Rhizobia

Root nodules, each containing billions of Rhizobiaceae bacteria

Rhizobia are soil bacteria that fix nitrogen (diazotrophs) after becoming established inside root nodules of legumes (Fabaceae). In order to express genes for nitrogen fixation, rhizobia require a plant host; they cannot independently fix nitrogen. In general, they are Gram-negative, motile, non-sporulating rods.

History

The first known species of rhizobia, Rhizobium leguminosarum, was identified in 1889, and all further species were initially placed in the Rhizobium genus. However, more advanced methods of analysis have revised this classification, and now there are many in other genera. Most research has been done on crop and forage legumes such as clover, alfalfa, beans, peas, and soy; more research is being done on North American legumes.

The word rhizobia comes from the Ancient Greek ῥίζα, rhíza, meaning "root" and βίος, bios, meaning "life". The word rhizobium is still sometimes used as the singular form of rhizobia

Taxonomy

Rhizobia are a paraphyletic group that fall into two classes of the proteobacteria—the alpha- and beta-proteobacteria. As shown below, most belong to the order Rhizobiales, but several rhizobia occur in distinct bacterial orders of the proteobacteria.^[1][2][3]

α-proteobacteria Rhizobiales Bradyrhizobiaceae Bosea B. lathyri B. lupini B. robiniae Bradyrhizobium B. arachidis B. canariense B. cytisi B. daqingense B. denitrificans B. diazoefficiens B. elkanii B. huanghuaihaiense B. iriomotense B. japonicum B. jicamae B. lablabi B. liaoningense B. pachyrhizi B. rifense B. yuanmingense Brucellaceae Ochrobactrum O. cytisi O. lupini Hyphomicrobiaceae Devosia D. neptuniae Methylobacteriaceae Methylobacterium M. nodulans Microvirga M. lotononidis M. lupini M. zambiensis

Phyllobacteriaceae Aminobacter Aminobacter anthyllidis Mesorhizobium M. abyssinicae M. albiziae M. alhagi M. amorphae M. australicum M. camelthorni M. caraganae M. chacoense M. ciceri M. gobiense M. hawassense M. huakuii M. loti M. mediterraneum M. metallidurans M. muleiense M. opportunistum M. plurifarium M. qingshengii M. robiniae M. sangaii M. septentrionale M. shangrilense M. shonense M. tamadayense M. tarimense M. temperatum M. tianshanense Phyllobacterium P. ifriqiyense P. leguminum P. sophorae P. trifolii

Rhizobiaceae Rhizobium R. alamii R. alkalisoli R. cauense R. cellulosilyticum R. daejeonense R. etli R. fabae R. galegae R. gallicum R. giardinii R. grahamii R. hainanense R. halophytocola R. helanshanense R. herbae R. huautlense R. indigoferae R. leguminosarum R. leucaenae R. loessense R. lupini R. lusitanum R. mesoamericanum R. mesosinicum R. miluonense R. mongolense R. multihospitium R. nepotum R. oryzae R. petrolearium R. phaseoli R. pisi R. pusense R. qilianshanense R. sphaerophysae R. sullae R. taibaishanense R. tibeticum R. tropici R. tubonense R. undicola R. vallis R. vignae R. yanglingense

Shinella S. kummerowiae Sinorhizobium/Ensifer S. abri E. adhaerens S. americanum S. arboris S. chiapanecum S. fredii E. garamanticus S. indiaense S. kostiense S. kummerowiae S. medicae S. meliloti E. mexicanus E. numidicus E. psoraleae S. saheli E. sesbaniae E. sojae S. terangae Xanthobacteraceae Azorhizobium A. caulinodans A. doebereinerae

β-proteobacteria Burkholderiales Burkholderiaceae Burkholderia B. caribensis B. dolosa B. mimosarum B. nodosa B. phymatum B. sabiae B. tuberum Cupriavidus C. taiwanensis

These groups include a variety of non-symbiotic bacteria. For instance, the plant pathogen Agrobacterium is a closer relative of Rhizobium than the Bradyrhizobium that nodulate soybean (and may not really be a separate genus). The genes responsible for the symbiosis with plants, however, may be more closely related than the organisms themselves, acquired by horizontal transfer (via bacterial conjugation) rather than vertical gene transfer (from a common ancestor). Evidence suggests that "symbiosis islands" exist and share similarities to pathogenicity islands.^[4]

Importance in agriculture

Rhizobia nodules on Vigna unguiculata

Although much of the nitrogen is removed when protein-rich grain or hay is harvested, significant amounts can remain in the soil for future crops. This is especially important when nitrogen fertilizer is not used, as in organic rotation schemes or some less-industrialized countries.^[5] Nitrogen is the most commonly deficient nutrient in many soils around the world and it is the most commonly supplied plant nutrient. Supply of nitrogen through fertilizers has severe environmental concerns.

Rhizobia is “the group of soil bacteria that infect the roots of legumes to form root nodules”.^[6] Rhizobia are found in the soil and after infection, produce nodules in the legume where they fix nitrogen gas (N₂) from the atmosphere turning it into a more readily useful form of nitrogen (N). From here, the nitrogen is exported from the nodules and used for growth in the legume. Once the legume dies, the nodule breaks down and releases the rhizobia back into the soil where they can live individually or reinfect a new legume host.^[6]

Specific strains of rhizobia are required to make functional nodules on the roots able to fix the N₂.^[7] Having this specific rhizobia present is beneficial to the legume, as the N₂ fixation can increase crop yield.^[8] Inoculation with rhizobia tends to increase yield.^[9]

Legume inoculation has been an agriculture practice for many years and has continuously improved over time.^[8] 12-20 million hectares of soybeans are inoculated annually. The technology to produce these inoculants are microbial fermenters. An ideal inoculant includes some of the following aspects; maximum efficacy, ease of use, compatibility, high rhizobial concentration, long shelf-life, usefulness under varying field conditions, and survivability.^[8] Once this ideal inoculant in produced it is most likely in a liquid or powder form where it is applied directly to the seed before planting.^[10][11]

Many poor countries have trouble with the introduction of new crops and reaching attainable yield. As they introduce new crops into their soils, these inoculants may foster legume growth and success in the area, therefore giving farmers more options for planting. Using these inoculants provide many other benefits as well such as not having to use nitrogen fertilizers.^[12] As a result of the nodulation process, after the harvest of the crop there are higher levels of soil nitrate, which can then be used by the next crop, and little to no nitrogen fertilizer is needed.^[13] Many poor farmers do not have access to fertilizers, so the sustainability of rhizobial inoculum is an important aspect in saving money for the essentials.

It has also been stated that “cereals were healthier and higher yielding when grown after a legume”.^[6] Cereals are another major crop, so if integrated in a crop rotation with legumes, then rhizobium inoculation can be very beneficial to crops other than legumes.^[10] Increasing the possible amount of yield for multiple crops would help farmers very much in growing their current food production as well to help close the gap between current yield and potential yield.^[14]

Symbiotic relationship

Rhizobia are unique in that they are the only nitrogen-fixing bacteria living in a symbiotic relationship with legumes. Common crop and forage legumes are peas, beans, clover, and soy.

Infection and signal exchange

The symbiotic relationship implies a signal exchange between both partners that leads to mutual recognition and development of symbiotic structures. Rhizobia live in the soil where they are able to sense flavonoids secreted by the roots of their host legume plant. Flavonoids trigger the secretion of nod factors, which in turn are recognized by the host plant and can lead to root hair deformation and several cellular responses, such as ion fluxes. The best-known infection mechanism is called intracellular infection, in this case the rhizobia enter through a deformed root hair in a similar way to endocytosis, forming an intracellular tube called the infection thread. A second mechanism is called "crack entry"; in this case, no root hair deformation is observed and the bacteria penetrate between cells, through cracks produced by lateral root emergence. Later on, the bacteria become intracellular and an infection thread is formed like in intracellular infections.

The infection triggers cell division in the cortex of the root where a new organ, the nodule, appears as a result of successive processes.

Infection threads grow to the nodule, infect its central tissue and release the rhizobia in these cells, where they differentiate morphologically into bacteroids and fix nitrogen from the atmospheric, elemental N₂ into a plant-usable form, ammonium (NH₃ + H⁺ → NH₄⁺), using the enzyme nitrogenase. The reaction for all nitrogen-fixing bacteria is:^[15]

N₂ + 8 H⁺ + 8 e⁻ → 2 NH₃ + H₂

In return, the plant supplies the bacteria with carbohydrates, proteins, and sufficient oxygen so as not to interfere with the fixation process. Leghaemoglobins, plant proteins similar to human hemoglobins, help to provide oxygen for respiration while keeping the free oxygen concentration low enough so as not to inhibit nitrogenase activity. Recently, a Bradyrhizobium strain was discovered to form nodules in Aeschynomene without producing nod factors, suggesting the existence of alternative communication signals other than nod factors.^[16]

Nature of the Mutualism

The legume–rhizobium symbiosis is a classic example of mutualism—rhizobia supply ammonia or amino acids to the plant and in return receive organic acids (principally as the dicarboxylic acids malate and succinate) as a carbon and energy source. However, because several unrelated strains infect each individual plant, a classic tragedy of the commons scenario presents itself. Cheater strains may hoard plant resources such as polyhydroxybutyrate for the benefit of their own reproduction without fixing an appreciable amount of nitrogen.^[17] Given the costs involved in nodulation and the opportunity for rhizobia to cheat, it may be surprising that this symbiosis should exist at all.

The coevolution of cooperation and choice poses a veritable quandary: choice acts to reduce variation, and therefore removes the incentive for its own maintenance. If this is true, choice should be evolutionarily unstable. If rhizobia were perfect cooperators, choosy hosts would suffer adverse fitness consequences if, as with legumes, choice carries energy costs. The continued existence of legume-rhizobia symbiosis has significant parallels to the lek paradox, wherein female selection of showy mates is maintained among birds. Ironically, cheating itself may be a stabilizing force of cooperation. As with birds, variation is introduced in each generation of rhizobia through immigration, mutation, and gene transfer. When the amount of variation in a population is sufficiently high, mutualism is maintained even when choice is costly. In a roundabout way, cooperation owes its very existence to the recurring consequences of consistent parasitism.^[18]

The Sanctions Hypothesis

There are two main hypotheses for the mechanism that maintains legume-rhizobium symbiosis (though both may occur in nature). The sanctions hypothesis theorizes that legumes cannot recognize the more parasitic or less nitrogen fixing rhizobia, and must counter the parasitism by post-infection legume sanctions. In response to underperforming rhizobia, legume hosts can respond by imposing sanctions of varying severity to their nodules.^[19] These sanctions include, but are not limited to reduction of nodule growth, early nodule death, decreased carbon supply to nodules, or reduced oxygen supply to nodules that fix less nitrogen. Within a nodule, some of the bacteria differentiate into nitrogen fixing bacteroids, which have been found to be unable to reproduce.^[20] Therefore, with the development of a symbiotic relationship, if the host sanctions hypothesis is correct, the host sanctions must act toward whole nodules rather than individual bacteria because individual targeting sanctions would prevent any reproducing rhizobia to proliferate over time. This ability to reinforce a mutual relationship with host sanctions pushes the relationship toward a mutualism rather than a parasitism and is likely a contributing factor to why the symbiosis exists.

The Partner Choice Hypothesis

The partner choice hypothesis proposes that the plant uses prenodulation signals from the rhizobia to decide whether to allow nodulation, and chooses only noncheating rhizobia. There is evidence for sanctions in soybean plants, which reduce rhizobium reproduction (perhaps by limiting oxygen supply) in nodules that fix less nitrogen.^[21] Likewise, wild lupine plants allocate fewer resources to nodules containing less-beneficial rhizobia, limiting rhizobial reproduction inside. This is consistent with the definition of sanctions, although called "partner choice" by the authors.^[22] However, other studies have found no evidence of plant sanctions, and instead support the partner choice hypothesis.^[23][24] While both mechanisms no doubt contribute significantly to maintaining rhizobial cooperation, they do not in themselves fully explain the persistence of the mutualism. The partner choice hypothesis is not exclusive from the host sanctions hypothesis, as it is apparent that both of them are prevalent in the symbiotic relationship.^[25]

Evolutionary History

The symbiosis between nitrogen fixing rhizobia and the legume family has emerged and evolved over the past 65 million years.^[26] Rhizobia provide valuable organic nitrogen to the plant in exchange for carbon generated from the plant’s photosynthesis. This exchange increases the relative fitness of both species.^[27] Although evolution tends to swing toward one species taking advantage of another in the form of noncooperation in the selfish-gene model, management of such symbiosis allows for the continuation of cooperation.^[28] When the relative fitness of both species is increased, natural selection will favor the symbiosis.

To understand the evolutionary history of this symbiosis, it is helpful to compare the rhizobia-legume symbiosis to a more ancient symbiotic relationship, such as that between endomycorrhizae fungi and land plants, which dates back to almost 460 million years ago.^[29]

Endomycorrhizal symbiosis can provide many insights into rhizobia symbiosis because recent genetic studies have suggested that rhizobia co-opted the signaling pathways from the more ancient endomycorrhizal symbiosis.^[30] Bacteria secrete Nod factors and endomycorrhizae secrete Myc-LCOs. Upon recognition of the Nod factor/Myc-LCO, the plant proceeds to induce a variety of intracellular responses to prepare for the symbiosis.^[31]

It is likely that rhizobia co-opted the features in already place for endomycorrhizal symbiosis, because there are many shared or similar genes involved in the two processes. For example, the plant recognition gene, SYMRK (symbiosis receptor-like kinase) is involved in the perception of both the rhizobial Nod factors as well as the endomycorrhizal Myc-LCOs.^[32] The shared similar processes would have greatly facilitated the evolution of rhizobial symbiosis, because not all the symbiotic mechanisms would have needed to develop. Instead the rhizobia simply needed to evolve mechanisms to take advantage of the symbiotic signaling processes already in place from endomycorrhizal symbiosis.

Other diazotrophs

Many other species of bacteria are able to fix nitrogen (diazotrophs), but few are able to associate intimately with plants and colonize specific structures like Legume nodules. Bacteria that do associate with plants include the actinobacteria Frankia, which form symbiotic root nodules in actinorhizal plants, and several cyanobacteria (Nostoc) associated with aquatic ferns, Cycas and Gunneras. Free-living diazotrophs are often found in the rhizosphere and in the intercellular spaces of several plants including rice and sugarcane, but in this case the lack of a specialized structure results in poor nutrient transfer efficiency compared to legume or actinorhizal nodules.

References

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4. Sullivan, John, T. (11 December 1997). "Evolution of rhizobia by acquisition of a 500-kb symbiosis island that integrates into a phe-tRNA gene". PNAS. 95 (9): 5145–5149. doi:10.1073/pnas.95.9.5145. PMC 20228. PMID 9560243.
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8. Catroux, Gerard; Hartmann, Alain; Revillin, Cecile (2001). Trends in rhizobium inoculant production and use. Netherlands: Kluwer Academic Publishers. pp. 21–30.
9. Purcell, Larry C.; Salmeron, Montserrat; Ashlock, Lanny (2013). "Chapter 5" (PDF). Arkansas Soybean Production Handbook - MP197. Little Rock, AR: University of Arkansas Cooperative Extension Service. p. 5. Retrieved 21 February 2016.
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11. Bennett, J. Michael; Rhetoric, Emeritus; Hicks, Dale R.; Naeve, Seth L.; Bennett, Nancy Bush (2014). The Minnesota Soybean Field Book (PDF). St Paul, MN: University of Minnesota Extension. p. 79. Retrieved 21 February 2016.
12. Stephens, J.H.G; Rask, H.M (2000). Inoculant production and formulation. Saskatoon: MicroBio RhizoGen Corporation. pp. 249–258.
13. Taylor, Adam. "Opening New Markets and Increasing Canadian Exports Generates Jobs—Creating Benefits for Middle-Class Families: Report". Government of Canada.
14. Shercan, D.P; Karki, K.B (n.d.). Plant nutrient management for improving crop productivity in Nepal. Nepal: Nepal Agriculture Research Council. p. 3.
15. The Nitrogen cycle and Nitrogen fixation, Jim Deacon, Institute of Cell and Molecular Biology, The University of Edinburgh http://www.biology.ed.ac.uk/archive/jdeacon/microbes/nitrogen.htm
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21. Kiers ET, Rousseau RA, West SA, Denison RF 2003. Host sanctions and the legume–rhizobium mutualism. Nature 425 : 79-81
22. Simms; et al. (2006). "An empirical test of partner choice mechanisms in a wild legume-rhizobium interaction". Proc. Roy. Soc. B. 273 (1582): 77–81. doi:10.1098/rspb.2005.3292.
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26. Herendeen, Patrick (1999). "A Preliminary Conspectus of the Allon Flora from the Late Cretaceous (Late Santonian) of Central Georgia, U.S.A". Annals of the Missouri Botanical Garden. 86 (2): 407–471. doi:10.2307/2666182. JSTOR 2666182.
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29. Martin, Parniske (2008). "Arbuscular mycorrhiza: the mother of plant root endosymbioses". Nature Reviews Microbiology. 6 (10): 763–775. doi:10.1038/nrmicro1987. PMID 18794914.
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Further reading

• Jones, KM; Kobayashi, H; Davies, BW; Taga, ME; Walker, GC; et al. (2007). "How rhizobial symbionts invade plants: the Sinorhizobium–Medicago model". Nature Reviews Microbiology. 5 (8): 619–33. doi:10.1038/nrmicro1705. PMC 2766523. PMID 17632573.

External links

• Legume sanctions maintain Rhizobium mutualism
• Current list of rhizobia species
• Nitrogen Fixation and Inoculation of Forage Legumes