Microbiological strategies for enhancing biological nitrogen fixation in nonlegumes

In an agro-ecosystem, industrially produced nitrogenous fertilizers are the principal sources of nitrogen for plant growth; unfortunately these also serve as the leading sources of pollution. Hence, it becomes imperative to find pollution-free methods of providing nitrogen to crop plants. A diverse group of free-living, plant associative and symbiotic prokaryotes are able to perform biological nitrogen fixation (BNF). BNF is a two component process involving the nitrogen fixing diazotrophs and the host plant. Symbiotic nitrogen fixation is most efficient as it can fix nitrogen inside the nodule formed on the roots of the plant; delivering nitrogen directly to the host. However, most of the important crop plants are nonleguminous and are unable to form symbiotic associations. In this context, the plant associative and endophytic diazotrophs assume importance. BNF in nonlegumes can be encouraged either through the transfer of BNF traits from legumes or by elevating the nitrogen fixing capacity of the associative and endophytic diazotrophs. In this review we discuss mainly the microbiological strategies which may be used in nonleguminous crops for enhancement of BNF.     

Export of nitrogenous compounds due to incomplete cycling within biological soil crusts of arid lands

Second only to water among limiting factors, nitrogen controls the fertility of most arid regions. Where dry and wet depositions are weak, as in the western US deserts, N inputs rely heavily on biological N(2) fixation. Topsoil cyanobacterial communities known as biological soil crusts (BSCs) are major N(2) fixation hot spots in arid lands, but the fate of their fixed N remains controversial. Using a combination of microscale and mesoscale process rate determinations, we found that, in spite of theoretically optimal conditions, denitrification rates in BSCs were paradoxically immaterial for nitrogen cycling. Denitrifier populations within BSCs were extremely low. Because of this absence of denitrification, and because of the limitation of respiration and ammonia oxidation by diffusive O(2) supply, we could demonstrate that BSCs function as net exporters of ammonium, nitrate and organic N to the soils they cover, in approximately stoichiometrically equal proportions. Overall export rates during periods of biological activity are in the range of tens to hundreds of mumol-N m(-2) h(-1), commensurate with those of N(2) fixation. These results explain the long-term dependence of BSCs on N(2) fixation, confirm their role in landscape fertility, and provide a robust argument for conservation of these endangered communities.     

Crucial role of (homo)glutathione in nitrogen fixation in Medicago truncatula nodules

Legumes form a symbiotic interaction with bacteria of the Rhizobiaceae family to produce nitrogen-fixing root nodules under nitrogen-limiting conditions. We examined the importance of glutathione (GSH) and homoglutathione (hGSH) during the nitrogen fixation process. Spatial patterns of the expression of the genes involved in the biosynthesis of both thiols were studied using promoter-GUS fusion analysis. Genetic approaches using the nodule nitrogen-fixing zone-specific nodule cysteine rich (NCR001) promoter were employed to determine the importance of (h)GSH in biological nitrogen fixation (BNF). The (h)GSH synthesis genes showed a tissue-specific expression pattern in the nodule. Down-regulation of the γ-glutamylcysteine synthetase (γECS) gene by RNA interference resulted in significantly lower BNF associated with a significant reduction in the expression of the leghemoglobin and thioredoxin S1 genes. Moreover, this lower (h)GSH content was correlated with a reduction in the nodule size. Conversely, γECS overexpression resulted in an elevated GSH content which was correlated with increased BNF and significantly higher expression of the sucrose synthase-1 and leghemoglobin genes. Taken together, these data show that the plant (h)GSH content of the nodule nitrogen-fixing zone modulates the efficiency of the BNF process, demonstrating their important role in the regulation of this process.     

Population genetic structure of wild rice Oryza glumaepatula distributed in the Amazon flood area influenced by its life-history traits

Wild plant species develop their own way of living to adapt to the specific environment of their habitats. Their life-history traits strongly affect the genetic structure of the population. The wild species Oryza glumaepatula Steud. growing in the Amazon basin seems to have characteristic life-history traits suited for the flood condition. At the vegetative growth stage, the culms frequently break at internodes. With no roots anchoring on the ground, plant bodies floating in the water move downriver by water current and wind. To examine the association between the life-history traits and genetic population structure of Amazonian O. glumaepatula , we analysed allozyme variability at 29 loci of 16 enzymes using 37 populations from five regions. Allozymes were not so variable (total gene diversity H _E = 0.044) compared with Asian wild rice, O. rufipogon Griff. The bottleneck effect and rare opportunity of interspecies gene flow may prevent the development of allozyme variability. Population genotypes tended to be differentiated among geographically isolated regions. Observed heterozygosities were much lower than expected heterozygosities, or gene diversity ( H _O = 0.003 for whole population) and F _IS over polymorphic loci was 0.931, indicating that O. glumaepatula has developed an inbreeding system. But, the intrapopulation gene diversity ( H _S) was higher than interpopulation gene diversity ( D _ST), as generally observed in outbreeding populations. The migration ability of O. glumaepatula makes long-distance seed dispersal possible. This might have led to frequent gene flow among populations.     

Nitrogen fixation and denitrification in plankton and periphyton of the Kama River Basin watercourses

The results of studies on molecular nitrogen fixation and denitrification by plankton, epiphytone, and epilithone in a number of natural and anthropogenic waterways of the Kama River Basin are presented for summer and autumn periods of 2005. The nitrogen fixation and denitrification was quantified by acetylene gas chromatography. The phototrophic anoxybiotic purple non-sulfurous bacteria, in particular, of Rhodopseudomonas, Rhodobacter and Rhodocyclus genera, play a significant role in active denitrification in the presence of sulfide-free flow water in the Kama River and its inflows in epilithone, algal-bacterial mats, and biological sewage disposal plants in Perm. On the other hand, the heterocyst cyanobacteria of Anabaena and Aphanizomenon genera had a leading position in active N₂ fixation in the plankton and periphyton of the lower river streams.     

Flood pulse influence and anthropic impact on the chemical composition and energy content of Oryza glumaepatula in an Amazonian lake.

The aim of this research was to study the flood pulse influence and the anthropic impact caused by bauxite tailings on the chemical composition of O. glumaepatula in Batata lake (PA, Brazil). Sampling was carried out in stands of O. glumaepatula in the low-water, filling, high-water, and drawdown periods in impacted and natural areas of Batata lake. During the low-water and drawdown periods the stands of O. glumaepatula were exposed, and in the filling and high-water periods the water depth was respectively 1.4 and 3.8 m. The collected material was dried at 70 degrees C, ground, and concentrations of total phosphorus, total nitrogen, organic carbon, and energy content were determined. The results indicate that the biomass increase, caused by the rise in water level, has a dilution effect on nitrogen and phosphorus concentrations in O. glumaepatula. The energy contents did not present significant differences in any of the studied periods. The results suggest that from the low water to filling period, nitrogen becomes more limiting to O. glumaepatula in the impacted area, whereas phosphorus becomes more limiting in the natural area. The population of O. glumaepatula contributes to the recovery of the impacted area of Batata lake as the detritus from this species accumulates over the sediment. This accumulation impedes future re-suspension of the bauxite tailings and increases the organic matter and nutrient concentrations in the impacted sediment.     

Influence of Algal Community Structure on Denitrification Rates in Periphyton Cultivated on Artificial Substrata

We conducted a field survey of periphyton cultivated on benthic mesh installations in freshwater aquatic systems, including two constructed wetlands and a pond, and also studied periphyton grown on a benthic mesh in laboratory mesocosms. The objectives of this study were to (1) determine if periphyton cultivated on benthic mesh denitrifies at higher rates than the underlying sediments and (2) determine if denitrification rates within periphyton vary with characteristics such as algal and bacterial community structure and biomass. We measured denitrification potential rates of field and laboratory periphyton by the acetylene inhibition method. We characterized algal community composition by algal identification and bacterial community composition by terminal restriction fragment length polymorphisms. Periphyton collected on benthic mesh from our field sites denitrified at significantly higher rates than the underlying sediments, regardless of sampling site or season. Results from both our field survey and laboratory studies indicated a significant, positive correlation between diatom presence and denitrification rate. In our laboratory studies, we found that periphyton with the highest diatom abundance showed the highest denitrification rates as well as a distinct bacterial community composition. These results suggest a synergistic relationship between diatoms and denitrifying bacteria that warrants further study.     

Trials to create artificial nitrogen-fixing symbioses and associations using in vitro methods: An outlook

Summary Biological nitrogen fixation is the most important process in which some prokaryotic organisms fix N₂ into ammonium. From an agricultural standpoint, biological nitrogen fixation (BNF) is critical because industrial production of nitrogen fertilizers seldom meets agricultural demands. To increase the BNF is one of the main challenges for the future. There are different possibilities for extending biological nitrogen fixation to the economically important plants. One of the possibilities is to create new artificial systems between diazotrophic bacteria and different higher plants. This is the main topic of the present review article which discusses the establishment of new associative and/or symbiotic systems, via introduction of diazotrophic bacteria into the roots by different methods; and incorporation of nitrogen-fixing bacteria in the entire plant by in vitro methods, through the establishment of intracellular endosymbioses via induced uptake of bacteria by plant protoplasts (endocytobiosis), and establishment of intercellular associations by forced introduction of bacteria into the plant tissues (exocytobiosis). The common characteristic of the methods to create artificial plant-microbe systems for atmospheric nitrogen fixation is the use of in vitro plant systems: cells, tissues and organ cultures. The review pays particular attention to new bacterial inoculation procedures for introduction of the diazotrophic bacteria inside the plant tissues.     

Old trees contribute bio-available nitrogen through canopy bryophytes

Symbiotic cyanobacteria??bryophyte associations on the forest floor are shown to contribute significantly to stand-level nitrogen budgets through the process of biological nitrogen fixation (BNF), but few studies have considered the role of canopy bryophytes. Given the high biomass of epiphytic bryophytes in many tree species of the North American temperate rain forest, we suggest that canopy bryophytes may contribute substantially to stand-level N dynamics. We confirm the presence of cyanobacteria and measure rates of BNF at three heights (0, 15 and 30 m) in Sitka spruce trees across three watershed estuaries of Clayoquot Sound, British Columbia, Canada. This study is the first to report BNF by cyanobacteria associated with epiphytic and forest floor bryophytes in the coastal temperate rain forest of North America. Cyanobacteria density was significantly greater in epiphytic bryophytes compared to mosses on the forest floor, and rates of BNF were highest at 30 m in the canopy. The majority of total stand-level BNF (0.76 kg N · ha^-1 · yr^-1) occurs in the canopy, rather than on the forest floor (0.26 kg N · ha^-1 · yr^-1). We suggest that BNF by cyanobacterial-bryophyte associations in the canopy of coastal temperate rain forests is a unique source of ecosystem N, which is dependent on large, old trees with high epiphytic bryophyte biomass.     

Biological Nitrogen Fixation in Two Tropical Forests: Ecosystem-Level Patterns and Effects of Nitrogen Fertilization

Humid tropical forests are often characterized by large nitrogen (N) pools, and are known to have large potential N losses. Although rarely measured, tropical forests likely maintain considerable biological N fixation (BNF) to balance N losses. We estimated inputs of N via BNF by free-living microbes for two tropical forests in Puerto Rico, and assessed the response to increased N availability using an on-going N fertilization experiment. Nitrogenase activity was measured across forest strata, including the soil, forest floor, mosses, canopy epiphylls, and lichens using acetylene (C₂H₂) reduction assays. BNF varied significantly among ecosystem compartments in both forests. Mosses had the highest rates of nitrogenase activity per gram of sample, with 11 ± 6 nmol C₂H₂ reduced/g dry weight/h (mean ± SE) in a lower elevation forest, and 6 ± 1 nmol C₂H₂/g/h in an upper elevation forest. We calculated potential N fluxes via BNF to each forest compartment using surveys of standing stocks. Soils and mosses provided the largest potential inputs of N via BNF to these ecosystems. Summing all components, total background BNF inputs were 120 ± 29 μg N/m²/h in the lower elevation forest, and 95 ± 15 μg N/m²/h in the upper elevation forest, with added N significantly suppressing BNF in soils and forest floor. Moisture content was significantly positively correlated with BNF rates for soils and the forest floor. We conclude that BNF is an active biological process across forest strata for these tropical forests, and is likely to be sensitive to increases in N deposition in tropical regions.     

一株高效甘蔗内生固氮菌NN08200的鉴定及其对甘蔗的促生长作用

【背景】生产上过高的氮肥投入是我国农业可持续发展的重要限制因子之一。利用生物固氮是减少氮肥施用量最为有效的途径,植物内生固氮菌资源的挖掘和利用对我国农业可持续发展具有重要实践意义。【目的】筛选高效甘蔗内生固氮菌,并对其联合固氮效率及促生长功能进行评价。【方法】从广西甘蔗茎基部组织分离筛选到一株内生固氮菌株NN08200,利用乙炔还原法测定固氮酶活性,通过菌落PCR扩增nif H基因确定菌株为固氮菌;通过菌株培养性状和菌体形态观察、Biolog细菌鉴定系统和16SrRNA基因序列分析确定该菌株的分类;采用盆栽接种测定菌株对甘蔗的实际促生长作用,并利用15N同位素稀释法测定其相对固氮效率。【结果】菌株NN08200的固氮酶活性达到2445nmolC2H4/(h·m L),菌株的nif H基因长度为339bp,与甘蔗内生固氮醋酸杆菌Gluconacetobacter diazotrophicus PAL5菌株的nif H相似性达99%;根据菌株培养性状和菌体形态观察、Biolog细菌鉴定系统和16SrRNA基因序列分析结果,菌株NN08200属于泛菌属(Pantoeasp.)细菌;盆栽接种菌株NN08200能显著提高甘蔗幼苗的株高和干重,15N同位素分析结果表明接种该菌株甘蔗植株的根、茎和叶从空气中获得氮素的百分率分别为7.49%、15.02%和10.79%,其联合固氮效率显著优于甘蔗内生固氮模式菌株G. diazotrophicus PAL5,利用后者接种的甘蔗根、茎和叶从空气中获得氮的百分率分别为3.53%、9.44%和4.87%。【结论】菌株Pantoea sp. NN08200是高效甘蔗内生固氮菌,其固氮促生长效果明显高于G. diazotrophicus PAL5菌株,可望研发成为优良固氮微生物肥料生产菌种,并可进一步用于甘蔗联合固氮菌作用机理的相关研究。     

Denitrification and Nitrogen Fixation in Alaskan Continental Shelf Sediments

Rates of nitrogen fixation and denitrification were measured in Alaskan continental shelf sediments. In some regions, rates of nitrogen fixation and denitrification appeared to be equal; in other areas, rates were significantly different. Potential rates of denitrification were found to be limited primarily by the available nitrate substrate. Major regional differences in rates of denitrification were not statistically significant, but significant differences were found for nitrogen fixation rates in different regions of the Alaskan continental shelf. Estimated net losses of nitrogen from Bering Sea sediments were calculated as 1.8 × 10¹² g of N/yr. Experimental exposure of continental shelf sediments to petroleum hydrocarbons reduced rates of nitrogen fixation and denitrification in some cases but not others. Long-term exposure was necessary before a reduction in nitrogen fixation rates was observed; unamended rates of denitrification but not potential denitrification rates (NO₃⁻ added) were depressed after exposure to hydrocarbons.     

Simultaneous measurement of denitrification and nitrogen fixation using isotope pairing with membrane inlet mass spectrometry analysis

A method for estimating denitrification and nitrogen fixation simultaneously in coastal sediments was developed. An isotope-pairing technique was applied to dissolved gas measurements with a membrane inlet mass spectrometer (MIMS). The relative fluxes of three N(2) gas species ((28)N(2), (29)N(2), and (30)N(2)) were monitored during incubation experiments after the addition of (15)NO(3)(-). Formulas were developed to estimate the production (denitrification) and consumption (N(2) fixation) of N(2) gas from the fluxes of the different isotopic forms of N(2). Proportions of the three isotopic forms produced from (15)NO(3)(-) and (14)NO(3)(-) agreed with expectations in a sediment slurry incubation experiment designed to optimize conditions for denitrification. Nitrogen fixation rates from an algal mat measured with intact sediment cores ranged from 32 to 390 microg-atoms of N m(-2) h(-1). They were enhanced by light and organic matter enrichment. In this environment of high nitrogen fixation, low N(2) production rates due to denitrification could be separated from high N(2) consumption rates due to nitrogen fixation. Denitrification and nitrogen fixation rates were estimated in April 2000 on sediments from a Texas sea grass bed (Laguna Madre). Denitrification rates (average, 20 microg-atoms of N m(-2) h(-1)) were lower than nitrogen fixation rates (average, 60 microg-atoms of N m(-2) h(-1)). The developed method benefits from simple and accurate dissolved-gas measurement by the MIMS system. By adding the N(2) isotope capability, it was possible to do isotope-pairing experiments with the MIMS system.     

Robust biological nitrogen fixation in a model grass–bacterial association

Nitrogen‐fixing rhizobacteria can promote plant growth; however, it is controversial whether biological nitrogen fixation (BNF) from associative interaction contributes to growth promotion. The roots of Setaria viridis, a model C₄ grass, were effectively colonized by bacterial inoculants resulting in a significant enhancement of growth. Nitrogen‐13 tracer studies provided direct evidence for tracer uptake by the host plant and incorporation into protein. Indeed, plants showed robust growth under nitrogen‐limiting conditions when inoculated with an ammonium‐excreting strain of Azospirillum brasilense. ¹¹C‐labeling experiments showed that patterns in central carbon metabolism and resource allocation exhibited by nitrogen‐starved plants were largely reversed by bacterial inoculation, such that they resembled plants grown under nitrogen‐sufficient conditions. Adoption of S. viridis as a model should promote research into the mechanisms of associative nitrogen fixation with the ultimate goal of greater adoption of BNF for sustainable crop production.     

一种高效研究大豆根瘤共生固氮的营养液栽培体系

为建立一种既可高效结瘤固氮, 又具有一定产量的大豆(Glycine max)营养液栽培系统, 设计并进行了2个试验。首先在不同供氮条件下, 研究了接种根瘤菌对大豆的结瘤状况、固氮能力、生物量及产量的影响。结果表明, 供氮过高或过低, 均影响大豆生长、产量形成及根瘤固氮; 并且植物生长所需的最适供氮水平远高于生物固氮所需的最适供氮水平。此外, 大豆生物固氮活性最高的时期在生殖期第一期(R1期)之前。由此推断, 大豆R1期前, 供应较低的氮, 有利于根瘤形成及固氮; 而从R1期起, 应提高供氮水平, 以促进植物生长及产量的形成。在此基础上开展第2个试验, 对供氮条件进行了优化处理(即R1期前低氮供应、R1期开始中氮供应)。结果表明, 与持续供应高氮相比, 优化供氮处理不仅可获得较多固氮酶活性较高的大根瘤, 还能保持较好的生长、获得更高的百粒重及维持80%左右的产量。研究结果不仅可为高效研究大豆根瘤共生固氮提供营养液配方, 还可为大豆高产高效栽培提供试验依据。     

Denitrification potential of a river floodplain during flooding with nitrate-rich water: grasslands versus reedbeds

Denitrification is a major mechanism for nitrogen removal from nitrogen-rich waters, but it requires oxygen-poor conditions. We assessed denitrification rates in nitrate-rich but also oxygen-rich river water during its stay in a floodplain. We measured diurnal oxygen fluctuations in floodwater along the river Rhine, and carried out an experiment to assess denitrification rates during day, evening and night. Denitrification in floodwater and flooded sediment were measured, comparing activity of periphyton and sediment from agricultural grasslands and reedbeds. Floodwater along the river Rhine was oxygen-saturated (> 10 mg O₂/L) during the day, but oxygen largely disappeared during the night (0.4–0.8 mg O₂/L). Independent of oxygen concentrations, denitrification in surface water alone hardly occurred. In flooded sediments, however, denitrification rates were much higher (1.1–1.5 mg N m^–2 h^–1), particularly at dark and oxygen-poor conditions (nighttime). In the experimental jars, reedbed-periphyton bacteria achieved similar denitrification rates as bacteria in sediment, but overall periphyton denitrification was of minor importance when calculated per square meter. Apart from oxygen levels, maximum denitrification appeared to be regulated by nitrate diffusion from water into the sediment, as the maximum quantity of N denitrified in the sediment equalled the quantity of N lossed from the surface water. Assessed 24-hr denitrification rates in the flooded floodplains (c. 15 mg N m^–2 d^–1) were similar in grasslands and reedbeds, and were rather low compared to rates in other floodplains.     

Nitrogen fixation in rice systems: state of knowledge and future prospects

Rice is the most important cereal crop. In the next three decades, the world will need to produce about 60% more rice than today's global production to feed the extra billion people. Nitrogen is the major nutrient limiting rice production. Development of fertilizer-responsive varieties in the Green Revolution, coupled with the realization by farmers of the importance of nitrogen, has led to high rates of N fertilizer use on rice. Increased future demand for rice will entail increased application of fertilizer N. Awareness is growing, however, that such an increase in agricultural production needs to be achieved without endangering the environment. To achieve food security through sustainable agriculture, the requirement for fixed nitrogen must increasingly met by biological nitrogen fixation (BNF) rather than by using nitrogen fixed industrially. It is thus imperative to improve existing BNF systems and develop N₂-fixing non-leguminous crops such as rice. Here we review the potentials and constraints of conventional BNF systems in rice agriculture, as well as the prospects of achieving in planta nitrogen fixation in rice.     

Abundance of microbial genes associated with nitrogen cycling as indices of biogeochemical process rates across a vegetation gradient in Alaska

Nitrification and denitrification processes are crucial to plant nutrient availability, eutrophication and greenhouse gas production both locally and globally. Unravelling the major environmental predictors for nitrification and denitrification is thus pivotal in order to understand and model environmental nitrogen (N) cycling. Here, we sampled five plant community types characteristic of interior Alaska, including black spruce, bog birch, tussock grass and two fens. We assessed abundance of functional genes affiliated with nitrification (bacterial and archaeal amoA) and denitrification (nirK/S and nosZ) using qPCR, soil characteristics, potential nitrification and denitrification rates (PNR and PDR) and gross mineralization rates. The main chemical and biological predictors for PNR and PDR were assigned through path analysis. The potential N cycling rates varied dramatically between sites, from some of the highest (in fens) to some of the lowest (in black spruce) measured globally. Based on path analysis, functional gene abundances were the most important variables to predict potential rates. PNR was best explained by bacterial amoA gene abundance followed by ammonium content, whereas PDR was best explained directly by nosZ gene abundance and indirectly by nirK/S gene abundance and nitrate. Hence, functional gene abundance is a valuable index that integrates recent environmental history and recent process activity, and therefore is a good predictor of potential rates. The results of this study contribute to our understanding of the relative importance of different biological and chemical factors in driving the potential for nitrification and denitrification across terrestrial ecosystems.     

水氮处理下不同品种水稻根系生长分布特征

为明确不同栽培条件下水稻(Oryza sativa)根系生长分布特征, 通过不同水氮处理和不同品种的水稻桶栽试验, 采用内置根架法, 于拔节期和抽穗期取样, 获取根系总干重(TRW)、不定根数(ARN)以及各类根(不定根、细分枝根和粗分枝根)的形态指标(长度、表面积和体积), 并分析植株根系生长状况和根系分布特征。结果显示: (1)各试验条件下抽穗期各项根系指标较拔节期均呈增长趋势。同一时期, 各项根系指标在3个施氮水平间均差异显著, 且随施氮量的增加而增加。不同水分处理下, 两个时期的ARN在湿润灌溉(W2)与保持水层(W1)之间差异均不显著, 而其他指标上W2处理均显著最高; 干旱处理 (W3)下, 仅拔节期的TRW和粗分枝形态指标与W1处理接近, 而在其他指标上均显著最低。不同品种间, ‘扬稻6号’ (V3)的各项根系指标均最高, 而‘日本晴’ (V1)和‘武香粳14’ (V2)间差异不显著。(2)各试验条件下, 抽穗期较拔节期根系下扎生长比例增加, 多分布于表层(0-5 cm)土中; 减少氮素和水分供应可提高根系在5 cm以下土层中的分布比例, 且分枝根反应最为明显; 品种V1和V2的深扎根性较V3明显。结果表明, 合理施氮与控水可优化水稻不同类型根的生长与分布特征, 但需考虑不同品种之间的差异。     

The barrier to radial oxygen loss from roots of rice (Oryza sativa L.) is induced by growth in stagnant solution

The present report describes experiments in which the effects of growth in aerated and stagnant nutrient solutions on adventitious root porosities and patterns of radial O2 loss (ROL) from the roots of four genotypes of rice (Oryza sativa L.) were evaluated. The genotypes studied are usually cultivated in farming systems which differ markedly in their degree of soil waterlogging and flooding. Rice genotypes were found to differ in the constitutive porosity (% gas space) of their adventitious roots when grown in aerated solutions (lowest was 16%, highest was 30%), and the roots grown in stagnant conditions had porosities between 28% and 38%. ROL from the adventitious roots raised in aerated solution increased with distance behind the tip in three of the four genotypes; whereas for roots raised in stagnant solution, ROL decreased with distance behind the tip which is indicative of a high resistance to diffusion between the aerenchyma and external medium. For example, at 35 mm behind the root tip the ROL from roots of the 'deepwater' cultivar grown in stagnant conditions was 0.7% of the rate of its aerated roots, for the 'lowland' cultivar it was 5.6%, and for one of the 'upland' cultivars it was 43.6%. Thus, the barrier to ROL from the adventitious roots in three of the four genotypes was induced by growth in stagnant nutrient solution. A low rate of ROL from the basal zones of roots in an O2-free environment is of adaptative value since longitudinal diffusion of O2 to the root apex would be enhanced which, in turn, enables greater penetration of roots into anaerobic soils.