of calcium phosphate only in Casiguran fine sand. The application of phosphorus at the rate of 100 pounds per acre produced better tillering, more penicles and higher straw and grain productions in Luisiana, Casiguran and Guadalupe only where the level of soluble phosphate was very low in pots where no phosphorus was applied. This study, thus, indicated the necessity of phosphorus fertilization in low land rice for soils which are low in phosphorus and high in active iron and aluminium.     

Contribution of basal portion of shoot to N2 fixation associated with wetland rice

Summary Oryza sativa grown in flooded soil were transferred to water culture solution and acetylene reduction activities (ARA) of intact plants and rootless plants were measured for 5 h. Relative rate of ARA associated with the rootless wetland rice plant as compared with an intact plant varied from 8 to 100 percent, depending on the growth stage and varieties of rice and highest at the early stage (3 weeks after transplanting) for all varieties tested (IR26, Latisail, Khao Lo, and JBS236). ARA of shoots was associated with basal parts of the shoots about 3 cm from the base of wetland cultivated rice andOryza australiensis. Phyllospheric ARA was negligible except for senescent outer leaf sheaths. Microaerophilic N₂-fixing bacteria also inhabited basal parts of shoots (outer leaf sheaths and stems) of wetland rice. These findings suggest that N₂-fixation is partly associated with the shoots of wetland rice plants.     

Nitrogen fixation by non-legumes in tropical agriculture with special reference to wetland rice

Summary Of the 143 million hectares of cultivated rice land in the world, 75% are planted to wetland rice. Wet or flooded conditions favour biological nitrogen fixation by providing (1) photic-oxic floodwater and surface soil for phototrophic, free-living or symbiotic blue-green algae (BGA), and (2) aphotic-anoxic soil for anaerobic or microaerobic, heterotrophic bacteria. TheAzolla-Anabaena symbiosis can accumulate as much as 200 kg N ha^–1 in biomass. In tropical flooded fields, biomass production from a singleAzolla crop is about 15 t fresh weight ha^–1 or 35 kg N ha^–1. Low tolerance for high temperature, insect damage, phosphorus requirement, and maintenance of inoculum, limit application in the tropics. Basic work on taxonomy, sporulation, and breeding ofAzolla is needed. Although there are many reports of the positive effect of BGA inoculation on rice yield, the mechanisms of yield increase are not known. Efficient ways to increase N₂-fixation by field-grown BGA are not well exploited. Studies on the ecology of floodwater communities are needed to understand the principles of manipulating BGA. Bacteria associated with rice roots and the basal portion of the shoot also fix nitrogen. The system is known as a rhizocoenosis. N₂-fixation in rhizocoenosis in wetland rice is lower than that ofAzolla or BGA. Ways of manipulating this process are not known. Screening rice varieties that greatly stimulate N₂-fixation may be the most efficient way of manipulating the rhizocoenosis. Stimulation of N₂-fixation by bacterial inoculation needs to be quantified.     

Root growth response of rainfed lowland rice to aerobic conditions in northeastern Thailand

Background and aims

Rice plants alternately experience anaerobic and aerobic conditions during their life cycle in rainfed lowlands. Each condition affects root growth differently. Our objective was to clarify the specific rice root response to aerobic conditions in rainfed lowlands.

Methods

At the Ubon Ratchathani Rice Research Center in northeastern Thailand, we obtained root samples from 17 ‘Surin1’ (Thai variety) BC₃-derived lines and 7 CT9993-5-10-1-M × IR62266-42-6-2 doubled-haploid lines from flooded and non-flooded paddy fields at the reproductive stage in 2010 and 2011.

Results

In the non-flooded trial, rice was grown aerobically by draining the perched water; soil moisture at a depth of 20 cm fluctuated between ?10 and ?30 kPa. Deep rooting was likely promoted under aerobic conditions, but slightly drier soils under longer dry spells seemed to restrict root penetration, as the topsoil rapidly hardened during dry spells of only a few days. Fine-root development in the topsoil was inhibited under aerobic conditions.

Conclusions

Even without drought stress, rice roots respond significantly to the disappearance of standing water in rainfed lowlands via deep rooting and root branching. We identified one promising ‘Surin1’ BC₃-derived line showing an adaptive response of deep rooting under aerobic conditions, which can be used as a breeding material for rainfed lowland rice in Thailand.     

Effects of Exogenous Factors on Enzymes of Carbon Metabolism in Thermoacidophilic Bacteria of the GenusSulfobacillus

Aerobic thermoacidophilic chemolithotrophic bacteria Sulfobacillus thermosulfidooxidans1269^Tand Sulfobacillus thermosulfidooxidanssubsp. asporogenes41 were shown to be resistant to stress factors, including high concentrations of Zn²⁺(0.8 M) and supraoptimal concentrations of H⁺(pH 1.2). The growth and biomass gain rates decreased, but bacteria retained their functions. The activity of nearly all enzymes involved in carbon metabolism decreased. Glucose was primarily metabolized via the Entner–Doudoroff pathway. The activity of tricarboxylic acid cycle enzymes decreased compared to that in cells grown under normal conditions. After saturation of the growth medium with 5 vol % CO₂, sulfobacteria utilized glucose by the Embden–Meyerhof and pentose phosphate pathways under mixotrophic conditions.     

Assay of nitrogen supplying capacity of tropical rice soils

Summary The nitrogen supplying capacity of 39 wetland rice soils evaluated by two anaerobic incubation methods and six chemical methods was compared with N uptake of IR 26 rice grown on these soils under flooded conditions in a greenhouse pot study. The uptake of N by rice correlated highly with the N supplying capacity determined by anaerobic incubation methods involving incubation of soils at 30°C for 2 weeks (r=0.84^**) or at 40°C for 1 week (r=0.82^**) as well as with the organic carbon (r=0.82^**) and total N (r=0.84^**) contents of soils. Among the chemical indexes, available N determined by the oxidative release of soil N by alkaline permanganate, acid permanganate, acid dichromate and hydrogen peroxide also provided good index of soil N availability to rice. According to these results soil organic carbon and total N contents seem to be good indexes of available nitrogen in tropical wetland rice soils.     

15N2 incorporation by rhizosphere soil influence of rice variety,organic matter and combined nitrogen

Summary Heterotrophic nitrogen fixation by rhizosphere soil samples from 20 rice cultivars grown under uniform field conditions was estimated employing¹⁵N-tracer technique. Rhizosphere soil samples from different rice cultivars showed striking differences with regard to their ability to incorporate¹⁵N₂. Rhizosphere samples from rice straw-amended (3 and 6 tons/ha) soil exhibited more pronounced nitrogen-fixing activity than the samples from unamended soil; while the activity of the rhizosphere samples from soils receiving combined nitrogen (40 and 80 kg N/ha) was relatively low. However, the inhibitory effect of combined nitrogen was not expressed in the presence of rice straw at 6 tons/ha. Results suggest that plant variety, application of combined nitrogen and organic matter influence the rhizosphere nitrogen fixation.     

Rice yield in relation to electroultrafiltration extractable soil potassium

Summary Potassium in wetland rice soils from five different locations in the Philippines was analyzed using the electroultrafiltration (EUF) technique and by extraction withN NH₄ acetate (pH 7). The soils contained low exchangeable K and responded to K application. The K soil test values were calibrated against the rice response to K application under field conditions. EUF extractable soil K correlated highly significantly with the rice yield response to K fertilizer, whereas the NH₄ acetate extractable K (exchangeable K) did not. Under limiting K supply in soils, rice yield depends more on the EUF-K than on the exchangeable K. Maximum grain yields were obtained when the EUF-K values after harvest and before wetland preparation were above 30 ppm K.     

Inhibition of methanogenesis by sulphate reducing bacteria competing for transferred hydrogen

A methanogenic bacterial consortium was obtained after inoculation of benzoate medium under N₂/CO₂ atmosphere with intertidal sediment. A hydrogen donating organotroph andMethanococcus mazei were isolated from this enrichment. H₂-utilising sulphate reducing bacteria were isolated under H₂/CO₂ in the absence of organic electron donors. TheMethanococcus was able to produce methane in yeast extract medium under N₂/CO₂ if the H₂ donating organism was present, and sulphate reduction occurred if the hydrogen utilising sulphate reducing bacteria were grown with the H₂ donating organism. The ability of the H₂ utilising sulphate reducing bacteria to inhibitMethanococcus competitively was shown in cultures containing both of these H₂ utilising bacteria.Abbreviations HDO hydrogen donating organism - SRB sulphate reducing bacteria - HSRB hydrogen utilising sulphate reducing bacteria     

Error caused by carbon dioxide in determination of ammonium by direct steam distillation of tropical wetland rice soils

Summary A study was made with eight Philippine wetland rice soils to quantify the possible error caused by the CO₂ evolved during direct distillation of soil suspensions in aerobic and anaerobic conditions with MgO. The error caused by CO₂ was eliminated by absorbing the ammonia distilled in H₂SO₄, which was gently boiled to derive off the CO₂ absorbed. The possible error caused by CO₂ was not eliminated when boric acid was used for absorbing ammonia. The difference in NH₄ ⁺ values determined by using sulfuric acid and boric acid methods gave an estimate of the error caused by CO₂. It was found that CO₂ evolved caused negative error in the NH₄ ⁺ values obtained using the direct distillation of soil suspensions with MgO in presence of KCl. The magnitude of error was higher and significant with anaerobic soil samples but this error was negligible with aerobic soils.     

Effects of Soil and Water Content on Methyl Bromide Oxidation by the Ammonia-Oxidizing Bacterium Nitrosomonas europaea

Little information exists on the potential of NH₃-oxidizing bacteria to cooxidize halogenated hydrocarbons in soil. A study was conducted to examine the cooxidation of methyl bromide (MeBr) by an NH₃-oxidizing bacterium, Nitrosomonas europaea, under soil conditions. Soil and its water content modified the availability of NH₄⁺ and MeBr and influenced the relative rates of substrate (NH₃) and cosubstrate (MeBr) oxidations. These observations highlight the complexity associated with characterizing soil cooxidative activities when soil and water interact to differentially affect substrate and cosubstrate availabilities.     

Effect of Xanthobacter,isolated and characterized from rice roots,on growth of wetland rice

With an autotrophic, N-free medium, Xanthobacter populations were isolated from the roots of wetland rice grown under field conditions. Xanthobacter populations ranged from 3.2×10⁴ to 5.1×10⁵ colony-forming units (cfu) g^-1 of root and averaged 47-fold higher on the root or rhizoplane than in the neighbouring nonrhizosphere. Characterization studies indicated dissimilarities in carbon utilization and motility among the isolated Xanthobacter strains and other recognized Xanthobacter species. Under gnotobiotic conditions, the population of one isolate, Xanthobacter sp. JW-KR1, increased from 10⁵ to 10⁷ cfu plant^-1 1 d after inoculation when a rice plant was present, but declined to numbers below the limit of detection (<10⁴ cfu assembly^-1) after 3 d in the absence of a plant. Scanning electron microscopy revealed Xanthobacter as pleomorphic forms on the rhizoplane. To assess the effect of Xanthobacter on plant growth, rice plants were grown under greenhouse conditions in plant assemblies containing sand and half-strength Hoagland's nutrient solution with and without nitrogen. Plants were either inoculated with 10⁵ cfu Xanthobacter g^-1 of sand or left uninoculated. After 40 d, plants without nitrogen showed no significant differences in top or root dry weight, plant height, root length, or number of tillers or leaves, whether the plants were inoculated or uninoculated. However, when nitrogen was added, inoculated plants had a significantly larger top dry weight (15%) and number of leaves (19%) than uninoculated plants. Under conditions of added and no added nitrogen, acetylene reduction assays showed Xanthobacter sp. JW-KR1 produced <0.1 (below detection limit) and 7 nmol C₂H₄ plant^-1 h^-1, respectively. Under the conditions studied, the results suggest that both Xanthobacter and wetland rice derive some benefits from their association.     

Enumeration of Free-Living Aerobic N2-Fixing H2-Oxidizing Bacteria by Using a Heterotrophic Semisolid Medium and Most-Probable-Number Technique

A heterotrophic semisolid medium was used with two sensitive assay methods, C₂H₂ reduction and O₂-dependent tritium uptake, to determine nitrogenase and hydrogenase activities, respectively. Organisms known to be positive for both activities showed hydrogenase activity in both the presence and absence of 1% C₂H₂, and thus, it was possible to test a single culture for both activities. Hydrogen uptake activity was detected for the first time in N₂-fixing strains of Pseudomonas stutzeri. The method was then applied to the most-probable-number method of counting N₂-fixing and H₂-oxidizing bacteria in some natural systems. The numbers of H₂-oxidizing diazotrophs were considerably higher in soil surrounding nodules of white beans than they were in the other systems tested. This observation is consistent with reports that the rhizosphere may be an important ecological niche for H₂ transformation.     

土壤氧气可获得性对双季稻田温室气体排放通量的影响

为探讨土壤氧气可获得性(SOA)对双季稻田温室气体排放的影响,利用静态箱气相色谱法对多种管理措施影响下稻田温室气体排放通量和土壤氧化还原电位(Eh)、pH值及田间淹水深度(H)等3种SOA因子进行了观测。结果表明,甲烷(CH₄)排放最集中的Eh值、pH值和H范围分别为-100-0mV、5 < pH < 6和1-5cm,3个范围内分别观测到48.8%、61.1%和77.0%的CH₄排放,其中H对CH₄排放影响最明显,单独由其就可解释37.8%的CH₄排放通量(P < 0.0001)。对于氧化亚氮(N₂O),观测到较多的负通量,其纯排放最密集的3种SOA因子的范围分别是:0-100mV、5 < pH < 6和1-5cm,而200-300mV是其排放的临界Eh范围,高于此范围N₂O排放极少。厌氧的反硝化过程是双季稻田N₂O产生的主导过程。可为水稻田温室气体排放机理研究提供基础数据。     

H2 oxidation,O2 uptake and CO2 fixation in hydrogen treated soils

In many legume nodules, the H₂ produced as a byproduct of N₂ fixation diffuses out of the nodule and is consumed by the soil. To study the fate of this H₂ in soil, a H₂ treatment system was developed that provided a 300 cm³ sample of a soil:silica sand (2:1) mixture with a H₂ exposure rate (147 nmol H₂ cm^–3hr^–1) similar to that calculated exist in soils located within 1–4 cm of nodules (30–254 nmol H₂ cm^–3hr^–1). After 3 weeks of H₂ pretreatment, the treated soils had a K_m and V_max for H₂ uptake (1028 ppm and 836 nmol cm^–3 hr^–1, respectively) much greater than that of control, air-treated soil (40.2 ppm and 4.35 nmol cm^–3 hr^–1, respectively). In the H₂ treated soils, O₂, CO₂ and H₂ exchange rates were measured simultaneously in the presence of various pH₂. With increasing pH₂, a 5-fold increase was observed in O₂ uptake, and CO₂ evolution declined such that net CO₂ fixation was observed in treatments of 680 ppm H₂ or more. At the H₂ exposure rate used to pretreat the soil, 60% of the electrons from H₂ were passed to O₂, and 40% were used to support CO₂ fixation. The effect of H₂ on the energy and C metabolism of soil may account for the well-known effect of legumes in promoting soil C deposition.     

Distribution of Hydrogen-Metabolizing Bacteria in Alfalfa Field Soil

H₂ evolved by alfalfa root nodules during the process of N₂ fixation may be an important factor influencing the distribution of soil bacteria. To test this hypothesis under field conditions, over 700 bacterial isolates were obtained from fallow soil or from the 3-mm layer of soil surrounding alfalfa (Medicago sativa L.) root nodules, alfalfa roots, or bindweed (Convolvulus arvensis L.) roots. Bacteria were isolated under either aerobic or microaerophilic conditions and were tested for their capacity to metabolize H₂. Isolates showing net H₂ uptake and ³H₂ incorporation activity under laboratory conditions were assigned a Hup⁺ phenotype, whereas organisms with significant H₂ output capacity were designated as a Hout⁺ phenotype. Under aerobic isolation conditions two Hup⁺ isolates were obtained, whereas under microaerophilic conditions five Hup⁺ and two Hout⁺ isolates were found. The nine isolates differed on the basis of 24 standard bacteriological characteristics or fatty acid composition. Five of the nine organisms were isolated from soil around root nodules, whereas the other four were found distributed among the other three soil environments. On the basis of the microaerophilic isolations, 4.8% of the total procaryotic isolates from soil around root nodules were capable of oxidizing H₂, and 1.2% could produce H₂. Two of the Hup⁺ isolates were identified as Rhizobium meliloti by root nodulation tests, but the fact that none of the isolates reduced C₂H₂ under the assay conditions suggested that the H₂ metabolism traits were associated with various hydrogenase systems rather than with nitrogenase activity. Results from this study support the concept that H₂ evolution by alfalfa root nodules has a significant effect on the surrounding microenvironment and influences the number and diversity of bacteria occupying that region.     

Effects of short-term drainage and aeration on the production of methane in submerged rice soil

Emission rates of CH₄ were measured in microcosms of submerged soil which were planted with rice. Drainage of the rice microcosms for 48 h resulted in drastically decreased CH₄ emission rates which only slowly recovered to the rates of the undrained controls. Drainage also resulted in drastically increased sulphate concentrations which only slowly decreased to nearly zero background values after the microcosms were submerged again. The mechanisms responsible for the decrease of CH₄ production by aeration were investigated in slurries of a loamy and a sandy Italian rice soil. Incubation of the soil slurries under anoxic conditions resulted first in the reduction of nitrate, sulphate and ferric iron before CH₄ production started. Incubation of the soil slurries for 48 h under air resulted in immediate and complete inhibition of CH₄ production. Although the soil slurries were then again incubated under anoxic conditions (N₂ atmosphere), the inhibition of CH₄ production persisted for more than 30 days. The redox potential of the soil increased after the aeration but returned within 15 days to the low values typical for CH₄ production. However, the concentrations of sulphate and of ferric iron increased dramatically after the aeration and stayed at elevated levels for the period during which CH₄ production was inhibited. These observations show that even brief exposure of the soil to O₂ allowed the production of sulphate and ferric iron from their reduced precursors. Elevated sulphate and ferric iron concentrations allowed sulphate-reducing and ferric iron-reducing bacteria to outcompete methanogenic bacteria on H₂ as common substrate. Indeed, concentrations of H₂ were decreased as long as sulphate and ferric iron were high so that the Gibbs free energy of CH₄ production from H₂/CO₂ was also increased (less exergonic). On the other hand, concentrations of acetate, the more important precursor for CH₄, were not much affected by the short aeration of the soil slurries, and the Gibbs free energy of CH₄ production from acetate was highly exergonic suggesting that acetotrophic methanogens were not outcompeted but were otherwise inhibited. Aeration also resulted in increased rates of CO₂ production and in a short-term increase of N₂O production. However, these increases were < 10% of the decreased production of CH₄ and did not represent a trade-off in terms of CO₂ equivalents. Hence, short-term drainage and aeration of submerged paddy fields may be a useful mitigation option for decreasing the emission of greenhouse gases.     

Autecology of Bradyrhizobium japonicum in soybean-rice rotations

The effect of rice culture on changes in the number of a strain of soybean root-nodule bacteria, (Bradyrhizobium japonicum CB1809), already established in the soil by growing inoculated soybean crops, was investigated in transitional red-brown earth soils at two sites in south-western New South Wales. At the first site, 5.5 years elapsed between the harvest of the last of four successive crops of soybean and the sowing of the next. In this period three crops of rice and one crop of triticale were sown and in the intervals between these crops, and after the crop of triticale, the land was fallowed. Before sowing the first rice crop, the number of Bradyrhizobium japonicum was 1.32×10⁵ g^–1 soil. The respective numbers of bradyrhizobia after the first, second and third rice crops were 4.52 ×10⁴, 1.26×10⁴ and 6.40×10² g^–1 soil. In the following two years the population remained constant. Thus sufficient bradyrhizobia survived in soil to nodulate and allow N₂-fixation by the succeeding soybean crop. At the second site, numbers of bradyrhizobia declined during a rice crop, but the decline was less than when the soil was fallowed (400-fold cf. 2200-fold). Multiplication of bradyrhizobia was rapid in the rhizosphere of soybean seedlings sown without inoculation in the rice bays. At 16 days after sowing, their numbers were not significantly different (p<0.05) from those in plots where rice had not been sown. Nodulation of soybeans was greatest in plots where rice had not been grown, but yield and grain nitrogen were not significantly different (p<0.05). Our results indicate that flooding soil has a deleterious effect on the survival of bradyrhizobia but, under the conditions of the experiments, sufficient B. japonicum strain CB 1809 survived to provide good nodulation after three crops of rice covering a total period of 5.5 years between crops of soybean.     

Close Association of Azospirillum and Diazotrophic Rods with Different Root Zones of Kallar Grass

The populations of diazotrophic and nondiazotrophic bacteria were estimated in the endorhizosphere and on the rhizoplane of Kallar grass (Leptochloa fusca) and in nonrhizosphere soil. Microaerophilic diazotrophs were counted by the most-probable-number method, using two semisolid malate media, one of them adapted to the saline-sodic Kallar grass soil. Plate counts of aerobic heterotrophic bacteria were done on nutrient agar. The dominating N₂-fixing bacteria were differentiated by morphological, serological, and physiological criteria. Isolates, which could not be assigned to a known species, were shown to fix nitrogen unequivocally by ¹⁵N₂ incorporation. On the rhizoplane we found 2.0 × 10⁷ diazotrophs per g (dry weight) of root, which consisted in equal numbers of Azospirillum lipoferum and Azospirillum-like bacteria showing characteristics different from those of known Azospirillum species. Surface sterilization by NaOCI treatment effectively reduced the rhizoplane population, so that bacteria released by homogenization of roots could be regarded as endorhizosphere bacteria. Azospirillum spp. were not detected in the endorhizosphere, but diazotrophic, motile, straight rods producing a yellow pigment occurred with 7.3 × 10⁷ cells per g (dry weight) of root in the root interior. In nonrhizosphere soil we found 3.1 × 10⁴ nitrogen-fixing bacteria per g. Diazotrophs were preferentially enriched in the Kallar grass rhizosphere. In nonrhizosphere soil they made up 0.2% of the total aerobic heterotrophic microflora, on the rhizoplane they made up 7.1%, and in the endorhizosphere they made up 85%. Owing to high numbers in and on roots and their preferential enrichment, we concluded that diazotrophs are in close association with Kallar grass. They formed entirely different populations on the rhizoplane and in the endorhizosphere.