Problems of the Acetylene Reduction Technique Applied to Water-Saturated Paddy Soils

The acetylene reduction assay for the measurement of N₂ fixation in a water-saturated paddy soil is limited by the slow diffusion of acetylene and ethylene. In laboratory incubation tests, vigorous shaking after the assay period is needed to release ethylene into the gas within the assay vials. Shaking prior to the incubation is also effective for dissolving acetylene in the water-saturated soil. However, a water-saturated soil depth of less than 10 mm during incubation is recommended. In field assays, some amounts of ethylene remain in the water-saturated soil phase of the acetylene reduction assay chamber, but stirring the water-saturated soil before sampling reduces the amount of ethylene remaining in soil. Evidence of a downward movement of acetylene and an upward movement of ethylene through rice plants was obtained. Because of the rapid transfer of acetylene to rice plant roots, an in situ acetylene reduction assay covering a rice hill is likely to detect nitrogen fixation in the proximity of roots where acetylene is easily accessible. Acetylene introduction to the water-saturated soil phase prior to assay did not greatly increase the acetylene reduction rate. Carbon dioxide enrichment in the assay chamber did not enhance nitrogen fixation in a paddy including rice and algae during a 1-day cycle.     

Nitrogenase activity in the rhizosphere of sugar cane and some other tropical grasses

Summary Roots of sugar cane had considerable nitrogenase activity and produced up to 5 n moles ethylene/h/g root by the reduction of acetylene. The rhizosphere soil and soil mid-way between the cane rows also reduced acetylene.Beijerinckia indica was abundant on roots and in the soil. Nitrogenase activity was also associated with roots ofPanicum maximum,Pennisetum purpureum andCymbopogon citratus.     

Endogenous ethylene production is a potential problem in the measurement of nitrogenase activity associated with excised corn and sorghum roots

Endogenous ethylene production was evaluated as a source of ethylene during acetylene reduction assays with freshly collected roots of field-grown corn, Zea mays L. cv Funks G-4646, and sorghum, Sorghum bicolor (L.) Moench. cv CK-60A. Ethylene production was not detected when roots were incubated in air without acetylene. The presence of endogenous ethylene production was confirmed when roots were incubated anaerobically and in the presence of 40 millimolar sodium hydrosulfite. Ethylene oxidase activity was also associated with excised roots. The rate of ethylene oxidation was higher than the rates of ethylene accumulation during either acetylene reduction assays or anaerobic incubations. These results indicate that the procedure of incubating roots of grasses in air to monitor endogenous ethylene production is not a valid control in acetylene reduction studies with grasses. The presence of endogenous ethylene production during acetylene reduction assays was demonstrated by using either CO to inhibit nitrogenase activity or chloramphenicol to inhibit nitrogenase synthesis in freshly excised roots.     

Immediate acetylene reduction by excised grass roots not previously preincubated at low oxygen tensions

Excised roots of Spartina alterniflora Loisel. and corn reduced acetylene in air without the previously reported period of zero activity lasting 8 to 18 hours. The profiles of acetylene-dependent ethylene accumulation by excised roots and intact plants of S. alterniflora were similar. No significant change in the number of bacteria associated with the roots was detectable during the assay. Most of the nitrogenase activity was detected in the roots and rhizomes of the plants. The salt marsh sediment also was capable of reducing acetylene. Additional damage to roots by washing and cutting increased the rate of acetylene reduction with samples incubated in air. Low concentrations of nitrate significantly inhibited the nitrogenase activity associated with the sediment and excised roots, but not with intact plants. Rates of acetylene reduction by excised corn roots were low. Oxidation and endogenous production of ethylene in the absence of acetylene were negligible. Measurements made with excised grass roots as described probably reflect the occurrence and magnitude of nitrogenase activity associated with the plants in the field.     

Endorhizal and Exorhizal Acetylene-reducing Activity in a Grass (Spartina alterniflora Loisel.)-Diazotroph Association

Earlier studies indicated that bacteria responsible for nitrogenase activity of some grasses are located inside the roots. Those studies were conducted with excised roots in which a long, unexplained “lag phase” occurred before initiation of nitrogenase activity. When hydroponically maintained Spartina alterniflora Loisel. was incubated in a two-compartment system with acetylene, ethylene was produced following, at most, a 2-hour lag in both the upper (shoot) and lower (roots + water) phases. Ethylene production in the upper phase not attributable to leaf-associated acetylene-reducing activity or to diffusion of ethylene from around the roots is considered to represent “endorhizal acetylene-reducing activity,” the internally produced ethylene diffusing into the upper phase via the lacunae. Ethylene produced in the lower phase is designated “exorhizal acetylene-reducing activity.” The endorhizal acetylene-reducing activity, in comparison to exorhizal activity, was relatively insensitive to additions of HgCl₂, NH₄Cl, or carbon sources to the lower phase. Post-lag acetylene-reducing activity of roots excised from plants growing in soil responded to additions in a manner similar to that of endorhizal acetylene-reducing activity, whereas post-lag acetylene-reducing activity of rhizosphere soil responded in a manner similar to that of exorhizal acetylene-reducing activity.     

Acetylene reduction by nitrogen-fixing blue-green algae

Summary Known nitrogen-fixing species of blue-green algae are capable of reducing acetylene to ethylene, but acetylene is not reduced by Anacystis nidulans, which does not fix nitrogen. Cycad root nodules which contain blue-green algae as endophytes reduce acetylene. Acetylene reduction is inhibited by carbon monoxide. Nitrate or ammonium-nitrogen has no immediate effect on algae reducing acetylene, but algae grown on nitrate-nitrogen gradually lose their capacity to reduce acetylene. Nitrate-nitrogen also inhibits heterocyst formation in these algae and there is a fairly direct correlation between the abundance of heterocysts in a particular sample and its capacity to reduce acetylene. Aphanizomenon flosaquae reduces acetylene and fixes nitrogen in unialgal culture and there is strong presumptive evidence that these reductions are carried out by the alga rather than by associated bacteria. The molar ratios of ethylene: ammonia produced vary within the range 1.4–1.8.     

Estimation of nitrogenase using a colorimetric determination for ethylene

Ethylene is measured by oxidizing it to formaldehyde and determining the formaldehyde colorimetrically. The assay is applied to estimation of nitrogenase in nodulated legume roots by measuring the ethylene produced from acetylene.     

Non-symbictic nitrogen fixation in the rhizosphere of Digitaria smutzii stent

Summary The acetylene reduction technique was used to test for the activity of nitrogenase in the rhizosphere of Digitaria smutzii grown in a solodic soil, and in the same soil in the absence of grass. The tests were made in McCartney bottles and regression analysis was used to compare rates of ethylene production. Roots with rhizosphere soil attached, exposed to acetylene and incubated anaerobically for 34 hours produced ethylene at a mean rate of 29 n moles C₂H₄/g root/h. No significant activity was detected under anaerobic conditions in the unplanted soil. Under aerobic conditions, significant but very low rates of ethylene production were observed in both the presence and absence of grass. Temperature treatments within the range 20°–32°C had no significant effect on rates of ethylene production.     

Field technique using the acetylene reduction method to assay nitrogenase activity and its association with the rice rhizosphere

Summary A plastic bag, used as a field assay chamber, assisted in the investigation of the acetylene reduction method as a technique to measure nitrogen-fixing activity in rice paddy fields. A study of the change in volume of the plastic bag and of the loss of acetylene and ethylene from the bag provided evidence that this plastic bag method was feasible in field assays. The field assay of rice at the grain ripening stage showed that the nitrogen-fixing activity increased linearly after a lag phase of 0 to 3 hours during a time-course experiment.Detachment of the aerial part of the rice plant from its root remaining in the field did not affect the nitrogen-fixing activity of the root.The markedly higher nitrogen-fixing activity in the planted areas of the field compared with the nonplanted areas between the plant rows indicated that the nitrogenase activity in the field is associated with the roots of the rice plant. re]19751204     

Nitrogen-fixing pseudomonads isolated from roots of plants grown in the canadian high arctic

Root-associated bacteria capable of reducing acetylene to ethylene (biological nitrogen fixation) were isolated from various native plants grown in the Canadian High Arctic. All the strains belonged to the genus Pseudomonas but varied in several physiological characteristics. The rates of acetylene reduction at 14 or 20 degrees C were higher than at 25 or 9 degrees C. Six strains reduced acetylene at 4 degrees C. All the strains exhibited chemotaxis to l-asparagine in semisolid agar at 4 to 25 degrees C. Eleven strains colonized roots of canola (Brassica campestris cv. Tobin) in field soil at population densities of log 4.3 to log 5.1 CFU/g of fresh root at 14 degrees C and log 4.0 to log 5.2 CFU/g of fresh root at 25 degrees C. Some of these nitrogen-fixing pseudomonad strains demonstrated a competitive advantage for root colonization over other rhizosphere bacteria at low temperatures. The combined capabilities of nitrogen fixation and root colonization by diazotrophic pseudomonads may be useful for the development of a biofertilizer inoculant for temperate and cold regions.     

Effect of Waterlogged Soil Conditions on the Production of Ethylene and on Water Relationships in Tomato Plants

The roots of tomato plants (Lycopersicon esculentum Mill., cv.Moneymaker) were exposed to low concentrations of oxygen bywaterlogging the soil or by growing the plants in nutrient solutionflushed with nitrogen gas. After 24 h, the rate of ethyleneproduction by the petioles, main stem, and shoot apex was increasedby 4–6-fold and the petioles developed epinastic curvatures.Removing the roots did not reproduce these responses. The amountsof ethylene produced by shoot tissues in response to physicalwounding was greatly increased by waterlogging the soil. The production of ethylene by roots was suppressed by the absenceof oxygen. When the roots were transferred back to an aerobicenvironment ethylene production quickly exceeded that observedin roots maintained continuously in aerobic conditions. The enhanced rate of ethylene production in the shoots occurredin the absence of increased water stress as measured with aleaf pressure chamber; leaf water potentials were increasedrather than decreased by waterlogging for 30 h or more. Thiswas associated with stomatal closure and reduced transpiration.Resistance to water flow through the plant increased as transpirationdecreased in response to waterlogging. However, at similar ratesof transpiration, resistance was normally lower in waterloggedplants than in controls.     

Effects of ethylene on root extension and nodulation of pea (Pisum sativum L.) and white clover (Trifolium repens L.)

Summary Low concentrations of ethylene, comparable with those known to occur in anaerobic soil, inhibited extension of pea roots to a similar extent to that previously reported for barley. Thus pea appeared to be less sensitive to ethylene than some non-leguminous dicotyledons. Perfusion of 10ppm of ethylene through the soil around the roots of pea and white clover resulted in reduced shoot dry weight. Nodulation, and the nitrogenase activity of those nodules which did form, was also greatly reduced. The ecological consequences of the sensitivity of nodulation and nitrogen fixation to ethylene, and the possible significance for quantitative studies involving the acetylene-reduction assay, are discussed.     

An increase in nitrogen content of Setaria italica and Zea mays inoculated with Azospirillum

Bacteria belonging to the genus Azospirillum isolated from Cynodon dactylon roots in Israel were compared with Azospirillum brasilense from Brazil and California for their ability to fix nitrogen in association with grasses under greenhouse conditions. The plants were grown in a system which avoided cross inoculation from the inoculated soil to the control, while maintaining the natural soil microflora and humidity level in the soil close to field capacity. The organisms tested significantly increased the dry weight of Zea mays and Setaria italica leaves, the total nitrogen content of these leaves (as measured by the Kjeldahl method), and supported acetylene reduction in intact nonsterile systems as compared with the noninoculated controls. Ethylene production in intact systems could be detected after 6h and was linear for 72h, providing a constant soil temperature (28-32 degrees C) was maintained.     

西南喀斯特石漠化环境适生植物构树细根、根际土壤化学计量特征

为了解西南喀斯特石漠化适生植物构树(Broussonetia papyrifera)对贫瘠土壤养分环境的适应策略, 及其细根、根际土壤的化学计量特征对石漠化等级的响应, 该研究以西南喀斯特石漠化环境适生植物构树为研究对象, 运用生态化学计量学方法, 开展不同等级石漠化环境构树细根、根际土壤有机碳(C)、全氮(N)、全磷(P)、全钾(K)、全钙(Ca)及全镁(Mg)养分含量特征及C、N、P化学计量特征研究。结果表明, 除Ca含量外, 喀斯特石漠化环境适生植物构树细根、根际土壤的养分含量均处于较低水平; 细根N:P为12.59, 表明构树生长受N和P共同限制; 随着石漠化等级的增加, 细根C、N含量和C:N、C:P呈先降后升的变化趋势, K、P含量是则表现为先升后降, Ca、Mg含量和N:P无明显变化规律; 不同等级石漠化环境中的构树根际土壤N、P、K、Ca含量呈不同的变化趋势, 而C、Mg含量及C、N、P化学计量特征的变化较不显著; 细根与根际土壤的化学计量特征之间存在显著的相关性, 二者的C、P、Ca、Mg含量、C:N、C:P分别对应呈显著正相关关系, 而N含量呈极显著负相关关系; 细根的K含量则较为稳定, 几乎不受根际土壤养分的影响。     

Nitrate inhibition of nodulation can be overcome by the ethylene inhibitor aminoethoxyvinylglycine

Previously, we reported (a) a positive correlation between the nitrate concentrations in growth medium and ethylene evolved from uninoculated and inoculated alfalfa (Medicago sativa) roots and (b) a negative correlation between ethylene evolution and nodulation. Here, we report that the inhibitory effect of NO₃⁻ on nodulation of alfalfa can be eliminated by the ethylene inhibitor aminoethoxyvinylglycine (AVG). This effect was probably related to the strong inhibition (90%) of ethylene biosynthesis caused by AVG in these inoculated and NO₃⁻-treated roots. These results support our hypothesis that the inhibitory effect of NO₃⁻ is mediated through the phytohormone ethylene. A possible role of endogenous ethylene in the autoregulation of nodulation also is discussed. AVG at 10 micromolar significantly (P < 0.05) increased total nitrogenase activity (acetylene reduction) in 2.5 and 5 millimolar NO₃⁻-fed plants probably as a result of the very high stimulation of nodulation.     

Root distribution in an Amazonian seasonal forest as derived from δ13C profiles

Carbon isotope ratios of the main stem in trees, saplings, and seedlings were correlated with their main stem diameter in an Amazonian seasonal forest. This correlation became the basis of using carbon isotope ratios of roots from various levels of the soil profile in order to determine root distribution from emergent, canopy and subcanopy trees, saplings and herbaceous understorey plants. It was observed that the distribution of roots in the soil profile is horizontally and vertically heterogeneous. Pockets of roots from saplings or herbaceous understorey plants were found as deep as 4 m and pockets of roots from emergent trees were found as shallow as 1 m depth.     

Influence of Acetylene on Growth of Sulfate-Respiring Bacteria

At a concentration of 20% of the atmosphere of the culture flasks, acetylene inhibited growth and carbon dioxide production by Desulfovibrio desulfuricans and Desulfovibrio gigas. The bacteria did not reduce acetylene to ethylene, and neither acetylene dicarboxylic acid nor ethylene was inhibitory. At 10%, acetylene was partially inhibitory for the desulfovibrios. At 5%, acetylene impeded the rate but did not limit the extent of growth and catabolism of the desulfovibrios. Desulfotomaculum ruminis was affected only negligibly, if at all, by acetylene and ethylene at any of these concentrations.     

Effects of applying etnylene to the root system of Zea mays on growth and nutrient concentration in relation to flooding tolerance

Previous studies have shown increases in the concentration of ethylene in the soil and roots of plants when the soil is water saturated (flooded). In Zea mays L. this occurs in association with an overall reduction in growth but without extensive foliar senescence and in conjunction with the development of an adventitious root system. We have assessed the possibility that ethylene may be involved in these responses to flooding. Mixtures of the gas in air were therefore supplied to the roots and stem-base of Z. mays growing in nutrient solution.
Seven or 14 d exposure to ethylene (1 or 5 νl 1⁻¹) inhibited seminal root elongation and growth in dry weight and accelerated the emergence of adventitious roots, although their final length and dry weight were depressed. Leaf extension was inhibited by 0.1,1.0 or 5.0 μl 1⁻¹ ethylene around the roots; leaves extending rapidiy at the start of treatment were the most sensitive. Final shoot fresh and dry weights were depressed by the gas but tie shootrroot dry weighl ratio and percentage dry matter were not affected greatly. Leaf chlorosis was not observed but the concentration of phosphorus in the shoots was 26 to 31% below normal.
When aeration of the nutrient solution was stopped, the concentration of dissolved oxygen declined and the concentration of ethylene in the roots increased. Similar changes occur in response to soil flooding. Root and shoot growth was slowed by non-aeration although the shootroot dry weight ratio remained unchanged. The phosphorus concentration of the shoots was depressed but there was little chlorosis or leaf death. The similarity in these respects between the effects of ethylene and non-aeration suggests that in flooded Z. mays , ethylene contributes to their development by accelerating the emergence of adventitioos roots, inhibiting phosphorus accumulation in the shoots and by a non-toxic inhibition of plant growth.     

Soil physical strength rather than excess ethylene reduces root elongation of Eucalyptus seedlings in mechanically impeded sandy soils

Seedling establishment in heavily compact soils is hampered by poor root growth caused by soil chemical or physical factors. This study aims to determine the role of ethylene in regulating root elongation through mechanically impeded sandy soils using Eucalyptus todtiana F. Muell seedlings. Concentrations of ethephon (1, 10, and 100???M) were added to non-compact soils, and endogenous ethylene production from seedling roots was compared to ethylene production of roots grown in physically compacted field soils (98.6?% sand). The ethylene-inhibitor 3,5-diiodo-4-hydroxybenzoic acid (DIHB) (0.1???M) was included for each treatment to counteract the negative effects of excess ethylene or compact soils on root elongation. Root elongation was reduced in high ethylene soils by 49?% and high bulk density soils by 44?%. Root ethylene production increased ninefold in roots grown in the high ethylene environment (100???M), but decreased 80?% in compact soils. The use of DIHB did not alter root length and produced varying results with respect to ethylene production, suggesting an interaction effect involving high amounts of soil ethylene. While ethylene regulates root growth, the physical strength of sandy soils is the major factor limiting root elongation in mechanically impeded soils.     

A Major Error in the Acetylene Reduction Assay: Decreases in Nodular Nitrogenase Activity Under Assay Conditions

Observations on both attached nodulated roots and detached noduleshave revealed that nitrogenase activity in many legume speciesdeclined rapidly in the presence of acetylene, with a concurrentreduction in respiration. The reduction began within a few minutesof exposure to acetylene and continued for 30–60 min beforea new steady-state was attained. A similar decline in H₂ evolutionand respiration was observed when N₂ was replaced with argonor helium. This suggests that the decrease is linked to thecessation of ammonia production. Measurements of ¹⁵N₂ uptakedemonstrated that it is the pre-decline rather than final rateof ethylene production which represents the real rate of nitrogenaseactivity. The implications of these findings for the interpretationof acetylene reduction and hydrogen evolution data are considered. Key words: Roots, Acetylene, Nitrogenase activity