B. Ben Bohlool

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.

Fast-Growing Rhizobia Isolated from Root Nodules of Soybean

Fast-growing rhizobia have been isolated from soybean root nodules collected in China. These new isolates are physiologically distinct from slow-growing soybean rhizobia. They formed effective nitrogen-fixing associations with wild soybean and an unbred soybean cultivar from China, but were largely ineffective as nitrogen-fixing symbionts with common commercial cultivars of soybeans.

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.

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.

Biochemical Characterization of Fast- and Slow-Growing Rhizobia That Nodulate Soybeans

Fast-growing, acid-producing soybean rhizobia were examined to determine their biochemical relatedness to each other, to typical slow-growing Rhizobium japonicum strains, and to other fast-growing species of Rhizobium. Although both the fast- and slow-growing soybean rhizobia were positive for catalase, urease, oxidase, nitrate reductase, and penicillinase, the fast-growing strains grouped with other fast-growing species of Rhizobium in that they tolerated 2% NaCl, were capable of growth at pH 9.5, utilized a large variety of carbohydrates (notably disaccharides), and produced serum zones in litmus milk. In addition, these fast-growing strains were similar to other fast-growing species of Rhizobium in that they produced appreciable levels of β-galactosidase and nicotinamide adenine dinucleotide phosphate-linked 6-phosphogluconate dehydrogenase but had no detectable hydrogenase activity. The fast-growing soybean rhizobia share symbiotic host specificity with Bradyrhizobium japonicum, but appear to be related biochemically to the other fast-growing species of Rhizobium.

PHENOLOGY, GROWTH, AND YIELD OF FIELD-GROWN SOYBEAN AND BUSH BEAN AS A FUNCTION OF VARYING MODES OF N NUTRITION

Abstract In field trials conducted at four sites in Hawaii, soybean (Glycine max) and bush bean (Phaseolus vulgaris) were either inoculated with homologous rhizobia, fertilized at high rates with urea, or left unamended. Crop phenology was assessed every few days. Rates of biomass and N accumulation and components of yield were measured five times during each crop cycle to assess the extent to which: (i) crops relying on soil, symbiotic, or fertilizer N differed in their growth characteristics; (ii) mode of N nutrition affected the timing of developmental stages; and (iii) effects of N nutrition on crop growth and development were related to final yield. While all measured variables differed significantly between sites, the effect of changing N source on these variables, in N limited environments, was consistent across sites. Rate and extent of node production, crop growth and yield were increased in symbiotic and N-fertilized crops as compared to unamended, non-fixing crops, while reproductive development was protracted. Extended time required to reach reproductive maturity was attributable to an increase in seed fill duration as time to flowering was not affected. Development and yield of N2-fixing crops were similar but not equivalent to those of N-fertilized crops. To produce reliable yield estimates, legume growth simulation models must be able to accurately simulate crop growth and phenology. The present data indicate that information relating to source and supply of N must be incorporated before such models can be used to generate reliable yield estimations. Results of these trials also provide a valuable dataset for calibrating model subroutines for inorganic nitrogen uptake and nitrogen fixation in soybean and bush bean growth under field conditions and adjusting model coefficients for tropical environments.

Serological differentiation of Azospirillum species belonging to different host-plant specificity groups

The results of the cross reactions of the 27 strains of Azospirillum spp. with 4 fluorescent antibodies (FA) show a neat differentiation between the two species. A. lipoferum represents a more homogenous group in respect to FA reactions and highly fluorescent preparations were obtained with strains from a large scope origin against Sp59 FA, the type strain. In contrast A. brasilense contains at least three sub groups in respect to FA reactions. The first includes all denitrifing strains (nir+) which react with FA from Sp7 the type strain. None of the nir- strains reacted strongly with Sp7 FA. One part of the A. brasilense nir- group which includes the strains isolated from well sterilized rice and wheat roots (Sp 107, 107 st, 106 and 109 st) reacts with FA of their reference strain Sp107 but not with that of Sp28 FA. The strains isolated from unsterilized roots and soils reacted with SP28 FA and not with that of Sp107 FA. In addition there were 3 strains (Sp A4, 34 and 67) which reacted with neither of the FAs.

Influence of acid soil on nodulation and interstrain competitiveness in relation to tannin concentrations in seeds and roots of Phaseolus vulgaris

Abstract The relationship between acid soils and (1) early nodulation in common beans, (2) interstrain competitiveness of Rhizobium leguminosarum bv. phaseoli strains and (3) tannin concentration in roots, were evaluated in two experiments. Ten Phaseolus vulgaris cultivars of different seed tannin content were grown in pots in a greenhouse in soil at pH 4.5 or 5.5, inoculated with equal amounts of strains CIAT 899 and KIM5. At pH 5.5, on all cultivars strain KIM5 occuppied a greater proportion of nodules than strain CIAT 899. This was reversed at pH4.5. Nodule number and nodule weight, 21 days after inoculation, were strongly reduced but root tannin concentrations doubled at pH 4.5. Nodule nmbers were negatively correlated with tannin concentration in roots at both pH values. Seed tannin content was negatively correlated with early nodulation, but this was statistically significant only at pH 5.5. Nodulation of cultivars with high seed tannin contents was generally more acid tolerant than of those with low seed tannin content. In a second experiment, six Phaseolus vulgaris cultivars were planted at two field sites with acid soil and one site with neutral soil. In the field, as in the pots, nodule numbers at early stages of plant development were reduced by 30–50% in the acid soils compared to the neutral soil. In contrast, root tannin content in acid soils was twice that in the neutral soil. There were significant cultivar and cultivar by location effects. Strains of rhizobia were tested for tolerance to bean seed tannin extracts, to seed diffusates and for survival on seed surfaces. The Rhizobium strains were sensitive to tannine extracts, with obvious strain differences. Sensitivity of most strains was greater at acidic pH. Seed diffusates, in contrast, did not inhibit growth of most strains.

THE ECOLOGY OF RHIZOBIUM IN THE RHIZOSPHERE: SURVIVAL, GROWTH AND COMPETITION

The excitement over prospects for genetic improvement of Rhizobium has, unfortunately, overshadowed the need for an understanding of its ecology. It must be kept in mind, however, that in order for any “superbug” engineered in the laboratory to have an economic impact, it must be able to withstand the stresses of the soil, survive and grow in the rhizosphere, and compete successfully with well-adapted indigenous rhizobia for nodulation of the appropriate host.

NON-DESTRUCTIVE CHLOROPHYLL ASSAY FOR SCREENING OF STRAINS OF BRADYRHIZOBIUM JAPONICUM

Abstract In greenhouse-grown soybeans [ Glycine max (L.) Merr. cv Clark] supplied with different amounts of combined nitrogen (0. 75. 150, 300 or 600 mg N as NH 4 NO) 3 . the chlorophyll content of leaf discs was significantly correlated with shoot dry weight ( r = 0.90; P r = 0.88; P Bradyrhizobium japonicum strains of varying effectiveness, there was a highly significant ( P r = 0.89); shoot total N ( r = 0,88): shoot N-fixed ( r = 0.88); nodule dry weight ( r = 0.85); and total nitrogenase activity ( r = 0.79). Effectiveness ranking of strains of B. japonicum based on chlorophyll estimates and shoot total N were similar. In field-grown soybean plants, the chlorophyll content of leaf discs was also significantly ( r = 0.92; P l ) correlated with N accumulated in the plant tops. Our data support the use of leaf chlorophyll content as an indirect measure of N status of greenhouse and field-grown soybeans and the effectiveness ranking of strains of rhizobia.