Application of epicatechin and protocatechuic acid in prevention and control of citrus huanglongbing
By applying epicatechin and protocatechuic acid agents to citrus trees, the shortcomings of existing technologies in the prevention and control of citrus Huanglongbing (HLB) have been overcome, achieving efficient and environmentally friendly control of HLB and significantly improving the health of citrus trees.
Patent Information
- Application Number
- CN202511559731.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-29
- Publication Date
- 2026-01-13
AI Technical Summary
Existing technologies are not effective in controlling citrus Huanglongbing (HLB). They rely on agronomic management and chemical pesticides, which cause environmental pollution. Furthermore, the development of antibiotic resistance makes control difficult, and there is a lack of efficient and environmentally friendly control methods.
Epicatechin and protocatechin acid are used as agents and are applied to citrus trees by injection. Combined with acceptable pesticide adjuvants, they synergistically enhance the disease resistance of citrus trees and inhibit the reproduction and spread of citrus Huanglongbing fungus.
It significantly inhibits the growth of citrus Huanglongbing pathogens, improves the health of citrus trees, has a good synergistic effect and a sustained control effect, and has the potential for environmentally friendly and efficient application.
Smart Images
Figure CN121312601A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant disease control technology, specifically involving the application of epicatechin and protocatechuic acid in the control of citrus Huanglongbing (HLB). Background Technology
[0002] Citrus Huanglongbing (HLB) is a devastating disease threatening the global citrus industry, caused by Candidatus Liberibacterasiaticus (CLAs). In the field, it is primarily transmitted by the citrus psyllid, a vector insect. HLB causes symptoms in citrus trees such as yellowing leaves, deformed fruit, and root necrosis, ultimately leading to tree death. Currently, control measures for HLB remain limited, mainly relying on agronomic management and the application of chemical pesticides to control citrus psyllids. However, these methods are not entirely effective and pose environmental pollution problems. Therefore, developing efficient and environmentally friendly control methods is an urgent challenge.
[0003] Although antibiotics (such as ampicillin) have shown some inhibitory effects against citrus Huanglongbing (HLB), their widespread application is hindered by the development of drug resistance and regulatory restrictions. Therefore, the development of novel, highly effective, low-toxicity control agents that target CLas and activate the plant's immune system is urgently needed. HLB-resistant varieties of citrus may contain substances with good inhibitory functions against HLB pathogens, representing an important resource for overcoming the disease. Currently, researchers have screened a large number of disease-resistant genes from different resistant varieties, but there is still a significant gap between these genes and their practical application in production. Encouragingly, in recent years, researchers have discovered that chlorogenic acid has a good inhibitory effect on HLB pathogens by comparing the secondary metabolites of HLB-resistant and susceptible citrus varieties. To better address the harm of HLB to the entire citrus industry, it is necessary to develop more substances with antibacterial properties.
[0004] Epicatechin (EC) belongs to the flavanol class of compounds. It is readily soluble in water and abundant in plants such as tea leaves and berries. It can indirectly enhance free radical scavenging ability by increasing the activity of antioxidant enzymes, exhibiting good antioxidant effects. It also possesses various functions such as antibacterial activity and immune regulation. Protocatechuic acid (PCA) is a phenolic compound widely found in plants, possessing various biological activities, such as antioxidant, antibacterial, and anti-inflammatory properties. To date, there have been no reports on the use of epicatechin and protocatechuic acid for the control of citrus Huanglongbing (HLB). Summary of the Invention
[0005] In order to overcome the shortcomings of the prior art, the present invention applies epicatechin and protocatechuic acid to the prevention and control of citrus Huanglongbing, in order to inhibit the reproduction of pathogens and enhance the disease resistance of citrus trees through their natural biological activity mechanism, thereby effectively controlling the spread of citrus Huanglongbing.
[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: This invention employs a method most closely resembling the field transmission pathway of citrus Huanglongbing (HLB), specifically through the feeding of infected citrus psyllids to transfect HLB pathogens. This method aims to identify disease-resistant substances by utilizing HLB-carrying citrus psyllids to feed on resistant and susceptible citrus varieties, thereby identifying metabolic substances with resistance activity against HLB in resistant and susceptible varieties. The study found that after inoculation with HLB-carrying citrus psyllids, a natural organic compound called epicatechin (ECC) was detected in both the resistant varieties Australian finger lemon and rough-skinned lemon.
[0007] The first aspect of this invention provides the application of epicatechin and / or protocatechuic acid in the control of citrus Huanglongbing (HLB).
[0008] A second aspect of the present invention provides a medicament for controlling citrus Huanglongbing (HLB), the medicament comprising epicatechin.
[0009] A third aspect of the present invention also provides a medicament for controlling citrus Huanglongbing (HLB), the medicament comprising protocatechuic acid.
[0010] A fourth aspect of the present invention also provides a medicament for controlling citrus Huanglongbing (HLB), the medicament comprising epicatechin and protocatechuic acid.
[0011] Preferably, the agent further includes excipients acceptable in the pesticide field.
[0012] More preferably, excipients acceptable in the pesticide field include solvents, emulsifiers, dispersants, wetting agents, fillers, and stabilizers. Solvents aid dissolution, emulsifiers promote compatibility, dispersants prevent agglomeration, wetting agents aid adhesion, fillers increase volume, and stabilizers preserve efficacy.
[0013] The fifth aspect of this invention provides a method for controlling citrus Huanglongbing (HLB), specifically: injecting a solution containing epicatechin and / or protocatechuic acid into the citrus tree using a syringe.
[0014] Preferably, the effective concentrations of epicatechin and protocatechuic acid are both 0.5-4 mg / mL. Higher concentrations do not significantly affect the efficacy, while lower concentrations will reduce the effectiveness.
[0015] More preferably, the effective concentration of epicatechin and / or protocatechuic acid is 1 mg / mL. At this concentration, epicatechin and protocatechuic acid exhibit the best control efficacy against citrus Huanglongbing (HLB) with minimal impact on the tree.
[0016] Preferably, the injection site is 5-15 cm above the ground on the main trunk of the citrus tree.
[0017] Preferably, the volume of the injection solution is: 100-150 mL for young trees and 200-400 mL for mature trees; the number of injections is: one injection three months after the first injection.
[0018] One week after injection, the Huanglongbing (HLB) content in citrus trees drops sharply, and the effect lasts for 2-3 months. After 2 months, the HLB content in citrus trees rises slightly, but still maintains a certain level of control. A second injection of diluted epicatechin and protocatechuic acid solution after 3 months can maintain the HLB content at a low level. By applying epicatechin and protocatechuic acid solution twice, infected citrus trees can quickly recover their vigor and maintain yield. Combined with fertilization and other cultivation measures, year-round production can be achieved with HLB-infected trees, and tree vigor can be gradually restored.
[0019] Preferably, the citrus fruits include mandarin oranges, tangerines, pomelos (such as honey pomelos), etc.
[0020] Compared with the prior art, the beneficial effects of the present invention are: To demonstrate the value of epicatechin and protocatechuic acid in the control of citrus Huanglongbing (HLB), this invention, through systematic experimental research and application verification, found that both epicatechin and protocatechuic acid can significantly inhibit the growth of HLB pathogens and exhibit good synergistic effects. Compared with the use of epicatechin or protocatechuic acid alone, the combined use of epicatechin and protocatechuic acid can significantly improve the control effect of HLB. Therefore, epicatechin and protocatechuic acid can effectively control the spread of citrus HLB, significantly improve the overall health of citrus trees, and have excellent application prospects and promotional value.
[0021] Furthermore, given the natural, environmentally friendly, and highly effective characteristics of epicatechin and protocatechuic acid, they demonstrate significant application potential in the control of citrus Huanglongbing (HLB) and are expected to become new key tools in this field. This discovery not only provides new ideas for the green control of HLB but also offers strong technical support for the sustainable development of the global citrus industry, which is of great significance for ensuring the stable development of the industry. Attached Figure Description
[0022] Figure 1 Comparison of epicatechin content in citrus varieties resistant to Huanglongbing (HLB); Figure 2 To investigate the short-term antibacterial effect of different concentrations of protocatechuic acid on Huanglongbing (HLB) of citrus tangerines; Figure 3 To investigate the short-term antibacterial effect of different concentrations of epicatechin on Huanglongbing (HLB) of citrus tangerines; Figure 4 The long-term antibacterial effect of two concentrations of protocatechuic acid solution on Huanglongbing (HLB) of citrus tangerine. Figure 5 The long-term antibacterial effect of two concentrations of epicatechin solution on Huanglongbing (HLB) of citrus tangerine. Figure 6 Growth of the tea branch citrus 60 days after protocatechuic acid injection; Figure 7 Growth of the tea branch citrus 60 days after injection of epicatechin; Figure 8 Growth diagram of pomelo trees infected with Huanglongbing after initial injection of a mixture of protocatechuic acid and epicatechin; Figure 9 This is a growth chart of pomelo 60 days after injection of a mixture of protocatechuic acid and epicatechin. Detailed Implementation
[0023] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.
[0024] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.
[0025] Example 1: Comparative Analysis of Epicatechin Content between Citrus Huanglongbing Resistant and Susceptible Varieties Currently, research on the development of substances resistant to citrus Huanglongbing (HLB) is relatively scarce, with existing studies mostly focusing on directly comparing the differences between HLB-resistant and susceptible varieties. Therefore, this invention employs the most realistic field transmission route for HLB—namely, using infected citrus psyllids to feed on and transfect the HLB pathogen—to directly identify substances in resistant and susceptible citrus varieties that exhibit resistance to HLB. The study found that after inoculation with HLB-carrying citrus psyllids, a natural organic compound called epicatechin (ECC) was detected in both the resistant varieties Australian finger lemon and rough-skin lemon. Specific research details are as follows: One hundred citrus psyllids, previously raised on Huanglongbing-infected trees and testing positive for Huanglongbing (confirmed by quantitative real-time PCR sampling, with the detection method referring to the literature "Huang Jiaquan, Li Li, Wu Fengnian, Zheng Zheng, Deng Xiaoling. (2022). Proliferation and pathogenicity of Huanglongbing carrying different prophages in citrus psyllids. Chinese Journal of Agricultural Science, 2022, 55(04):719-728"), were released onto resistant Australian finger lemon and susceptible Satsuma mandarin orange for feeding. After 15 days, leaves and fruits from both treatments were collected. Metabolomics analysis was performed to determine and compare the leaf metabolomes of the resistant Australian finger lemon and the susceptible Satsuma mandarin orange. A total of 322 metabolites were found, of which 126 were differential metabolites. Five substances were associated with resistance to Huanglongbing in citrus, including chlorogenic acid, isochlorogenic acid, and epicatechin, etc. Figure 1 Therefore, further research will be conducted on the role of epicatechin in the prevention and control of citrus Huanglongbing (HLB).
[0026] Example 2: Control effects of epicatechin and protocatechuic acid on Huanglongbing (HLB) of citrus branches. In the spring of 2024, 250 mL solutions of epicatechin or protocatechuic acid (purchased from Shanghai Maclean Biochemical Technology Co., Ltd., purity ≥99%, solvent: water) at concentrations of 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mg / mL were injected into the trunks of 5-year-old *Citrus citrus* trees infected with Huanglongbing (HLB) using a syringe (one injection per tree). The injection site was 10 cm above the ground on the main trunk of the citrus tree. Before injection, the trees were numbered, and leaves were collected. The specific collection criteria were: select mature branches from each *Citrus citrus* tree, collect one leaf from each branch, and collect a total of 12 leaves per tree. Leaves were collected from both the outer and inner branches of the tree, ensuring even collection. After leaf collection, the leaves were washed and dried, the midrib was cut, and 0.1g of DNA was extracted from each sample. The content of *Citrus citrus Huanglongbing* was detected by quantitative real-time PCR (the detection method was based on the literature "Li Zhipeng, Guan Wei, Huang Yang, Yang Yuwen, & Zhao Tingchang. (2019). Study on dynamic changes of *Citrus citrus Huanglongbing* content in the host. Journal of Fruit Science, 36(11), 1540-1548"). Leaves from mature branches were collected one week after injection, and the leaf collection method was the same as before injection. DNA was extracted from the samples again, and the CT value of pathogen content was detected (the pathogen content is expressed as CT value, and its value is negatively correlated with the pathogen content). Four replicates were set for each concentration. The detection results showed that ( Figure 2 , 3Compared with the control (injected water), all concentrations of epicatechin or protocatechuic acid solutions could reduce the content of Huanglongbing bacteria in citrus, but the effects of 1 mg / mL and 2 mg / mL were the most prominent (the greater the increase in CT value after 7 days, the better the antibacterial effect).
[0027] Example 3: Long-term control effects of epicatechin and protocatechuic acid on Huanglongbing (HLB) of citrus tangerines. In the spring of 2025, 250 mL of epicatechin or protocatechuic acid solutions at concentrations of 1.0 and 2.0 mg / mL were injected into the trunks of 5-year-old citrus trees infected with Huanglongbing (HLB) using a syringe. The injection site was 10 cm above the ground on the main trunk. The HLB content was measured on days 7, 14, 30, 60, and 90. On day 90, epicatechin or protocatechuic acid solutions at the same concentrations were injected again, and the HLB content was measured on days 30, 60, and 90. The detection method was the same as in Example 1. Five citrus trees were used in each experimental group. The results showed that ( Figure 4-7 ), epicatechin or protocatechuic acid solutions of 1.0 and 2.0 mg / mL can effectively reduce the content of citrus Huanglongbing pathogen. On day 90, it was found that the control effect of 1.0 mg / mL treatment began to decline, while the content of citrus Huanglongbing pathogen could be maintained at a low level by injecting epicatechin and protocatechuic acid solutions again.
[0028] Example 4: The control effect of epicatechin and protocatechuic acid on Huanglongbing of pomelo In the spring of 2025, a 1.0 mg / mL epicatechin solution and a 1.0 mg / mL protocatechuic acid solution (volume ratio 1:1) were mixed. 300 mL of this mixture was injected via trunk injection into 4-year-old pomelo trees infected with Huanglongbing (HLB) (using 300 mL of a 1.0 mg / mL epicatechin solution and a 1.0 mg / mL protocatechuic acid solution as a control). The injection site was 10 cm above the ground on the main trunk of the citrus tree. The HLB content in the citrus trees was measured on days 30, 60, and 90. The detection method was the same as in Example 1. Each experiment was repeated with 5 pomelo trees. The results were shown in Table 1. Figure 8-9 A mixed solution of 1.0 mg / mL epicatechin and 1.0 mg / mL protocatechuic acid can significantly reduce the content of Huanglongbing pathogen in pomelo and citrus, and has a significant synergistic effect compared with the use of epicatechin or protocatechuic acid alone.
[0029] Table 1. Antibacterial effects of the combination of catechin and protocatechin on Huanglongbing (HLB) of pomelo and citrus. The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.
Claims
1. Application of epicatechin and / or protocatechuic acid in the control of citrus Huanglongbing (HLB).
2. A pesticide for controlling citrus Huanglongbing (HLB), characterized in that, The drug includes epicatechin.
3. A pesticide for controlling citrus Huanglongbing (HLB), characterized in that, The pharmaceutical agent includes protocatechuic acid.
4. A pesticide for controlling citrus Huanglongbing (HLB), characterized in that, The pharmaceutical agent includes epicatechin and protocatechuic acid.
5. A pesticide for controlling citrus Huanglongbing according to any one of claims 2-4, characterized in that, The agent also includes excipients acceptable in the pesticide field.
6. A method for controlling citrus Huanglongbing (HLB), characterized in that, The solution containing epicatechin and / or protocatechuic acid was injected into the citrus tree using a syringe.
7. A method for controlling citrus Huanglongbing according to claim 6, characterized in that, The effective concentrations of epicatechin and protocatechuic acid are both 0.5-4 mg / mL.
8. A method for controlling citrus Huanglongbing according to claim 6, characterized in that, The injection site is 5-15 cm above the ground on the main trunk of the citrus tree.
9. A method for controlling citrus Huanglongbing according to claim 6, characterized in that, The dosage of the injection solution is: 100-150 mL for young trees and 200-400 mL for mature trees; the number of injections is: after the first injection, inject again 3 months later.
10. A method for controlling citrus Huanglongbing according to claim 6, characterized in that, The citrus fruits include tea tangerines, sugar tangerines, and pomelos.