Radial gas diffusion from roots of rice (Oryza sativa L.) and Kallar grass (Leptochloa fusca L. Kunth), and effects of inoculation withAzospirillum brasilense Cd

Cortical root air space (aerenchyma) helps rice and Kallar grass to survive flooding conditions. The dependence of the oxygen concentration in the rhizosphere on the root aerenchyma volume, the plant age,-species and plant respiration is described. Additionally diffusional effects of different types of gases are evaluated. Inoculation of the rhizosphere with the micro-aerobically N₂-fixing microorganismAzospirillum brasilense Cd brought about an increased oxygen concentration in the rhizosphere by the factor 3.3 for rice and 5.3 for Kallar grass. This effect is thought to be due to enhanced root cell wall permeability probably caused by IAA-like phytohormones released by the bacteria.     

Improvement of degraded physical properties of a saline-sodic soil by reclamation with kallar grass (Leptochloa fusca)

A field experiment was conducted to evaluate the effectiveness of growing salt tolerant plants to improve the physical characteristics of a saline-sodic soil. Kallar grass [Leptochloa fusca (L.) Kunth], a species tolerant to salinity, sodicity and alkalinity, was irrigated for five years with poor quality ground water (EC = 0.14 S m^–1, SAR_adj=19.3, RSC = 9.7 meq L^–1). The soil physical properties of plant available water, saturated hydraulic conductivity, structural stability, bulk density and porosity were determined at the end of each year. The growth of kallar grass for three years significantly improved the physical properties of the soil and these were maintained with further growth of grass up to five years. Kallar grass significantly increased plant available water with time (r=0.97^**). The available water was highly correlated (r=0.92^**) with increases in soil organic matter content, porosity (r=0.99^**) and other physical properties. Soil hydraulic conductivity increased substantially with time from 0.035 to 55.6 mm d^–1 in the topsoil (0–20 cm) in five years and was significantly correlated with porosity, water retention, structural stability and organic matter content of soil. The soil structural stability index improved significantly from 32 to 151 with kallar grass and showed greater increases in the surface soil than at depth. The cropping of kallar grass resulted in a linear increase of soil organic matter content (r=0.92^**) which improved porosity and other soil physical properties (r0.82^*). This study confirmed that kallar grass is effective for rehabilitation and restoration of soil fertility in saline-sodic areas on a sustainable basis.     

Nitrogen fixation associated with non-legumes in agriculture

Summary This review examines the nitrogen cycle in upland agricultural situations where nonlegume N₂-fixation is likely to be important for crop growth. Evidence for associative fixation is adduced from accumulation of N in the top 15 cm soil under grasses, from N balances for crop production obtained from both pot and field experiments, in tropical and temperate environments, measurements of nitrogen (C₂H₂ reduction) activity, uptake of¹⁵N₂ by plants and¹⁵N isotope dilution. Factors influencing the activity such as the provision of carbon substrate by the plant and the efficiency of its utilisation by the bacteria, plant cultivar, soil moisture and N levels, and inoculation with N₂-fixing bacteria are discussed. Crop responses to inoculation withAzospirillum are detailed. The breakdown of crop residues, particularly straw, can support large levels of N₂-fixation. Cyanobacteria as crusts on the soil surface also fix nitrogen actively in many environments. Fixation by the nodulated, non-legume treesCasuarina andParasponia has beneficial effects in some cropping systems in Asia. I conclude that nonlegume N₂-fixation makes a significant contribution to the production of some major cereal crops in both temperate and tropical environments.     

Quantification of N2-fixation and survival of inoculated diazotrophs associated with roots of Kallar grass

White clover plants were grown for 97 days under two temperature regimes (20/15°C and 8/5°C day/night temperatures) and were supplied with either small amounts (a total of 80 mg N pot^–1) of ammonium (NH ₄ ⁺ ) or nitrate (NO ₃ ^– ) nitrogen, or received no mineral N and relied on N₂ fixation. Greatest growth and total leaf area of clover plants occurred in N₂ fixing and NO ₃ ^– -fed plants grown at 20/15°C and poorest growth occurred in NH ₄ ⁺ -fed plants grown at 8/5°C. Nodule mass per plant was greater at 8/5°C due to increased nodule numbers rather than increased dry weight per nodule. This compensated to some extent for the reduced N₂-fixing activity per unit dry weight of nodule tissue found at the low growth temperature up to 116 d after sowing, but thereafter both activity per nodule dry weight and activity per plant were greater at the low temperature. Highest nitrate reductase activity (NRA) per g fresh weight and total activity per leaf, petiole or root occurred in NO ₃ ^– -fed plants at 8/5°C. Low growth temperature resulted in a greater partitioning of total plant NRA to the roots of NO ₃ ^– -fed plants. The results are considered in relation to the use of N fertiliser in the spring under field conditions.     

Biochemical changes during composting of kallar grass (Leptochloa fusca L. Kunth)

Summary Biochemical changes during composting of kallar grass (Leptochloa fusca L. Kunth) were studied for twelve weeks. Some criteria based on cation-exchange capacity, acid-hydrolysable carbon, lignin-humus complex, humic acid and cumulative CO₂-C evolution were established for assessing the stability of kallar grass compost. Sequence of changes in different chemical and biological parameters indicated that the compost was stabilized in six weeks.

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Resumen Se han estudiado los cambios bioquímicos que tienen lugar durante y el compostaje del kallar (Leptochloa fusca L. Kunth) durante un periodo de 12 semanas. Se establecen criterios para valorar la estabilidad del compuesto, basados en: la capacidad de intercambio catiónico, el carbono ácido hidrolizable, el complejo ligno-húmico, los ácidos húmicos, y la evolución acumulativo C-CO₂. La secuencia de cambios en los distintos parámetros químicos y biológicos indican que el compuesto se estabilizó en 6 semanas.

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Résumé Les modifications biochimiques au cours du compostage du kallar-grass (Leptochloa fusca, L. Kunth) ont été étudiées pendant douze semaines. Plusieurs caractéristiques, comprenant la capacité d'échange de cations, l'hydrolyse acide du carbone, le complexe lignine-humus, les acides humiques et le dégagement de CO₂ on été établies pour évaluer la stabilité du compost de kallar-grass. La séquence de ces différents paramètres chimiques et biologiques montre que le compost est stable au bout de six semaines.

     

Nitrogen fixation in Pseudomonas stutzeri

A recently developed oxygen gradient system and a complex medium were used to isolate a microaerobically N₂-fixing heterotrophic bacterium from the rhizosphere of a high fixing Sorghum nutans cultivar. The isolate was identified as nif(+) phenotype of Pseudomonas stutzeri on the basis of cultural, physiological and biochemical characteristics, including DNA/DNA hybridization. N₂ fixation was demonstrated by assimilation of ¹⁵N₂ into cellular protein; the physiology of nitrogen fixation was studied. The isolate contains one 30 MD plasmid and can be cured with associated loss of N₂ fixation capability.Dedicated to Prof. Dr. W. Nultsch on the occasion of his 60th birthday     

Growth and solute composition of the salt-tolerant kallar grass [Leptochloa fusca (L.) Kunth] as affected by nitrogen source

A study was conducted to elucidate the effect of N form, either NH₄ ⁺ or NO₃ ^–, on growth and solute composition of the salt-tolerant kallar grass [Leptochloa fusca (L.) Kunth] grown under 10 mM or 100 mM NaCl in hydroponics. Shoot biomass was not affected by N form, whereas NH₄ ⁺ compared to NO₃ ^– nutrition caused an almost 4-fold reduction in the root biomass at both salinity levels. Under NH₄ ⁺ nutrition, salinity had no effect on the biomass yield, whereas under NO₃ ^– nutrition, increasing salinity from 10 mM to 100 mM caused 23% and 36% reduction in the root and shoot biomass, respectively. The reduced root growth under NH₄ ⁺ nutrition was not attributable to impaired shoot to root C allocation since N form did not affect the overall root sugar concentration and the starch concentration was even higher under NH₄ ⁺ compared to NO₃ ^– nutrition. The low NH₄ ⁺ (2 mM) and generally higher amino-N concentrations in NH₄ ⁺- compared to NO₃ ^–-fed plants indicated that the grass was able to effectively detoxify NH₄ ⁺. Salinity had no effect on Ca²⁺ and Mg²⁺ levels, whereas their concentration in shoots was lower under NH₄ ⁺ compared to NO₃ ^– nutrition (over 66% reduction in Ca²⁺; over 20% reduction in Mg²⁺), but without showing deficiency symptoms. Ammonium compared to NO₃ ^– nutrition did not inhibit K⁺ uptake, and the K⁺-Na⁺ selectivity either remained unaffected or it was higher under NH₄ ⁺ than under NO₃ ^– nutrition. Results suggested that while NH₄ ⁺ versus NO₃ ^– nutrition substantially reduced root growth, and also strongly modified anion concentrations and to a minor extent concentrations of divalent cations in shoots, it did not influence salt tolerance of kallar grass.     

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.     

Comparative water-use efficiency of Sporobolus arabicus and Leptochloa fusca and its relation with carbon-isotope discrimination under semi-arid conditions

Water-use efficiency (WUE) of Leptochloa fusca (L.) Kunth (Kallar grass) and Sporobolus arabicus Boiss. was determined under different soil moisture regimes. Plants grown in lysimeters were subjected to three soil moisture regimes, viz. well-watered (100%), medium-watered (75%), and low-watered (50%) of total available water (TAW). The soil moisture was restored on alternate days by adding the required volume of water on the basis of neutron moisture meter readings taken from neutron access tubes installed in each lysimeter. The grasses were harvested after suitable intervals (4 months) to obtain maximum biomass. Leaf samples collected at each harvest were analyzed for carbon-isotope discrimination (¹³C) with an isotope ratio (¹³C/¹²C) mass spectrometer. Results indicated significant differences in WUE of both grasses subjected to different water regimes. Sporobolus arabicus showed higher WUE than Kallar grass. However, Kallar grass showed better value of yield response factor (k _y = 0.649) compared with Sporobolus (k _y = 1.06) over the entire season. The data confirm that these grasses can be grown successfully in water-limited environments by selecting an optimum soil moisture level for maximum biomass production. The mean carbon-isotope discrimination (¹³C) of Kallar grass (–14.4) and Sporobolus (–12.8) confirm that both are C₄ plants. The carbon-isotope discrimination () was significantly and negatively correlated with WUE of the two species studied. The results of the present study confirm that ¹³C or of leaves can be used as good predictor of WUE in some C₄ plants.     

Effect of salinity and inoculation on growth,nitrogen fixation and nutrient uptake ofVigna radiata (L.) Wilczek

This study reports the effect of salinity and inoculation on growth, ion uptake and nitrogen fixation byVigna radiata. A soil EC_e level of 7.5 dS m⁻¹ was quite detrimental causing about 60% decline in dry matter and grain yield of mungbean plants whereas a soil EC_e level of 10.0 dS m⁻¹ was almost toxic. In contrast most of the studied strains of Rhizobium were salt tolerant. Nevertheless, nodulation, nitrogen fixation and total nitrogen concentration in the plant was drastically affected at high salt concentration. A noticeable decline in acetylene reduction activity occurred when salinity level increased to 7.5 dS m⁻¹.     

Effects of sulphur nutrition on growth and nitrogen fixation of pea (Pisum sativum L.)

A S-deficient soil was used in pot experiments to investigate the effects of S addition on growth and N₂-fixation in pea (Pisum sativum L.). Addition of 100 mg S pot⁻¹ increased seed yield by more than 2-fold. Numbers of pods formed were the most sensitive yield component affected by S deficiency. Sulphur addition also increased the concentration of N in leaves and stems, and the total content of N in the shoots. The amounts of N fixed by pea were determined at four growth stages from stem elongation to maturity, using the ¹⁵N dilution technique. Sulphur addition doubled the amount of N fixed at all growth stages. In contrast, leaf chlorophyll content and shoot dry weight were increased significantly by S addition only after the flowering and pod fill stage, respectively. Pea roots were found to have high concentrations of S, reaching approximately 10 mg g⁻¹ dry weight and being 2.6–4.4 times the S concentration in the shoots under S-sufficient conditions. These results suggest that roots/nodules of pea have a high demand for S, and that N₂-fixation is very sensitive to S deficiency. The effects of S deficiency on pea growth were likely to be caused by the shortage of N, due to decreased N₂-fixation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Nitrogen fixation (15N dilution) with soybeans under Thai field conditions

Controlled environment and field studies were conducted to determine relationships between various measurements of N₂ fixation using soybeans and to use these measures to evaluate a number ofBradyrhizobium japonicum strains for effectiveness in N₂ fixation in Thai soils.¹⁵N dilution measurements of N₂ fixation showed levels of fixation ranging from 32 to 161 kg N ha⁻¹ depending on bacterial strain, host cultivar and location. Midseason measures of N₂ fixation were correlated with each other, but not related measures taken at maturity. Ranking ofB. japonicum strains based on performance under controlled conditions in N-free media were highly correlated with rankings based on soybean seed yields and N₂ fixation under field conditions. This study showed that inoculation of soybeans with effectiveB. japonicum strains can result in significant increases in yield and uptake of N through fixation. The most effective strains tested for use in Thai conditions were those isolated from Thai soils; however, effective strains from other locations were also of benefit.     

Nitrogen fixation associated with a hotspring green alga

A unicellular alga which can grow in the light without a combined nitrogen source was isolated from a hot spring. The cells were almost spherical, usually 5–10 m in diameter. Absorption spectra of the watersoluble pigments and of the acetone-extracted ones revealed the existence of chlorophyll a and b and the absence of phycobilins. Thin sections examined by electron microscopy revealed an eukaryotic organization with features typical of the coccoid green algae (the Chlorococcales). Cells divided by internal cytokinesis and subsequent liberation of daughter cells from the parental wall, in a way similar to Chlorella. The alga reduced acetylene to ethylene and incorporated ¹⁵N₂ into cell protoplasm when incubated in a low oxygen atmosphere. Nitrogenase activity was light-dependent, microaerophilic and thermophilic. Although the association of symbiotic nitrogen fixing prokaryotes with the cells may still be possible, any such organisms have not so far been detected.Abbreviations Used DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Chl chlorophyll - MBM modified Bristol medium - TLC thin layer chromatography     

Occurrence of effective nitrogen-scavenging bacteria in the rhizosphere of kallar grass

Bacteria occurring in high numbers on the rhizoplane of kallar grass grown at a natural site in Pakistan were effective scavengers of traces of combined nitrogen from the atmosphere. Bacteria grew under appropriate conditions in nitrogen-free semi-solid malate medium in the form of a typical subsurface pellicle which resulted in a significant nitrogen gain in the medium within 3 to 4 days of incubation; this could be also measured by¹⁵N-dilution. Bacteria grew and incorporated nitrogen under an atmosphere containing NH₃ and N₂O. A rapid and strong binding of strain W1 to roots of kallar grass grown in hydroponic culture was found by using a³²P-tracer technique. We obtained no evidence for diazotrophy of our strains because they failed to grow on nitrogen-free media when gases of high purity were used. No¹⁵N₂ was incorporated when bacteria were grown on¹⁵N₂ although a nitrogen gain was found, no acetylene reduction was observed and no homology with DNA containing sequences ofnifHDK structural genes for the nitrogenase components fromKlebsiella pneumoniae were detected. Owing to close contact of these bacteria with roots of kallar grass, utilization of scavenged nitrogen by the plant may have to be taken into account.     

Nitrogen fixation in a large shallow lake: rates and initiation conditions

The fixation of molecular nitrogen (N₂fix) by cyanobacteria in situ and in PO₄-P enrichment experiments was investigated in large shallow Lake Võrtsjärv in 1998–2000. In this lake, N₂fix started when TN/TP mass ratio was about 20, which is much higher than Redfield mass ratio 7. The rate of N₂fix varied between 0.81 and 2.61 gN l^–1 d^–1 and maximum rate (2.61 gN l^–1 d^–1) was measured in 15.08.2000. In L. Võrtsjärv a lag period of a couple of weaks occurred between the set-up of favourable conditions for N₂fix as the appearance of N₂-fixing species and depletion of mineral nitrogen, and the real N₂fix itself. However, if the favorable conditions for N₂fix occurred in the lake, N₂fix started after enrichment with PO₄-P in mesocosms even then when no N₂fix was detected in the lake. N₂fix in mesocosms was also more intensive than in lake water. In our experiments PO₄-P concentrations higher than 100 gP l^–1started to inhibit N₂fix.     

In vitro propagation of a Saccharum officinarum (L.) and Sclerostachya fusca (Roxb.) A. Camus hybrid

Summary Callus induction and plant differentiation were obtained in an intergeneric hybrid of Saccharum officinarum and Sclerostachya fusca. The sub clones showed morphological variation. Chromosome numerical variation was not observed but structural aberrations were noticed in some sub clones. The study indicates the use of tissue culture technique for inducing intergeneric gene transfer in Saccharum hybrids.     

Establishment of inoculatedAzospirillum spp. in the rhizosphere and in roots of field grown wheat and sorghum

Summary Four field experiments were carried out with wheat or sorghum in different regions of Brazil. The aim was to study the establishment of inoculatedAzospirillum strains, marked with resistance to various antibiotics, in the rhizosphere and in roots. The levels of the various antibiotics were chosen according to the resistance of the indigenousAzospirillum population.Azospirillum brasilense strains Sp 107 and Sp 245 could be established in all three wheat experiments and predominated within theAzospirillum population in washed, and especially in surface sterilized, roots. Strains Sp 7 and Cd established poorly in wheat roots.Azospirillum lipoferum Sp S82 represented 72% of the root isolates from sorghum inoculated with this strain. This strain and naturalAzospirillum infection became concentrated in the upper parts of the root system. Improved methods for root surface sterilization in which the absence ofAzospirillum on the root surface was established by pre-incubating roots with paraffin-capped ends in NFb medium confirmed the establishment of inoculatedAzospirillum strains within sorghum roots in the field.     

Nitrogen fixation in perennial forage legumes in the field

Nitrogen acquisition is one of the most important factors for plant production, and N contribution from biological N₂ fixation can reduce the need for industrial N fertilizers. Perennial forages are widespread in temperate and boreal areas, where much of the agriculture is based on livestock production. Due to the symbiosis with N₂-fixing rhizobia, perennial forage legumes have great potential to increase sustainability in such grassland farming systems. The present work is a summary of a large number of studies investigating N₂ fixation in three perennial forage legumes primarily relating to ungrazed northern temperate/boreal areas. Reported rates of N₂ fixation in above-ground plant tissues were in the range of up to 373 kg N ha^–1 year^–1 in red clover (Trifolium pratense L.), 545 kg N ha^–1 year^–1 in white clover (T. repens L.) and 350 kg N ha^–1 year^–1 in alfalfa (Medicago sativa L.). When grown in mixtures with grasses, these species took a large fraction of their nitrogen from N₂ fixation (average around 80%), regardless of management, dry matter yield and location. There was a large variation in N₂ fixation data and part of this variation was ascribed to differences in plant production between years. Studies with experiments at more than one site showed that also geographic location was an important source of variation. On the other hand, when all data were plotted against latitude, there was no simple correlation. Climatic conditions seem therefore to give as high N₂ fixation per ha and year in northern areas (around 60°N) as in areas with a milder climate (around 40°N). Analyzing whole plants or just above-ground plant parts influenced the estimate of N₂ fixation, and most reported values were underestimated since roots were not included. Despite large differences in environmental conditions, such as N fertilization and geographic location, N₂ fixation (Nfix; kg N per ha and year) was significantly (P<0.001) correlated to legume dry matter yield (DM; kg per ha and year). Very rough, but nevertheless valuable estimations of Nfix in legume/grass mixtures (roots not considered) are given by Nfix = 0.026DM + 7 for T. pratense, Nfix = 0.031DM + 24 for T. repens, and Nfix = 0.021DM + 17 for M. sativa.     

The effect of moisture stress on stolon and adventitious root development in white clover (Trifolium repens L.)

Summary Humidity, at the young nodes of white clover stolones, varied by enclosing nodes in the atmosphere above a range of saturated solutions, inhibited root initiation at 85% RH or less. The threshold humidity for root initiation increased to about 93% on young nodes subject to moisture stress or old nodes on well watered plants in which root initiation had been previously suppressed by low humidity.Roots at old nodes and at the three youngest on stolons were either subject to moisture stress or adequately watered. Growth of young roots and N₂-fixation were more adversely affected by the direct effects of drought than by subjecting old roots to drought. Although old roots under stress affected new root growth and N₂-fixation, length of roots and lateral root number were little affected. By contrast stolon growth was affected more by stress to old roots than to young nodes, although after 6 weeks the contribution made by young roots to stolon growth was almost as high as old roots.The data suggest that deep roots at old nodes will allow clover stolons to grow during drought due to the high acropetal movement of water but initiation of roots and functioning of young roots at the soil surface will be adversely affected, with possible implications on the persistence of clover.     

Effect of varying periods of inoculation ofAzolla pinnata R. Brown on its growth,nitrogen fixation and response to rice crop

Summary Inoculation of water fernAzolla pinnata R. Brown (Bangkok isolate) at the rate of 500kg fresh weight ha⁻¹ in rice fields at weekly intervals after planting in addition to 30 kg N ha⁻¹ as urea showed a decrease in its growth and N₂-fixation with delay in application. Use of Azolla up to 3 weeks after planting (WAP) during wet and 4 WAP during dry season produced significantly more grain yield than 30 kg N ha⁻¹, whereas its application upto one WAP produced more grain yield than 60 kg N ha⁻¹. Grain yield with Azolla applied at the time of planting was similar to that of 60 kg N treatment during the wet season. Higher grain yields in zero and one WAP Azolla treatments resulted due to increase in both number of panicles m⁻² and number of grains/panicle while the subsequent Azolla inoculations increased grain yield mainly by producing more number of grains/panicle. Dry matter and total N yields at maturity of rice crop were more with Azolla application upto 3 WAP during wet and 2 WAP during dry season while the reduction in sterility (%) was observed upto one WAP over 30 kg N ha⁻¹ during both seasons. Number of tillers m⁻² and dry matter production at maximum tillering and flowering were more than 30 kg N ha⁻¹ with the use of Azolla upto one WAP. Increased grain N yield was observed with the use of Azolla upto 4 WAP during two seasons whereas straw N yield increased upto one WAP during wet and 2 WAP during dry season.