According to statistics, the average major crop loss globally caused by fungal diseases accounts for about 10%-15% of the total yield, and the annual direct economic loss is as high as hundreds of billions of dollars . There are many types of fungal diseases, accounting for more than 70%-80% of all plant diseases, and there are more than 20,000 kinds of fungi in nature that can infect various plants and cause diseases . If uncontrolled, plant fungal diseases can cause catastrophic losses to crop yields. In 1847, the late blight disease caused by the fungus P. infestans resulted in a massive reduction in potato yields, ultimately leading to more than one million deaths from starvation in Ireland . Fungal diseases are widespread, and due to their latent infection characteristics, they can harm fruits, leaves, roots and other plant parts, and reduce the yield or commercial value of crops, seriously affecting the safety of agricultural production.
1.The current status and control strategies of powdery mildew
Common vegetable and fruit powdery mildew pathogens are Erysiphe cucurbitacearum and Sphaerotheca cucurbitae, both of which belong to the ascomycete . Generally, the symptoms start with small yellow spots on the leaves, and then develop into round or oval spots with a white powdery mold layer on the surface. The mildew spots are dispersed separately at the early stage, and then combined into a large mold spot, which can even cover the whole leaf, seriously affecting photosynthesis, disturbing the metabolism, causing early senescence of the crop, and reducing the yield. Powdery mildew can infect a wide range of crops such as melons, vegetables, fruits, food crops, flowers, and trees. It is spreaded widely, and has over 650 species by now all over the world. . Powdery mildew is a multi-cycle disease with short incubation period and high reproduction rate. The spores can be widely transmitted by air, infecting the crops repeatedly , and very difficult to control.
With the adjustment of agricultural structure, and the adoption of planting technology in artificial environment facilities like greenhouses, the concentrated environment with warm and high humidity has further expanded the losses caused by powdery mildew. According to statistics, powdery mildew has become one of the main targets of strawberry disease control. Powdery mildew causes about 10%-20% yield reduction every year, and when it breaks out, it can reach more than 50% or even total yield loss. In Xinjiang province, one of China's main grape-producing regions, powdery mildew has spread throughout the region since it was first discovered in 1956. Affected by climate conditions in 2009, the damage caused by powdery mildew outbreak in grapes in Xinjiang was as high as 40%, and some orchards were destroyed. Wheat is one of the most important food crops in the world. In recent years, as the climate changes in some areas, increased rainfall and rising temperature have caused the increasing damage of powdery mildew. In severe cases, the yield reduction can reach more than 40%, which seriously endangers food safety.
Fig.1: Symptoms of powder mildew on strawberry and cucumber
At present, the chemical fungicides are mainly used for the control of powdery mildew, including triazole fungicides (difenoconazole, penicillin, tebuconazole, fluconazole, etc.), pyraclostrobin, and mancozeb, fluazinam, triadimefon and other chemicals used alone or in combination. Among them, mancozeb, propineb and ect. are mainly used as preventive and protective products, which can not kill the mycelium that invades the leaves. Systemic fungicides such as difenoconazole, flusilazole, triadimefon, myclobutanil, and tetrafluconazole can inhibit crop growth to a certain extent, and long-term use will reduce plant resistance to pathogenic fungus. Now the frequent use and increased concentration of the chemicals have become the main measures to deal with highly resistant powdery mildew. The continuous use of a single chemical causes increased the resistance year by year. At the same time, the negative impacts such as increased costs of chemical control, decreased crop quality, and increased chemical residue in crops have become more and more serious.
Therefore, developing new bio-control products and methods is one of the main effective ways to control powdery mildew. With the rapid development of science and technology, through in-depth research on natural plant source extraction and formulation technology, plant metabolites are more and more popular because of their good control efficiency, fast degradation in nature, and not easy to cause disease resistance. They are more and more used in plant disease control. Cnidium monnieri is a traditional Chinese medicine, which is the dry and mature fruit of Cnidium monnieri, a plant of the family Apiaceae. The main active ingredients are Osthol and other coumarin compounds. Cnidium monnieri has anti-arrhythmia, anti-virus, anti-tumor and other effects, and is widely used in the treatment of various skin diseases such as vitiligo and psoriasis .
Fig. 2: Cnidium monnieri plant and chinese medicine
2.The history of the research and use of Cnidium extract-Osthol
Cnidium monnieri has been recorded in ancient herbal medicine books, such as Compendium of Materia Medica and Shennong Bencao Jing (Shennong Emperor's Classic of Materia Medica). Shennong Bencao Jing recorded that Cnidium monnieri had curaitve effects on "women’s swelling and pain in the vagina, men’s impotence, damp itching, remove numbness, sharpen joints, and epilepsy and wounds." Modern medicine shows that Osthol has the effects of antispasmodic, hypotensive, antiarrhythmic, immune function enhancing and broad-spectrum antifungal .
As early as 1984, Xiang Rende and other researchers from the Nanjing Institute of Materia Medica in China separated and purified 8 monomeric compounds from Cnidium monnieri extracts by column chromatography, which are Palmitic acid, Osthol, bergamot bergapten, bornyl isovalarate, Cnidiadin, isopimpinellin, and etc. According to the activity assay against Trichophyton, Osthole has the strongest antifungal activity .
Osthole belongs to coumarin compounds, the core structure of which is composed of benzene ring and pyrone ring. As a member of coumarin compounds, osthole has not only the core structure of coumarin, but also the isopentenyl structure. The compounds with isopentenyl structure play an important role in disease control .
Fig.3: Osthole CAS: 484-12-8
At the same time, a large number of research reports have shown that osthole also has certain insecticidal effects in agriculture and has high inhibitory effects on certain pathogenic fungi. It is mainly used to prevent and control powdery mildew on traditional Chinese medicines such as ginseng, coptis, honeysuckle and chrysanthemum, and also can prevent gray mold and anthracnose. The research data of Yan Qingping  showed that osthole has a strong inhibitory effect on the spore germination of strawberry powdery mildew, and 60 μg/ml concentration of osthole can completely inhibit the spore germination of powdery mildew after 24 hours of treatment. Fuhui  and other researchers found that the control efficiency of 1% osthole emulsion 16.7-25μg/ml on strawberry powdery mildew was 95.15%-98.13%.
Relying on China's rich medicinal plant resources and application technology, Chengdu Newsun Crop Science Co., Ltd, a company focusing on innovative research and development of green agricultural biotechnology, has successfully developed a new natural compound osthole after years of research and development and used it for controlling powdery mildew and other diseases, which makes outstanding contributions to the global organic and green agricultural production.
3.The latest research progress of osthole
(1)Registration information of Osthole. According to the China Pesticide Information Network, in October 2017, Chengdu Newsun Crop Science Co., Ltd. (hereinafter referred to as "Chengdu Newsun") obtained ICAMA registration of 0.4% Osthole AS from the Ministry of Agriculture and Rural Affairs of China (registration number: PD20172589). The registered target disease is strawberry powdery mildew.
Fig. 4 Registration information of 0.4% Osthole AS
(2)Mode of actions of 0.4% CE-Osthole AS for antifungi. After testing and analysis, osthole has a significant control effect on powdery mildew. It can strongly inhibit the germination of powdery mildew spores, destroy the mycelium and sporangia, and cause the mycelium break and lose its ability to infect. At the same time, it has the effect of eradicating the mycelia that have formed the penetration peg.
Fig. 5 Optical microscope data of powdery mildew mycelium
After 90 hours of applying 0.4% CE osthole AS, the sporangia of powdery mildew pathogen were ruptured and inactivated. For crop leaves that had been infected with white powder and formed the penetration peg, it was significantly observed that the white powder penetration peg was eradicated after applying 0.4% CE osthole AS.
Fig. 6: Scanning electron microscope data of powdery mildew penetration peg
(3)Research on extraction technology of CE osthole. After years of scientific research, Chengdu Newsun adopts the original plant-source co-extraction technology, which has more abundant active components and obvious synergistic effects in the product, and significantly increases the efficiency of Cnidium monnieri extract against diseases. And further reduction of the application cost effectively promotes the use of osthole in agriculture.
According to relevant reports, the traditional extraction methods of osthole include reflux, ultrasound, supercritical, continuous dynamic counterflow and etc. In contrast, co-extraction technology (CE) is based on the theory of traditional Chinese medicine and uses modern extraction technology to maximize the extraction of various active ingredients and beneficial components. It solves the problems of single extraction component, low product efficiency, and high application cost.
Fig. 7: Research on prescription co-extraction technology
0.4% CE osthole AS has more abundant extract components, realizes maximum extraction of various coumarin active ingredients, reduce production energy consumption, and have more significant synergistic effects between components, good affinity with plants, wide control spectrum, and high safety.
Fig.8: Various active ingredients of 0.4%CE osthole AS
(4)Research on the formulation technology of 0.4% CE osthole AS. Utilizing modern pesticide formulation technology, precise formula development has been carried out to deal with the multiple and complex components extracted from Cnidium monnieri, optimizing the ratio of raw materials for the formulation, and using multiple light scattering tests to develop the best formulation with stable storage ability. At the same time, according to the target pests and crops, through the selection of surfactant, combined with nano-emulsification technology, dynamic and static surface tension optimization, contact angle test optimization and other technologies, the properties of the formulation including wetting, spreading, film-forming adhesion, moisturizing and anti-evaporation are improved, increase the retention of spray liquid on leaves, increase the adhesion and coverage of active ingredients on the plant leaves and pests, improve the field performance, and realize the improvement of the efficacy of the botanical pesticide.
In order to ensure the stability of various active ingredients, the polymer adhesive components were screened and applied to improve the ability of the pesticide to wrap quickly at the recommended concentration, and reduce the surface tension of the pesticide solution. Data shown that the spreadability is super than other similar products, which is helpful for the pesticides spread and adhension, and increases the product efficacy. Through repeated experiments, the performance of the formulation has been improved, and the appearance of the product has been achieved as a homogeneous liquid, which is automatically dispersed in water at the nanoscale; it has excellent wetting and penetrating properties, and has remarkable wetting and penetrating properties after being diluted by 300 times. Multiple light scattering shows that the product has good stability, consistent in phases, and does not have any particle impurities or precipitation.
Fig.9: Multiple Light Scattering Spectrum of 0.4%CE osthole AS
(5)Research on the application technology of 0.4% CE osthole AS. The successful development of the natural botanical fungicide 0.4% CE osthole AS will help to effectively alleviate the problems of low efficiency and resistance in powdery mildew control. At present, the long-term dependence on the chemical fungicide has increased the application frequency and the cost, resulting in serious resistance and excessive chemical residues. Compared with traditional control technologies, natural botanical fungicides have the advantages of easy degradation in nature, low toxicity, synergistic effect of multiple components, less resistance, and safe to plants.
At the same time, in order to solve the problem of short and slow performance of botanical pesticides in the field, and give full play to their advantages, Chengdu Newsun has established an active control system of "prevention-preventive treatment-treatment" of biopesticides, namely before and at the early stage of the disease, biopesticides are mainly used; during the outbreak period, biopesticides are used together with chemical pesticides to suppress the infection of the disease to the greatest extent, reduce the index of disease occurrence, and reduce the use of chemical pesticides.
Through indoor experiments, it was found that the inhibitory rate of 0.4% CE osthole AS diluted by 300 times on powdery mildew was above 80%. Preventive effects at the early stage of the disease, 0.4% CE osthole AS can achieve a significant control effect.
Fig.10: Indoor test results of 0.4% CE osthole AS on cucumber powdery mildew
At the middle and late stages of disease occurrence, that is, the outbreak period, use the core control method of "botanical pesticide + chemical pesticide", on the premise of improving the control effects,can reduce the use of chemical pesticide, decrease chemical residues and resistance problem, and improve the quality of agricultural products and the agricultural eco-environment, and helps in maintaining biodiversity and reducing environmental pressure.
The results of a large number of field experiments show that use 0.4% CE osthole AS in combination with chemical fungicides such as 25% Bupirimate, 30% Difenoconazole + Kresoxim-methyl and ect. can reduce the use of chemical fungicides by 10-30% and achieved the results of chemical use reduction and control efficiency enhancement.
In 2021, the powdery mildew disease was seriously infected melons in Yandian Town, Shen County, Liaocheng, China. The use of 0.4% CE osthole AS 400 times + 30% Difenoconazole + Kresoxim-methyl 1,500 times / 25% Bupirimate 600 times showed significant synergistic effect, reduced 30% dose of the chemical fungicide, and the control effect reached more than 80%, and the hyphae of melon powdery mildew turned black and dry and became agglomerates.
Fig.11: Field results of 0.4% CE osthole AS combined with 25% Bupirimate
In the same year, strawberry powdery mildew occurred seriously in Linyi City, China. After treatment with 0.4% CE osthole AS 500 times + 30% Difenoconazole + Kresoxim-methyl 1000 times, the white powder on strawberry fruits was wiped off.
Fig.12: Field results of 0.4% CE osthole AS combined with 30% Difenoconazole + Kresoxim-methyl
Strawberry powdery mildew broke out in Jiande City, Hangzhou, China. Use 0.4% CE osthole AS 400 times + 25% Ethirimol 750 times to prevent and control powdery mildew. After 2 days of application, the white powder on the leaves was wiped off. At 6 days after application, the control efficacy was 80%, and the synergistic effect was significant.
Fig.13: Field results of 0.4% CE osthole AS combined with 25% Ethirimol
0.4% CE osthole AS not only has good antifungi activity, but also has good product safety, and is safe to young strawberry fruits, leaves, branches and other fruits and vegetables.
Fig.14: Safety test of 0.4% CE osthole AS
4.The industrialization potential of the medicinal plant Cnidium chinensis
As one of the earliest used medicinal plants, Cnidium chinensis was mostly wild in the early years. With the continuous development of modern pharmacology, more medicinal values of Cnidium chinensis have been discovered, and people's demand for Cnidium chinensis is increasing year by year. Cnidium chinensis is mainly planted in Jiangsu, Anhui, Shandong, Hubei, Hebei and other provinces. In addition, it is also planted in a small areas of Guangxi, Sichuan, Shaanxi, Shanxi, Henan, Zhejiang and etc. It is distributed in most parts of China. Cnidium chinensis has the characteristics of easy planting and easy management. It grows fast and is not affected by weeds. It can harvest two times per year and is easy to plant. Artificial planting solves the problem of insufficient supply of Cnidium chinensis medicinal materials in the market, which has laid a solid foundation for the industrialization of Cnidium chinensis. In recent years, with the global development of biosynthesis technology, the synthesis of active plant metabolites through eukaryotic factory technologies such as yeast and plant stem cells has become a hot research direction. It is foreseeable that in the future, it will be possible to produce osthole through large-scale bioreactors, which will further promote the in-depth industrial application of osthole.
The history of control of powdery mildew can be traced back to 100 years ago, and the organic sulfur represented by sulfur products and other broad-spectrum fungicides were gradually replaced by the Triazole fungicides such as Triadimefon, Myclobutanil, Hexaconazole, Difenoconazole, Flusilazole and etc. Subsequently, new types of active ingredients represented by Azoxystrobin, Enestroburin, and Kresoxim-methyl showed higher effects and gradually replaced triazole fungicides. With people's emphasis on biosafety and food safety, research and development of new botanical fungicides and their application technologies are an inevitable trend in the development of green agricultural inputs now. 70% to 80% of crop diseases worldwide are caused by fungi, among which powdery mildew is one of the diseases with the largest infection area and the most difficult control. The market capacity of the products for powder mildew control exceeds 1 billion US dollars.
The launch of the new natural botanical pesticide CE osthole, because of its outstanding effects, low application cost, high safety, no residue, and more friendly to the environment, solves the problems of rapid increase of traditional chemical fungicide resistance, low control effect, high pesticide residues and environmental unsecure, providing solid technical support for food safety and agricultural product quality and safety, maintaining biodiversity, reducing environmental pressure, and contributing to the realization of agricultural value improvement and sustainable agricultural development, and promoting healthy growth of bioagriculture.
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