Overview
The wettable powder formulation of our genetically engineered Bacillus subtilis (the ideal strain) is a key solution for managing Coffee Leaf Rust (CLR). The wettable powder has been chosen as the most suitable application method as it is expected to give optimal results with our Biocontrol Agent (BCA),
Our genetically engineered B. subtilis, utilizing a dual-promoter system for Iturin A production, offers both preventative and curative action against CLR, unlike traditional fungicides like copper fungicides or Bordeaux mixture, which are primarily preventative.
Preventative Action: The constitutive promoter ensures continuous, baseline production of Iturin A, even before the infection starts, creating a protective barrier on coffee plants that prevents fungal spores from establishing. This provides ongoing protection without the need for constant reapplication.
Curative Action: The inducible promoter kicks in under specific environmental triggers, like infection stress, leading to a surge in Iturin A production. This rapid increase targets and disrupts existing fungal cells, effectively acting as a cure once the pathogen has infected the plant.
Outlined below are the key aspects of the product (in comparison with existing products) including the performance metrics production methodology, formulation, application guidelines, and cost analysis of the product are stated below.
For our implementation, cumate will be added into our formulation of wettable powder which will be taken up by our chassis passively. The formulation (chassis + Cumate) will be sprayed onto the coffee leaves in the plantation to distribute the chassis for its antifungal action against the HV. Once the Cumate has degraded or washed away the bacteria will no longer be able to survive in that environment. Cumate is a biodegradable inexpensive lab chemical. It is degraded by several microorganisms especially alphaproteobacterial like Rhodococcus,Pseudomonas, and Sphingomonas and is not toxic to the environment. [3][4][5]
Performance Metrics
There are three major divisions of control measures currently in practice to mitigate the impact of CLR. The performance metrics of all three are presented to demonstrate the benefits of wettable powder.
Chemical Fungicides
The existing chemical control for Coffee Leaf Rust includes copper products (contact-preventive), systemic triazoles (preventive–curative), and systemic strobilurins (rust spore eradicator). Copper-based fungicides are very effective and have a tonic effect, but are costly and must be present on the leaf before the infection occurs. The copper accumulates in the soil, reaching levels toxic to the surrounding biome. This is an unavoidable problem, as it is necessary to have a positive charge to bind with sites capable of interfering with the fungus’s metabolism and killing it. Therefore, a suspension with 1000 L of water must contain 1.2 kg of the active ingredient Cu2+ [1]. Protective dithiocarbamate fungicides are also adequate for the control of coffee rust. However, under the heavy rains of many coffee-producing regions, their residues do not adhere as well as those of the copper-containing fungicides. H.vastatrix is genetically diverse, with more than 40 different races identified and more constantly appearing, making antifungal resistance a serious concern.
The carboxamide molecules in use are benzovindiflupir, fluxapyroxade, and thifluzamide. The triazole molecules currently in use include cyproconazole, epoxiconazole, flutriafol, tebuconazole, and triadimenol, whereas, among others.
| Fungicide | Active Components | Restricted Entry After Usage (h) |
|---|---|---|
| Priaxor Xemium | Fluxapyroxad 14.33%, Pyraclostrobin 28.58% | 12 |
| OxiDate 2 | Hydrogen Dioxide 27.1%, Peroxyacetic Acid 2% | 1 |
| Kocide 3000 | Copper Hydroxide 46.1% | 48 |
| Badge X2 | Copper Oxychloride 23.83%, Copper Hydroxide 21.49% | 24 |
Resistant cultivars
T A cross between C. arabica and C. canephora, known as Hibrido de Timor, and its derivatives, has been widely used as a resistant hybrid but isn't resistant to all races of rust. Other resistant varieties, such as robusta hybrids and arabica hybrids, are only resistant to some rust races and come with the downside of inferior cup quality. H.vastatrix is genetically diverse, with more than 40 different races identified and more constantly appearing. Due to the fungi’s rapid rate of evolution, the resistance genes of the plant have often been rendered ineffective, which, when coupled with the issues of replacing entire fields of plants and taking huge losses till they reach maturity, has rendered this method obsolete. Additionally, spreading a single resistant variety globally significantly lowers genetic diversity and increases the chance of the rust overcoming said resistance, among other long-term ecological issues.
The carboxamide molecules in use are benzovindiflupir, fluxapyroxade, and thifluzamide. The triazole molecules currently in use include cyproconazole, epoxiconazole, flutriafol, tebuconazole, and triadimenol, whereas, among others.
Agronomic practices
Sanitation, pruning, weed control, fertilization, and the regulation of shade have all been used with varying degrees of success as none significantly challenge the fungus’s survival to the required degree.
| Control Method | Disease Severity Reduction | Reference |
|---|---|---|
| Extract of rust-infected coffee leaf and extract of Solanum lycopersicum infected with Crinipellis perniciosa | 31% and 27%, respectively | [2] |
| Extracts of Cinnamomum verum and Citrus sinensis | 90% and 92%, respectively | [2] |
| Aqueous extracts of coffee leaves | >97% | [2] |
| Greenforce Cuca | R 48% | [2] |
| Fitoforce Full: product of the coffee industry + P2O5 (15.1%) and Cu (2.85%) | 47% | [2] |
| Acibenzolar-S-methyl (ASM) | 53% | [2] |
| ASM | 12.10% | [2] |
| K phosphonate 1: P2O5 (35%) and K2O (25%); K phosphonate 2: P2O5 (33.6%) and K2O (29.0%) | 47% and 74% | [2] |
| Mn phosphonate: P2O5 (51.0%) and Mn (9.7%) | 62% | [2] |
| Cu phosphonate: P2O5 (20.3%) and Cu (4.0%) | 37% | [2] |
| Plants sprayed with Melaleuca alternifolia | 20.9% Reduction | [2] |
| Seven bacterial isolates (Bacillus sp.—B10, B25, B157, B175, B205, and B281; Pseudomonas sp.—P286) | The isolate B157 reduced the intensity of rust and was as effective as copper hydroxide | [2] |
| Entomopathogenic and mycoparasitic fungus Lecanicillium lecanii | Significant suppression of H. vastatrix | [2] |
| Trifloxystrobin + tebuconazol | 90% | [3] |
| Bayleton | 71.84% | [4] |
| Bordeaux mixture | 53.37% | [4] |
APPLICATION AND DOSAGE
Components of wettable powder
The wettable powder formulation is prepared with various components, that consider compatibility with the bacterial species to increase shelf life, bioactivity, and overall product quality. There are six major components of the formulation incorporated in an estimated ratio as given below:
1. Biocontrol/Microbial Agent: The microbial agent must be kept under optimal conditions to ensure the desired result. Protection against chemical or physical damage during formulation is essential for maintaining its viability.
2. Carriers: The carrier is an inert material that evenly distributes the microbial agent. It plays the role of a solid diluent in the formulation and has no separate effect on the soil or plant environment. The optimum carrier can be considered based on the suspension rate and cost.
3. Dispersants:The dispersants ensure the even distribution of the powder in water or solvents, enhancing the formation of a stable dispersion. It is used to reduce solid or liquid particles in the composition dispersion system to be assembled, and when preparing wetting powder, adding dispersant makes it easy to form dispersion liquid and suspension, and keep the metastable function of dispersion.
4. Wetting agent: The wetting agents are the surfactants that reduce the surface tension between powder and water for easy dissolution and effective application. The main function of this wetting agent is to easily soak the solid material of the composition in water/solvent.
Some wetting agents also exhibit dispersing properties, while some dispersants also have wetting capabilities. Therefore, both the suspension rate and wetting time are taken into account during the screening of dispersants and wetting agents. A higher suspension rate indicates better dispersion, while a shorter wetting time reflects better wettability. The optimal ratio is determined by assessing both factors, with the ideal combination being the one that achieves the maximum suspension rate and the shortest wetting time for the specific formulation.
5. Stabilizers: The stabilizers contribute to making the composition a homogeneous mixture by not letting any components settle down increasing stability. It ensures the protection of microbes from oxidative damage and any environmental stress during storage, hence increasing the shelf life. It also enhances dispersion ability and adhesion to the plant leaf surface.
6. UV Protectant: Protects the microbial agent against UV radiation when exposed to sunlight which might affect its efficacy. It also prevents the UV rays from degrading the components, preserving the integrity of all active ingredients.
The following are the potential compounds or materials used as additives, and other agents required to achieve the optimum formulation for the product.
| Component | Estimated Proportion (%) | Role |
|---|---|---|
| Bacillus subtilis spores | 50-60 | Active ingredient; provides antifungal activity. |
| Inert carriers | 30-40 | Ensures ease of application and mixing. |
| Dispersing agent | 1-5 | Facilitates proper suspension in water for spraying. |
| Inducing agent (Methyl cumate powder) | TBD | Induces the kill-switch mechanism of the bacteria. |
| Anti-caking agent | Trace | Prevents clumping during storage. |
Formulation Breakdown
The composition of the proposed formulation for our wettable powder needs to be optimized with each component present in precise proportions to serve its intended purpose. The proportions for optimal production are given below.
| Component | Estimated Proportion (%) | Role |
|---|---|---|
| Bacillus subtilis spores | 50-60 | Active ingredient; provides antifungal activity. |
| Inert carriers | 30-40 | Ensures ease of application and mixing. |
| Dispersing agent | 1-5 | Facilitates proper suspension in water for spraying. |
| Inducing agent (Methyl cumate powder) | TBD | Induces the kill-switch mechanism of the bacteria. |
| Anti-caking agent | Trace | Prevents clumping during storage. |
Cumate function in Kill Switch doctumentation here
The estimated proportions of the wetting agent, stabilizer, and UV protectant will be determined after further experimentation and field testing.
PRODUCTION
The production process for the Bacillus subtilis wettable powder focuses on achieving the optimal spore density, quality control, and ensuring that the final product remains stable during storage and effective when dispersed in the field. The following steps outline the production of the wettable powder.
1. Spore Density Optimization: To ensure effective application in the field, the wettable powder must maintain a high spore density. This allows for a high concentration of Bacillus subtilis in each application, maximizing its ability to combat CLR.
| Parameter | Optimal Conditions |
|---|---|
| Spore Concentration | Target: TBD, after further experimentation |
| Field Application | Concentration should be 103-104 CFU/mL less than target concentration of dry wettable powder - for example, if target concentration is 1×1011 CFU/g of wettable powder, then final suspension should reach 1×107 - 1×108 CFU/mL upon dilution in water. |
| Fermentation Process | 24–48 hours of Bacillus subtilis growth at 30–37°C, ensuring high sporulation rates. |
2. Production Steps and Process Flow
1. Fermentation Process
After the fermentation period, the spores are harvested using centrifugation or filtration to separate them from the culture medium.
2. Harvesting
Bacillus subtilis is cultured in bioreactors under optimized conditions (pH 6.5-7.0, temperature 30-37°C) for 24-48 hours to induce sporulation.
3. Drying Process
The spore concentrate undergoes either spray drying or freeze drying to remove moisture while maintaining spore viability.
4. Formulation
The dried spores are blended with inert carriers (e.g., kaolin clay or silica) to create the wettable powder formulation. Dispersing agents such as sodium lignosulfonate are added to ensure smooth suspension in water during application. Cumate (p-isopropyl benzoate) is a biodegradable, membrane-permeative chemical that can easily enter bacterial cells without transporters. It is non-toxic to Bacillus subtilis and is not known to have any negative effects on the coffee plant itself. To trigger the kill switch mechanism in the bacteria, it can be added to the wettable powder formulation in the form of methyl cumate powder. (See Formulation in detail above)
A. Dry mixing and Addition of Additives
Dry mixing is a critical process where each component of the formulation—whether it's the active ingredient or inert materials like carriers, dispersants, or wetting agents is finely pulverized to achieve a consistent particle size. This fine powder ensures uniform distribution of all ingredients when applied or suspended in water and helps achieve homogeneity. Specific additives are incorporated to enhance the overall performance of the wettable powder.
B. Sieving
Sieving is the process of passing the mixed powder through a fine mesh or sieve to ensure uniform particle size. This is crucial for maintaining the quality, consistency, and effectiveness of the application.
5. Quality Control
The final wettable powder is tested for spore density, homogeneity, and viability to ensure that the product meets field-use standards.
6. Packaging
The product is packaged in moisture-resistant bags with proper labeling, including storage instructions to ensure a long shelf-life (12-24 months under optimal conditions).
A Brief Outline of the Production Steps
| Process | Description |
|---|---|
| Spray Drying/Freeze Drying | Removes moisture while maintaining spore viability |
| Spore Purity Testing | Ensures target spore density (CFU/g) in the final powder |
| Quality Control | Each batch undergoes testing for spore viability and dispersibility in water. |
| Packaging and Labeling | Packaged in moisture-resistant bags, labeled with appropriate storage instructions (cool, dry place). Shelf-life of up to 24 months with proper storage. |
WETTABLE POWDER APPLICATION GUIDELINES
This powder must be dispersed into the water before use and applied using appropriate equipment to ensure even distribution over the coffee plant foliage. Below are the proposed guidelines for the correct use of the wettable powder product. After further experimentation and field studies, exact parameters (spore concentration, frequency of application, etc.) will be defined.
| Parameter | Details |
|---|---|
| Frequency | Application every few days, particularly during the growing season, for a given period. Increase frequency during periods of high humidity or rainfall to prevent fungal spread. |
| Timing | Apply at the early stages of CLR detection or as a preventive treatment before the rainy season. Avoid applying during heavy rains to prevent runoff. |
| Water Suspension | Suspend a few grams of wettable powder per liter of water in a spray tank. Agitate constantly to maintain a uniform suspension during application. |
| Application Method | Use manual or mechanical sprayers to distribute the suspension across foliar surfaces evenly. Ensure thorough coverage. |
| Tank Mixing | Ensure the wettable powder is thoroughly mixed in the water with constant agitation to avoid clumping and settling at the bottom of the tank. |
| Spray Volume | Use approximately 500-1000 liters of suspension per hectare depending on the density of the coffee plant foliage. |
Note: Ensure that the lower surfaces (abaxial) of the leaves are adequately covered. Apply early in the morning or late in the afternoon to reduce the risk of evaporation and maximize the product's effect.
Cost Effectiveness: Comparative Breakdown of Wettable Powder, Traditional Fungicides, and Bordeaux Mixture
| Component | Traditional Fungicides (Copper-Based) | Bordeaux Mixture | Wettable Powder (Bacillus subtilis) |
|---|---|---|---|
| Active Ingredient Cost | Expensive copper compounds like copper oxychloride, are prone to market fluctuations [9][10] | A mixture of copper sulfate and lime is less expensive but still involves copper mining and extraction costs [9][11] | Cost-effective spores, easily produced via fermentation [11] |
| Carrier/Fillers | Synthetic fillers with potential toxicity and environmental impact [9] | Lime serves as the main filler, which is cheaper but requires careful handling to avoid plant toxicity [11] | Inert, organic carriers like talc or clay, eco-friendly [11] |
| Surfactants/Dispersants | Synthetic surfactants are needed for better adhesion and spread [9] | Lime acts as a natural surfactant, but it can reduce efficacy during rain or irrigation [11] | Natural, biodegradable surfactants that assist in the even distribution of spores [11] |
| Application Frequency | Frequent reapplications are required due to wash-off and degradation [9][10] | Frequent reapplications, especially after rain, due to lime’s poor resistance to washing [11] | Fewer applications are needed due to spore longevity and field persistence [11] |
| Environmental Costs | High soil degradation, and copper toxicity, requires environmental remediation [9][10] | High copper residue in soil, potential lime overuse affecting pH, leading to soil degradation over time [11] | Biodegradable, no soil degradation, potential subsidies for eco-friendly practices [11] |
| Long-Term Yield Impact | Pathogen resistance can develop, requiring more expensive treatments or higher doses [9][10] | Long-term use leads to copper accumulation, affecting soil and plant health negatively [11] | Stable and consistent efficacy, lower likelihood of pathogen resistance, ensuring better long-term yields [11] |
| Storage and Handling | Requires careful handling due to chemical composition [9][10] | Lime and copper components are corrosive, need special equipment for application [11] | Easy to store and handle, with no corrosive effects, spores have longer shelf life [11] |
Financial Comparison
| Factor | Traditional Fungicides (Copper-Based) | Bordeaux Mixture | Wettable Powder (Bacillus subtilis) |
|---|---|---|---|
| Initial Cost | High due to copper pricing and synthetic additives | Moderate, but the price of copper sulfate is still a concern | Low to moderate, due to local and renewable production of bacterial spores |
| Application and Labor Cost | High due to frequent reapplications and chemical handling | High due to frequent reapplications, especially after rain | Low, thanks to fewer applications and easier handling processes |
| Environmental Cost | Significant long-term environmental costs, copper toxicity remediation | Soil degradation, pH imbalance, copper accumulation | Minimal environmental costs, no toxicity or soil degradation |
| Long-Term Financial Viability | Unsustainable with the development of pathogen resistance | Moderate, but unsustainable in the long-term due to copper build-up | Highly sustainable, with stable costs over time and government incentives for biocontrol usage |
Product Summary:
This Bacillus subtilis wettable powder is an eco-friendly, biocontrol solution designed specifically to combat Coffee Leaf Rust (CLR). Its high spore density ensures potent activity against Hemileia vastatrix, while its formulation as a wettable powder allows for easy integration into standard agricultural practices. With proper application techniques and storage, the product offers long-term viability and effectiveness in the field.
By following the outlined application guidelines, and production steps, and ensuring proper formulation with the right inert carriers, the wettable powder can be successfully deployed to protect coffee crops from devastating rust infections.
References
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