Significant Yield Increase in Phaseolus vulgaris by Inoculation with a Rhizobium leguminosarum

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Significant Yield Increase in Phaseolus vulgaris Obtained by Inoculation with a Trifolitoxin-producing, Hup⁺ Strain of Rhizobium leguminosarum bv. phaseoli

A. Leonardo Iniguez, University of Wisconsin-Madison, Department of Agronomy, 1575 Linden Dr., Madison 53706; Eduardo A. Robleto, University of Nevada-Las Vegas, Department of Biological Sciences, Las Vegas 89154; Angela D. Kent, University of Wisconsin-Madison, Department of Civil and Environmental Engineering, 1415 Engineering Hall, Madison 53706; and Eric W. Triplett, University of Florida, Department of Microbiology and Cell Science, 1052 Museum Road, Gainesville 32611-0700

Abstract

The construction of a stably maintained, broad host range plasmid (pHUTFXPAR) carrying genes that enhance nodulation competitiveness through trifolitoxin production and efficient nitrogen fixation by oxidation of the H₂ evolved from nitrogenase was reported by Kent et al. (7). Here the field testing of the efficacy of this construct is reported. pHUTFXPAR was inserted into Rhizobium leguminosarum bv. phaseoli 127K105a by conjugation. Three field experiments were conducted to test the ability of pHUTFXPAR to enhance the grain yield response provided by 127K105a on Phaseolus vulgaris. In 2000, the first trial was conducted at a University of Wisconsin field site in Madison. The second and third trials were performed at two Madison locations in 2002. All field experiments were conducted with 12 replicates of three treatments including uninoculated and inoculation with either 127K105a or 127K105a(pHUTFXPAR). The pooled dry seed weight from both years and both locations show significant 12 and 14% yield increases from the plants inoculated with 127K105a(pHUTFXPAR) compared to the uninoculated and the 127K105a treatments, respectively.

Introduction

According to historical records, the use of leguminous plants in agriculture can date as far as 5th millennium B.C. (5). Since then, the agricultural use of these plants can be found throughout different geographical and cultural regions of the world (5). The discovery of the association between nitrogen-fixing bacteria and leguminous plants led to better understanding of the tremendous impact of legumes in agriculture (2,13). Since their discovery many of these symbiotic bacteria have been isolated and their nitrogen-fixing abilities have been characterized (8).

Over the last two decades, a number of strategies have been proposed to enhance the ability of root nodule bacteria to provide increased amounts of fixed N to its legume host (8). Among these strategies is the construction of inoculum strains that recycle the H₂ produced as an oligate product of the nitrogenase reaction (8,12). At least 25% and as much as 60% of the energy consumed in nitrogen fixation is wasted in the production of H₂ (1). A schematic is provided that describes the benefits of the hydrogen uptake process (Fig. 1). Some strains of Bradyrhizobium japonicum are known to be able to oxidize the H₂ produced during the nitrogenase reaction. As a result, much of the energy consumed in the production of H₂ is recovered. Those strains that possess this activity are referred to as uptake hydrogenase positive or Hup⁺. The presence of the Hup⁺ phenotype in B. japonicum can increase soybean yield by as much as 17% (6). As a result, the Hup⁺ phenotype is common among B. japonicum strains used as commercial inocula in North America. However, the Hup⁺ phenotype is very rare among strains used to inoculate other legumes such as common bean, alfalfa, or peas (8).

Fig. 1. Schematic depicting the process of hydrogen uptake conferred by pHUTFXPAR in 1278K105a. Hydrogen is an obligate product of the nitrogenase reaction. H₂ is oxidized to protons and electrons. Through electron transport, ATP is recovered and is available for bacterial and/or plant metabolism. The addition of genes to Rhizobium that provides this hydrogen uptake phenotype results in increased bean production.

Another equally important problem that must be resolved before any practical benefits can be obtained from improved root nodule bacteria is the inability of inoculum strains to compete with native root nodule bacteria for root nodulation. One strategy to alleviate this problem concerns an anti-rhizobial peptide called trifolitoxin. Under field conditions, an inoculum strain that produces this peptide is capable of inducing more nodules than strains that do not produce this peptide (9).

Kent et al. (7) reported the construction of plasmid pHUTFXPAR that possesses genes coding for increased nodulation competitiveness through trifolitoxin production and increased yield through the Hup⁺ phenotype. Here, the pHUTFXPAR plasmid is added to Rhizobium leguminosarum bv. phaseoli 127K105a and used as an inoculum strain to test its ability to enhance the yield of common bean, Phaseolus vulgaris, under field conditions. The competitiveness of this inoculum strain is ensured by the addition of the trifolitoxin production and resistance genes added to pHUTFXPAR (9,10). Furthermore, addition of the partitioning locus from the broad host range plasmid RK2 to pHUTFXPAR ensures that this plasmid will be maintained, and the Hup⁺ phenotype expressed, in root nodules.

Testing the Effect of pHUTFXPAR on Yield Response Provided by 127K105a on Phaseolus vulgaris

Strain and plasmids. The plasmid used in this work, pHUTFXPAR, was constructed and inserted in Rhizobium leguminosarum bv. phaseoli 127K105a as described by Kent et al. (7). The strains used in this work were Rhizobium leguminosarum bv. phaseoli 127K105a and Rhizobium leguminosarum bv. phaseoli 127K105a(pHUTFXPAR). Trifolitoxin production by 127K105a(pHUTFXPAR) was confirmed using the bioassay described previously (7). Bacterial cultures were stored as frozen stock solutions at -75°C in 15% glycerol. Bacterial cultures used for seed Inoculation were grown in Bergernsen's synthetic agar medium (3). The growth medium was supplemented with streptomycin (50 mg/ml) for the culture of 127K105a. Streptomycin (50 mg/ml) and kanamycin (100 mg/ml) were added to the medium for the culture of 127K105a(pHUTFXPAR).

Preparation of inoculum and seed coating. Three inoculum treatments, uninoculated, 127K105a, and 127K105a(pHUTFXPAR), were used. Growth of bacterial cell culture and inoculation of Phaseolus vulgaris cv. Maverick Pinto were done as described previously (9).

Experimental plot and sites. During the 2000 planting season, a field trial was conducted at a University of Wisconsin site on the Madison campus. Two field trials were conducted in 2002 at the University of Wisconsin-Madison campus and at the West Madison field station of the University of Wisconsin. Their respective soil characteristics are shown in Table 1. No soil amendments were used for these experiments. Seeds were planted by hand at a rate of 6 seeds per foot, in rows of 5 ft. There were two rows per replicate for a total of 12 replicates per treatment. The treatments were separated by rows of uninoculated borders and they were randomized in a complete block design for both years. Weeds were removed about 4 times during all planting seasons at both locations.

Table 1. Soil Characteristics where of the locations where the experiments were conducted.

Year	Locations	pH	% Organic matter	P concn (ppm)	K concn (ppm)	Texture	Previous crop
2000^a	Herric Dr.	6.6	6	4^b	210	Silt loam	Corn
2002^a	Herric Dr.	6.6	6	4^b	210	Silt loam	Corn
2002	West Madison	6.9	2.6	22	110	N/A	Corn

^a No change in soil characteristics were observed during the two planting seasons.

^b This phosphorous concentration was calculated in solution.

Harvesting and yield measurement. Fifty plants per replicate were harvested at maturity and dried at 60°C for one week, followed by mechanical thrashing for seed collection. Cleaned seeds were dried at 60°C to 3% water content and dry seed weight was adjusted to a final 13% moisture to estimate fresh grain weight.

Data analysis. Minitab 12.23 for Windows was used to perform analysis of variance with a 95% level of confidence. The Least Significant Difference (LSD) method was used to compare the means of all treatments.

Results. All plants in all treatments and at all sites were nodulated. Furthermore, the uninoculated plants showed no signs of nitrogen deficiency. Plants inoculated with 127K105a showed slightly higher yield increases but these were statistically insignificant at the 95% confidence level (Fig. 2). The addition of pHUTFXPAR to 127K105a increased yield by 14% compared to the uninoculated treatment, significant at the 95% confidence level (Fig. 2). Addition of pHUTFXPAR to 127K105a also caused a 12% yield increase over plants inoculated with wild type 127K105a (Fig. 2).

Fig. 2. Comparison of dry seed weight (bu/acre) of Phaseolus vulgaris under three inoculant treatments: Uninoculated, wild type 127K105a, and 127K105a(pHUTFXPAR). This comparison combines the grain yield collected over the 2000 and 2002 planting seasons. During the 2002 planting seasons the experiments were carried out at two different locations. From left to right the first two treatments show dashed bars indicating that their yields are not significantly different while the solid bar is significantly different from all other treatments.

Discussion

There are very few reports in the literature of increased legume yield by the addition of specific genes to root nodule bacteria. Addition of a second copy of dctAB and low constitutive expression of nifA to a strain of Sinorhizobium meliloti increased alfalfa yield significantly over a four-year period (4,11). Evans et al. showed that the Hup⁺ phenotype in B. japonicum could significantly increase soybean yield (6). Here we show that the addition of the hydrogen uptake genes from B. japonicum to R. leguminosarum bv. phaseoli can result in substantial yield increases in common bean, Phaseolus vulgaris. As common bean is a major protein source for well over a billion people in Africa and Latin America, this result is significant to agriculture.

The fact that all plants in this study were nodulated shows that indigenous rhizobia were present in the field. As no nitrogen was applied to the plots and as the plants showed no nitrogen deficiency symptoms, the indigenous rhizobia appeared to fix nitrogen at reasonable levels. The inoculum strain without pHUTFXPAR did not provide a yield benefit to the plants. This can be attributed to either or both of the following two factors. First, the indigenous rhizobia may have been better competitors for nodulation than 127K105a. Second, strain 127K105a may not be able to fix more nitrogen than the indigenous rhizobia. The addition of pHUTFXPAR to 127K105a did cause a substantial yield increase may be attributed to increased nodulation competitiveness provided by the TFX production phenotype. This increase may also be attributed to the uptake hydrogenase phenotype that recycled the H₂ produced during the nitrogenase reaction. It is also possible that both phenotypes are required. Past work showed that the addition of the TFX phenotype alone to the inoculum strain increased nodulation competitiveness but had no influence on bean yield (9). For this reason, the yield increases observed here are believed to be the result of the addition of the uptake hydrogenase genes, which were absent in the previous study (9). However, this was not tested directly in these experiments.

The use of a plasmid to add these genes to inoculum strains has a number of implications. First, given that the yield enhancement is based on the addition of a broad host range, stable plasmid to an inoculum strain, it would be very straightforward to apply this technology to any bean-nodulating strain and even nodulating strains of other grain legumes. Second, this plasmid is not self-mobilizable. This minimizes, but does not eliminate, the possibility that this plasmid could move to indigenous bacteria in the soil. To date, we have been unable to detect the movement of this plasmid to other bacteria in the field. If this plasmid did move to indigenous rhizobia, it would probably improve the nitrogen fixation ability of the indigenous strains. However, if it were to transfer to a non-nitrogen-fixing strain of Rhizobium, it would make the strain more competitive for nodulation versus those strains that are sensitive to trifolitoxin.

Further research is warranted to determine the success of this technology across the broad range of environmental conditions used for the culture of common bean around the world. Various bean nodulating strains may be used in different parts of the world in order to provide an inoculum well adapted to specific soil conditions. As the Hup⁺ phenotype has now been shown to be useful in increasing the yield of soybean and common bean, it is reasonable to expect that other grain legumes might benefit from this technology as well.

Acknowledgments

We thank Nitragin, Inc. (Milwaukee, WI), and the University of Wisconsin-Madison College of Agricultural and Life Sciences for providing support for this work. We also thank the technical assistance provided by Patrick Riggs and Nick Simon.

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