R. D. Holsten

Bio: R. D. Holsten is an academic researcher from DuPont. The author has contributed to research in topics: Nitrogenase & Azotobacter. The author has an hindex of 2, co-authored 2 publications receiving 2103 citations.

The Acetylene-Ethylene Assay for N2 Fixation: Laboratory and Field Evaluation

Abstract: The methodology, characteristics and application of the sensitive C(2)H(2)-C(2)H(4) assay for N(2) fixation by nitrogenase preparations and bacterial cultures in the laboratory and by legumes and free-living bacteria in situ is presented in this comprehensive report. This assay is based on the N(2)ase-catalyzed reduction of C(2)H(2) to C(2)H(4), gas chromatographic isolation of C(2)H(2) and C(2)H(4), and quantitative measurement with a H(2)-flame analyzer. As little as 1 mumumole C(2)H(4) can be detected, providing a sensitivity 10(3)-fold greater than is possible with (15)N analysis.A simple, rapid and effective procedure utilizing syringe-type assay chambers is described for the analysis of C(2)H(2)-reducing activity in the field. Applications to field samples included an evaluation of N(2) fixation by commercially grown soybeans based on over 2000 analyses made during the course of the growing season. Assay values reflected the degree of nodulation of soybean plants and indicated a calculated seasonal N(2) fixation rate of 30 to 33 kg N(2) fixed per acre, in good agreement with literature estimates based on Kjeldahl analyses. The assay was successfully applied to measurements of N(2) fixation by other symbionts and by free living soil microorganisms, and was also used to assess the effects of light and temperature on the N(2) fixing activity of soybeans. The validity of measuring N(2) fixation in terms of C(2)H(2) reduction was established through extensive comparisons of these activities using defined systems, including purified N(2)ase preparations and pure cultures of N(2)-fixing bacteria.With this assay it now becomes possible and practicable to conduct comprehensive surveys of N(2) fixation, to make detailed comparisons among different N(2)-fixing symbionts, and to rapidly evaluate the effects of cultural practices and environmental factors on N(2) fixation. The knowledge obtained through extensive application of this assay should provide the basis for efforts leading to the maximum agricultural exploitation of the N(2) fixation reaction.

Stable Isotopes in Plant Ecology

Abstract: ▪ Abstract The use of stable isotope techniques in plant ecological research has grown steadily during the past two decades. This trend will continue as investigators realize that stable isotopes can serve as valuable nonradioactive tracers and nondestructive integrators of how plants today and in the past have interacted with and responded to their abiotic and biotic environments. At the center of nearly all plant ecological research which has made use of stable isotope methods are the notions of interactions and the resources that mediate or influence them. Our review, therefore, highlights recent advances in plant ecology that have embraced these notions, particularly at different spatial and temporal scales. Specifically, we review how isotope measurements associated with the critical plant resources carbon, water, and nitrogen have helped deepen our understanding of plant-resource acquisition, plant interactions with other organisms, and the role of plants in ecosystem studies. Where possible we also...

Global inputs of biological nitrogen fixation in agricultural systems

Abstract: Biological dinitrogen (N2) fixation is a natural process of significant importance in world agriculture. The demand for accurate determinations of global inputs of biologically-fixed nitrogen (N) is strong and will continue to be fuelled by the need to understand and effectively manage the global N cycle. In this paper we review and update long-standing and more recent estimates of biological N2 fixation for the different agricultural systems, including the extensive, uncultivated tropical savannas used for grazing. Our methodology was to combine data on the areas and yields of legumes and cereals from the Food and Agriculture Organization (FAO) database on world agricultural production (FAOSTAT) with published and unpublished data on N2 fixation. As the FAO lists grain legumes only, and not forage, fodder and green manure legumes, other literature was accessed to obtain approximate estimates in these cases. Below-ground plant N was factored into the estimations. The most important N2-fixing agents in agricultural systems are the symbiotic associations between crop and forage/fodder legumes and rhizobia. Annual inputs of fixed N are calculated to be 2.95 Tg for the pulses and 18.5 Tg for the oilseed legumes. Soybean (Glycine max) is the dominant crop legume, representing 50% of the global crop legume area and 68% of global production. We calculate soybean to fix 16.4 Tg N annually, representing 77% of the N fixed by the crop legumes. Annual N2 fixation by soybean in the U.S., Brazil and Argentina is calculated at 5.7, 4.6 and 3.4 Tg, respectively. Accurately estimating global N2 fixation for the symbioses of the forage and fodder legumes is challenging because statistics on the areas and productivity of these legumes are almost impossible to obtain. The uncertainty increases as we move to the other agricultural-production systems—rice (Oryza sativa), sugar cane (Saccharum spp.), cereal and oilseed (non-legume) crop lands and extensive, grazed savannas. Nonetheless, the estimates of annual N2 fixation inputs are 12–25 Tg (pasture and fodder legumes), 5 Tg (rice), 0.5 Tg (sugar cane), <4 Tg (non-legume crop lands) and <14 Tg (extensive savannas). Aggregating these individual estimates provides an overall estimate of 50–70 Tg N fixed biologically in agricultural systems. The uncertainty of this range would be reduced with the publication of more accurate statistics on areas and productivity of forage and fodder legumes and the publication of many more estimates of N2 fixation, particularly in the cereal, oilseed and non-legume crop lands and extensive tropical savannas used for grazing.

Biological invasion by Myrica faya in Hawai'i: plant demography, nitrogen fixation, ecosystem effects

Abstract: Myrica faya, an introduced actinorhizal nitrogen fixer, in invading young volcanic sites in Hawaii Volcanoes National Park. We examined the population biology of the invader and ecosystem-level consequences of its invasion in open-canopied forests resulting from volcanic cinder-fall. Although Myrica faya is nominally dioecious, both males and females produce large amounts of fruit that are utilized by a number of exotic and native birds, particularly the exotic Zosterops japonica. In areas of active colonization, Myrica seed rain under perch trees of the dominant native Metrosideros polymorpha ranged from 6 to 60 seeds m{sup {minus}2} yr{sup {minus}1}; no seeds were captured in the open. Planted seeds of Myrica also germinated an established better under isolated individuals of Metrosideros than in the open. Diameter growth of Myrica is > 15-fold greater than that of Metrosideros, and the Myrica population is increasing rapidly. Rates of nitrogen fixation were measured using the acetylene reduction assay calibrated with {sup 15}N. Myrica nodules reduced acetylene at between 5 and 20 {mu}mol g{sup {minus}1} h{sup {minus}1}, a rate that extrapolated to nitrogen fixation of 18 kg ha{sup {minus}1} in a densely colonized site. By comparison, all native sources of nitrogen fixation summed to 0.2 kg ha{sup {minus}1}more » yr{sup {minus}1}, and precipitation added < 4 kg ha{sup {minus}1} yr{sup {minus}1}. Measurements of litter decomposition and nitrogen release, soil nitrogen mineralization, and plant growth in bioassays all demonstrated that nitrogen fixed by Myrica becomes available to other organisms as well. We conclude that biological invasion by Myrica faya alters ecosystem-level properties in this young volcanic area; at least in this case, the demography and physiology of one species controls characteristics of a whole ecosystem.« less

N2-Fixation in Field Settings: Estimations Based on Natural 15N Abundance

Abstract: This paper reviews a growing body of literature on the use of variations in the natural abundance of 15N to estimate the fractional contribution of N2-fixation to N2-fixing systems. This method is based on the small difference in 15NN abundance which frequently occurs between N derived from N2-fixation and N derived from other sources. The requirement of the method is that this difference be significant. Whether this requirement is met is site specific and must be empirically established at each site of interest. Advantages and disadvantages of this method are compared with those of more conventional methods. Sources of error, including heterogeneity of 15NN abundance of non-atmospheric N sources are considered. Tests of the method, under both greenhouse and field conditions, are described. Estimates based on this method compare favourably with other methods for field evaluation of N2-fixation, provided that the site and the sampling strategy are appropriate for application of the method. Applications of the method in several ecosystems are described.

Applications of the acetylene-ethylene assay for measurement of nitrogen fixation

Abstract: A comprehensive report of the acetylene reduction assay for measurement of N2 fixation is presented. The objective is to facilitate the effective use and identify some potential limitations of the method. The report is based on more than 200 accounts of the use of this technique in 15 countries during the last 5 years. Methods of measurement of N2 fixation are compared. Nomenclature, e.g., N2[C2H2] fixed, is introduced to identify values of N2 fixation determined by C2H2-C2H2 assay. The biochemical basis of the assay is described along with relevant characteristics including Km, C2H2/N2 conversion factor, and specific N2[C2H2]-fixing activities obtained with various systems. Effects of combined nitrogen, temperature, light, pO2, N2, pC2H2 and water on activity are summarized. Available methods for sample preparation, assay chamber, gas phase, assay condition, termination of reaction, C2H4 analysis and expression of results are compared. The many uses of the C2H2-C2H4 assay for investigations of the biochemistry of nitrogenase and physiology of N2-fixing organisms, definition of N2-fixing organisms and measurement of field N2 fixation by legume, non-legume, soil, marine, rhizosphere, phylloplane and mammalian samples are tabulated.