Distribution of Hydrogen-Metabolizing Bacteria in Alfalfa Field Soil

H₂ evolved by alfalfa root nodules during the process of N₂ fixation may be an important factor influencing the distribution of soil bacteria. To test this hypothesis under field conditions, over 700 bacterial isolates were obtained from fallow soil or from the 3-mm layer of soil surrounding alfalfa (Medicago sativa L.) root nodules, alfalfa roots, or bindweed (Convolvulus arvensis L.) roots. Bacteria were isolated under either aerobic or microaerophilic conditions and were tested for their capacity to metabolize H₂. Isolates showing net H₂ uptake and ³H₂ incorporation activity under laboratory conditions were assigned a Hup⁺ phenotype, whereas organisms with significant H₂ output capacity were designated as a Hout⁺ phenotype. Under aerobic isolation conditions two Hup⁺ isolates were obtained, whereas under microaerophilic conditions five Hup⁺ and two Hout⁺ isolates were found. The nine isolates differed on the basis of 24 standard bacteriological characteristics or fatty acid composition. Five of the nine organisms were isolated from soil around root nodules, whereas the other four were found distributed among the other three soil environments. On the basis of the microaerophilic isolations, 4.8% of the total procaryotic isolates from soil around root nodules were capable of oxidizing H₂, and 1.2% could produce H₂. Two of the Hup⁺ isolates were identified as Rhizobium meliloti by root nodulation tests, but the fact that none of the isolates reduced C₂H₂ under the assay conditions suggested that the H₂ metabolism traits were associated with various hydrogenase systems rather than with nitrogenase activity. Results from this study support the concept that H₂ evolution by alfalfa root nodules has a significant effect on the surrounding microenvironment and influences the number and diversity of bacteria occupying that region.     

Effect ofAzospirillum inoculation on nitrogen fixation and growth of several winter legumes

Summary Inoculation of naturally nodulatedPisum sativum L. (garden pea) withAzospirillum in the greenhouse caused a significant increase in nodule numbers above controls. Field inoculation of garden peas in the winter 1981–1982 andCicer arietinum L. (chick pea), in winter 1982–1983, withAzospirillum one week after plant emergence, produced a significant increase in seed yield, but did not affect plant dry matter yield. ForVicia sativa L. (vetch) grown in soil in the greenhouse and in the field for forage, winter 1980–1981, inoculation significantly increased dry matter yield, %N, N-content, and acetylene reduction (nitrogen fixation) activity. InHedysarum coronarium L. (sulla clover), winter 1981–1982, inoculated with both its specificRhizobium (by the slurry method) andAzospirillum, 7 days after emergence, there was an increase in acetylene reduction above controls inoculated withRhizobium alone. These results suggest that it is possible, under conditions tested in this work, to increase nodulation, nitrogen fixation, and crop yields of winter legumes by inoculation withAzospirillum.     

Uptake Hydrogenase Activity Determined by Plasmid pRL6JI in Rhizobium leguminosarum Does Not Increase Symbiotic Nitrogen Fixation

We examined three groups of wild baboons (Papio cynocephalus) in Amboseli National Park, Kenya, to determine the prevalence of aerobic antibiotic-resistant fecal bacteria in nonhuman primates with and without contact with human refuse. Using standard isolation and replica plating techniques, we found only low numbers of antibiotic-resistant gram-negative enteric bacteria in two groups of baboons leading an undisturbed existence in their natural habitat and having limited or no contact with humans. However, resistance was significantly higher among enteric bacteria from the third group of baboons living in close proximity to a tourist lodge and having daily contact with unprocessed human refuse. Conjugation studies and analysis of the cell DNA by gel electrophoresis showed that in many cases resistance was plasmid-borne and transferable. These data suggest that wild nonhuman primates in frequent contact with human debris have a higher proportion of antibiotic-resistant enteric bacteria than do conspecifics without this contact. The findings further suggest that such groups of wild animals may constitute a heretofore overlooked source of antibiotic resistance in the natural environment.     

Promotion of nod Gene Inducers and Nodulation in Common Bean (Phaseolus vulgaris) Roots Inoculated with Azospirillum brasilense Cd

Inoculation of Phaseolus vulgaris with Azospirillum brasilense Cd promoted root hair formation in seedling roots and significantly increased total and upper nodule numbers at different concentrations of Rhizobium inoculum. In experiments carried out in a hydroponic system, A. brasilense caused an increase in the secretion of nod gene-inducing flavonoids, as was observed by nod gene induction assays of root exudates fractionated by high-performance liquid chromatography. Possible mechanisms involved in the influence of A. brasilense on this symbiotic system are discussed.     

Field application of vesicular-arbuscular mycorrhizal fungi improved garlic yield in disinfected soil

 The production of certified garlic propagation material requires measures to be taken against pathogenic nematodes. Methyl bromide (MB) may be used for this purpose, but is known to cause stunting in Allium spp. Vesicular-arbuscular mycorrhizal (VAM) fungal inoculum was applied to the planting furrow after MB treatment. VAM-inoculated plants were larger, had more green leaves, an increased photosynthesis rate, especially at low light intensities, and higher fresh and dry weights than plants in uninoculated plots. The mean bulb weights from uninoculated and VAM-treated plots were 27 g and 51 g respectively. The native or an improved VAM population should be reintroduced after soil disinfection to ensure satisfactory garlic yields. Accepted: 15 January 1997     

Identification of a novel genetically controlled step in mycorrhizal colonization: plant resistance to infection by fungal spores but not extra-radical hyphae

Vesicular arbuscular mycorrhizal fungi infect plants by means of both spores and vegetative hyphae at early stages of symbiosis. Using 2500 M2 fast-neutron-mutagenized seeds of the miniature tomato (Lycopersicon esculentum) cultivar, Micro-Tom, we isolated a mutant, M161, that is able to resist colonization in the presence of Glomus intraradices spores. The myc(-) phenotype of the mutant was stable for nine generations, and found to segregate as a single Mendelian recessive locus. The mutant exhibited morphological and growth-pattern characteristics similar to those of wild-type plants. Alterations of light intensity and day/night temperatures did not eliminate the myc(-) characteristic. Resistance to mycorrhizal fungal infection and colonization was also evident following inoculation with the fungi Glomus mosseae and Gigaspora margarita. Normal colonization of M161 was evident when mutant plants were grown together with arbuscular mycorrhizal-inoculated wild-type plants in the same growth medium. During evaluation of the pre-infection stages in the mutant rhizosphere, spore germination and appressoria formation of G. intraradices were lower by 45 and 70%, respectively, than the rates obtained with wild-type plants. These results reveal a novel, genetically controlled step in the arbuscular mycorrhizal colonization process, governed by at least one gene, which significantly reduces key steps in pre-mycorrhizal infection stages.     

Investigation of Four Classes of Non-nodulating White Sweetclover (Melilotus alba annua Desr.) Mutants and Their Responses to Arbuscular-Mycorrhizal Fungi

The nitrogen-fixing symbiosis between Rhizobiaceae and legumes is one of the best-studied interactions established between prokaryotes and eukaryotes. The plant develops root nodules in which the bacteria are housed, and atmospheric nitrogen is fixed into ammonia by the rhizobia and made available to the plant in exchange for carbon compounds. It has been hypothesized that this symbiosis evolved from the more ancient arbuscular mycorrhizal (AM) symbiosis, in which the fungus associates with roots and aids the plant in the absorption of mineral nutrients, particularly phosphate. Support comes from several fronts: 1) legume mutants where Nod(-) and Myc(-) co-segregate, and 2) the fact that various early nodulin (ENOD) genes are expressed in legume AM. Both strongly argue for the idea that the signal transduction pathways between the two symbioses are conserved. We have analyzed the responses of four classes of non-nodulating Melilotus alba (white sweetclover) mutants to Glomus intraradices (the mycorrhizal symbiont) to investigate how Nod(-) mutations affect the establishment of this symbiosis. We also re-examined the root hair responses of the non-nodulating mutants to Sinorhizobium meliloti (the nitrogen-fixing symbiont). Of the four classes, several sweetclover sym mutants are both Nod(-) and Myc(-). In an attempt to decipher the relationship between nodulation and mycorrhiza formation, we also performed co-inoculation experiments with mutant rhizobia and Glomus intraradices on Medicago sativa, a close relative of M. alba. Even though sulfated Nod factor was supplied by some of the bacterial mutants, the fungus did not complement symbiotically defective rhizobia for nodulation.     

Enhanced levels of free and protein-bound threonine in transgenic alfalfa (Medicago sativa L.) expressing a bacterial feedback-insensitive aspartate kinase gene

Threonine, lysine, methionine, and tryptophan are essential amino acids for humans and monogastric animals. Many of the commonly used diet formulations, particularly for pigs and poultry, contain limiting amounts of these amino acids. One approach for raising the level of essential amino acids is based on altering the regulation of their biosynthetic pathways in transgenic plants. Here we describe the first production of a transgenic forage plant, alfalfa (Medicago sativa L.) with modified regulation of the aspartate-family amino acid biosynthetic pathway. This was achieved by over-expressing the Escherichia coli feedback-insensitive aspartate kinase (AK) in transgenic plants. These plants showed enhanced levels of both free and protein-bound threonine. In many transgenic plants the rise in free threonine was accompanied by a significant reduction both in aspartate and in glutamate. Our data suggest that in alfalfa, AK might not be the only limiting factor for threonine biosynthesis, and that the free threonine pool in this plant limits its incorporation into plant proteins.     

Effect of Azospirillum inoculation on some growth parameters and n-content of wheat,sorghum and panicum

The potential of the nitrogen fixing bacteriumAzospirillum brasilense to enhance development and increase growth of several gramineae was investigated. In both sterilized and non-sterilized systems heading and flowering occurred earlier in the inoculated plants as compared to the noninoculated ones. Total shoot and root weights, total-N content, plant height and leaf length were significantly increased by inoculation.From the Department of Agricultural Botany.