This project aims to develop an efficient, low-cost, integrated solution for the early detection and control of
Figure 1 Bacterial wilt infection in crops
Bacterial wilt, caused by the soil-borne pathogen Ralstonia solanacearum, can infect over 400 crops including tomatoes, peppers, tobacco, and bananas, making it a devastating global disease [1]. Prevalent in warm, humid regions worldwide, it is particularly severe in Southern China (e.g., Guangxi, Guangdong, Fujian, Hainan). The persistent rainfall during the "Dragon Boat Water" period and typhoons from April to September in Guangdong create ideal conditions for outbreaks [2]. Initially, plants wilt at midday and recover in the morning and evening, making early detection difficult. Subsequently, infected plants rapidly die within days, potentially causing yield losses of 16%-41% in tomatoes, 64%-65% in potatoes, and 60%-90% in eggplants [3].
Figure 2 Tomato plants affected by bacterial wilt (Source: Wikipedia)
Ralstonia solanacearum is a Gram-negative, aerobic, short rod-shaped bacterium. Its optimal growth temperature is 28-33°C and optimal pH is 6.6 [4]. Ranked second among the top 10 important plant pathogenic bacteria [5], it is one of the world's most destructive plant pathogens. It spreads via fertilizer, water, infected soil, seeds, farming practices, and underground pests. Typically entering through root wounds or stem lenticels, it can latently colonize the rhizosphere for days or weeks in warm, humid conditions. Once the population reaches a threshold sufficient to block the plant's vascular system, plants suddenly wilt, by which time the disease is widespread and difficult to control [6].
Figure 3 Ralstonia solanacearum under electron microscope (Source: Wikipedia)
The pathogenicity of R. solanacearum is precisely regulated by a Quorum Sensing (QS) mechanism [7]. During infection, the PhcB protein continuously synthesizes specific Acyl-Homoserine Lactone (AHL) signal molecules [8]. At low cell densities, AHL concentration remains at baseline. As the pathogen proliferates, AHL accumulates significantly in the environment [9][10]. This increase in AHL concentration directly correlates with disease progression: high AHL levels signal that the bacterial population has reached a critical density, triggering synchronized virulence gene expression, leading to vascular blockage and tissue rot [11].
Figure 4 Secretion of AHL by Ralstonia solanacearum in infected plants
The project inspiration came from a team member whose family grows tomatoes for a living. This year, their tomato field was again struck by bacterial wilt. Initially, the plants wilted during the day and recovered at night, leading the family to believe it was just water stress. However, within just 7-8 days, previously healthy plants completely withered and died, leaving no chance for intervention. Bacterial wilt has become the "number one killer" of tomato plants, spreading so rapidly that farmers describe it as "more aggressive than cancer." Therefore, early detection and control of bacterial wilt are critically important.
Various detection methods for bacterial wilt exist, each with advantages and limitations. Traditional methods (e.g., symptom observation) are simple and low-cost but subjective and incapable of early diagnosis. Immunological methods (e.g., ELISA) require laboratory equipment [12]. Molecular biology methods like PCR/qPCR are sensitive and accurate but time-consuming, require expensive equipment, involve complex operations, and are difficult to popularize [13][14]. LAMP technology is suitable for field application but still faces challenges like complex primer design and aerosol contamination risk [15]. Emerging technologies like bioluminescent reporter systems and hyperspectral imaging rely on expensive equipment, complex algorithms, or genetic modification, currently limiting their use to research and keeping them distant from field application [16][17].
Figure 5 Comparison of Current Detection Methods for Bacterial wilt
To enable more intuitive and rapid detection of plant disease, GEC-Guangzhou has designed an early detection device for bacterial wilt. This device operates through the synergy of three core systems: First, a highly sensitive detection system identifies trace amounts of the pathogen's specific signal molecules in the soil for early disease warning. Second, a unique visual signal system converts the invisible chemical signal into a visible colorimetric reaction, requiring no complex instruments and being extremely user-friendly. Finally, a precise treatment system non-invasively activates the plant's own immune system, enabling green control of the disease. This device combines the three major advantages of being early, non-invasive, and convenient, offering a completely new disease management strategy for agricultural production.
Figure 6 The Three Core Systems
E. coli BL21, one of the most commonly used prokaryotic expression hosts, has the T7 RNA polymerase gene integrated into its genome, enabling efficient adaptation and driving of the T7 expression system for rapid, high-level expression of foreign proteins [18]. Additionally, BL21 grows rapidly, has simple and easily controllable culture conditions, making it one of the most reliable and powerful chassis microorganisms for both basic research and industrial production.
Figure 7: BL21 Escherichia coli (Source: Wikipedia)
In the engineered E. coli BL21, the constitutive promoter J23100 continuously expresses the QscR protein. When AHL signal molecules are absent from the environment, the QscR protein specifically binds to the operator site of the PA1897 promoter, inhibiting transcription of the downstream red fluorescent protein (RFP) reporter gene; the cells produce no fluorescence [19]. Once AHL signal molecules released by R. solanacearum enter the cell, they bind to QscR, forming a QscR-AHL complex. This causes a conformational change in the protein, dissociating it from the PA1897 promoter. This derepression activates the PA1897 promoter, driving high-level expression of the RFP gene, thereby producing a red fluorescent signal detectable by instruments. This achieves sensitive and specific reporting of pathogen presence. This system makes the bacteria "flash a red light" upon detecting the pathogen, enabling early warning.
Figure 8 Detection and Alarm System
Detecting the red fluorescence (RFP) from System 1 necessarily relies on specialized equipment like fluorescence microscopes or portable fluorometers. While this is not an issue in the laboratory setting, for farmers or field technicians needing to make quick decisions, the high cost of equipment, inconvenience of carrying it, and operational complexity make it difficult to popularize.
To make the alarm signal more obvious and eliminate dependence on complex equipment, System 2 visualizes the signal, creating a visible pigment precipitate. Indigo is an insoluble blue precipitate that serves as a natural dye. It is non-toxic and harmless, forming stable blue spots inside or around the cells, or turning the entire colony blue. The signal does not fade, is intense in color, clearly visible even in outdoor sunlight, and the results can be stored long-term.
Figure 9 Chemical structure of indigo
In the engineered bacteria, indigo synthesis relies on two heterologous enzymes we introduced: TnaA (tryptophanase) and FMO (flavin-containing monooxygenase). They work synergistically to catalyze the endogenous substrate L-tryptophan from the E. coli stepwise, ultimately producing indigo. Specifically, TnaA first catalyzes the decomposition of tryptophan to produce indole. FMO then oxidizes indole to indoxyl, which spontaneously oxidizes and polymerizes in air, finally forming insoluble blue indigo precipitate [20]. This colorimetric mechanism allows the engineered bacteria to display a distinct, visible blue color upon perceiving the pathogen signal. This reaction offers strong signal intensity, high stability, and is irreversible, significantly enhancing the portability, practicality, and field applicability of the detection system, making it highly suitable for rapid field testing.
Figure 10 Visual Signal System
Salicylic acid (SA) is a key endogenous hormone that can activate the plant's own broad-spectrum immune responses. It does not directly kill bacteria but induces Systemic Acquired Resistance (SAR) in the plant, prompting the whole plant to synthesize pathogenesis-related proteins and strengthen cell walls. This effectively inhibits the spread and damage of R. solanacearum within the vascular system, delaying symptom onset and reducing overall disease severity. It represents a green control strategy based on host immunity [21].
Figure 11 Chemical structure of salicylic acid
The system uses the QscR-AHL complex-mediated promoter regulation as the core switch: The genes for isochorismate synthase (ICS) and isochorismate pyruvate lyase (IPL) are tandemly cloned downstream of the PA1897 promoter, constructing a "PA1897-ICS-IPL" expression unit [22]. Here, PA1897 acts as an inducible promoter specifically activated by the QscR-AHL complex. Its transcriptional activity strictly depends on the concentration of AHL signal molecules in the environment. When pathogen infection releases AHL, QscR binds AHL to form an active complex. By binding to cis-acting elements in the PA1897 promoter region, it initiates the coordinated expression of downstream ICS and IPL, achieving "on-demand" salicylic acid synthesis.
Figure 12 Treatment System
The core target customers for this solution are large-scale producers of high-value cash crops, mainly including large-scale planting enterprises, farms, agricultural cooperatives, and professional growers. They intensively cultivate susceptible crops like tomatoes, peppers, eggplants, potatoes, and bananas, especially in hot and humid regions like Guangdong, Hainan, and Guangxi where bacterial wilt is prevalent. They have an urgent need for early disease detection to avoid large-scale yield reduction and economic losses. They desperately require a practical tool that can overcome laboratory dependence and enable rapid, on-site detection to guide control measures. The visually detectable, low-cost, integrated solution offered by this project precisely meets their core demand for improving disease management capabilities.
Figure 13 Target Customers
The system's use involves two core steps, detection and treatment, both performed within a safe, enclosed operating environment to prevent direct environmental release of the engineered bacteria.
First, the detection step: The user collects a liquid sample from the target soil area using a dedicated water sample collector. If the area is infected by bacterial wilt, the sample will contain AHL signal molecules released by the pathogen. The collected sample is automatically deposited into a disposable sterile vacuum blood collection tube prefilled with lyophilized engineered bacteria powder from our project. The tube is then placed on a specially designed, portable 3D-printed shaker for incubation. If AHL is present in the sample, it activates the sensing system of the engineered bacteria, causing them to rapidly produce visible indigoidine pigment. The user can determine the presence of bacterial wilt infection risk in that soil area based solely on the color change, without needing specialized instruments.
Subsequently, the treatment step: For samples testing positive, the system offers a targeted treatment strategy. A portion of the positive sample is added to another specialized reaction device containing a different strain of engineered bacteria. This strain is designed to sense AHL and, in response, initiate the synthesis of salicylic acid (SA). Stimulated by AHL, this strain efficiently produces salicylic acid. The user can subsequently extract and purify salicylic acid from the reaction solution and apply it to the original soil sampling area. This activates the plant's systemic resistance, achieving precise and ecological control of bacterial wilt in that area.
Figure 14 Usage Process
In practical production, the project's main task is to mass-produce the two functional bacterial strains through large-scale fermentation, prepare them into lyophilized powder formulations, package them into dedicated disposable collection tubes, and ultimately launch them as a storable, ready-to-use detection product.
The specific production process includes: First, separately cultivating the sensing reporter strain (for generating the indigo signal) and the salicylic acid synthesis strain (for subsequent treatment) on a large scale in strictly controlled bioreactors. After harvesting the bacterial cells via high-speed centrifugation, they are mixed with protective agents (e.g., trehalose, skimmed milk) and processed into highly active, stable lyophilized powder through freeze-drying. Subsequently, under aseptic conditions, a quantified amount of the lyophilized powder is aliquoted into disposable sterile vacuum blood collection tubes, which are immediately vacuum-sealed with nitrogen gas flushing to ensure long-term stability during transportation and storage.
The final product – the ready-to-use detection tube – requires the user only to inject the soil liquid into the tube using the sampler; results are visible to the naked eye after brief incubation. This process translates synthetic biology technology into a standardized, scalable biological product, ensuring product consistency and reliability, suitable for disease monitoring and precise control applications in large-scale agricultural production.
Figure 15 Production Process of Engineered Bacteria
The core design of this project highly prioritizes biosafety, preventing the environmental release risk of engineered bacteria from the source. Initially, directly applying engineered bacteria to the soil for in-situ detection was considered. However, due to prudent consideration of the potential ecological impact of genetically modified organisms (GMOs) spreading in the natural environment, we completely abandoned that plan in favor of the current fully enclosed, controllable detection format.
The E. coli BL21 engineered strain we selected is itself a safe, non-pathogenic, non-toxigenic strain. The final product strictly encapsulates this strain within disposable collection tubes. The bacteria are in a dormant state after deep freeze-drying, and the entire operation is completed within the sealed tube. After detection, all materials can be uniformly inactivated by high-temperature treatment, ensuring the engineered bacteria never contact the external environment.
This design strictly adheres to the biosafety principles of "preventing escape, preventing spread, and ensuring inactivation." While fully leveraging the advantages of synthetic biology technology, it also fulfills the commitment to environmental responsibility, complying with the safe application standards for agricultural microbial technology.
Figure 16: Engineered bacteria in sealed disposable tubes (powder shown for illustration only).
Figure 17 Project Advantages
We provide an innovative, integrated detection and control solution for the global challenge of bacterial wilt that is early, low-cost, on-site implementation, and highly safe. Utilizing synthetic biology, the project constructs an intelligent engineered bacterial system capable of precise pathogen sensing, visual signal reporting, and on-demand production of plant immunity activators. This shifts disease management from passive remediation to active early warning and green intervention. The core technology operates entirely within a closed system, eliminating the risk of environmental release of genetically engineered organisms, combining technical advancement with ecological safety. It offers a new paradigm for the green and sustainable management of field crop diseases, holding significant value for ensuring food security, reducing pesticide use, and promoting the development of smart agriculture.
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