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Project / Description
Description
Microbial biocontrol to protect cacao from Phytophthora, reduce chemical use, and support smallholder farmers. Built to be safe for farmers and ecosystems.
Problem & Impact
Cocoa crops worldwide are being devastated by black pod disease caused by Phytophthora species, leading to over 30% yield losses, threatening farmer livelihoods and the global chocolate supply.
Project Goal
Engineer Bacillus subtilis to detect Phytophthora and secrete antimicrobial peptides, creating a sustainable biocontrol spray that protects cocoa plants.
Why This Project?
Current fungicides are expensive, environmentally damaging, and losing effectiveness. A biological alternative offers farmers a safer, more resilient solution.
Technical Approach
Using a biosensing system that detects reactive oxygen species and elicitins, B. subtilis acts only in the presence of Phytophthora, ensuring targeted defense.
Abstract
Cocoa plants, the source of our beloved chocolate, are facing a serious threat. Across the world, farmers are experiencing devastating crop losses due to various climate change and pathogenic diseases. The most prevalent one is the black pod disease, caused by several Phytophthora species. In some areas, yield losses exceed 30%, which gravely impacts the livelihoods of millions and endangers the future of one of the world’s most loved foods [1].
As fungicides lose efficacy and the impact of climate change grows, there is an urgent need for alternative solutions. To address this problem, we are developing a microbial biocontrol agent by engineering Bacillus subtilis. The bacteria are modified to secrete antimicrobial peptides (AMPs) upon detection of Phytophthora species, preventing black pod disease.
Importance of Cocoa
Consumed by millions, cocoa can be found in many forms in food, beverages and even in pharmaceuticals. It is a major agricultural product that is exported to all corners of the world. In 2024, global production was estimated to be around 4.37 million tons [2]. Globally, Africa accounts for the largest share of cocoa production, followed by the Americas and Asia [3].
Cocoa plays a crucial role in providing employment for smallholder farmers and across the value chain. Globally, around 5-6 million farmers depend almost entirely on cocoa for their livelihoods, with cocoa contributing 60-90% of household income that is used to support their families, purchase food, and pay for education [3]. Moreover, cocoa plays an important role in the economies of the producing countries and provides a significant income stream for local governments and is a major contributor to their GDP. For Ivory Coast, its importance is the most apparent, as cocoa and derived products represent 30% of its total exports [4].
However, cocoa trade is not exclusively important for countries where cocoa is grown. The Netherlands and Belgium import large volumes of cocoa, process it, and then re-export the intermediate or finished products [5]. However, an increasing share of cocoa processing is now shifted to producing countries themselves, as their governments aim to retain more value [6].
Challenges in the Cocoa Industry
Central to the cocoa industry is the role of smallholder farmers, who are responsible for over 90% of global cocoa production [7]. Yet, the economic benefits of the cocoa value chain are distributed unequally. While the downstream sector captures 79.4% of the industry's value, cocoa farmers only receive 6.6% [8]. Despite the global chocolate industry’s value exceeding $130 billion [9], the farmers who produce it remain in poverty, with low living standards [10].
In addition, cocoa crops are highly susceptible to climate change. As a result, the risk of crop destruction and income loss rises sharply. Meanwhile, global demand for chocolate continues to grow. This mismatch between increasing demand and declining production capacity poses a real threat to the supply chain. Sustainable solutions are urgently needed to safeguard the future of cocoa (Figure 1) [11].
The Problem of Phytophthora
Phytophthora, derived from the Greek words phyto and phthora, literally translates to ‘plant destroyer’. This name reflects its devastating impact on agriculture. Phytophthora species are oomycetes, which resemble fungi but are phylogenetically distinct. These pathogens are infamous for causing a wide range of diseases in plants. Their ability to spread rapidly via water and soil, coupled with the production of resilient spores, makes control extremely difficult once an outbreak begins [12].
An infamous example of its devastating effects was the Irish Potato Famine between 1845 and 1852, caused by Phytophthora infestans. This pathogen destroyed more than 70% of the potato harvest for seven consecutive years, causing the death of over 1 million people and forcing another 2 million to emigrate [13].
Nine species that have been found to cause black pod disease are: P. palmivora, P. megakarya, P. citrophthora, P. megasperma, P. arecae, P. heveae, P. capsici, P. tropicalis, and P. cocoaicola. However, P. palmivora and P. megakarya are the most devastating species for cocoa plants. The disease is characterized by lesions on pods that turn dark brown or black [12]. As such, black pod disease costs the global cocoa industry an estimated 423 million USD annually [14], causing the loss of approximately 30% of cocoa pods and 10% of cocoa trees (Figure 2) [15].
Current Solutions and Shortcomings
Phytophthora’s mode of action is based on suppressing the plant’s immune system during an initial biotrophic phase. This is achieved through the secretion of effector proteins that interfere with host defense signaling pathways. Once established it switches to a necrotrophic phase [17].
Copper-based fungicides have been the conventional treatment for black pod disease control in agriculture for years, primarily due to their broad-spectrum action and their affordability. They kill germinating spores and disrupt multiple metabolic pathways in a wide range of microbes, including fungi, bacteria, and oomycetes [17]. However, the disadvantages of copper-based fungicides are becoming increasingly apparent. Prolonged use leads to copper accumulation in the environment, which disrupts beneficial microbial communities [17].
On the other hand, systemic fungicides, which have been available for about 40 years, penetrate plant tissues and target specific biochemical processes within pathogens. Their specificity means they are generally more effective against pathogens but are also more vulnerable to resistance development and are considerably more expensive. This issue was recognized before metalaxyl was widely used in cocoa, as resistant Phytophthora strains had already emerged in other crops. To minimize resistance risk, spray mixtures combining systemic and multi-site fungicides are used for dual protection [17].
Cutting and pruning is a common practice among cocoa farmers. It consists of removal of affected plant tissues. The approach is used not only to eliminate the tissues affected by disease, but it also allows light to penetrate the tree canopy. While pruning has shown some success in managing plant health, it does not provide sufficient protection for cocoa crops on its own [18].
To combat the disease, breeding for resistance remains the most promising long-term solution. Researchers are developing reliable screening methods and using DNA markers to accelerate breeding programs. Another approach involves identifying naturally resistant cocoa trees in regions with high disease pressure, leveraging the genetic diversity within cocoa plantations to develop more resilient crop varieties [19]. However, breeding programs are inherently slow, often taking many years for the new varieties to be fully adoptable. This is a time that the cocoa farmers and industry cannot afford to lose (Figure 3).
Our Solution
As awareness about the disastrous effects of some agrochemicals on the environment increases, the industry has been looking for alternatives. Due to stringent regulations, the cost of developing a new pesticide reaches an estimated 350 million dollars and can take up to 10 years [20]. An alternative is the development of biological pesticides, whose market has grown faster than that of chemical pesticides [21].
Our project focuses on engineering B. subtilis to secrete antimicrobial peptides (AMP) directly onto the cocoa leaf and pod surface. These peptides are known to be active against Phytophthora, thus inhibiting growth or killing the pathogen, and preventing black pod disease (Figure 4).
Project Inspiration
Our primary inspiration for this project was to address a challenge that threatened the livelihoods of farmer communities in the Global South. Phytophthora species cause devastating losses not only in cocoa, but also in other key crops such as potato and tomato. By targeting this widespread pathogen, we aim to develop a solution that could potentially protect cocoa and the communities that depend on it. Additionally, current control methods are costly, pollute the environment, and work insufficiently in the long term. By developing a biological approach, we aim to offer farmers a more sustainable, effective, and accessible solution that protects their crops, while safeguarding the environment.
The Chassis
For our project, we have chosen B. subtilis as a chassis, due to its GRAS status, biocontrol potential, and the engineering options.
B. subtilis is one of the most well-studied Gram-positive bacteria. It is known as a model organism due to its ease of cultivation, fast growth, and natural competence. It is a rod-shaped aerobic microorganism that forms endospores when exposed to stress [22], [23]. B. subtilis is found most abundantly in soil and in the gastrointestinal tracts of animals [24].
In agricultural settings, B. subtilis can be used as a biocontrol agent or biostimulant due to its antibiosis, competition for nutrients and antibiotic production [25]. Recently, B. subtilis strains have been observed to associate with Theobroma cocoa. Endophytic strains of B. subtilis promote plant growth by increasing biomass and influencing leaf development. Additionally, B. subtilis strains isolated from the rhizosphere were found to produce antimicrobials that potentially increase cocoa’s resilience against pathogens [24], [26].
Following sequencing of the full B. subtilis genome in 1997 [27], the bacterium received increased attention in both basic and translational research. The availability of the various well-characterized DNA parts, like ribosome binding sites (RBSs), promoters, regulatory elements, greatly facilitates the assembly of tightly controlled constructs for B. subtilis. Over the years, numerous genetic toolkits enabling efficient DNA engineering of B. subtilis were released [28], [29], [30], [31], [32]. We were specifically interested in the SubtiToolKit, with its collection of genetic parts and utilization of Golden Gate Assembly method to finalize the construct [29].
The Sensing System
With PhytoBlock, we aim to selectively target Phytophthora species. Therefore, we designed a biosensing system that senses the presence of Phytophthora by integrating two signals that occur upon infection. As it is an AND gate, it will only be activated when both inputs are present.
The first input for our logic gate is the presence of reactive oxygen species (ROS), a strong plant defense signal [33]. For ROS detection, we selected bacterial promoters responsive to oxidative stress [33]. Our second is a group of highly conserved proteins secreted by Phytophthora during the early stages of infection, called elicitins [34]. Elicitins are detected by a receptor-based module inspired by plant elicitin receptors [35]. By focusing on a conserved region of these receptors, our system remains robust across different elicitins and Phytophthora species [36].
Intracellularly, these two signals are combined via a split T7 RNA polymerase, a well-characterized part in literature [37], [38]. This minimizes false positives from general plant stress and makes PhytoBlock a reliable system to sense impending attacks of Phytophthora.
Our AMPs
The effector molecules of our system are AMPs. These are small, bioactive molecules that form an essential component of innate defense across diverse life forms. A defining characteristic of plant AMPs is their cysteine-rich nature, in which multiple disulfide bonds stabilize the peptide structure [39], [40]. AMPs are classified into structural families based on sequence similarity, cysteine motifs, disulfide connectivity, and tertiary folding. The principal families include thionins, defensins, hevein-like peptides, knottins, lipid transfer proteins, α-hairpinins, and snakins [39].
In general, AMPs exert their function by targeting and disrupting cellular membranes by creating pores, lying on the lipid bilayer surface in a carpet-like manner, or acting as detergent molecules [41]. The lack of a particular proteinaceous target makes it harder for the affected organisms to develop resistance to AMPs. Additionally, some AMPs, like human cathelicidin AMP LL-37, apart from their ability to lyse membranes, inhibit the activity of intracellular proteins [42]. Large online repositories, like APD [43] or DBAASP [44], gather extensive data about natural and synthetic AMPs, allowing to screen for particular properties or targeted organisms.
We selected our antimicrobial peptides (AMPs) based on their low complexity in heterologous production, as they are synthesized on ribosomes and do not require post translational modifications. In addition, they show no toxicity toward humans or common production hosts and have demonstrated inhibitory effect against Phytophthora species. More information about our AMPs can be found on the Part page.
Secretion of AMPs
Our genetic constructs are assembled with Golden Gate assembly in Escherichia coli, amplified, purified and subsequently transformed into Bacillus subtilis. For this design, the AMP is fused to a secretion tag (ST) and expressed under the control of a xylose-inducible promoter. The secretion tag directs the AMP through the native B. subtilis secretion pathways, after which it is cleaved off, releasing the mature AMP into the medium.
An alternative version of this cloning strategy allows for direct purification of the AMP from cell lysates. In this approach, the secretion tag is replaced with a fusion tag such as a His-tag, SUMO tag, or thioredoxin, allowing purification of the fusion protein using a Ni-NTA affinity column.
Formulation of Our Spray
Biocontrol sprays can be applied on various parts of the plant. These include foliar spraying, soil treatment, seed coating, and seed priming [45]. For example, Pseudomonas spp. and B. subtilis have been reported to have antagonistic properties against Phytophthora species [26], [46], [47]. Such applications highlight the potential of bacterial biocontrol agents as a persistent and environmentally safe alternative for managing foliar pests and pathogens across different crops.
Previous studies have demonstrated the potential of B. subtilis as a biocontrol agent against plant pathogens. In these studies, the formulation was typically prepared as a wettable powder that consists of endospores mixed with clay powder and a dispersing agent. This preparation was user-friendly, as it could be dissolved in water and sprayed onto plants. Based on available literature and existing commercial biocontrol products, we estimate that approximately 1.7 kg/ha will have to be applied on a weekly basis.
Our earlier conversations with farmers from West Africa, as well as companies in the cocoa industry, have taught us about the logistical challenges they face. Since the plantations are located deep in forests, it is highly impractical to carry large volumes of liquid pesticides or fungicides. Furthermore, storage of heat-labile chemicals will prove challenging in tropical regions. Therefore, our solution will be provided as a powder of B. subtilis spores. This formulation can be easily transported and activated when needed, by dissolving it in water or a readily available nutrient medium. This would significantly reduce the burden on farmers while enabling effective field application.