Testing biostimulants at Insight Plant Health: phosphate solubilization
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).
Be careful what you claim.
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!
