A fermented Chinese medicine extract preparation for preventing and treating apricot fire blight and its preparation method

Abstract

The present invention provides a Chinese medicine extract fermentation preparation for preventing and treating apricot fire blight, wherein the Chinese medicine extract fermentation preparation is prepared by inoculating plant lactobacillus into a mixed solution containing a Chinese medicine extract and auxiliary materials for fermentation to obtain a fermentation product liquid; wherein the Chinese medicine includes honeysuckle, coptis root, phellodendron, forsythia, sophora flavescens, gallnut, purslane, cyathula officinalis, and liquorice; and the auxiliary materials include red sugar, peptone, corn steep liquor, and calcium carbonate. The Chinese medicine extract fermentation preparation prepared by the present invention is based on the biological control technology of microorganisms, has the advantages of safety, no residue, and environmental friendliness, and will not continuously enhance the drug resistance of pathogens while preventing and treating apricot fire blight on pear trees, which has a wide range of practical value for preventing and treating related diseases caused by apricot fire blight pathogens.

Description

Technical Field

[0001] The invention belongs to the technical field of plant microbial preparations, and specifically relates to a traditional Chinese medicine extract fermentation preparation and a preparation method and application thereof. Background Art

[0002] Fire blight Erwinia amylovora ) is a devastating bacterial disease that seriously harms the production of Rosaceae fruit trees such as pears, apples, hawthorns, and crabapples. It is listed as a Class I crop disease and agricultural plant quarantine pest in my country. The disease first occurred in New York State, USA in 1780, and has now spread to more than 60 countries and regions. At present, there is a lack of effective drugs and single prevention and control measures for apricot fire blight, which is difficult to prevent and control. It still continues to occur and spread in many countries. Safety prevention and control is a global problem. At present, the use of chemical agents is the most common means of prevention and control of apricot fire blight. Agricultural streptomycin was once considered the best agent for the prevention and control of apricot fire blight, but its frequent, large-scale and continuous use has caused the pathogen's resistance to drugs to continue to increase and the prevention effect to decrease; at the same time, agricultural streptomycin has a series of problems such as the risk of enrichment in the human body through the food chain and environmental pollution. my country stopped registering it in June 2016 and withdrew from the pesticide market in April 2018. There is currently no better alternative.

[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. Aureobasidium pullulans ) and bacteriophage as active ingredients (Blossom Protect and AgriphageTM Fire Blight), which have good control effects on fire blight. Foreign patent EP14798394.4 discloses that Lactobacillus acidophilus strains are used to control fire blight. The high antagonistic activity of the strain is partly attributed to the production of peptides and non-peptide compounds that inhibit the growth of pathogens (including Escherichia coli). In China, Klebsiella ( Klebsiella sp ) has a good control effect on apricot fire blight (CN112322561A), Bacillus ( Becillus sp ) and Lactobacillus plantarum have a significant inhibitory effect on apricot fire blight (CN112725215A), but there are no registered biocontrol products for the prevention and control of apricot fire blight in China. Summary of the invention

[0004] In view of this, the present invention provides a Chinese medicine extract fermentation preparation, which is prepared by inoculating plant lactobacillus into a mixed solution containing a Chinese medicine extract and auxiliary materials and fermenting the resulting fermentation product liquid. Dripping the Chinese medicine extract fermentation preparation on pear trees can have a significant effect in preventing and treating pear fire blight.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: A Chinese medicine extract fermentation preparation for preventing and treating fire blight of apricot, wherein the Chinese medicine extract fermentation preparation is prepared by inoculating plant lactobacillus into a mixed solution containing a Chinese medicine extract and auxiliary materials and fermenting the resulting fermentation product liquid, and the OD of the Chinese medicine extract fermentation preparation is 600 Greater than 1.2, viable bacteria count greater than 1.0×10 6 cfu / mL; Wherein, the plant lactobacillus includes plant lactobacillus A with a preservation number of CCTCC No: M 2019026 and plant lactobacillus B with a preservation number of CGMCC 1.2158; The Chinese medicines include honeysuckle, coptis root, phellodendron, forsythia, sophora flavescens, gallnut, purslane, cyathula officinalis, and liquorice; The auxiliary materials include brown sugar, peptone, corn steep liquor and calcium carbonate.

[0006] Furthermore, the volume ratio of the Lactobacillus plantarum inoculum to the mixed solution is 3-8%; Among them, the inoculation ratio of Lactobacillus plantarum A and Lactobacillus plantarum B is 1:1.

[0007] Furthermore, the mass ratio of honeysuckle, coptis chinensis, phellodendron chinense, forsythia suspensa, sophora flavescens, gallnut, purslane, cyathula capitata, and licorice in the traditional Chinese medicine is 2:2:2:2:2:2:2:2:1:1.

[0008] Furthermore, the addition amounts of brown sugar, peptone, corn steep liquor and calcium carbonate in the auxiliary materials are 5-8%, 1-2%, 0.5-1% and 0.05-0.1% of the mass of the traditional Chinese medicine extract, respectively.

[0009] A method for preparing the above-mentioned Chinese medicine extract fermentation preparation comprises the following steps: S1. Weigh each Chinese medicine according to the mass ratio, add water for extraction, and filter to obtain the Chinese medicine extract; S2, adding auxiliary materials to the above-mentioned traditional Chinese medicine extract according to the mass ratio, sterilizing, cooling, and obtaining the fermentation liquid; S3, inoculating Lactobacillus plantarum into the fermented liquid according to the inoculation ratio and inoculation amount, and fermenting to obtain a fermented preparation of a Chinese medicine extract.

[0010] Furthermore, the specific extraction steps in step S1 are as follows: first add 5 to 7 times the total mass of the traditional Chinese medicine for extraction for 1 to 2 hours, filter to obtain filtrate I and filter residue, then add 7 to 9 times the mass of water to the filter residue for extraction for 0.5 to 1 hour, filter to obtain filtrate II, and combine filtrate I and filtrate II to obtain the traditional Chinese medicine extract.

[0011] Furthermore, the specific fermentation conditions in step S3 are as follows: temperature 40-45° C., rotation speed 150-200 r / min, ventilation volume 1:0.5, and fermentation time 48-72 h.

[0012] Application of the above-mentioned Chinese medicine extract fermentation preparation in preventing and treating pear fire blight of pear trees.

[0013] Furthermore, the specific application steps are as follows: The Chinese medicine extract fermentation preparation is added with water to prepare a drip solution, and then injected into the pear tree by dripping to achieve the prevention and treatment of pear fire blight. Among them, the mass ratio of the Chinese medicine extract fermentation preparation to water is (1~3): (100~200), the dripping temperature of the dripping solution is 40~45℃, the dripping speed is 5~8mL / min, and the dripping time is 7~10d.

[0014] Furthermore, the dosage of the Chinese medicine extract fermentation preparations of different tree ages for dripping is as follows: For trees under 10 years old, the dosage of fermented Chinese medicinal extract is 10g; for trees between 10 and 20 years old, the dosage of fermented Chinese medicinal extract is 20g; for trees over 20 years old, the dosage of fermented Chinese medicinal extract is 30g.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The invention uses Chinese herbal medicine as the main material, ferments the extract thereof, and finally obtains a Chinese herbal medicine extract fermentation preparation, which has the advantages of safety, no residue, and environmental friendliness, and prevents and treats pear fire blight on pear trees without increasing the drug resistance of pathogens. Treating the diseased pear trees with the Chinese herbal medicine extract fermentation preparation at a temperature of 40-45°C by dripping can significantly solve the mortality rate of fragrant pear trees due to fire blight, and the cure rate is as high as 99.2%. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a graph showing the inhibitory effect of the fermented Chinese medicine extract preparations prepared in Example 1 and Comparative Examples 1-2 of the present invention on the pathogenic bacteria of Fire Blight. 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 fire blight was provided by the School of Life Sciences of Wuhan University.

[0019] Lactobacillus plantarum A with the deposit number of CCTCC No: M 2019026 was provided by Wuhan Wuerli Bioengineering Co., Ltd.

[0020] Lactobacillus plantarum B with the deposit number of CGMCC 1.2158 was purchased from the General Microbiology Center of China National Committee for the Collection of Microorganisms.

[0021] The specific raw materials not described in the present invention are all existing materials and can be directly purchased from the market.

[0022] The fermented Chinese medicine extract preparation for preventing and treating fire blight of apricot provided by the present invention is specifically prepared as follows: S1. Weigh honeysuckle, coptis chinensis, phellodendron, forsythia suspensa, sophora flavescens, gallnut, purslane, cyathula capitata, and liquorice in order at a mass ratio of 2:2:2:2:2:2:2:1:1 and mix; then add 5-7 times the mass of water of the total mass of the Chinese medicine for soaking for 1-2 h, filter to obtain filtrate I and filter residue; then add 7-9 times the mass of water to the filter residue for soaking for 0.5-1 h, filter to obtain filtrate II, combine filtrate I and filtrate II to obtain the Chinese medicine extract.

[0023] S2. Add brown sugar, peptone, corn steep liquor and calcium carbonate in the order of 5-8%, 1-2%, 0.5-1% and 0.05-0.1% of the mass of the traditional Chinese medicine extract, mix them, and sterilize them at 121°C for 20 minutes to obtain the fermentation liquid.

[0024] S3, inoculating Lactobacillus plantarum at 3-8% of the volume of the fermented liquid, wherein the Lactobacillus plantarum is prepared by mixing Lactobacillus plantarum A and Lactobacillus plantarum B in a volume ratio of 1:1 after pure culture in MRS liquid medium; setting fermentation conditions: temperature 40-45°C, rotation speed 150-200 r / min, ventilation volume 1:0.5, fermentation time 48-72h; detecting OD during fermentation 600 Greater than 1.2, viable bacteria count greater than 1.0×10 6 cfu / mL, and the fermentation is completed to obtain the Chinese medicine extract fermentation preparation.

[0025] The Chinese medicine extract fermentation preparation prepared by the present invention can be used to prevent and treat pear fire blight of pear trees, and the specific operation is as follows: The Chinese medicine extract fermentation preparation and water are prepared into a drip solution in a mass ratio of (1-3): (100-200), and injected into pear trees at a drip temperature of 40-45°C, a drip speed of 5-8mL / min, and a drip time of 7-10d to achieve the prevention and control of pear fire blight.

[0026] Among them, the dosage of Chinese herbal extract fermentation preparation for dripping is as follows according to the different ages of trees: 10g of Chinese herbal extract fermentation preparation for each tree under 10 years old, 20g of Chinese herbal extract fermentation preparation for each tree between 10 and 20 years old, and 30g of Chinese herbal extract fermentation preparation for each tree over 20 years old.

[0027] The invention uses Chinese herbal medicine as the main material, first extracts it to obtain a Chinese herbal medicine extract, then adds fermentation auxiliary materials, inoculates plant lactobacillus for fermentation, and finally obtains a Chinese herbal medicine extract fermentation preparation. The Chinese herbal medicine extract fermentation preparation is based on the biological control technology of microorganisms, has the advantages of safety, no residue, environmental friendliness, etc., and will not continuously enhance the drug resistance of pathogens while preventing and controlling pear fire blight on pear trees.

[0028] Example 1 This embodiment provides a fermented Chinese medicine extract preparation for preventing and treating fire blight, and the preparation method is as follows: S1. Weigh 2 parts each of honeysuckle, coptis root, phellodendron, forsythia, sophora flavescens, gallnut, purslane, 1 part each of cyathula and liquorice in order by mass, and mix them; then add water 5 times the total mass of the Chinese medicine for immersion for 2 hours, filter, and obtain filtrate I and filter residue; then add water 8 times the total mass of the Chinese medicine to the filter residue for immersion for 1 hour, filter, and obtain filtrate II; combine filtrate I and filtrate II to obtain the Chinese medicine extract.

[0029] S2. Obtain the percentage mass of the Chinese medicine extract according to step S1, weigh 5% brown sugar, 1% peptone, 1% corn steep liquor, and 0.1g calcium carbonate in sequence, add them to the above Chinese medicine extract and mix them, then sterilize at 121°C for 20min, cool down after completion, and obtain the fermentation liquid.

[0030] S3, inoculate Lactobacillus plantarum according to 5% of the volume of the fermented liquid, wherein Lactobacillus plantarum A with a preservation number of CCTCC No: M 2019026 and Lactobacillus plantarum B with a preservation number of CGMCC 1.2158 are mixed in a volume ratio of 1:1 after pure culture in MRS liquid medium. Set the fermentation conditions: temperature 42°C, speed 180r / min, ventilation volume 1:0.5, fermentation time 60h; after fermentation, the traditional Chinese medicine extract fermentation preparation is obtained, and the OD of the traditional Chinese medicine extract fermentation preparation prepared by testing is 600 is 1.5, and the number of viable bacteria is 2.0×10 6 cfu / mL.

[0031] Comparative Example 1 This comparative example provides a fermented Chinese medicine extract preparation for preventing and treating fire blight of apricot. The preparation method is basically the same as that of Example 1, except that only Lactobacillus plantarum A with a preservation number of CCTCC No: M 2019026 is inoculated for fermentation, and the rest remains unchanged.

[0032] Comparative Example 2 This comparative example provides a fermented Chinese medicine extract preparation for preventing and treating fire blight of apricot. The preparation method is basically the same as that of Example 1 except that only Lactobacillus plantarum B with a preservation number of CGMCC 1.2158 is fermented, and the rest remain unchanged.

[0033] Furthermore, in order to understand the inhibitory effect of the Chinese medicine extract fermentation preparations prepared in the above examples and comparative examples on the pathogenic bacteria of fire blight of apricot, the following tests were also conducted: Take the activated apricot fire blight pathogen solution and spread it on the plate, use a hole puncher (diameter 5mm) to punch holes on the plate with bacterial culture medium, cover it with a lid, and culture it for 2~3 days. Observe the presence and size of the antibacterial circle around the hole. The antibacterial results are shown in Figure 1 (Among them, the left hole is added with the fermented Chinese medicine extract of Example 1, the upper right hole is added with the fermented Chinese medicine extract of Comparative Example 1, and the lower right hole is added with the fermented Chinese medicine extract of Comparative Example 2.

[0034] It can be seen from the figure that the antibacterial effect of the Chinese medicine extract fermentation preparation provided in Example 1 is significantly higher than that of the Chinese medicine extract fermentation preparations provided in Comparative Examples 1 and 2; the antibacterial effect of Lactobacillus plantarum used alone is lower than the technical effect of combined use.

[0035] Furthermore, in order to understand the control effect of the Chinese medicine extract fermentation preparation prepared in Example 1 on diseased pear trees, the following experiments were also conducted: The Chinese medicine extract fermentation preparation prepared in Example 1 was added with water at a mass ratio of 2:150 to prepare a drip solution, and the diseased pear trees were treated according to the following groups, and 10 diseased pear trees were randomly selected in each group. The specific results are shown in Table 1. Among them, the dosage of the Chinese medicine extract fermentation preparation was as follows according to the age of the tree: 10 g of the Chinese medicine extract fermentation preparation was used for each tree under 10 years old, 20 g of the Chinese medicine extract fermentation preparation was used for each tree between 10 and 20 years old, and 30 g of the Chinese medicine extract fermentation preparation was used for each tree over 20 years old.

[0036] Group 1: The drip solution was heated to 42°C and kept warm, then dripped into the diseased pear trees at a rate of 6 mL / min for 8 days.

[0037] Group 2: The drip solution was dripped into the diseased pear trees at room temperature at a rate of 6 mL / min for 8 days.

[0038] Group 3: The drip solution was used to irrigate the roots of the diseased pear trees for 8 days.

[0039] Group 4: The drip solution was sprayed on the diseased pear trees for 8 days.

[0040] Table 1 The cure of diseased pear trees in different treatment groups

[0041] From the results in Table 1, it can be seen that the use of the Chinese medicine extract fermentation preparation prepared by the present application and the corresponding method to prevent and control pear fire blight can significantly reduce the incidence rate and increase the pear yield; compared with Group 1 and Group 2, 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 trees, inhibit the activity of drugs, etc., resulting in poor effects; compared with Group 2 and Group 3, it can be seen that the use method of the Chinese medicine extract fermentation preparation will also lead to differences in prevention and control. Irrigation and spraying may make the contact between the fermentation preparation and the trees not direct enough, resulting in poor effects and thus limiting the absorption and utilization of the agent by the trees. Among them, irrigation has the worst effect.

[0042] In addition, through the long-term garden prevention and control experience of the inventor, it is concluded that when the Chinese medicine extract fermentation preparation prepared by the present invention is used to prevent and control pear fire blight of pear trees, the diseased pear trees need to be dripped twice a year, and it is not a one-time solution; the first time is in early to mid-March, and the second time is in early to mid-June. In this way, the prevention and control effect can achieve the best effect, and the occurrence of pear fire blight can be almost completely prevented and controlled.

[0043] Other raw materials or structures not specifically described in the present invention already exist in the prior art and can be directly purchased from the market.

[0044] 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 fermented Chinese medicine extract preparation for preventing and treating fire blight, characterized in that: The Chinese medicine extract fermentation preparation is prepared by inoculating plant lactobacillus into a mixed solution containing the Chinese medicine extract and auxiliary materials and fermenting the resulting fermentation product liquid; Wherein, the plant lactobacillus includes plant lactobacillus A with a preservation number of CCTCC No: M 2019026 and plant lactobacillus B with a preservation number of CGMCC 1.2158; The Chinese medicines include honeysuckle, coptis root, phellodendron, forsythia, sophora flavescens, gallnut, purslane, cyathula officinalis, and liquorice; The auxiliary materials include brown sugar, peptone, corn steep liquor and calcium carbonate.

2. The Chinese medicine extract fermentation preparation according to claim 1, characterized in that: The volume ratio of the Lactobacillus plantarum inoculum to the mixed solution is 3-8%; Among them, the inoculation ratio of Lactobacillus plantarum A and Lactobacillus plantarum B is 1:

1.

3. The Chinese medicine extract fermentation preparation according to claim 1, characterized in that: The mass ratio of honeysuckle, coptis chinensis, phellodendron chinense, forsythia suspensa, sophora flavescens, gallnut, purslane, cyathula capitata and liquorice in the traditional Chinese medicine is 2:2:2:2:2:2:2:2:1:

1.

4. The Chinese medicine extract fermentation preparation according to claim 1, characterized in that: The addition amounts of brown sugar, peptone, corn steep liquor and calcium carbonate in the auxiliary materials are 5-8%, 1-2%, 0.5-1% and 0.05-0.1% of the mass of the traditional Chinese medicine extract, respectively.

5. A method for preparing the fermented Chinese medicine extract preparation according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. Weigh each Chinese medicine according to the mass ratio, add water for extraction, and filter to obtain the Chinese medicine extract; S2, adding auxiliary materials to the above-mentioned traditional Chinese medicine extract according to the mass ratio, sterilizing, cooling, and obtaining the fermentation liquid; S3, inoculating Lactobacillus plantarum into the fermented liquid according to the inoculation ratio and inoculation amount, and fermenting to obtain a fermented preparation of a Chinese medicine extract.

6. The preparation method according to claim 5, characterized in that: The specific extraction steps in step S1 are as follows: first add 5 to 7 times the total mass of the traditional Chinese medicine for extraction for 1 to 2 hours, filter to obtain filtrate I and filter residue, then add 7 to 9 times the mass of water to the filter residue for extraction for 0.5 to 1 hour, filter to obtain filtrate II, and combine filtrate I and filtrate II to obtain the traditional Chinese medicine extract.

7. The preparation method according to claim 5, characterized in that: The specific fermentation conditions in step S3 are as follows: temperature 40-45° C., rotation speed 150-200 r / min, ventilation volume 1:0.5, and fermentation time 48-72 h.

8. Use of the fermented preparation of the Chinese medicine extract according to any one of claims 1 to 4 in preventing and treating fire blight of pear trees.

9. The use according to claim 8, characterized in that: The specific application steps are as follows: The Chinese medicine extract fermentation preparation is added with water to prepare a drip solution, and then injected into the pear tree by dripping to achieve the prevention and treatment of pear fire blight. Among them, the mass ratio of the Chinese medicine extract fermentation preparation to water is (1~3): (100~200), the dripping temperature of the dripping solution is 40~45℃, the dripping speed is 5~8mL / min, and the dripping time is 7~10d.

10. The use according to claim 9, characterized in that: The dosage of Chinese medicine extract fermentation preparations for instillation at different ages is as follows: For trees under 10 years old, the dosage of fermented Chinese medicinal extract is 10g; for trees between 10 and 20 years old, the dosage of fermented Chinese medicinal extract is 20g; for trees over 20 years old, the dosage of fermented Chinese medicinal extract is 30g.

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