Croda: How to select the correct components for your biopesticide-based products

Carlos Eduardo Oliveira da Silva, Lead Researcher at Croda explores various methods that are available to assess the compatibility of formulation components with microorganisms, to produce highly stable biological products. Methods discussed include Croda’s new patented capability, highlighting how this provides additional information to make more informed decisions about formulation design.

For more in-depth information about these methods join one of Croda’s free, live webinars starting on 18 October 2021. Click here to register for the event.

The use of beneficial microorganisms to control pests and diseases in agriculture is well known and well established in current global agriculture. The use of biological control agents (BCAs) in agriculture dates back centuries with one of the first uses in China almost 5000 years ago, where citrus growers used predator ants (Oncophylla smaradina) to control other ants which were attacking the citrus tree foliage. The first application of virus and fungi to control pest insects is thought to have been introduced between XVIII and XIX centuries.

Since then, the use of BCAs has become more intensive and in some cases they control pests and diseases better than traditional pesticides. The improvement of formulation design and the development of new application technologies, among other factors, has contributed to the global biopesticide market growing to US$4.3 billion in 2020, with the projection to reach more than US$ 8 billion by 2025. However, even with these advancements, there is still a need for innovation in this area to produce successful products that are accepted by the market. The analysis method to assess the effects of formulation components (and the formulation itself) on microorganisms’ survival is one of the most important steps in the biological-based product development program.

Specifically in this article, we are going to discuss how to evaluate the effect formulation components have on microorganism survival. There are several methods to do this ranging in complexity, ease of use and level-of-detail obtained, each one with its advantages and disadvantages.

1. Inhibition zone method or Kirby-Bauer test

In the inhibition zone method or Kirby-Bauer test, it is possible to analyze the effects of different components on microorganisms in a quick and easy way (with some adaptation depending on the product characteristics). Example results obtained using the Kirby-Bauer test are presented in figure 1.

Figure 1. Results obtained using Kirby-Bauer method to evaluate the effects of different component on Bacillus sp. in Muller-Hinton-Agar (MHA) media. A is the control sample (only sterilized water), B shows a mild negative effect and C shows a severe negative effect (Source: Internal data).

This method is widely used in microbiology laboratory routines due to it being fast and easy to perform. However, the inhibition zone, regardless of the size, does not provide information related to the kind of effect. For example, in this specific case it doesn’t determine if the component is bactericidal or bacteriostatic. Additionally, this method only provides visual information meaning that more comprehensive conclusions must be correlated with previous information related to the microorganism and/or application technology that will be used in the field. This test is useful to indicate a good starting point to begin the formulation screening program. We use it at Croda to determine, in a simple way, which products can be used in the formulation development process.

2.    The colony-forming unit (CFU) method

Another method well known and largely used is the CFU method. This method is used to estimate the number of viable cells (bacteria or fungi) to determine the concentration of microorganism in a sample and to infer the capacity of microorganism to produce colonies in a specific environmental condition. For instance, the results using the CFU method can be observed in figure 2.

 

Figure 2. Results obtained using the colony-forming unit method (CFU). In this image, it is possible to observe how the different dispersant purity grade can impact the number of colonies of Trichoderma asperellum after three days of incubation in potato-dextrose-agar (PDA) media at 25℃ (Source: Internal data).

The CFU method presents several advantages; it is easy to implement (no sophisticated equipment or specific devices are required), the results can be obtained after a few hours (depending on the kind of microorganism and/or strain you are analyzing) and it is widely used by the market to assess the shelf-life of commercial products. This method only provides an estimate of viable cells because the colonies can rise from one or more grouped cells, providing a non-accurate value of how many cells are viable in a sample. Viable cells that agglomerate can be miscounted as a single colony which is one reason why results are expressed in CFU/mL (or grams) instead cells/mL (or grams).

3. The conidia germination method (or direct viability) method

We also use the conidia germination method (or direct viability) which is specifically used for fungi conidia. When compared with the two previous analyses, this method provides an accurate value regarding the number of viable conidia in a sample. Moreover, using this method, it is possible to determine the germinated conidia, non-germinated but activated conidia (that is viable conidia but not germinated due the short period of incubation) and unviable conidia, all in a single evaluation. Using this method, it is possible to collect precise information about how the components (or complete formulation) are affecting fungi survival. The results using this method can be obtained after a few hours (depending on specie or strain you are analyzing) and it is possible to evaluate a long-term effect of different components on conidia viability. In some cases, this method can determine if the component has biocide or biostatic effects over time. Example results using this method are presented in figure 3.

 

Figure 3. Results obtained using the conidia germination method (or direct viability method) for Trichoderma asperellum after 15 hours at 25℃ in potato-dextrose-agar (PDA) media. Here, it is possible to observe how the regular (A) and high-purity grade surfactant (B) affected the conidia viability. The red arrows show the unviable conidias and the green arrows highlight the germinated conidia and viable non-germinated conidia.

Using this method, it is possible to observe visually, how the formulation components can affect the fungi conidia viability, providing an accurate result about the component and its effects on fungi conidia, once the result is expressed in percentage of viable conidia. One of the disadvantages of this method is the time taken, mainly during the evaluations. To perform the evaluation, it is necessary to use an optical microscope with the magnification capacity of at least 100 times with an embedded high-definition camera. It also requires precise environmental control during the incubation period as any deviation in temperature during this stage can drastically influence the results.

4. Croda’s new PrecisionBio™ method

It is important to evaluate, not only the germination capacity, but also the conidia vigor. Some products can specifically affect the conidia vigor, affecting the fungi performance when applied in the field. To evaluate this aspect, we developed a software called PrecisionBio™. This digital capability processes images obtained via the optical microscope to determine the conidia vigor over time. Figure 4 displays results obtained using the PrecisionBio™ software.

 

Figure 4. PrecisionBio™ software interface and the results obtained after image processing. In A, the PrecisionBio software user interface, in B, raw data related to conidia vigor over 180 days after formulation process and in C, one of the possibilities to analyze the data set obtained using the software (as example, it was calculated the area below curve of conidia vigor. The column with different letter is statistically different by  Dunnett test with α = 0,05).

Using the software, it is possible to determine the number of germinated conidia and also, a value related to conidia vigor. With the raw data, it is possible to analyze how each formulation (or component itself) can affect individually the conidia vigor over time. Additionally, it is possible to apply some statistical tools to define which component was less harmful to fungi conidia. One of the advantages of using this method, is to generate new information regarding the effects of components on conidia in a more convenient way, using the same images which were obtained in the conidia germination method. With more information about the behaviour of the conidia, when studied with common formulation aids and other inerts, the more assertive your formulation development program becomes, allowing you to save time during your study.

Closing remarks

In general, all methods presented here have their advantages and disadvantages. In contrast, all methods are complementary, but not comparable between each other. Using all these different methods, allow accurate information to be obtained regarding how microorganism can behave when exposed to different components. We recommend using all the methods discussed here in formulation development programs at different stages, allowing quick identification of which components are potentially causing issues in the formulation design, saving time and resource.

All the methods mentioned in this article are used in our microbiology laboratory for internal projects, mainly during formulation development projects. We can also combine these microbiology evaluations with our other capabilities which include in vivo tests (performed in our new Product Validation Centre) and application simulation studies (using our new spray chamber capability) to produce successful microbial pesticides that deliver optimum performance in the field.

To know more about our microbiology studies or other capabilities, contact us on cropcare@croda.com

Additionally, for more in-depth information about these methods join one of Croda’s free, live webinar starting on 18 October 2021. Click here to register for the event.