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Background Research

About Plant Nematode Diseases

Plant nematode is one of the important pathogens causing crop diseases in China. It is a serious threat to China's wheat, rice ,corn and other food and economic crops and the safety of production of important diseases. For example, Soybean cyst nematode disease occurs in an area of 1.33×10⁶ hm² per year, which can cause soybean yield reduction of 5%-10%, and the serious occurrence of plot yield reduction of more than 30%, or even no harvest. Losses caused by soybean cyst nematode disease amounted to more than 600 million yuan. With global climate change, cultivation system reform and the rapid development of large-scale, mechanised and high-value agriculture, plant nematode diseases are in a serious trend of occurrence, and will rise to be the second largest plant disease in China. Common nematode control methods, such as chemical control and land management, have problems such as pollution of the environment and poor quick-acting properties.There are 435 pesticide products registered in 2022 in China, of which only 9 are microbial pesticides, and there are few pesticide products for nematode control.In addition, the world's population has increased significantly, and the amount of arable land is decreasing year by year.The demand for food has increased dramatically due to the continuous growth of the population.The pressure on limited land resources has increased substantially. At the same time, we have also had a comprehensive understanding of biopesticides.

About Biopestiside

Policy Perspective: The government is increasingly supporting biopesticides. In July 2021, the Ministry of Agriculture and Rural Affairs introduced four favorable policies for biogenic pesticides, which have spurred the development of biopesticides and promoted green and sustainable growth in the pesticide industry. The "14th Five-Year National Pesticide Industry Development Plan," issued in January 2022, also emphasized the critical role of biopesticides in optimizing pesticide varieties, advancing green development, and ensuring the quality of agricultural products and environmental safety. It explicitly called for the development and transformation of the biogenic pesticide industry.

Economy Perspective: The market potential for biopesticides is substantial. From 2020 to 2025, the global biopesticide market is projected to grow at a compound annual growth rate of 10%. By 2029, the market size is expected to reach $1.87 billion, indicating a promising future for the industry.

Social Perspective: Social factors such as population growth, shrinking arable land, and increasing environmental pollution are driving the growing demand for microbial pesticides, fostering positive momentum in the biopesticide industry. Additionally, we designed a survey to assess public awareness of biopesticides. Using a snowball sampling method, we collected over 400 responses. The results revealed that public knowledge of biopesticides is extremely low, with only 3.4% of respondents being highly informed.In addition to quantitative research, we also conducted qualitative studies. We visited a village in Hunan Province and interviewed the villagers about the current situation of nematode diseases in the area. We found that the village chief had little knowledge of root-knot nematode disease and was only aware of pine wilt disease through online news. To our surprise, nematode infestations were not uncommon in this village, yet the villagers attributed issues such as plant wilting, slow growth, and reduced yield solely to a lack of water and nutrients, without a scientific understanding of plant nematode diseases. These quantitative and qualitative findings have also provided direction for our subsequent HP work.

Figure 1. Level of public understanding of biopesticides.

Technical Perspective:The field of biopesticides is evolving rapidly with continuous research and innovation. Technological advancements have made biopesticides more efficient and diverse, with the application of new technologies such as microbial engineering, gene editing, and synthetic biology. The integration of nanotechnology and precision agriculture further enhances the application efficiency and stability of biopesticides. Production technologies for biopesticides are becoming increasingly mature, leading to improved production efficiency and quality control, reduced costs, and the promotion of large-scale production. Application technologies are also advancing, with the use of drones, satellite remote sensing, and other methods enabling precise application in farmlands, reducing usage while enhancing pest control effectiveness. These technological developments make biopesticides more feasible for large-scale agricultural production.

Project Prototype

In selecting the chassis organism, we chose Bacillus velezensis. The bacteria is widely found in soil, plant rhizospheres, and various natural environments, exhibiting significant plant growth-promoting and biocontrol characteristics. Therefore, we believe Bacillus velezensis is the ideal choice for our project.

The engineered bacteria derived from Bacillus velezensis, named "Nematode Buster", primarily resides at the roots of plants, forming a biofilm. Its main function is to sense and attack nematodes, thus protecting the plants. When the engineered bacteria detect an increase in salicylic acid concentration due to nematode damage, they synthesize virus-like particles (VLPs).These particles carry nematode-resistant protein Cry6Aa2 and shRNA targeting nematode infection of plants and their own movement, thus achieving the effect of killing nematodes.

Figure 2. Circuit Design for Nematode Buster.

Project Optimisation

For the selection of the chassis organism, we chose Bacillus subtilis. The bacteria is widely distributed in soil, air, and water and it is already used as biopesticides or biofertilizers to promote plant growth and enhance crop disease resistance. Moreover, it is harmless to humans and animals, making it widely applicable in agriculture, the food industry, and medicine. Therefore, we believe that Bacillus subtilis is the ideal choice for the chassis organism of our project.

The Plant Guard is engineered from Bacillus subtilis is primarily designed to enhance plant immunity and defend against nematodes. Similar to the mechanism of Nematode Buster, the engineered bacteria detect an increase in salicylic acid levels caused by nematode damage to plants and begin synthesizing virus-like particles (VLPs). These VLPs contain RNA that targets plant immune regulation. Additionally, the VLPs carry externally attached plant immunity-activating proteins, which aid in inducing plant immune responses.

To prevent the unnecessary leakage of engineered bacteria, we have designed a suicide module for the engineered bacteria.For the suicide module design, we employed the photo-toxic protein Killer Red to execute the suicide function of the engineered bacteria. Research indicates that Killer Red is a red fluorescent protein that can produce reactive oxygen species (ROS) under yellow-green light (540-585 nm), facilitating the self-destruction of the engineered bacteria. Based on its characteristics, our engineered bacteria can be killed under both artificial green light and natural sunlight, ensuring complete safety.

In addition, we have optimized and improved certain details of the circuit to enhance the insecticidal efficiency of the engineered bacteria.

Figure 3. Circuit Design for Bacillus Gemini.

Plant Guard and Nematode Buster can eliminate nematodes through dual channels, to achieve better extermination effects. Ultimately, the combination of Plant Guard and Nematode Buster has formed the HZAU-China 2024 project—Bacillus Gemini.

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Improvements

During our discussions with farmers, we introduced our project to the local agricultural technicians in the village. During this period, the local technicians raised concerns about the safety of the engineered bacteria. We responded by explaining that our engineered bacteria are pro-biotic strains friendly to the environment, vegetation, and humans, so there is no need to worry about negative impacts from the bacteria.

Additionally, during our preliminary background research, we discovered that some farmers had a limited understanding of plant nematode diseases,and some of them were even unaware of the issue. Therefore, we conducted an offline agricultural knowledge session for local farmers. Using promotional brochures we created, the session presented agricultural production knowledge in simple and easy-to-understand language, focusing on how to identify nematode infestations in plants and environmentally friendly control methods. After listening to our explanation of plant nematode diseases, the farmers expressed that they had learned a lot and felt more confident in handling similar problems in the future. The village committee leaders remarked that through this knowledge session, they saw university students' concern for agricultural issues and their sense of responsibility in building new rural areas.

Figure 4. Team members are talking to farmers working in the field about nematodes.

In discussions with experts, Professor Shunping Yan pointed out that salicylic acid levels vary between plants in normal and stress conditions. Therefore, we needed to adjust the sensitivity of NahR to salicylic acid to enable it to respond appropriately under different concentrations. To address this, we conducted mutations and screened 16 NahR sequences with increased substrate affinity, achieving optimization of NahR.

During our discussions with the staff from the Agricultural and Rural Affairs Bureau, we inquired about the root depth of locally grown crops to simplify the TAA diffusion model. Based on their experience, staff from the Bureau of Agriculture and Rural Affairs informed us that the root depth is approximately 20 cm. Therefore, we simplified the TAA diffusion source to a sphere with a radius of 10 cm, improving the accuracy of our predictions. Meanwhile,we learned that farmers consider several factors when selecting pesticides for agricultural production, including the speed of action, insecticidal effectiveness, cost-effectiveness, and ease of application. Additionally, with the increasing enforcement from environmental protection agencies in recent years, farmers are also incorporating environmental protection into their pesticide selection criteria. Based on these considerations, we summarized our product design goals as: green, simple, and efficient.So,we shared our idea of developing a lyophilized powder formulation with the technical personnel from the Agricultural and Rural Affairs Bureau, who recognized the potential of this product format. They indicated that with the expanding scale of greenhouse production, easy-to-use biopesticides have a promising market outlook.

Figure 5. Team Members Visiting the Agricultural and Rural Affairs Bureau and Environmental Protection Bureau for Discussions.

To confirm the feasibility of our idea to create a lyophilized powder formulation, we visited Abclonal Technology. Abclonal Technology is a company focused on the research, development, production, and sales of antibodies, with rich experience in production. We went to their production and R&D base to observe their advanced manufacturing machinery. When we presented our idea of producing engineered bacterial agents as lyophilized powders to their technical staff, they analyzed the technical feasibility and stated that lyophilized powders are indeed a highly suitable format for our project product, offering good operability and effectiveness.

Figure 6. Team Members are engaging in Discussions with Staff from Abclonal Technology.

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