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Legume Inoculation and Nitrogen Fixation


by ABM Inoculants, printed with permission

By volume, about 80% of the air we breathe is nitrogen. Over ever acre of land there occurs approximately 35,000 tons of this free nitrogen. This nitrogen is in a free state and is useless to plants unless it can be combined with other elements. This combining or conversion can be made by certain bacteria living in the soil in association of leguminous plants. This source of nitrogen can be utilized for more successful stands, better soil improvements, improved crop quality and higher yields by inoculating legumes seed with the proper bacteria at the time of seeding.

There are approximately 12,000 species of legumes and approximately 50 species are grown commercially in the US. Legumes can be one of our most economical and efficient sources of protein for livestock. Legumes provide not only protein but phosphorous, calcium, and minerals. The protein content is directly related to the high nitrogen content.

Life in the soil is quite diverse ranging from microscopic single-celled organisms to large burrowing animals. Some examples are:

  • VERTIBRATES: (Gophers, Moles, and Reptiles)
  • ARTHROPODS: (Ants, Mites, Millipedes, Spiders, and woodlice)
  • MOLLUSKS: (Slugs, Snails)
  • WORMS: (Earthworms, Nematodes)
  • PROTAZOA: (Amoebae, Ciliates, and Flagellates)
  • ROOTS OF HIGHER PLANTS: (Trees, Shrubs and Crops)
  • ALGAE: (Blue, Green and Diatoms)
  • FUNGI: (Molds, Yeast)
  • ACTINOMYCETES: (Many kinds)
  • BACTERIA: (The simplest form of plants)

Bacteria are single-celled plants and exceed all other soil organisms in numbers and kind. There may well be over one ton of bacteria in an acre of soil. Most bacteria are rod shaped, and approximately one micron in diameter and up to two or three microns in length (1 micron = 1/25,000 of an inch). Soil bacteria can be divided into two large groups based on their energy requirements. Bacteria that get their energy from oxidation of inorganic compounds or elements, their carbon from carbon dioxide and their nitrogen from inorganic compounds are said to be autotrophic bacteria. Heterotrophic bacteria obtain their energy and carbon needs from complex organic substances. These heterotrophic bacteria are further divided into those requiring fixed nitrogen and those utilizing free nitrogen or the fixers. Nitrogen fixing bacteria are either non-symbiotic or symbiotic. It is the symbiotic nitrogen fixing bacteria that we want in the inoculation of legumes. Bacteria in this group are in the genus Rhizobium.

In the process of symbiotic nitrogen fixation, the Rhizobium bacteria in the soil make contact with the root hairs of the legume plant. This causes the root hairs to curl. An infection thread is formed on the root through which the bacteria migrate to the center of the root. Inside the root the bacteria multiply and form bodies called bacteroids. The enlargement that occurs in the root as a result of this growth is the nodule we see on the roots of legume. The bacteroid receives the nitrogen from the bacteria that has been fixed from the free nitrogen in the air. The host plant can also benefit from increased water uptake and nutrient absorption due to the surface area of the root caused by the nodule. Associated plants are benefited by the nitrogen that is excreted from the nodule and when the nodules disintegrate and decompose. The nodules disintegrate rapidly at the time of seed formation.

The absence of nodules on the roots would indicate that the plant derives all of its nitrogen from the soil. Nodules on the root are a sign that the plant may be benefiting from fixed nitrogen.

Nodules that are pink or red inside are active in producing nitrogen for the host plant. Nodules that are white, green, or brown is a sign they are producing little or no nitrogen. Not all Rhizobium are beneficial in the fixation process. Some forms of Rhizobium infect the root and form small white nodules but do not fix any nitrogen. It has been estimated that approximately 25% of the native Rhizobium present are beneficial nitrogen-fixing bacteria. This leaves 75% present questionable. This gives another good reason to use fresh inoculant on the seed at planting time.

We must remember that bacteria are microscopic one-celled plants. They have the same requirements for temperature, moisture, nutrients, etc., as do higher plant forms. It is for this reason native populations of bacteria can be low. Extreme cold temperatures in winter, high summer temperatures, low organic matter content, low rainfall, and the drying out of soil after a crop is harvested and irrigation ceases are not conductive to a favorable microbial population. Therefore to insure that adequate opportunity for inoculation to occur, all legumes should be inoculated at time of seeding.

Cross-Inoculation Groups

Legume bacteria are highly selective and not all bacteria will function on all legumes. It is important to obtain a culture of the kind of bacteria for the legume you intend to plant. These different kinds of bacteria are placed into groups called cross-inoculation groups. There are eight main cross-inoculation groups. These groups and some of the more important legumes in each group are listed as follows:

Alfalfa Group
Common Name Scientific Name
Alfalfa Medicago Name
White sweet clover Melilotus alba
Spotted Bur-clover M.arabica
Hubam sweet clover M. alba annua
Black Medic M. lupulina
Yellow sweet clover M. Officinalis
Yellow alfalfa M. falcate


Clover Group
Common Name Scientific Name
Alsike clover Trifilium hybridum
Ladino clover T. repens(gigantum)
Hop clover T. agrarium
Sibthrop Suckling clover T. dubium
Sub clover T. subterraneum
Schreb Field Clover T. procumbens
Strawberry clover T. fragiferum
Red Clover T. pretense
Berseem clover T. alexandrinum
White Clover T. repens
Zigzag clover T. medium


Pea and Vetch Group
Common Name Scientific Name
Field Peas Pisum arvense
Narrowleaf vetch V. angustifolia
Garden pea P. satvum
Purple vetch V. atropurpurea
Austrian winter pea P. sativum(var.arvense)
Sweet pea Lathyrus odoratus
Common vetch Vicia sativa
Rough pea L. hirsustus
Hairy vetch V. villosa


Cowpea Group
Common Name Scientific Name
Cowpea Vigna sinensis
Lima bean Phaseolus lunatus
Common lespedeza Lespedza striata
Korean lespedeza L. stipulacea
Wild-indigo Baptisia tinctoria
Alyeeclover Alysicarpus vaginalis
Hairy indigo Indigofera hirsute


Bean Group
Common Name Scientific Name
Garden beans, kidney Phaseolus vulgarius
Scarlet runner bean P. coccineis
bean, navy bean,Pinto bean (multiflorus)


Lupine Group
Common Name Scientific Name
Blue lupine Lupinus Angustifolius
White lupine L. albus
Washington lupine L. polyphyllus
Yellow lupine L.luteus


Soybean Group
Common Name Scientific Name
All varieties of soybeans Glycine max (Soja max)


Specific Strain Group
Common Name Scientific Name
Birdsfoot trefoil Lotus corniculatus
Siberian pea-shrub Caragana aborescens
Big trefoil L. uliginosus
Sainfoin Onobrychis viciaefolia
Leadplant Amorpha canescens
Crown vetch Coronilla varia
Cicer milkvetch Astralagus cicer

When working with inoculants, some important items to remember are:

  1. Use a viable inoculant for the species of legume you are going to seed.
  2. Mix only the amount if inoculant and seed that you plan to seed at that time.
  3. Protect inoculated seed from high temperature and sunlight.
  4. Plant seed as soon as possible after inoculation.

It has become popular to buy seed that has already been inoculated by the seed dealer. If such seed is purchased, remember the basic rules in handling the inoculant. Don't expose the pre-inoculated seed to extremes in temperature. Store seed in a cool, dry place. If you have any doubts as to the viability of the inoculant, or the seed is not planted as scheduled – RE-INOCULATE.

Do not become confused with chemical seed treatments and inoculation of legume seed. Chemical seed treatments are applied to control fungi, insects, and injurious soil organisms. Most seed disinfectants are toxic to legume bacteria. Read the label of any chemical seed treatment product before use.

Complete fertilizers should not be allowed to come in direct contact with inoculated legume seeds. Phosphates are not as harmful as nitrogen or potassium fertilizers. Normally if the concentration of fertilizers does not injure germination, it will not ordinarily harm legume bacteria.

Bacteria may be aerobic or anaerobic

  • Aerobic – Requires gaseous oxygen
  • Anaerobic – Do not require gaseous oxygen

Bacteria may be autotrophic or heterotrophic

  • Autotrophic – Utilize carbon dioxide or carbonates as sources of carbon and oxidize inorganic elements or compounds as energy source.
  • Heterotrophic – Utilize organic materials to obtain energy and are incapable of utilizing inorganic compounds.

Bacteria may be non-symbiotic or symbiotic

  • Non-symbiotic – Free living
  • Symbiotic – Living in association with another organism for mutual benefit.

The bacteria associated with nitrogen fixation in legumes are aerobic, heterotrophic, symbiotic organisms.

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