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.

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Soybean (Glycine max (L.) Merr.) is a subtropical legume that requires root zone temperatures (RZTs) in the 25 to 30$ sp circ$C range for optimal symbiotic activity. The inability of soybean to adapt to cool soil conditions limits its development and yield in short season areas. In particular, nodulation and N$ sb2$ fixation by this subtropical crop species is sensitive to cool (RZT). The objectives of this thesis were to determine whether or not PGPR could be used to help overcome the low RZT inhibition of soybean nodulation, to improve soybean nitrogen fixation and yield under field conditions and to determine the methods by which such increases occurred. The work reported in this thesis has demonstrated that PGPR can increase early season nodulation and total seasonal nitrogen fixation and yield of soybean growing in an area with cool spring soils. The ability of PGPR to stimulate soybean nodulation and growth was shown to be related to their ability to colonize soybean roots, and this was shown to be related to RZT. All steps in early nodulation were stimulated by the presence of PGPR. The beneficial effects of PGPR are exerted through a diffusible molecule excreted into the growth medium. The addition of genistein, a plant-to-bacteria signal molecule already shown to stimulate soybean N$ sb2$ fixation at low RZT, plus PGPR causes increases in soybean nodulation, N$ sb2$ fixation, and growth that were greater than those caused by the addition of PGPR alone, but only at 25 and 17.5$ sp circ$C, and not at 15$ sp circ$C RZT.