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William R. Graves and Lorna C. Wilkins

A laboratory exercise for illustrating aspects of biological nitrogen fixation (BNP) to students in plant science courses is described. Surface-sterilized seeds of black locust (Robinia pseudoacacia L.) and soybean (Glycine max Merill) were sown together in plastic containers filled with a sterile, soilless medium. Containers were assigned randomly to treatments designed to show how inoculation with two strains of rhizobial bacteria and application of nitrate affect root nodulation and plant growth. Results demonstrated that BNF occurs in diverse legumes, that legumes vary in the strains of rhizobia with which they associate, that nodulation is inhibited by nitrate, and that dependency on BNP can reduce growth compared with plants provided nitrate.

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Lih-Yuh Yueh and David L. Hensley

The influence of 12 pesticides on C2H4 reduction and modulation of soybean (Glycine max L. Merr.) and lima bean (Phaseolus lunatus L.) was evaluated. All except diazinon were innocuous at 3× the label rate. Diazinon decreased C2H4 reduction of soybean 2 days after application, but not after 7 days or at normal label rates. Nitrogen fixation of excised nodules imbibed with diazinon indicated that it may have directly affected nitrogenase function. Soybean nodule numbers were decreased by application of 3× rates of methomyl and trifluralin, but lima bean nodule numbers were decreased only by trifluralin. Trifluralin also depressed soybean but not lima bean modulation at label rates. Methomyl did not affect soybean modulation at label rate. Both chemicals were non-toxic to Rhizobium sp. in a disc inhibition study.

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Raymond Baptiste, Lurline Marsh, Dyremple Marsh, and David Trinklein

Low temperatures adversely affect legume- Rhizobium symbiosis in the temperate regions. Plant growth and N-fixation of two pigeonpea and two cowpea genotypes were examined at three temperature regimes (20/10 C, 30120 C and 38/25 C day/night). Sterilized seeds were inoculated with broth culture containing approximately 1 × 109 cells ml-1 of Bradyrhizobium USDA 3278, 3458 and 3472. Nitrogen fixation by pigeonpea was inhibited at 20/10 C. Cowpea IT82E-16 inoculated with USDA 3458 at 20/10 C produced the greatest amount of nodules. Inoculation had no effect on Nitrogenase activity in pigeonpea. Pinkeye Purple Hull inoculated with USDA 3472 at 20110 C had the highest Nitrogenase activity. These results indicate a wide degree of variability among genotypes and Bradyrhizobium in their response to temperature.

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Stephanie Rademan and Dyremple Marsh

A wide range of methodologies, ranging from Leonard jar to growth pouch have been used to investigate the nitrogen fixation process in leguminous crops. The effectiveness of most of these research methods have been questioned. Problems encountered vary from difficulty in root separation to water log conditions. This study was undertaken to determine the effect of different growing media on nodule development and harvestability. Black and Red seed coat kidney bean were surface sterilized and inoculated with the Rhizobium phaseoli strain UMR 1899. Seeds were planted in 8.5 cm diameter sterile clay pots containing the respective growing medium. These growing media were sand, Promix GM, Promix BX, and fritted clay. The black seed coat kidney bean had higher germination rate under all media for all dates recorded. Black kidney bean grown in sand and fritted clay had plant heights significantly greater than ones grown in the other media on the third harvest date. Nodule activities as measured by shoot dry weight and nodule number were significantly higher in both beans grown in fritted clay than in other media. Promix GM plants with dry weight of .45g for the black bean and .32g for the red beans were the lowest. Nodule separation from the growing media was easiest when plants were grown in sand, however, this was not significantly different from that of plants grown in fritted clay.

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Dyremple B. Marsh, Wayne McLaughlin, and James S. Beaver

Methods to improve the grain yield of red kidney bean without the addition of commercially fixed nitrogen will have significant benefits to farmers in Jamaica and other tropical regions. Red kidney beans provide a major portion of the dietary protein for most families in these regions. Our experimental objective was to evaluate the nitrogen fixing capabilities of several breeding lines of Phaseolus vulgaris when inoculated with Rhizobium strains isolated from Jamaican soils. Surface sterilized seeds of 11 Phaseolus lines were inoculated with inoculum prepared from 5 day old Rhizobium YEM mixture. Rhizobium used were T2 and B17 from Jamaica and UMR 1889. The greenhouse study was arranged as a completely randomized design. Bean lines 9056-101, 9056-98B, 8954-5 and 8954-4 showed improved nodulation and N2 fixation when inoculated with UMR 1899. The combination of breeding line 8954-5 and Rhizobium strain B17 produced the highest nodule number and shoot dry weight of 193 and 0.72 g, respectively. The Rhizobium strain B17showed some ability to compete successfully for nodule sites against known effective strains.

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M. Tiffany Laws and William R. Graves

Symbiotic associations between Alnus maritima (Marsh.) Muhl. ex Nutt. (seaside alder) and actinomycetes in the genus Frankia Brunchorst result in root nodules in which atmospheric nitrogen (N) is fixed. The economic and environmental benefits of N fixation have led to interest in inducing root nodules during production of A. maritima. Because woody plants produced in nurseries typically are provided N fertilizer, our objectives were to determine how applied N influences nodulation of A. maritima and to characterize how short-term changes in root-zone N affect the function of nodules. Potted seedlings were grown in perlite that was inoculated with 30 mL of soil from the root zones of mature plants in their native habitat on the Delmarva Peninsula. Each pot was drenched once daily for 10 weeks with nutrient solution that contained ammonium nitrate at 10 concentrations from 0 to 8 mm. Plants that received no ammonium nitrate formed the most nodules, and nodulation decreased linearly as ammonium nitrate increased from 0.25 to 4 mm. Plants treated with ammonium nitrate at 4 or 8 mm formed nearly no nodules, while ammonium nitrate at 0.5 mm resulted in vigorous plants with an average nodule count of 70. In a second experiment, a population of nodulated seaside alders was established by irrigating seedlings in inoculated perlite once daily with 0.5-mm ammonium nitrate for 6 weeks. Plants then were provided ammonium nitrate at 0.5, 2, or 4 mm for 2 weeks. Acetylene-reduction assays showed suppressed nodule activity among plants provided 2- and 4-mm ammonium nitrate. Daily irrigation of those plants with N-free solution subsequently led to a rapid depletion of root-zone N and to a concomitant resurgence of nodule activity. These results demonstrate that N fertilization can be managed to promote nodulation of A. maritima and show that decreased nodule activity caused by short-term increases in root-zone N is reversible.

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Lin Wu and Armando Torres

The effect of tall fescue turf on growth, flowering, modulation, and nitrogen fixing potential of Lupinus albifrons Benth. was examined for greenhouse and field grown plants. No allelopathic effect was observed for lupine plants treated with tall fescue leachates. The nitrogen-fixing potential measured by nodule dry weight and acetylene reduction rates was not significantly affected by either tall fescue turf or low nitrogen fertilization. Both the greenhouse and field studies showed that the growth, sexual reproductive allocation, and number of inflorescences were significantly reduced when lupine plants were grown with tall fescue. The root length densities of tall fescue turf and lupine monoculture were measured. The tall fescue turf had 20 times higher root length density (20 cm cm-3 soil) than the lupine plants monoculture. This suggests that intense competition at the root zone may be a dominant factor which limits the growth of the lupine plants. The reproductive characters of the lupine plants was improved by phosphorus fertilization. Transplanting of older lupine plants into the turf substantially alleviated the tall fescue turf competitive effect.

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G.M. Sajid and W.F. Campbell

Evolution of hydrogen gas (H2) during N2 reduction in root nodules results in inefficient use of energy needed for N2 fixation. Cultivars of chickpea (Cicer arietinum L.) were inoculated with Rhizobium strains with and without genes for uptake hydrogenase (Hup) activity. H2 evolution, acetylene reduction activity, and uptake hydrogenase (Hup) activity were assayed on the resulting nodules. The Hup strains produced higher plant yields than the Hup+ strains. The +N controls produced significantly higher yields than the –N controls and plants inoculated with Rhizobium strains. Hydrogen uptake activity by Rhizobium strains was influenced by the cultivar characteristics. Expression of the plasmid-borne hup genes (pHU52) of Bradyrhizobium japonicum was modified by the host cultivar. The average nodule fresh weight and shoot and root dry weights of the cultivars significantly increased following inoculation with the transconjugant Hup+ Rhizobium strain. Thus, biological N2 fixation may be enhanced by selecting Rhizobium strains that are appropriately matched to the particular cultivar. Incorporation of transconjugant Hup+ genes can increase rhizobial activity.

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James D. McCurdy, J. Scott McElroy, and Elizabeth A. Guertal

nodulation and nitrogen fixation by white clover Plant Soil 41 509 519 Davies, A. Evans, E. 1990 Axillary bud development in white clover relation to defoliation and shading treatment Ann. Bot. (Lond.) 55 171 178 Dudeck, A.E. Peacock, C.H. 1983 Rate, method

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William R. Graves, Sandra R. Anfinson, and Kathryn K. Lappegard

Scotch laburnum [Laburnum alpinum (Mill.) Bercht.], Amur maackia (Maackia amurensis Rupr. & Maxim.), and Chinese wisteria [Wisteria sinensis (Sims) Sweet] were inoculated with compatible rhizobia and treated with leaching fractions (LF) of 0, 0.2, and 0.4 using fertilizer solutions with 3.6 and 10.7 mol N/m3 for 10 weeks. LF did not affect plant dry mass, leaf area, or stem length. Growth was higher among plants provided 10.7 mol N/m3, but only plants provided 3.6 mol N/m3 formed root nodules. We conclude that growth is not reduced by eliminating leaching during the first 10 weeks of seedling development, and that application of 10.7 mol N/m3 prevents nodulation of these species.