Date of Award

5-1-2014

Degree Name

Doctor of Philosophy

Department

Plant Biology

First Advisor

Klubek, Brian

Abstract

Soybean has a strong demand for nitrogen that can be acquired from atmosphere for vegetative growth and seed production through the symbiosis with the soil bacterium Bradyrhizobium japonicum (B. japonicum). However, the native soil bradyrhizobia may be ineffective in nitrogen fixation and the greatest limiting factor in increasing symbiotic nitrogen fixation is the inability to influence the infection of soybean roots by a desired strain of B. japonicum due to competition from the native bradyrhizobia. Previous studies have demonstrated the efficacy of a co-inoculum seed treatment on the symbiotic competency of the soybean cultivar LS90-1920 in greenhouse and field trials. The co-inoculation by the soil bacterium Streptomyces kanamyceticus (S. kanamyceticus) strain ATCC 12853 and strains of B. japonicum more efficient in nitrogen fixation and resistant to the antibiotics kanamycin and neomycin may have an advantage over the native bradyrhizobia regarding soybean root infection (Gregor et al., 2003). However, inconsistent inoculation responses in field trials and low efficacy in nodule competency by selected Bradyrhizobium japonicum (B. japonicum) co-inocula were observed under greenhouse conditions. These results were attributed to insufficient population size or growth of viable co-inocula associated with the seed treatments. This recent study showed that the nodulation response of LS90-1920 to B. japonicum strains KNI-1 and KNI-3 is independent of the inoculum dose and age of the broth culture. Iron supplement to the inoculum nutrient solution significantly increased the total biomass of nodules formed by strain KNI-1 but not by strain KNI-3 on a per plant basis and had no effect on the nodule number regardless of B. japonicum strain. In the glass bead viability study, the effect of inoculum nutrient solution concentration on the viability of bacterial co-inocula is species-specific and influenced by seed coating material. The growth of Pseudomonas putida strains displayed a dependency on the concentration of the inoculum nutrient solution with graphite or vermicompost as the seed coating material treatment or with activated charcoal treatment associated with 0.1% or 1.0 % inoculum nutrient solution. The seed coating material treatments of vermicompost and graphite promote stronger growth of S. kanamyceticus strain ATCC 12853 than the activated charcoal treatment. After a six-day incubation at 28oC, a 1.0 % inoculum nutrient solution maintained the highest viable populations of co-inocula with activated charcoal and a 0.1% inoculum nutrient solution was most effective in the maintenance of the co-inocula population when graphite or vermicompost was employed as the seed coating material. By applying the appropriate level of inoculum nutrient solution, the viability of a selected B. japonicum KNI strain and co-inocula remained stable for six days in activated charcoal and graphite treatment regardless of the number of applied co-inocula. However, the vermicompost treatment did not maintain the viable populations of the B. japonicum KNI strains and P. putida strain G11-32 but support the vigorous growth of S. kanamyceticus strain ATCC 12853 and P. putida strain 17-29. Greenhouse studies employing sterilized vermiculite as a soybean growth medium showed no significant differences in nodule competency by the inoculum/seed coating treatments associated with B. japonicum strain KNI-1. However, the co-inoculum treatments significantly increased either the total nitrogenase activity (B. japonicum strain KNI-3 with S. kanamyceticus strain ATCC 12853) or the nodule number (B. japonicum strain KNI-3 with S. kanamyceticus strain ATCC 12853 and P. putida strain 17-29) versus the singular inoculum treatment of strain KNI-3. Soil-pot studies under the same greenhouse conditions showed no significant differences in the nodule competency between the inoculum treatment of B. japonicum strain KNI-3, the co-inoculum treatment of strain KNI-3 and S. kanamyceticus, and the non-inoculated control regardless of seed coating material. However, co-inoculation of emergence-promoting rhizobacteria (Pseudomonas putida strain 17-29 and G11-32) with strain KNI-3 and S. kanamyceticus strain ATCC 12853 may improve the total nitrogenase activity and specific nitrogenase activity, depending on the seed coating material and soil type. The treatment with activated charcoal employed as a seed coating material and the co-inocula of strain KNI-3, S. kanamyceticus strain ATCC 12853 and P. putida strains 17-29 or G11-32 showed significantly higher total nitrogenase activity (Stoy silt loam) and specific nitrogenase activity (Drummer silty clay loam) versus the non-inoculated control. For the Bethalto silty clay loam, the same co-inoculum treatment associated with graphite and vermicompost as the seed coating material significantly increased the total nitrogenase activity. The seed coating treatment by activated charcoal enhanced nodulation competency for both the 2010 and 2011 field trials resulting in higher grain yield, seed nitrogen content, and seed protein content versus the seed coating treatment by graphite. No significant differences by the inoculum treatments were determined.

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