E. L. Schmidt

Lectins: a possible basis for specificity in the rhizobium-legume root nodule symbiosis

Soybean lectin labeled with fluorescein isothiocyanate combined specifically with all but 3 of 25 strains of the soybean-nodulating bacterium Rhizobium japonicum. The lectin did not bind to any of 23 other strains representative of rhizobia that do not nodulate soybeans. The evidence suggests that an interaction between legume lectins and Rhizobium cells may account for the specificity expressed between rhizobia and host plant in the initiation of the nitrogen-fixing symbiosis.

The Immunofluorescence Approach in Microbial Ecology

Fluorescent markers appropriately conjugated to antibody proteins provide the basis for a method to visualize those antibodies as they participate in antigen-antibody reactions. The method is referred to as the fluorescent antibody (FA) or immunofluorescence (IF) technique; it has been in widespread and successful use in medical microbiology and in pathology as a highly sensitive and specific cytochemical staining procedure for many years. Most current applications of the technique are for the localization of cellular and viral antigens in tissues and for the rapid detection and identification of infectious agents.

Quantitative Autecological Study Of Microorganisms In Soil By Immunofluorescence

The fluorescent antibody (FA) technique makes possible an autecological approach to microbial ecology because it permits specific microorganisms to be seen and identified in their natural habitats. A method is reported which further extends the FA technique to the quantification of specific bacteria in aquatic and terrestrial ecosystems. Procedures were developed for the most difficult FA-quantification problem, that of the microorganism growing in soil. The protocol that was evolved included: release of bacteria from soil in a dispersed suspension; flocculation of soil colloids out of suspension; concentration, on a special membrane filter, of the bacteria remaining in a known volume of suspension; staining with appropriate homologous FA; and enumeration of reactive cells by incident light immunofluorescence microscopy. Data are reported on the quantification of several bacteria in soil. FA-counts of Rhizobium japonicum, strain USDA 110, agreed well with viable plate counts as the population developed in sterilized soil. Growth of the same organism in nonsterile soils could be followed only by FA-counts. Growth rates and population features for nonsterile soils differed greatly from those observed in sterile soil or laboratory cultures. Escherichia coli die-off was studied quantitatively in normal, nonsterile soil by selective plating, and by the FA-membrane filter count. Agreement between the two methods was good, but FA counts were high at certain stages, probably due to the rapid accumulation of recently-dead cells. As a final example Nitrobacter was enumerated by specific FA-membrane filter counts during nitrification in a partially sterilized soil to relate the dynamics of growth to the conversion of nitrite to nitrate.

Nonspecific Staining: Its Control in Immunofluorescence Examination of Soil

Gelatin preparations were used to treat soil slides prior to addition of fluorescent antibody. Nonspecific staining was avoided, with no detectable interference to specific staining. Gelatin-rhodamine conjugates served to counterstain as well as to prevent nonspecific staining.

Limiting Factors for Microbial Growth and Activity in Soil

Over the last decades important progress has been made in our knowledge of microbial enzyme machinery and its manipulation, especially in the field of free-living and symbiotic nitrogen fixation, allowing the possible development of new microbial strains or new plant-microorganisms systems in order to improve the quantity and quality of crop yields. However, it must be remembered that the soil microorganism involved in plant associations functions in the complex environment made up of the soil and the lower atmosphere. Thus, the growth and activity of any given soil microorganism depend not only on its genetic characteristics but also on a complex of factors constituting its environment, which finally governs the expression of its intrinsic capabilities. Since the impact of environmental factors on soil microorganisms is often still poorly understood, it appeared necessary to review current concepts concerning this aspect of microbial ecology, focusing attention on the nature and role of limiting factors.

EVIDENCE FOR DOUBLE INFECTION WITHIN SOYBEAN NODULES

Rhizobium japonicum strains 117 and 138 (USDA) were used in equal proportions to inoculate soybean seedlings. Preparations of individual nodules were stained with strain-specific fluorescent antibodies and rhizobia present were identified by direct microscopic examination using transmitted tungsten (darkfield) light and reflected near-ultraviolet light singly and in combination. At an inoculation density of 108 rhizobia per plant, 32 percent of the nodules contained both strains. The percent double infection decreased as inoculum density decreased. Strain 138 generally predominated over strain 117 in accounting for the majority of the nodules that contained only one strain, and cells of 138 were generally numerically predominant in nodules that contained both strains. Streak plate cultures of selected nodule slurries provided data that fully reinforced the fluorescent-antibody data.

Detection of Aspergillus flavus in Soil by Immunofluorescent Staining

Strains of Aspergillus flavus grown in soil in the presence of buried slides could be detected on the slides by fluorescent antibody techniques. Staining with A. flavus antiserum labeled with fluorescein, followed by examination with fluorescence microscopy revealed characteristic fluorescence at sites of distribution of the homologous fungus. The specificity of the reaction and the absence of nonspecific absorption of antibody to soil materials suggest that the method may be useful in studying the ecology of the soil microflora.

Absorption and translocation of mammalian viruses by plants. I. Survival of mouse encephalomyelitis and poliomyelitis viruses in soil and plant root environment.

Abstract The rates of degradation at 30° of strains FA and GD VII mouse encephalomyelitis viruses and of types 1 (Mahoney) and 2 (YSK) polioviruses in liquid and sand suspensions and in soil were determined. Mouse encephalomyelitis virus, strain FA, survived 3–4 weeks in sterile or nonsterile soil; virus in sterile or nonsterile liquid suspension survived for from 7 to 12 days. Soil pH influenced survival of GD VII virus which persisted for 3–4 weeks in alkaline soil, at least 5 weeks in neutral soil, but could not be recovered from acid soil after incubation for 24 hours. Type 1 poliovirus was recoverable from sterlle soil for 6–7 weeks, and from nonsterile soil for 2–3 weeks. Soil and soil clays adsorbed and held firmly large quantities of poliovirus. In the environment of roots of tomato plants cultivated hydroponically: (a) types 1 and 2 polioviruses were inactivated at similar rates, and (b) type 1 virus from cell culture was inactivated more rapidly than virus in monkey brain suspension. Monkey brain type 1 virus was inactivated more rapidly in the environment of pea roots than in that of tomato roots.

Recent Advances in the Ecology of Rhizobium

Microbial ecology deals with the interactions between microorganisms and their environment. These interactions regulate the biochemical transformations carried out by microorganisms in nature and hence are of great theoretical and practical importance. Information on the ecology of those microorganisms with the ability to transform atmospheric nitrogen into biologically useful nitrogen is particularly critical to the goal of enhancing biological nitrogen fixation. This review will concentrate on the ecology of the nitrogen-fixing soil bacteria of the genus Rhizobium prior to its entry into the root of a crop legume host plant.

Nitrobacter in Mammoth Cave

Mammoth Cave, a large natural limestone cavern formed 20 to 30 million years ago in rocks laid down during the Mississippian Period, lies in westcentral Kentucky and borders on the western coal basin and the Mississippian Plateau. Historical1y, over 1800 tons of nitrate sediments were mined from Mammoth Cave prior to and during the War of 1812, and were subsequently processed for gunpowder. The extensiveness of the operation is substantiated by the large number of mining archeological artifacts that remain in the cave (Faust, 1967). Although the mechanism of saltpetre formation, CaNOJ, in cave ecosystems is unknown, various hypotheses have been suggested for saltpetre formation. Brown (1809) suggested that nitrates are leached into the cave sediments through drainage water since high concentrations of nitrates are sometimes found in cavernous sandstone rock. Priestley (1809) on the other hand suggested that weak nitrous acid produced in the atmosphere resulted in the deposition of saltpetre. Generally, it is thought that nitrate deposits in caves are formed by the degradation of bat guano (Clark, 1924); Hess (1900) reported, however, that deposits of nitrate extended over five miles into the cave, and such distances are not usually traversed by bats. Faust (\ 967) suggested that saltpetre formation was mediated by free-living (non-symbiotic) nitrogen fixing bacteria capable of fixing atmospheric nitrogen and using carbon dioxide as the sole energy source with the concomitant formation of CaNOJ. Yet, such an organism has never been reported nor isolated. Thus, the mode of formation of such large saltepetre deposits within Mammoth Cave and the role of bacteria in their formation remains unclear. Cave ecosystems provide the microbial ecologist with a selective natural