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Rhizobia Inoculation for Organic Farming Systems

 Author: Rebecca Fudge, PhD Candidate, Plant and Microbial Biology, University of Minnesota

Rhizobia inoculation can ensure that you are getting as much nitrogen as possible from your legumes. In this article, you will learn how inoculants work, how to apply them, and how to choose the right inoculant for your farm.

Nitrogen is critical for plants’ growth and development. Most plants take up nitrogen from the soil, but the legume family of plants can derive nitrogen directly from the air (and remember: air is almost 80% nitrogen gas!) Legumes can’t do this alone, however. They must associate with soil bacteria called rhizobia to engage in the process of biological nitrogen fixation. In this process, rhizobia form specialized organs on the legume’s roots called nodules, which are ideal environments for the conversion of atmospheric nitrogen into a nitrogen form the plant can use. This makes legumes valuable sources of protein (think of soybeans, lentils, chickpeas, and more) as well as valuable sources of soil nitrogen fertility. 
The process of biological nitrogen fixation in a legume-based cropping system. Image Credit: Rebecca Fudge, created in BioRender

Are my legumes fixing nitrogen?

To check whether your grain, forage, or cover crop has been successfully nodulated, you should check the roots for nodules. Nodules that are actively fixing nitrogen will be pink, because of a substance that rhizobia produce called leghemoglobin, which keeps the nodule a low-oxygen environment. Inactive, ineffective nodules will be white in color. They are a sign that the rhizobia in your field are not providing sufficient nitrogen for your plants’ growth and development.
An active, pink nodule on a hairy vetch plant. Note that nodules are not always spherical in shape. Image Credit: Rebecca Fudge

When should I inoculate my legumes?

Rhizobia are common soil bacteria and can persist in the soil for a long time. However, there are many different rhizobia species (as of the most recent classification, 238 species to be exact). There is a high degree of specificity in the legume-rhizobia relationship, so not all rhizobia can form nodules all legumes. For example, the Bradyrhizobium japonicum species that nodulates soybeans is not an appropriate partner for field pea, which forms associations with Rhizobium leguminosarum. It is important to choose the right inoculant for the legume species you are planting, or nodules will not form (See Table 1). Note that some inoculant companies now include multiple rhizobia species in an inoculant, so that the mixture is appropriate for more than one legume.

Rhizobia Species

Legume Partner(s)

Ensifer meliloti


Bradyrhizobium japonicum


R. trifolii

Red clover, crimson clover, white clover

R. leguminosarum

Pea, bean, and true vetch 

Mesorhizobium loti

Birdsfoot trefoil

Table 1. Common legumes for the Upper Midwest and their rhizobia partners.

If you previously inoculated and grew a legume in a field, that field likely contains rhizobia. Rhizobia can survive for years in soil when they are in their dormant state, and then they can be ready to form nodules once a legume is planted. It takes about 1 million rhizobia cells per seed for effective nodulation to occur, so the field must also have a sufficiently large rhizobia population. Unfortunately, there is no easy way to test whether rhizobia are in your field, other than planting a legume and checking to see if it forms nodules. Therefore, it is recommended to apply inoculant when growing a legume for the first time.

It is also important to keep in mind that the rhizobia that persist in the field are not always the most effective at performing biological nitrogen fixation. Rhizobia evolve quickly because genes can pass from one bacteria to another. Often, they evolve to better colonize plant roots (since the plant sends a lot of nutrients to them when they are in the nodules), but they don’t always have the best genes for fixing nitrogen. As a result, it is useful to keep inoculating with recommended rhizobia strains, because rhizobia that have persisted in your fields might not be the most effective.

Cold Effects on Biological Nitrogen Fixation

Unfortunately for those of us in the Upper Midwest, it is well-documented that cold temperatures have a negative effect on biological nitrogen fixation. Rhizobia rely on an enzyme called nitrogenase to fix nitrogen, which does not function as efficiently at low temperatures. These concerns mostly apply to winter annual cover crops, which are planted in the fall. As long as inoculation occurs within the legume’s optimal temperature range (for most cold-hardy cover crops, this is about 15°C/60°F), nodules will form and rhizobia will fix nitrogen before the onset of winter dormancy.

Some research suggests that inoculation with rhizobia actually helps plants survive better in the cold. A recent study on alfalfa showed that when plants were exposed to a cold snap (-6°C/21°F) for 8 hours, plants with active nodules (i.e. pink nodules that are fixing nitrogen) survived at a much higher rate than plants with no nodules or with inactive nodules (white nodules that aren’t fixing nitrogen) (Liu et al. 2019). This is possibly because rhizobia help plants store useful compounds that enable them survive the cold. Even though cold temperatures make it more challenging for rhizobia to do their job, it is very helpful to inoculate legumes that must face the cold!

How do I inoculate?

Inoculants can come in several forms, but the most common is a bacteria-infused peat. While it may just look like a dusty substance to the naked eye, there are indeed rhizobia in there! Because you’re dealing with living organisms, you s

hould treat your inoculant with care. The manufacturer will provide directions to store your inoculant successfully; usually a cool and dry place, such as a refrigerator, is optimal. Inoculants come with expiration dates, which should be adhered to – applying an expired inoculant will likely not result in the nodulation you want.

It is common on small-scale organic farms to mix the peat-based inoculant with seeds prior to planting. To do this, you will need to combine seeds with the inoculant in a large space (such as on a tarp or in a tub) to ensure each seed gets sufficiently coated in the inoculant. Follow the manufacturer’s instructions on how much inoculant to apply, especially if you have never inoculated crops in that field before.

Organic farmers should pay careful attention to whether their inoculant is certified by the Organic Materials Review Institute (OMRI). OMRI-certified inoculants cannot contain genetically-modified organisms, and OMRI also prohibits certain practices (such as using sewage sludge) in inoculant production. You can find all the OMRI-approved inoculants on their website.

Sources Cited:
Liu, Y. S., J. C. Geng, X. Y. Sha, Y. X. Zhao, T. M. Hu, and P. Z. Yang. 2019. Effect of rhizobium symbiosis on low-temperature tolerance and antioxidant response in alfalfa (Medicago sativa l.). Frontiers in Plant Science 10:1–13.

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