Genetic material

 

Pathogen-free seeds of two soybean cultivars (‘Tropical G. max - 4E’ = TGX -4E and non-tropical ‘Soma’) were obtained from the International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria and the Crop Breeding Institute (CBI), Harare, Zimbabwe, respectively.

 

Two rhizobial strains of Bradyrhizobium japonicum (designated I and II) and cowpea type (designated III and IV), were used in the study. The B. japonicum strains were obtained from a commercial outlet in Gainesville, Florida and the cowpea-type strains were obtained from Dr. P. Singleton at NifTAL, Hawaii, USA. Each strain was maintained in the laboratory by sub-culturing periodically (every 60 days) on yeast-mannitol agar (YMA) growth medium described by Vincent (1970). The cultures were streaked directly on YMA plates (15.0 x 100.0 mm) using the method described by Somasegaran and Hoben (1994) and incubated at room temperature.

 

Inoculum was prepared by excising (under sterile conditions in the laboratory) two pieces of agar (20 x 20 mm) supporting rhizobial colonies from the subculturing dishes that were then placed in bottles containing 300 ml sterile distilled water. Two drops of the surfactant Tween® 80 (polyoxyethylene sorbitan monooleate) were added to each bottle before shaking vigorously to disperse the rhizobial cells. Each bottle was wrapped with aluminum foil to protect rhizobia from inactivation by UV light.

 

 

Planting procedure and evaluation of N₂-fixation

 

The planting procedure involved placing one seed in a hole (1 cm deep) in the center of a Conetainer® (Stuewe and Son, Inc., USA) measuring 4 cm in diameter x 20.5 cm in length filled with moist sterile washed sand. After adding inoculum (0.1 ml), each seed was immediately covered with the sand. The resulting plants were grown in the greenhouse with day/night temperatures of 28/20^oC without supplemental light in Gainesville, Florida, USA.

 

One week after emergence 0.1 g of microelement fertilizer (3.72% Fe, 9.28% Mg, 0.002% Mo and 2.32% Mn) was applied to each plant. Two weeks after emergence and at weekly intervals thereafter, 0.1 g of nitrogen-free (0-10-20) fertilizer was applied to each plant.

 

Six weeks after emergence, each plant was classified as either vigorous with dark green leaves indicating effective nodulation and N₂-fixation (+) or stunted with chlorotic yellow leaves indicating ineffective nodulation and no N₂-fixation (-). In each cultivar x strain combination, five plants (replications) were inoculated. One uninoculated (control) plant was included to assess effective nodulation.

 

Nodules from plants with green leaves and yellow leaves were harvested. Each nodule was washed and carefully rinsed with water before excision into halves in order to determine the color of the nodular tissue and relate it to the leaf color of the plant.

 

After six weeks of growth under nitrogen-free conditions, all the uninoculated (control) plants were distinctly chlorotic, yellow and stunted. Rhizobial strain I induced effective nodules on both cultivars (Table 1). However, 'Soma' responded differentially to the strains. The cowpea-type rhizobial strain IV, was compatible with the cultivar TGX-4E but not with cultivar Soma (Table 1). This indicated that major cultivar and rhizobial differences were present and could be detected by this sensitive greenhouse conetainer method.

 

 

 

Table 1. The effectiveness of nodulation in early juvenile plants of two soybean cultivars grown in nitrogen-free medium inoculated individually with rhizobial strains. (+ = dark green leaves on normal, N₂-fixing plant; - = yellow leaves on chlorotic, nonfixing plant).

 

             

 

 

 

Chlorotic plants with yellow leaves, were visually distinguishable from the vigorous plants with dark green leaves (Figure 1). Because the plants were grown in a nitrogen-free medium, the available nitrogen as indicated by the dark green leaves, was derived from the N₂-fixation process. Nitrogenous compounds resulting from N₂-fixation are exported from root nodules in the form of ureides (allantoin and allantoic acids) and translocated to the leaves where they are catabolized (Winkler et al. 1987) and used for the biosynthesis of chlorophyll and other proteins essential for photosynthesis.

 

 

 

 

Figure 1. Differences in leaf color indicating differences in N₂-fixation effectiveness among early juvenile soybean plants grown in nitrogen-free medium inoculated with a cowpea type rhizobial strain IV.

 

 

 

All the inoculated plants had nodules regardless of cultivar or rhizobial strain. The morphology, size and exterior color of intact nodules were generally indistinguishable. However, the cross-sections of nodules indicated clear differences in the color of nodular tissue between effective and ineffective nodules (Figure 2). The ineffective nodules were white to light green inside while the effective nodules were characteristically pinkish-brown indicating differences in N₂-fixation capabilities between the two nodule types.

 

 

 

 

      

 

Figure 2. Intact and cross-section of root nodules from early juvenile soybean plants grown in a nitrogen-free medium and inoculated with a cowpea-type rhizobial strain IV. a = root nodules from plants with dark green leaves indicating effective nodulation and b = root nodules from plants with light yellow leaves indicating ineffective nodulation.

 

 

In summary, our results demonstrated a simple, inexpensive and reproducible method for identifying early juvenile soybean genotypes effective in N₂-fixation under nitrogen-free growth conditions. The method would be useful in plant breeding programs concerned with rapid screening of soybean for N₂-fixation effectiveness.

   

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Vincent JM (1970). A manual for the practical study of root nodule bacteria. IBP Handbook No. 15. Oxford, Blackwell Scientific Publications, Oxford, England.

 

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