Innovative Natural Products — CE Berberine Registered For the First Time in The World to Control Citrus Moss Disease

Moss disease is a kind of parasitic disease on the leaves and stems of fruit trees, which is mainly composed of algae and many kinds of microorganisms. Citrus is a kind of fruit tree which is seriously harmed, and its output will be affected in a large scale when the moss disease is serious. As one of the most important cash crops in the world, citrus is planted in tropical and subtropical regions. In recent years, the world citrus planting area has been growing steadily. According to the data of the Food and Agriculture Organization of the United Nations (FAO), from 2000 to 2015, the area increased from 876.73 hm2 to 1343.27 hm^2 [1]. At the same time, because of the increasing benefits of citrus in recent years, more and more people are engaged in the citrus planting industry, and the citrus planting density is increased. According to the survey statistics, the outbreak rate of citrus moss disease has shown an upward trend in recent years. In China alone, about 13 million mu (15 mu = 1 ha) of citrus orchards are affected by the moss disease every year, accounting for 30% of the total area of citrus.

Citrus Moss Disease is easy to breed in humid and rainy environment. When it comes on, it mainly harms the branches, leaves, fruits and other parts of citrus, weakens the photosynthesis rate of plants, reduces the photosynthesis products, and directly affects the color, appearance and internal quality of fruits. The moss disease on the ground will seize the fertilizer and water and block the infiltration of water, affect the exchange of gas in the soil, seriously affect the growth and development of fruit trees, leading to a significant reduction in citrus yield.

At present, the prevention and control of moss disease mainly depends on chemical pesticides such as ethylicin, ambam, peroxyacetic acid, and some adjuvant products without relevant qualifications. However, these products have not been officially registered on the target of moss disease, and there are potential safety risks. In addition, these chemicals themselves have strong irritating smell, poor compatibility, easily phytotoxicity, and uneven quality levels. With the increasing concern for environmental issues, the gradual increase in the area of organic green agriculture and the upgrading of consumer demand, the global market has become increasingly demanding on the quality, quality safety, use safety, production traceability and other requirements of fruits. Therefore, it is urgent to develop new, safe, efficient and environmentally friendly natural biological pesticides to control moss disease.

1. Studies on Berberine

Berberine (Ber), is a kind of quaternary ammonium isoquinoline alkaloid extracted and separated from the medicinal plant Coptis Chinensis. It is a yellow needle like crystal with bitter taste. It mainly exists in the Ranunculaceae plants Coptis chinensis, Phellodendron chinense and Sankezhen, with the content of about 10%, and exists in the form of hydrochloride. Coptis chinensis has a long history of being used as medicine in China. The earliest literature record is Shennong's Herbal Classic ^[2]. According to its historical records and data, Berberine has been deeply studied in the medical field today, and it has been found that it can treat various infectious diseases, mainly digestive tract bacterial infections, respiratory tract infections, dermatological infections, etc., and can widely inhibit pathogenic microorganisms ^[3-4].

It has been reported that Berberine can prevent the reproduction of cholera, typhoid and dysentery bacteria at very low concentrations. It also has the effect of resisting gram-positive bacteria such as Staphylococcus aureus and Streptococcus and gram-negative bacteria such as Typhoid bacteria, Shigella dysentery bacteria and gonococcus. In addition, it has antibacterial properties against Bacillus cereus, Bacillus subtilis, Bacillus diphtheriae, Escherichia coli, Pneumococcus, Shigella Newcastle, Streptococcus pyogenes, Mycobacterium tuberculosis and other animal pathogens, as well as Candida, Cryptococcus, Yeast and other fungi. With the rapid development of science and technology, it is found that Berberine is resistant to Staphylococcus aureus, mainly because it interferes with bacterial protein biosynthesis by causing changes in ribosome and cell wall ^[2].

However, the function of Berberine is not only limited to the antibacterial effect. Many researchers have found that Berberine has pharmacological effects in the treatment of tumors, diabetes, cardiovascular diseases, hyperlipidemia, viral infection, cerebral ischemic injury, mental diseases, Alzheimer's disease and osteoporosis. It can be seen that the physiological function of Berberine is very rich, and its potential development and application value is very great. In the past ten years, more than 2000 articles have reported the clinical efficacy, pharmacological mechanism, absorption metabolism, structure activity relationship and other research results of Berberine. Among them, the more systematic research is the structure-activity relationship research on antibacterial, antimalarial, antiarrhythmic and anti-tumor ^[5]. However, there are few reports on the research and application as pesticides, which has great potential for development in the future.

In the past ten years, with the in-depth study of Berberine in the agricultural field, it was found that Berberine has a significant role in the prevention and control of citrus moss disease, creating a new research and application direction of Berberine in the pesticide field. It also makes full use of the advantages of Chinese plant resources, breaks the tradition and continues to innovate in the agricultural field, and uses advanced biotechnology to bring Berberine, a new natural product, into the field of plant protection. According to the data, Chengdu Newsun Crop Science Co., Ltd. (hereinafter referred to as "Chengdu Newsun"), a biotechnology enterprise from China, based on the innovative research and development of efficient Co-Extraction technology of Coptis chinensis, has solved the problems of single active ingredient of traditional Coptis chinensis extract products and poor effect on agricultural disease prevention and control, and has innovatively developed "0.5% CE Berberine AS", which can effectively control the occurrence of citrus moss disease, It is more conducive to ensuring the quality of fruit, reducing the use of traditional chemical pesticides and reducing the dependence on chemical pesticides.

Fig. 1: Medicinal parts of Coptis Chinensis

2. The latest research progress of Berberine in controlling citrus moss disease

1) Study On The Mechanism Of Berberine

With the rapid development of biological science and technology, through the high-throughput sequencing technology (Illumina Miseq), researchers in Chengdu Newsun have studied the composition, structure and diversity of microbial communities in the citrus leaves with moss disease, and found that there are more than 100 species of pathogenic phyllospheric microorganisms attached to the citrus leaves with moss disease (Fig. 2), and the possible pathogen is the uncultured Apatococcus sp, Heterochlorella luteoviridis, Cyphellophora sessionis, C. gloeosporioides, etc. It can be inferred from the pathogen group that moss disease is caused by multiple algae and fungi ^[6-7].

Fig. 2: Species analysis of phyllosphere microorganisms of citrus moss disease leaf

The experimental data showed that Berberine could seriously inhibit and destroy the algae and their symbiotic bacteria attached to the leaves and branches. Berberine compounds have a certain inhibitory effect on the activities of topoisomerase I (TOP I) and topoisomerase II (TOP II), and it is found that Berberine has a strong binding effect with DNA ^[8]. In addition, Berberine can inhibit the synthesis of ergosterol, increase the permeability of cell membrane, thus causing the leakage of substances in cells, such as nucleic acid and protein, and at the same time, cause the cell membrane potential depolarization and lipid peroxidation, so that the active molecules can enter the algal cytoplasm without obstacles ^[9], rapidly inhibit the growth and reproduction of algae, make the algal organelles lose their functions, and photosynthesis is blocked and eventually die.

Fig. 3: Trypan Blue staining showed cell rupture after 5 days treated with 0.5% CE Berberine

(the left picture is CK, while the right one is treated by 0.5% CE Berberine)

The covering layer of citrus moss disease on the leaves was made into suspension, and mixed with 0.5% CE Berberine AS for culture. After 5 days of culture, it was stained with Trypan Blue. According to the principle of Trypan Blue staining, when cells were damaged or died, Trypan Blue could penetrate the denatured cell membrane, combine with the disintegrated DNA, and make it colored. It can be clearly seen that after treated with 0.5% CE Berberine AS, all cells will be stained for 5 days, and 0.5% CE Berberine AS will cause cell death by damaging the cell membrane of moss disease, so as to achieve the effect of clearing moss disease (Fig. 3).

2) Study on extraction technology of Berberine

Based on the pharmacological characteristics of Berberine itself, after years of research and development, Chengdu Newsun has greatly excavated the active substances in the roots of Coptis chinensis through the Co-Extraction technology. Through the substantial optimization of the process, it has obtained more beneficial ingredients. With this technology, other active components are retained as much as possible while ensuring the content of indicated components . According to the retrieval of China Pesticide Information Network, this technical achievement was registered as a 0.5% Berberine AS approved by ICAMA of the Ministry of Agriculture and Rural Affairs of the People’s Republic of China in April 2022, becoming the first plant pesticide officially registered to prevent and control moss in the world (Fig. 5).

Fig. 5: Registration information of Berberine against citrus moss

3) Research progress of 0.5% CE Berberine

Supported by advanced extraction process and supplemented by excellent formulation technology, it can reduce the surface tension of product, promote the penetration of the product to the covering layer of algal pathogenic mixture, and enhance the efficacy of active ingredients (Fig. 6). The dispersibility in water is good, and the solution after dispersion is transparent, in a homogeneous state, which can be quickly wetted and permeated. Multiple light scattering stability data show that 0.5% CE berberine AS has good stability (Fig. 7), fully meeting various field application environments.

Fig. 6: Dynamic surface tension test of 0.5% CE Berberine AS with 150 times dilution

Fig. 7: 0.5% CE Berberine AS formulation performance

4) Study on application technology of 0.5% CE Berberine AS

The 0.5% CE Berberine AS exclusively launched by Chengdu Newsun, which is extracted from plants and used on plants, is an important technology for the prevention and control of Citrus Moss Disease. With the help of innovative technology, the rapid effect of the product is excellent, which takes effect in 5-7 days. Pharmaceutical grade ingredients have very low toxicity, and the control effect is excellent. The maximum control effect in the field can reach more than 95%, which is comparable to ethylicin. At the same time, 0.5% CE Berberine AS, as a natural product, is a botanical bio-fungicide. On the basis of excellent control effect, it is safer to use on different crops, without damaging fruits and leaves, no volatile pungent odor, while with quick effect and environment friendly.

Fig. 8: The activity test of 0.5% CE Berberine AS on citrus moss with 5 days after treated

The activity of 0.5% CE Berberine AS was determined by a rapid and stable in vitro detection method. With the analysis and comparison of 5 days after treated, it was obvious that the attachment layer of moss disease on citrus leaves was dead and white. The significant control effect was comparable to chemical product ethylicin.

Fig. 9: Field experimental data of the effect of 0.5% CE Berberine AS on citrus moss

0.5% CE Berberine AS was used in Meishan city, Sichuan Province, China to prevent and control citrus moss at the initial stage. The moss gradually turned white 5 days after treatment, and the control effect reached 70.37% with 10 days after treatment. The control effect reached 98.58% with 12 days after treatment. It completely killed and shed the attachment layer of citrus moss, and did not cause damage to the leaves. Compared with the ethylicin, there was no peculiar smell (Fig. 9).

Fig. 10: Control Citrus Moss Disease by 0.5% CE Berberine AS in Jitan Town, Ganzhou City

During the high incidence period of Citrus Moss Disease in Jitan Town, Ganzhou City, Jiangxi Province, China, the whole tree trunk was covered with a thick layer of moss disease. After 10 days of applying 0.5% CE Berberine diluted 200 times, the pathogenic algae were destroyed, died and fell off. The control effect was significant, reaching 97%. The branches and trunks of fruit trees were not damaged, and there was no peculiar smell compared with ethylicin (Fig. 10)

Fig. 11: Control Tea Moss Disease by 0.5% CE Berberine Aqueous Solution in Shangba Town, Enshi City

The tea moss disease is severe occurrence in Shangba Town, Enshi City, Hubei Province, China. After spraying 0.5% CE Berberine diluted 200 times in the high incidence period for 14 days, all the moss become yellow and white, and the moss attached to the root also turns yellow and dead. The control effect is significant (Fig. 11)

Fig. 12: Control Citrus Moss Disease by 0.5% CE Berberine AS in Heshan Town, Pujiang County, Sichuan Province

During the high incidence period of citrus moss in Heshan Town, Pujiang County, Sichuan Province, after spraying 0.5% CE Berberine diluted 150 times for 5 days, the green algae moss on the citrus leaves become white and wither. The control effect is significant, reaching 98.7%. The control effect is the same as that of the ethylicin, which immediately solves the problem that a large number of moss adhere to affect the photosynthesis of citrus.

Fig. 13: The safety test for two varieties of citrus by different concentrations of 0.5% CE Berberine AS after 7 days of treatment

Fig. 14: The safety test for two varieties of citrus "Chunjian" and "Dekopon"by different concentrations of 0.5% CE Berberine as after 12 days of treatment

The safety test of 0.5% CE Berberine AS was carried out in Meishan City, Sichuan Province, China. The safety test for two varieties of citrus, "Chunjian" and "Dekopon", was carried out at the fruit enlargement stage and the tender leaf stage. The results showed that at high or low concentrations, 0.5% CE Berberine AS had no damage to citrus fruits and tender leaves, and was extremely safe.

The above field bioassay results showed that 0.5% CE Berberine AS had excellent control effect on citrus moss disease regardless of the region, crop position, initial and high incidence periods, and its control effect reached the level of chemical pesticides. It has no pungent smell, is friendly to the applicator, and has good adhesion. At the same time, according to the safety test, under the recommended concentration, it is safe to use it during the sprouting, flowering and young fruit stages of crops, and has no effect on the young buds, flowers and fruits enlargement. It is safe and environmentally friendly to non target organisms such as natural enemies.

For the application of biopesticides, the best using time is before or at the beginning of the occurrence of diseases or insects, which can not only maximize the prevention and control effect and value of biopesticides, but also minimize the impact of pests and diseases on agriculture. In the period of high incidence of diseases and pests, using biopesticides and chemical pesticides together by tank mixing, due to the huge difference in the action mechanism of the two kinds of pesticides, the synergistic effect is more significant. While reducing the usage amount of chemical pesticides and slowing down the chemical pesticide resistance, the quality and safety of agricultural products are improved, the agricultural ecological environment is improved, and the environmental pressure is reduced. this can maintain biodiversity and promote the value of agricultural industry.

3. Gene editing technology effectively promotes the healthy development of Coptis chinensis industry

Coptis chinensis Feinch is a perennial herb of Ranunculaceae, which is mainly distributed in the alpine mountains of Sichuan, Guizhou, Yunnan, Hubei and other provinces in China. It is one of the rare Chinese medicinal materials in southwest China. As Coptis chinensis grows slowly and is difficult to cultivate, it is not easy to popularize. In addition, some regions have been digging for a long time without planting, resulting in resource depletion and increasing market supply tension. In order to promote the industrial development of Coptis chinensis, a high-quality seed and seedling breeding base shall be established according to the development of science and technology, and diversified production methods shall be provided for the industrial development of Coptis chinensis. On the basis of mastering the variety resources of Coptis chinensis, excellent lines with high quality and high yield shall be screened by studying and analyzing the genetic correlation between individual traits and yield and quality of each line of Coptis chinensis population. At the same time, with the help of the mature TDNA fragment gene integration technology, the individual traits are improved, high-quality varieties are cultivated, the purity of varieties is improved, and the product quality is optimized. Accelerate the promotion of high-quality seedlings by means of tissue culture and other technical means ^[10]. These technologies and methods have provided a strong guarantee for the healthy development of Coptis chinensis industry.

According to incomplete statistics, as of the end of 2021, moss disease has a market size of nearly 1 billion Yuan in China, and is expected to have a market size of nearly 3 billion Yuan in the world, and is growing rapidly at an average compound growth rate of 10% every year. Relying on rich natural resources and rising biotechnology level, Chengdu Newsun has innovatively developed a natural botanical fungicide - 0.5% CE Berberine AS with the characteristics of safety, efficiency and no residue, which can not only solve the problems of lower photosynthetic efficiency, poor tree growth, tree vigor decline, quality and yield decreasing that caused by moss disease in agricultural production, but also help to improve the quality and safety of agricultural products, delay chemical pesticide resistance, reduce dependence on chemical pesticides and maintain biodiversity. At the same time, it is conducive to enhancing the competitiveness of agricultural products, promoting the upgrading of pesticide industrial structure and promoting the green and sustainable development of agriculture, which has important economic and social significance.

References:

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[3] Qian Zhang, Xiangshu Piao, Research Progress In Bacteriostasis of Berberine [J] Chinese Journal of Animal Husbandry, 2010 (3): 4

[4] Boli, Weiliang Zhu, Kaixian Chen, Research Progress Of Berberine and Its Derivatives [J] Journal of Pharmacy, 2008, 43 (008): 773-787

[5]Inna, I, Severina, et al. Transfer of Cationic Antibacterial Agents Berberine, Palmatine, and Benzalkonium Through Bimolecular Planar Phospholipid Film and Staphylococcus aureus Membrane[J]. IUBMB Life, 2001, 52(6):321-324.

[6] Leiyang, Haijian Yang, Xunlan Li, Effects of Moss Breeding on the Biodiversity of Citrus Leaves [J] Plant Protection, 2019, 45 (6): 9

[7] Gustavs L , Schumann R , Karsten U , et al. Mixotrophy in the terrestrial green alga Apatococcus lobatus (Trebouxiophyceae, Chlorophyta)[J]. Journal of Phycology, 2015:n/a-n/a.

[8] Yongyang, Zhiying Lei, Fangping Wu, Research Progress on Antibacterial Effect of Berberine [J] Advances in Modern Biomedicine, 2010 (9): 3

[9] Ruyi Wang, Screening of Botanical Pesticides and Study on The Mechanism of Berberine Against Alternaria Solani [D] Beijing University of Chemical Technology, 2018

[10] Shengwang Pan, Haiyang Wang, Factors Restricting the Industrialization of Coptis chinensis and Development Countermeasures -- Take Shizhu County As An Example [J] Journal of Southwest Agricultural University: Social Science Edition, 2004, 2 (4): 56-58

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