Reducing development time of microbial formulation with Atlox™ BS-50

The biological market is increasing year by year, and the formulation industry must keep pace with this growth by delivering innovation and contributing to faster development, thereby streamlining processes within the biological industry. To help bring products to market at a faster pace, Croda launched Atlox™ BS-50, a new product specifically designed for biologicals, with the purpose of optimising the formulation process.

Atlox™ BS-50 is a ready-to-use powder delivery system for spore forming microbes. Its simplicity in use means only the addition of the microbe is required to complete a wettable powder (WP) formulation, reducing formulation development time.  Components of Atlox™ BS-50 have been carefully optimised by Croda’s scientists to provide the best performance and have undergone viability testing to ensure they are compatible with common microbes, and therefore providing versatility for its use.

Atlox™ BS-50 can incorporate fungi and bacteria propagules such as Trichoderma spp., Metarhizium spp., Beauveria bassiana and Bacillus spp. providing microorganism stabilisation while also enhancing physicochemical stability in the application tank.

Formulation stability improvement with Atlox™ BS-50

In terms of physicochemical properties, in Croda’s research formulations with Atlox™ BS-50 demonstrated good efficiency in maintaining solid particles in suspension, generating more homogeneous spray mixtures. A formulation with 30% Trichoderma asperellum resulted in 78% suspensibility. This is an important characteristic for WP formulations, contributing to effective field application by preventing nozzle clogging, maintaining uniformity in the tank mix, obtaining better distribution of active ingredients in the spray, and consequently increasing the chances of achieving the target. The formulation also overcomes the high hydrophobicity of the spores, allowing them to wet and therefore be incorporated into water.

Image 1: A 30 wt% Trichoderma asperellum formulation with Atlox™ BS-50 (a) and the dilution in water (b)

Biological performance improvement with Atlox™ BS-50

Microorganisms naturally tend to lose their viability over time due to their metabolic activity, which is directly affected and accelerated by the conditions of the system and the type of propagule. In this sense, the formulation needs to act by providing a suitable micro-environment, protecting the microorganisms physically and chemically to maintain their integrity and lower metabolic activity to avoid an abrupt loss of their viability.

To showcase the biological performance of Atlox™ BS-50 a Trichoderma asperellum case study is presented here, consisting of 30 wt% conidia formulated with 70 wt% Atlox™ BS-50. The results are compared to pure conidia and a benchmark composition made of common powder formulation components on the market. Over the testing period (0 to 180 days), the samples were assessed to obtain biological parameters such as the number of colony-forming units (CFU), viability, and vigour (using Croda’s PrecisionBio™ software).

CFU results

CFU testing 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.

The Trichoderma asperellum samples underwent CFU testing with the results displayed in Figure 1. Trichoderma asperellum formulated with Atlox™ BS-50 exhibited a more gradual decline in the CFU number over time compared to pure conidia and the benchmark, indicating that Atlox™ BS-50 provides an environment where conidia stability is better sustained. 

Figure 1. Graph comparing the colony-forming units (CFU) obtained from Trichoderma asperellum formulation with and without Atlox™ BS-50 over time (0-180 days), compared to a benchmark. Note: The difference in the value on the first assessment is attributed to a dilution that occurred during the formulation process.

Conidial viability results

Conidia germination tests determine accurate values regarding the number of viable conidia in a sample. Examining the conidial viability results in Figure 2, the formulation with Atlox™ BS-50 promoted a lower decrease in conidia viability, while the pure conidia and benchmark showed a more pronounced decrease. These results are particularly significant in terms of field performance. A higher number of viable conidia available in the formulation consequently increases the possibility of the product reaching the target in the field. Additionally, the physiochemical properties of Atlox™ BS-50 enhance the distribution of propagules in the application tank, contributing to an improved distribution and coverage and the chances of reaching the target.

Figure 2. Graph comparing conidia viability obtained from Trichoderma asperellum formulation with and without Atlox™ BS-50 over time (0-180 days), compared to a benchmark.

The average general viability of conidia over the 180 days corroborates the results observed in the Figure 3. The highest percentage of viable conidia at the end of the testing period was from the formulation with Atlox™ BS-50, at around 81.3%, while the pure conidia and benchmark obtained 61.7% and 73.3% respectively.

Figure 3. Long-Term comparison of average Trichoderma asperellum conidia viability after 180 days. *Bars with the same letter are considered equal according to Tukey test (p< 0.05).

The data highlights the effectiveness of the Atlox™ BS-50 formulation in reducing the natural decrease in microorganism viability inside the package, contributing to the benefits obtained in its stabilisation over time.

Conidia vigour results

It is important to evaluate, not only the germination capacity, but also the conidia vigour. Some products can specifically affect the conidia vigour, affecting the microbe performance when applied in the field. To evaluate this aspect, Croda developed a software called PrecisionBio™. This digital capability processes images obtained via the optical microscope to determine the conidia vigour over time. Using the software, it is possible to determine the number of germinated conidia and, a value related to conidia vigour.

Based on the data obtained using Croda’s PrecisionBio™ software, it was possible to create a vigour trend for each sample (Figure 4).

Figure 4. Area under the curve of conidia vigour (AUCCV) and vigour conidia over 180 days obtained from Trichoderma asperellum formulated with and without Atlox™ BS-50 compared to pure conidia.

Conidia formulated with Atlox™ BS-50 presented the highest conidia vigour when compared with the benchmark and pure conidia. Additionally, the drop in conidia vigour throughout the 180 days, was reduced when the conidia was formulated with Atlox™ BS-50. This effect is represented by the area under the curve of conidia vigour (AUCCV). In this regard, the Atlox™ BS-50 presented the highest value (15125,85), followed by pure conidia (11471,85) and benchmark (11265,75). The conidia vigour information is extremely important in field conditions, especially when considering microorganisms that will act against pathogens and/or insects through competition or parasitism, allowing the biopesticide active to rapidly establish in target/soil, providing an improvement in its efficacy. 

The pictures in Figure 5 show the microscopy images of the samples on day 0 and after 180 days, showing the difference that was quantified by the vigour data mentioned previously.

Figure 5. Images comparing Trichoderma asperellum conidia germination obtained over 180 days, from formulations with Atlox™ BS-50, a benchmark, and pure conidia.

Concluding remarks

Atlox™ BS-50 simplifies formulating microorganisms as only the addition of the microbe is required to complete a wettable powder (WP) formulation, therefore reducing development time. It is compatible with common microbes, such as Trichoderma spp., Metarhizium spp., Beauveria bassiana and Bacillus spp., therefore providing versatility for its use. The case study presented using Trichoderma asperellum exemplifies the great potential of Atlox™ BS-50 as a powder delivery system that provides exciting results in a number of CFU, viability, and vigour studies after 180 day stability period.

To find out more about Atlox™ BS-50, click here.

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