Inoculation, Nodulation, Nitrogen Fixation and Transfer
Dr. Twain J. Butler - Assistant Professor, Texas A&M - Stephenville
Dr. Gerald Evers - Professor, Texas A&M - Overton


Nitrogen is the most limiting nutrient for plant growth. A legume plant´s ability to use nitrogen from the air is the best-known benefit of growing legumes but the least understood. Approximately 79% of the air is nitrogen gas. However, it is not in a form that plants can use. In reality it is not the plant that removes nitrogen from the air but Rhizobium bacteria, which live in small tumor like structures on the legume, plant roots called nodules. These bacteria can take nitrogen gas from the air in the soil and transform it into ammonium (NH4), which can be used by the plant. This ammonium is the same form as in ammonium nitrate (34-0-0) and ammonium sulfate (21-0-0) fertilizer.

The nitrogen fixation (N2-fixation) process between the legume plant and rhizobia bacteria is referred to as a symbiotic (mutually beneficial) relationship. Each organism receives something from the other and gives back something in return. Rhizobia bacteria provide the legume plant with nitrogen in the form of ammonium and the legume plant provides the bacteria with carbohydrates as an energy source.

Seed Inoculation

Inoculation is the process of adding the proper Rhizobia bacteria to the legume seed so that N2-fixation can occur. Most legume species have a specific rhizobia strain that maximizes N2-fixation. There are numerous strains of native Rhizobium bacteria that occur naturally in different soils. Some of these rhizobia strains are capable of infecting a given legume species but will vary in their efficiency to fix nitrogen. Ineffective strains will form many small nodules on the legume root but fix little or no nitrogen. Effective rhizobia strains that fix high rates of nitrogen form fewer but larger nodules that have dark pink or red centers (due to leghemoglobin present). To ensure that an effective rhizobia strain is present when planting a legume species, the seed are inoculated (Rhizobium bacteria applied on the seed) before planting.

Legumes can be categorized by their inoculant groups (Table 1), however, these are only general guidelines. Most inoculant companies mix different inoculums to give a broader range of effectiveness.

Table 1. Inoculant Groups
Groups Species
I. Alafalfa Group
      A. Type A Inoculant Alfalfa, yellow sweet clover, and white sweetclovers
      B. Type N Inoculant Barrel, black, burr, botton, and spotted burr medics
II. Clover Group
      A. Type B Inoculant Ball, red, and white clovers
      B. Type O Inoculant Arrowleaf clover
      C. Type R Inoculant Berseem, crimson, and Persian clovers
      D. Type WR Inoculants Rose and subterranean clovers
III. Pea and Vetch Group
      A. Type C Inoculant Austrian winter pean, Caley pea, rough pea and vetches
IV. Bean Group
      A. Type D Inoculant Garden bean, kidney bean, navy bean, and pinto bean
V. Soybean Group
      A. Type S Inoculant soybeans
VI. Cowpea Group
      A. Type EL Inoculant Alyceclover, beggarweed, cowpeas, indigo, kudzu,
partridge pea, lespedeza, tick clover, Desmodium sp.
      B.  Type P Inoculant Peanuts
      C.  Type GU Inoculant Guar
VII. Lupine Group
      A. Type H Inoculant All Lupines and seradella
VIII. Other
      A. Type K Inoculant Birdsfoot trefoil
      B. Type M Inoculant Crownvetch
      C. Type F Inoculant Sainfoin

When purchasing inoculant be sure the legume species you want to plant is listed on the package and that the expiration date has not passed. The inoculant should be purchased when buying the legume seed several weeks in advance of the estimated planting date. This allows time for the retailer to order the seed and/or inoculant if not kept in stock. Ground peat moss is used as a carrier for the bacteria by inoculant companies. There are several brands of inoculant. The most effective ones are those which have a large number of rhizobia per gram of inoculant and contain a sticker that helps hold the inoculant to the seed such as HiStick and Pelinoc-Pelgel. Rhizobia bacteria are very susceptible to high temperatures. Be sure the inoculant is kept in a cool dry location away from direct sunlight. Most inoculant companies recommend their products be kept in a refrigerator until used except for HiStick, which can be kept at room temperature. It is desirable to drill the inoculated seed in the soil to help protect the bacteria from the sun and high temperatures.


Nodulation is a highly specific process that is extremely complex. The first step in the establishment of symbiotic N2-fixation is the attachment of host-specific Rhizobia bacteria to the root hair tips. Legume roots exude flavenoids (specific phenolic compounds) which attract and stimulate bacteria growth around the rhizosphere. The Rhizobia use flavenoids to stimulate the nod gene (symplasmid gene) to stimulate this process known as nodulation. Rhizobia bacteria secrete Indole acetic acid (IAA), which acts like an auxin stimulating cell division in the root causing root hair curling and deformation called a nodule.

Legume roots secrete sugar-binding proteins (lectins), which facilitate the binding of bacteria to the root hairs. An infection thread, which is an internal tubular extension of the plasma membrane grows from the infection site to other cells and fuses with the host cell plasma membrane by degrading the cell wall. This allows for the exchange and essentially allows the bacteria and root to act as one unit.

Poor nodulation may occur even if good seed inoculation practices were used. Rhizobia bacteria begin dying as soon as the inoculated seed are planted. The longer the seed lies in the soil before germination, the fewer viable rhizobia are present. If regular inoculant is just applied to the seed with water, buttermilk, or Coke as a sticker, the bacteria may only survive in the soil for about a week. Inoculant containing a sticker or that is coated on the seed provides more protection for the bacteria, which improves its survival to about 3 weeks. It is difficult to introduce a new legume species into a pasture that has had a native, naturalized, or different legume species growing on it for several years. The rhizobia strain infecting the previously grown legume species will have built up a large soil population over the years. Just because of greater numbers, the resident rhizobia strain may occupy most of the infection sites on the new seeded legume and prevent infection by the introduced rhizobia strain.

Nitrogen Fixation and Transfer

Nitrogen fixation is the conversion of atmospheric nitrogen to ammonium nitrogen in the presence energy (ATP) and the nitrogenase enzyme complex. The bacteria encode an enzyme complex called nitrogenase (Mo-Fe, Fe-S protein), which is made up of Molybdenum, Iron, and Sulfur. This enzyme complex is actually responsible for N2-fixation.

N2 + 8e- + 8H+ +16Mg-ATP Nitrogenase    2NH3 + 16 ADP + 16 Pi +H2

Rate of N2-fixation is directly related to legume plant growth rate. Anything that reduces plant growth such as drought, low temperature, limited plant nutrients, or disease will also reduce N2-fixation. Maintaining sufficient leaf area in a legume stand to intercept most of the sunlight is also critical to maintaining a high growth rate to support N2-fixation. When the legume plant matures and dies, nodules on the root system decompose and release the rhizobia into the soil. If the same legume species is planted again the following year or volunteers from seed produced the previous year, sufficient numbers of rhizobia are usually present to provide good nodulation.

The ammonium form of nitrogen is incorporated into organic acids in the root. These compounds (amides ­ temperate legumes and ureides in tropical legumes) are transported via the xylem to the plant. The primary pathways for nitrogen transfer from the legume to the soil are through grazing livestock and decomposition of dead legume plant material. The root system and unused leaves and stems of annual legumes die at plant maturity and are decomposed by soil microbes over time. Nitrogen contained in this plant material is released over time and is available to other plants. However, because this nitrogen is not available until after the legume dies only grasses that follow the legume growing season can use it.

When legume forage is consumed by grazing livestock most of the nitrogen in that forage passes through the animal and is excreted in the urine and feces. Unfortunately about 50% of the nitrogen in the urine is lost through volatilization. Another problem is the distribution of feces and urine on the pasture. With continuous grazing at low stocking rates, much of the animal excreta is concentrated around local areas. Animal excreta distribution is improved with rotational grazing systems where stock density is higher.

The quantity of nitrogen fixed by legumes can range from none to over 175 pounds per acre. Factors that influence the quantity of nitrogen fixed are the level of soil nitrogen, the rhizobia strain infecting the legume, amount of legume plant growth, how the legume is managed, and length of growing season. If given a choice, a legume plant will remove nitrogen from the soil before obtaining nitrogen from the air through N2-fixation. A legume growing on a sandy soil very low in nitrogen will get most of its nitrogen from the air while a legume growing on a fertile river bottom soil will get most of its nitrogen from the soil. General estimates of the amount of nitrogen fixed in the eastern half of Texas range from 50 to 100 lb N/acre for annuals and about 150 lb N/acre for alfalfa.

Table 2. Published Nitrogen fixation estimates for winter annual legumes.
Species Scientific name Annual Nitrogen Production Per Acre
Arrowleaf clover Trifolium vesiculosum Savi 113
Ball clover Trifolium nigrescens L. 84
Berseem clover Trifolium alexandrinum L. 243-357
Black medic Medicago lupulina L. 85
Burr medic Medicago polymorpha L. 131
Button medic Medicago orbicularis L. 111
Crimson clover Trifolium incarnaturm L. 138
Rose clover Trifolium hirtum All. 87
Red clover Trifolium pratense L. 100-128
Subterranean clover Trifolium subterraneum L. 143-175
White clover Trifolium repens L. 37-348
Alfalfa Medicago sativa 132
Austalian winter pea Pisum arvense L. 150
Hairy vetch Vicia villosa Roth. 80-89
Woolly-pod vetch Vicia dasycarpa Ten. 230