Thesis (M.S.)--University of Missouri-Columbia, 2008. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on July 8, 2009) Includes bibliographical references.

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Soybean [Glycine max (L.) Merr.] is a tropical to subtropical legume that requires root zone temperatures (RZTs) in the 25 to 30°C range for optimal symbiotic activity. The inability of soybean to adapt to cool soil conditions limits its development and yield in short season areas. In particular, nodulation and nitrogen fixation by this crop species is sensitive to cool RZTs. The isoflavone genistein, which is the most effective plant-to-bacterium signal in the soybean nitrogen fixation symbiosis, has been used to pretreat Bradyrhizobium japonicum inocula. This resulted in increased soybean nodulation and nitrogen fixation in several studies, indicating that genistein preincubated inocula could overcome low RZT inhibition of plant growth and yield. The effectiveness of isoflavones was found to vary among soybean cultivars. Some legume cultivars apparently supply limiting amounts of the flavonoids. The objective of this thesis was to determine how soybean cultivars of different maturity groups would respond to genistein incubation of B. japonicum prior to inoculation. Two field experiments were conducted in 1997 and 1998 involving eleven soybean cultivars of three maturity groups organized in a randomized complete block design. Cells of B. japonicum, treated with genistein or not, were applied onto seeds in the furrow at the time of planting. The results of this study indicated that genistein application increased nodule number and nodule dry matter per plant, increased plant nitrogen content, grain protein and grain yield of all cultivars. There was no interaction between maturity group and genistein application, and there was no correlation between maturity groups and increase in nodulation, total biomass, nitrogen content and yield due to genistein treatment. Thus, responses of soybean cultivars of different maturity groups to genistein treatment are not related to maturity and/or yield potential.

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In the N$ sb2$ fixing legume symbiosis, suboptimal root zone temperatures (RZTs) not only decrease N$ sb2$ fixation. but reduce the formation and development of nodules. The purpose of this thesis was to elucidate the mechanism by which suboptimal RZTs affect nodulation and nodule development in legumes, such as soybean (Glycine max (L.) Merr.) and to attempt to find ways to overcome this inhibition. Initial studies characterized the RZT response in soybean plants inoculated with Bradyrhizobium japonicum. In plants grown at RZTs from 25 to 17$ sp circ$C, the time between soybean inoculation with B. japonicum and the beginning of N$ sb2$ fixation increased by 2.5 days for every $ sp circ$C decrease, whereas below 17$ sp circ$C RZT each $ sp circ$C appeared to delay the onset of N$ sb2$ fixation by 7 days. RZTs less than 17$ sp circ$C strongly inhibited the nodulation process and, as a result also sharply decreased N$ sb2$ fixation per plant. The greater sensitivity below 17$ sp circ$C is due to events related to, or occurring before infection initiation. Coinoculation of soybean with B. japonicum and other microorganisms beneficial to legumes, either vesicular-arbuscular (VA) mycorrhizae or plant growth promoting rhizobacteria (PGPR), increased soybean nodulation and N$ sb2$ fixation, but these increases were temperature dependent. Vesicular-arbuscular mycorrhizal colonization had a negative effect on nodule establishment below 18.5$ sp circ$C RZT, but a positive one above this RZT. At each temperature tested some PGPR increased the amount of fixed N and number of nodules formed, whereas some decreased the level of these variables. The most stimulatory strain at each temperature was: 15$ sp circ$C-Serratia proteamaculans 1-102, 17.5$ sp circ$C - S. proteamaculans 1-102 and Aeromonas hydrophila P73, and 25$ sp circ$C - S. liquefaciens 2-68. / Because our research indicated that an event before infection thread initiation was most sensitive, and because the first known step in establishment of the symbiosis is production of a plant-to-bacterial signal molecules. I tested whether the poor nodulation at suboptimal RZTs was related to disruption of plant-to-bacterium signalling. Inocula bacteria were preincubated with genistein, a major isoflavonoid signal molecule in soybean. This shortened the period between inoculation and root hair curling, and hastened the onset of N$ sb2$ fixation under both controlled environment and field conditions. At 15 and 17.5$ sp circ$C RZTs, 20 and 15 $ mu$M genistein was found to reduce the inhibition of suboptimal RZTs, increase nodulation, and accelerate the onset of ${ rm N} sb2$ fixation. When applied to the plant rhizosphere in the field, genistein also reduced the inhibitory effects of cold spring soils on nodulation and N$ sb2$ fixation. Direct measurements of genistein accumulation in soybean roots indicated that, with decreasing RZTs, genistein accumulation decreased. B. japonicum USDA110 containing plasmid ZB977 with nodY-lacZ fusion genes incubated with genistein under different temperatures indicated that higher genistein concentrations and longer incubation times were required to activate the lacZ gene to a maximum level under low incubation temperature. Overall, these findings suggested that plant-to-bacteria signal molecules such as genistein may be an important limiting factor in the nodulation of legume plants at low RZT.

This study was undertaken to characterize the nod and sdh operons of Bradyrhizobium japonicum and Sinorhizobium meliloti. Ten putative B. japonicum mutants with altered nod gene induction characteristics were isolated by screening mutants for genistein-independent nod gene expression. The mutants were found to have higher nodY expression than the wild-type in the presence of genistein. The increased sensitivity of all mutants to genistein was more apparent under suboptimal inducer concentration (0.1muM) and/or temperature (15°C). The expression of nodY gene induction was determined for five strains (Bj30050, 53, 56, 57, 58) under different temperature and inducer conditions. These five strains were also found to produce more lipochitooligosaccharide than the wild-type, at both 25°C and 15°C. Three of the ten mutant strains (including Bj30056 and 57) were unable to fix nitrogen with soybeans grown at optimal temperatures. Based on nodY gene expression and symbiotic phenotype the B. japonicum mutants were classified into three groups. / A molecular genetic approach was taken to investigate the regulation of expression of succinate dehydrogenase (SDH) in S. meliloti. The sdhCDAB genes encoding SDH were shown by RT-PCR to be co-transcribed and thus constitute an operon. The transcriptional start site and putative promoter region of the first gene in the operon, sdhC , were identified by 5'-RACE and DNA sequence analysis. Transcriptional lacZ fusions to sdhC indicated that expression of the operon is regulated by carbon source in the growth medium but not by growth phase. The highest expression of the sdh operon was observed in cells grown with acetate, arabinose and glutamate, as sole carbon sources, and the lowest expression was observed in cells grown with glucose and pyruvate as sole carbon sources. / Also presented is the isolation and characterization of the first defined sdh mutant in a rhizobial species. The mutants helped demonstrate that the total lack of SDH activity would be lethal to S. meliloti cells. Symbiotic phenotype of the mutants indicated that SDH is required for N2-fixation.

Soybean (Glycine max L. Merr.) and Bradyrhizobium japonicum can form a nitrogen fixing symbiosis. This symbiosis is important for most sustainable agriculture systems. This thesis examines two ways to enhance nodulation and nitrogen fixation by this symbiosis: coinoculation of plant growth promoting bacteria (PGPB) with B. japonicum, and addition of RNA to a bradyrhizobial culture medium. The optimal coinoculation dose of Serratia proteamaculans 1--102 and S. liquefaciens 2--68 was determined as 108 cells per plant under both optimal and suboptimal root zone temperatures (RZTs). Nodulation dynamics studies indicated that coinoculation of these two PGPB caused earlier nodule initiation and a higher nodulation rate, contributing to the higher nodule number and nodule weight. The coinoculation also increased nitrogen fixation efficiency under both optimal and suboptimal RZTs. A novel inducible activator only produced by the bacteria after addition of flavonoids to the culture system was prepared and evaluated in greenhouse and field experiments. Fourteen non-bradyrhizobial endophytic bacteria (NEB) were isolated from the surface sterilized root nodules, and three of these, designated NEB4, NEW and NEB17, showed soybean plant growth promotion under both greenhouse (with controlled RZTs) and field conditions. Alone, they were neither nodule inducers nor nitrogen fixers. Biolog tests and partial 16S rRNA gene sequence analyses placed the three strains in genus Bacillus: NEB4 and NEB5 are B. subtilis and NEB17 B. thuringiensis. Bradyrhizobium species grow slowly, making the culture process long and the cost of inoculant production higher. Addition of commercial yeast RNA to the bacterial culture medium accelerated the bacterial growth rate, shortened the culture time and increased the lipo-chitooligosaccharide (LCO) yield in flask cultures. Inoculation experiments in the greenhouse also showed that bradyrhizobial inoculant produced in the presence of RNA had better symb

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