Why Implementation Matters
The value of technology lies not only in laboratory discoveries but also in solving real-world problems. In the field of citrus aphid control, while chemical pesticides can suppress pests in the short term, they lead to increased resistance and ecological damage. Existing biological control solutions, due to their slow effectiveness, operational complexity, and high costs, are difficult to popularize. To address these challenges, we have advanced APHiGO from laboratory research to field application. Throughout this process, we remain committed to translating green technology into tangible outcomes, ensuring scientific effectiveness while prioritizing the operational habits and practical needs of farmers, so that the technology can truly be adopted in the field.
From Lab to Field
To translate laboratory research into practical orchard pest control, we have developed three types of biological control agents targeting citrus aphids: RNA dry powder for trunk injection, engineered Metarhizium capsules for foliar spraying, and environment-responsive Bacillus subtilis slow-release microcapsules. Each formulation is tailored to specific orchard conditions and farmers' usage habits, ensuring seamless transition from lab to field. Click the boxes below to explore the composition and application methods for each agent.
RNA biopesticide
We have chosen to prepare the RNA pesticide in a lyophilized powder form to address the challenge of RNA molecules' instability and rapid degradation in liquid form. This lyophilized formulation creates a stable amorphous glass-like protective layer on the surface of the RNA molecules, enabling long-term storage and transportation at room temperature without relying on complex cold-chain systems. This significantly enhances the product's practicality and commercialization potential, transforming it from a laboratory technology into a stable, convenient green pest control tool that can be easily accessed by farmers in the field.
Fig 1. Appearance diagrams of RNA dry powder pesticide and trunk injector
Composition and Proportions
▼- Artificial miRNA: 1% (w/w). The active ingredient, designed to target the citrus aphid's specific genes.
- Trehalose: 5% (w/w). Trehalose is a non-reducing disaccharide that forms an amorphous glass-like structure during lyophilization, interacting with RNA to prevent structural degradation and stabilize the nucleic acid molecule.
- Mannitol: 93.899% (w/w). Mannitol is a commonly used excipient that crystallizes during lyophilization to form a solid formulation structure. It improves the mechanical strength of the lyophilized product, creating a regular crystalline microstructure that facilitates rapid sublimation and ice removal. Mannitol acts as a bulking agent, providing structural support.
- TE Buffer Solution: Tris:EDTA = 10:1, corresponding to a solid content of 0.1% (w/w). Tris helps maintain the pH of the solution, while EDTA chelates metal ions that typically activate RNases. EDTA protects RNA from degradation.
- Isothiazolinone: 0.001%, a fungicide used to prevent microbial contamination of the formulation once rehydrated.
Laboratory Preparation of RNA Pesticide Dry Powder (2g)
▼I. Calculation of Component Quantities
| Component | Mass Percentage (% w/w) | Mass Required for Preparing 2g Dry Powder (g) |
|---|---|---|
| Artificial miRNA | 1.00% | 0.0200 |
| Trehalose | 5.00% | 0.1000 |
| Mannitol | 93.90% | 1.87798 |
| Tris-HCl | ~0.091% | 0.00182 |
| EDTA Disodium | ~0.009% | 0.00018 |
| Isothiazolinone | 0.00% | 0.00002 |
II. Experimental Preparation
1. Equipment: Analytical balance, pH meter, magnetic stirrer, vortex mixer, -80°C freezer or liquid nitrogen, freeze dryer, sterile vials or lyophilization bottles.
2. Reagents and Materials: Ultrapure water (RNase-free), Tris-HCl, EDTA, trehalose, mannitol, artificial miRNA (dsRNA), isothiazolinone stock solution (e.g., 1000 ppm).
3. Environmental Requirements: Clean the work surface with RNase decontaminant, wear gloves and a mask, and operate in a sterile environment as much as possible to prevent microbial and RNase contamination.
III. Experimental Steps
1. Prepare TE Buffer (1L, 10mM Tris-HCl, 1mM EDTA, pH 8.0): Weigh 1.211 g Tris-base and 0.372 g Na₂EDTA·2H₂O, dissolve in approximately 800 mL ultrapure water. Adjust the pH to 8.0 with HCl. Add ultrapure water to a final volume of 1L. Sterile filter (0.22μm filter membrane) and store at room temperature or 4°C.
2. Prepare the Master Mix: Weigh 1.878 g solid mannitol and 0.100 g trehalose, add approximately 25 mL of TE buffer to a centrifuge tube, and vortex or gently stir until all solids are completely dissolved, forming a clear solution. Add 0.020 g of accurately weighed artificial miRNA powder to the solution. Then add 20 μL of the 1000 ppm isothiazolinone stock solution, gently mix. Finally, adjust the total volume of the mixture to 30 mL with TE buffer.
3. Pre-Freezing: Dispense the prepared mixture into pre-weighed lyophilization bottles. Place the containers with the solution into a freezer and freeze rapidly at -80°C until the solution is completely frozen into a solid.
4. Freeze-Drying: Immediately transfer the fully frozen samples to the pre-chilled chamber of the freeze dryer. Execute the freeze-drying program. After the freeze-drying process is complete, quickly seal the containers under a dry inert gas (nitrogen).
Application in Tree Trunk Injection Technology:
To ensure that the RNAi signal can efficiently and stably reach its target site, this RNA pesticide dry powder is designed in combination with the tree trunk injection technology. Farmers can reconstitute the precise amount of dry powder in clean water and use a trunk injector to directly deliver the pesticide into the citrus tree's vascular system. This application method allows the artificial miRNA to be transported with the tree's transpiration stream and photosynthetic products to newly growing shoots, leaves, and other aphid-infested areas. This method not only avoids the degradation from UV radiation and washing by rain that leaf spraying faces but also significantly extends the effective duration of the RNA pesticide. Additionally, it minimizes environmental drift and exposure to non-target organisms, making it a key component in achieving large-scale, precise application of RNA interference technology in perennial woody crops.
Engineered Metarhizium Oil Suspension Capsule
We have chosen to prepare the engineered Metarhizium as an oil suspension capsule. Traditional spore powders are prone to environmental degradation, while liquid formulations have a short shelf life and are inconvenient for storage and transportation. This design utilizes a stable oil phase environment created by sunflower seed oil as the carrier and antioxidants, while integrated UV protectants provide crucial protection for the spores during field application. More importantly, encapsulation simplifies the preparation process to a straightforward "one capsule, one bucket of water" operation, completely eliminating farmers' concerns about not knowing how to use or being reluctant to use microbial pesticides. This greatly enhances the user experience and compliance. This formulation transforms cutting-edge synthetic biology achievements into a stable, efficient, and convenient standardized product.
Fig 2. Concept Map of the Use of Metarhizium anisopliae Oil Suspension Capsules for Engineering
Composition and Proportions
▼- Active Ingredient: Engineered Metarhizium conidia powder, 10.0% (w/w). Metarhizium anisopliae is a genetically modified strain expressing lethal dsRNA targeting aphids, which simultaneously triggers RNA interference during infection, enhancing lethality.
- Plant-based Carrier Oil (Sunflower Seed Oil): 83.0% (w/w). As the continuous phase of the oil dispersion, the carrier oil helps maintain the hydrophobicity of the conidia and improves leaf adhesion after spraying.
- Antioxidant (BHT, 2,6-Di-tert-butyl-4-methylphenol): 0.1% (w/w). Used to prevent oxidation and deterioration of organic components in the oil phase, protecting spore viability.
- Surfactant (Tween-80, Polysorbate 80): 2.0% (w/w). A non-ionic emulsifier that ensures the formulation can be easily diluted in water and forms a stable emulsion, improving spray coverage.
- UV Protection Additive (Humic Acid): 0.5% (w/w). Effectively protects the light-sensitive conidia from UV damage.
Laboratory Formulation (Final Weight: 2g)
▼I. Material Preparation
| Component | Mass/Volume | Mass Percentage (w/w) |
|---|---|---|
| Dry powder of conidia of engineered Metarhizium anisopliae | 0.200 g | 10.00% |
| Sunflower seed oil | 1.660 mL | 83.00% |
| BHT | 0.002 g | 0.10% |
| Tween-80 | 0.040 mL | 2.00% |
| Humic acid | 0.010 g | 0.50% |
II. Experimental Preparation
Equipment: Analytical balance, vortex mixer, ultrasonic disruptor or high-speed homogenizer, 1-5 mL small centrifuge tubes, light-protective containers.
Environmental Requirements: Ensure the work surface is clean. The process should be performed in a light-protected, sterile environment as much as possible to prevent premature spore germination or contamination.
III. Procedure
1. In a light-protected small centrifuge tube, measure approximately 1.5 mL sunflower seed oil. Accurately add 0.002 g BHT and 0.010 g humic acid powder, then add 0.040 mL of Tween-80 surfactant. Finally, accurately weigh 0.200 g of engineered Metarhizium spore dry powder, and slowly, in batches, add the spore powder to the oil phase mixture while continuously vortexing or gently stirring.
Bacillus subtilis Slow-Release Formulation
This system utilizes genetically engineered Bacillus subtilis as a biological sensing platform, enabling it to detect the main component—sucrose—secreted by aphids in their honeydew. When the engineered bacteria sense the sucrose signal, the sucrose-inducible promoter inside the bacteria is activated, driving the expression of downstream genes and ultimately producing volatile signaling molecules such as methyl salicylate (MeSA).
Design: Sodium Alginate and Boronic Acid Polymer-Based Bacillus subtilis Slow-Release Formulation
Main Principles:
- Sodium Alginate: A stable base with excellent compatibility.
Sodium alginate (SA) is a natural polysaccharide that is widely used in biomedical and food industries due to its excellent biocompatibility, low toxicity, and degradability. Its molecular chain is rich in repeating units of guluronic acid (G) and mannuronic acid (M), which can combine with other substances to form stable three-dimensional gel networks.
- Boronic Acid (PBA): Smart-trigger unit.
Boronic acid (PBA) is a Lewis acid that functions by specifically recognizing and reversibly binding to molecules containing cis-diol groups (such as sucrose in aphid honeydew). Before the sugar signal arrives, the boronic acid group can form a reversible, dynamic covalent bond with the sugar acid residues in the alginate chain that contain diols. This is a dynamic equilibrium process, where the formation and breaking of bonds occur simultaneously. This dynamic crosslinking network provides some structural support for the gel.
The sugar molecules, including sucrose, in aphid honeydew contain cis-diol groups that can bind with boronic acid. When these sugar molecules diffuse into the gel network, they "compete" with sodium alginate for the boronic acid groups. Due to the difference in affinity, the external sugar molecules replace sodium alginate, forming new boronic ester bonds with boronic acid. When a large amount of external sugar molecules enters and competes for binding, the original dynamic crosslinked network made up of boronic acid-alginate ester bonds is disrupted. This breaks the structural integrity of the network, weakening its physical barrier and causing rapid swelling and disintegration of the gel, thus enabling the rapid release of the encapsulated Bacillus subtilis.
Fig 3. Mechanism diagram of Bacillus subtilis slow-release preparation
Final Product: The final product is a lyophilized microsphere containing Bacillus subtilis (as per literature), sealed in moisture- and light-protective containers. Farmers add the appropriate amount of dry powder (into a spray tank) and dissolve it completely to form a uniform bacterial suspension. Using conventional agricultural sprayers, they can evenly spray the reconstituted suspension on the citrus tree's leaves and surface.
Field Application Scenarios
To bridge the gap between laboratory research and practical application, we have developed the CitrusShield platform, enabling intelligent monitoring and timely intervention for orchard pest control. The core module of the platform consists of engineered Bacillus subtilis sustained-release formulations pre-applied in citrus orchards. These microorganisms persistently colonize leaf surfaces and, upon detecting sucrose signals from aphid honeydew during feeding, release methyl salicylate (MeSA) as an indicator of pest presence.
A smart sensor network deployed across the orchard continuously monitors environmental temperature, humidity, MeSA concentration, and carbon dioxide levels, transmitting the data to a cloud-based analysis system to support reliable pest population assessments. Farmers can access real-time data and historical trends via a WeChat mini-program, which also sends alerts for abnormal fluctuations, ensuring timely awareness of orchard conditions.
Based on monitoring data trends, farmers can make scientifically informed control decisions: when MeSA levels are low, indicating the latent phase of aphid infestation, RNAi formulations can be applied via trunk injection for preventive control. When MeSA concentrations rise persistently alongside abnormal CO₂ levels, Metarhizium formulations are recommended for emergency eradication. In the future, as field data and algorithmic models accumulate, the mini-program will integrate intelligent decision-making capabilities to automatically generate tiered control recommendations, empowering farmers to manage orchard pests with greater precision and efficiency.
Figure 1 - CitrusShield Smart Ecosystem
From Lab to Application
In transitioning laboratory achievements into field-ready products, we have faced multifaceted challenges spanning scientific, regulatory, economic, and social dimensions.
- Regulatory Compliance & Approval:
As China currently lacks specific regulations for RNA-based pesticides, we authored Regulatory Analysis of RNA Pesticides in China and the RNA Pesticide Industry Development Bluebook to outline domestic and international policies and propose context-appropriate strategies. Through consultations with industry experts such as Xie Ning, we have assessed policy risks to ensure both the scalability and compliance of our technologies. - Cost-Effective & Sustainable Production:
High laboratory synthesis costs and complex processes posed barriers to economic viability. By adopting cell-free in vitro transcription for RNA synthesis, we achieved scalable production. Further optimization of lyophilized powder processes and formulation ratios provided a practical economic foundation for translating lab research into field-applicable products. - Public Acceptance:
Concerns among farmers and consumers regarding synthetic biology initially hindered product adoption. To address this, we organized 13 targeted science outreach sessions, participated in 8 large-scale exchange events, and reached over 18,000 individuals through online and offline channels. In collaboration with other teams, we also compiled the Debunking Synthetic Biology Misconceptions handbook to enhance public understanding and support sustainable technology adoption.
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By integrating regulatory analysis, process optimization, and public education, we have established an end-to-end framework from laboratory research to field application—effectively addressing key barriers and laying a solid foundation for future commercialization.
Future Outlook
Looking to the future, the promotion of CitrusShield will be based on the established technological foundation and field validation results. Building on the three core formulations — trunk-injected RNA dry powder, engineered Metarhizium capsules, and Bacillus subtilis intelligent slow-release microcapsules — we will steadily advance the following work:
Within the regulatory framework, we will systematically conduct regional adaptability demonstrations and commercialization exploration, allowing more farmers to experience the practical value of the intelligent monitoring and precise intervention system firsthand. At the same time, we will continue optimizing the technology by developing a regionalized RNAi molecular library and improving predictive models to enhance the stability and effectiveness of the formulations in different production areas and against various aphid strains. In addition, we are actively advancing cross-crop research on RNAi technology platforms, exploring feasible pathways to extend its application to pest control in other crops, laying the foundation for future technology reuse and system expansion.
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