A kind of prevention and control method of grapefruit disease

Abstract

The invention relates to the technical field of plant disease prevention and control, and specifically discloses a method for preventing and controlling grapefruit diseases. Specifically, the invention proposes a prevention and control method of spraying eucalyptus leaf extract or ginkgo fruit extract on grapefruits that are diseased in the young fruit stage, wherein the disease is black spot disease caused by Phyllosticta citriasiana, and the preparation method of the extract comprises the following steps: using an immersion method, using 95% ethanol as a solvent for immersion, filtering, and concentrating to obtain the extract. Experiments of the invention show that the eucalyptus leaf extract can effectively prevent and control the grapefruit black spot disease caused by the infection of Phyllosticta citriasiana, and the prevention efficiency is as high as 82.26%. The research provides a green and sustainable approach for the prevention and control of grapefruit black spot disease, and is expected to reduce the occurrence of the disease.

Description

Technical Field

[0001] The invention relates to the technical field of screening botanical antibacterial agents, and in particular to a botanical antibacterial agent for preventing and treating grapefruit black spot disease. Technical Background

[0002] Pomelo black spot disease is a disease that infects young fruits and shows symptoms in the mature stage. According to the symptoms of the affected fruits, it can be divided into black star type and black spot type. The black star type is mainly characterized by the initial appearance of small reddish-brown spots on the affected fruits, which then expand to a round shape with bulges around them, then turn reddish-brown to dark brown, with a sunken gray-brown center and many small black spots scattered on it. It only infects the epidermis. The black spot type is mainly characterized by the initial appearance of light yellow or orange-yellow spots on the affected fruits, which gradually expand to become dark brown to black irregular spots, slightly sunken, with many small black spots scattered in the center. In severe cases, the spots can be connected into pieces or even expand to the entire fruit surface. Black spot disease seriously affects the appearance quality of pomelo fruit and affects the storage and transportation of pomelo fruit.

[0003] In recent years, the incidence of pomelo black spot disease caused by Asian citrus leaf spot mold (Phyllosticta citriasiana) has continued to rise. The pathogen mainly invades the new shoots, tender leaves, young fruits (green fruits) and mature fruits of pomelo, causing symptoms such as black stars, brown spots and black spots on the surface of the infected peel, which seriously damages the commercial value of pomelo. What is particularly tricky is that the pathogen has a latent characteristic and can hide in the peel for a long time, which makes it difficult for conventional fungicides to effectively eradicate the pathogen. At the same time, the long-term use of a single type of fungicide, such as mancozeb, methyl thiophanate, copper preparations, etc., is not only easy to cause pathogens to develop drug resistance, but the long-term use of copper preparations will also damage the environment and may cause drug damage, leaving difficult-to-eliminate drug spots on the peel, further affecting product quality. Therefore, it is urgent to carry out green prevention and control work, and effective measures need to be taken to meet this challenge.

[0004] As an emerging alternative, botanical fungicides are mainly derived from plant extracts and have natural antibacterial activity. Compared with traditional chemical synthetic fungicides, botanical fungicides show lower toxicity and fewer side effects, and are less irritating to humans and the environment. More importantly, many botanical fungicides have broad-spectrum antibacterial effects, can inhibit the growth of a variety of pathogens, and are not likely to cause bacterial resistance, providing a new approach to solving the problem of pomelo black spot disease. However, there are relatively few studies on botanical fungicides related to pomelo, and it is urgent to strengthen related research and development work in order to provide strong support for the sustainable development of the pomelo industry. Summary of the invention

[0005] The main purpose of the present invention is to provide a grapefruit disease, specifically a control method for black spot disease, to solve the problems existing in the background technology.

[0006] In order to achieve the above objectives, the present invention provides the following technical solutions:

[0007] On the one hand, the present invention provides a method for preventing and controlling grapefruit diseases, which comprises spraying eucalyptus leaf extract or ginkgo fruit extract on grapefruits that are diseased in the young fruit stage, wherein the disease is black spot caused by Phyllosticta citriasiana, and preparing the extract by an immersion method, using 95% ethanol as a solvent for immersion, filtering, and concentrating to obtain the extract.

[0008] Furthermore, the eucalyptus leaf extract is used in a concentration of not less than 60 mg / L.

[0009] Furthermore, the present invention provides the use of eucalyptus leaf extract or ginkgo fruit extract for preventing and controlling plant diseases caused by Phyllosticta citriasiana. The preparation method of the extract is: using an immersion method, using 95% ethanol as a solvent for soaking, filtering, and concentrating to obtain the extract.

[0010] Furthermore, the plant disease in the application is grapefruit black spot disease.

[0011] On the other hand, the present invention provides an application of a fungicide in preventing and controlling infection by Phyllosticta citriasiana, wherein the active ingredient of the fungicide is eucalyptus leaf extract or ginkgo fruit extract, and the preparation method of the extract is: using an immersion method, using 95% ethanol as a solvent for soaking, filtering, and concentrating to obtain the extract.

[0012] Furthermore, in the fungicide, the concentration of eucalyptus leaf extract is not less than 6.02 mg / L, and the optional fungicide includes stabilizers, synergists, preservatives, colorants or emulsifiers in addition to the active ingredients.

[0013] The technical effects achieved by the present invention are:

[0014] The results of the indoor fungicide toxicity determination of the present invention show that the four botanical fungicides, namely, ginkgo fruit extract, privet fruit extract, Magnolia officinalis bark extract and eucalyptus leaf extract, have a certain inhibitory effect on the pathogenic bacteria P. citriasiana of grapefruit black spot disease, and the order of their strength is eucalyptus leaf extract> privet fruit extract> ginkgo fruit extract> Magnolia officinalis bark extract. The indoor prevention effect determination shows that the four botanical fungicides all have a relative prevention and control function on grapefruit black spot disease. When the drug concentration is 30 mg / L, the prevention and control effects are eucalyptus leaf extract> ginkgo fruit extract> privet fruit extract> Magnolia officinalis bark extract. The field prevention effect test shows that different concentrations of eucalyptus leaf extract and ginkgo fruit extract have the effect of preventing and controlling grapefruit black spot disease. When the concentration reaches 60 mg / L, the prevention and control effect of eucalyptus leaf extract is the best, and the relative prevention effect reaches 82.26%. In addition, through the observation of the field test, the eucalyptus leaf extract did not cause drug damage to grapefruit at different gradient concentrations, and no drug spots were produced on the surface of the grapefruit. DETAILED DESCRIPTION

[0015] The present invention is further described below by examples. It should be emphasized that these examples are only used as examples to illustrate the principles and effects of the present invention and do not constitute a limitation on the scope of protection of the present invention. In the following examples, the experimental methods mentioned are conventional methods generally used in the art unless otherwise specified. Similarly, the materials, reagents, etc. used in the examples can be obtained through conventional commercial channels unless otherwise specified.

[0016] Table 1 Medicinal plants tested

[0017]

[0018] Example 1 Determination of the toxicity of the tested fungicides against Phyllostomia citri Asiatica

[0019] 1 Test materials

[0020] Test pathogen: The test bacteria is Phyllosticta citriasiana (P. citriasiana), a common pathogen of grapefruit black spot disease, which was isolated, identified and preserved from diseased grapefruit.

[0021] Test culture medium: Potato sucrose medium (PDA): Add 200g of fresh potato to 1000mL of pure water and boil for 30min, then filter with 3 layers of gauze, add pure water to make the volume to 1000mL, then add 20g of glucose and 20g of agar powder, dissolve and divide into portions, sterilize under high pressure at 121℃ for 20min, cool and place in a constant temperature incubator at 37℃ for 24h to ensure sterile growth before use.

[0022] Preparation method of extracts of medicinal plants: ethanol immersion extraction method is adopted, and the solvent used is 95% ethanol. The specific method is: all plant samples are fully crushed with a grinder to make plant dry powder, 50g of plant dry powder is taken, 250mL of ethanol is added at a ratio of 1:5 and soaked for 2 days, filtered with slow filter paper and funnel, and soaked three times, the filtrate is combined, and concentrated on a RE-52A rotary evaporator (speed: 80rpm, time about 30 minutes) to obtain a concentrated solution with a mass concentration of 1g / mL of plant extract (that is, 1mL of extract contains 1g of plant dry powder sample), about 50mL, and stored in a 4℃ refrigerator for later use.

[0023] 2 Pathogen activation

[0024] Take out the freeze-dried tube of pathogenic bacteria from the -80℃ refrigerator, and after it adapts to room temperature, use a sterile pipette to absorb 0.3ml of sterile water, drop it into the freeze-dried tube, and gently shake it until it is completely dissolved in the sterile water to form a uniform bacterial suspension. Use a sterile pipette tip to absorb all the bacterial suspension and inoculate it on a fresh and strictly sterilized PDA medium using the plate streak method. After the inoculation is completed, place the PDA slant in a 25℃ constant temperature box and culture it in the dark for 5 days before use.

[0025] 3. Preparation of bacterial suspension

[0026] The activated P. citriasiana hyphae were placed on PDA medium at 25°C for 2 weeks. After a large number of conidia were formed, the colonies on the medium were scraped and placed in sterile water in a conical flask. The mixture was shaken thoroughly and prepared to a concentration of 1×10 6 / mL spore suspension.

[0027] 4. Determination of toxicity of indoor fungicides

[0028] The growth rate method was used to determine the inhibitory effect of fungicides on P. citriasiana. The four test agents were prepared into drug-containing culture medium plates of different concentrations (10 mL per plate). Each test agent was treated with 5 concentrations. The blank control (CK) was a PDA culture medium without drugs. Each treatment was repeated 3 times. Use a puncher to punch out bacterial blocks with a diameter of 0.5 cm and move them to the center of the drug-containing culture medium plate. The inoculated plates were placed in a 25°C incubator for culture. The growth of the colonies was observed regularly. After 7 days, the diameter of the colonies was measured by the cross method to calculate the inhibition rate. Inhibition rate (%) = [(control group colony diameter - colony diameter after agent treatment) / (control group colony diameter - bacterial cake diameter)] × 100%.

[0029] In order to further quantify the toxic effects of the four botanical fungicides on P. citriasiana, we converted the concentration (mg / L) of each agent into a logarithmic value (x), and converted the corresponding three-repetition average of the mycelial growth inhibition rate into a probability value (y). Subsequently, statistical software was used to perform regression analysis on these data and fit the toxicity regression equation of the four botanical fungicides on P. citriasiana. Finally, the EC50 value of each agent was obtained by solving the equation, that is, the concentration of the agent required to achieve a 50% growth inhibition rate. The smaller the EC50 value, the stronger the toxicity of the agent to P. citriasiana and the more significant the antibacterial effect.

[0030] 5 Results of indoor toxicity test

[0031] The growth inhibition results and toxicity of the four botanical fungicides on P. citriasiana are shown in Table 2. As the concentration of the fungicide increases, the antibacterial effect becomes more obvious. Among them, the eucalyptus leaf extract has the best inhibitory effect on P. citriasiana. When the concentration reaches 10 mg / L, the inhibition rate reaches 85.98%. The second is Ligustrum lucidum fruit extract. When the concentration reaches 10 mg / L, the inhibition rate is 71.26%; Magnolia officinalis bark extract has the worst inhibitory effect on P. citriasiana. When the concentration reaches 10 mg / L, the inhibition rate is only 60.88%. The experimental results show that the EC of the four agents on P. citriasiana 50 The toxicity of eucalyptus leaf extract to P. citriasiana was the strongest. 50 The value was 6.02 mg / L, followed by Ligustrum lucidum fruit extract, EC 50 The value was 7.29 mg / L; the toxicity of Ginkgo biloba extract and Magnolia officinalis bark extract decreased in turn, EC 50 They are 8.1 and 9.25 mg / L respectively.

[0032] Table 2 Growth inhibition effect and toxicity analysis of botanical fungicides on P. citriasiana

[0033]

[0034]

[0035] Example 2 Indoor efficacy determination of botanical fungicides against grapefruit black spot disease 1 Preparation of bacterial suspension

[0036] The preparation of PDA culture medium refers to the method described in Example 1 "1. Test materials". The preparation of bacterial suspension refers to Example 1 "3. Preparation of bacterial suspension". The concentration of pathogenic bacteria spore solution is 1×10 6 Pieces / mL.

[0037] 2 Indoor protection effectiveness determination

[0038] Take healthy and fresh pomelo fruit in the billiard stage, disinfect the surface with 75% alcohol, rinse with sterile water 3 times, dry naturally, and use the puncture inoculation method (each puncture site has 10 puncture points), use a sterile cotton swab to apply P. citriasiana spore liquid to the fruit wound and the surrounding fruit surface, and use an equal amount of clean water as a control, each treatment is repeated 5 times, and after smearing, put it in a plastic box and place it in a 28℃ constant temperature box. Ginkgo fruit extract, Ligustrum fruit extract, Magnolia officinalis bark extract and eucalyptus leaf extract are prepared into the required agent stock solution, and different concentrations of fungicides are sprayed on the surface of pomelo fruit using a constant sprayer, about 10ml per fruit each time, and sterile water is set as a blank control. During the test, the drug was applied 4 times, with an interval of 3 days between each application. The disease of the fruit was observed one week after the last injection, and the incidence rate and disease index were statistically analyzed.

[0039] Incidence rate (%) = (number of lesions at the puncture site on the leaf or fruit ÷ total number of puncture sites on the leaf or fruit) × 100; Disease grading standard: 0 level means no lesions at the puncture site on the leaf or fruit; 1 level means 1 to 3 lesions at the puncture site; 2 level means 4 to 6 lesions at the puncture site; 3 level means 7 or more lesions at the puncture site; Disease index (%) = (0 × n0 + 0.25 × n1 + 0.5 × n2 + 0.75 × n3) ÷ n × 100, n0 represents the number of leaves or fruits at level 0, n1, n2, n3, and so on; n is the total number of leaves or fruits at each level. Relative prevention effect (%) = (disease index of control group - disease index of treatment group) / disease index of control group × 100.

[0040] 3 Indoor protection effectiveness test results

[0041] The results of indoor control efficacy determination (Table 3) show that the four tested agents can effectively reduce the incidence and alleviate the incidence of grapefruit black spot disease. The grapefruit treated with 30 mg / L eucalyptus leaf extract had an incidence of 26.99%, a disease index of 13.93, and a relative control effect of 83.68%. Among the four agents, the control effect was the most significant; the grapefruit treated with 30 mg / L ginkgo fruit extract had an incidence of 35.86%, a disease index of 16.93, and a relative control effect of 80.17%, which was relatively significant; followed by the grapefruit treated with 30 mg / L Ligustrum lucidum fruit extract, with a relative control effect of 63.77%; the grapefruit treated with 30 mg / L Magnolia officinalis bark extract had an incidence of 39.11%, a disease index of 35.32, and a relative control effect of 58.63%, which was the worst among the four plant-derived agents.

[0042] Table 3 Results of indoor protective efficacy of the tested agents on pomelo fruit

[0043]

[0044] Example 3 Field efficacy determination of botanical fungicides against grapefruit black spot disease 4 Preparation of bacterial suspension

[0045] The preparation of PDA culture medium refers to the method described in Example 1 "1. Test materials". The preparation of bacterial suspension refers to Example 1 "3. Preparation of bacterial suspension". The concentration of pathogenic bacteria spore solution is 1×10 6 Pieces / mL.

[0046] 5 Field control effectiveness determination

[0047] Healthy and disease-free pomelo trees were selected as test plants. Fruits of the same size were selected during the fruiting period for prevention efficacy determination. The inoculation method was the same as in Example 2. P. citriasiana spores were applied to the fruit wound and the surrounding fruit surface with a sterile cotton swab. Eucalyptus leaf extract and ginkgo fruit extract were prepared into the required drug stock solution. Different concentrations of fungicides were sprayed on the surface of the pomelo fruit using a constant sprayer. Each fruit was sprayed with about 15 ml of the drug each time, and sterile water was set as a blank control. During the test period, the drug was applied 4 times, with an interval of 3 days between each application. After each application, a special bag for pomelo fruit was put on to prevent interference from other diseases and insects. The disease condition of the fruit was observed one week after the last application, and the incidence rate and disease index were statistically analyzed.

[0048] The fruit disease classification standard is as shown in Example 2.

[0049] 6 Field control results

[0050] The results of the field pesticide control efficacy comparison test showed that the treatment with 60 mg / L eucalyptus leaf extract had the best control effect, with an incidence rate of only 21.96%, a disease index of 14.98, and a relative control effect of 82.26%; the treatment with 60 mg / L ginkgo fruit extract had a lower control effect, with an incidence rate of 32.86, a disease index of 25.14, and a relative control effect of 70.07%.

[0051] Table 4 Field efficacy test results of the tested agents against grapefruit black spot disease

[0052]

[0053] Field trials showed that 60 mg / L eucalyptus leaf extract had the best protective effect on grapefruit with black spot disease, with a relative protective effect of 82.16. Therefore, eucalyptus leaf extract can be used as a recommended agent to prevent grapefruit black spot disease in this study.

[0054] Black spot disease caused by Asian citric acid is one of the important diseases that seriously affects the commodity value and export earnings of grapefruit. At present, there is a lack of effective tools for green and sustainable control in the technology for preventing and controlling grapefruit black spot disease. In this study, the results of indoor toxicity test showed that four botanical fungicides, namely ginkgo fruit extract, Ligustrum lucidum fruit extract, Magnolia officinalis bark extract and eucalyptus leaf extract, had a certain inhibitory effect on the pathogen of grapefruit black spot disease, P. citriasiana, and the order of their strength was eucalyptus leaf extract > Ligustrum lucidum fruit extract > ginkgo fruit extract > Magnolia officinalis bark extract. Indoor prevention effect test showed that all four botanical fungicides had relative prevention and control functions against grapefruit black spot disease. When the drug concentration was 30 mg / L, the prevention and control effects were eucalyptus leaf extract > ginkgo fruit extract > Ligustrum lucidum fruit extract > Magnolia officinalis bark extract. Field efficacy tests showed that different concentrations of eucalyptus leaf extract and ginkgo fruit extract were effective in preventing and controlling grapefruit black spot disease. When the concentration reached 60 mg / L, eucalyptus leaf extract had the best prevention and control effect, with a relative prevention efficiency of 82.26%.

[0055] Through a series of rigorous indoor toxicity evaluations, indoor control effect tests, and field control effect verification, this study successfully selected agents with good control effects on grapefruit black spot. This discovery not only opens up a new direction for grapefruit disease prevention and control, and provides strong support for reducing the occurrence of diseases, but also the selected fungicides are all derived from nature and have environmentally friendly characteristics.

[0056] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent transformations made using the contents of the present invention specification under the concept of the present invention, or direct / indirect applications in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A method for preventing and controlling grapefruit diseases, characterized in that: Eucalyptus leaf extract is sprayed on pomelo fruits that are diseased in the young fruit stage, wherein the disease is black spot caused by Phyllosticta citriasiana. The extract is prepared by: using an immersion method, using 95% ethanol as a solvent for immersion, filtering, and concentrating to obtain the extract; the use concentration of the eucalyptus leaf extract is not less than 60 mg / L.

2. Use of eucalyptus leaf extract for preventing and controlling plant diseases caused by Phyllosticta citriasiana, characterized in that: The preparation method of the extract is: adopting an immersion method, using 95% ethanol as a solvent for immersion, filtering, and concentrating to obtain the extract.

3. The use according to claim 2, characterized in that: The plant disease is grapefruit black spot disease.

4. Use of a fungicide in preventing and controlling infection by Phyllosticta citriasiana, characterized in that: The active ingredient of the fungicide is eucalyptus leaf extract. The preparation method of the extract is: adopting an immersion method, using 95% ethanol as a solvent for soaking, filtering, and concentrating to obtain the eucalyptus leaf extract. In the fungicide, the concentration of the eucalyptus leaf extract is not less than 6.02 mg / L.

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