Method for preventing and treating citrus liberobacter asiaticum through combination of biological agent and heat preservation cover

Abstract

The invention provides a method for preventing and treating citrus huanglongbing by combining a biological agent with a heat preservation cover, which comprises the following steps: S1, mixing honeysuckle, coptis chinensis, cortex phellodendri, fructus forsythiae, radix sophorae flavescentis, gallnut, purslane, radix cyathulae and liquorice, adding water for extraction, and filtering to obtain a traditional Chinese medicine extracting solution preparation; s2, spore flora, lactic acid bacteria flora and saccharomycetes are taken and mixed, water is added, the mixture is stirred to be uniform, and a compound bacteria preparation is obtained; s3, a heat preservation cover is formed around the infected citrus tree, the temperature is increased to 40-50 DEG C, and heat preservation is conducted; and S4, firstly injecting the compound bacteria preparation into the infected citrus by adopting instillation, then injecting the traditional Chinese medicine extracting solution preparation on the next day, and circularly instilling the two preparations for 3-4 times, so that the aim of preventing and treating the citrus huanglongbing can be fulfilled. The method provided by the invention is good in control effect, has the advantages of safety, no residue, environmental friendliness and the like on the whole, and is beneficial to vigorous popularization and application.

Description

Technical Field

[0001] The invention belongs to the technical field of fruit tree disease prevention and control, and particularly relates to a method for preventing and controlling citrus Huanglongbing disease by combining a biological agent with a heat preservation cover. Background Art

[0002] Citrus Huanglongbing ( Citrus Huanglongbing, The pathogen of HLB is believed to be a phloem-restricted α-Proteobacterium ( Alphaproteobacteria ) Gram-negative bacteria. According to the sequence characteristics of the pathogen's 16SrDNA and β-operon genes, as well as the transmission medium and the thermosensitivity of the pathogen, the citrus Huanglongbing pathogen can be divided into Asian species ( Candidatus Liberibacter asiaticus , Clas), African species ( Candidatus Liberibacter African , Claf) and American species ( Candidatus Liberibacter americanus, There are three species of HLB: HLB, Clam, and Clam. Among them, CLas is the most widely distributed in the world and causes the greatest losses to the citrus industry. The pathogen of citrus Huanglongbing in my country is also this species. The content of HLB pathogen is highest from October to December every year, and lowest from March to May. The spatial distribution of HLB pathogen in host plants is: seed coat>leaf midrib≈branch>fruit stalk≈orange peel>mesophyll>fruit peel≈fruit flesh>trunk. HLB is mainly transmitted by diseased seedlings and citrus psyllids, and can infect almost all citrus plants, causing devastating effects on the world's citrus industry. Its destructive power has far exceeded any other citrus disease. Recent research results show that citrus Huanglongbing is an immune disease triggered by Bacillus phloem. Traditional methods of citrus Huanglongbing prevention and control mainly use chemical pesticides to kill the transmission medium, causing soil pollution and reduced fertility.

[0003] In recent years, the biopesticide industry, which has the advantages of safety, no residue, and environmental friendliness, has continued to grow and become the focus of international green pesticide development. Microbial-based biological control technology is considered to be one of the main development directions for controlling bacterial diseases of crops in the future. For example, patent document CN108617696A records a composite microbial agent for citrus Huanglongbing, which is compounded with Bacillus thuringiensis, Beauveria bassiana, Trichoderma harzianum, and Actinomycetes Frankia in a mass ratio of (1.5-2.5): (2.5-3.5): (3-5): (0.5-1.5). Although the composite microbial solution has a balancing effect on microorganisms in the soil, due to the lack of a step to disinfect microorganisms, harmful bacteria such as Gram-negative bacteria that cause Huanglongbing still have the possibility of causing disease, outbreaks, and infections. Patent document CN106857141A describes a method for preventing and treating citrus Huanglongbing, which uses quicklime to adjust soil acidity, uses organic biological fertilizers to improve soil structure and quality, and further uses biological bacteria to combine with plant immune inducers to promote citrus plant root vitality and improve citrus disease resistance, thereby efficiently and greenly preventing and treating citrus Huanglongbing and promoting the healthy growth of citrus plants. However, when using quicklime to prevent and treat Huanglongbing, quicklime can only generate heat and kill microorganisms on the surface of the ground when it comes into contact with water, and it is difficult to penetrate into the soil. Summary of the invention

[0004] In view of this, the present invention provides a method for preventing and controlling citrus Huanglongbing by using a biological agent in combination with a heat preservation cover. The biological agent in the present invention comprises two types: a traditional Chinese medicine extract preparation and a microbial preparation. By injecting the diseased citrus trees under heat preservation conditions, the Huanglongbing can be killed better, the mortality rate of citrus trees due to Huanglongbing can be reduced, and the citrus trees will not be harmed.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: A method for preventing and controlling citrus Huanglongbing by using a biological agent and a heat preservation cover comprises the following steps: S1. Mix honeysuckle, coptis root, phellodendron, forsythia, sophora flavescens, gallnut, purslane, cyathula officinalis, and liquorice, add water for extraction, and filter to obtain a Chinese medicine extract preparation; S2, mix the spore bacteria, lactic acid bacteria and yeast, add water and stir evenly to obtain a composite bacterial preparation; S3. Form a heat shield around the infected citrus trees, raise the temperature to 40-50°C and keep it warm; S4. Use drip to first inject the compound bacterial preparation into the infected citrus, and then inject the Chinese medicine extract preparation every other day. The two preparations are dripped in a cycle for 3 to 4 times to achieve the purpose of preventing and controlling citrus Huanglongbing disease.

[0006] Furthermore, in step S1, the mass ratio of honeysuckle, coptis root, phellodendron, forsythia, sophora flavescens, gallnut, purslane, cyathula and licorice is 2:2:2:2:2:2:2:2:1:1.

[0007] Further, the extraction steps are as follows: Mix the Chinese medicines according to the mass ratio, add water 5 to 7 times the total mass of the Chinese medicines, soak for 1 to 2 hours, filter, and obtain filtrate I and filter residue; take the filter residue, add water 7 to 9 times the mass of the filter residue, soak for 0.5 to 1 hour, filter, and obtain filtrate II; combine filtrate I and filtrate II to obtain the Chinese medicine extract preparation.

[0008] Furthermore, in step S2, the mass ratio of spore bacteria, lactic acid bacteria, and yeast was 1:2:0.5, and the activity of each bacterium was 1.0×10 10 cfu / g~8.0×10 11 cfu / g.

[0009] Furthermore, the mass ratio of the total mass of various bacteria after mixing in the composite bacterial preparation to water is (1~3) :100.

[0010] Furthermore, the spore-forming bacteria group is a mixture of Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, and Bacillus megaterium; The lactic acid bacteria group is a mixture of Lactobacillus casei, Pediococcus acidilactici, Enterococcus faecalis and Enterococcus faecium; The yeast is Saccharomyces boulardii.

[0011] Furthermore, the mass ratio of Bacillus thaliana, Bacillus licheniformis, Bacillus coagulans and Bacillus megaterium in the spore-forming bacteria group is 1:2:1:0.5; The mass ratio of Lactobacillus casei, Pediococcus acidilactici, Enterococcus faecalis and Enterococcus faecium in the lactic acid bacteria group is 1:1:0.5:0.5.

[0012] Furthermore, the protective cover in step S3 is made of nylon film, and the heat preservation is carried out by an industrial heater.

[0013] Furthermore, the specific injection conditions of the Chinese medicine extract preparation in step S4 are as follows: heating to 40-50°C before injection, injection at a rate of 5-8 mL / min, injection volume of 1000 mL per plant under 10 years old, 1200 mL per plant between 10 and 20 years old, and 1500 mL per plant over 20 years old; The specific injection conditions of the composite bacteria preparation are as follows: heat to 40-50°C before injection, inject at a rate of 3-6mL / min, and use 10g of composite bacteria per plant for trees under 10 years old, 20g per plant for trees between 10 and 20 years old, and 30g per plant for trees over 20 years old.

[0014] In some specific embodiments, preferably, the specific injection conditions of the Chinese medicine extract preparation in step S4 are as follows: heating to 45° C. before injection and injecting at a speed of 6 mL / min; The specific injection conditions of the composite bacterial preparation are as follows: heat to 45°C before injection and inject at a rate of 5 mL / min.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses the prepared Chinese medicine extract preparation, the composite bacteria preparation and the heat preservation cover in combination to inject the infected citrus trees, which can significantly reduce the mortality rate of citrus trees due to Huanglongbing, and does not harm the citrus trees; it has the advantages of safety, no residue, and environmental friendliness. After the inventor's long-term garden prevention and control, the method provided by the present invention can reduce the incidence of Huanglongbing from the original 10.0% to less than 1%; in addition, the citrus yield is increased from the current average of 1500kg / mu to 1826kg / mu, increasing the yield by more than 20%. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a graph showing the inhibitory effects of different preparations of the present invention on the pathogen of citrus Huanglongbing. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0018] Sources and physical and chemical parameters of key test materials: The pathogen of citrus Huanglongbing was provided by the School of Life Sciences of Wuhan University.

[0019] The strains used were purchased from JuneYao RunYing Biotechnology (Shanghai) Co., Ltd. The specific details are as follows: Bacillus subtilis: strain number BS-GA28, viable cell count 8.0×10 11 cfu / g, production date 20220301; Bacillus licheniformis: strain number BL-GA26, viable count 5.0×10 11 cfu / g, production date 20220301; Bacillus coagulans: strain number BC-G44, viable count 1.0×10 11 cfu / g, production date 20220301; Bacillus megaterium: strain number BM-GA53, viable cell count 1.0×10 10 cfu / g, production date 20220301; Lactobacillus casei: strain number LC-G11, viable count 4.0×1011 cfu / g, production date 20220301; Pediococcus acidilactici: strain number PA-G73, viable count 2.0×10 11 cfu / g, production date 20220301; Enterococcus faecalis: strain number EP-GA65, viable count 2.0×10 11 cfu / g, production date 20220301; Enterococcus faecium: strain number SF-GA12, viable count 2.0×10 11 cfu / g, production date 20220301; Saccharomyces boulardii: strain number SD-G19, viable count 2.0×10 10 cfu / g, production date 20220301.

[0020] The specific raw materials not described in the present invention are all existing materials and can be directly purchased from the market.

[0021] The present invention provides a method for preventing and treating citrus Huanglongbing by using a biological agent in combination with a heat preservation cover, and the specific steps are as follows: S1. Preparation of Chinese medicine extract preparations: weigh honeysuckle, coptis root, phellodendron, forsythia, sophora flavescens, gallnut, purslane, cyathula capitata, and liquorice in a mass ratio of 2﹕2﹕2﹕2﹕2﹕2﹕1﹕1, mix them, add 5-7 times the total mass of the mixed Chinese medicine in water, soak for 1-2 h, filter to obtain filtrate I and residue; take the residue, add 7-9 times the mass of water in water, soak for 0.5-1 h, filter to obtain filtrate II; combine filtrate I and filtrate II to obtain the Chinese medicine extract preparation.

[0022] S2. Preparation of composite bacterial preparation: Weigh spore-forming bacteria, lactic acid bacteria, and yeast in a mass ratio of 1:2:0.5 (the activity of each bacterium is 1.0×10 10 cfu / g~8.0×10 11 cfu / g), and then add warm water according to the mass ratio of the total mass of various bacteria mixed to water (1-3) ﹕ 100, stir evenly, and then the composite bacteria preparation is obtained.

[0023] Among them, the spore-forming bacteria group is composed of Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans, and Bacillus megaterium mixed in a mass ratio of 1:2:1:0.5; the lactic acid bacteria group is composed of Lactobacillus casei, Pediococcus acidilactici, Enterococcus faecalis, and Enterococcus faecium mixed in a mass ratio of 1:1:0.5:0.5; and the yeast is Saccharomyces boulardii.

[0024] S3. Installation of insulation cover: Use nylon film to cover the infected citrus fruits, put a thermometer and industrial heater at the bottom to maintain the temperature inside the protective cover at 40~50℃.

[0025] S4. Reagent injection: First, heat the Chinese medicine extract preparation to 40~50℃ and inject at a rate of 5~8mL / min. The injection volume is 1000mL per plant under 10 years old, 1200mL per plant between 10 and 20 years old, and 1500mL per plant over 20 years old. The next day, heat the composite bacteria preparation to 40~50℃ and inject at a rate of 3~6mL / min. The dosage of the composite bacteria is 10g per plant under 10 years old, 20g per plant between 10 and 20 years old, and 30g per plant over 20 years old. Cycle the above two preparations for 3~4 times to achieve the purpose of preventing and controlling citrus Huanglongbing disease.

[0026] The present invention adopts the form of combined use of traditional Chinese medicine extract, composite bacteria preparation and heat preservation cover, which can significantly reduce the mortality rate of citrus trees due to Huanglongbing disease without causing harm to the citrus trees; it has the advantages of being safe, residue-free and environmentally friendly as a whole.

[0027] Example 1 This embodiment provides a method for preventing and controlling citrus Huanglongbing by combining a biological agent with a heat preservation cover, and the specific steps are as follows: S1. Preparation of Chinese medicine extract preparation: Weigh 200g of honeysuckle, 200g of coptis, 200g of phellodendron, 200g of forsythia, 200g of sophora flavescens, 200g of gallnut, 200g of purslane, 100g of cyathula and 100g of liquorice respectively, mix (a total of 1600g), add 9600g of water, soak for 1.5h, filter to obtain filtrate I and residue; take the residue, add water 8 times the mass of the residue, soak for 0.8h, filter to obtain filtrate II; combine filtrate I and filtrate II to obtain the Chinese medicine extract preparation.

[0028] S2. Preparation of composite bacterial preparation: Weigh 1 part of Bacillus subtilis, 2 parts of Bacillus licheniformis, 1 part of Bacillus coagulans, and 0.5 parts of Bacillus megaterium respectively and mix them to form a spore bacteria group; then weigh 1 part of Lactobacillus casei, 1 part of Pediococcus acidilactici, 0.5 parts of Enterococcus faecalis, and 0.5 parts of Enterococcus faecium respectively and mix them to form a lactic acid bacteria group.

[0029] The above-mentioned spore bacteria, lactic acid bacteria, and Saccharomyces boulardii were mixed in a mass ratio of 1:2:0.5 (a total of 3.5 g), 300 g of warm water was added, and the mixture was stirred evenly to obtain a composite bacterial preparation. S3. Installation of insulation cover: Use nylon film to cover the infected citrus fruits, put a thermometer and industrial heater at the bottom to maintain the temperature inside the protective cover at 45℃.

[0030] S4. Reagent injection: First, heat the Chinese medicine extract preparation to 45℃ and inject at a rate of 6mL / min. The injection volume is 1000mL per plant under 10 years old, 1200mL per plant between 10 and 20 years old, and 1500mL per plant over 20 years old. The next day, heat the composite bacteria preparation to 45℃ and inject at a rate of 5mL / min. The dosage of the composite bacteria is 10g per plant under 10 years old, 20g per plant between 10 and 20 years old, and 30g per plant over 20 years old. Cycle the above two preparations three times to achieve the purpose of preventing and controlling citrus Huanglongbing disease.

[0031] Comparative Example 1 This comparative example provides a method for preventing and treating citrus Huanglongbing disease, and its steps and methods are basically the same as those in Example 1, except that only the Chinese medicine extract preparation is injected, and the composite bacteria preparation is not injected, and the rest remains unchanged.

[0032] Comparative Example 2 This comparative example provides a method for preventing and treating citrus Huanglongbing disease, and its steps and methods are basically the same as those of Example 1, except that only the composite bacteria preparation is injected, and the traditional Chinese medicine extract preparation is not injected, and the rest remains unchanged.

[0033] Comparative Example 3 This comparative example provides a method for preventing and controlling citrus Huanglongbing disease, and its steps and methods are basically the same as those in Example 1, except that the installation of the heat preservation cover in step S3 is omitted, and the injection is only performed at room temperature (25°C), and the rest remain unchanged.

[0034] Comparative Example 4 This comparative example provides a method for preventing and controlling citrus Huanglongbing disease, and its steps and methods are basically the same as those in Example 1, except that: in step S2, only the spore-forming bacteria group is used to prepare the composite bacterial preparation, and the rest remain unchanged.

[0035] Comparative Example 5 This comparative example provides a method for preventing and treating citrus Huanglongbing disease, and its steps and methods are basically the same as those in Example 1, except that: in step S4, the Chinese medicine extract preparation and the composite bacteria preparation are only injected once, and no cyclic injection is performed, and the rest remain unchanged.

[0036] Comparative Example 6 This comparative example provides a method for preventing and treating citrus Huanglongbing disease, and its steps and methods are basically the same as those in Example 1, except that: in step S4, the temperature of the Chinese medicine extract preparation and the composite bacteria preparation is heated to 60°C, and the rest remains unchanged.

[0037] The methods of Example 1 and Comparative Examples 1-5 were used to control citrus trees in highly infected areas, and the final results are shown in Table 1.

[0038] Table 1 Final control effect of Example 1 and Comparative Examples 1-5

[0039] As shown in Table 1, the prevention and control of citrus Huanglongbing by the method of the present application can significantly reduce the incidence rate and increase the yield of citrus. Compared with Comparative Examples 1 and 2, the lack of composite bacterial preparations and Chinese medicine extract preparations respectively, although the overall prevention and control effect is also there, the effect will be worse. Compared with Comparative Examples 3 and 5, it can be seen that temperature is also one of the key factors affecting the prevention and control effect. Too low temperature may affect the absorption of the number, inhibit the activity of the drug, etc., resulting in poor effect, while too high temperature may affect the tolerance of the tree, resulting in partial inactivation of the drug activity, etc., resulting in poor effect. Compared with Comparative Example 4, it can be seen that the type of bacteria in the bacterial preparation will also affect the prevention and control effect, and the effect of a single bacterial species will obviously deteriorate. Compared with Comparative Example 5, it can be seen that the number of injections also affects the prevention and control effect. Prevention and control requires long-term and multiple injections to achieve better results, not once and for all. The feature of the present application is that the composite bacterial preparation and the Chinese medicine extract preparation are combined to prevent and control at a specific temperature (40~50℃) and multiple cyclic injections, which can achieve good results.

[0040] Furthermore, in order to understand the inhibitory effects of the above-mentioned Chinese medicine extract preparation and compound bacterial preparation on the pathogenic bacteria of citrus Huanglongbing, the following tests were also conducted: Take the activated citrus Huanglongbing pathogen solution and spread it on the plate, put the filter paper soaked with the drug on the plate, cover it with a lid, and culture it for 2-3 days. Observe the presence and size of the antibacterial circle around the filter paper disc and calculate the antibacterial rate. The antibacterial rate results are shown in Figure 1 , Table 2. The preparations soaked in each filter paper are as follows: 1. The upper filter paper is soaked in a Chinese medicine extract + a compound bacteria mixed preparation; 2. The right filter paper is soaked in a Chinese medicine extract preparation; 3. The left filter paper is soaked in a compound bacteria preparation; 4. The lower filter paper is soaked in a sterilized culture medium (i.e., a blank control) as a control group.

[0041] Inhibition rate = [(diameter of inhibition zone in treatment group - diameter of inhibition zone in blank group) / diameter of inhibition zone in treatment group] × 100%.

[0042] Table 2 Antibacterial results of each experimental group

[0043] From the results in Table 2, it can be seen that although the Chinese medicine extract preparation and the composite bacteria mixed preparation provided by the present invention can both inhibit the citrus Huanglongbing pathogen when used alone, the antibacterial effect of the preparation formed by mixing the Chinese medicine extract and the composite bacteria is more obvious.

[0044] Depend on Figure 1(The upper filter paper is soaked in drug extract + compound bacteria mixed preparation; the right filter paper is soaked in drug extract preparation; the left filter paper is soaked in compound bacteria preparation; the lower filter paper is soaked in sterilized culture medium) It can be seen that: the antibacterial zone diameter formed by Chinese medicine extract + compound bacteria mixed preparation is the largest and the effect is the best; the antibacterial zone diameter of the Chinese medicine extract preparation is second; the antibacterial zone diameter of the compound bacteria preparation is the smallest and the effect is the worst.

[0045] The above are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preventing and controlling citrus Huanglongbing by using a biological agent in combination with a heat preservation cover, characterized in that: The following steps are involved: S1. Mix honeysuckle, coptis root, phellodendron, forsythia, sophora flavescens, gallnut, purslane, cyathula officinalis, and liquorice, add water for extraction, and filter to obtain a Chinese medicine extract preparation; S2, mix the spore bacteria, lactic acid bacteria and yeast, add water and stir evenly to obtain a composite bacterial preparation; S3. Form a heat shield around the infected citrus trees, raise the temperature to 40-50°C and keep it warm; S4. Use drip to first inject the compound bacterial preparation into the infected citrus, and then inject the Chinese medicine extract preparation every other day. The two preparations are dripped in a cycle for 3 to 4 times to achieve the purpose of preventing and controlling citrus Huanglongbing disease.

2. The method according to claim 1, characterized in that: In step S1, the mass ratio of honeysuckle, coptis root, phellodendron, forsythia, sophora flavescens, gallnut, purslane, cyathula and liquorice is 2:2:2:2:2:2:2:2:1:

1.

3. The method according to claim 2, characterized in that The extraction steps are as follows: Mix the Chinese medicines according to the mass ratio, add water 5 to 7 times the total mass of the Chinese medicines, soak for 1 to 2 hours, filter, and obtain filtrate I and filter residue; take the filter residue, add water 7 to 9 times the mass of the filter residue, soak for 0.5 to 1 hour, filter, and obtain filtrate II; combine filtrate I and filtrate II to obtain the Chinese medicine extract preparation.

4. The method according to claim 1, characterized in that In step S2, the mass ratio of spore bacteria, lactic acid bacteria, and yeast was 1:2:0.5, and the activity of each bacterium was 1.0×10 10 cfu / g~8.0×10 11 cfu / g.

5. The method according to claim 4, characterized in that The mass ratio of the total mass of various bacteria after mixing in the composite bacterial preparation to water is (1~3) :

100.

6. The method according to claim 4, characterized in that The spore-forming bacteria group is a mixture of Bacillus subtilis, Bacillus licheniformis, Bacillus coagulans and Bacillus megaterium; The lactic acid bacteria group is a mixture of Lactobacillus casei, Pediococcus acidilactici, Enterococcus faecalis and Enterococcus faecium; The yeast is Saccharomyces boulardii.

7. The method according to claim 4, characterized in that The mass ratio of Bacillus oxalis, Bacillus licheniformis, Bacillus coagulans and Bacillus megaterium in the spore-forming bacteria group is 1:2:1:0.5; The mass ratio of Lactobacillus casei, Pediococcus acidilactici, Enterococcus faecalis and Enterococcus faecium in the lactic acid bacteria group is 1:1:0.5:0.

5.

8. The method according to claim 1, characterized in that: The protective cover in step S3 is made of nylon film, and the heat preservation is carried out by an industrial heater.

9. The method according to claim 1, characterized in that: The specific injection conditions of the Chinese medicine extract preparation in step S4 are as follows: heating to 40-50°C before injection, injecting at a speed of 5-8 mL / min, and the injection volume is 1000 mL per plant under 10 years old, 1200 mL per plant between 10 and 20 years old, and 1500 mL per plant over 20 years old; The specific injection conditions of the composite bacteria preparation are as follows: heat to 40-50°C before injection, inject at a rate of 3-6mL / min, and use 10g of composite bacteria per plant for trees under 10 years old, 20g per plant for trees between 10 and 20 years old, and 30g per plant for trees over 20 years old.

10. The method according to claim 1, characterized in that The specific injection conditions of the Chinese medicine extract preparation in step S4 are as follows: heating to 45° C. before injection and injecting at a rate of 6 mL / min; The specific injection conditions of the composite bacterial preparation are as follows: heat to 45°C before injection and inject at a rate of 5 mL / min.

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