**Competition for Nodulation**

Julieta Pérez-Giménez, Juan Ignacio Quelas and Aníbal Roberto Lodeiro *IBBM-Facultad de Ciencias Exactas, Universidad Nacional de La Plata-CONICET Argentina* 

#### **1. Introduction**

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Nitrogen (N) is the nutrient that most often becomes limiting for plant growth. Soybean may obtain this nutrient from the air, thanks to its ability to perform a symbiosis with bacteria of the genera *Bradyrhizobium* (*B. japonicum, B. elkanii, and B. liaoningense*), *Sinorhizobium* (*S. fredii* and *S. xinjiangense*) and *Mesorhizobium* (*M. tianshanense*). These bacterial species are collectively known as soybean-nodulating rhizobia, but only *B. japonicum*, *B. elkanii*, and *S. fredii* were used as commercial inoculants for soybean crops, with *B. japonicum* being the most widely employed. In this symbiosis the rhizobial partner reduces the atmospheric N2 to NH3 in a reaction catalyzed by the nitrogenase enzymatic complex, while the plant partner supplies the C sources that provide the energy required for the N2 reduction reaction. Since atmospheric N2 is an unlimited source of N, the process of N2 fixation is of great potential for sustainable agriculture, and in the special case of legumes, the symbiosis is so efficient that in hydroponic culture the plant may satisfy all its N needs without resorting to any other N source. In addition, this symbiosis is a biological process that does not require fossil energy consumption, and does not leak any contaminant byproduct to the biosphere. Therefore, the inoculation of legume crops with selected rhizobial strains of high N2 fixation performance is an extended practice in agriculture since decades ago. In parallel, the industry of inoculants is very active, commercializing a variety of formulations with different strains and combinations with other plant-promoting rhizobacterial species such as *Azospirillum brasilense* or *Pseudomonas fluorescens*. For the farmers, inoculating a legume crop with active rhizobia is a simple procedure, and its economic cost is much lower than applying chemical fertilizers. All these advantages are, however, obscured by the fact that in field crops the symbiotic N2 fixation seldom provides the expected results, and the plants may consume the N from the soil.

Several factors account for this low performance of N2 fixation in field crops. In energetic terms, N2 fixation is more costly for the plant than soil N uptake and therefore soil N is preferred when this source is not limiting, or when N2 fixation is inefficient (Salon et al., 2009). This may be appreciated if one takes into account that the symbiosis only occurs in a specialized organ known as root nodule. It is there where the rhizobia differentiate into the state able to reduce N2 −the bacteroid− and where the O2 concentration is lowered at levels compatible with nitrogenase activity (Patriarca et al., 2004). Therefore, rhizobia must infect the roots and trigger the development of nodules, which finally will be occupied by the rhizobia. During the earliest steps of nodule development and root infection (Ferguson et al., 2010), the plant-rhizobia relationship is more similar to a pathogenesis than to a mutualistic symbiosis: rhizobia invade plant tissues, consume plant energy resources, induce a tumor-like development, and proliferate inside plant cells, without any benefit for the plant until nodules are completely developed and N2 fixation starts. Indeed, rhizobial strains with low N2-fixing efficiency or unable to fix N2 trigger typical plant defense reactions and lead to a weakening of the plant. In such a scenario, N removal from soil may be quite significant. It has been estimated that in soils with low N content, a good N2-fixing symbiosis may provide 70 % of N that plant needs (i.e. 70 % of all plant N coming from the air) while an inefficient symbiosis only provides 20-30 % of N (Unkovich & Pate, 2000). Therefore, the difference between a good and a bad symbiosis would be around 40 % plant N being obtained from the air or the soil, respectively. N contents of soybean grains are around 2.5 % w/w, depending on the cultivar, all this N being removed from the ecosystem at grain harvest. Hence, considering a mean yield of 2,500 kg ha-1 the difference between a good and a bad N2-fixing symbiosis equals 25 kg N ha-1 that are respectively conserved or removed from the soil each year.

In soybean-producing countries like Argentina, these crops are grown in soils with low N content, either because the soils were previously cropped with species of high soil N demand, such as wheat or corn, or because they are in marginal areas. Therefore, N2 fixation becomes a key input of sustainable soybean cropping, because this species has also a high N demand, and thus, the N that cannot be provided by N2 fixation must be supplied as chemical fertilizer, which involves many environmental problems and has a higher cost.

The efficiency of the N2-fixing symbiosis depends on many factors. Of primary importance are the total number of nodules formed in each plant, and the N2-fixing activity of each nodule. As mentioned before, nodulation has an energetic cost to the plant and therefore the number of nodules cannot be too high. Instead, an optimal number of nodules able to provide the necessary amount of fixed N2, with a reasonable energetic cost involved in its maintenance, is regulated by the plant. In this way, once a given number of active nodules are established, the plant progressively inhibits the formation of new nodules (see below). This indicates that both the plant genotype (by its ability to assimilate fixed N2 and its ability to control the number of nodules) and the rhizobial genotype (by its N2-fixing activity) are key determinants of the symbiosis performance. However, being a biological process, the environment also plays a fundamental role, not only by its influence on the activity of each partner, but also by its interaction with both genotypes.

Competition for nodulation between inoculated rhizobia and different rhizobial strains resident in the soil is a striking example of this complexity. Normally, the soils are populated with rhizobia either from the indigenous bacterial population or introduced by the inoculants used in previous crop seasons. Since rhizobia are soil bacteria, they readily adapt to a new soil and exchange genetic material with the local soil microbiota. However, in the soil there is not a high selective pressure for high N2 fixation performance and therefore, genetic drift leads to dispersion of the N2-fixing potential among different genotypes with diverse efficiencies. Therefore, the soil rhizobial population is often of high efficiency to nodulate the plants, but of medium to low efficiency to fix N2. Hence, the competition of this population for plant nodulation may prevent the newly inoculated strains to occupy a significant proportion of the nodules, leading to lack of N2 fixation response to inoculation (Toro, 1996). To get an approximation to the problem, we can consider that each nodule contains a clone of bacteria derived from a single precursor bacterium that initiated the root infection. Occasionally, two bacterial clones may share a nodule, but it is extremelly unfrequent to find nodules with more than two clones. Given that a single soybean plant might possess, at most, in the order of 102 nodules at maturity,

induce a tumor-like development, and proliferate inside plant cells, without any benefit for the plant until nodules are completely developed and N2 fixation starts. Indeed, rhizobial strains with low N2-fixing efficiency or unable to fix N2 trigger typical plant defense reactions and lead to a weakening of the plant. In such a scenario, N removal from soil may be quite significant. It has been estimated that in soils with low N content, a good N2-fixing symbiosis may provide 70 % of N that plant needs (i.e. 70 % of all plant N coming from the air) while an inefficient symbiosis only provides 20-30 % of N (Unkovich & Pate, 2000). Therefore, the difference between a good and a bad symbiosis would be around 40 % plant N being obtained from the air or the soil, respectively. N contents of soybean grains are around 2.5 % w/w, depending on the cultivar, all this N being removed from the ecosystem at grain harvest. Hence, considering a mean yield of 2,500 kg ha-1 the difference between a good and a bad N2-fixing symbiosis equals 25 kg N ha-1 that are respectively conserved or

In soybean-producing countries like Argentina, these crops are grown in soils with low N content, either because the soils were previously cropped with species of high soil N demand, such as wheat or corn, or because they are in marginal areas. Therefore, N2 fixation becomes a key input of sustainable soybean cropping, because this species has also a high N demand, and thus, the N that cannot be provided by N2 fixation must be supplied as chemical fertilizer, which involves many environmental problems and has a higher cost. The efficiency of the N2-fixing symbiosis depends on many factors. Of primary importance are the total number of nodules formed in each plant, and the N2-fixing activity of each nodule. As mentioned before, nodulation has an energetic cost to the plant and therefore the number of nodules cannot be too high. Instead, an optimal number of nodules able to provide the necessary amount of fixed N2, with a reasonable energetic cost involved in its maintenance, is regulated by the plant. In this way, once a given number of active nodules are established, the plant progressively inhibits the formation of new nodules (see below). This indicates that both the plant genotype (by its ability to assimilate fixed N2 and its ability to control the number of nodules) and the rhizobial genotype (by its N2-fixing activity) are key determinants of the symbiosis performance. However, being a biological process, the environment also plays a fundamental role, not only by its influence on the activity of each

Competition for nodulation between inoculated rhizobia and different rhizobial strains resident in the soil is a striking example of this complexity. Normally, the soils are populated with rhizobia either from the indigenous bacterial population or introduced by the inoculants used in previous crop seasons. Since rhizobia are soil bacteria, they readily adapt to a new soil and exchange genetic material with the local soil microbiota. However, in the soil there is not a high selective pressure for high N2 fixation performance and therefore, genetic drift leads to dispersion of the N2-fixing potential among different genotypes with diverse efficiencies. Therefore, the soil rhizobial population is often of high efficiency to nodulate the plants, but of medium to low efficiency to fix N2. Hence, the competition of this population for plant nodulation may prevent the newly inoculated strains to occupy a significant proportion of the nodules, leading to lack of N2 fixation response to inoculation (Toro, 1996). To get an approximation to the problem, we can consider that each nodule contains a clone of bacteria derived from a single precursor bacterium that initiated the root infection. Occasionally, two bacterial clones may share a nodule, but it is extremelly unfrequent to find nodules with more than two clones. Given that a single soybean plant might possess, at most, in the order of 102 nodules at maturity,

removed from the soil each year.

partner, but also by its interaction with both genotypes.

this is the order of magnitude of the bacterial individuals that can survive the root penetration. However, in soils with several years of soybean cropping there may be in the order of 105 to 107 soybean-nodulating rhizobia colonizing the proximity of the root −the rhizosphere− and therefore, for each bacterial cell that succeeds in penetrating the root, there are 100 that remain outside. Considering that nodules are protected environments for rhizobia, a harsh competition is established among rhizospheric rhizobia to gain access to the root nodules. In this process, the bacterial genotypes will have a prominent role in defining their competitiveness, but also the plant genotype will dictate how and when the bacteria will be allowed to penetrate the roots, the interaction between the bacterial and plant genotypes will determine which strains will be favored, and the interaction of the environment with both genotypes will determine the relative advantages for each bacterial strain.

Soybean seeds are inoculated with around 105-107 rhizobia seed-1, but more than 80 % of the rhizobia die within the first 2 h after inoculation (Streeter, 2003). From the survivors, only a small percentage reaches the rhizosphere after sowing (López-García et al., 2002). Thus, given the above figures, obtaining around 10 % of all the nodules occupied by the inoculated strain, as is currently accomplished in soybean crops, may be considered quite successful, but still it is completely insufficient to get a significant increase in plant N2 fixation above the levels obtainable without inoculation. Therefore, to get a significant proportion of plant N coming from the air it is imperative to improve inoculant's competitiveness to obtain significantly higher percentages of nodules occupation, and active research about this problem is being done since decades. In this chapter we will provide a look on the methods employed to study the problem of competition for nodulation, the bacterial and plant traits that may influence the competition, the ecological aspects that modulate the plant and rhizobia interaction, and how these factors may be managed in order to profit the symbiosis to increase soybean crop yields.
