Symbiotic Associations Between Actinorhizal Plants And Frankia

Symbiotic associations are formed between the bacterial genus Frankia (gram positive, high G+ C, filamentous bacterium) and many nonleguminous plant species widely distributed among eight different families and commonly referred to as actinorhizal species (Table 14.10). Well-known plant genera include Alnus, Casuarina, Myrica, Hippophae, Elaeagnus, Ceanothus, and Purshia. These plants are distributed worldwide and tend to be woody shrubs or trees that colonize N-limited landscapes that are usually in the primary stages of recovery from some disturbance (wild fire, landslide, flood, and logging). These plants colonize sites that range widely in latitude, elevation, and water and nutrient availability, and the amounts of N2 fixed vary considerably (< 10 to 300 kg N ha-1 year-1 (Table 14.5)).

In contrast to the first isolation of rhizobia over 100 years ago (1888), Frankia isolates were first obtained in culture in 1978 (Callaham et al., 1978). Frankia has continued to be a notoriously difficult bacterium to isolate from nodules and to study in the laboratory; yet, considerable information has been obtained about it by using molecular biological methods that circumvent the need for obtaining pure cultures. Phylogenetic analyses of 16S rDNA and glutamine synthetase gene sequences of Frankia strains indicate that the genus belongs to three phylogenetically distinct clades or clusters (Fig. 14.8), Clawson et al., 2004). Clade I is represented

"Higher' Hamamelididae

Comptonia Myrica Alnus Casuarina Allocasuarina Gymnostoma Coriaria Datisca Elaeagnus Hippophae Shepherdia Colletia Discaria Retanilla Talguenea

Trevoa Ceanothus Dryas Purshia Cowania Chamaebatia Cercocarpus

Plants Frankia symbionts

FIGURE 14.8 Relationships between actinorhizal host genera and three phylogenetically distinct clades of Frankia with emphasis on the mode of root infection that initiates nodule formation. On the left of the diagram, thick lines denote plant genera in which root infection occurs via root hairs, thin lines denote plant genera in which infection occurs via intercellular penetration of the root surface. On the right side of the diagram, solid lines indicate the presence of typical Frankia strains in nodules of a plant genus. Dashed lines indicate that Frankia clade III strains are rarely found in nodules on these plant genera. Reprinted from Clawson et al. (2004) by permission of the publisher; copyright (2004) Elsevier.

"Higher' Hamamelididae

exclusively by microsymbionts that have been detected in nodules of a wide variety of hosts, but never obtained into culture. Clade II isolates are associated with a few genera of actinorhizal plants (e.g., Casuarina, Alnus, Myrica) that establish their symbioses through a root-hair infection process similar to that of legumes. Clade III strains tend to range widely across hosts that primarily practice the intercellular infection process.

There are many similarities between legumes and actinorhizals in the mode of plant infection, symbiosis establishment, and N assimilation, but there are also many differences. Whereas legume nodules represent stem-like organs, with a peripheral vascular system and nodule primordia that originate in the root cortex, acti-norhizal nodules consist of modified lateral roots, possess a central vascular system, and arise from nodule primordia originating in the root pericycle. Recently, it was shown that the expression of some nodulin genes associated with BNF and N metabolism is more similar among legumes and intracellularly infected actinorhizal plant species than among the different phylogenetic groups of actinorhizal plants (Pawlowski and Bisseling, 1996). In Frankia, N2 fixation occurs in swellings located at the tips of filaments referred to as vesicles. In the symbiotic state, vesicles are produced in large numbers, with vesicle shape and the degree of septation differing among different host plant species. For example, spherical-septated vesicles are found in Alnus and elongated-nonseptated vesicles in Coriaria. Interestingly, vesicles are not found in infected cells of Casuarina nodules.

Similar to the legume-rhizobia associations, some actinorhizal plants such as Casuarina and Myrica produce an O2-transporting hemoglobin that correlates with the presence of an O2 diffusion barrier surrounding infected cells. Most acti-norhizals do not produce hemoglobin, however, and it is generally felt that the vesicle wall of the Frankia itself is the major diffusion barrier to O2. In Coraria, infected cells are surrounded by a suberized periderm layer whose thickness varies with O2 concentration.

biotechnology of bnf

With the world's population projected to increase to 8.3 billion by 2025, and the accompanying demand for plant nutrient N, there will continue to be a need to increase the contribution of BNF to food and fiber production. As mentioned earlier in this chapter, rates of BNF under field conditions vary widely, and there are many environmental/agronomic factors generally associated with plant nutrition, water availability, and plant disease that hinder N2-fixing species from reaching their yield potential under field conditions. We should not overlook the fact that some undesirable attributes of N2-fixing species, such as toxin production, along with ethnical/historical biases, disfavor their use as major food and fiber sources in some parts of the world. The Leguminosae is an enormous plant family distributed worldwide, with 16,000 to 19,000 species in about 750 genera (Allen and Allen, 1981). Despite this wealth of diversity, very few legumes have been successfully domesticated and used worldwide by humans for agricultural purposes. For many years attempts to expand the agricultural use of leguminous species beyond their regions of origin had a checkered history because the appropriate rhizobia were not always present in the soil. As a consequence, it has been standard practice for many years to inoculate legume seed with appropriate rhizobia before planting a legume species in regions where (a) it has not been grown previously, (b) the native flora of the region did not contain legumes that were close relatives of the crop, and (c) an interval of several years exists between the use of a legume in the rotation. Another example of a situation in which inoculation is recommended includes replacement of an older established cultivar of a legume species with a new disease-resistant variety. The new variety might not form a highly effective symbiotic association with native rhizobial strains that were effective on the older cultivar. During the 20th century, agronomists and soil microbiologists recognized that many environments in which crop production and soil quality could benefit tremendously from BNF contain conditions hostile for survival of rhizobia. As a result, inoculant-quality rhizobial strains are usually prescreened for their ability to tolerate low pH, high aluminum, high temperature, and desiccation and to be genetically stable. Nonetheless, despite our best efforts, we have not yet been able to overcome the facts that many soils contain indigenous or naturalized populations of rhizobia that are suboptimally effective at BNF on agronomically important legumes and that it is extremely difficult to displace them with superior N2-fixing strains. As scientists make more progress in elucidating the molecular signals needed for nodulation by rhizobia and infection by mycorrhizal fungi, and with the genomic sequences of more bacteria and plants becoming available, it is not outside the realm of possibility that we will learn how to develop other symbiotic/ endophytic associations between plants and N2-fixing bacteria. The example of sugarcane and Glu. diazotrophicus gives hope for a future that includes N2-fixing cereals and other food and fiber crops.


The authors acknowledge the contributions of photographs and figures from many colleagues who are research specialists in various aspects of BNF. It would have been very difficult to produce this chapter without their assistance.

references and suggested reading

Allen, O. N., and Allen, E. K. (1981). "Leguminosae: a Source Book of Characteristics, Uses, and Nodulation." Univ. of Wisconsin Press, Madison.

Belnap, J., and Lange, O. L. (2003). "Biological Soil Crusts: Structure, Function and Management." Ecological Studies Series 150. Springer-Verlag, New York.

Callaham, D., Del Tredici, P., and Torrey, J. G. (1978). Isolation and cultivation in vitro of the actino-mycete causing root nodulation of Comptonia. Science 199, 899-902.

Clawson, M. L., Bourret, A., and Benson, D. R. (2004). Assessing the phylogeny of Frankia-actinorhizal plant nitrogen fixing nodule symbioses with Frankia 16SrRNA and glutamine synthetase gene sequences. Mol. Phylogenet. Evol 31, 131-138.

Dean, D. R., and Jacobson, M. R. (1992). Biochemical genetics of nitrogenase. In "Biological Nitrogen Fixation" (G. Stacey, R. H. Burris, and H. J. Evans, eds.), pp. 763-834. Chapman & Hall, New York.

Evans, H. J., and Barber, L. E. (1977). Biological nitrogen fixation for food and fiber production.

Science 197, 332-339.

Galloway, J. N., Aber, J. D., Erisman, J. W., Seitzinger, S. P., Horwath, R. W., Cowling, E. B., and

Cosby, B. J. (2003). The nitrogen cascade. Bioscience 53, 341-356. Hill, S. (1992). Physiology of nitrogen fixation in free-living heterotrophs. In "Biological Nitrogen Fixation" (G. Stacey, R. H. Burris, and H. J. Evans, eds.), pp. 87-134. Chapman & Hall, New York. Kennedy, C., Lee, S., Sevilla, M., Meletzus, D., Gunapala, N., Gardiol, A., and Davidson, S. (2000). Analysis of genes for nitrogen fixation and studies of plant growth enhancement in the dia-zotrophic endophyte of sugarcane, (Glucon)acetobacter diazotrophicus. In "Nitrogen Fixation: from Molecules to Crop Productivity" (F. O. Pedrosa, M. Hungria, G. Yates, and W. E. Newton, eds.), pp. 401-404. Kluwer Academic, Dordrecht. Limpens, E., and Bisseling, T. (2003). Signalling in symbiosis. Curr. Opin. Plant Biol. 6, 343-350. Limpens, E., Franken, C., Smit, P., Willemse, J., Bisseling, T., and Geurts, R. (2003). Lys M domain receptor kinases regulating rhizobial nod factor-induced infection. Science 302, 631-633. Merrick, M. J. (1992). Regulation of nitrogen fixation genes in free-living and symbiotic bacteria. In "Biological Nitrogen Fixation" (G. Stacey, R. H. Burris, and H. J. Evans, eds.), pp. 835-876. Chapman & Hall, New York. Parniske, M., and Downie, J. A. (2003). Locks, keys and symbioses. Nature 425, 569-570. Paul, E. A., and Clark, F. E. (1996). Closing the nitrogen cycle. In "Soil Microbiology and

Biochemistry." 2nd ed. Academic Press, San Diego. Pawlowski, K., and Bisseling, T. (1996). Rhizobial and actinorhizal symbioses: what are the shared features. Plant Cell 8, 1899-1913. Radutolu, S., Madsen, L., Madsen, E. B., Felle, H. H., Umehara, Y., Grenlund, M., Sato, S., Nakamura, Y., Tabata, S., Sandal, N., and Stougaard, J. (2003). Plant recognition of symbiotic bacteria requires two lys M receptor-like kinases. Nature 425, 585-592. Reinhold-Hurek, B., and Hurek, T. (1998). Life in grasses: diazotrophic endophytes. Trends Microbiol. 6, 139-144.

Spaink, H. P. (2000). Root nodulation and infection factors produced by rhizobial bacteria. Annu. Rev. Microbiol. 54, 257-288.

Sylvester, W. B., and Musgrave, D. R. (1991). Free-living diazotrophs. In "Biology and Biochemistry of Nitrogen Fixation" (M. J. Dilworth and A. R. Glenn, eds.), pp. 162-186. Elsevier, New York. Urquiaga, S., Cruz, K. H. S., and Boddy, R. M. (1992). Contribution of nitrogen fixation to sugar cane:

nitrogen-15 and nitrogen-balance estimates. Soil Sci. Soc. Am. J. 56, 105-114. Vitousek, P. M., Aber, J., Horwath, R. W., Likens, G. E., Matson, P. A., Schindler, D. W., Schlesinger, W. H., and Tilman, D. (1997). Human alterations of the global nitrogen cycle: causes and consequences. Issues Ecol. 1, 1-16. Wagner, G. M. (1997). Azolla—a review of its biology and utilization. Bot. Rev. 63, 1-26. Waters, J. K., Hughes, B. L., Purcell, L. C., Gerhardt, K. O., Mawhinney, T. P., and Emerich, D. W. (1998). Alanine, not ammonia, is excreted from N2-fixing soybean nodule bacteroids. Proc. Natl. Acad. Sci. USA 95, 12038-12042. Weaver, R. W., and Danso, S. K. A. (1994). Dinitrogen fixation. In "Methods of Soil Analysis," Part 2, "Microbiological and Biochemical Properties" (R. W. Weaver, J. S. Angle, and P. J. Bottomley, eds.), pp. 1019-1045. Soil Sci. Soc. Am., Madison, WI. Young, J. P. W. (1992). Phylogenetic classification of nitrogen fixing organisms. In "Biological Nitrogen Fixation" (G. Stacey, R. H. Burris, and H. J. Evans, eds.), pp. 43-86. Chapman & Hall, New York.

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