Fixing our Perspective on N-Fixing – FULL ARTICLE

Fixing our perspective on N-fixing

A summary of recent multi-state studies by university researchers (Performance of Selected Commercially Available Asymbiotic N-fixing Products in the North Central Region) reports a low rate of positive benefits using commercial biological N-fixing products marketed to US row crops.  

Unbiased, third-party studies like these are critical for new technologies – like biological N-fixing products.  However, it is important to not prematurely say these products “don’t work” before better understanding 1) why these outcomes are occurring in the field and 2) are we considering the correct success metrics? 

While limited information was available on how the various field trials were conducted in the university research studies, delivery of the biological N-fixing products involved either liquid in-furrow (LIF) with starter fertilizer (Envita® and ProveN® 40), foliar (F) application at V2-3 (Envita®) or V4-V8 (UtrishaTM N) with non-chlorinated water, or seed treatment (ST) of a dry powder (MicroAZ-ST) (Table 1).   

Table 1.  Biological N-fixation products evaluated in the university research trials. 

Product Mfg Bacteria Strain Guaranteed Analysis Formulation Application Rate Delivered With
Envita® Azotic Gluconacetobacter diazotrophicus 1×107 CFU/mL Liquid 3-4 oz/5 gpa (LIF)2-10 oz/10-20 gpa (F) 7-23-5 starter/water10 oz/acre (water)
ProveN® 40 Pivot Bio Klebsiella variicolaKosokonia sacchari ≥1×108 CFU/mL≥1×108 CFU/mL Liquid 12 oz/5 gpa (LIF) Varied starters(10-34-0, 7-22-5,6-24-6)
UtrishaTM N Corteva Methylobacterium symbioticum 3×107 CFU/g Wettable Powder 5 oz/10-75 gpa (F) 10 gpa (water)
MicroAZ-ST TerraMax Azospirillum lipoferumAzospirillum brasilense 5×105 CFU/g Dry Powder 4 oz/cwt (ST) Dry seed treatment

Out of 53 corn experiments on biological N-fixing products, 51 experiments were reported as having no significant yield increase with use of product over N rate alone.  The N rate benefit from the two studies finding significant yield improvement ranged from 12 to 20 pounds N/acre.  Before concluding that there is “no additional benefit” to the use of biological N-fixing products, we need to dig deeper and understand why there may be a lack of benefit occurring.

A lack of positive benefit in the field is often attributed to application of the product where it is not needed (the system), exposure to adverse soil conditions (the environment), and/or poor application practice (delivery).  Loss of microbe viability and presence of contaminants in the manufactured product also contributes to poor performance in the field. 

Evaluating field efficacy of microbial inoculants is technically challenging.  Multi-year, multi-site field data with appropriate levels of replication are needed to obtain adequate statistical significance; to detect a 10% difference in yield, between 9 and 28 replicates has been recommended (Leggett et al, 2015).  Comparisons between trials is further complicated due to the wide variability in the methods used, field conditions, and formulation and application details.  Rather than applying a strict probability significance threshold, a Bayesian approach to the statistical analysis has been recommended to quantify the likelihood for differences between treatments and their levels of uncertainty (Ellison, 2004). It is also important to include both negative controls (no inoculation, no N fertilizer) and positive controls (no inoculation with N fertilizer) in field trials. 

For biological N-fixing products, while yield remains a key metric, consideration of net return and return on investment (ROI) also needs to be taken into account, particularly when considering reduction in N rates.  While not statistically significant, several of the university research studies showed either maintenance of yield or slight yield increases at reduced N rates using the biological N-fixing products. 

Beck’s PFR Proven multi-year, multi-site studies are conducted on production acres and have found similar yield maintenance at reduced N rates with biological N-fixing products, showing the potential for some products to maintain yield and improve profitability.  Tables 2 and 3 show examples from both the university research and Beck’s PFR studies for the Envita® product applied in-furrow to corn during the 2020 and 2022 seasons, respectively.  With reduction in N rates by 25 pounds/acre and greater, net returns and ROI were increased in many cases over the high N rate control

Shifting our perspective on yield expectations to net returns may provide better success measures for evaluating the benefits of biological N-fixing products.  Additional benefits for decreasing fertilizer N rates relate to environmental and sustainability goals.  Also, other measures besides yield are important for comparisons, such as nutrient content in grain and increase in biomass. 

No doubt, future work to improve consistency and performance of biological N-fixing products is needed.  Better understanding of the system, environment, and delivery are essential to better inform practices being used in the field.   

Table 2. Comparison of biological N-fixation studies evaluating Envita® at reduced N rates (2020).

Total Nitrogen (units/ac)



Bu/ac difference

Net Return


2020 Purdue University corn N rate trial with Envita®













No Envita®











No Envita®










2020 Beck’s PFR multi-location corn N-fixation study with Envita®













No Envita®










Corn $3.72/bu. UAN $0.44/unit N. Envita® $398.00/gal ($9.95/ac).

Purdue University study – Envita® was applied to corn with long-term no-till management. No details on Envita® application were provided. 

Beck’s PFR study – Envita® was applied to corn with conventional tillage, previous crop soybean; starter 10 gal 28-0-0 2×2.  No details on Envita® application were provided.

Table 3. Comparison of biological N-fixation studies evaluating Envita® at reduced N rates (2022).

Total Nitrogen (units/ac)



Bu/ac difference

Net Return


2022 Michigan State University N-fixation product trial with Envita®













No Envita®











No Envita®










2022 Beck’s PFR multi-location corn N-fixation study with Envita®













No Envita®











No Envita®










Corn $6.11/bu. UAN $1.04/unit N. Envita® $518.00/gal ($12.95/ac).

Michigan State University study – Envita® was applied in-furrow to corn at planting at 3.2 oz/ac. A base rate of 60 pound N/ac was applied in 2×2 placement at planting; additional 50 pounds N/ac or 120 pounds N/ac applied side-dress application at V3-V4. No details on Envita® application were provided.

Beck’s PFR study – Envita® was applied to corn with either conventional or no-till management, previous crop soybean; starter 18.5 gal 30-0-0 or 20 gal 28-0-0 2×2.  Envita® was applied with starter (2.5 gpa water + 2.5 gpa 6-24-6). 

History of N-fixing

Microbial products have enormous potential to provide alternative, more sustainable approaches for agriculture while protecting the environment.  However, what has been proven in research is not fully translating to consistent, beneficial results in the field – which is causing some to question their effectiveness. 

Symbiotic biological N-fixing products (referred to as inoculants) for legumes date back 120 years (“Nitragin”; Nobbe and Hiltner 1896).  The biological N-fixing process occurs spontaneously due to the native rhizobia population in the soil; however, some regions do not have naturalized populations and farmers commonly apply inoculants.  Soybean inoculation is more common in South America than in the United States.  In Brazil and Argentina, nearly all soybeans are inoculated with elite N-fixing Bradyrhizobium strains. In the United States, only about 15% of farmers use soybean inoculants.  Factors that have contributed to less use of soybean inoculants in the United States include planting soybean varieties with limited response to inoculation and limited yield increases in soils with soybean history, due to the established symbiotic bacteria populations in the soil (Graham et al, 2004).

Nitrogen fixation in cereal crops (specifically corn) has been actively researched for several decades.  Several new non-symbiotic N-fixing products (Table 1) were recently introduced to the US corn market.   Together these new microbial inoculants are impacting several million acres, only a small fraction of the ~90 million acres of corn planted in the United States and ~500 million acres planted worldwide each year.   

Why are biological N-fixing products so successful in Brazil? 

Several key factors have contributed to the widespread use of inoculants in Brazil:

1.       Intense research: Brazil has a long history of intense research with soybean inoculants, leading to the identification of four elite Bradyrhizobium strains most effective with soybean cultivars and soil conditions experienced in Brazil (Dobbereiner and Duque, 1980).  Success with soybean inoculation led to demand for microbial inoculants for additional crops, with focused development of elite Azospirillum strains for use in cereal crops (Santos et al 2021). 

2.       Commercial standards: From the beginning, Brazil created legislation defining the commercial standards with regards to concentration (minimally 109 CFU/mL), purity, shelf-life guarantee (greater than 6 months), and the absence of biological contaminants in the products at the 10-5 dilution.  The maintenance of cell concentration and ensuring product quality contributes to achieving the desired performance.

3.       Strict quality control: Brazil created government programs to perform official quality control analyses of commercial N-fixing products (de Souza et al 2019). 

Regulation of commercial biofertilizer products varies widely worldwide.  Brazil, France, and Canada have mandatory product registration, with samples analyzed randomly every year.  Commercial standards for seed treatment require at least 105 to 106 CFU per seed (Hermann and Lesueur 2013).  Presently, no microbial inoculant regulation exists in the United States; however, a regulatory framework is being developed under the Plant Biostimulants Act of 2023. 

The System

Nitrogen is one of the most crucial elements for biological systems and is required in large amounts by plants for efficient growth.  Farmers use more than 100 million metric tons of N fertilizer each year, which consists of ammonia, nitrates, and other nitrogen-containing compounds.  Increasing use of N fertilizers in agriculture is negatively affecting the environment and soil through soil acidification, pollution of groundwaters by nitrates, and nitrous oxide gas emissions.  These concerns, along with a growing demand for ‘greener’, more sustainable agricultural practices are driving growth of the Ag biologicals market.

How does biological N-fixation work?

A small group of soil bacteria known as diazotrophs are capable of reducing atmospheric dinitrogen (N2) into forms of ammonia (NH3) the plant can use.  Biological N-fixation is an energy expensive process for the bacteria, requiring 16 ATP molecules to break down a single N2 molecule.  The reaction is catalyzed by the enzyme complex nitrogenase.  Nitrogenase is extremely oxygen sensitive and inactivates in aerobic environments. 

Challenges with biological N-fixation largely stem from the energy expense to the bacteria, coupled with oxygen sensitivity of the bacteria’s nitrogenase complex required for reducing dinitrogen to ammonia. This oxygen sensitivity further complicates energy requirements of the bacteria as energy is most efficiently produced by the bacteria using aerobic respiration in the presence of oxygen (the “oxygen paradox”; Marchal and Vanderleyden 2000). 

Current US agriculture practices depend on high N fertilizer applications.   Excessive use of N fertilizers leads to soil acidification (low pH levels) creating an unfavorable environment for soil microbes.  Further, conventional tillage practices to disrupt the soil environment for better aeration create increased oxygen levels in the soil that can inhibit nitrogenase activity. 

Soybean (legume) inoculants are the most well established biological N-fixing bacteria.  Legumes fix atmospheric nitrogen through a highly specific symbiotic relationship with rhizobia in its root nodules. Nodule formation depends on the exchange of signals between the host plant and rhizobia, leading to bacteria infection in the nodule where they are protected from oxygen diffusion.   Rhizobia strains vary in their nodulation ability, N-fixation efficiency, compatibility with different cultivars, and ability to adapt to different environmental conditions, leading to variability in efficacy across different locations (Thilakarathna and Raizada, 2017).  Nonetheless, bacteria that associate with legumes are reasonably efficient at fixing nitrogen with rates of 50-465 kg N ha-1 yr-1 (Pankievicz et al 2019).

Non-symbiotic bacteria are less efficient at fixing nitrogen.  These bacteria are either free-living or endophytic and reside in the rhizosphere at the surface or interior of the plant root.  For non-legumes, to limit oxygen diffusion, either viscous root exudates produced by the plant host and/or exopolysaccharides or biofilms produced by bacteria on root surfaces can increase efficiency of nutrient exchange.  Further, endophytic bacteria appear promising candidates as they colonize the intercellular spaces of plant tissue, so fixed nitrogen may be released directly to the plant without competition like in the rhizosphere.  

Many diazotrophs also promote plant growth through other mechanisms such as phytohormone synthesis, phosphate solubilization, and the acquisition of other nutrients like calcium, potassium, iron, copper, magnesium and zinc (Bashan and De-Bashan, 2010).  Azospirillum spp. is one of the most studied free-living N-fixing bacteria.  Azospirillum-plant interactions involve multiple, beneficial mechanisms including phytohormone production, stimulation of root growth, and N fixation (Cassan et al 2020).  With general plant growth promotion, when multiple mechanisms are claimed for microbial inoculants, it becomes more difficult to separate out inoculation responses in field settings.

Regardless of the claims, a microbial inoculant will only be beneficial if it is targeting a significant limitation in the soil where it is applied.  In the case of biological N-fixation, if plant growth is limited when N is applied, other factors are limiting plant growth and an N-fixing inoculant may not be beneficial.  Routine soil tests measuring available soil N can be used to indicate potential for biological N-fixing products to provide benefit.

The Environment

Most microbial products commercially available in the United States have been developed for a mass market.  No microbial inoculant is effective in all environments.  Focused regional development and selection of adapted microbe strains for various environmental stress factors may support improvements in field performance (see Why are biological N-fixing products so successful in Brazil?)

Plants actively recruit beneficial microbiomes using complex chemical ‘cross-talk’ and molecular signaling between the microorganisms and plant roots.   Success-limiting factors in the environment include the plant-microbe specificity (‘cross-talk’), variable soil conditions (moisture, temperature, nutrient levels, pH, salinity, soil type), and interactions with the native microbiome (competition, predation, antagonism). 

Survival and persistence of microbes in the soil has been shown to be affected by extreme soil pH, drought, nutrient deficiencies, salinity/alkalinity, extreme temperatures, and toxicities (Hungria and Vargas, 2000).  In general, microbe survival and concentration are favored by neutral pH, adequate moisture, adequate organic carbon, and low salinity (Thilakarathna and Raizada, 2017).  Inoculant response will be affected by resource availability and the extent of native species diversity and concentration in the soil.  Competitive pressure is less likely to be detrimental to the inoculant if host specificity is strongly expressed and they are applied at high concentrations close to the plant roots or seed at planting.  

Therefore, key to success for microbial inoculants in the field is survival, establishment, colonization, and interaction of the microbe with the plant in the rhizosphere.  The rhizosphere is the area of soil immediately surrounding the root, which is directly influenced by the plant root exudates.  Root exudates serve as food for the microorganisms, and include a range of organic acids, amino acids, sugars and other small molecules.  Depending on the plant species and environment, differences in the chemical composition of the exudates will attract a particular microbiome to the rhizosphere (Mavrodi et al 2021).  And similarly, there are mechanisms of the microorganisms that allow recognition of plant molecules in order to more effectively survive and colonize the rhizosphere.  

The concept of the ‘disease triangle’ can be similarly applied to beneficial microbes (‘inoculant triangle’; Figure 1), in which the plant host, beneficial microbe, and environment must all interact positively to achieve the desired outcomes in plant growth promotion and performance.

Figure 1. The Inoculant Triangle

While certain conditions promote microbe survival in the soil and rhizosphere, greater yield responses to inoculants have been shown under harsher environment conditions for the plant.  Recent meta-analyses in Brazil (Barbosa et al 2021; Barbosa et al 2022) found the critical factors with greatest response to inoculation are related to low productive environments, including sandy soils, low N levels, water deficiency, and tropical climate.  A global meta-analysis (Schutz et al 2018) similarly found superior biofertilizer performance in dry and tropical climates.  Soil and environmental conditions in the United States differ relative to tropical environments. 

No-tillage and higher soil organic matter (SOM) were also found to support greater responses to inoculation (Barbosa et al 2021, 2022).  Under no-tillage, high soil moisture content, low soil temperature, high carbon content, and preservation of soil aggregates all favor soil microbial activity.  Stress factors such as water deficiency and high temperatures can be exaggerated under conventional tillage practices due to the lack of plant residues on the soil surface.  


Microbial inoculants have the best chance to survive and compete in the soil environment if they are applied at high concentrations, in a highly viable form, close to the plant roots or seed at planting. 

Keeping living microorganisms highly viable in a commercial product and supply chain is technically very challenging, particularly for non-spore forming gram-negative bacteria (which encompass all biological N-fixing bacteria).  Typically, greater than 99% (i.e., 2 log reduction or greater) of viable microbes die or become compromised during processing, shipping and storage in the supply chain.  Consequently, by the time the product is used, many commercial products likely contain far fewer numbers of the claimed microbe than product labels suggest. 

Quality standards are critical to ensure that microbial products reaching the farmer include high concentrations of highly viable microbes and no contamination.  Presence of contaminates can have a negative effect on the microbial inoculant concentration, and therefore, product performance.  Some inoculant products currently on the market contain numerous different microorganisms, sometimes with low numbers (<105 CFU/mL) of each strain.  Maintaining quality control of microbial consortia at high concentrations without contamination is technically very challenging.   

In Brazil, where commercial standards exist, a minimum concentration of 109 CFU/mL is required.  A recent assessment of commercial soybean inoculants in Brazil found high quality inoculants with concentrations frequently >1010 CFU/mL and only 2.2% of samples had detectable contaminants (de Souza et al 2019).    Another study of products from the United States and several other countries, where commercial standards currently do not exist, found only 37% of the inoculants were not contaminated, and 40% did not contain any of the claimed strains, but only contaminates (Herrmann et al 2015).   Clearly, absence of regulations is impacting commercial microbial inoculant quality, and ultimately performance and adoption by farmers in the United States and worldwide.

Why is starting with a high concentration of microbes important?

Delivery of a high concentration of microbes is important to overcome inoculant losses during application and in the soil environment so that there is sufficient survival of cells for long enough periods of time to establish and colonize the emerging plant root system. 

Limited data exist about the adequate inoculum rates to maximize the benefits of biological N-fixation.  In Brazil, inoculum rates of 106 CFU/seed are required for soybean inoculants, supported by field trials evaluating the impact of inoculum rate on nodulation number, grain yield, and N content in grains (Hungria et al 2017).  Optimal Azospirillum spp. concentrations of 105 to 106 CFU/mL were determined in greenhouse trials of wheat based on root surface area increases (Bashan 1986). 

Table 4 presents results from a microplot trial of corn evaluating inoculum rates of 104, 106, 108 CFU/seed.  Root colonization was measured at V3 and V9 stages of plant development and found to increase with increasing inoculum rate to levels of ~3×108 CFU/g and 3×106-1×107 CFU/g root dry weight in the rhizosphere at V3 and V9 stages, respectively.  These results support previous studies indicating at least 106 CFU/seed is a reasonable target for application.  Considerably lower colonization was achieved at a lower inoculum rate of 104 CFU/seed.  As the plant matures, better understanding of the inoculants’ persistence and continued benefit to the plant is needed. 

Table 4. Root colonization of Pseudomonas spp. at V3 and V9 stages of corn growth at three inoculum rates: 104, 106, 108 CFU/seed.  Inoculum was added as a fresh liquid culture. Root colonization (CFU/g dry soil or root weight) was measured from bulk soil (soil attached to the root), rhizosphere (wash one from root), and rhizoplane (wash two from root) of plant samples. 



12 Days Post Planting (V3)

27 Days Post Planting (V9)

104 CFU/seed

106 CFU/seed

108 CFU/seed

104 CFU/seed

106 CFU/seed

108 CFU/seed

Bulk Soil






















Assuming no loss during seed application, achieving 106 CFU/seed requires at least 2-3×109 CFU/mL of starting inoculant concentration at typical application rates of 2-3 oz/cwt seed (Table 5).   To achieve 105 CFU/seed, at least 2-3×108 CFU/mL is needed.  While most soybean inoculants containing Bradyrhizobium spp. contain >2×109 CFU/mL, other biological N-fixing and plant growth promoting products have considerably lower guaranteed concentrations (see Table 1).    

Table 5.  Theoretical amount of microbial inoculant applied per seed for a range of inoculant concentrations at typical application rates.


            Application Rate


Inoculant Concentration (CFU/mL)





























































*Represented as CFU/seed

What happens to the inoculant once applied?

Plant growth-promoting inoculants typically have the greatest effect when applied early in plant development.  When applied with the seed, there is a period of time before the seed germinates that the inoculant must be able to survive before plant root exudates become available.  Therefore, inoculants are best introduced when there is adequate moisture and temperature conditions for expedient seed germination. Further, applying inoculants along with a protective, nutritive resource (e.g., complex nutrient medium) can aid inoculant survival until seed germination.

Effective attachment (or “root colonization”) to the plant roots allows the microorganisms to more readily exert their beneficial mechanisms towards the plant.  Root colonization by microorganisms involves several steps to take place, starting with initial adherence, followed by cellular multiplication and formation of a biofilm, and then subsequent movement of cells to another site on the root to start the process over.  Microorganisms that have cellular structures such as pili or flagella aid in adherence and movement along the root.  Also, prolific biofilm production aids microorganisms to survive different environmental stresses and maintain high levels of cells attached to the roots.  

Figure 2 shows examples of corn root colonization by Pseudomonas spp. and Bacillus spp. using a replica printing method (Leben 1983). Generally, Pseudomonas is considered a typical rhizosphere microorganism known for their ability to competitively colonize plant roots.  Bacillus preferentially occur in the bulk soil.  Bacillus are endospore-forming bacteria, which requires the bacteria to germinate into a vegetative cell to multiple and colonize the plant root.  As Figure 2 shows, most of the Bacillus cells remained on the inoculation line in spore form and did not colonize the root, while Pseudomonas cells colonized along the entire tap root.  These results are consistent with Dijkstra et al (1987), who found stronger colonization potential for P. fluorescens (up to 107 CFU/cm root) than for B. subtilis (up to 105 CFU/cm root).  A minimal colonization threshold of 105 cells per gram of root has been suggested for the induction of plant systemic resistance mechanisms by Pseudomonas (Thomashow and Bakker, 2015).

Figure 2. Root colonization of corn seed by Bacillus and Pseudomonas strains.  Strains were inoculated on a non-nutritive agar with corn seed (left); after seed germination (right), replica plating shows  Pseudomonas cells colonized along the tap root, while Bacillus cells colonized the top of the seed or did not colonize at all and remained on the inoculation line.  

Why is formulation so important?

Appropriate formulation is required for a high quality product.  Microorganisms need to be well protected to survive in high numbers in the formulation under variable and potentially harsh conditions during storage.  During storage, high temperature is typically the most detrimental condition for the microorganisms. 

Formulations are broadly divided into those using solid carriers or liquid formulations.  Choice of formulation depends on the microorganism, cost, shelf-life and application requirements among other factors.  No formulation is perfect and each has its advantages and disadvantages (Figure 3). 

Figure 3. Different formulation types have advantages and disadvantages.  

Historically, peat is the most commonly used carrier for microbial inoculants, given its low cost and protective, nutritive environment for the microorganisms.   The main drawback of peat is the variability in its quality and composition, leading to differences in its capacity to support cell survival.  Liquid formulations tend to have a shorter shelf-life, given that metabolic activity of microbial cells continues during storage; but can be produced in high concentrations and are easy to handle and apply.  Dry formulations (e.g., powders and granules) generally have longer shelf-life as the microbes are forced into a dormant state during controlled drying; however, dessication of microbial cells during drying can cause high levels of cell death, particularly for non-spore forming microbes.  Advancements in drying technologies and formulation are leading to more dry microbial inoculants being developed, with high concentration and longer shelf-life of 1 year and greater. 

Recent meta-analyses (Barbosa et a 2021, Barbosa et al 2022) of microbial inoculants in Brazil found peat-based inoculants had slightly higher yield responses compared to liquid inoculants.  Peat or similar carrier materials can provide protection and nutrients to the microorganisms for survival during application and in the soil environment.  However, liquid formulations make up over 80% of the market in South America because liquids are easier to produce by industry and easier to use by farmers (Cassan et al 2020).   Application of liquid inoculants to seed involves drying of the microbe solution in an uncontrolled environment, causing subsequent dessication loss of cells which could impact performance.  Application of dry inoculants avoids dessication loss during application, if the dry carrier can provide a protective environment. 

How are microbial inoculants applied?

Microbial inoculants are typically applied to row crops at planting either by seed treatment or in-furrow delivery.  Seed treatment is most widely used on a commercial scale because it is suited to management practices and requires less material than in-furrow delivery.  Foliar application to coincide with certain plant development stages is also increasing in practice. 

Nearly all corn seed is treated with fungicide and insecticide by the manufacturer.  For soybean, wheat and other crops, use of pesticide seed treatment is variable and typically done at the retailer or on-farm using a range of different seed treatment equipment.  Addition of the microbial inoculant to the seeds can occur either as an overtreatment or at the same time as pesticide seed treatment.  Pre-inoculation of seeds weeks (or months) in advance is desired by farmers given the tight planting window.

The potential for long-term viability of cells on inoculated seeds depends on the microorganism, cell protectant in the formulation, and pesticide compatibility.  Typical cell protectants and additives include: sugars (saccharides, polysaccharides), phosphates (mono-, dipotassium-), glycerol, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), alginate, and xanthan gum.  Recent pre-inoculation studies of Bradyrhizobium with cell protectant, with and without pesticide seed treatment, found approximately 2 log loss (i.e., 99% loss) of cells from the applied amount of 1×106 CFU/seed to the recovered amount of 1×104 CFU/seed after storage for 15 to 30 days (Hungria et al 2020; Sartori et al 2023) before planting.  With pre-inoculation even several weeks in advance of planting, considerable cell loss on-seed can translate to more variable and potentially poor performance in the field.  Therefore, seed treatment of biological N-fixing products should occur as close to planting as possible. 

Most commercial seed treaters include an inoculant port to keep the microorganisms separate from the chemical pesticides, colorants, and any other liquid additives being applied. The liquid is sprayed by an atomizer onto the seeds; distribution and drying of the material occurs during tumbling of the seeds in a drum and/or during conveyance on the seed tender.  Inoculants are typically applied separately through an inoculant port as an aqueous suspension or with cell protectants and additives.  During drying, microorganisms experience the negative effects of dessication, as well as any toxicity from the chemicals on the seed. 

In-furrow delivery provides some advantages to the microorganisms in avoidance of harsh conditions during drying in a seed treater, but requires larger quantities of inoculant compared to on-seed application.  Typically, inoculants are added directly to the in-furrow tank, which may include a mixture of starter fertilizer, fungicide, and/or insecticide.  The in-furrow liquid stream places the microbes in the general area around the seed, but also wastes considerable inoculant applied between seeds.  Recent advancements in liquid in-furrow equipment benefit application of microbial inoculants through multiple jet lines for separation of microorganisms from other liquids (e.g., FurrowJet®, Precision Planting) and on/off valve switching for liquid reduction and more precise application with seed (e.g., 360 DASHTM, 360 Yield). 

All tank mix components need to be evaluated with the microbial inoculant for compatibility.   Table 6 provides example log loss of cells after six hours exposure to different commonly used starter fertilizers and water used in the tank mix.  Low salt index starter fertilizers with a salt index <20 are necessary to minimize osmotic shock to microorganisms.  Generally, 10-34-0 with a salt index of 20 is too harsh for many microorganisms.  Diluting 10-34-0 by at least one-half can improve compatibility.  Lower salt index starter fertilizers (such as 6-24-6 with a salt index of 11.5) are recommended.   Some water sources may contain too high of chlorine levels (> 2 ppm) and/or minerals that are detrimental to the microorganisms.   As shown in Table 6, each bacteria strain has different compatibility responses and needs to be evaluated on a case by case basis.

Table 6.  Compatibility of Pantoea spp. strains to starter fertilizers after 6 hours exposure to tank mix. Represented as log loss, where 1 log loss=90% reduction in cells, 2 log loss=99% reduction in cells, etc. 

Pantoea strain 10-34-0 10-34-0 (half strength) 6-24-6 Locally sourced water
3BB1 1.04 0.23 0.23 0.08
IALR 611 0.06 0.12 0.20 1.43
IALR 325 0.69 0.15 0.26 0.56

Foliar application is receiving increasing interest in biological N-fixation, particularly for endophytic bacteria for their ability to colonize intercellular spaces in plant tissue for N-fixation.  Compared to more precision applications with seed treatment and in-furrow delivery, a downside to foliar application is the greater amount of material needed to spray leaf surfaces.   In addition, exposure to UV radiation and dessication during drying on the leaf surface may cause microbe losses before the cells can colonize the plant tissue.  

Technical breakthroughs in delivery of biologicals are just beginning.  Layering seed lubricants with biologicals is a great example of a different way to deliver microbial inoculants.   Application of dry microbial products with the seed lubricant is advantageous because it fits with existing practice for seed singulation and avoids microbe dessication losses typically experienced with liquid seed treatment.  Two new products were introduced into the US market in 2023 using seed lubricants to deliver dry microbial products: BioWakeTM Seed Lubricant (AMVAC, Newport Beach CA) and Hopper ThrottleTM (Meristem Crop Performance, Powell, OH). 

The Future

As farmers struggle with rising input costs, unstable supply chains, environmental concerns and tightening restrictions on fertilizer use, agriculture stands at an inflection point.  Transformative change in how we produce more food with less chemicals is gaining momentum.  Ag biologicals have made rapid advancements driven by changing market pressures and technological advancements.  But Ag biologicals require a more holistic, systems-based approach for successful use compared to traditional Ag-chem practices.    

Farmer’s trust in Ag biologicals has been a major roadblock for widespread adoption.  Based on CropLife’s 2023 Biologicals Survey, 54% of leading Ag retailers indicated a lack of trust in product performance as the key barrier to adoption of biologicals, up considerably from 41% in 2022. Nonetheless, the Mckinsey report (2022) shows continued growing momentum for biologicals with more than 20-30% of large farms (>5,000 acres) indicating they use or plan to use biologicals. 

For continued momentum and wider adoption to occur in the United States, several key areas need to be addressed:

·   A regulatory framework that includes commercial standards for concentration, purity, and shelf-life guarantees. 

·   Monitoring of product quality post-manufacturing in the supply chain to ensure commercial standards are being met. 

·   Focused education for farmers, channel and university partners that more clearly communicates what the biological does, when and where it will work, and best practices for delivery.  

·   More field studies and meta-analyses that take into consideration methods used, field conditions, formulation and application details to support better understanding of when, where, and how biologicals work best. 

·   Advancement in tools to assist with in-field verification that the biological products are working.  


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