Asymbiotic nitrogen-fixing bacteria from the surfaces of nodules and roots of legumes

Abstract

EVANS, H. J:, N. E. R. CAMPBELL, and S. HILL. 1972. Asymbiotic nitrogen-fixing bacteria from the surfaces of nodules and roots of legumes. Can. J. Microbiol. 18: 13-21. Facultative anaerobic asymbiotic N2-fixing bacteria have been identified consistently on the surfaces of roots and nodules of soybean plants cultured under laboratory and greenhouse conditions. An examination of a limited number of samples of alfalfa and clover plants from natural soils also has revealed similar types of bacteria. Most of the isolates investigated were members of the Klebsiella-Aerobacter group. Serological tests of 11 isolates indicated different degrees of relationship to Klebsiellapneumoniae. Rates of growth and N2 fixation in anaerobic cultures were markedly stimulated by a sterile extract of soybean nodules. Bacteria of the type identified on root surfaces of legumes were found often as contaminants of legume seeds and in most cases were not removed by surface sterilization treatments.EVANS, H. J., N. E. R. CAMPBELL et S. HILL. 1972. Asymbiotic nitrogen-fixing bacteria from the surfaces of nodules and roots of legumes. Can. J. Microbiol. 18: 13-21. Des bactkries facultatives anaerobies qui fixent l'azote asymbiotiquement ont kt6 constamment identifiees sur les surfaces des racines et des nodules des plantes de la five soja, lesquelles 6taient cultivkes sous des conditions de laboratoire et de serre. Un examen d'un nombre limite dY6chantillons de plantes de luzerne et de trbfle des sols naturels a aussi rdvelC des types semblables de bact6ries. La plupart des souches Ctudites sont membres du groupe Klebsiella-Aerobncter. Des tests s6rologiques sur 11 souches indiquent diffkrents degres de correlation avec Klebsiella pnelrmoniae. Les taux de croissance et de fixation de I'azote en cultures ana6robies sont grandement stirnu16 par unextrait sterile des nodules de la fbve soja. Les batteries des types identifiks sur les surfaces des racines des ICgumineuses sont souvent trouvCes comme contaminants des graines des 1Cgumineuses, et dans la plupart des cas elles ne sont pas enlevees par traitements de stkrilisation de la surface.The occurrence of bacteria other than rhizobia in and on the surfaces of legume roots and nodules has been reported by many investigators. Fred et al. (7) summarized pertinent literature from 1888 to 1928 in which 23 different investigations revealed several different bacterial contaminants in legume nodules. These authors concluded that Bacillus radiobacter (now classified as Agrobacteriurn radiobacterj was the most frequently encountered nodule contaminant. Wilson et al. (12,20) have investigated the problems of culturing leguminous plants under conditions that exclude all microorganisms other than rhizobia. In their experiments seeds were surface sterilized, plants were cultured in sterile glass jars, and all manipulations were conducted in sterile inoculation chambers. Despite these precautions an examination at harvest revealed that about 50% of the cultures contained 1Received March 26. 1971. z~h i s re...

Relation between Glutamine Synthetase and Nitrogenase Activities in the Symbiotic Association between Rhizobium japonicum and Glycine max

“…Since the bacteroid tissue of legume root nodules is the site BISHOP, GUEVARA, ENGELKE, AND EVANS for N2 fixation (3) and since crude extracts of bacteroids exhibit relatively low glutamine synthetase activity (5,8), it seemed logical to investigate the response of the bacteroid enzyme to NH4+. One major difficulty in experiments of this type is the control of contamination of bacteroid suspensions from large populations of bacteria on nodule surfaces (9). Contamination was monitored in the experiment described in Table I.…”

“…Effect of NH4+ on Activity and Adenylylation of Glutamine Synthetase in Bacteroid Suspensions Bacteroid suspensions were prepared from soybean root nodules as described under "Materials and Methods" and Na2SO4 or (NH4)2SO4 was added as indicated. Suspensions were prepared from surface-disinfected nodules (9), 5000 units of penicillin G were added/ml of suspension, and bacterial contamination after 10 hr did not exceed 0.05% of the cell mass. (21) containing 30 mm Na2SO4 (control), and the second group was subirrigated with 20 liters of nitrogenfree nutrient solution containing 30 mm (NH4)2SO4 (NH4+-treated roid suspensions and nodulated roots are incubated in the absence of a source of combined nitrogen.…”

“…The sedimented bacteroid fraction was washed by resuspension in 12 ml of sucrose-phosphate buffer and collected by centrifugation at 6,000g for 15 min. When large preparations of bacteroids were required, the relative proportions of sucrosephosphate buffer and PVP were the same as described above, but a blender was used for the maceration process in place of a mortar and pestle (Table I), and the nodules were surfacesterilized by the sodium hypochlorite method utilized by Evans et al (9). Bacteroids were prepared as described above and were resuspended in 100 ml of sterile sucrose-phosphate buffer.…”

Nitrogen Fixation and a Nitrogen-Fixing Bacterium From the Roots of Eragrostis Ferruginea

“…Nitrogen-fixing bacteria isolated from the rhizospheres are diverse; e.g., facultative anaerobes such as Bacillus polymyxa, Bacillus macerans, and Enterobacter cloacae from wheat (16) and Klebsiella-Aerobacter group from legumes (17), and aerobic bacteria such as Spirillum lipoferum from pasture grass Digitaria decumbens (13), Azotobacter paspali from Paspalum notatum (10) and Beijerinckia indica from sugar cane (18) have been reported. The isolate, ER201, described in this report, although its taxonomic position is still uncertain, is a strict aerobe and resembles Azotobacter in morphological appearance.…”

Inhibition of Protein Synthesis by D-threo-chloramphenicol in the Laboratory and Nodule Forms of Rhizobium lupini

“…Studies of the metabolism of bacteroids are hampered by the difficulty in isolating bacteroids relatively free from fast-growing contaminating organisms, as it is very difficult to produce legume seedlings which are bacteria free (Evans, Campbell & Hill, 1971). Therefore care was taken to minimize the numbers of contaminating bacteria and to monitor the numbers of these bacteria throughout the incubation periods.…”