Rhizobia are soil bacteria that fix nitrogen (diazotrophs) after becoming established inside root nodules of legumes (Fabaceae). Rhizobia require a plant host; they cannot independently fix nitrogen. In general, they are Gram-negative, motile, non-sporulating rods.
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, and soy; recently, more work is occurring on North American legumes.
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.
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).
Importance in agriculture
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. 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 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.
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:
- 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.
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. Given the costs involved in nodulation and the opportunity for rhizobia to cheat, it may be surprising that this symbiosis should exist at all.
There are two competing hypotheses for the mechanism that maintains legume-rhizobium symbiosis (though both may occur in nature). The sanctions hypothesis suggests the plants police cheating rhizobia. Sanctions could take the form of reduced nodule growth, early nodule death, decreased carbon supply to nodules, or reduced oxygen supply to nodules that fix less nitrogen. 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. 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 just given, although called "partner choice" by the authors. However, other studies have found no evidence of plant sanctions, and instead support the partner choice hypothesis. While both mechanisms no doubt contribute significantly to maintaining rhizobial cooperation, they do not in themselves fully explain the persistence of the mutualism.
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.
The symbiosis between nitrogen fixing rhizobia and the legume family has emerged and evolved over the past 65 million years. 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. 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.
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. 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.
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. 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.
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.
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