Breeding for Better Nitrogen Fixation in Grain Legumes: Where do the Rhizobia Fit In?

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Peter H. Graham, Department of Soil, Water, and Climate, University of Minnesota, St Paul, MN 55108; Mariangela Hungria, Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA) Soja, Londrina, PR, Brazil; and Becki Tlusty, Department of Soil, Water, and Climate, University of Minnesota, St Paul, MN 55108

Abstract

All of the elements needed to significantly enhance N₂ fixation in grain legumes by plant breeding are currently available, but attention to this problem has been limited. This paper considers genetic variation in traits associated with nodulation and N₂ fixation and how they might be utilized. It also considers the role of rhizobia in an effective grain-legume breeding program.

Elements of an Effective Applied Breeding Program

An effective applied breeding program must be based on certain elements. These include:

· traits whose improvement will enhance crop production and/or profitability;

· genetic variation in the trait to be advanced;

· identification of parents differing in that trait;

· understanding of trait genetics;

· formulation of appropriate breeding strategy; and

· development of a field-based, simple, and inexpensive method of progeny selection.

In the case of breeding for enhanced nitrogen (N₂) fixation in grain legumes, all of these elements have been demonstrated, yet progress in all but a few cases has been limited. This paper evaluates the genetic variation for nitrogen fixation in grain legumes, the practices needed for significant gains through plant breeding, and above all, the role of the rhizobia in effective improvement in this trait.

Symbiotic Nitrogen Fixation in Legumes

Essentially all agriculturally important legume species have the ability to symbiose with a group of bacteria collectively known as rhizobia. In this symbiosis the bacteria derive energy from the host for growth and N₂ fixation, and are protected from external stresses; the host accesses a form of nitrogen it could not otherwise utilize. Worldwide some 44 to 66 million tons of N₂ are fixed annually, providing nearly half of all the nitrogen used in agriculture. The quantity of nitrogen needed for agriculture is projected to increase in the period to 2030 (36), and in the USA could lead to greater environmental pollution. Reduced dependence on fertilizer N and attention to farming practices that favor the more economically viable and environmentally prudent N₂ fixation will benefit both agriculture and the environment (37).

Currently, the contribution of N₂ fixation to the production of grain legumes in the USA is consistently less than what could be achieved. Only 15% of farmers use inoculants, while the percentage N derived from fixation in soybean and common bean is thought to have declined from 65% to only 54%, and from 44% to 37%, respectively since 1985 (38). A recent report even suggests that corn-soybean production in the American Midwest is not sustainable, and depends on government subsidy (29). Factors that have contributed to a reduced dependence on N₂ fixation include the availability and low cost of N fertilizers and manures, the use of plant varieties limited in their ability to fix N₂ in symbiosis, and edaphic constraints that include soil acidification, drought, and shortage of specific nutrients (14).

Rates of N₂ fixation vary with plant species and environment, but with grain legumes potential rates of as much as 0.9 to 1.8 lb/acre per day have been suggested (10) -- a rate that would satisfy essentially all of the plant’s nitrogen needs. Unfortunately, this level of N₂ fixation is rarely achieved, though benefits from a program aimed at enhanced N₂ fixation in grain legumes can be dramatic. This is evident in the case of soybeans in Brazil where yields have increased more than 4-fold since 1968, and where average yields in the Cerrado now outstrip those achieved in the USA (20). In this region more than 90% of farmers use inoculants and over 60% repeat inoculate. Percent N derived from fixation in studies conducted across Brazil varies from 69% to 94%, with an economic return of a dependence on symbiotic N₂ fixation in soybean of $1.95 billion (US) annually (20,21). Similar responses to inoculation of soybean have been obtained recently in parts of the American Midwest (J. Beuerlein, personal communication, J. E. Kurle, personal communication) (Fig. 1), and are beginning to stir greater interest in the inoculation of this and other crops.

Fig. 1. Response to inoculation in soybean, Staples, MN, 2003. (a) Inoculated (centre row) and uninoculated soybean plants 55 days after seeding. Yield differences between inoculated and uninoculated plots in this experiment varied with strain, inoculant formulation, and pesticide seed treatment, but under optimum conditions exceeded 1200 lbs/acre. (b) Early crown nodulation of inoculated soybean plants 55 days after seeding.

Cultivar Variation in Traits Associated with N₂ Fixation

Cultivar variation in traits associated with N₂ fixation has been demonstrated in essentially every legume studied to date. Selected important examples include clover (27), soybean (16,28), common bean (13,31), and alfalfa (6,22). Further, these differences have been shown using a range of different traits each associated with N₂ fixation. They include cultivar differences in nodule number and mass, speed of nodulation, lateral root nodulation post flowering, N accumulation, acetylene reduction activity, allantoic acid production, and nodule enzyme production and function.

Not all of these cultivar differences would be of value in a breeding program. Commonly, for example, N₂ fixation levels are correlated with time to flowering, but in the northern USA at least, the use of cultivars with a longer pre-flowering period is only useful if overall time to maturity can be held constant. Similarly, nodule number and mass are usually inversely related, with an ineffectively nodulated cultivar having many small nodules (27). Nodule mass per plant is a more useful indicator of symbiotic potential.

Nodulation and N₂ fixation in legumes are generally thought to be quantitatively inherited traits (3). Because of this we have emphasized the identification of parents that differ in the contributions each might bring to the symbiosis. We have conceptualized these as nodule mass, activity (carbohydrate supply and form, specific nodule activity, or nodule enzyme function), and duration (senescence, lateral root nodulation) functions. Traits used in the parental selection process need not necessarily be ones that can also be applied at a field scale. For example, the ability to produce high levels of specific nodule enzymes could be an important factor in parental selection, but difficult and expensive to use in screening segregating populations.

Fedorova et al. (8) reported more than 300 tentative consensus sequences in nodule libraries of Medicago truncatula that are not found in other libraries prepared from this host. Additional gene products are undoubtedly needed for infection and nodule initiation. While many of these will not vary with legume or cultivar, the probability is that future studies will identify many additional traits useful in characterizing host variability in nodulation and N₂ fixation.

Breeding and Selection for Enhanced N₂ Fixation in Grain Legumes

A number of different breeding approaches have been used to improve N₂ fixation levels in legumes. They include backcross-inbred methods for population development (3); recurrent selection for enhanced nitrogen fixation (7); bidirectional selection for specific nodule enzymes (6); and simple, double, and three-way crosses (M. Hungria, personal communication). All have succeeded in raising levels of N₂ fixation. In fact, even selection for crop yield without particular attention to N₂ fixation can enhance total N and N₂ fixed (5). While a number of different traits should be considered in careful parental selection, initial field screening of the progeny can be simply and inexpensively undertaken. We have selected common bean varieties for superior N₂ fixation using low N soil conditions and only seed yield and plant biomass N as selection criteria. The latter trait is included to distinguish between superior N₂ fixation levels and high harvest N index. Both Elisondo Barron et al. (7) and Hungria and Bohrer (19) point to losses in N₂-fixing ability in breeding populations evaluated on the basis of seed yield or seed N alone.

Heritability estimates in fixation studies vary with trait measured, but range from 0.22 to 0.76. A problem in many of the studies cited above is that few progeny perform better than the superior N₂-fixing parent. This is not surprising if N₂ fixation is quantitatively inherited, and if parental selection is based mainly on nodule mass or overall N₂ fixation. Transgressive segregation toward markedly superior N₂ fixation will require the pyramiding of genes contributing to nodule mass, activity, and duration, and approaches that parallel those used in yield enhancement. At higher levels of crop production, competition between seeds and nodules for nutrients and energy could mean that one couldn’t be improved without decline in the other. Fortunately, that time does not appear to be near. In the meantime a priority must be the development of markers with which superior nodulation or N₂ fixation can be dissected. To this point, there has been a surprising dearth of studies on marker-assisted selection for traits associated with nodulation and N₂ fixation.

Where do the Rhizobia Fit In?

Ability to nodulate and fix N₂ must be a part of the breeding program for all agronomically important legumes. At some stage, all breeding lines need to be tested in low N soils, and those that are weak to average in N₂ fixation discarded. Inoculant strains and inoculation practices used in these trials need to be the best available, and any variable likely to reduce the potential for nodulation and N₂ fixation (seed treatment, soil compaction, inappropriate N fertilization, and the limited availability of fixation-specific nutrients including P, Ca, Fe, and Mo) avoided or corrected.

In an age of rapidly changing, often bioengineered varieties, rhizobial strain selection needs to be revisited. Hungria et al. (21) reviewed changes in the inoculant strains used for soybean in Brazil since 1960. These authors noted that soils in much of Brazil were initially devoid of rhizobia, and were generally inoculated with strains from the USA and Australia. More recently, variants of these initial inoculant strains have been recovered from soil and found to be better adapted to local soil conditions, more competitive, and higher yielding than the original inoculant strains. In one study, plants inoculated with a variant of Bradyrhizobium japonicum CB1809 outyielded those inoculated with the wild type strain by more than 600 lb/acre. Similar studies need to be undertaken in the USA, where some inoculant strain recommendations have not been changed in 20 years.

Three additional areas stand out as warranting particular research attention:

· host-Rhizobium interaction, and accounting for it in breeding programs;

· differences in root colonization and saprophytic competence;

· stress tolerance and its impact on host-strain interaction.

Host-Rhizobium interaction. Host-Rhizobium interaction, with some strains performing better with specific cultivars than with others, has been evident in some, but not all germplasm evaluation studies. Mytton et al. (26) with Vicia faba found that host-Rhizobium interaction explained 74% of the variation in symbiotic response. In contrast, Roskothen (33) found limited host-strain interaction in Vicia faba, attributing this difference in results to the limited diversity of rhizobia used in the earlier study. It is our experience that host or strain effects can be expected where the plants or strains being evaluated come from different geographical areas. Bernal and Graham (2) found a local common bean cultivar to recover different bean rhizobia from soil than an introduced one, while Bernal (1) noted that cultivars derived from different races of bean in Latin America differed in speed of nodulation with rhizobia from the Andean region. Similar results have been obtained with different introductions of Glycine max into Africa (25). Implicit in these results is the need to screen all potential inoculant strains against all compatible hosts.

Root colonization and saprophytic competence. Gibson et al. (9) and others have reported instances in which inoculant strains, applied under near-optimum conditions, did not persist in the soil, while McDermott and Graham (24) noted that soybean inoculant rhizobia were competitive in nodule formation in the crown region of the plant near where they were placed, but failed to colonize the root system. These studies suggest problems in rhizosphere colonization and saprophytic competence, which might be overcome by a better understanding and management of legume rhizodeposition. This would require both a detailed study of cultivar variation in rhizodeposition and of rhizobial differences in root colonization. Results obtained in these studies might also contribute to a better understanding of the basis for preferential nodulation by specific cultivars (32). Recent advances in understanding microbial community structure in the rhizosphere should facilitate such a study. Grayston et al. (15) have already noted differences in rhizosphere community structure associated with plant improvement, while Siciliano et al. (34) found that Pseudomonas inoculants used in phytoremediation had significant impacts on root associated microbial communities.

Stress tolerance and its impact on host -strain interaction. Soil acidity, drought, low soil P, or high soil N levels can all affect symbiosis between host and rhizobia, influencing rhizobial survival in soil, the host, or the process of nodulation itself (11). The clearest example of how this can affect plant breeding is with soil pH. Differences in both host and rhizobial strain tolerance to soil pH exist in Medicago (17,18) and Phaseolus (12,39), and can interact to enhance plant establishment and growth. In the case of Medicago, this has meant the introduction of annual species into more than 800,000 acres of Australia where the soils were previously too acid for the growth of many medic species. In the acid soils of Brazil, Rhizobium tropici can account for 97% of the nodules associated with beans. For both pH and low soil P, cultivar use can significantly affect strain recovery from soil (I. Christiansen and P. H. Graham, personal communication). Careful selection of inoculant strain is essential for any legume breeding nursery likely to be grown under stress.

Crop Improvement: A Dying Breed

A recent paper in Nature (23) laments the decline in public and institutional plant breeding in the face of genetic engineering encroachments into this field. Yet with few exceptions, what might molecular approaches achieve with N₂ fixation that traditional breeding methods might not also do? Rengel (30) reviews some molecular options, but discusses mainly supernodulation, opine exudation, and flavonoid expression. More likely to have success are studies such as those of Tesfaye et al. (35) with over-expression of malate dehydrogenase, a trait that could affect not only aluminum and low P tolerance, but also levels of N₂ fixation (C. P. Vance, personal communication). Could improvements in malate dehydrogenase expression also be achieved exploiting cultivar differences and conventional approaches?

Conclusion

Legume breeding programs must give greater emphasis to the symbiosis between host and rhizobia. Practical approaches to enhanced nitrogen fixation and improved tolerance to edaphic constraints would permit a lower cost and a more sustainable form of agriculture, but have largely been bypassed in the rush to study more molecular aspects. We have previously paraphrased a comment by Catroux et al. (4) that again seems appropriate here: “We enter the era of biotechnology knowing more and more about the growth of legumes at the gene level, but in only a few cases able to effectively translate this information into major gains in productivity.”

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