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Introduction

According to the State of Food Security and Nutrition in the World (SOFI) report, about 735 million people faced hunger in 2022^[1]. To solve these series of problems, agricultural development is one of the most powerful tools to end extreme poverty, boost shared prosperity, and feed a projected 9.7 billion people by 2050. Agriculture is also crucial to economic growth: accounting for 4% of global gross domestic product (GDP) and in some least developing countries, it can account for more than 25% of GDP^[2].

Tomato is a particularly important crop in agriculture worldwide. FAO data shows that the global tomato harvest area and total tomato production in 2021 were about 5.17 million hectares and 189 million tons respectively. Among them, China's tomato planting are reaches 1.14 million hectares, and its annual output ranks first in the world, reaching 68 million tons, accounting for 11.2% of the national vegetable output. The gross production value of tomatoes in the world is $100 million, of which the value in China is $35.5 million^[3].

Figure 1. Data on tomato production.

Furthermore, tomato is one of the most common foods in our daily life, with the nature of fruits and vegetables. It can appear in a variety of forms on the recipe, such as tomato scrambled eggs, tomato hot pot, tomato sauce, tomato salad, etc., has become an indispensable food on the table of people all over the world.

Problems

Plant fungal diseases are one of the main culprits affecting the quality and yield of crops. Their existence has greatly impacted food health and agricultural development, and may even further increase the degree of hunger. These diseases are caused by fungi, which secrete a variety of pathogenic factors to attack the host when they infect crops, and can spread widely before effective measures are taken.

Figure 2. Problems caused by plant fungal diseases

Botrytis cinerea is a kind of worldwide pathogen of gray mold. It can infect the roots, leaves, flowers and fruits of plants, causing diseases on more than 500 plant species, including more than 200 crop species^[4]. B. cinerea is a plant pathogen suitable for growing in low temperature and high humidity. In agricultural production, the temperature in the greenhouse is usually maintained at 15-20°C, with high relative humidity, which is easy to cause a large range of gray mold. B. cinerea is one of the most damaging post-harvest pathogens in many agricultural products^[5]. In the process of storage and transportation after harvest, B. cinerea will accelerate the deterioration of crops, resulting in serious logistics losses.

In nature, B. cinerea resist stress through overwinter sclerotium and conidium in soil and host plant tissues. Sclerotium is mainly formed in hosts that are about to die and lack nutrients. The blackened outer skin and β-glucan protect the internal mycelium, which enhances the resistance of sclerotium to adversity. It protects sclerotium from dryness, ultraviolet radiation, and long-term bacterial attack. Sclerotium is the main form of B. cinerea to overwinter and survive adversity. It can survive for about 11 months in the soil. In suitable conditions for germination, sclerotium, and conidium will germinate and infect the host. Conidia spread with air flow, rain or dew drops, tools and clothing in farming operations, and often invade from the host's weakened organs, tissues or wounds, causing disease. There is a latent period from attachment to infection. The mycelium propagates on the disease residue, the disease spot expands, kills the host, and after the host nutrients are absorbed, B. cinerea will produce a large number of conidium in the infected site through a space conidium pedoides under signal transduction and regulation. After the conidia mature and fall off, they spread and infect through rain, irrigation in fields or protected areas, and wind, forming an infection cycle.^[6].

Figure 3. The infection cycle of B. cinerea.

At present, chemical fungicides are the main means to control B. cinerea. AAlthough it can inhibit diseases efficiently, it is also easy to endow B. cinerea with resistance, which weakens the control effect in the long run. Chemical control is mainly divided into two types. One is protective fungicides with preventive effects, such as mancozeb and chlorothalonil, used before the onset of the disease^[7]. The other is therapeutic fungicides such as pyrimethanil and procymidone that are used after the onset of the disease^[8]. The two fungicides have their own advantages, but the common problem between them is that the control effect becomes worse after a period of use, that is, B. cinerea has resistance. Moreover, chemical fungicides are not conducive to environmental protection, and the pesticide residues brought by it will harm the health of humans and animals.

Our solution: PolycoBead

We designed the product PolycoBead, which is a bead that is packaged into a fixed volume. The bead contain two main components: RNAi preparations and engineered bacteria that can produce agents that induce plant immunity.

• RNAi biopesticides: The main component is CPP-shRNA, whose shRNA can trigger the RNAi process, so as to specifically target and silence the key genes and survival genes of B. cinerea to inhibit its infection to tomato. CPP is the cell membrane penetrating peptide that binds to shRNAs to help shRNAs more stably exist in the natural environment and be more easily absorbed by cells.


• Engineering Bacillus subtilis: As a common biocontrol bacterium in agriculture, B. subtilis is able to competitively attach to plant roots and protect plants from infection by other pathogens. We engineered B. subtilis WB600 to express the 22-amino acid sequence of the N-terminal of P. aeruginosa flagellin protein (Flg22), which is a pattern signaling molecule, can be bound by pattern recognition receptors on the surface of tomato cells, thereby inducing the plant's natural immune system to improve resistance to gray mold.

Figure 4. PolycoBead.

The beads are designed to solve the following problems:

• - RNA pesticides are easy to degrade and not easy to preserve: Due to the presence of nucleases and other factors in the environment, our shRNA products are extremely easy to degrade, and may degrade during our production and transportation, resulting in weakened effects. The presence of a bead membrane helps insulate RNA from the environment and prevent RNA degradation.


• - Due to the phenomenon of pesticide abusion, many farmers do not know how much pesticide is appropriate to use. The net weight of each coagulated bead is about 1.5g, containing 10mg of RNA, and every 2 coagulated beads are mixed with 10L of water, which can treat about 100 square meters of tomato fields ( a density of about 100-120 plants with 50-60 fruits) to avoid the abuse of pesticides.

Figure 5. The use of PolycoBead.

More details can be viewed in Design and Proof of concept

Practical Application

RNAi biopesticide is a new type of pesticide based on RNAi technology, known as "another technological revolution in the history of pesticides", is currently one of the most promising crop protection products. RNAi pesticides have significant advantages over traditional pesticides:

• --They are highly specific and do not affect non-target organisms.


• --No environmental toxicity, can be degraded in natural ways.


• --Can be developed for all pest species, short development cycle, flexible target change.


• --The amount used is very low, with 2-10g per hectare of crop control.

By specifically targeting the fungal mRNA sequence, the process of RNAi can eventually trigger the degradation of mRNA, resulting in a silencing effect on the interested gene. In the nascent field of RNAi pesticides, several giant companies have made great strides. Bayer has applied for the first exogenous application of dsRNA biocide, which is expected to be available in 2024; GreenLight Biosciences (has been acquired) has reduced the price of dsRNA to $0.50 per gram, which is very promising in terms of product commercialization.

Plant immune-inducing agents, also known as plant vaccines, are a new type of biopesticides, which combine the theories of plants, pests and diseases and biopesticides, and are currently a hot research direction in the field of biopesticides in the world. Plant vaccines do not directly kill pathogens, including pathogenic bacteria and pests, but stimulate the immune system in the plant through induction preparations to improve the antibacterial and insect-resistant ability of plants, which is different from traditional pesticides. At the same time, plant vaccines can also stimulate a series of metabolic regulation systems in plants, promote plant tissue growth, and ultimately achieve the purpose of increasing production and preserving quality. Since the end of the 20th century, countries around the world have officially registered a variety of plant immune-inducing drugs, and some pesticide giant companies and research institutes have carried out the industrial production of plant immune-inducing drugs. At present, plant vaccines available on the market include oligosaccharide inducing factors, protein elicitors, and probiotics.

As of March 2019, the number of registered products of biochemical pesticides, microbial pesticides and botanical pesticides approved and registered in China is about 1,200, accounting for only 4% of the total number of registered pesticides in China, which is much lower than that of chemical pesticides^[9]. As of September 2023, only 44 plant immune-inducing agents have been registered, and the total number of registered pesticides is 45,733, accounting for only 0.962%, and their use and acceptance are also low^[10].

Policy is the prerequisite for technology commercialization. If we want to promote our products to further expand the test scale and enter the market, we must clearly clarify the legal norms that our products need to comply with. During the investigation, we found that there are no relevant laws and regulations on RNA pesticides in China. Therefore, we combined our own experimental basis and the existing standards of various countries to write a proposal for China's RNA industry standards, and submitted it to the relevant departments, hoping to promote the early implementation of China's RNA pesticide regulations. In addition, we hope to form an RNA enterprise alliance (Though-Oriented Modernization of Agriculture-Targeted Organization, TOMATO) to drive the transformation of traditional chemical pesticide companies to adapt to future trends in biopesticides.

More details can be viewed in integrated human practices and entrepreneurship.