New technology to control grey mold with outstanding effects and no resistance problem

1. Introduction

Fungicides are the chemicals that effectively control or kill pathogens - bacteria, fungi and algae - that are harmful to plants. Globally, it is generally used as a general term for chemicals to control various pathogens ^[1]. All kinds of plant pathogens are one of the main factors causing plant diseases, which have caused huge economic losses to agricultural production. Fungal diseases are the largest category of plant diseases, accounting for 70%~80% of plant diseases, and the disease occurs in a wide area, extremely harmful, and difficult to control ^[2].

Fig1. Common fungal diseases

Fungal diseases have caused huge economic losses to garden plants and crops around the world. Botrytis cinereal, one of the main fungal diseases, can harm more than 470 plants such as Solanaceae, Cucurbitaceae, and Rosaceae ^[3], causing plant leaves drop, fruits and vegetables rot, and agricultural products quality decline. This disease causes more than 10 billion U.S. dollars economic losses worldwide every year. For the grape wine industry, the annual wine production can be reduced by up to 20% due to Botrytis cinereal outbreak. Grapes are also often infected by Botrytis cinereal during storage, and the post-harvest losses can be as high as 50% in severe cases ^[4]. In China, there are large planting areas of strawberry, but due to the damage of Botrytis cinerea, the production will generally be reduced by 20% to 30%, and be as high as more than 50% in severe cases. This has caused economic losses to fruit farmers which cannot be underestimated. Botrytis cinerea is an important disease in tomato cultivation, which can cause 20% to 30% reduction in tomato yield, and even as high as 50%. Among ornamental plants in gardens, roses are also severely damaged during long-distance refrigerated transportation. According to the investigations, more than 70% of post-harvest losses can be caused under extreme conditions. The Fusarium head blight (FHB) that occurs on wheat, also known as scab or tombstone, causes reduction in grain weight, low germination rate and vigor, low flour production and quality and low economic value. Generally, the infection rate of FHB in a severe year can reach 50-100% with yield loss 10-20%. In recent years, the climate change in some major production areas also leads to outbreak of powdery mildew in wheat, and in severe cases, the yield reduction can reach more than 40%. With the adjustment of agricultural planting structure and the use of greenhouses, the concentrated planting environment with warm and high humidity makes fungal diseases more difficult to control. Fungal diseases are very easy to outbreak in artificial climate environments. In China, powdery mildew causes strawberry production reduce by 10% to 20% every year, and reach more than 50% or even 100% when it breaks out.

For a long time, the control of plant fungal diseases has mainly relied on chemical fungicides, such as Difenoconazole, Myclobutanil, Tebuconazole, Triflumizole, Pyraclostrobine, Mancozeb, Fluazinam, Triadimefon, Pyrimethanil, Procymidone, Iprodione, and etc., which are widely used to control fungal diseases of crops. The preventive and protective fungicides like mancozeb have limited control effects, and cannot meet the needs of growers for disease control when they are used alone. The systemic fungicides like Difenoconazole not only inhibit the infection of pathogenic fungi, but also affect the physiological indicators of crops, e.g. inhibit chlorophyll synthesis and plant growth, and reduce the resistance of plants to pathogenic fungi. At the same time, continuous use of chemical fungicides has further caused problems such as resistance problem, chemical residues, and environmental pollution ^[6]. The control of fungal diseases will enter into a vicious circle that the issues of the environmental impact, crop health and disease control restrict each other.

According to market research, the global fungicide market keeps developing in recent years. Due to the increasing resistance of pathogenic fungi to chemical fungicides year by year, the market has a continuous demand for new products. According to the reports, in 2012-2013, the resistance of Botrytis cinerea to Carbendazim in Hubei Province of China increased by 63.6% ^[7]. The resistance of Botrytis cinerea to Carbendazim in Liaoning, Hebei, Beijing, Anhui and Sichuan provinces increased by 53.6%-99.0% ^[8]. On the other hand, the adjustment of the global crop planting structure, the increased areas and types of vegetables, fruits and various economic crops boost demand for fungicide products, so the fungicides have a huge market potential in the future. At the same time, with people's increasing concern about environmental issues and the gradual increase in the area of organic agriculture, the demand for natural crop protection inputs with low toxicity and low resistance in the global market is increasing. It is urgent to develop a bio-pesticide that is environmentally friendly, highly efficient, low-toxic, not easy to cause resistance problem, and can promote crop growth.

2. Research status on Reynoutria Japonica Houtt. Extract

Reynoutria japonica Houtt. is a traditional Chinese medicinal plant found in eastern China, central and southern China, Liaoning, Shaanxi, Gansu, Sichuan, Guizhou, Yunnan provinces. According to Chinese traditional medicine record, its roots and rhizomes are used in medicine, which have the effects of activating blood and relieving pain, clearing heat and dampness evil, quenching thirst and phlegm ^[9]. Studies have shown that Reynoutria japonica Houtt. is rich in active ingredients such as Polydatin, resveratrol, emodin and physcion ^[10]. Its extract has anti-cancer function with a significant inhibitory effect on the proliferation of tumor cells, and can greatly slow down the growth rate of tumor tissue ^[11-12]. At the same time, the secondary metabolites of Reynoutria japonica Houtt have natural resistance ability to diseases and pests, so there is very few diseases and pests occur during the growth of Reynoutria japonica Houtt, and almost do not need pest control ^[13].

Fig2. Medicinal plant Reynoutria japonica Houtt and its medicinal parts

Based on the characteristics of secondary metabolites of Reynoutria japonica Houtt and their pharmacological effects, Reynoutria japonica Houtt is used to control plant diseases in agriculture. As one of the most abundant active ingredients among the secondary metabolites of Reynoutria japonica Houtt, physcion has high activities against plant diseases such as powdery mildew, downy mildew, gray mold and anthracnose.

Fig3. Physcion CAS: 521-61-9

3. Research progress on industrialization of Reynoutria japonica Houtt extract

(1) Registration status of Reynoutria japonica Houtt extract

According to the report of AgroPages, Prime Source division of Albaugh, LLC has launched the fungicide Azoxy Bio (Azoxystrobin and Reynoutria sachalinensis extract), which has been registered with the US Environmental Protection Agency in the spring of 2022, is a systemic fungicide that can not only prevents and controls major diseases in lawn and ornamental plants, but also promotes the plant immune system and achieves good disease resistance management.

It is known that the new bio fungicide registered by Chengdu Newsun Crop Science Co., Ltd. (hereinafter referred to as "Chengdu Newsun") - Reynoutria japonica Houtt extract for the prevention and control of gray mold will also be registered and realizes industrialization soon.

(2) Mechanism of actions of Reynoutria japonica Houtt extract of Chengdu Newsun

With the experimental tests, 0.4% Reynoutria japonica Houtt extract had significant inhibitory and destructive effects on powdery mildew spore germination. After 96h of treatment, through trypan blue staining and appearance morphological observation, large number of sporangia were stained with the structure destroyed, and lost the germination vigor.

Fig4 Lab test results of 0.4% Reynoutria japonica Houtt extract on powder mildew spores after 96 hour of treatment

According to the experiments, it was showed that 0.4% Reynoutria japonica Houtt extract not only had excellent control efficacy, but also promoted crop growth and alleviated the pesticide stress on crop growth. With the recommended concentration, the leaf length and width increased 1.26 cm and 2 cm respectively.

Fig5. Lab test results of Reynoutria japonica Houtt extract on crop growth

(3) Research on the extraction technology of Reynoutria japonica Houtt extract

After years of research and development, Chengdu Newsun first proposed the product development idea of co-extraction technology and the concept of the active ingredients complex of natural extracts. By controlling the key extraction process, the bioactive secondary metabolites in the natural extracts are controlled in a targeted manner. Through the co-extraction technology, the active substances in the rhizome of Reynoutria japonica Houtt were extracted at the greatest extent and obtained the active ingredients complex including emodin, physcion, chrysophanol, Rheic acid and resveratrol, which solves the problems of single composition in botanical pesticides, low control efficiency and high use cost, effectively promotes the use of Reynoutria japonica Houtt extract in agriculture.

Fig6. Research on co-extraction technology and active ingredients complex of Reynoutria japonica Houtt extract

Through mixing, crushing, screening, reflux heating, filtration, clarification, concentration and other process technologies of medicinal raw materials, multiple continuous extraction is carried out. The active ingredients complex in medicinal plant raw materials are purified and precipitated at different stages, which significantly improves the utilization rate of raw materials, reduces production costs, and significantly increases market competitiveness. With the rich active ingredients complex, 0.4% Reynoutria japonica Houtt extract has significant synergistic effect and wide control spectrum, improves crop immunity and promotes crop growth.

Fig7. Various natural active ingredients in 0.4% Reynoutria japonica Houtt extract

(4) Advanced formulation technology of 0.4% Reynoutria japonica Houtt extract

Using advanced suspension formulation technology, the precise formula development are carried out for Reynoutria japonica Houtt extract containing multiple components and complex compositions, optimize the ratio of raw materials for formulation, and combine multiple light scattering tests to develop the best formula with stable storage property. At the same time, according to the target pests and target crops, through surfactant prescription screening, combined with dynamic and static surface tension optimization, contact angle test optimization and other technologies, improves the wetting and spreading, film adhesion, moisturizing and anti-evaporation properties, increases the retaintion of spray solution on leaves, the adhesion and coverage of active ingredients to the surface of crops and pests, improves the control efficacy and field results of the botanical pesticides.

Fig8. Appearance and water dispersion performance of 0.4% Reynoutria japonica Houtt extract

Through repeated experiments and exploration, the performance of the formulation was improved, and the appearance of the product was a homogeneous liquid, and the automatic nano-scale dispersion in water was achieved. After 500 times dilution, it has significant wettable and permeable properties.

Fig9. Dynamic surface tension data of 0.4% Reynoutria japonica Houtt extract

The multi-dimensional stability data showed that the 0.4% Reynoutria japonica Houtt extract formulation had good stability, and there was no obvious precipitation in the product after hot storage, which is fit for the various field environments.

Fig10. Multiple light scattering spectra of 0.4% Reynoutria japonica Houtt extract

(5) Research on the safety of 0.4% Reynoutria japonica Houtt extract

After research, 0.4% Reynoutria japonica Houtt extract has low toxicity to the organisms such as birds, silkworms and bees. The results of toxicological tests showed that it is slightly toxic to rats, non-irritating to the eyes and the skin of white rabbits, and no skin allergy reaction on guinea pig. According to the toxicity grading standard of pesticides, 0.4% Reynoutria japonica Houtt extract suspension is classified as slightly toxic pesticide.

The lab results of the safety test of 0.4% Reynoutria japonica Houtt extract on cucumber seedlings during the growth period showed that there was no harmful effect after 5 days of treatment with the recommended dose, 200% and 300% of the recommended doses (125 times, 166 times, 250 times and 500 times dilution).

Fig11. Lab safety test of 0.4% Reynoutria japonica Houtt extract on cucumber

The field safety test of 0.4% Reynoutria japonica Houtt extract on strawberry during flowering showed that with the recommended dose, 200% and 300% of the recommended doses (diluted 125 times, 250 times and 500 times), there was no damage on the old leaves, young leaves and flowers of strawberries.

Fig12. Field safety test of 0.4% Reynoutria japonica Houtt extract on strawberry

At the recommended dose, 0.4% Reynoutria japonica Houtt extract is safe to use at bud and flowering stages, and has no harmful effects on buds and flowers. It is suitable for comprehensive management of plant disease prevention and control, providing a solid guarantee for food safety and the quality and safety of agricultural products.

(6) The application of 0.4% Reynoutria japonica Houtt extract

0.4% Reynoutria japonica Houtt extract has a good control efficacy on powdery mildew. The lab test showed that 0.4% Reynoutria japonica Houtt extract with 500 times dilution and one application had 99.4% control efficacy on cucumber powdery mildew after 15 days of treatment, and there was no significant difference with the chemical fungicides.

Fig13. Lab test data of 0.4% Reynoutria japonica Houtt extract on cucumber powder mildew

0.4% Reynoutria japonica Houtt extract also had a good control efficacy on cucumber gray mold. The test results showed that 0.4% Reynoutria japonica Houtt extract can reach 67.4% efficacy, which had similar results as chemical fungicide.

Fig14. Lab test data of 0.4% Reynoutria japonica Houtt extract on cucumber grey mold

The field test results of 0.4% Reynoutria japonica Houtt extract on chives grey mold showed that the efficacy of 0.4% Reynoutria japonica Houtt extract with 500 times dilution was 56.25%, while the efficacy of chemical fungicide Pyrimethanil 1000 times and 1500 times dilution were 50.57% and 33.52% respectively, and there was no significant difference between Reynoutria japonica Houtt extract and chemical fungicide.  At the same time, 0.4% Reynoutria japonica Houtt extract 500 times dilution mixed with 70% dose of Pyrimethanil, the control efficacy can increase by 20%  with the significant synergistic effect.

Fig15. Field test result of 0.4% Reynoutria japonica Houtt extract on chives grey mold

In summary, 0.4% Reynoutria japonica Houtt extract has a good control efficacy on gray mold and powdery mildew. Before the disease occurs, spray with 500 time dilution can effectively prevent the occurrence of disease. At the early stage of the disease, mixed with 70% dose of chemical fungicide, can effectively control the spread of diseases, at the same time, it has a significant effect on promoting crop growth.

4. The industrial development potential of the medicinal plant Reynoutria japonica Houtt

Reynoutria japonica Houtt, a perennial herbaceous plant of the family Tateaceae, is mainly distributed in East China, Central South China, Liaoning, Shaanxi, Gansu, Sichuan, Guizhou, Yunnan and other places. Reynoutria japonica Houtt grows quickly, is easy to plant and manage. At present, large-scale artificial planting has been realized, and solved the problem of insufficient supply of raw materials in the market, which has laid a solid foundation for the industrialization of Reynoutria japonica Houtt. In recent years, with the development of the cutting-edge biotechnologies such as molecular breeding and synthetic biology, the establishment of high-quality seedling breeding bases and the production of secondary metabolites of Reynoutria japonica Houtt through microorganisms, plant stem cells and other biosynthetic technologies have become popular research directions. It is foreseeable that in near future, the industrialization and development of Reynoutria japonica Houtt will be diversified, master the variety resources of Reynoutria japonica Houtt, and screen out high-quality, high-yield and excellent strains through the genetic correlation between individual traits, yield and quality of each strain of Reynoutria japonica Houtt population. With the help of the mature TDNA fragment gene integration technology, individual traits will be improved, breed high-quality varieties, improves the purity of the arieties, and optimizes the product quality. Supporting the enterprises to promote environmentally friendly and low-toxicity botanical pesticides is conducive to improving product competitiveness and promoting the upgrading of pesticide industry structure, which is of great economic significance.

5. Prospects

According to the statistics, the insecticides, fungicides and herbicides, as the main products of pesticides, accounts for more than 95% of the global pesticide market. According to the "In-depth Survey and Investment Trend Forecast Report on the Status of China's Fungicide Industry 2022-2029", the market size of China's fungicide increased from 4.03 billion yuan in 2017 to 5.7 billion yuan in 2020. At present, among the major pesticide products in China, fungicides following insecticides and herbicides, accounting for 22.13% ^[2]. In recent years, gray mold has become an important, globally distributed fungal disease. Among them, the gray mold caused by Botrytis cinerea (B. cinerea) is the most widely distributed, which can harm more than 200 crops such as vegetables, fruit trees and flowers. In recent years, with the continuous expansion of the cultivation area of the protected lands, the damage of gray mold has become increasingly serious. According to the Molecular Plant Pathology, Botrytis cinerea ranks the second among the top ten plant pathogenic fungi that cause economic losses worldwide, only following rice blast ^[15]. Chengdu Newsun makes full use of China's biological resource advantages, and innovatively develops 0.4% Reynoutria japonica Houtt extract to control gray mold, which has the characteristics of outstanding effects, low application cost, remarkable effect on promoting crop growth, high safety, no residue, and more friendly to the environment, which solves the pain points of rapid increase of resistance of traditional chemical fungicides, low control efficacy, high pesticide residue, impact on crop growth and environmental insecurity, and meets the urgent needs of industry development. It not only effectively controls the occurrence of diseases, but also helps to restore and rebuild the ecosystem with natural regulation as the core from the macro and micro levels, and realizes the balance of plant micro-ecosystems and the harmonious coexistence between man and nature.

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