**4. Ecological aspects of the plant-rhizobia interaction**

Rhizobia are world-wide distributed bacteria and therefore traits for adaptation to almost every environment where agriculture is carried out may be found. In addition, local populations of rhizobia are not restricted to nodulate the indigenous legume species. Horizontal gene transfer of rhizobial symbiotic genes was documented not only for those rhizobial species that carry this information in transmisible symbiotic plasmids (Torres-Tejerizo et al., 2011) but also for those species that carry this information in the bacterial chromosome (Gomes-Barcellos et al., 2007; Sullivan & Ronson, 1998), and therefore the ability to nodulate a newly introduced legume species is rapidly acquired by the local population. A mathematical model was developed to simulate the propagation of horizontally transferred symbiotic genes to a local non-symbiotic population and the prediction is that such genes can be fixed in the local population in a few generations (Provorov & Vorobyov, 2000). Nevertheless, the major richness in rhizobial genotypes for a given legume species is often found in the centers of origin of that species. Soybean is originated in Asia and therefore, it may be presumed that most of the soybean-nodulating rhizobia are originated in this same region. Therefore, searches for new soybean-nodulating strains with special adaptations were conducted there. A recent survey was performed in soils from four different regions in China (Thomas-Oates et al., 2003). Many fast-growing rhizobia were isolated and classified according to various physiological, biochemical and genetic characteristics, to build a catalogue of fast-growing soybean-nodulating strains with different adaptations that can be used according to local requirements.

Competitiveness of inoculant strains for nodulation of certain plant cultivars in special areas may benefit from such collections. Nevertheless, it is important to understand how the peculiarities of a region may influence competitiveness. It is not a simple task to predict the most limiting factors in a given environment. Sometimes these factors are climate and soil characteristics that can be readily noted, but in many instances there are environmental influences that are hard to identify. These influences may take place at the onset of root infection or during rhizosphere colonization. Rhizosphere is a complex and dynamic habitat (Hinsinger et al., 2005; Watt et al., 2006) that requires a particular approach for its analysis. New cell labeling and microscopy tools are expanding our knowledge on rhizosphere events, since now it is possible to follow a single living cell in real time in the rhizosphere. The knowledge that we are gaining thanks to these methodologies will bring new ideas and applications in the near future.

Although rhizosphere colonization is of prime importance in competition for nodulation, there are reports indicating that the relationship is not so straightforward. In two pea fields (named sites I and II) inoculated with *R. leguminosarum* bv *viceae* it was found that, although the rhizobial indigenous populations were similar, the inoculant resulted more competitive at site I but less competitive at site II, even when at this site the inoculum was applied at very high concentration; however, in laboratory tests of competition of the inoculant against the dominant strain from site II, both resulted equally competitive (Meade et al., 1985). Strains of *R. leguminosarum* bv *trifolii* that were the most abundant nodule occupiers in fieldgrown subclover were not the most competitive in laboratory experiments carried out in Leonard jars with perlite-vermiculite as substrate (Leung et al., 1994a). In particular, there were four isolates, classified according to their electrophoretic types (ET), which were the most abundant in the field crops. Two of them, named ET 2 and 3, were studied in more detail. Although the ET 3 isolate was in general more competitive than the others, the authors recovered some isolates that, although were rarely present in the field nodules, were more competitive than ET 3 in the laboratory. Regarding the isolate ET 2, although it was found in a large proportion of field nodules, it had a low competitiveness in laboratory. The authors attributed these behaviors to a series of factors, among them the obvious possibility that the environment exerted a decisive influence in the field, but also considered more subtle alternatives, such as a possible non-random distribution in the soil of the more competitive, yet rare isolates. Similarly, a dominant serotype of *R. leguminosarum* bv *trifolii*  was found in field nodules of subclover, although there were 13 distinct serotypes in that site. However, the rhizosphere effect, i.e. the ratio between rhizosphere and non rhizosphere population densities increased much more among the rare serotypes than in the dominant one in spring, while this effect was similar in other seasons (Leung et al., 1994b). Moreover, in a study carried out by Laguerre et al. (2003) in France, a difference between fave bean and pea was observed for the relationship of dominance in soil or nodules in the *R. leguminosarum* bv *viceae* population. While the success in nodule occupancy of rhizobial genotypes in fava bean was mainly determined by the rhizobial symbiotic genotype independently of the soil conditions, in pea there was a stronger influence of the rhizosphere colonization ability (linked to the genomic background but not necessarily to the symbiotic phenotype) on the competition for nodulation. These results underscore the complexity of the environmental effect on genotypic expression, which is differentially exerted on a given rhizobial population according to the plant genotype, the season, and the soil structure.

It has been argued that rhizobia from soil or rhizosphere are more competitive than rhizobia from rich broths due to a physiological state induced by nutrients limitation, which

Competitiveness of inoculant strains for nodulation of certain plant cultivars in special areas may benefit from such collections. Nevertheless, it is important to understand how the peculiarities of a region may influence competitiveness. It is not a simple task to predict the most limiting factors in a given environment. Sometimes these factors are climate and soil characteristics that can be readily noted, but in many instances there are environmental influences that are hard to identify. These influences may take place at the onset of root infection or during rhizosphere colonization. Rhizosphere is a complex and dynamic habitat (Hinsinger et al., 2005; Watt et al., 2006) that requires a particular approach for its analysis. New cell labeling and microscopy tools are expanding our knowledge on rhizosphere events, since now it is possible to follow a single living cell in real time in the rhizosphere. The knowledge that we are gaining thanks to these methodologies will bring new ideas and

Although rhizosphere colonization is of prime importance in competition for nodulation, there are reports indicating that the relationship is not so straightforward. In two pea fields (named sites I and II) inoculated with *R. leguminosarum* bv *viceae* it was found that, although the rhizobial indigenous populations were similar, the inoculant resulted more competitive at site I but less competitive at site II, even when at this site the inoculum was applied at very high concentration; however, in laboratory tests of competition of the inoculant against the dominant strain from site II, both resulted equally competitive (Meade et al., 1985). Strains of *R. leguminosarum* bv *trifolii* that were the most abundant nodule occupiers in fieldgrown subclover were not the most competitive in laboratory experiments carried out in Leonard jars with perlite-vermiculite as substrate (Leung et al., 1994a). In particular, there were four isolates, classified according to their electrophoretic types (ET), which were the most abundant in the field crops. Two of them, named ET 2 and 3, were studied in more detail. Although the ET 3 isolate was in general more competitive than the others, the authors recovered some isolates that, although were rarely present in the field nodules, were more competitive than ET 3 in the laboratory. Regarding the isolate ET 2, although it was found in a large proportion of field nodules, it had a low competitiveness in laboratory. The authors attributed these behaviors to a series of factors, among them the obvious possibility that the environment exerted a decisive influence in the field, but also considered more subtle alternatives, such as a possible non-random distribution in the soil of the more competitive, yet rare isolates. Similarly, a dominant serotype of *R. leguminosarum* bv *trifolii*  was found in field nodules of subclover, although there were 13 distinct serotypes in that site. However, the rhizosphere effect, i.e. the ratio between rhizosphere and non rhizosphere population densities increased much more among the rare serotypes than in the dominant one in spring, while this effect was similar in other seasons (Leung et al., 1994b). Moreover, in a study carried out by Laguerre et al. (2003) in France, a difference between fave bean and pea was observed for the relationship of dominance in soil or nodules in the *R. leguminosarum* bv *viceae* population. While the success in nodule occupancy of rhizobial genotypes in fava bean was mainly determined by the rhizobial symbiotic genotype independently of the soil conditions, in pea there was a stronger influence of the rhizosphere colonization ability (linked to the genomic background but not necessarily to the symbiotic phenotype) on the competition for nodulation. These results underscore the complexity of the environmental effect on genotypic expression, which is differentially exerted on a given

rhizobial population according to the plant genotype, the season, and the soil structure.

It has been argued that rhizobia from soil or rhizosphere are more competitive than rhizobia from rich broths due to a physiological state induced by nutrients limitation, which

applications in the near future.

predispose the rhizobia to seek for plant root infection. Such physiological conditioning was observed for processes that may be related with rhizosphere colonization and nodulation, such as motility and roots infectivity (Lodeiro et al., 2000). However, nutrients limitation achieved by suspending in poor media rhizobia previously grown in rich broths is different than nutrients limitation achieved by rhizobia themselves at stationary growth phase, when nutrients from the broth are exhausted. In the last case, infectivity is diminished (López-García et al., 2001). Several reports indicated that strains isolated from highly competitive soil rhizobial populations frequently lack their superior competitiveness in laboratory tests or when reintroduced into soil containing an established population. In turn, in several instances an apparently poor competitor for nodulation in laboratory experiments resulted very competitive in soil (Lochner et al., 1989 and references therein). Hence, López-García et al. (2002) tested directly this concept on the basis of competition between two nearly isogenic *B. japonicum* strains (Fig. 1). They established a population of strain LP 3004 (USDA 110 Sm-resistant) in vermiculite pots by inoculating the pots with the bacteria in N-free plant nutrient solution and leaving the pots without plants for at least 1 month in the greenhouse. After an initial period of cell divisions, the rhizobial population stabilized in around 106 rhizobia ml-1 without decaying along this period. After this incubation, when the nutrient-limited physiological state seemed to be acquired by this rhizobial population,

Fig. 1. Demonstration that the rhizobial position in the rooting substrate is determinant for competition for nodulation. Two nearly isogenic strains (indicated in red and green) were inoculated either on the seeds and the rooting substrate or mixed homogeneously before added to the pots. As result, the strain inoculated on the seeds occupied few nodules, despite being intrinsically more competitive. For futher details see text.

the pots were divided in two groups. To one of them soybean plantlets were planted and inoculated on the seedlings with around 108 rhizobia plant-1 of the strain LP 3001 (USDA 110 Sp-resistant) freshly obtained from a rich broth. From the other half of the pots the rhizobia were removed, suspended in N-free plant nutrient solution, and mixed there with another aliquot of rich broth-grown LP 3001 in 1:1 relationship (106 rhizobia ml-1 of each strain). This mixture was added to fresh vermiculite pots, and soybean plantlets were planted. After 20 days in the greenhouse the nodules were recovered and their contents were identified according to their antibiotic resistances. As a result, it was observed that in the pots where the rich broth rhizobia were inoculated on the seedlings, these rhizobia formed around 20 % of the nodules, but in the pots where both strains were homogeneously mixed before pouring them into the vermiculite, the rhizobia from the rich broth formed more than 80 % of the nodules. This experiment demonstrated that the superior competitiveness of the established population is not caused by a nutrient-limited physiological state, but simply by the better position that they had in the vermiculite with respect to the growing roots: the authors also observed that at field capacity the movement of the rhizobia in the vermiculite is very scarce, which was recently corroborated with non-flagellated mutants (Althabegoiti et al., 2011), and supported the idea that the initial cells that colonize the rhizosphere do not arrive swimming but are "scavenged" by the displacement of the growing roots.

Life in the soil is nevertheless very important for the rhizobia. They may persist even in the absence of legume crops, and it was observed that *Bradyrhizobium sp. (Lotus)* retains its nodulation, competition, and N2 fixing characteristics even after 10 years in the soil (Lochner et al., 1989). If we consider a soybean crop season, and take into account that nodules start to senesce at grain filling, we can estimate that the rhizobia are into the nodules for less than 40 % of a year; the other 60 % of the time they have to survive in the free-living state in the soil. During this period the rhizobia have to face diverse threats, including UV irradiation, temperature changes, predation, drought, flooding, etc. Since these microorganisms are unable to sporulate, a preferred state of endurance is the biofilm (Danhorn & Fuqua, 2007). To this end, the cell surface components play a major role, but flagella are lost and biofilm rhizobia are not motile, which may in part explain the lack of effects of motility on competition for nodulation in non-flooded soils. In addition, plant lectins may help in developing the biofilms. It was observed that soybean lectin enhances the biofilm formation by *B. japonicum* in a way that is dependent on the presence of the receptor EPS molecule in the bacteria (Pérez-Giménez et al., 2009). Since this process seems not related with plant infection, it was argued that lectin-assisted biofilm formation may favor *B. japonicum*  biofilms in the vicinity of decaying soybean roots or even on dead roots, where soybean lectin may have been released. This is supported by the observation that soybean lectin is remarkable stable, being unaltered even after a week of incubation at 70 oC. Thus, this enhancement of biofilms formation where soybeans were recently cultivated may keep a localized high rhizobial population for the next nodulation cycle, thus explaining the heterogeneities of rhizobial distribution in the soil previously postulated by Leung et al. (1994).

#### **5. Conclusion**

Competition for nodulation still remains a very complex and largely unknown phenomenon, yet a very important issue for N2 fixation technology. Nevertheless, understanding of this phenomenon has advanced in the last years, and several measures to improve competitiveness of rhizobial inoculated strains may be proposed. Among that are

the pots were divided in two groups. To one of them soybean plantlets were planted and inoculated on the seedlings with around 108 rhizobia plant-1 of the strain LP 3001 (USDA 110 Sp-resistant) freshly obtained from a rich broth. From the other half of the pots the rhizobia were removed, suspended in N-free plant nutrient solution, and mixed there with another aliquot of rich broth-grown LP 3001 in 1:1 relationship (106 rhizobia ml-1 of each strain). This mixture was added to fresh vermiculite pots, and soybean plantlets were planted. After 20 days in the greenhouse the nodules were recovered and their contents were identified according to their antibiotic resistances. As a result, it was observed that in the pots where the rich broth rhizobia were inoculated on the seedlings, these rhizobia formed around 20 % of the nodules, but in the pots where both strains were homogeneously mixed before pouring them into the vermiculite, the rhizobia from the rich broth formed more than 80 % of the nodules. This experiment demonstrated that the superior competitiveness of the established population is not caused by a nutrient-limited physiological state, but simply by the better position that they had in the vermiculite with respect to the growing roots: the authors also observed that at field capacity the movement of the rhizobia in the vermiculite is very scarce, which was recently corroborated with non-flagellated mutants (Althabegoiti et al., 2011), and supported the idea that the initial cells that colonize the rhizosphere do not

arrive swimming but are "scavenged" by the displacement of the growing roots.

**5. Conclusion** 

Life in the soil is nevertheless very important for the rhizobia. They may persist even in the absence of legume crops, and it was observed that *Bradyrhizobium sp. (Lotus)* retains its nodulation, competition, and N2 fixing characteristics even after 10 years in the soil (Lochner et al., 1989). If we consider a soybean crop season, and take into account that nodules start to senesce at grain filling, we can estimate that the rhizobia are into the nodules for less than 40 % of a year; the other 60 % of the time they have to survive in the free-living state in the soil. During this period the rhizobia have to face diverse threats, including UV irradiation, temperature changes, predation, drought, flooding, etc. Since these microorganisms are unable to sporulate, a preferred state of endurance is the biofilm (Danhorn & Fuqua, 2007). To this end, the cell surface components play a major role, but flagella are lost and biofilm rhizobia are not motile, which may in part explain the lack of effects of motility on competition for nodulation in non-flooded soils. In addition, plant lectins may help in developing the biofilms. It was observed that soybean lectin enhances the biofilm formation by *B. japonicum* in a way that is dependent on the presence of the receptor EPS molecule in the bacteria (Pérez-Giménez et al., 2009). Since this process seems not related with plant infection, it was argued that lectin-assisted biofilm formation may favor *B. japonicum*  biofilms in the vicinity of decaying soybean roots or even on dead roots, where soybean lectin may have been released. This is supported by the observation that soybean lectin is remarkable stable, being unaltered even after a week of incubation at 70 oC. Thus, this enhancement of biofilms formation where soybeans were recently cultivated may keep a localized high rhizobial population for the next nodulation cycle, thus explaining the heterogeneities of rhizobial distribution in the soil previously postulated by Leung et al. (1994).

Competition for nodulation still remains a very complex and largely unknown phenomenon, yet a very important issue for N2 fixation technology. Nevertheless, understanding of this phenomenon has advanced in the last years, and several measures to improve competitiveness of rhizobial inoculated strains may be proposed. Among that are the manipulation of host-controlled restriction of nodulation, the genetic manipulation of the plant and bacterial partners, selection of superior strains, improvement of inoculant formulations by manipulating the culture media and the physiological and metabolic state of the bacteria, and the improvement of inoculant application technologies, particularly with in-furrow inoculation. These methods, as well as the new developments that are in progress, are necessary for the sustainable agriculture of the future.

#### **6. Acknowledgment**

The work of the authors is funded by Agencia Nacional de Promoción de la Investigación Científica y Tecnológica (ANPCyT), Argentina. JPG and JIQ are fellows of Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina. ARL is member of the scientific career of CONICET.

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