Study On Rhizobium Inoculants

Rhizobium leguminosarum bv.trifolii TA1 and Mesorhizobium huakuii 7653R were selected to draw growth curves in six rhizobium culture media of YMA, BSE, TY, SM, PA and Alrarez mannital medium (AMM). Results demonstrated that the rhizobia tested grew fast in BSE and YMA and nearly fail to grow in PA. Strain TA1 shown only a little growth in AMM,but 7653R grew in AMM even faster than in SM. Although strains in BSE have the fastest growth ,but it is not suitable for industry fermentation because its complicated procedures and shortage of bean sprout in summer. In comparison, YMA was easy to prepare and easy to control its quality ,so that it was the best medium for industry production of rhizobium inoculants.The growth and its corresponding OD growth curves of strains tested in YMA were made on the same graph to show their relations . The generation time (GT)of 7653R in YMA was 6.6 hours. The generation time of TA1 in YMA were 2.9 hours . Both of solid and liquid concentrated inoculants were made based on the growth curve analysis, and viability of the inoculants after 7 days, 15days, 30days, 60days, 90days, 120days, 150days and 180 days' storage were analyzed.Peat , vermiculite and pearlite were used as carriers to make solid inoculants after adjustment on pH value and moisture content. The viability was compared after storage at room temperature from March to September in Wuhan .Relsults demonstrated that peat was the best ,pearlite the second, and vermiculite was the worst . Results of viable number tests demonstrated that the viable numbers of three solid inoculants in the first 30 days could maintain at rather high levels and declined gradually in the following five months. Viable number of 7653R peat-based inoculant could increased to the maximal of 3.7 ×10¹⁰/gram and TA1 vermiculite-based inoculant could be up to the maximal of 7 × 10⁸/gram.The decrease in viable number of two strains tested was the lowest in peat, lower in vermiculite and the highest in pearlite .Concentrated liquid rhizobium inoculant was made and the viable numbers were tested after the storage in 4℃ refrigerator at different period .Relsults demonstrated that viable cells of 7653R were higher than that of TAl.The viable numbers were all declined following the storange time. There were obvious differences between cells stored in vacuum and in N₂ gas.The viable number of TA1 was higher in vacuum is than in N2.But for 7653R viable number in N₂ gas was higher than in vacuum.The viable cells of TA1 in N₂ gas will drop to about 1/10 of the original.Three bacteriostatic agents of sodium benzoate - P-Hydroxybenzoic acid and Ethyl-P-hydroxybenzoate were used to increase the viable cell percentage in concentrated liquid inoculants. Suitable concentrations of bacteriostatic agents were 30ppm for sodium benzoate ^ 5-7ppm for P-Hydroxybenzoic acid and 2-5ppm for Ethyl-P-hydroxybenzoate . Their protective effect will decreased when the concentrations tested were higher or lower than the suitable concentrations.Rhizobia cells will even be killed if the concentrations used increased to a certain comparatively high levels. Trehalose was selected as a protective agent according to the date obtained. Four trehalose cencentrations of 0.1% , 0.59^ 1% and 2% were compared with distilled water and sodium chloride solution. Results demonstrated that the protective effect was increased with the concentrations tested. So that in the next experiment, trehalose concentrations of 1%, 2%, 3%s 4% and 5% were again tested and foune that the 4-5% trehalose was the best concentration.