Becker Underwood

February 2003

What is inoculation?

How does nitrogen fixation work?

Why and when is inoculation necessary?

What are the benefits of inoculation?

How does the nitrogen cycle work?

Do legumes always nodulate?

What is the best way to evaluate nodulation?

What are the yield benefits of inoculating?

Are inoculants and chemical seed treatments compatible?

What makes one inoculant different from another?

Are all inoculant products the same strength or potency?

Does application method affect performance?

Other related nodulation data

Can inoculants be used in organic production?

Are inoculants genetically enhanced?

Rhizobia inoculant specificity information

Do inoculants require special storage or handling?

What is inoculation? [Back to Top]

The process of inoculation is the application of commercially available rhizobia bacteria to legume seed or into soil where a legume will be planted. Rhizobia are the active ingredient in all legume inoculant products. The presence of rhizobia is necessary for a legume to be able to convert atmospheric nitrogen into a usable form in the absence of readily available nitrogen from fertilizers or manure. This process is referred to as nitrogen fixation.

How does nitrogen fixation work? [Back to Top]

Nitrogen fixation is the utilization of the free gaseous nitrogen in the air by soil residing bacteria in cooperation with legume plants to produce usable nitrogen products, which plants and other organisms can use to grow and to produce protein, fiber and seed. For clarity, the term “usable” nitrogen can be substituted for “fixed” nitrogen. Plants use this fixed nitrogen in the same manner they would use commercial fertilizers or nitrogen in manure. Legume growers see fixation in their soybeans, peanuts, clovers, alfalfas, dry beans and other legume field crops, but nitrogen fixation is a universal phenomenon. Wherever nitrogen fixation occurs, it is mediated by the enzyme nitrogenase. Rhizobia symbiosis via the nitrogenase enzyme converts gaseous nitrogen to ammonia, which is then assimilated into amino acids and subsequently these acids are transported throughout the plant. Most of this goes to the above ground parts of the plants where the plant uses it to make protein, nucleic acids and other necessary nitrogen containing compounds needed for plant growth and seed production. These processes are no different than when the plant assimilates ammonia fertilizer. An added advantage particular to legumes is the amount of fixed nitrogen, which is returned to the soil at the end of the crop year. For example, soybeans leave from 55 to 120 pounds per acre of usable nitrogen for next year’s crop, while alfalfa is estimated to leave from 120 to 300 pounds per acre of usable nitrogen after plow-down.

• Soil that has not hosted the specific legume.
• Soil that has not hosted the specific legume for more than 4 years. (CRP land, for example.)
• Soil pH of less than 5.8. (The pH should be adjusted by liming prior to inoculation.)
• Soil pH of more than 8.5.
• Soil organic matter of less than 1%.
• Drought or flooding.
• Topsoil conditions exceeding 80° F.
• Soil erosion.
• Use of soil treatments and chemicals.

As technology and selection of rhizobia strains improve, new, more efficient strains of rhizobia should be used to replace native strains , which may be residing in the soil. It has been suggested that rhizobia become ‘lazy’ or less effective as they reside in the soil for extended periods. Thus, there is the potential benefit of adding high levels of newly selected strains of rhizobia each time a legume is planted by using commercial inoculant products. It is important to note that the presence of a nodule does not assure nitrogen fixation. Native rhizobia become ineffective over time but will still nodulate the legume. You can evaluate nodules by examining the color of the nodule at different times during the growing season.

What are the benefits of inoculation? [Back to Top]

Seventy-nine percent of the air we breathe is made up of nitrogen and inoculated legumes are able to convert and use this “free” nitrogen. Properly nodulated legumes can leave 55 to 300 pounds of nitrogen per acre in the soil. The exact amount depends on effectiveness of the nitrogen fixation process, type of legume, length of time the legume is grown, soil nutrient levels and nitrogen already available. The nitrogen provided by inoculated legumes grown in crop rotation helps boost yield and lower fertilizer costs for corn or small grains – in an environmentally safe manner.

Fertilizer savings:

Due to the nitrogen-fixing ability of legumes inoculated with rhizobia, the need for commercial nitrogen fertilizer is virtually eliminated. Seventy-nine percent of the air we breathe is made up of nitrogen and inoculated legumes are able to convert and use this ‘free’ nitrogen.

Higher yields:

All legumes average more pounds per acre when properly nodulated, which increases total crop production and income. For example, studies of soybeans on ground thought not to need inoculant have shown an average increase of 2+ bushels per acre.

Effectiveness of rhizobia strains:

Rhizobia can become lazy over time and fix less nitrogen even though they remain good nodulators. Continuous use of fresh rhizobia will maximize yield benefits, as these rhizobia will out-compete the indigenous rhizobia for root nodulation.

Added nitrogen to the soil:

Properly nodulated legumes add 55 to 300 pounds of nitrogen per acre to soil. The exact amount depends on effectiveness of the nitrogen fixation process, type of legume, length of time grown, soil nutrient levels, and the nitrogen already available.

Benefits rotated crops:

Nitrogen provided by inoculated legumes grown in crop rotation helps boost yield and lower fertilizer costs for corn and/or small grains.

Economical and safe:

Inoculation is a low cost way to ensure nodulation with the proper strain of nitrogen-fixing rhizobia bacteria for increased yield in an environmentally safe manner.

Improved soil conditions:

Legume plants decompose rapidly, leaving organic matter in the soil, which improves its physical, chemical and biological condition.

Up-to-date strains:

Rhizobia strains used in inoculation products today are the most effective available.

How does the nitrogen cycle work? [Back to Top]

The schematic chart on the following page gives a visual representation of how nitrogen moves through the soil, and through the plants and animals dependent on the soil. This is an idealized system, for in actual farm situations, there are a number of additions (fertilizer for the plants and purchased feed and supplements for the animals) and there are a number of subtractions and/or ‘leaks.’ The primary subtraction is the harvest and removal of the grain crop or animal which removes nitrogen from the farm. Soil erosion provides the major ‘leak’ of nitrogen plus all of the other nutrients. The major way of replacing the nitrogen is with purchased fertilizer or the nitrogen fixation supplied by legumes in normal crop rotations. Legumes, with their ability to use the air as their source of nitrogen, are a major source of nitrogen input to a farm’s nitrogen cycle. Legumes both contribute more to the organic matter pool than do nonlegumes during decomposition, and don’t use as much nitrogen from this pool while growing. Consequently, there is more nitrogen left for succeeding non-legume crops. It is important to note that an abundance of nitrogen fertilizer available to the legume seedling will affect nodulation onset. The legume will delay nodulation until it senses a lack of readily available nitrogen. Only then will it chemically signal the rhizobia to infect/invade the root hairs of the seedling.

Do legumes always nodulate? [Back to Top]

Large amounts of excess nitrogen in the soil, either from an applied fertilizer, manure, or residual left from previous years, can inhibit nodule formation on legume plants even though there are plenty of the appropriate rhizobia available in the soil. The plants will look green and healthy, at least through the first part of the growing season, until the soil nitrogen is used. The reason is that if a developing legume plant has the choice between readily available soil nitrogen and inoculant rhizobia bacteria, the plant chooses the soil nitrogen. In this regard, we suggest that not more than 40 pounds of available nitrogen be applied per acre when starter fertilizers are used with legumes.

What is the best way to evaluate nodulation? [Back to Top]

The late vegetative stage is a good time to dig up a few plants to look at the root structure and to evaluate nodulation. You can also check for early nodulation at about 3 weeks after emergence from the soil. Not all legumes (especially under dry or cool growing conditions) will show nodules at 3 weeks, but it is a reasonable time to start to examine the legume roots. Plan to use a garden spade rather than just pulling the plant out of the ground. Soaking the plants in a bucket of water will help remove excess soil clods without removing nodules. On edible beans, soybeans, peanuts, alfalfas, and other legumes, which have been planted in the spring, there should be several nodules located on or around the taproot. For the alfalfa and clovers, which have been growing for a year or two, the nodules will appear on the lateral roots and be long and slender in shape. When evaluating nodules, keep in mind you are looking for overall nodule mass not just quantity. For example, ten small nodules with a mass of 50 grams will be as effective as four large nodules with a mass of 50 grams if all of them are pink or red in color indicating active nitrogen fixation. Slice open a few nodules and check the color. Nodules actively fixing free atmospheric nitrogen to usable ammonia will range in color from pink to bright red. If the nodules are white, they are ineffective, or may not be developed yet to a stage at which they can fix nitrogen. To check if white nodules are immature or, in fact, ineffective examine the plant roots again a week later. This will usually give enough time for young nodules to mature into pink or red colored nitrogen-fixing nodules. If nodules are green (and usually soft), they are past their prime and have already contributed to the plant’s nitrogen economy. It has been observed that some of the newer strains of rhizobia have the ability to keep nodules pink or red (active) for longer periods of time. For early seeded peas, the nodules may be turning green in color during the summer months. This is something that will be observed on soybeans in August and September after the plants are well into the pod filling stage of growth.

What are the yield benefits of inoculating? [Back to Top]

The presence or extent of the yield increase will depend on many factors including soil type, legume type, and climatic and soil conditions. It is important to remember that nitrogen is only one factor in determining yield and does not substitute for other essential nutrients, disease problems, etc. For soybeans there can be greater than ten bushel per acre yield increases on ground, which has not grown soybeans before. On soils that have grown soybeans before and on which new improved rhizobia inoculant strains are used, expected yield increases are in the 2-bushel per acre range.

Are inoculants and chemical seed treatments compatible? [Back to Top]

Both seed treatments and rhizobium inoculants often are placed together on seed prior to planting. What affect does this have on the inoculant? If the pesticide, herbicide, or fungicide is too toxic, the rhizobia may be killed before they have a chance to form nodules. The compatibility of inoculants with seed treatments and with starter fertilizers has become a major question since the introduction and consequent popularity of liquid inoculants. The following is a set of general rules concerning this question.

1. Insecticides are more toxic than fungicides, which are more toxic than herbicides.

2. One of the reasons in-furrow inoculants were developed was to keep the inoculant away from chemically treated seed. Thus, granulated peat or liquid based in-furrow inoculant applications are always the first recommendation when pesticide seed treatments are used. This type of application keeps the inoculant bacteria out of any prolonged direct contact with the pesticide.

3. When an inoculant and a seed treatment are combined on the seed, keep exposure times as short as possible. The general recommendation is to plant the seed as soon as possible after the seed is treated and inoculated regardless of the maximum allowable time before re-inoculation is required. Check COMPATIBILITY section for details.

4. Liquid interfaces speed up any detrimental activity. Therefore, if the chemical is a liquid, it should be applied first and allowed to dry before the inoculant is applied.

5. Peat-based inoculants are more protective of the rhizobia than liquid based inoculants.

6. For seed pre-treated with a pesticide, the recommendations in order of priority are:

· A liquid or granular peat inoculant applied in-furrow (Rhizo-Flo™, NOD+™ or Frozen-Prep XP™).

· A high adhesion formulation peat (Soy-Sterile or Rhizo-Stick®).

· A liquid inoculant with the limitation mentioned in the COMPATIBILITY section.

Look for specific guidelines in the COMPATIBILITY section of this training manual or, if in doubt, call Becker Underwood at 800-232-5907 with questions concerning product compatibility. Below is a priority list for use of Becker Underwood’s Urbana inoculant formulations. This is especially important when inoculants are used under stress conditions. Mixing an inoculant with a chemical pesticide or applying to chemically treated seed are considered stress conditions for the rhizobia bacteria in the inoculant.

First recommendation:

· An in-furrow inoculant

Rhizo-Flo™ granular

NOD+™ or Frozen-Prep XP™ liquid inoculants

Second recommendation:

· Regular rate of a high adhesive inoculant Soy-Sterile Rhizo-Stick®

· NOD+™ or Frozen-Prep XP™ used as an adhesive for a humus-based seed applied inoculant (Soy- Sterile, Rhizo-Stick® or Traditional Humus)

Third recommendation:

· regular rates of liquid or humus-based inoculants in the planter box

What makes one inoculant different from another? [Back to Top]

Inoculant carrier types:

The rhizobia bacteria are usually produced, stored, and delivered in three different carriers:

A. Water

B. Peat

C. Clay

A. Water-based carriers are divided into two types:

· Frozen concentrates (Frozen-Prep XP™). Frozen-Prep XP™ is comparable to an orange juice concentrate in that it is thawed, diluted, and then used.

· Ready-to-use liquids (NOD+™).

B. Peat-based carriers are formulated in four forms:

· Rhizo-Flo™ is a granulated carrier for in-furrow application.

· Soy-Sterile is a sterilized carrier with high adhesion for seed application.

· Rhizo-Stick® is a non-sterilized carrier with high adhesion for seed application.

· Traditional peat inoculant is a non-sterilized carrier, with low adhesion for seed application unless applied as a slurry. Slurry application allows for 95%+ adhesion to the seed.

C. Clay-based carriers:

Clay-based product (Dormal® and Dormal Plus™) are primarily used for alfalfa and is designed to be a long-term pre-inoculant for alfalfa applied by the seed processor. These various forms of inoculants have been developed to adapt to the grower’s application equipment and specific agronomic conditions. Thus, the choice of inoculant carrier type is most often a question of what is compatible to the grower’s situation. However, the farmer sometimes has a decision to make with regard to the best carrier for his farm. To help make that choice, we have listed the advantages and disadvantages of each carrier type on the following page.

The goal of any rhizobia inoculant is to provide the proper specific strains of rhizobia in large numbers. An important secondary goal is to produce an inoculant, which coats the seed well or can be introduced into the soil in a precise manner via methods that are convenient and efficient for the farmer. Thus, inoculants, in a manner of speaking, become like different types of equipment or different formulations of fertilizer. Certain types of equipment work best for certain crops. Certain fertilizers (bulk, anhydrous ammonia, liquid slurry, or starter) work best according to the crop and management scheme. Similarly different inoculant carriers work best with different types of seed, soil, or planting equipment.

Are all inoculant products the same strength or potency? [Back to Top]

Throughout this training manual you will see reference to the potency or strength of inoculant products. Typically this refers to the amount of bacteria delivered by each product onto each seed or into the soil around each seed. In theory, the more bacteria delivered to the root zone at the time of application or planting, the more rhizobia available when the plant needs it to begin forming nodules. The practicality of this theory will, of course, vary by product, environmental conditions, delivery method and a host of other variables. Rhizobia per seed, or per area, are calculated based on five facts about each product and the seed it is going to be put upon.

· Weight or volume of the package

· Rhizobia per gram or milliliter of inoculant

· Application rate of the inoculant to the seed or the soil

· Adhesion level of the product to the seed, if applicable (usually dry seed applied products)

· Seeds per pound of the seed being planted

· Seeding rate per acre

For example, Rhizo-Stick® Package size in grams 2270 pounds x 454 grams Rhizobia per gram (2 x 108) 200,000,000 based on guarantee printed on the packaging Total rhizobia in package (4.54 x 1011) 454,000,000,000 Total amount of seed treated per package (pounds) 1,500 as listed on the packaging Seeds per pound 3,000 as listed on seed tag Total seed treated per package of inoculant 4,500,000 Divide total rhizobia in package by total seeds treated by package Based on seeds per pound 100,889 bacteria delivered per seed if all product adheres to the seed Adhesion level (usually quoted with peat products) 68% Bacteria delivered per seed based on actual adhesion 68,604 rhizobia per seed When comparing inoculant products from various manufacturers or distributors, make sure any calculations of rhizobia per seed are based on these facts. One way to manipulate the information is to use a different seed per pound basis, not to compare actual application rate or adhesion levels, etc. For example, if two products treat the same amount of seed and have the same rhizobia per gram guarantee, one might assume they are of the same strength in what they deliver for rhizobia per seed. However, if one product’s application rate is higher than another or the adhesion level of the two products is different, the rhizobia per seed will vary. Also, many companies use different seeds per pound to calculate this number. This will greatly affect the rhizobia per seed. For soybeans, Becker Underwood uses 3,000 seeds per pound as the general number.

Does application method affect performance? [Back to Top]

Becker Underwood recommends in-furrow application of inoculation products rather than a seed-applied application of a high quality inoculant in conditions of stress or first year legume production. Under the best of production circumstances, all inoculant products may perform similarly. However, in conditions of stress or in cases of first time legume production (former CRP land brought into legume production would be considered first year legume production), in-furrow application of a dry or a liquid inoculant is the best recommendation. The bacteria are applied directly into the root zone and are immediately protected by the soil when the furrow is closed. On the following page are results of plots from the University of Guelph, Guelph, Ontario Canada completed in 1999 on first year soybean ground. These plots were designed to evaluate nodulation counts only, but are a good example of why Becker Underwood has always recommended in-furrow applications in first year ground situations above other methods.

Soy-Sterile was referred to as “Sterile USDA” at the time of the study.

*Formerly Mega-Prep

In-furrow application may also be best if a chemical seed treatment is applied to the seed. Please see the COMPATIBILITY section of the training manual for more details.

Other related nodulation data . . . [Back to Top]

Virginia research shows that phosphorus and potassium affect nodulation and hence nitrogen fixation. Note how phosphorus and potassium increased nodule number, percent nitrogen in nodule and seed protein production.

Can inoculants be used in organic production? [Back to Top]

Currently Nod+ for Soybeans, as well as some our Dormal products are certified by OMRI. Other Becker Underwood products are currently being reviewed for possible OMRI submission and certification.

Are inoculants genetically enhanced? [Back to Top]

Becker Underwood’s Urbana product line was granted the right to produce and sell an enhanced strain of Rhizobia for alfalfa in 1997. The identity is listed as Strain PC2 Sinorhizobium meliloti. Two things were engineered into the PC2 strain; (1) An extra copy of the regulatory gene ‘nifA’ was added. This allows the bacterium to fix more nitrogen in conjunction with the host alfalfa plant; (2) A ‘dct’(dicarboxylic acid transport) gene was altered in a way that makes the bacterium more efficient in the use of the energy furnished by the alfalfa plant. The PC2 inoculant (in Becker Underwood’s exclusive Urbana Dormal PLUS™ carrier) is applied to the alfalfa seed prior to planting. It is not sprayed or applied in any fashion on the growing crop. Studies have shown that the bacteria do not migrate to the foliage part of the alfalfa. The EPA verified that the strain was safe after evaluating 8 years of laboratory and field studies. Sinorhizobium meliloti is a natural soil organism that has been known and studied for over 100 years. It is one of the species of the rhizobia genera, which contribute to the natural nitrogen fixed by legumes. A process, which makes legumes the mainstay of beneficial crop rotations. During these many years of study and commercial use, rhizobia have never been shown to cause problems for plants, animals, or people. No harmful or pathogenic components were added to the PC2 during the genetic alteration. Becker Underwood will use these new techniques with other rhizobia strains for other legumes as the technology proves itself. We will fully evaluate all strains in conjunction with universities and the EPA before releasing the strains, and will label all strains that are genetically engineered.

Credits:

University of Guelph, Guelp Ontario Canada

Footnotes:

1 Strains are to rhizobia what varieties are to field crops

2 Assumes a depth of 6.75 inches

3 Source: Albrecht, W.A. 1933. Inoculation Of Legumes As Related To Soil Acidity, Journal Of American

Society of Agronomy 25

Rhizobia inoculant specificity information – Inoculants are not interchangeable. [Back to Top]

There are many different species of rhizobia – Sinorhizobium sp., Rhizobium sp., and Bradyrhizobium sp. It is important to know that rhizobia are legume specific. For example, soybean inoculant (Bradyrhizobium japonicum) does not treat dry beans, which requires a different species of rhizobia (Rhizobium leguminosarum – biovar phaseoli). Click here for detailed listings of which legumes are treated by which rhizobia strain inoculant.

Do inoculants require special storage or handling? [Back to Top]

Inoculants do require more care than the average chemical or seed treatment product because they contain live bacteria susceptible to environmental conditions during storage. Generally, the following recommendations apply:

NOD+™, Rhizo-Flo™, Soy-Sterile, Rhizo-Stick®, Traditional Humus, Dormal® and Dormal Plus™ (PC2)

• Keep inoculant as cool as possible, 40-77°F.
• Do not expose to heat or direct sunlight.
• Do not store outside or unprotected from elements.
• Do not allow inoculant to freeze and thaw repeatedly.
• Do not allow product to get wet or expose product to extreme humidity.
• Do not leave in warehouse if fumigants are in use.
• Do not stack product more than two pallets high.
• Do not use if package seal is broken.
• Do not use after labeled expiration date.
• Destroy unused product after labeled expiration date.
• Product is appropriate for use only on legume seed listed on product label.

Frozen-Prep XP™

• Do not touch dry ice in shipping containers with bare hands.
• Remove Frozen-Prep XP™ from Styrofoam shipping container upon receipt and immediately place into a freezer.
• Keep frozen until use.
• Further transport of Frozen-Prep XP™ must be by a means sufficient to keep the product completely frozen until use by the producer.
• Do not use after labeled expiration date.
• Destroy unused product after labeled expiration date.
• Product is appropriate for use only on legume seed listed on product label.