The secondary metabolites of microbial biocontrol products: Reflections from an Analytical Chemistry Scientist on Regulatory Developments

By Stephen Pierson, Analytical Chemist, Certis Biologicals 

Microbes and the secondary metabolites they produce

In the world of crop protection, biocontrol products are looked upon as an environmentally friendly alternative to reduce the input of conventional (synthetic) pesticides on agriculture fields and non-agricultural ecosystems. Biological pesticides utilize microorganisms, fermentation by-products, pheromones or extracts of substances that occur naturally in our ecosystem to ward off pests and diseases. There are a variety of mechanisms in which microorganisms can function, but one of them is by producing secondary metabolites which can destabilize and kill both pathogens and insects. What makes these secondary metabolites unique is their specificity, a molecule that has been designed by the organism over several thousands of reproduction cycles to fight for its survival, against harmful organisms and help their allies growing in the soil. These secondary metabolites can range from small molecules to longer peptides, and their efficacy against plant pathogens are an important tool for growers. The production of these metabolites can be seen in both fermentation and under natural environmental conditions. For non-live biological products, generally, the organism is fermented to produce a concentrated amount of secondary metabolites and then it is killed or inactivated, leaving only the secondary metabolite(s) as the active ingredient(s). In the case of living products containing the viable microorganism, the organism can produce secondary metabolites in fermentation that continue to produce these secondary metabolites, if the pathogen is present, after they have been sprayed thus providing extended protection. This secondary metabolism can play a huge role in why certain strains of beneficial bacteria or fungus show higher efficacy on pathogens than other strains of the same organism. Due to evolution, some strains have specially curated their metabolism into making molecules which destroy pests that infect and jeopardize our food supply. Certis Biologicals’ Bacillus amyloliquefaciens strain D747, for example, has a nice profile of three different classes of secondary metabolite lipopeptides, iturins, fengycins, and surfactins that give our Double Nickel/Amylo-X products outstanding efficacy on a variety of fungal and bacterial plant pathogens. 

Advances in Analytical Technology

With major advances in analytical chemistry instrumentation and sample preparation techniques over the last 20 years, detecting even the smallest quantities of these metabolites has become possible. Today, mass spectrometry (MS) detection has become a mainstream analytical method which can routinely detect molecules at the low parts per billion (ppb) level, and in some cases can reach parts per trillion (ppt) limits of detection. For the non-scientist, the best way to describe the scale at which these instruments work is to look at the entire population of China, which has approximately 1.4 billion people, and being able to single out 1 person in that entire population. This is the power of these methods and detectors. MS detectors can work with a variety of analytical instruments but most commonly they are paired with high performance liquid chromatography (HPLC) and/or gas chromatography (GC) systems which help separate both polar and non-polar molecules before detection. 

Despite the power of HPLC-MS and GC-MS instruments, testing metabolites from complex matrices still provides an analytical challenge. Each different matrix can provide different interferences when detecting secondary metabolites at the ppb level which is why there are not established methods in place for extracting and analyzing these compounds. While this analytical power has allowed scientists and companies to learn more about their products and interactions these molecules may have, regulatory agencies have also placed biological products under higher scrutiny due to these testing capabilities, treating them more and more like conventional chemical pesticides. Since there are a variety of these secondary metabolites produced by organisms at, or above ppb levels, some regulatory agencies have set limits on certain metabolites without conclusive data determining that they are actually harmful. And often times, proving the negative – absence of hazard – is a limiting factor on bringing new microorganisms under development to market.

The relevance or non-relevance of the presence of these metabolites

Public literature reviews remain an essential part of regulators’ safety assessments for microbial products and their metabolites. In some cases, this data can be relevant, but in others the data is not representative of what the metabolite does in nature. Does the metabolite degrade? Is it soluble in water? How does it make it to the consumer after it has been diluted 1000-fold when sprayed onto the field? Is it appropriate to extrapolate cell-level effect data on a petri dish to the organism level? These are all questions that should be answered before putting a limit on the concentration of certain metabolites in the final product. Furthermore, it is very often the case that the metabolite of interest that is being regulated does not exist as a commercially available standard material, leading to very expensive costs procuring a non-certified standard material from an academic institution or elsewhere to quantify the amount of metabolite in the product. And in some cases, the metabolite that is under regulatory scrutiny may be the one that is responsible, or a major contributor, for the efficacy of the product. Limiting the level of such metabolites, in some cases without reasonable scientific evidence, is limiting the availability of biocontrol products to the growers.

The ″need to know″ and its implication on innovation

The time and expense it takes for companies to renew registrations, extend label uses, improve formulations, and register new products stifles innovation and threatens the food supply as plant-destroying pathogens and insects co-evolve and become harder to control. This evolution is part of the biology of the target pest/disease organism (host) and microbial-based products need to keep up with such evolution of the target to remain effective.

If the product that is being used naturally occurs in soil and/or in the plant, and has been used for several years (in some cases, several decades) for crop protection, without reported negative effects on people consuming food with these products applied, why are we not considering this as evidence that it is safe to use? Making it harder to register and renew these products for use on crops is impairing innovation and exhausting resources in development departments and regulators alike. Should we trust years of evidence that eating food treated with these products with a sample size of millions of people is safe, or should we trust an experiment done on a petri dish telling us that a naturally occurring metabolite might be harmful at the ppb level? I understand that regulation exists to protect the people, and it must exist, but the question we need to ask ourselves today is: has it gone too far? 

Note: Certis Biologicals strongly supports biocontrol regulations and regulatory data to demonstrate the safe use of biological products for crops, humans, and the environment. The opinions expressed herein are those of the author and do not reflect the opinions or views of Certis Biologicals, the website, or its affiliates.