Rhizobia

Rhizobia are soil-dwelling bacteria that form root nodules on legumes. These bacteria are diazotrophic, meaning they convert atmospheric nitrogen into ammonia, but they can only do this while living inside a legume’s root nodules—they cannot fix nitrogen on their own. Typically, rhizobia are gram-negative, motile, non-sporulating rods. They exist freely in the soil until they infect a legume root, triggering nodule formation. Inside these nodules, they turn nitrogen gas (N₂) into ammonia (NH₃), a form of nitrogen the plant can use. The legume then uses this nitrogen for growth. When the legume dies, the nodule breaks down, releasing the bacteria back into the soil, where they either live independently or infect another legume.

The first rhizobia species identified was *Rhizobium leguminosarum* in 1889, and all later species were initially grouped under the same genus. Most research has focused on crop and forage legumes like clover, alfalfa, beans, peas, and soybeans, though work on North American legumes is expanding.

Taxonomically, rhizobia are a paraphyletic group spread across two classes of Pseudomonadota: alphaproteobacteria and betaproteobacteria. Most belong to the order Hyphomicrobiales, but some appear in other Pseudomonadota orders. These groups also contain non-symbiotic bacteria; for instance, the plant pathogen *Agrobacterium* is more closely related to *Rhizobium* than to the *Bradyrhizobium* that nodulate soybeans.

In agriculture, much of the fixed nitrogen is removed when protein-rich grain or hay is harvested, but enough can remain in the soil to benefit future crops. This is especially valuable where nitrogen fertilizer is not used, such as in organic rotations or less-industrialized regions. Nitrogen is the most commonly deficient nutrient in many soils worldwide and the most frequently supplied plant nutrient, but fertilizer use carries serious environmental costs. Specific rhizobia strains are needed to form functional, nitrogen-fixing nodules on legume roots. Having the right strain boosts legume yield, and inoculation with rhizobia generally increases crop output. Rhizobia also help legumes resist insect herbivores, especially when multiple species are present. Legume inoculation has been practiced for many years and continues to improve. Each year, 12–20 million hectares of soybeans are inoculated. An ideal inoculant is effective, easy to use, compatible, contains high rhizobial numbers, has a long shelf life, works under various field conditions, and survives well. These inoculants can improve legume cultivation, and after harvest, soil nitrate levels rise, benefiting the next crop.

Rhizobia are unique as the only nitrogen-fixing bacteria that live symbiotically with legumes. Common crop and forage legumes include peas, beans, clover, and soy. This symbiosis is a classic mutualism: rhizobia supply ammonia or amino acids to the plant, and in return receive organic acids (mainly malate and succinate) as carbon and energy. However, because several unrelated strains infect each plant, a tragedy of the commons can occur. Cheater strains may hoard resources like polyhydroxybutyrate for their own reproduction without fixing much nitrogen. Given the costs of nodulation and the opportunity for cheating, the persistence of this symbiosis is notable.

The relationship begins with a signal exchange. The most studied mechanism is intracellular infection. Rhizobia live freely in soil until they detect flavonoids—compounds derived from 2-phenyl-1.4-benzopyrone—secreted by host plant roots. This triggers bacterial accumulation and attachment to root hairs. Flavonoids then promote the DNA-binding activity of NodD, a LysR-family regulator, which leads to secretion of nod factors once bacteria enter the root hair. Nod factors cause root hair curling and the formation of an infection thread—a cellulose-lined tube the bacteria travel through into root cells. The bacteria then infect neighboring cells, and continuous cell proliferation forms the root nodule. A second mechanism, crack entry, is used by rhizobia infecting aquatic hosts like *Sesbania rostrata*. Here, no root hair deformation occurs; instead, bacteria enter through cracks created by lateral root emergence. Inside the nodule, bacteria differentiate into bacteroids and fix nitrogen into ammonium using nitrogenase. Ammonium is converted into amino acids like glutamine and asparagine before being exported to the plant. In exchange, the plant supplies the bacteria with carbohydrates as organic acids and provides oxygen for cellular respiration.