Padma Somasegaran

Quantifying the Growth of Rhizobia

This chapter deals with routine enumeration techniques for pure cultures of rhizobia. The total or direct count is performed using the microscope. Optical density measurements are used to estimate the number of cells in broth culture. The viable count is accomplished through plating methods. The mean generation times of a Rhizobium sp. and a Bradyrhizobium sp. in broth culture are computed.

Oils as adhesives for seed inoculation and their influence on the survival of Rhizobium spp. and Bradyrhizobium spp. on inoculated seeds.

Mineral oil, peanut oil and soybean oil were compared with water and gum arabic for their suitability as adhesives for seed inoculation with peat inoculants. Inoculated seeds were stored at 4, 28 and 34°C, and sampled after 1, 3 and 9 days to determine the survival of rhizobia. Germination and nodulation tests were performed on the inoculated seeds. Results showed that oils were suitable adhesives for peat inoculants. Although the oils initially bound less inoculant to the seed, the number of surviving rhizobia was similar to that obtained by the gum arabic treatment after storage at 28 and 34°C for 3 and 9 days. An interesting finding of this experiment was that peanut and soybean oils were superior to gum arabic in supporting significantly higher numbers of chickpea rhizobia at 34°C. Inoculated seeds tested for germination and nodulation showed no adverse effects from the oil treatments. Oils hold good potential as adhesives for seed application in inoculation technology.

Collecting Nodules and Isolating Rhizobia

The purpose of this chapter is to become familiar with legumes in the field, examine their nodules, isolate rhizobia from nodules, and preserve the isolates. The subfamilies in the Leguminosae will be discussed and identifications will be made with the help of a botanical key. Nodules will be sectioned and examined. Simple stains of nodule smears will be examined under the microscope. Rhizobia will be isolated from nodules and grown on presumptive test media. The isolates will be authenticated on their original host plants and then preserved on ceramic beads.

Counting Rhizobia by a Plant Infection Method

The plant infection count, also known as the most-probable-number (MPN) count, is used to determine the number of viable and infective rhizobia in the presence of other microorganisms. The trap legume selected in the MPN method must belong to the same cross-inoculation group of legumes nodulated by the rhizobia under investigation. This indirect method is commonly used to determine the quality of inoculants produced from nonsterile carrier materials. It is also used to determine the number of rhizobia in the soil.

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.

Polyclonal antisera production by immunization with mixed cell antigens of different rhizobial species

Somatic antigens of Bradyrhizobium japonicum, Rhizobium sp. (Cicer arietinum) and Rhizobium sp. (Leucaena leucocephala) were prepared as standard, single-species type from cultured cells. Equal numbers of the cells of these rhizobia were then combined to obtain a mixed-rhizobial-species antigen preparation. Rabbits were immunized either with the standard, single-species type or with the mixed-rhizobial-species antigen preparations. The antisera developed from the mixed antigen immunization contained antibodies for all three rhizobial species, detectable at agglutination titres of over 800. The mixed-rhizobial-species antisera were made species specific by cross-absorption. The cross-absorbed and the mixed-rhizobial-species antisera were generally similar in quality for strain identification by agglutination, fluorescent antibodies, immunoblot and ELISA. A 66% reduction in cost was estimated for the production of antisera by immunization with mixed-rhizobial-species antigen.

Agglutinating Antigens from Root Nodules

This experiment is designed to demonstrate that the Bradyrhizobium japonicum bacteroids in soybean (Glycine max) nodules share common antigenic properties with its bacterial genotype in culture. Therefore, bacteroids from fresh, desiccated, or oven-dried nodules can be used directly for identifying the occupant strain by simple agglutination. This direct method eliminates the time-consuming steps of isolating the strain in pure culture prior to its use as an antigen in an agglutination reaction.

Producing and Applying Fluorescent Antibodies

With the fluorescent antibody (FA) technique, the antigen-antibody complex can be viewed directly under the microscope. This allows the researcher to detect and identify microorganisms simultaneously. FAs are useful for rhizobial strain identification in ecological research. In this chapter, antisera are purified by ammonium sulfate precipitations and dialysis. The protein content of the dialysate is determined. The immunoglobulin fraction is then conjugated with fluorescein isothiocyanate (FITC). The FITC-antibody conjugate is separated from the unreacted FITC by column chromatography (gel filtration). It is then used to identify rhizobia in nodules by the direct FA technique. A modification of this method, referred to as the indirect FA technique is described in Appendix 13.

Somatic Agglutination Reactions with Pure Cultures of Rhizobia

This chapter will describe relatively simple serological procedures for estimating the concentration of antibodies in a serum by agglutination. The rhizobial antigen is heat treated to inactivate the flagellar antigens and to illustrate the granular clumps characteristic of the insoluble somatic antigen reaction. The highest dilution of the serum at which positive agglutination occurs is used to define the titer.

Identifying Rhizobia by Immunoblot

The immunoblot technique is an enzymatic immunoassay for the detection of antigens at picogram (10–12) levels. Like the indirect enzyme-linked immunosorbent assay (ELISA), the method described here can be used with a commercially prepared secondary anti-body-enzyme conjugate. In principle, the ELISA and the immunoblot assay are similar. In the immunoblot assay, the antigens are immobilized on a nitrocellulose membrane and reacted with strain-specific (primary) antibodies. After a washing step, the fixed primary antibodies bound to the membrane are reacted with alkaline phosphatase conjugate of antibodies specific to the primary antibodies. Reaction with an enzyme substrate causes appearance of purple dots on a white background, indicating the presence of the rhizobial strain assayed. Like the ELISA, but unlike most other serological methods, the immunoblot assay can be used to analyze a large number of samples simultaneously. This makes this method especially useful for establishing strain occupancy in root nodules. In this chapter, the use of the immunoblot assay is demonstrated in a strain com-petition experiment between two strains of Bradyrhizobium japonicum.