Dave Greenshields 3/4/24 Dave Greenshields 3/4/24

How we evaluate nitrogen fixation at Insight Plant Health

Testing for nitrogen fixation

Nitrogen (N) fixation is the conversion of atmospheric nitrogen into ammonia, and is pivotal in sustaining ecosystems and in global food production. While most of the fertilizer N used for agriculture is derived through the energy-intensive Haber-Bosch process, N fixation can also be carried out naturally by certain bacteria and archaea. These N-fixing bacteria can be classified according to the strength of their associations with plants, which range from free-living, to associative, to symbiotic. Rhizobia are symbiotic N fixers that work with the plant to form specialized N-fixing organs called nodules on the roots of legumes. These symbioses are usually quite specific, where only a certain species of rhizobial bacteria can interact with a certain species of plant (e.g. Bradyrhizobium japonicum and soybeans or Mesorhizobium ciceri and chickpeas). Symbiotic N fixation has long been recognized as an agricultural tool, and rhizobial inoculants have been sold for over 100 years.

More recently, free-living associative N fixing bacteria have been introduced for use in agriculture (e.g. Azospirillum brasilense, Methylobacterium symbioticum, and Gluconacetobacter diazotrophicus). These associative bacteria form looser relationships with plants and are able to interact with a range of different plant hosts. The free-living N fixers don’t provide the same level of N to their associated plants as symbiotic rhizobia do, but they can provide some N, and can often also promote plant growth through other biostimulant activities.

Identifying and using the best-performing N-fixing strains in agriculture will lead to more fixed N being transferred to the crop. Similarly, selecting crop varieties with improved N fixation traits or incorporating high performance N-fixing species into a cropping system will also lead to more fixed N into the operation. At Insight Plant Health, we’ve helped several customers identify and characterize novel N-fixing bacteria to make new and improved inoculant products. We’re also working with the Saskatchewan Ministry of Agriculture and Saskatchewan Pulse Growers to identify high-performing rhizobia for fenugreek in Saskatchewan.

Using N isotopes to measure N fixation

Nitrogen makes up 78% of the atmosphere and exists in two stable isotopic forms, 14N and 15N. The isotopic composition of N is 99.634% 14N and 0.366% 15N. The constant ratio of 14N/15N in the atmosphere and other natural compounds provides a basis for using 15N2 or 15N-enriched fertilizers for quantifying the amount of N fixed by bacteria and transferred to plants. Neither 14N nor 15N are radioactive and therefore pose no health risks. Their stable nature allows experiments involving these isotopes to be run over long periods.

Incorporation and measurement of 15N into plant biomass

To evaluate the N-fixing ability of a particular plant-bacteria association, we use an 15N dilution protocol. We add a small amount of 15N-labelled fertilizer (15N-NH4NO3) to pots containing the test plants and to non-N-fixing reference plants (e.g. wheat). The plants are grown to just after the peak N-fixing stage (usually the pod-filling stage) and analyzed for N isotopes by mass spectrometry. IPH collaborates with University of Saskatchewan’s and University of California -Davis’s isotope labs for mass spectrometry analyses. The non-N-fixing plants have soil N and fertilizer N available to them. The fixing plants have soil N, fertilizer N and atmospheric N available to them. Because we added 15N-labelled fertilizer to the soil, the atmospheric N has a lower 15N signal than the soil and fertilizer N sources, and plants that fix the most atmospheric N have the lowest 15N signal. We calculate the percent N derived from atmosphere (%Ndfa) using an equation based on the 15N dilution in the N-fixing plants.

Initial screening of symbiotic bacteria for their ability to fix N in Leonard’s jar system. The left jar contains plants that were grown in no N growth medium, and seeds were not inoculated with the symbiotic bacteria (negative control). The middle plants were grown in a medium supplemented with N, and seeds were not inoculated. The right jar contains plants that were grown in no N medium, and seeds were inoculated with a novel symbiotic rhizobium.

*Variable nodule qualities produced by different symbiotic rhizobia.*

Evaluating N fixation in free-living bacteria using 15N

To show that a prospective inoculant or biostimulant product is able to fix N, we use a similar approach to evaluating symbiotic N fixation, but we grow the bacteria in pure culture in the lab instead of in soil in the greenhouse. To evaluate free-living N-fixers, the bacteria are first grown in an N-free medium with several media changes to ensure that all of the N in the culture is from the bacteria themselves. Once the culture has stopped growing, we incubate them them in a synthetic sterile environment enriched with 15N2. After incubating for two weeks in this 15N2-enriched environment, we collect the cells and analyze the 14N/15N isotope ratio by mass spectrometry. Known positive controls (e.g. Azospirillum brasilense) and negative controls e.g. Escherichia coli) are used to compare the novel bacteria.

*Experimental setup for evaluating free-living bacteria (left) and enriching the environment with* 15N2 gas (right).

Insight Plant Health has experience testing N fixation ability and efficiency in rhizobia, free-living bacteria, and legume plants. We can help support your product development, registration, and sales by providing concrete numbers to back up product claims.

Dave Greenshields 2/25/24 Dave Greenshields 2/25/24

Testing biostimulants at Insight Plant Health: phosphate solubilization

Phosphate solubilization testing

Over the last several years, regulators in the EU and US have sought to define and regulate biostimulants as a separate group of products based on their activity. The Technical Committee on Plant Biostimulants of the European Committee for Standardization uses this definition: Plant biostimulants are products, based on substances and/or microorganisms, stimulating plant nutrition processes independently of the product’s nutrient content with the sole aim of improving one or more of the following characteristics of the plant: nutrient use efficiency; tolerance to abiotic stress; or crop quality traits; and may be applied to plants or soils. In the US, the guidance more or less the same except where a product claims its biostimulant activity as a result of plant growth regulation, the product is regulated as a pesticide and requires a pesticide registration under the EPA’s Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA).

In both the US and EU, substances or organisms that increase the availability of phosphorus (P) to plants are considered biostimulants, and it is therefore important to establish whether a product can make this claim by showing that it increases P availability and how it accomplishes that feat. At Insight Plant Health, we’ve helped several customers show that their biological products increase the availability and plant uptake of P. This is how we do it.

In vitro phosphate solubilization

The simplest way to screen for organisms capable of solubilizing phosphate is to grow them on rock phosphate and see if it can break down that rock. I’ve seen this demonstrated at trade shows, where a sales rep will mix powdered rock phosphate with a solution containing a compound purported to be produced by their microbial product. They shake the jar and voila! The powdered phosphate is dissolved into the solution.

To accomplish this same test in the lab, we make petri dishes containing calcium phosphate, which increases the opacity of the agar. When an organism is actively solubilizing the phosphate in the agar, it creates a clearing zone that can be seen by eye.

*Clearing zones around a phosphate solubilizing microbe.*

The clearing zones that microbes make on calcium phosphate plates can be measured and provide a quantitative indicator of the strength of their P solubilizing ability. To better quantify solubilization activity, however, we generally use a liquid assay. In these assays, a known amount of calcium phosphate is added to a liquid medium where the organism of interest is incubated over a set period of time. The cells and insoluble phosphate are spun down in a centrifuge, and the soluble phosphate is measured in the supernatant with a spectrophotometer assay. When compared to a non-solubilizing or heat killed control, we can determine the amount of phosphate solubilized over the period of time. Performing the assay several times over the course of the experiment gives the amount of phosphate solubilized per unit time (i.e. the speed of solubilization) and weighing the biomass or counting the CFUs in the medium can give an amount of phosphate solubilized per unit of microbe (i.e. the efficiency of solubilization).

Testing the solubilization of insoluble inorganic phosphate by a biostimulant product only provides part of the picture of how a product could increase the availability of soil P. In soils with high organic matter content or soils fertilized with manure, much of the phosphate is unavailable to plants because it remains bound in inaccessible detritus and organic macromolecules. The process of organic phosphate mineralization involves the enzymatic hydrolysis of organic P compounds by soil microorganisms that produce phosphatases and phytases. These enzymes break down organic molecules, such as phytate, phospholipids, nucleic acids, and phosphoproteins, into simpler forms of P, including orthophosphate ions that can be taken up by plants. Production of these enzymes is part of a common microbial phosphate starvation response, and we measure them in the lab using enzyme activity assay kits designed for the purpose.

Measuring changes in phosphate availability and plant uptake

To get an idea of how a particular biostimulant product changes the availability of phosphates in the soil, we use a variation on the Hedley fractionation method (which was published while Hedley was a postdoc at the University of Saskatchewan - small world!). In pot experiments, we compare the water extractable P fraction to the Olsen extractable P fraction to the total P in samples with and without the biostimulant product to see how the product influences changes in phosphate availability. In the absence of plants, these assays are straightforward and provide an indication of the product's efficacy in situ. However, many microbial biostimulants are symbionts and may need the presence of a host plant to grow and reproduce in the soil. In these cases, the presence of the plant can confound the discrete separation of the P pools in the above experiment, as the plant is actively taking up phosphate with and without the help of the biostimulant.

*The phosphorus cycle in agriculture*

By Welcome1To1The1Jungle at English Wikipedia, CC BY 3.0, https://commons.wikimedia.org/w/index.php?curid=48498089

To more directly measure a biostimulant product’s ability to influence plant P uptake, we measure P concentrations in the plants themselves. Unsurprisingly, the biggest effects are usually seen when the experiment is conducted with low levels of available P, but we’ve worked with a couple of customer products that increase plant P uptake significantly in highly fertilized soils as well. For both the soil and plant assays, we’ve had success with both in-house methods and subcontracted ICP- mass spectrometry through the Saskatchewan Research Council.

OK, but how does the biostimulant product solubilize (or mineralize) P?

It’s all very well to say that a product is a P solubilizer, and many products make that claim, but few of the associated tech sheets and websites describe a mode of action (MOA) to support it. There are a number of ways that a biostimulant can increase available P and plant P uptake, and at Insight Plant Health, we have helped customers define their P solubilization MOA in both registered products and products still under development. In some cases, a microbial product may lower the pH of its environment to free up bound phosphate. Microbes that solubilize soil P often secrete small molecules like organic anions and siderophores that solubilize phosphate bound to metals (like calcium, iron and aluminum) in the soil. Measurement of organic anions like gluconate, citrate, and oxalate in either the soil solution or in vitro under phosphate starvation can show whether these compounds contribute to the solubilization MOA. Similarly, siderophore assays can also gauge the contribution of these metal-binding compounds to P solubilization. Where an enzymatic means of P mineralization is responsible for a product’s activity, we measure phosphatases and phytases produced by the microbes in pot experiments or in vitro. Whether a product's MOA comes down to organic anions, siderophores, enzymes or simple acidification, we’ve also had success correlating those changes with changes in gene expression in the microbe, which can help to bolster the specific MOA claim.

One area that’s a bit controversial from a regulatory perspective is improved phosphorus nutrition through plant growth promotion. Every spring, X (Twitter) is flooded with side-by-side images of control plants with small roots beside plants treated with a biostimulant that have noticeably larger roots. To follow the EPA guidance in the US, these products should be registered through the EPA FIFRA route if they are sold with a claim of plant growth promotion. However, by sticking with a claim of P solubilization and having the data to back it up, the product can be exempted from EPA FIFRA registration as an inoculant or soil amendment. In March, 2023, a new bill, The Plant Biostimulant Act of 2023, was introduced in the US senate that would define biostimulants and exclude them from regulation under FIFRA. Regardless of the regulatory path chosen for a product, it’s never a bad idea to know exactly how that product works. This foundation helps to sell the product to the sales team, and it gives them a solid message to relay to your customers.

Contact Insight Plant Health today to help develop the real story behind your product!

Dave Greenshields 2/16/24 Dave Greenshields 2/16/24

Making disease inoculum for field trials

Every growing season, new crop varieties and crop protection products are tested in field trials across Canada. To score disease resistance in new varieties and to test the efficacy of new crop protection products, researchers often introduce the diseases of interest to their test plots using lab-produced disease inoculum. At Insight Plant Health, we manufacture hundreds of kilograms of disease inoculum every spring for customers across Canada. This is how we do it.

Isolating the pathogens

During the summer, our lab receives various crop samples for disease identification. In the course of identification, we often isolate the pathogen in pure culture by incubating parts of the diseased plant on agar followed by successive subculturing until a single fungal or bacterial strain is isolated. We supplement these in-house isolates with disease isolates from other labs or from the customers themselves if they have a particular strain they’re interested in. In practice, all of the strains we produce for field trials have come from our own isolations or other companies because governments and universities needlessly prohibit most uses of their strains with restrictive material transfer agreements. Because the pathogens will be released back into the environment, we follow the Code of Practice for Plant Pathogen Trials, which is a set of rules developed by the Canadian Phytopathological Society and CropLife that outlines how to safely produce and use disease inoculum in field and greenhouse trials. All of the strains we use have been identified by DNA barcoding, which is a standard method for identifying and classifying living things.

*Various fungi isolated from diseased plants at Insight Plant Health*

Preparing the substrate

Once we have the isolate in hand and a customer orders the inoculum, we prepare growth substrates to produce the pathogen at scale. At Insight Plant Health, we produce both liquid and solid disease inoculums, but in this post, I’ll describe the production of solid inoculum.

For field scale trials, we generally use grain as the growth substrate. For most of the pathogens we grow, we use a blend of wheat and corn, although some prefer corn or wheat alone, while others grow better on rye or barley. The first step in preparation of the substrate is to soak the grain in water. The soaking time depends on the grain, but overnight soaking works in most cases. Once soaked, the excess water is removed from the grain by sieving and the grain is transferred into autoclavable growth bags.

*Soaked grains in autoclavable growth bags*

We have tried a number of different growth bags for inoculum production, but have had the best success with Unicorn brand mushroom spawn bags. These bags are fitted with a filter patch that allows for gas exchange without contaminating the grain inside. Once the bags are filled with grain, they are transferred to an autoclave for sterilization. The grain bags are autoclaved for 40 minutes at 121°C and 15 psi to ensure that the grain is free from any potential contaminants ahead of inoculation with the pathogen. Autoclaving also has the advantage of killing all of the seeds, which prevents the inoculum from making a big mess of the customers’ plots.

Inoculation and growth

Once the grain has cooled to room temperature, it is ready for inoculation with the pathogen. Depending on the isolate, the grain can either be inoculated using a liquid culture or several petri dishes chopped up into small pieces. The petri dish method tends to lead to less contamination. The inoculated growth bags are sealed with a heat sealer.

*Chopped up agar containing the pathogen spread into the sterilized grain*

The growth bags inoculated with the pathogen are left to grow either in the lab or growth chamber, depending on whether growth or development of the pathogen is encouraged by light. We track the colonization of the grain over time, and generally let the growth go until the starch from the seed is thoroughly colonized by the pathogen when a seed is split open. This complete colonization usually takes 2-4 weeks.

Drying and packaging

The colonized grain has to be dried before it can be used by the customer. To get the excess water from the grain, we cut the bags open and leave them in the biosafety cabinet overnight with the fan running. We then transfer the colonized grain into trays and store them in a portable enclosure with a dehumidifier running until they are <15% moisture.

Once dry, the grain is packaged in vacuum bags or pails with silica gel packs to keep it dry and then shipped out to customers for field application.