Inoculation of Pulse Crops

Pulse Crops

Pulse crops are annual grain legumes that produce large seeds traditionally used for human food or livestock feed. The pulse crops grown in Saskatchewan include: pea, lentil, chickpea, faba bean, dry bean, soybean, chickling vetch (grass pea) and lupin.

Pulse crops have the ability to obtain much of their nitrogen requirement from the air within the soil surrounding their roots. This is done when the plant forms a mutually beneficial relationship with soil bacteria called Rhizobium.

Nitrogen Fixation

Nitrogen (N) fixation is the process whereby legume crops and specific Rhizobium bacteria (rhizobia) work together to make nitrogen from the soil air surrounding the roots available for use by the plant. Soon after the crop germinates, rhizobia enter the root hairs. Once inside, the bacteria penetrate further into the root through an infection thread. The rhizobia rapidly multiply within the root and the plant responds by forming specialized structures called nodules, in which the rhizobia are contained. The process of root infection and nodule formation is referred to as "nodulation." It may take three to four weeks after seed germination before nodulation is evident on the plant roots.

Air held in small pores in the soil contains approximately 80 per cent nitrogen in a gaseous form (N2). In this form, the nitrogen is not available for plant use. However, the Rhizobium bacteria in the root nodules can "fix" this form of nitrogen gas by binding it with hydrogen and forming ammonium (NH4), which is available for use by plants. The nitrogen fixation process requires a considerable amount of energy which is provided by the plant. The plant also provides nutrients and water to the rhizobia in the nodules and, in return, the rhizobia provide fixed nitrogen to the plant.

The amount of nitrogen fixed varies with the type of crop, crop health, the supply of nitrogen already available in the soil, and other environmental conditions. Under ideal conditions, pulse crops can fix as much as 50-80 per cent of their total nitrogen requirement, with the remaining nitrogen coming from soil or fertilizer sources. Pulse crops can be ranked according to their estimated ability to fix nitrogen: faba bean > pea > chickling vetch > chickpea > lentil > soybean > lupin> dry bean.

Inoculation

Products containing Rhizobium bacteria are called nitrogen inoculants. Inoculation is the process of introducing the appropriate Rhizobium bacteria to the soil in numbers sufficient to ensure successful nodulation. This is done by coating the seed with a liquid or peat-based powder inoculant, or by treating the soil with a granular or liquid inoculant. Commercial inoculants, when properly applied, ensure that every plant is exposed to a sufficient number of bacteria to initiate nodulation and start the nitrogen fixation process. Rhizobium bacteria are not very mobile in the soil, and thus, the inoculant must come in contact with the developing seedling for infection of root hairs to occur. Specific pulse crops require specific Rhizobium species for nodulation. For example, a Rhizobium species capable of nodulation in lentil and pea crops is not capable of nodulation in chickpea (Table 1). If the wrong Rhizobium species is used, inoculation will have no beneficial effect. Soils commonly lack sufficient numbers of the correct Rhizobium bacteria to optimize the nitrogen fixation process.

Table 1. Rhizobium species required for legume crops

Scientists have selected efficient strains (see Inoculant Strain) of Rhizobium for each pulse crop. For example, a Rhizobium strain may be able to produce nodules in both lentil and pea, but may be much more effective in pea. Soils with a history of pulses may contain residual bacteria. Residual Rhizobium bacteria can produce nitrogen-fixing nodules. However, bacteria that survive in the soil for a number of years may not be present in sufficient numbers, may be inefficient fixers of nitrogen, or may be slow to colonize the roots. For these reasons, most experienced pulse crop growers inoculate each time they plant a pulse crop.

Fertilizer and Inoculation

A soil test is the best tool to provide guidelines for crop fertility needs. Pulse crop seedlings use nitrogen from the top 15-30 cm (6-12 in.) of the soil until nitrogen fixation starts. Nitrogen below this depth will not be available to small seedlings. Nitrogen fixation may take three to four weeks to become well established.

Nitrogen is necessary for high pulse crop yields, but generally, nitrogen fertilizer application is not required. When properly inoculated with the appropriate Rhizobium inoculant, pulse crops can derive a significant portion of their nitrogen requirement through fixation. The remaining required nitrogen comes from what is available in the soil at seeding (i.e. soil test levels of nitrogen) and nitrogen that is released from the soil and plant residue decomposition during the growing season. Although nitrogen fixation generally is sufficient to meet crop needs, some growers provide supplemental nitrogen to their pulse crops prior to the onset of nitrogen fixation by applying low rates of starter nitrogen. In soils with available nitrogen levels less 11 kgN/ha (10 lb.N/acre), early plant growth may be slow and seedlings may appear yellow due to nitrogen deficiency. This early nitrogen deficiency can be corrected by providing low levels of starter nitrogen at seeding. This can alleviate early nitrogen deficiency and speed vegetative growth but does not necessarily translate into improved yield.

Nodule formation and fixation are very sensitive to external nitrogen sources including fertilizers and available soil nitrogen. When the supply of nitrogen available from the soil and fertilizer increases, the amount of nitrogen fixed by the plant decreases. Low levels of available nitrogen may have little impact on nodulation and fixation; however, when the combined levels of available soil and fertilizer nitrogen reach approximately 40 kgN/ha (35 lb.N/acre), any additional nitrogen will reduce nodulation and fixation. Combined levels of soil and fertilizer nitrogen greater than 55 kgN/ha (50 lb.N/acre) can dramatically delay nodulation and reduce or eliminate fixation.

Research supported by the Canada-Saskatchewan Agri-Food Innovation Fund was carried out over three years in the Black soil zone at Indian Head and Melfort to test the effectiveness of using starter nitrogen in dry pea. The results showed that starter nitrogen rates above 40 kgN/ha (35 lb.N/acre) caused plant numbers to be reduced due to the toxic effect of the fertilizer on pea seedlings. Total crop dry matter weight increased with increasing fertilizer rates up to 40 kg/ha (35 lb./acre) actual nitrogen. However, starter nitrogen did not increase grain yield except when no inoculant was used.

Pulse crops grown in the Brown and Dark Brown soil zones and in fields with no previous history of the crop could benefit by the addition of low rates of starter nitrogen, as these soils do not have high levels of organic matter or residual Rhizobium bacteria to colonize secondary roots and overcome initial deficiencies.

Starter nitrogen should not be placed with the seed to avoid seedling injury. Starter fertilizer such as monoammonium phosphate (example: 12-51-0) provides small amounts of nitrogen, and, depending on the soil test, may provide all the required starter nitrogen.

Pulse crops must have phosphorus for plant development, nodule formation, and nitrogen fixation. A soil test will provide a guideline for phosphorus requirements. Pulses are sensitive to seed-placed phosphate (P₂O₅) fertilizer. Sensitivity is greater for pea, followed by chickpea, lentil, dry bean, and faba bean. If possible, fertilizer should be side-banded for pea, chickpea, lentil and dry bean to avoid reduction of plant stands. The use of a wider spread in the seed row will also reduce seedling injury. Some pulse crop growers apply extra P₂O₅ to the previous year's cereal crop to increase available phosphorus for the pulse crop and avoid the risk of seedling injury. A phosphorus solubilizing inoculant like JumpStart® can be used to increase the availability of soil phosphate to crops. Where narrow openers are used and the risk of seedling injury is greater from seed-placed P₂O₅ fertilizer, a phosphorus solubilizing inoculant can be used to provide some "starter" phosphate.

Dry bean - fertilizer and inoculation

Generally, dry beans are poor fixers of nitrogen in comparison to pea, lentil, faba bean, and chickpea. Research completed on inoculated dry bean by the University of Saskatchewan and Agriculture and Agri-Food Canada (AAFC), Morden Research Station, indicated that there is a positive yield response to the application of starter nitrogen at rates of 25-75 kgN/ha (22-67 lb.N/acre). The research also indicated differences between bean varieties with respect to their ability to fix nitrogen. For these reasons, the current recommendation for non-irrigated dry bean production in Saskatchewan is to inoculate the crop (Figure 1) and use 55 kgN/ha (50 lb.N/acre) starter nitrogen, broadcast or side-banded.

Checking Nodulation

Nodule formation may begin approximately two weeks after crop emergence, but will likely take three to four weeks under most growing conditions. The number of nodules and rate of fixation will increase as the plant grows, and normally reaches a maximum at the mid-flowering stage to correspond to the time when the plant needs the most nitrogen. After flowering, the number of nodules and the amount of fixation declines as the plant progresses to maturity.

Successful nodulation and fixation can be determined by carefully digging up (not pulling) plants from several areas of the field. The nodules are delicately attached to the roots and can be easily brushed off. Gently brush or wash the soil from the roots and look for clumps of nodules on the roots (Figure 2). If the inoculant was seed-applied, most nodules will form on the primary root near the crown. Nodules forming on secondary roots away from the primary root are most likely the result of residual Rhizobium bacteria in the soil. If a soil applied granular or liquid inoculant was used, nodules form on primary and secondary roots throughout the treated layer of soil.

Carefully slice open the nodules. The interior of nodules that are actively fixing nitrogen are pink to beef-red in colour (Figure 3). This colour is due to the presence of a substance called leghemoglobin, an iron-containing pigment that must be present for active fixation. If the inside of the nodule is brown, white or green, the nodule is not effective.

Poor fixation can lead to symptoms of nitrogen deficiency, including the yellowing of the leaves at the base of the plant prior to flowering, and poor plant development. A nitrogen deficiency is often shown by the older leaves at the bottom of the plant turning yellow.

Reduced plant growth and yield can occur if there are too few actively fixing nodules. Scientists assess fixation by determining nodule number, size, weight and colour, and in some cases, the distribution of nodules throughout the root system. Plants with greater nodule number, size, and weight are considered to be the highest nitrogen fixers. Comparisons of fixation from one field to another, or within the same field, must be done using the same crop and variety and must be at the same crop stage.

Failure of Nodulation

If careful inspection of the pulse crop root reveals little or no nodulation, inoculation may have been ineffective because of adverse conditions such as dry topsoil. If the field has a history of the same pulse crop, residual Rhizobium bacteria present in the soil may initiate enough nodulation to meet nitrogen requirements for a moderate yield.

If further inspection proves that nodulation has not occurred, and deficiency symptoms appear, a top-dressed application of nitrogen fertilizer may be an option. Limited information is available for recommendations for Saskatchewan growers, so the decision on the amount and formulation of fertilizer to be applied should be made after careful consideration.

Submit crop tissue samples to a soil test laboratory to determine nutrient levels present in the plant. The laboratory will provide instructions for the proper collection and packaging of the samples. Tissue samples should be taken as soon as possible in the growing season and prior to first bloom. The laboratory will work with the producer to determine the course of action to be taken, based on local moisture conditions and target yields.

Fertilizer applications should take place as early in the growing season as possible. Supplemental nitrogen should not be applied later than approximately six weeks after emergence of the crop, as it may cause excessive vegetative growth, poor pod set, and delayed maturity. In research carried out on pea and chickpea in Montana in 1999 and 2000, fertilizer was applied after crop emergence to overcome nitrogen deficiency due to inoculant failure. Their conclusions indicated that yield loss due to inoculant failure could be prevented by applying fertilizer within six weeks of seeding, coinciding with the 9-12 node stage of pea and the 10-13 node stage of chickpea.

Causes of N Fixation Failure

Nitrogen fixation is a biological system that requires the plant and Rhizobium bacteria to work in balance. Conditions that cause nitrogen fixation failure include:

• Using the wrong species of Rhizobium for the crop seeded, or using inadequate rates of inoculant. Calibrate seeding equipment using inoculated seed as it may have slower flow rates than seed that has not been inoculated.
• Delays between inoculation and seeding. The bacteria die rapidly when exposed to temperature extremes and drying conditions. Ideally, inoculate only enough seed that can be planted within six hours. Under cool, dark storage conditions, bacteria can survive on seed for about one to two days, depending on the inoculant formulation. Any further delay will require retreating. Check product labels for manufacturer's instructions - some liquid products must be reapplied if the treated seed is held longer than six hours.
• Improper inoculant storage conditions. High temperatures or exposure to sunlight can kill the bacteria. This can occur in a short period of time, such as during transport or inside a warm vehicle. Inoculants should always be kept cool and out of direct sunlight.
• Environmental conditions (cool soil, waterlogged soil, drought stress or salt stress) that slow crop development and reduce the ability of the plant to supply energy and nutrients for nodule formation and fixation. Plant stress may result in delayed nodule formation and cause the fixation process to stop.
• Using chlorinated water when applying sticker inoculant.
• Low available phosphate levels can slow seedling development and reduce the ability of the plant to fix nitrogen.
• Dry soil during the first two weeks after seeding can lead to desiccation (drying out) of the inoculant and death of the Rhizobium.
• Native Rhizobium bacteria can cause colonization of the roots; however, the native Rhizobium may be inefficient nitrogen fixers. In some cases, nodules form, but do not have the pink or red interior and are not effective.
• Shallow seeding into soil that dries out periodically after seeding may lead to the desiccation of the Rhizobium.
• High levels of available soil nitrogen (more than 55 kgN/ha or 50 lb.N/acre) cause the crop to preferentially use nitrogen from the soil.
• Failure to use a sticker with a peat powder inoculant may limit the number of bacteria on the seed coat and thus reduce nodulation. Many peat powder inoculants come with a pre-formulated sticker, a ready to use product.
• Blending granular inoculant with fertilizer in the seeder tank. Live bacteria counts of granular inoculant blended with 11-51-0 fertilizer were reduced by 93 per cent after three hours.
• Bridging of granular inoculants in the seeder tank can lead to uneven application (Figure 4). Follow manufacturer's label directions to avoid bridging.

Inoculant Formulations

1. Peat based powder - a fine powder containing a specific number of Rhizobium bacteria per gram. An additional solution is used to stick the inoculant directly to the seed. Commercial sticker products are available. Stickers can also be prepared by making a 10 per cent solution of corn syrup, table sugar, or honey in water. Powdered milk can also be an effective sticker. However milk-replacer products containing antibiotics will kill the Rhizobium bacteria.
2. The sticker should be dribbled or sprayed onto the surface of the seed. After the seed is moistened, the inoculant is applied following the manufacturer's instructions. Small amounts of seed can be inoculated in a tub or cement mixer. Larger amounts require the sticker and inoculant to be slowly added to the seed as it flows into an auger. Ensure the auger is emptied before it is stopped to prevent build-up of accumulated sticker. If bridging problems occur, allow the seed to dry in a cool place for an hour before filling the seed tank.
3. Peat based powder with pre-formulated sticker - a fine powder with the sticker already added. The inoculant must be applied directly to the seed by shaking it from the bag onto the seed as it is being augered, or as the seed tank is being filled. Seed may also be moistened with water before applying inoculant. Empty the auger to reduce inoculant build-up.
4. Liquid based - the liquid inoculant is applied directly to the seed as it is being augered. Empty the auger to avoid inoculant build-up.
   Research at AAFC Brandon and AAFC Indian Head investigated the use of liquid-based inoculants soil applied in the seed row or as a sideband using conventional liquid fertilizer equipment (see Inoculant Formulation Comparisons). Some liquid inoculants are registered for use in a seed row application. Check labels for application rates and handling instructions. Caution must be taken to avoid the use of chlorinated water as it will kill the Rhizobium bacteria.
5. Granular based - contain specific numbers of Rhizobium bacteria on a peat-based or clay-based granule. The product can be metered directly into the furrow with the seed, allowing very accurate measurement and application. A separate tank and meter is required.
6. The granules must NOT be mixed with granular fertilizer. Granular inoculants are subject to moisture build-up within the seeder tank that leads to bridging and compaction. To avoid bridging, tanks should not be filled more than one-half full, and product should not be transported in the tank. If granular products are left in the tank overnight, considerable condensation can occur that can result in reduced flowability and plugging. Therefore, ensure that the tank is aerated (lid open) and kept out of direct sunlight and heat. Follow manufacturer's directions.
7. Pre-inoculated seed - seed is treated with a peat-based inoculant encapsulated with a seed coating at a commercial facility prior to purchasing the seed. No further inoculation is required. Viability of pre-inoculated seed is dependent on time and storage conditions. Always follow label instructions carefully.

Inoculant Strain

Inoculants are available in either mixed-strain or single-strain formulations. Mixed-strain inoculants may contain bacteria effective in initiating nitrogen fixation in more than one pulse crop or may contain two or more strains specific to a single pulse crop. An example of a mixed-strain inoculant is a product that is very effective in both pea and lentil. Single-strain inoculants contain Rhizobium particularly effective in one pulse crop. Always use an inoculant suitable for the pulse crop being grown.

Inoculant Formulation Evaluations

Research completed at the University of Saskatchewan evaluated the performance of inoculant formulations in chickpea. Peat-based and liquid inoculants were applied directly to the seed, and granular inoculants were applied either in the seed row or placed in a side-band, 2.5 cm (1 in.) to the side, and at depths of either 2.5 or 8 cm (one or three in.) below the seed. Results indicated that inoculation using granular formulations was as good as, or better than, other formulations. The peat-based powder and liquid formulations performed as well as the granular formulation in some instances, especially when soil moisture was not limited. Studies carried out in drier soil conditions favoured granular products. Granules placed below and to the side of the seed in moist soil may result in better Rhizobium survival and enhanced fixation relative to seed-placed inoculant. Granular inoculants promoted nodule development on lateral roots, and the test results indicated that yields were improved by the presence of sufficient nodulation on lateral roots.

Research at AAFC Indian Head and AAFC Melfort evaluated inoculant formulations in pea. When soil moisture conditions were below normal, the granular formulation performed better than the peat-based powder, which performed better than the liquid formulation. In these conditions, the soil applied inoculant was favoured. When soil moisture was normal and soil organic matter levels exceeded six per cent, no difference was found among formulations.

In studies carried out at the Canada-Saskatchewan Irrigation Diversification Centre at Outlook, dry bean was found to fix lower levels of nitrogen compared to other pulse crops. However, the highest bean yields were obtained with granular inoculant treatments compared to other inoculant formulations.

In research conducted at AAFC Brandon and AAFC Indian Head, liquid inoculant formulations were tested for their performance as a soil treatment when dribbled into the seed row or as a sideband. Treatments tested included a liquid formulation suspended in a water solution and applied to the soil, a granular formulation applied to the soil, a liquid formulation applied to the seed, and an untreated check. The soil-applied liquid and granular formulations performed equally, and all formulations and application methods increased seed yield over the non-inoculated check. Soil-applied liquid formulations require further investigation, especially in drier areas and low organic matter soils.

It is important to note that all inoculants will deliver ample viable Rhizobium bacteria when the directions for use are followed and the product is applied properly. Although studies indicate that granular inoculants perform as well as or better than the peat-based powders or liquid products, granular products are more costly and require an additional tank and meter on seeding equipment. Experienced pulse crop producers routinely achieve successful nodulation and fixation using the lower cost seed-applied inoculants. Some growers use a liquid/peat inoculant combination on first time pulse crop fields and in adverse soil moisture or temperature conditions, where the liquid is applied first and used as the sticker for the peat powder.

Storage

Inoculants contain living bacteria and should be kept cool and out of direct sunlight. An unheated building will provide good temporary storage at the time of seeding. A refrigerator is ideal for prolonged storage. Repeated freezing and thawing of the inoculant will reduce the viability of the Rhizobium and should be avoided. Most inoculant manufacturers do not recommend freezing products for long-term storage, although products that inadvertently freeze during storage in early spring will not likely lose viability. If a product is frozen, it should remain frozen until used. Thaw slowly out of direct sunlight and consult the product label or the manufacturer for directions.

Inoculant product packaging displays an expiry date after which the number of live bacteria is no longer guaranteed, and the product may no longer ensure adequate nodulation. Always check the product expiry date at time of purchase.

Inoculants sold in Canada are registered under The Fertilizers Act administered by the Canadian Food Inspection Agency (CFIA).

Inoculants for Organic Crop Production

A major source of nutrients, especially nitrogen, in organic production systems is derived from green fallowing a legume crop. Pulse crops are very important for their ability to fix nitrogen, diversify crop rotations, and improve soil tilth and biological activity. Some inoculants meet the standards for certified organic crop production. There may, however, be restrictions on certain inoculant and organic producers should check with their certification agency for a list of approved inoculant products.

Safety Precautions

Nitrogen fixing inoculants for pulse crops are generally non-toxic and non-hazardous. However, the use of standard protective equipment, including gloves, goggles, and an approved respirator is recommended.

Seed Treatment Fungicides

For information on available seed treatment fungicides please consult the current version of the Guide to Crop Protection.

Note: Seed treatment fungicides vary with regard to their impact on the viability of nitrogen fixing and phosphate inoculants. Always read and follow product labels, or contact your inoculant manufacturer for guidelines on the proper use of combinations of fungicides and inoculants.