Nucleotide sequence of the fixABC region of Azorhizobium caulinodans ORS571: similarity of the fixB product with eukaryotic flavoproteins, characterization of fixX, and identification of nifW.

Summary The nucleotide sequence of a 4.1 kb DNA fragment containing the fixABC region of Azorhizobium caulinodans was established. The three gene products were very similar to the corresponding polypeptides of Rhizobium meliloti. The C-terminal domains of both fixB products displayed a high degree of similarity with the -subunits of rat and human electron transfer flavoproteins, suggesting a role for the FixB protein in a redox reaction. Two open reading frames (ORF) were found downstream of fixC. The first ORF was identified as fixX on the basis of sequence homology with fixX from several Rhizobium and Bradyrhizobium strains. The second ORF potentially encoded a 69 amino acid product and was found to be homologous to a DNA region in the Rhodobacter capsulatus nif cluster I. Insertion mutagenesis of the A. caulinodans fixX gene conferred a Nif^– phenotype to bacteria grown in the free-living state and a Fix^– phenotype in symbiotic association with the host plant Sesbania rostrata. A crude extract from the fixX mutant had no nitrogenase activity. Furthermore, data presented in this paper also indicate that the previously identified nifO gene located upstream of fixA was probably a homologue of the nifW gene of Klebsiella pneumoniae and Azotobacter vinelandii.     

Nutrient transfer between the root zones of soybean and maize plants connected by a common mycorrhizal mycelium

The objective of the study was to determine whether nutrient fluxes mediated by hyphae of vesicular-arbuscular mycorrhizal (VAM) fungi between the root zones of grass and legume plants differ with the legume's mode of N nutrition. The plants, nodulating or nonnodulating isolines of soybean [ Glycine max (L.) Merr.], were grown in association with a dwarf maize ( Zea mays L.) cultivar in containers which interposed a 6-cm-wide root-free soil bridge between legume and grass container compartments. The bridge was delimited by screens (44 μm) which permitted the passage of hyphae, but not of roots and minimized non VAM interactions between the plants. All plants were colonized by the VAM fungus Glomus mosseae (Nicol. & Gerd.) Gerd. and Trappe. The effects of N input to N-sufficient soybean plants through N₂-fixation or N-fertilization on associated maize-plant growth and nutrition were compared to those of an N-deficient (nonnodulating, unfertilized) soybean control. Maize, when associated with the N-fertilized soybean, increased 19% in biomass, 67% in N content and 77% in leaf N concentration relative to the maize plants of the N-deficient association. When maize was grown with nodulated soybean, maize N content increased by 22%, biomass did not change, but P content declined by 16%. Spore production by the VAM fungus was greatest in the soils of both plants of the N-fertilized treatment. The patterns of N and P distribution, as well as those of the other essential elements, indicated that association with the N-fertilized soybean plants was more advantageous to maize than was association with the N₂-fixing ones.     

Effect of salinity on growth of four strains of Rhizobium and their infectivity and effectiveness on two species of Acacia

Two Rhizobium strains (WU1001 and WU1008) were isolated from nodules of Acacia redolens growing in saline areas of south-west Australia, and two strains selected from the University of Western Australia's culture collection (WU429 isolated from A. saligna and WU433 from A. cyclops). The growth of each in buffered, yeast extract mannitol broth culture was largely unaffected by salt up to 300 mM NaCl. A slight increase in lag time occurred at concentrations of 120 mM NaCl and above, but cell number at the static phase was not affected. Each of the four Rhizobium strains tested accumulated Na⁺ but showed decreasing levels of sugar with increasing salt in the external medium. Amino acid levels also increased, in some cases by more than tenfold. However, the relative proportion of each remained fairly constant in the bacteria, irrespective of salt treatment. Only trace quantities of proline were detected and there was no increase in this amino acid with salt. Acidic amino acids (glutamate and aspartate) remained as a constant proportion.Rhizobium strains WU429, WU1001 and WU1008 produced effective nodules on both A. cyclops and A. redolens grown in sand with up to 80 mM NaCl (added in nutrient solutions free of nitrogen). Strain WU433 was highly infective on both Acacia species tested at low salt concentrations (2–40 mM NaCl), but infection was sensitive to salt levels at 120 mM NaCl and above. Nodules formed with strain WU433 were, however, ineffective on both A. redolens and on A. cyclops and showed nil or negligible rates of acetylene reduction at all salt concentrations. Strains WU429, WU1001 and WU1008 in combination with a highly salt-tolerant provenance of A. redolens formed symbioses which did not vary significantly in nodule number and mass, specific nodule activity or total N content irrespective of salt level up to 160 mM NaCl. On a more salt sensitive provenance of A. redolens and on A. cyclops the infectivity and effectivity of the Rhizobium strains tested usually decreased as the external salt concentration increased. These data are interpreted to indicate that tolerance of the legume host was the most important factor determining the success of compatible Rhizobium strains in forming effective symbioses under conditions of high soil salinity.     

Nodulation of lucerne (Medicago sativa L.) in an acid soil: Effects of inoculum size and lime-pelleting

The effects of inoculum level and lime-pelleting were studied in an acid soil with respect to the nodulation and growth of lucerne (Medicago sativa cv Resis) and the population dynamics of Rhizobium meliloti. In small root-boxes (rhizotrons), the in-situ survival of inoculated rhizobia was studied in the micro-environment around the seed for a period of 12 days after sowing. During the initial 24 hours, a strong increase in rhizobial numbers was measured, concomitantly with the development of roots. As a result of lime-pelleting, rhizobial numbers were higher only at 3 days after sowing (P<0.05). Later, this difference diminished steadily. Addition of lime did not increase the adhesion of the rhizobia to the seedling tap root. Plant responses to inoculation were studied in pots. To obtain optimal nodulation, the soil had to be neutralized around the seed with lime and at least 10⁵ cells of R. meliloti were required. With more than 10⁵ rhizobia per seed, lime-pelleting increased the number of crown-nodulated seedlings from 24% to 77%. Higher numbers of rhizobia could not compensate the effect of lime. A strong correlation was found between crown nodulation, nitrogen content and dry weight of the shoots.     

Biology of nitrogen-fixing Rhizobacteria

In non-legumes associative nitrogen-fixing system, several genera of rhizobacteria have been reported. The object of this paper is to summarize the current understanding of how rhizobacteria adhere to the root surface of non-legumes especially rice and other cereal crops. Evidence for involvement of rice lectin in adhesion will be reviewed. An emphasis will be placed on theKlebsiella R15 ammonium assimilation system in free-living state and in associative state with rice seedlings. Nitrogenase and glutamine synthetase (GS) activities of associativeKlebsiella increased significantly in the rhizosphere of rice comparing to the free-living state. In rice, the soluble form of GS specific activity appear to be slightly lower than in rice root in the absence of bacteria. These results suggest that nitrogen-fixing activity has been enhanced during association. The dinitrogen fixed should be changed to amino acids via GS-GOGAT pathway in bacteria. Transfer of fixed nitrogen and assimilation in the rice plant is the problem that needs to be solved in order to improve the efficiency of associative nitrogen fixation.     

In vitro micropropagation and plant establishment of muscadine grape cultivars (Vitis rotundifolia)

Shoot apical meristems were used to establish regenerative axillary bud cultures of 9 muscadine grape cultivars. Meristems taken from 10 cm long shoots had less contamination (3%) and a higher survival rate (94%) than those from shorter or longer shoots. Of media tested, MS, 1/2 MS, and C₂D resulted in equivalent shoot proliferation rates, whereas, WPM produced stunted shoots. When pooling results for 3 cultivars, 5, 10 and 20 M BA and 5 M TDZ produced the highest average number of shoots per cultured apex (3.4–3.8). However, shoots produced with TDZ were stunted and did not root well. For rooting of shoots directly in potting mix, a rooting powder pretreatment significantly increased the number of roots per shoot but did not affect percent rooting or root length. For rooting in vitro, 1 M NAA significantly increased all parameters measured. Although more shoots rooted in vitro than in vivo (77% vs. 46%), the latter was judged preferable since acclimatized plants were produced in less time and a major culture step was eliminated. Significant differences among cultivars were noted for measured responses in all experiments.Abbreviations BA benzyladenine - Kin kinetin - MS Murashige & Skoog (medium) - NAA naphthaleneacetic acid - TDZ thidiazuron - WPM woody plant medium     

High frequency of plant regeneration from hypocotyl explants of Brassica carinata A. Br.

An efficient tissue culture system for high frequency of plant regeneration from hypocotyl explants of Brassica carinata was developed via manipulation of culture medium and selection of explants. Explants grown on medium containing combinations of 2 mg l^-1 BA and 0.01 mg l^-1 NAA or 4 mg l^-1 kinetin and 0.01 mg l^-1 2,4-D regenerated shoots at 100% frequency. High frequency shoot regeneration occurred only from explants originating from 6 to 7-day-old but not younger or older seedlings. Explants showed higher regeneration capacity at the distal end than the proximal end, and the upper segment was more regenerative than the lower segment of hypocotyl. Regenerants were rooted on half-strength growth regulator-free medium, acclimatized and developed into normal, fertile plants.Abbreviations BA benzyladenine - 2-4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - MS Murashige & Skoog     

Freeze-preservation of buds from Scots pine trees

A procedure has been developed for freeze-preservation of buds of the Scots pine (Pinus sylvestris L.). Instead of liquid nitrogen, cold storage in –80°C was used. The partly dormant material used in the experiments was obtained directly from a natural stand in Northern Finland and no prefreezing or cryoprotectants for preconditioning were used. Cooling velocity was 1°C/min up to a terminal freezing temperature of –39°C, after which the buds were immersed in liquid nitrogen at –196°C for 10 minutes. The material was then transferred to a deepfreezer at –80°C and stored up to 6 months. After rapid thawing, the buds were sterilized and their viability was tested by FDA staining and by culturing meristems on 1/2 MS medium for at least two weeks. All the freezing experiments were performed during March and April. The best survival of buds (90–100%) was achieved at the beginning of April, after which a pronounced decline in survival occurred obviously due to a rise in the water content of the buds.