Solvay: Enhanced Adjuvants & Co-formulants Toolbox for Microbial Biopesticides

Through the use of living organisms, natural substances or semiochemicals, biopesticides prevent or reduce damage from pests and pathogens, emerging as one of the most promising tools for sustainable agriculture. They complement the use and reduce the risk of resistance to synthetic agrochemical actives, thus strengthening integrated pest management (IPM) adoption. Growth drivers include consumer demand for organic and residue-free food, increased environmental, biodiversity and health concerns with some conventional pesticides, associated with a rising regulatory pressure on agrochemicals. In this context, European Union’s Farm to Fork strategy and the Brazil National Bio-Input Program set clear targets to favor this transition towards mitigation of conventional agrochemicals usage. Among biological control (Biocontrol) agents, the use of  beneficial microorganisms represent more than 58% of the total market share of biopesticides¹.

However, the global rate of adoption of microbial Biocontrol based solutions remains relatively low in comparison with conventional pesticides, with often less consistent biological efficacy reported in the field, in adverse conditions. Classical root causes analysis include lack of stability of the product during storage, less active material reaching the target and rapid degradation of the biopesticide once applied related to environmental factors. In this context, optimized formulation strategies are a key lever to enhance global bioefficacy of such biopesticides.

Adequate co-formulant selection is especially vital as it can lead to:

- Improved shelf life, with enhanced microorganisms viability

- Satisfactory wetting & homogeneous dispersion upon formulation dilution without agglomeration (no nozzles blocking etc.)

- Spray droplet size control for optimal coverage (foliar & soil)

- Improved wetting, adhesion, humectancy and rainfastness on target

- Improved viability/ survival rate of microorganism coated on seed

- Increased microbial activity against targeted pests and pathogens, and improved bioavailability of beneficial metabolites

There is however no universal solution for microbial biopesticides formulation: each beneficial microorganism species has different sensitivities to chemicals, water, temperature, environmental factors such as UV, drought and humidity. Depending on the mode of action of the microbial biopesticide and the surface targeted, the functionalities required for the final formulation may vary considerably, involving for instance the need for adjuvants bringing rainfastness, humectancy, increased wetting & solubilization.  For Bacillus subtilis or B. amyloliquefaciens biopesticides for example, the selection of co-formulants is crucial to achieve a good bioavailability and improve the solubilization of the lipopeptides cocktail produced by the bacteria during the fermentation process, for an adequate antifungal activity. Additionally, the specific strains and fermentation processes can have a substantial impact on final broth composition, impacting formulation methodology and biological performance.  

In this work, more than 60 co-formulants or adjuvants were evaluated to assess their impact on the viability of four key microorganisms either beneficial fungi or bacteria, among which two biofungicides (Bacillus subtilis (CCT0089), Trichoderma harzianum (CCT4790)) and two bioinsecticides (Bacillus thuringiensis (CCT2335), Beauveria bassiana (CCT3161)).

The selection of co-formulants was made, based on the functionality they can bring to the targeted microbial biopesticide, the relevant formulation type, EPA NOP regulation compliance criteria for some of them, and no or low hazards considerations.  The compatibility with the four microorganisms strains was investigated at several concentrations of adjuvants, from 1 to 5% (w/w) in water via zone of inhibition tests, an adapted agar disk-diffusion methodology (Balouiri et al. 2016)²: six blank disks of 6mm of diameter, saturated with each adjuvant solution were placed in a petri dish previously inoculated with 0.5ml of microorganism’s cells. Water and hydrogen peroxide (50%) were used as positive and negative control respectively. If the co-formulant inhibited the growth or killed the microorganisms, the radial length of the inhibition zone around the disk was measured. This methodology aims at anticipating potential viability issues, once microorganism strains are activated and germinate, upon formulation dilution and application.

Figure 1. Zone of inhibition tests with Bacillus thuringiensis: left side, adjuvant with no negative impact on microorganism growth; right side, adjuvant with significant inhibition on microorganism growth. *The bottom right disk in both plates represents the positive control (water), therefore, no inhibition was expected.

Table 1 shows an extraction of results obtained for 20 co-formulants:

Table 1. Zone of inhibition test, compatibility of co-formulants with 4 microorganisms
Dark green: no inhibition zone with 1% or 5% adjuvant; light green: slight inhibition with 5% adjuvant, no inhibition with 1% adjuvant; orange: significant inhibition observed at 1 and 5% adjuvant (radius inhibition zone [2 - 11mm]); nt: not tested
*compliant with EPA’s National Organic Program (NOP):  CAS# under  List 4A or List 4B, thus OMRI eligible

Interestingly, Soprophor^® BSU, 4D/384 and 796/P grades, Antarox^® 25-R-2, L-62 EO/PO copolymers, Antarox^® B600, Supragil^® RM/210-EI & Geropon^® T36 which are wetting or dispersing agents for wettable powders (WP), Water Dispersible Granules (WDG) or Suspension Concentrates/Flowable Concentrates (SC/FS), display a good biological compatibility with tested microorganisms strains. For B. bassiana, which needs typically to be formulated in a water-free medium such as a vegetable Oil Dispersion (OD) to keep the fungal conidia in dormant state for a suitable shelf-life, Alkamuls^® T20, RC or OL 40 are good emulsifiers options.

Geronol^® Odessa 01, a versatile ready-to-use vegetable oil dispersion basis, containing emulsifier & dispersant system, as well as rheology modifier for adequate stabilization, is also a particularly interesting formulation solution for B. bassiana or T. harzianum fungi.

After this first biocompatibility assessment, specific adjuvants were further tested to determine if they could favor microorganism growth upon dilution, and finally, application and efficacy trials were conducted. In the following example, a microbial growth study in presence of AgRHEA™ SticGuard, a natural retention and rainfastness agent based on guar gum, is presented: the microorganisms were separately inoculated in the experimental broth containing 1% (w/v) of AgRHEA™ SticGuard and the control media (without AgRHEA™ SticGuard). All flasks were incubated at 30°C, 150 rpm, for 96h, with sampling every 24h for colony counting or dry weight measurements. The growth rate was calculated and compared with control experiments. As shown on Figure 2, in presence of 1% (w/v) of AgRHEA™ SticGuard, a significant increase of microbial growth was observed for all microorganisms tested, indicating a viability enhancing effect.

Figure 2. Relative increase of bacteria or fungi growth rate with AgRHEA™ SticGuard.

The improvement of retention and rainfastness with AgRHEA™ SticGuard was further investigated through bioefficacy trials, in collaboration with BIOTRANSFER research service company. A commercial biofungicide based on a strain of Bacillus amyloliquefaciens was applied at a rate of 2.5kg/ha (300L/ha spray volume) with/without AgRHEA™ SticGuard at a dosage of 0.07%(w/v) or with a commercial synthetic latex at 0.1%(w/v) on grapevine leaves, with 6 replicates for each series. An artificial rain of 20mm or 40mm was then applied for some samples, prior to inoculation with calibrated suspension (10⁵ spores/ml) of Botrytis cinerea strain. 4 & 6 days after inoculation, the disease intensity was calculated in % of the total area of the leaf analyzed. The biological efficacies were determined from the AUDPC³ values, expressed in % vs untreated control.

As shown in Figure 3, compared with the control (without adjuvant addition) and the commercial benchmark, AgRHEA™ SticGuard not only promoted a better efficacy in absence of rain (from 12 to 22%), but also revealed a more important “synergetic boosting” effect in presence of mild rain (20-mm). This effect is considered to be related to the moisture-facilitated growth of Bacillus amyloliquefaciens or a redistribution of the product by the rain. Furthermore, under heavier rain simulation (40-mm), the biological efficacy of the reference system without adjuvant was annihilated, probably related to the complete wash-off of the biopesticide solution, while it was exceptionally well retained in presence of adjuvants, especially with AgRHEA™ SticGuard.

Figure 3. Adjuvant effect on biological efficacy of a commercial WDG Bacillus amyloliquefaciens with/without 2 rainwashing amounts (20-mm, 40-mm)

In summary, thanks to the multi-disciplinary approach of microbiology, phys-chem, and agro formulation expertises, a series of co-formulants/adjuvants were identified as interesting candidates for the formulation design of microbial pesticides. A natural biodegradable polymer AgRHEA™ SticGuard was evidenced to bring both bioefficacy enhancement and rainfastness improvement on microorganisms.

Additional technologies within the SOLVAY toolbox are also available to solve for example wetting/agglomeration/aggregation issues observed with microorganisms & their metabolites upon dilution. For on-seed application of biologicals, AgRHO^® BIOBINDER microplastic-free binder solutions or AgRHO^® S-Boost biostimulants range for synergistic combination with microorganisms are technologies that pave the way for enhanced biological efficacy in a sustainable way.


References
[1] Agrow Biopesticides, 2019
[2] Balouiri M, Sadiki M, Ibnsouda SK 2016. Methods for in vitro evaluation antimicrobial activity: A review. J Pharm Biomed Anal. 6(2): 71-79.
[3] AUDPC: Area Under Disease Progress Curve, Madden et al. 2007.

This article will be published in AgroPages '2021 Biologicals Special' magazine to be published this June.

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