Introduction

Our global food production system is facing several sustainability problems. Going forward, the world may face wide reaching threats to longevity with the growing population expected to require at least 50% more food by 2050. This would require agricultural land of nearly twice the size of India and lead to a 275% above target contribution to greenhouse gas emissions by agriculture (World Resources Institute—https://research.wri.org/wrr-food). Already today, climate change is impacting the crops, with, e.g., plants being extremely vulnerable to heat waves and leading to new plant pathogens to which no tools have been developed yet. At the same time, an estimated 30% of all the food produced is still wasted along the food supply chain.

Building a resilient and sustainable food production system can be seen as one of the most important responsibilities of the current generation. Taking on this task, the European Green Deal initiative is pushing toward an unprecedented change in agricultural practices, for instance by proposing to reduce chemical pesticides use with 50% by 2030. In addition, other initiatives such as the EU Biodiversity strategy and the Farm to Fork strategy are calling for respect and restauration of our European biodiversity and sustainable food production systems.

The problem with chemicals

Conventional chemical pesticides are generally considered as a likely source of human health and environmental risks (Samada and Tambunan 2020). In 2018, 4.1 million tons of food protection products, which are currently mostly chemical based, were used to preserve the production of our plant-based food (http://www.fao.org/faostat/en/?#data).

Furthermore, an analysis performed by the European Food Safety Authority (“EFSA”) on selected food products showed that 40% of the tested food samples in 2018 contained one or more pesticide residues. For the category of fruits and tree nuts, almost 60% of the samples showed detectable residues with more than 4% above maximum authorized levels (EFSA,. https://www.efsa.europa.eu/en/interactive_pages/Pesticides_report_2017). As a consequence, it is no surprise that the collective sentiment of the broad population is one of the aversion against chemical pesticides.

Highly regulated territories are now banning chemical pesticides with increased pace and so depleting the toolbox of the farmer. The need for alternative solutions is, therefore, urgent and pronounced.

The need for sustainable solutions

The resilient food production system of the future will require to work on different key elements, such as the best genetic techniques, land and water management and switching to more plant-based diets. A key driver, however, is the development of effective biocontrol products, for which the requirements are high. They should be effective, have no harmful effects to humans and the environment, leave no chemical residues and act in a specific way.

Many innovative approaches to search for these effective biocontrols have been initiated. The biological food protection market has been growing at rates well above the conventional chemical food protection market, and for the past 10 years at rates of around 15% per year, expected to reach $7.7 billion, by 2025. Although, the current existing biocontrol products do have generally a cleaner safety profile and are well accepted in the market, most of them are still unable to match the efficacy and consistency of conventional chemical alternatives.

Using its proprietary technology platform: The AGROBODY™ Foundry, Biotalys aims to develop products that will help reduce the agricultural environmental footprint, optimize the use of our natural resources and provide healthy and safe choices for consumers. AGROBODY™ biocontrols demonstrate a biological-like clean human and environmental safety profile, due to their intrinsic rapid biodegradability, while providing conventional chemical-like performance, thereby addressing a key shortcoming of most biological food protection products that are typically less consistent and effective when compared to conventional chemical food protection products. In addition, this technology platform enables the identification of novel modes of action where conventional chemical innovation has been challenged over the past decade and where biological products do not usually provide a clear and single type of mode of action.

Biotalys’ AGROBODY Foundry™ platform allows rapid identification, development and production of novel antibody-derived protein-based biological food protection (or “biocontrol”) product candidates that have the potential to address a broad range of biological food threats with novel modes of action. Antibody-derived proteins have already demonstrated their potential as therapeutic molecules across multiple pharmaceutical applications. Biotalys is demonstrating the potential of this technology across multiple food and agriculture applications.

AGROBODY™ bioactive and AGROBODY Foundry™

The basis of the AGROBODY Foundry™ platform was originally discovered at the Vrije Universiteit Brussel (VUB) in Belgium (Hamers-Casterman et al. 1993). The invention is based on the observation that Camelidae, the family which includes camels, llamas and alpacas, in addition to generating conventional antibodies, also possess a different class of antibodies (Fig. 1). These antibodies lack the CH1 domain and the light chains, but still have the full antigen-binding capacity of conventional antibodies. In these “heavy-chain only” antibodies, antigen binding occurs through a single variable domain (“VHH”) which is the smallest functional fragment of a naturally occurring heavy-chain antibody.

Fig. 1
figure 1

Conventional antibody (left), heavy-chain antibody (middle) and AGROBODY.™ bioactive (right)

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The AGROBODY™ bioactive

Endowed by natural evolution, antibody-derived proteins exhibit unique biophysical, biochemical and pharmacological characteristics and have received considerable academic and industry-based research over the past 30 years in the field of drug development for human (Van Bockstaele et al. 2009) and animal use, but also in their development for diagnostic tools (Huang et al. 2010), biosensors and applications in plant and food protection (Vincke and Muyldermans 2012, Hemmer et al. 2017, Kourelis et al. 2023).

Specific applications in plant and food protection have been limited to academic research activities in view of the much greater importance of the production cost and scale in the agricultural and food industry. Biotalys has now developed VHH for use in the agricultural and food industries, dubbing them as AGROBODY™ bioactives.

AGROBODY Foundry™, a unique and scalable proprietary technology platform for effective, environmentally safe and clean protein-based biocontrol solutions, with multiple possible applications

Biotalys’ AGROBODY Foundry™ platform is a unique and scalable technology platform that allows the development of AGROBODY™ bioactive protein-based biocontrol product candidates to target multiple indications, such as bunch rot, powdery mildew, etc., with novel modes of action. These novel modes of action make it less likely for a target organism to develop resistance, compared to the speed of resistance developed against widely used conventional chemical food protection products.

Due to their small size and specific structure and properties, AGROBODY™ bioactives are ideal building blocks for the generation of novel protein-based biocontrol products. They have multiple advantages:

  1. (a)

    They are produced by fermentation in simple microorganisms such as yeast followed by filtration steps, thus limiting the amount of energy and waste from the production.

  2. (b)

    They are designed to be applied by growers or industry professionals like a conventional chemical food protection product without the need to change farm equipment or to adapt distribution channels for specific temperature conditions as could be the case for certain microbial biocontrol products that require a more controlled environment.

  3. (c)

    They are designed to be easily introduced in growers’ integrated pest management (IPM) programs as alternatives to existing conventional chemical food protection products or to improve resistance management.

  4. (d)

    They are designed to be as effective and consistent as conventional chemical food protection products when used in the framework of an IPM program, but as harmless as microbial food protection products.

  5. (e)

    They are designed to be safe for growers and consumers, allowing a rapid re-entry in the field and short pre-harvest intervals.

  6. (f)

    They are by nature biodegradable in the environment in which they are used. Their stability is fine-tuned during the R&D process to allow maximum efficacy prior to their natural degradation into their amino acid building blocks, which potentially represent a source of nutrients for plants and microorganisms.

  7. (g)

    They are specific to the target diseases or pests. The mode of action and the spectrum of activity can be tuned during the R&D process, avoiding undesired impact on beneficial organisms and ecosystem.

Conventional chemical and microbial food protection product R&D platforms often require intensive scouting and screening in the research phases across large numbers of possible new lead candidates to find candidates that are effective against specific insects, fungi or microbes. Biotalys’ AGROBODY Foundry™ platform, in contrast, offers the advantage of generating AGROBODY™ bioactives specifically against the targeted insect, fungi or microbe. AGROBODY™ bioactives are designed to act against a given target through the immunization process, which has the potential to provide in a single step a broad range of active protein-based bioactives with different modes of action. By leveraging the natural and potent immune system of llamas, the probability of generating biologically active biocontrol product candidates with diverse modes of action and targeted biological activity is increased, clearly differentiated from existing chemical and microbial R&D platforms in the industry. As it comes to the scale-up production of the product, multiple fermentation hosts (yeasts and filamentous fungi) can be used, developed and engineered to produce AGROBODY™ bioactives at scale, leveraging scientific progress in synthetic biology (De Groeve et al. 2023). As compared to the multi-step chemical synthesis approach for conventional chemical pesticides, the one-step fermentation approach represents an effective approach to produce at industrial scale.

EVOCA™

To identify an active anti-fungal VHH (i.e., an AGROBODY™ bioactive), llama’s was immunized with a Folch lower phase lipid extract (Folch et al. 1957) from Fusarium oxysporum. VHH-encoding cDNA was amplified from peripheral blood mononuclear cells (PBMCs) purified from llama blood samples and cloned into a phagemid vector. The required VHHs are then enriched using the phage display technology allowing selection of antigen-binding VHH from the collection of millions of different VHH present in the library. After multiple rounds of panning, a selection of VHHs were produced in a microbial host for further characterization.

In order to screen for AGROBODY™ bioactives active against the plant pathogen Botrytis cinerea, purified VHH were tested for their antifungal activity in an in vitro antifungal assay. The purified VHH were, therefore, mixed with Botrytis cinerea spores in liquid medium and incubated for 72 h at 20 °C. Growth was continuously monitored using the IncuCyte S3 Live-Cell Analysis System (Sartorius GmbH, Göttingen, Germany), and confluence compared to non-treated samples was evaluated (Fig. 2). IC50 values (referring to the concentration (µM) inhibiting 50% of spore germination and/or mycelial growth) were determined using three replicates over three independent experiments and compared to a negative control (irrelevant VHH).

Fig. 2
figure 2

Comparison of the antifungal activity of the AGROBODY.™ bioactive in EVOCA™ and the irrelevant VHH (negative control) against Botrytis cinerea spores (n = 3)

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The AGROBODY™ bioactive that was further developed into EVOCA™ showed an IC50 value of 0.76 ± 0.10 µM. The IC50 value in this experiment is the inhibitory concentration of the AGROBODY™ bioactive at which 50% of the cell density relative to the untreated control is reached. Microscopical phenotypic observations show rupture of the spore wall during germination upon treatment with this AGROBODY™ bioactive (Fig. 3). We hypothesize that this effect is caused by an interaction of the AGROBODY™ bioactive with lipids in the cell membrane, affecting cell membrane integrity and impacting spore germination and hyphal growth, ultimately resulting in a fungicidal effect.

Fig. 3
figure 3

Fungicidal activity of the AGROBODY™ bioactive that was further developed into EVOCA™, visualized under the microscope over a time series as indicated in the pictures (up to 5 days after incubation). A Non-treated Botrytis cinerea spores. B Botrytis cinerea spores treated with the AGROBODY™ bioactive at a concentration of 20 µM

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Field trial data

After in vitro characterisation, EVOCA™ was tested in more than 500 field trials (efficacy trials and regulatory trials) over multiple seasons under different environmental conditions for product development and positioning, comparing its performance to conventional chemical and biological crop protection products. Trials were conducted by renowned specialized independent contract research organizations, according to industry standards and European and Mediterranean Plant Protection Organization guidelines (to comply with EU regulatory requirements) (Pscheidt and Ocamb 2023). The analysis of the robust data package provided the required insights for product positioning and developed user recommendations for growers under local and season-dependent environmental conditions and disease pressure.

EVOCA™ field trial results in grapes

EVOCA™ was tested in grapes field trials against two key diseases: bunch rot (Botrytis cinerea) and powdery mildew (Uncinula necator). Figure 4 shows the results over 26 trials where EVOCA™ with adjuvant replaced the chemical standard (cyprodinil + fludioxonil) at flowering in an IPM program. The program containing EVOCA™ and adjuvant at flowering performed at least as good as the full chemical program, while also reducing chemical pesticides residues on the harvested grapes.

Fig. 4
figure 4

Botrytis cinerea control on bunches in vines from 8 US trials in 2021 and 18 US trials in 2022. Untreated controls mean no treatment at timings A (flowering), B (bunch closure) and C (veraison). Chemical products were used at recommended rates. 95% confidence intervals vs. untreated control are [37.6–66.2%] for the 2021 chemical fungicide rotation, [62.7–80.2%] for the 2021 EVOCA substitution at flowering, [57.2–69.2%] for the 2022 chemical fungicide rotation and [63.4–73.6%] for the 2022 EVOCA substitution at flowering. (*) indicates a statistically significant difference between treatment programs. Meta-analysis p-value: 0.0019

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Figure 5 shows the result of four trials in EU of a standalone program of EVOCA™ compared to a biological and chemical reference (also standalone) against powdery mildew.

Fig. 5
figure 5

(left) Summary of EVOCA™ field trial in grapes against powdery mildew and (right) picture of damage caused by powdery mildew. Results are from three trials with 10 sprays and one trial with nine sprays. Synthetic reference: TOPAZE (100 g/l penconazole) was applied at 0.25 L/ha. Synthetic fungicide rotation means a program according to local practices, up to six different compounds. Biological references: Taegro (13% W/W Bacillus amyloliquefaciens strain FZB24), applied at 0.37 kg/ha; or Romeo (94.1% W/W Saccharomyces cerevisiae), applied at 0.25 kg/ha

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When used to protect vines against powdery mildew, EVOCA™ demonstrated its intrinsic activity. Standalone applications of EVOCA™ can meet or exceed the suppression of leaf powdery mildew observed from standalone applications of leading biologicals on the market.

EVOCA™ field trial results in fruity vegetables

In the context of the results of the vegetables field trials, Evoca™ has been tested against powdery mildew in tomato and cucurbits.

Figure 6 shows the results of EVOCA™ at two doses, in comparison with chemical and biological references in a standalone program. EVOCA™ performance for the proposed regulatory rate (375 g ai/ha LWA) reaches 79% disease control, comparable to the level of powdery mildew control provided by the biological reference Bacillus pumilus. In comparison with the standalone synthetic references, EVOCA™ outperformed metrafenone, however, underperformed versus the difenoconazole + fluxapyroxad premixture.

Fig. 6
figure 6

(left) Summary of EVOCA™ field trials in tomato against powdery mildew from 3 EU trials in 2022 and (right) picture of damage caused by powdery mildew on tomato leaves. Results are from 5 weekly sprays (ABCDE) starting before the first infection. Synthetic fungicide rotation: trial 1: (AB) sulfur/(C) bupirimate/(DE + difenoconazole + fluxapyroxad); trial 2: (AC) metrafenone/(B) azoxystrobin/(D) penconazole/(E) boscalid + pyraclostrobin; trial 3: (AB) difenoconazole + fluxapyroxad/(BD) penconazole. Synthetic reference: trial 1: Dagonis (50 g/l difenoconazole + 75 g/l fluxapyroxad), 0.6 l/ha; trials 2&3: Vivando (500 g/l metrafenone), 0.3 L/ha. Biological reference: trials 1&2&3: Sonata (1.38%W/W Bacillus pumilus QST 2808), 8 à 10 l/ha. Product names: Thiopron ( 825 g sulphur/l), 6 l/ha; Nimrod 250 EW (250 g bupirimate /L), 2 l/ha; Dagonis (50 g/l difenoconazole + 75 g/l fluxapyroxad), 0.6 l/ha; Vivando (500 g metrafenone /l), 0.3 l/ha; Ortiva (250 g/l azoxystrobin), 80 ml/100 l; Topaz or Penkona 100 EC (100 g/l penconazole), 0.5 l/ha and Signum (26.7%W/W boscalid + 6.7%W/W pyraclostrobin), 100 g/100 l. All products were used at the recommended dose rate

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Figure 7 shows the results of EVOCA™ at two dosages, in comparison with chemical and biological references in a standalone program. EVOCA™ performance for the proposed regulatory rate (375 g ai/ha LWA) reaches 73% disease control, comparable to the level of powdery mildew control provided by the biological references.

Fig. 7
figure 7

(left) Summary of EVOCA™ field trial in cucumber against powdery mildew from 4 EU trials in 2022 and (right) picture of damage caused by powdery mildew on cucumber leaves. Results are from 5 weekly sprays (ABCD (E*)) starting before the first infection. Synthetic reference fungicides: ABCD(E)* difenoconazole + fluxapyroxad (2 trials); ABCDE metrafenone (1 trial); ABCDE penconazole (1 trial). Synthetic fungicide rotation: AB sulfur/C bupirimate/DE dimethomorph + pyraclostrobin (1 trial); ACE difenoconazole + fluxapyroxad/BD penconazole (1 trial)*; AC difenoconazole + fluxapyroxad/BD penconazole (1 trial); AD myclobutanil/BE penconazole/C boscalid + pyraclostrobin (1 trial). Biological reference: ABCD(E)* Bacillus pumilus (3 trials); ABCDE potassium hydrogen carbonate (1 trial). Product names and dose rates: Thiopron ( 825 g sulphur/l), 6 l/ha; Nimrod 250 EW (250 g bupirimate /l), 1 l/ha; Dagonis (50 g/l difenoconazole + 75 g/l fluxapyroxad), 0.6 l/ha; Vivando (500 g metrafenone /l), 0.2 l/ha; Topaz or Penkona 100 EC (100 g/l penconazole), 0.5 l/ha; Signum (26.7%W/W boscalid + 6.7%W/W pyraclostrobin), 1.5 kg/ha LWA; Cabrio duo (72 g dimethomorph + 40 g pyraclostrobin/l), 280 g ai/ha; Systhane 20 EW (200 g myclobutanil/l), 0.15 l/ha LWA; Sonata (1.38%W/W Bacillus pumilus QST 2808), 8 à 10 l/ha and Karma (85%W/W potassium hydrogen carbonate), 1.2 kg/ha LWA. (*) one trial received only 4 sprays

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Field trials with EVOCA™ consistently demonstrate a potential best in class product with an efficacy comparable to conventional chemical reference products under low to moderate disease pressure when used in an IPM program.

Safety of EVOCA™

Apart from its performance, various studies were performed by external, accredited laboratories to assess the safety profile of EVOCA™. The results of the study confirm that EVOCA™, as a result of its design, has a beneficial toxicity profile and is safe for humans, mammals and the environment.

Toxicity profile of EVOCA™

The safety of EVOCA™ for mammals, including humans, was assessed as per industry standard, through six toxicological effect studies in rats (so-called “six-pack” studies). These relate to exposure to the product through oral intake, dermal exposure, inhalation, skin irritation, eye irritation and skin sensitization. Each of the endpoints in these studies (indicator for the toxicity level, Table 1) shows that no classification is required.

Table 1 Endpoints of six-pack study with EVOCA™
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Ecotox profile of EVOCA™

EVOCA™ was also assessed on its (lack of) toxic effect on beneficial plants and organisms and the environment (so-called “ecotox”), such as aquatic life (fish, water insects and algae), bees and birds. Each of these ecotox studies shows the beneficial profile of the product for the environment, considerably more beneficial than the existing chemical products (Table 2).

Table 2 Ecotox profile of EVOCA™. National depending legislation instructs how these results need to be used for the purpose of risk assessments and labeling of the final product. For example, in Europe, the CLP-classification principles do apply (ECHA, https://echa.europa.eu/documents/10162/23036412/clp_en.pdf/58b5dc6d-ac2a-4910-9702-e9e1f5051cc5), while in the US, EPA’s guidelines for ecological risk assessment do apply (EPA, Guidelines for Ecological Risk Assessment (epa.gov))
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Biodegradability testing of the active substance of EVOCA™ and residues in soil and water

The active substance of the EVOCA™ is a protein fragment (“AGROBODY™ bioactive”) of 13.2 kDa. Biotalys has performed various studies on the biodegradability of the purified protein of EVOCA™, the technical grade active substance (TGAS) and the dry technical grade active substance whereby the water was extracted.

As expected, the results of each of these studies show readily biodegradability of the tested substance. Therefore, due to the nature of the protein fragment and its characteristics regarding its fate and behavior in the environment (readily biodegradable), the existence of relevant residues of the active substance in the soil and water derived from the application of EVOCA™, and any adverse impacts on soil microorganisms are considered as very unlikely.

In conclusion: EVOCA™ as the first biofungicide from Biotalys’ AGROBODY Foundry™ platform, has the potential to be a differentiating biocontrol for several reasons:

  • Best-in-class plant safe formulation, similar to conventional chemical plant protection products in terms of product quality, shelf-life and mixing and spraying attributes

  • Activity confirmed against known fungicide resistant Botrytis isolates, making EVOCA™ a good tool to manage resistance in Botrytis in the context of an IPM program (data not shown).

  • New modes of action/new Fungicide Resistance Action Committee (“FRAC”) code: Group 51 under F10. FRAC codes are important tools for growers to select different modes of action for use in a given season to optimize product rotation. A new mode of action and FRAC code provides more flexibility and options to manage resistance.

  • The ready biodegradability prevents accumulation in soil and the environment.