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.
William R. Graves and Lorna C. Wilkins
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.
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.
Dyremple B. Marsh and Luther Waters Jr.
Zinc concentration, nitrogen fixation (C2H2 reduction), nodulation and dry-matter distribution in the early maturing cowpea [Vigna unguiculata (L.) Walp] line Mn 13 were examined at 5 levels of Zn (0.0, 0.6, 1.5, 2.5 and 5 ppm) under field and greenhouse conditions. Significant increases in nodule number, nodule dry weight, and acetylene reduction occurred when plants received the higher Zn levels. In all plant parts sampled, there was increased Zn accumulation with increased Zn application, with roots having the maximum accumulation. Seeds per pod and seed yield (kg/ha) were highest at the higher applied Zn levels. The yield response to added Zn was reflected primarily by an increase in the number of seeds per pod. Zinc nutrition is important to the nodulation and fixation processes, and it may affect both Rhizobium nutrition and dry-matter accumulation.
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.
K. W. Zary and J. C. Miller Jr.
Diurnal and seasonal patterns of nitrogen fixation N2(C2H2) in southernpea were delineated, using 7 genotypes which differed in potential to fix nitrogen. Diurnal activity peaked at 1200 hours, at both 34 and 53 days after planting (DAP). Significant differences in total activity between genotypes were observed, with maximum activity generally coincident with full flowering. High fixing genotypes were higher in total nitrogenase activity throughout the growing season, than were the low fixing genotypes. Peak activity for the latter was found at 34 DAP, while the former peaked at 46 DAP. Mean nodule mass, nodule number and plant dry weight were greatest 53 DAP.
David W. Walker and J. Creighton Miller Jr.
Two greenhouse studies were conducted to investigate the relationship between water stress and N2 fixation among drought-resistant and susceptible cowpea [Vigna unguiculata (L.) Walp.] genotypes. In both experiments, seeds were planted in 7.6-liter black polyethylene pots containing composted sawdust medium and were inoculated with Rhizobium. Throughout the experiments, flowers were removed to maintain vegetative growth. Water stress treatments were imposed by withholding water, while the control plants were watered as needed. The treatments were applied 58 and 56 days after planting (DAP) in the first and 2nd experiments, respectively. In both experiments, leaf water potential (LWP), shoot fresh weight (SFW), shoot dry weight (SDW), root fresh weight (RFW), nodule fresh weight (NW), nodule number (NN), and plant specific activity (PSA) by both in situ and destructive acetylene reduction methods were measured. Repeated observations of in situ acetylene reduction were made 58, 63, and 71 DAP in the first experiment. All other variables were measured 77 to 78 DAP in the first experiment. Single observations of all variables, including in situ and destructive acetylene reduction were made 56, 67, and 81 DAP in the 2nd experiment. Results suggested that resistant genotypes are capable of maintaining LWP and biomass production (as measured by SDW and SFW) during water stress. In addition, the effect of water stress on N2 fixation was far greater than the influence of genotype when genotypes were selected for relative drought resistance. Path analysis revealed that LWP is correlated to N2 fixation in water-stressed plants, and improvement of plant water status via drought resistance should increase N2 fixation potential under drought conditions. Therefore, breeding for drought resistance in conjunction with N2 fixation may be more beneficial than breeding strictly for N2 fixation potential without regard for environmental adaptation. The in situ method of acetylene reduction was found to be useful for detecting physiological changes due to water stress and estimating its genotypic N2 fixation potential.
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.
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.
S. K. O’Hair and J. C. Miller Jr.
The effects of the cowpea strains of southern bean mosaic (SBMV-CS), cucumber mosaic (CMV-CS), and tobacco mosaic (TMV-CS) viruses on cowpea [Vigna unguiculata (L.) Walp] plant growth and N fixation parameters were evaluated in greenhouse and field experiments. TMV-CS infection resulted in the most severe symptoms and was associated with significant reductions in nitrogenase activity, total nodule weight, nodule number, and plant dry weight, in susceptible cultivars. Infection with CMV-CS was associated with significant reductions in nitrogenase activity, nodule number, and plant dry weight in field-grown ‘Mississippi Silver’. SBMV-CS infection was least severe and was associated with a significant reduction in nitrogenase activity and nodule weight in only 1 susceptible cultivar under field conditions.