Soybean evolved under warm conditions, and is physiologically adapted for warm conditions. The optimum temperature for soybean nodulation is 25-30°C. Southwestern Quebec is on the North American northern limit for soybean production. Soybean plants emerging in the region frequently turn light green once cotylendonary N reserves are exhausted, and remain that way until nodulation is complete (Zhang et al. 1995). We postulated that low soil temperatures inhibited soybean nodulation. We were able to show that the time from inoculation increased by about 2 days per °C decrease in temperature between 25 and 17°C, and by approximately a week per °C between 17 and 15°C (Zhang, Smith 1994; Zhang et al. 1995). Further, the time that was most sensitive to low temperature was the first 12 h after inoculation (Zhang, Smith 1994), a time when signal exchange was taking place.
Signal exchange begins with the secretion of phenolic compounds, flavonoids and isoflavonoids, by host plants (reviewed by Schlaman et al. 1998). These compounds induce the nod genes, resulting in the production of the bacteria-to-plant signals (Kondorosi 1992). These are lipo-chito-oligosaccharides (LCOs). These compounds play a key role in initiating the early events of the legume-rhizobia nitrogen-fixing symbiosis (Lhuissier et al. 2001). In this role they are referred to as Nod factor. Extensive work on LCOs has revealed that members of the group are responsible for the host specificity of rhizobia (Perret et al. 2000). LCOs invoke multiple physiological responses in the host: root hair deformation (Spaink et al. 1991; Prithiviraj et al. 2000a), induction of nodulin genes essential for infection thread formation (Horvath et al. 1993) and cortical cell division (Schlaman et al. 1998). Purified Nod factors per se are capable of initiating complete nodule structures at submicromolar concentrations in some legume-rhizobia systems (Denarie, Cullimore 1993).
We added plant-to-bacteria signal compounds (generally genistein) to bacteria used as inocula on soybean plants and found that this accelerated the very earliest stages of nodulation (Zhang, Smith 1994), leading to earlier subsequent nodule development and the onset of nitrogen fixation. As a result, plants inoculated with genistein treated Bradyrhizobium japonicum cells were larger and contained more nitrogen. While the optimum genistein concentration is known to be 5 pM at 25°C (Kosslak et al. 1987), at 15°C it was higher, at 20 pM (Zhang, Smith 1995). At the same time we were able to show that, at lower root zone temperatures soybean roots contained less genistein (Zhang, Smith 1996a).
Zhang and Smith (1996b, 1997) showed that incubation of B. japonicum with genistein, prior to application as an inoculant, or directly applied into the seed furrow at planting, increases soybean nodulation, N2 fixation and total N yield, when field conditions are such that they would normally delay or inhibit nodulation. This was caused by a shortening of the time between inoculation and when the infection thread reached the bottom of the root hairs.
Pan and Smith (2000a) showed that genistein treated B. japonicum 532C, with a genetic marker, had higher levels of nodule occupancy than the untreated cells under greenhouse conditions. Paau et al. (1990) reported that adding soybean meal to the fermentation medium can alter the nodule readiness of the rhizobia and that this has an effect on the competitiveness of the inoculant strains (McDemott, Graham 1990).
Soybean nodulation is also inhibited by mineral nitrogen (Zahran 1999). We were able to show that when N concentration in the rooting medium increased above 50 mg L"1, genistein concentrations in root systems decreased, and daidzein concentration in plant root systems decreased steadily as N application level increased from 0 to 150 mg L"1 (Zhang et al. 2000). Inoculation of soybean with B. japonicum that has been pre-activated with genistein and daidzein also improves plant nodulation and nitrogen fixation under levels of mineral nitrogen that are inhibitory to nodulation (Pan, Smith 2000b). Pan and Smith (2000b) found that the plants receiving pre-incubated B. japonicum cells had more nodules, nodule weight, and plant nitrogen content, especially in a low N containing sandy soil and where 20 kg N ha"1 were added as mineral fertilizer.
A company, Bios Agriculture Inc., has commercialized these findings. They have accumulated data from over 200 site-years in the Canadian soybean production areas, and in the northern tier states of the USA. In general, this technology results in a yield increase on the order of 10%, when applied to early seeded soybean crops.
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