Improvement of Rhizobium inoculants by mutation, genetic engineering and formulation.

The use of mutation and genetic engineering techniques have resulted in Rhizobium strains with improved characteristics. The latter approach can provide improvement and new traits not achievable previously. Enhanced commercial Rhizobium inoculants, however, still rely solely on traditional approaches including formulation improvement, mutation, and strain selection. The lack of contribution to the commercial product lines by genetic engineering is mostly due to the time delay and financial burdens cast by the regulatory policies rather than a lack of progress in the application of modern genetic technologies. Such constraints may lessen in time as the public becomes more educated about the technologies and the subsequent political pressure decreases.

Improvement of Rhizobium inoculants.

A practical approach was used to develop a Rhizobium (Bradyrhizobium) japonicum inoculant that increases soybean (Glycine max (L.) Merr.) yield in fields with indigenous Rhizobium populations, which typically outcompete strains present in existing commercial inoculants and therefore decrease the value of inoculant use. Field tests managed by several universities in the Mississippi delta region averaged a 169-kg/ha (P < 0.01) grain yield increase. The inoculant contains a mixture of mutants selected for increased nitrogen fixation ability. These mutants were derived from indigenous wild-type strains that are capable of high-level occupancy of nodules in soybean fields in the Mississippi delta region. To ensure microbiological purity, the inoculant is fermented directly in the point-of-use container with a vermiculite carrier (L. Graham-Weiss, M. L. Bennett, and A. S. Paau, Appl. Environ. Microbiol. 53:2138-2140, 1987). It should be possible to use this approach to produce more effective Rhizobium inoculants for any legume in any geographical area.

Production of bacterial inoculants by direct fermentation on nutrient-supplemented vermiculite.

When supplemented with a nutrient source and moisture, sterile finely ground vermiculite can be used to directly ferment bacterial cultures to prepare bacterial inoculants. All tested bacterial species, including Rhizobium japonicum, R. phaseoli, R. meliloti, R. leguminosarum, Bacillus megaterium, and several Pseudomonas strains, grew at least 10,000-fold in 1 week at room temperature. The final product was stable and had no special storage or handling requirements. Due to the unique properties of vermiculite, direct fermentation of bacteria on nutrient-supplemented vermiculite offers a reliable process for manufacturing bacterial inoculants.

Regulation of Nodulation by Rhizobium meliloti 102F15 on Its Mutant Which Forms an Unusually High Number of Nodules on Alfalfa.

A mutant (WL3A150) of Rhizobium meliloti 102F51 that elicits an unusually high number of nodules on its host, alfalfa (Medicago sativa), supports the idea that the host may rely on early bacteroid development in the nodule or on metabolites produced in the infection thread as one of the signals to control further nodulation. This mutant was initially isolated because of its Fix phenotype. It consistently formed many more nodules than all the other Fix mutants isolated from strain 102F51 (a total of 11 mutants). Nodules formed by this mutant were small and white and were indistinguishable in appearance from nodules formed by the other Fix mutants. An ultrastructural study of the nodules, however, showed that this mutant, although forming numerous infection threads, failed to develop into bacteroids. The ability of the mutant to form an unusually high number of nodules coulde be suppressed in a time-dependent manner by the presence of the wild type.

Agglutinin from Alfalfa Necessary for Binding and Nodulation by Rhizobium meliloti.

A protein that specifically agglutinates Rhizobium meliloti, the alfalfa root nodule endosymbiont, has been purified from alfalfa seed. Material cross-reactive to antiserum prepared against the purified agglutinin is present in all alfalfa varieties that were tested but is absent in corn and other legumes not nodulated by Rhizobium meliloti. Studies with nonnodulating mutants of this microorganism incapable of binding to alfalfa roots suggest that the agglutinin is responsible for specific recognition between Rhizobium meliloti and alfalfa and that this recognition is an essential step in nodule formation.

Host recognition in the Rhizobium-soybean symbiosis.

Polar binding of Rhizobium japonicum to roots and root hairs of Glycine soja (L.) Sieb. and Zucc. is specifically inhibited by d-galactose and N-acetyl-d-galactosamine, haptens of Glycine max seed lectin. A protein, immunologically cross-reactive with the G. max seed lectin, is present in G. soja seed extracts. Peptide mapping of the purified G. max and G. soja lectins indicates that the two are similar in structure. Soybean lectin can be localized on the surface of both G. max and G. soja roots by indirect immunolatex techniques. These observations indicate that the Rhizobium-binding lectin, previously isolated from seeds, also is present on the root surface-the site of the initial steps in the infection. This lectin is capable of binding Rhizobium japonicum to the root.

Developmental fate of Rhizobium meliloti bacteroids in alfalfa nodules.

Nitrogen-fixing bacteroids are degraded during nodule senescence. This is in contrast to recent implications that viable bacteroids can be released into soil from legume nodules. Rhizobia originating from persistent infection threads in senescing nodule plant cells seem to be the source of viable cells required for perpetuation of the Rhizobium spp. population in the soil. Our conclusions were derived from electron microscopic examination of stages of development and senescence of alfalfa root nodules.

Separation of algal mixtures and bacterial mixtures with flow-microfluorometer using chlorophyll and ethidium bromide fluorescence.

The applicability of flow-microfluorometer to separate microbial cells was demonstrated with algal and bacterial cells. Algal mixtures were sorted according to the natural chlorophyll fluorescence and the bacterial mixtures were sorted according to the fluorescence of ethidium bromide-stained nucleic acid.

DNA content of free living rhizobia and bacteroids of various Rhizobium-legume associations.

The DNA content of bacteroids from 22 different Rhizobium-legume associations was compared to that of the corresponding free living Rhizobium species using laser flow microfluorometry. In all 18 effective associations, the bacteroids had either similar or higher DNA content than the free living rhizobia. Bacteroid populations isolated from effective clover (Trifolium repens) and alfalfa (Medicago sativa) nodules had an average DNA content of >1.5-fold higher than free living R. trifolii and R. meliloti. These populations also contained a significant number of bacteroids with more than 3-fold the DNA content of the free living rhizobia. Populations isolated from effective nodules of winged beans (Psophocarpus tetragonolobus), peas (Pisum sativum), and mung beans (Phaseolus aureus) had an average DNA content of 1.1- to 1.5-fold higher than free living R. "cowpeas" and R. leguminosarum. Bacteroids from nodules of lupins (Lupinus angustifolius and L. minaretta), kidney beans (Phaseolus vulgaris), and soybeans (Glycine max), however, had similar DNA content to the free living forms. Two of the four associations which formed ineffective nodules contained bacteroids with lower DNA content than the free living rhizobia. The other two associations contained bacteroids with slightly higher or similar DNA content to the free living rhizobia. Nodules of the ineffective associations also did not contain leghemoglobin.

Studies on bacteroid size and nucleic acid content of alfalfa bacteroids fractionated by velocity sedimentation.

A velocity sedimentation procedure was described to fractionate bacteroids of alfalfa nodules into four subpopulations. Bacteroids in these subpopulations were different in size and nucleic acid content as determined by microscopy and flow-microfluorometry (FMF). The slowest-sedimenting bacteroids (fraction I) were small and resembled free-living Rhizobium meliloti both in size and nucleic acid content. The fastest-sedimenting bacteroids (fraction IV) were 2 to 3 times longer and contained 3 to 4 times more nucleic acid than the small bacteroids in fraction I and free-living R. meliloti. A positive correlation was established between bacteroid size and relative nucleic acid content of bacteroids in alfalfa nodules.

Development of Bacteroids in Alfalfa (Medicago sativa) Nodules.

The morphology, acetylene reduction capability, and nucleic acid content of bacteroids in different regions of alfalfa (Medicago sativa var. Buffalo) nodules were studied by electron microscopy, gas chromatography, and laser flow microfluorometry, respectively. Bacteroids in the nodule tips were small (1 to 2.5 micrometers in length), had low nucleic acid content, and contained distinct central nucleoids. These bacteroids were comparatively inactive in acetylene reduction in situ. Bacteroids in the middle regions of alfalfa nodules were greatly enlarged (5 to 7 micrometers in length), had relatively high nucleic acid content, and did not possess central nucleoids. The bacteroids were very active in acetylene reduction. Bacteroids in the basal nodule region also were enlarged and without distinct nucleoid regions, but had relatively low nucleic acid content and low in situ acetylene-reducing activity.

Flow-microfluorometric analysis of Escherichia coli, Rhizobium meliloti, and Rhizobium japonicum at different stages of the growth cycle.

The applicability of flow-microfluorometry (FMF) to the study of bacterial samples was investigated on cultures of Rhizobium meliloti, Rhizobium japonicum, and Escherichia coli using fluorescent and light-scattering signals. This technique which analyzes individual bacterial cells in a population was used to monitor the relative change in nucleic acid content and cell size during the growth cycle of the three microorganisms which were known to have different growth rates. Early log-phase E. coli cells contained at least eightfold more nucleic acid and were significantly larger than the stationary-phase cells. Cultures of early log-phase R. meliloti cells contained three to four-fold more nucleic acid and were slightly larger than cells in the stationary phase. Rhizobium japonicum had very little change in either parameter. In general, the amount of change in both cell size and nucleic acid content upon initiation of log-phase growth was related to the overall growt rate of the organisms, with E. coli experiencing the greatest change and R. japonicum the least. Results obtained by FMF analysis, therefore, were consistent with observations reported by earlier workers. Cultures of R. meliloti also were used to demonstrate that the intensity of the fluorescent signals was sensitive to digestion by DNase and RNase and to prolonged storage and fixation. The potential use of FMF in the study of microorganisms is discussed.

Comparison of nucleic acid content in populations of free-living and symbiotic Rhizobium meliloti by flow microfluorometry.

Populations of symbiotic Rhizobium meliloti extracted from alfalfa nodules were shown by flow microfluorometry to contain a significant number of bacteroids with higher nucleic acid content than the free-living rhizobia. Bacteroids with lower nucleic acid content than the free-living bacteria were not detected in significant quantities in these populations. These results indicate that the incapability of bacteroids to reestablish growth in nutrient media may not be caused by a decrease in nucleic acid content of the symbiotic rhizobia.