Agronomy Journal 94:870-875 (2002)
© 2002 American Society of Agronomy
SOYBEAN
Low Temperature–Tolerant Bradyrhizobium japonicum Strains Allowing Improved Soybean Yield in Short-Season Areas
Hao Zhanga,
Trevor C. Charlesc,
Brian T. Driscollb,
Balakrishnan Prithiviraja and
Donald L. Smith*,a
a Plant Sci. Dep., Macdonald Campus of McGill Univ., 21111 Lakeshore Rd., Ste-Anne-de-Bellevue, QC, Canada H9X 3V9
b Dep. of Nat. Resour. Sci., Macdonald Campus of McGill Univ., 21111 Lakeshore Rd., Ste-Anne-de-Bellevue, QC, Canada H9X 3V9
c Dep. of Biol., Univ. of Waterloo, Waterloo, ON, Canada N2L 3G1
* Corresponding author (dsmith{at}macdonald.mcgill.ca)
Received for publication May 21, 2001.
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ABSTRACT
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In short-season soybean [Glycine max (L.) Merr.] production areas, low soil temperature is potentially an important factor limiting soybean growth and yield. Some strains originating from cooler areas can cause more nodulation and nitrogenase activity under low-temperature conditions. We have attempted to find Bradyrhizobium japonicum strains that can fix more N than strain 532 C under low-temperature conditions. We selected 40 B. japonicum strains from the USDA collection based on their isolation from soils of northern locations. These 40 strains were tested for their ability to grow at a low (15°C) temperature, and the best two (USDA 30 and USDA 31) were selected for evaluation under field conditions. Inoculation with USDA 30 and USDA 31 resulted in greater soybean yields (an 8% increase, averaged over the 2 yr) than inoculation with 532 C. The increased yield was due to the formation of more pods per plant, and more seeds per plant, but not due to an increase in 100-seed weight. This indicated that the benefit caused by the superior strains occurred early in plant development, probably due to increased N fixation early in the growing season. This possibility was supported by the observations that leaf areas, grain protein production, and total protein levels for plants inoculated with USDA 30 and USDA 31 were greater than those inoculated with 532 C. These findings clearly demonstrate that inoculant strains likely to perform best in a given geographical area are those selected for the conditions prevalent in the area.
Abbreviations: OD620, optical density at 620 nm • YEM, yeast extract mannitol
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INTRODUCTION
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SOYBEAN is one of the most important crops cultivated in eastern Canada, with an annual production of approximately one million hectares. When in symbiotic association with Bradyrhizobium japonicum, soybean plants can fix up to 200 kg ha-1 yr-1 N (Smith and Hume, 1987), reducing the need for expensive and potentially environmentally damaging N fertilizer.
Rhizobia strains tend to perform best when they are used in environments similar to the ones from which they were isolated. Roughley (1970) found that the strain TA1, isolated from a comparatively cold environment, nodulated and formed bacteroids on sub clover (Trifolium subterraneum L.) at 7°C while strain SU297, isolated from a warmer environment, did not. Lipsanen and Lindstrom (1986) found close agreement between the nitrogenase activity of different strains of Rhizobium trifolii and their geographical origin and growth in pure culture at low temperatures. Prévost et al. (1987) found that the nitrogenase activities of five arctic rhizobia isolates were higher at low temperatures than those of temperate rhizobia when symbiotic with the legume sainfoin (Onobrychis viciifolia Scop.). Hume and Shelp (1990) found that inoculation of soybean with the B. japonicum strain 532 C, a Hup- strain, resulted in greater yields under Canadian conditions than inoculation with USDA 110, USDA 142, and USDA 143.
Soybean is a subtropical legume that requires root zone temperatures in the 25 to 30°C range for optimal symbiotic activity (Jones and Tisdale, 1921; Dart and Day, 1971). At lower temperatures, the expression of the nod genes is suboptimal, resulting in delays in the onset of nodulation (Zhang et al., 1996; Zhang and Smith, 1996). In regions such as Canada, low soil temperature is potentially a major factor limiting soybean growth and symbiotic N fixation (Whigham and Minor, 1978). The addition of genistein has proven to be an effective means of generating increases in N fixation and yield (Zhang and Smith, 1996). Zhang and Smith (1996) indicated that two strains of B. japonicum (USDA 110 and 532C) responded differently to signal molecule additions; addition of genistein-preincubated B. japonicum USDA 110 significantly increased the yield of soybean compared with USDA 110 alone while there was no difference between genistein treated and untreated B. japonicum 532 C under field conditions. Banfalvi et al. (1988) reported that some B. japonicum strains could express nod genes in the absence of genistein or related compounds. These observations led us to formulate the following hypotheses: (i) There is variability among B. japonicum strains for ability to nodulate soybean plants and increase soybean yield under field conditions in a short-season, cool spring area, and (ii) strains of B. japonicum selected for growth at low temperatures have the ability to overcome low root-zone temperature inhibition of soybean nodulation in the field in a short-season, cool spring area. Hence, the objective of this study was to evaluate selected strains of B. japonicum for their ability to improve soybean growth and yield in an environment with low soil temperatures during the early portion of the growing season.
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MATERIALS AND METHODS
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Strain Evaluation (Free-Living)
Thirty-eight strains were selected from the USDA collection based on isolation from northern locations in the USA. Forty (Table 1) B. japonicum strains (38 from the USDA collection plus USDA 110 and 532 C) were evaluated for their capacity to grow free-living at two temperatures (15 and 25°C) by streaking these cells on yeast extract mannitol (YEM) medium (Vincent, 1970) in petri dishes. The strains that were able to grow at 15°C were further evaluated for growth rate in liquid culture, in 20-mL test tubes containing 5 mL of YEM broth while shaken at 150 rpm. The optical density at 620 nm (OD620) was observed after 1, 3, 5, and 7 d, and the relative growth [e.g., ln (OD620) Day 7 - ln (OD620) Day 1/7 d] was calculated (Lynch and Smith, 1993). For each temperature, the experiment was arranged in a completely randomized design with four replicates for each strain, plus a control (uninoculated YEM broth).
Strain Evaluations under Field Conditions
Bacterial Material
The three strains that grew fastest at 15°C (USDA 30, USDA 31, and 532 C) were selected for subsequent field evaluation. The field experiment used these strains to test whether inoculating soybean with strains USDA 30 and USDA 31 would result in yield levels greater than those of 532 C. The inocula were produced by culturing the above three strains in YEM broth (Vincent, 1970) in 250-mL flasks shaken at 125 rpm at 25°C. When the subcultures reached mid-log phase, pure medium was used to dilute the inoculum to an OD620 of 0.08 (equivalent to 108 cells mL-1) (Bhuvaneswari et al., 1980).
Site Preparation and Field Layout
Each plot was 4.5 by 3.2 m and consisted of eight rows of plants, with 0.4 m between rows. The space between adjacent blocks was 1 m. The space between plots was 0.8 m. The seasonally available soil N was determined by including one plot of a non-nodulating Evans cultivar in each block. The experiments were carried out at the Lods Agronomy Research Center, McGill University, Macdonald Campus, Ste. Anne de Bellevue, QC, Canada. A mix of oat (Avena sativa L.) and barley (Hordeum vulgare L.) were grown on the 1998 site in 1997, and corn (Zea mays L.) was grown on the 1999 site in 1998. In each case, the stems and leaves were incorporated into the soil after harvest through fall plowing. In both years, K and P were provided by a spring application of a mixture of potash (110 kg ha-1 of 0–0–60) and triple superphosphate (220 kg ha-1 of 0–46–0) following the recommendations of a soil test. The soil was a Chicot light sandy loam (mixed, frigid Typic Hapludalf) in both 1999 and 1998.
Planting Methods
Seeds of the soybean cultivars Maple Glen and OAC Bayfield and a non-nodulating Evans line were surface-sterilized in sodium hypochlorite (2% solution) and then rinsed several times with distilled water. These seeds were planted on 15 May in 1998 and 18 May in 1999. Twenty milliliters of bacterial solution (OD620 = 0.08; approximately 10-8 cells mL-1) or YEM (control) per 1 m of row were applied evenly, by syringe, directly onto the seeds along the furrow. Alcohol sterilization of the implements was used to prevent cross contamination throughout planting and all subsequent data collection procedures. Following emergence, seedlings reached a stand of approximately 500000 plants ha-1 in all plots. Where stands were excessive, they were hand-thinned at the seedling stage.
Experimental Design
The field experiment was a 4 x 2 factorial organized in a randomized complete block design with four blocks. Treatments were comprised of factorial combinations of four inoculants (strains 532 C, USDA 30, USDA 31, and an uninoculated control) and two soybean cultivars (Maple Glen and OAC Bayfield).
Harvest and Data Collection
Daily average air temperature, average soil temperatures at a depth of 5 cm, and precipitation were recorded at the Macdonald Campus weather station (Fig. 1 and 2)
. The classification of soybean growth stages followed those of Fehr et al. (1971). To collect data on the development of leaf number and leaf area, plant samples (10 plants each) were harvested from each plot at four development stages: (i) V3, three nodes on the main stem with fully developed leaves beginning with the unifoliolate nodes (20 June in 1998 and 24 June in 1999); (ii) R1, one open flower at any node on the main stem (28 June in 1998 and 2 July in 1999); (iii) R4, pods 2 cm long at one of the four uppermost nodes on the main stem with a fully developed leaf (28 July in 1998 and 4 August in 1999); and (iv) R8, 95% of the pods having reached their mature pod color (14 August in 1998 and 18 August in 1999). Leaf number and leaf area per plant were determined using a Delta-T area meter (Delta-T Devices, Cambridge, UK). At harvest, mature pod number and seed number per plant were determined on 10 plants from each plot. The remainder of the plants in the plot were harvested with a small-plot combine (Wintersteiger, Salt Lake, UT) at harvest maturity and then oven-dried at 70°C for at least 48 h before being weighed for yield determination. The N contents of the seeds were determined by Kjeldahl analysis (Kjeltec system, Tecator AB, Hoganas, Sweden). Seed protein concentration was calculated by multiplying N concentration by 6.25.
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Fig. 1. The average daily temperature, soil temperature (at a depth of 5 cm), and precipitation during the 1998 soybean growing season (Ste. Anne de Bellevue, QC, Canada).
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Fig. 2. The average daily air temperature, soil temperature (at a depth of 5 cm), and precipitation during the 1999 soybean growing season (Ste. Anne de Bellevue, QC, Canada).
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Statistical Analysis
Results were analyzed statistically by analysis of variance using the Statistical Analysis System (SAS) computer package (SAS Inst., 1989). When analysis of variance showed significant treatment effects, the least significant difference (LSD) test was applied to make comparisons among the means at the 0.05 level of significance (Steel and Torrie, 1980). When differences occurred at levels of significance between 0.05 and 0.1, this is noted in the text.
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RESULTS
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Seasonal Temperatures
The seasonal soil-temperature data in 1998 and 1999 showed that the average daily root zone temperatures at a depth of 5 cm were below 25°C (Fig. 1 and 2). The air temperature data in 1998 was higher than that in 1999. These data demonstrated that low soil temperature is a limiting factor to soybean nodulation in Canada and may be the main limitation to N fixation and plant development in such areas.
Selection of Strains
Similar soil and air temperature measurements, in previous years, were the basis for our selection of 15 and 25°C as the two critical temperatures to evaluate and select strains that grow better under low temperatures. Our results indicated that only 532 C, USDA 30, USDA 31, and USDA 110 can grow well at 15°C in petri dishes and grew at measurable rates in liquid culture (Table 2), whereas other strains grew poorly or not at all at 15°C. All strains grew well at 25°C (data not shown).
Inoculation Effects on Plant Development
Inoculation with B. japonicum accelerated soybean leaf development and leaf area accumulation relative to the uninoculated control (Fig. 3)
. The increase was greatest at R4 (Harvest 3). The increase resulting from inoculation with USDA 30 and USDA 31 was greater than that resulting from inoculation with 532 C. Leaf number was not different among inoculation treatments (data not shown). Bayfield produced greater average leaf areas than Maple Glen. The difference between cultivars was greatest at the midpoint of plant development (Harvest 3).
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Fig. 3. The effects of inoculated strain on soybean (Maple Glen and Bayfield) leaf area per plant in (A) 1998 and (B) 1999. Each value is plotted as the mean ± standard error (n = 10). Plant samples were harvested from each plot at four development stages: (i) three nodes on the main stem with fully developed leaves beginning with the unifoliolate nodes (V3), (ii) one open flower at any node on the main stem (R1), (iii) pods 2 cm long at one of the four uppermost nodes on the main stem with a fully developed leaf (R4), and (iv) 95% of the pods having reached their mature pod color (R8).
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Inoculation Effects on Soybean Yield and Grain Protein Content
There were no interactions between the two factors tested. Inoculation with USDA 30 and USDA 31 resulted in higher grain protein and total protein levels than inoculation with 532 C (Tables 3 and 4). There were no differences between the cultivars for grain protein and total protein.
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Table 3. Main effects of Bradyrhizobium japonicum strain and soybean cultivar on soybean yield, grain protein, total protein, 100-seed weight, pod number per plant, and seed number per plant at the final harvest in 1998.
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Table 4. Main effects of Bradyrhizobium japonicum strain and soybean cultivar on soybean yield, grain protein, total protein, 100-seed weight, pod number per plant, and seed number per plant at the final harvest in 1999.
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Inoculation with 532 C, USDA 30, and USDA 31 increased grain yield by 9 to 22% over the uninoculated control (Tables 3 and 4). In both years, the grain yield increases associated with USDA 30 and USDA 31 were greater (6.2 and 7.7%, respectively, in 1998 and 9.2 and 8.7%, respectively, in 1999) than with 532 C (Tables 3 and 4). Bayfield produced more seed yield (9–13%) than Maple Glen. Generally, the inoculation treatments increased pod number by 10 to 18%, seed number by 5 to 10%, and 100-seed weight by 16 to 20%. In 1998, pod and seed numbers were greater for Bayfield than Maple Glen although this was not the case in 1999. There were no differences between Maple Glen and Bayfield for 100-seed weight (Tables 3 and 4).
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DISCUSSION
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Bradyrhizobium japonicum strain 532 C is used in nearly all Canadian inoculants. It was selected because of its superior performance in field experiments (Hume and Shelp, 1990). It replaced USDA 110, which had been widely used and is still used in the USA. Our observation that USDA 110 and 532 C were among the small group of strains that grew reasonably well, free-living, at 15°C suggests that the ability to grow well at low temperatures has been the reason for their success in the field. This is supported by the observation that USDA 30 and USDA 31 grew better, free-living at 15°C, than 532 C (Table 2). This suggests that wider screening for strains that grow well at low temperatures could lead to the identification of better strains.
The selection of bacterial strains adapted to prevailing conditions should minimize adverse environmental effects on legume nodulation and N fixation (Gibson, 1971). The lower temperature limit of a N fixation symbiosis is largely due to sensitivity on the part of the plant but can be modified by the strain of bacterial symbiont used (Lie, 1974). Nodulation and N fixation at a given temperature vary with bacterial strains (Dart et al., 1976; Gibson, 1971; Lindemann and Ham, 1979). A number of studies have attempted to correlate the environmental origins and growth in culture of rhizobial strains with their symbiotic effectiveness under low-temperature stress. Ek-Jander and Fahraeus (1971) found that the performance of rhizobial strains at low temperatures was influenced by their geographical origin. In our experiments, at 25°C, all 40 strains (Table 1) grew well while at 15°C, only B. japonicum 532 C, USDA 30, USDA 31, and USDA 110 grew at measurable rates (Table 2). Visual observation of nodulation in the noninoculated plots of potentially nodulating soybean cultivars demonstrated that there were B. japonicum cells in the soil of the field sites. However, the increases in yield-related variables due to inoculation showed that the native B. japonicum population was not sufficient to allow the same yield expression as inoculated treatments or that these B. japonicum strains were less suitable for N fixation in symbiosis with soybean than those added in the inocula.
In North American soybean production areas, such as those in Canada, low soil temperature is one of the main limitations to soybean N fixation (Zhang and Smith, 1996). However, strains 532 C, USDA 30, and USDA 31 were able to grow reasonably well at a relatively low temperature (15°C) while this was not true for other strains. We found that inoculation with strains USDA 30 and USDA 31 increased grain yield by 8.7 to 9.2% more than that of 532 C in 1998 (Table 3) and 6.2 to 7.7% more than that of 532 C in 1999 (Table 4). Inoculation with USDA 30 and USDA 31 increased grain protein more than inoculation with 532 C (P = 0.1 in 1998 and P = 0.09 in 1999) and total protein more than inoculation with 532 C (P = 0.09 in 1998 and P = 0.1 in 1999). Analyzing the yield components, we found that the pod number and seed number were increased more by USDA 30 and USDA 31 than by 532 C in 1998 (P = 0.05) and 1999 (P = 0.07). For 100-seed weight, there were no differences among strains USDA 30, USDA 31, and 532 C. Thus, the increase in grain yield was due to a combination of increased pod number and seed number. Given that pod number and seed number are determined shortly after flowering, and 100-seed weight is determined during the subsequent grain-filling period, the data suggest that the bulk of the benefit derived from inoculation with the two USDA strains was related to an early advantage, such as an earlier onset of N fixation. This was further supported by greater leaf areas for USDA 30– and USDA 31–inoculated plants than 532 C–inoculated plants (Fig. 3) and the greater seed and total protein levels.
Given that USDA 30 and USDA 31 were included in this work because of their greater ability to grow, free-living, at lower temperatures than the other 38 strains evaluated (Table 2), it seems likely that the better performance of plants inoculated with these strains was due to their greater adaptability to form the N-fixing symbiosis in the cool soil conditions of early season Canadian soybean production areas. The probable reason is that these two strains grow and enter into symbiosis more quickly than 532 C under low temperature. Prévost et al. (1987) reported that the nitrogenase activities of rhizobia isolated from cold areas were higher at low temperature than those from warm areas. Roughley (1970) also found that a strain isolated from a cold environment caused nodulation and formed bacteroids under low temperatures while a strain isolated from a warmer environment did not. The greater early development and yield due to these two strains led us to speculate that they improve nodulation, bacteroid formation, and nitrogenase activity relative to 532 C. This is supported by data that showed that using these two strains as inoculants increased grain protein content and total protein production more than 532 C (Table 3 and 4).
In summary, selecting strains tolerant of low temperatures identified B. japonicum strains better suited to soybean production in areas with cool spring soil conditions. More thorough screening of field isolates and those from culture collections may lead to the discovery of even better strains. In our work, inoculation with either USDA 30 or USDA 31 resulted in greater grain yields than inoculation with 532 C, which is currently used in most Canadian soybean production. Presumably, this was due to their better ability to grow, enter into symbiosis, and fix N in the cool soils of the early season. Thus, these stains are better adapted and perform better under cool-season conditions than 532 C and could be used in commercial inoculants in Canadian soybean production.
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REFERENCES
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ABSTRACT
INTRODUCTION
