Overcoming shelf-life hurdles: Solvay solutions for robust Oil Dispersions microbial biopesticides formulations

Under the global transition toward sustainable agriculture, biologicals become more and more important in the area of plant protection. Besides their eco-friendly and residual-free features desired in organic farming, they also complement the use of synthetic agrochemical active ingredients thus strengthening integrated pest management (IPM) adoption.

Among the different species of microbial biologicals, bacteria and fungi occupy an important place in today’s application area, e.g. Bacillus sp., Trichoderma sp., Beauveria bassiana, Paecilomyces lilacinus or Metarhizium anisopliae. For these biologicals to exert the best performance in foliar, soil or seed application, they are often processed and/or formulated to integrate desired properties when proposed into the market. Suitable formulations are homogeneous and stable mixtures of microbial actives, secondary metabolites in some cases, and inert ingredients which enable an easier-to-use and more efficacious product. Nevertheless, one of the major issues that slow down the global rate of adoption of microbial biopesticides is the limited shelf-life of concentrated formulations. Indeed, strong constraints on storage conditions, usually at low temperature, combined with the fear of final users for non consistent biological efficacy are the main limitations of such formulations.

Challenges in formulating fungi biopesticides

In order to prolong the shelf-life of microbial pesticides, a water-free medium is usually preferred to avoid premature germination. The most common way to achieve this is via the development of a dry formulation, such as wettable powders (WP) and water dispersible granules (WDG). However, although this may work well for most sporulating bacteria like Bacillus sp, many fungal spores are less robust toward the relatively high temperature during the industrial drying process.

 
In order to exclude water without going through tough physical conditions and facilitate product handling by final users,  liquid oil dispersion (ODs) form is often desired for fungal biopesticide formulations, usually based on white mineral oils or plant oils (e.g. soybean, rapeseed). An extra biological efficacy interest of combining entomopathogenic fungi with oils has also been highlighted in several studies [1-4], e.g. emulsified rapeseed oil was proven to interact synergistically with B. bassiana blastophores, resulting in increased target pest mortality. Hypotheses behind such improved efficacy include the prevention of beneficial fungal spores desiccation by thin oil layer, a better adhesion to lipophilic insect cuticles, or an easier dissemination of spores to infect susceptible parts of the insect body when formulated in oil [1-4].

Despite their proven benefit for biological efficacy enhancement, preparations based on oils remain complex and require emulsifiers, rheology modifiers, dispersants that often have detrimental effects on spore viability. Storage stability of fungal biopesticides over a long period of time at/above room temperature is a particular challenge due to the delicate nature of the fungal conidia. As expert in the field for over 40 years, Solvay is committed to provide innovative and high performance formulation aids and adjuvants to accelerate the global transition to sustainable agriculture, and has been developing specific solutions for OD formulation of fungal pesticides with good physical stability, shelf-life improvement and application characteristics.

Adequate emulsifiers for OD of fungal spores

The first challenge raised here for formulators would be to select mild co-formulants non-toxic to fungi (spores viability), with low water activity to avoid premature spore germination, so as to ensure both biological stability and targeted physical characteristics.

The compatibility of various emulsifiers was investigated with two conidial fungi Trichoderma harzianum (strain CCT 4790) and Beauveria bassiana (ATCC 7159) at several concentrations of adjuvants from 1 to 5% (w/w) in water via zone of inhibition tests, an adapted agar disk-diffusion methodology [5]. This methodology aims at anticipating potential viability issues, once microorganism strains are activated and germinate, upon formulation dilution and application. Among the ten derivatives tested, specific sorbitan esters or castor oil ethoxylated derivatives such as Alkamuls^® OL40, RC and VO2003 emerged as attractive emulsifiers technologies for these conidia species, Alkamuls^® VO2003 being a very versatile emulsifier technology towards a wide range of vegetable oils.

Table 1:  Promising emulsifiers based on ethoxylated sorbitan esters & ethoxylated castor oils for OD formulations of fungal spores.

AgRHEA™ OD-EASY, a robust ready-to-use solution for fungal biopesticides Oil Dispersions

The second series of challenges for formulators of microbial Oil Dispersions is to achieve on top of satisfying microorganism viability upon storage, a good physical storage stability of the formulation concentrate without major sedimentation or agglomeration (typically less than 15% syneresis). A good suspensibility and emulsion homogeneity upon dilution of the concentrate in water is also required, without particle aggregation which could cause nozzle clogging upon application.

AgRHEA™ OD-EASY was developed for such purposes, as an optimal all-in-one Oil Dispersion basis solution with a good cost-performance balance, including compatible emulsifying and dispersing agents, as well as a rheology modifier in a vegetable oil basis. This versatile solution provides formulations with a robust performance profile, extensive storage stability and homogeneous dilution characteristics.

Figure 1: An example of OD formulation of 20% of Beauveria bassiana in AgRHEA™ OD-EASY/soybean oil blend.

Figure 1 shows an example of OD formulation prepared by blending 20% of Beauveria bassiana (technical powder, 1.2x1011CFU/g) into a liquid carrier containing 45% of AgRHEA™ OD-EASY in soybean oil.

Microbial viability studies were carried out on Beauveria bassiana and Trichoderma harzianum by adding 5% of biological commercial products in 95% carriers (45% AgRHEA™ OD-EASY with 50% soybean oil) in a total amount of 20ml, and incubating each tube without agitation, according to the microorganisms specifications (Table 2). For comparison, soybean oil was used as a control. Each trial was performed in duplicates, and the viability evaluations were performed at 0, 7 days, 15 days, and monthly, homogenizing the tube before each sampling. To obtain a reliable quantification of the viable spores, serial dilution in 1% Tween 80 aqueous solution was performed, followed by plating of different dilutions in agar media. Agar plates were incubated until fungi colonies grew and could be counted. The final results were determined through the average of the counts of each microorganism (CFU/mL).

Table 2: Incubation specifications for each microorganism.

 [*] This study corresponds to an accelerated aging test, as in real conditions the formulated products would be stored in general under 4°C

Figure 2: Viability evolution (CFU/mL) of Beauveria bassiana and Trichoderma harzianum in different liquid carriers under accelerated conditions (25°C/30°C) over one month.

As shown in Figure 2, in both Beauveria bassiana and Trichoderma harzianum cases, the formulation in AgRHEA™ OD-EASY has the slowest microbial viability decay with time under the incubation conditions. At the end of one month, the viability stays above 107 CFU/mL in AgRHEA™ OD-EASY carrier, while decreases by 10 times in soybean oil (control). Another commercially known liquid carrier based on 2-ethyl hexyl lactate chemistry was also tested here, with a drastic detrimental effect on microorganism viability after one month storage. This superior shelf-life enhancement of different fungi conidia makes AgRHEA™ OD-EASY an ideal choice for various OD formulations of fungi.

Innovative bio-based liquid carriers for extended shelf life at room temperatures

Besides vegetable-oil-based solutions like AgRHEA™ OD-EASY, innovative bio-based liquid carriers are currently under development at Solvay, addressing an enlarged scope of fungal biopesticides with an even higher shelf-life enhancement target.

Microbial viability studies were carried out on Beauveria bassiana and Paecilomyces lilacinus according to the experimental procedures described before. From Figure 3 below, it can be seen that Carrier DV SolvExpe066 and Carrier DV SolvExpe067 showed excellent shelf-life enhancing properties across at least a 8-month span for both fungal biopesticides.

Figure 3: Viability evolution (CFU/mL) of Beauveria bassiana and Paecilomyces lilacinus in different liquid carriers under accelerated conditions (25°C) up to at least 8 months.

This paves the way for a new generation of concentrated formulations of beneficial fungi, with extended shelf-life at/above room temperature, enabling an easier handling of the product for final users and potentially better biological efficacy.

In summary, to tackle one of the key challenges in the adoption of microbial biopesticides accompanying today’s sustainable agriculture transition, different solutions for shelf-life enhancement of  fungal concentrated formulations (especially Oil Dispersions) were investigated at Solvay.

Three mild and versatile emulsifiers (Alkamuls^® OL40, RC and VO2003) were highlighted, showing good biocompatibility with major fungal biopesticides. Moreover, an all-in-one OD basis solution, AgRHEA™ OD-EASY, was developed for providing an extensive storage stability at/above room temperature and homogeneous dilution characteristics, while keeping a good cost-performance balance. Furthermore, other bio-based liquid carriers are currently under development,showing viability of Beauveria bassiana and Paecilomyces lilacinus in the formulations up to at least 8 months at room temperature.

Beyond solutions for shelf life enhancement, Solvay is committed to support biocontrol formulators in finding optimized formulation strategies for improved biological efficacy.  Additional technologies within the Solvay toolbox are available to improve wetting, retention/ rainfastness (AgRHEA™ SticGuard), dispersion homogeneity or to answer specific seed treatment requirements. For additional information please send your inquiry to our biocontrol technical expert, Ling Qi (ling.qi2@solvay.com).

References
[1] Prior et al., J. Invertebr. Pathol., 52 (1988), pp. 66-72
[2] Bateman et al., Ann. Appl. Biol., 122 (1993), pp. 145-152
[3] Ibrahim et al., Mycol. Res. 103 (7) : 901–907 (1999)
[4] https://doi.org/10.1016/j.biocontrol.2019.104106
[5] Balouiri M, Sadiki M, Ibnsouda SK 2016. Methods for in vitro evaluation antimicrobial activity: A review. J Pharm Biomed Anal. 6(2): 71-79.

The authors would like to thank Fernanda Costa, Anaïs Marquilly,  Mayara Gazeta, Vanessa Silva, Neilton Araújo, Marina Pessoa and Mayara Santa Fé for their contribution to the article.

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