CN114304146A - Xcn 1-containing microbial source sterilization ointment, and preparation method and application thereof - Google Patents
Xcn 1-containing microbial source sterilization ointment, and preparation method and application thereof Download PDFInfo
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- CN114304146A CN114304146A CN202111483446.0A CN202111483446A CN114304146A CN 114304146 A CN114304146 A CN 114304146A CN 202111483446 A CN202111483446 A CN 202111483446A CN 114304146 A CN114304146 A CN 114304146A
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Abstract
The invention relates to Xcn 1-containing microbial source sterilization ointment, a preparation method and application thereof, wherein the ointment contains Xcn1, a film forming material, a thickening agent, a penetration enhancer, a humectant and a solvent, and the content of Xcn1 serving as an effective component is more than 0.02%. The bactericidal ointment has good control effect on diseases of fruit trees such as apple canker (Cytosporanmandashurica), apple ring spot (Physiosporicola), kiwifruit canker (Pseudomonas syringaepv. actindiae) and the like. As an environment-friendly agricultural bactericide, the ointment can be directly used without dilution, and the influence on the environment is greatly reduced by adopting a method of smearing and applying the bactericide when in use. Compared with the existing sterilization ointment on the market, the ointment provided by the invention has the characteristics of less dosage and wider application range.
Description
Technical Field
The invention relates to the field of pesticide preparation processing and microbial pesticides, in particular to Xcn 1-containing microbial sterilization ointment, a preparation method and application.
Background
With the gradual enhancement of environmental protection consciousness and the improvement of quality safety standard of international agricultural products, people put forward higher requirements on research and development of new pesticides, and biological pesticides become the trend of pesticide development. Experimental research shows that the X.nematophila YL001 fermentation liquor has obvious bactericidal activity under the conditions of in vitro and in vivo, and the main active ingredient of the fermentation liquor is Xcn 1. Xcn1 has unique chemical structure, has strong inhibiting effect on plant pathogenic bacteria such as Actinidia chinensis planch, is not easy to generate drug resistance, and can be naturally degraded in environment. Therefore, Xcn1 is a pollution-free fungicide for controlling agricultural diseases, and development of commercial products is urgently required. However, as a secondary metabolite produced by a microorganism, the lower content of the fermentation broth is an important factor for limiting the direct utilization of Xcn1, and how to directly utilize Xcn1 at a lower cost becomes an important problem in the process of commercial development thereof.
The kiwifruit canker can be transmitted by grafting, pruning branches and the like, so that the branches and the stems can be canker and even the tree body can die, and a large amount of yield is reduced. The rainwater in the high-quality kiwi fruit main production area in China is more, and in the kiwi fruit grafting and trimming process, a pesticide which is resistant to rainwater scouring, capable of protecting wounds and isolating pathogens, long in lasting period, low in residue and easy to degrade is needed. The existing pesticide has the problems of easy washing by rain, short lasting period and the like in the using process, and the problems can be solved by using the paste.
The biopesticides mainly used in China, such as validamycin, zhongshengmycin, kasugamycin and the like, mainly have water aqua, wettable powder, soluble powder and the like, and in order to widen the application range of the biopesticides, more types of preparations are needed to meet the requirements in the medication process, and the paste is a feasible preparation for widening the application range of the biopesticides. At present, the domestic registered sterilizing paste (or pastes) comprise 1% tebuconazole paste, 3% thiophanate methyl paste, 5% thiophanate methyl paste, 2% oxine-copper paste, 3% imazalil paste, 5% captan paste, 3.3% methyl-naphthalene acetic acid paste and the like, and are mainly used for preventing and treating apple canker, no sterilizing paste for preventing and treating kiwifruit canker is available, and the sterilizing paste for preventing and treating kiwifruit canker has a very large potential market.
Disclosure of Invention
The invention aims to provide Xcn 1-containing microbial source sterilization ointment, a preparation method and application. The paste is a paste agricultural sterilization preparation prepared by adding a film-forming agent, a thickening agent, a humectant, a penetration enhancer and water into Xcn1 highly concentrated fermentation liquor. The application method is that the medicine is directly smeared on the cut or the scab of the trimmed branch, and the medicine dosage per square centimeter is 0.1-0.2 g.
The technical scheme of the invention is as follows:
an ointment containing Xcn1 for killing bacteria is prepared by removing sugar and protein from fermentation broth of xenorhabdus nematophila (Xenorhabdus snematophila) capable of generating Xcn1, concentrating, adding film-forming agent, humectant, thickener, penetration enhancer and water, and making into ointment;
optionally, the mass percentages of the raw materials contained in the paste are respectively as follows:
0.04-0.15 percent of Xcn1 concentrated fermentation liquor, 5.32 percent of film forming material, 2.52 percent of thickening agent, 1.96 percent of humectant, 2.46 percent of penetration enhancer and 100 percent of water supplement.
Optionally, the fermentation broth is a fermentation broth of xenorhabdus nematophila YL001(x.nematophila), and the specific preparation method comprises the following steps:
placing an X.nematophila YL001 strain in an LB culture medium, activating for 24 hours at 180rpm/min and 28 ℃ to prepare a seed solution, adding the seed solution into a PP3 culture medium according to the proportion of 10%, fermenting for 48 hours at 150rpm/min and 28 ℃, centrifuging the fermentation liquor for 20 minutes at 4 ℃ and 10000rpm/min, collecting the supernatant, adding 5.5g/L of aluminum sulfate octadecahydrate, rapidly stirring for 1min, standing for 6 hours, centrifuging, taking the supernatant, carrying out reduced pressure distillation and concentration to 1/4 times of the original volume, adding 80% ethanol, stirring for 10 minutes, refrigerating overnight at 4 ℃, centrifuging, taking the supernatant, carrying out reduced pressure distillation and concentration to 240 times, and obtaining the concentrated fermentation liquor containing 3.0% Xcn 1.
Optionally, the film forming agent is selected from one or a mixture of more than two of sodium carboxymethylcellulose, polyvinyl alcohol, polyethylene glycol, organic bentonite, diatomite, sodium alginate, liquid paraffin, gelatin, xanthan gum, polyvinylpyrrolidone and soluble starch.
Optionally, the humectant is one or a mixture of more than two of ethylene glycol, glycerol and vaseline.
Optionally, the thickener is one or a mixture of more than two of sodium carboxymethylcellulose, polyvinylpyrrolidone and sodium alginate.
Optionally, the penetration enhancer is one or a mixture of more than two of azone, polyoxyethylene lauryl ether and sec-octanol polyoxyethylene ether.
The preparation method of the Xcn 1-containing microbial source sterilization ointment comprises the steps of preparing Xcn 1-containing microbial source sterilization ointment, wherein the Xcn 1-containing microbial source sterilization ointment is prepared by using a preparation method of the Xcn 1-containing microbial source sterilization ointment;
the preparation method comprises the following steps:
adding polyvinyl alcohol into water, heating and dissolving, sieving by a 40-mesh sieve to remove insoluble substances, cooling to 65 ℃, adding sodium carboxymethylcellulose, fully stirring and dissolving, adding azone and glycerol into the mixture, uniformly mixing, cooling to 45 ℃, adding Xcn1 concentrated fermentation liquor, supplementing water to 100%, stirring at the stirring speed of 600-800 rpm for 20-30 minutes, and thus obtaining the microbial source sterilization paste containing 0.04-0.15% of Xcn 1.
Optionally, the preparation of the Xcn 1-containing concentrated fermentation broth comprises:
placing an X.nematophila YL001 strain in an LB culture medium, activating for 24 hours at 180rpm/min and 28 ℃ to prepare a seed solution, adding the seed solution into a PP3 culture medium according to the proportion of 10%, fermenting for 48 hours at 150rpm/min and 28 ℃, centrifuging the fermentation liquor for 20 minutes at 4 ℃ and 10000rpm/min, collecting the supernatant, adding 5.5g/L of aluminum sulfate octadecahydrate, rapidly stirring for 1min, standing for 6 hours, centrifuging, taking the supernatant, carrying out reduced pressure distillation and concentration to 1/4 times of the original volume, adding 80% ethanol, stirring for 10 minutes, refrigerating overnight at 4 ℃, centrifuging, taking the supernatant, carrying out reduced pressure distillation and concentration to 240 times, and obtaining the concentrated fermentation liquor containing 3.0% Xcn 1.
The Xcn 1-containing microbial source sterilization ointment is used for preventing and treating apple rot (Cytosporanmandanhucica), apple ring rot (Physalosporapicola) and/or kiwi canker (Pseudomonas syringaepv. actinoidiae);
the application method comprises the following steps:
the liniment is directly applied to the cut or the scab of the trimmed branch, and the dosage per square centimeter is 0.1-0.2 g.
The invention has the advantages and meanings that:
(1) xcn1 the paste is a novel microbial source bactericide, which is in line with the development trend of agriculture and pesticides and is a novel pollution-free pesticide preparation; compared with the sterilization paste existing in the market, the Xcn1 paste has the characteristics of less dosage, no residue and no pollution;
(2) xcn1 the fermentation liquid is obtained by fermenting the nematophilic pathogenic bacillus, the nematophilic pathogenic bacillus resource is abundant in our country, the fermentation liquid containing Xcn1 is easy to get, the mother drug cost is low; xcn1 the use of organic solvent is greatly reduced in the preparation process of the paste, the pollution in the preparation processing is reduced, and the influence on non-target organisms is also reduced; xcn1 the paste has simple preparation method, low processing cost, and suitability for industrial production.
Drawings
The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure without limiting the disclosure. In the drawings:
FIG. 1 is a response surface diagram of the influence of two factors of each auxiliary agent on the diameter of a bacteriostatic circle;
FIG. 2 is a high performance liquid chromatogram for detecting active ingredients of standard substance (top) and ointment (bottom) of 2Xcn 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the present invention will be described in further detail with reference to embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The Xcn 1-containing microbial source sterilization ointment and the preparation method thereof remove proteins and saccharides in xenorhabdus nematophila fermentation liquor based on a flocculation method and an organic solvent precipitation method, carry out reduced pressure distillation and concentration to prepare Xcn1 parent drugs, add a proper amount of film-forming agent, thickening agent, humectant, penetration enhancer and water, and evenly mix by a stirring method to prepare pasty agricultural sterilization preparations with different Xcn1 contents. An agricultural bactericidal ointment which is prepared by directly utilizing xenorhabdus nematophilus fermentation liquor and takes Xcn1 as a main bacteriostatic active component. The paste is a paste agricultural sterilization preparation prepared by adding a film-forming agent, a thickening agent, a humectant, a penetration enhancer and water into Xcn1 highly concentrated fermentation liquor. Xcn 1A highly concentrated fermentation broth is obtained based on a concentration technique to remove proteins and polysaccharides. The application aim of the invention is to provide a biological source sterilization ointment which can be directly used and can prevent and treat various branch diseases. The application method is that the medicine is directly smeared on the cut or the scab of the trimmed branch, and the medicine dosage per square centimeter is 0.1 to 0.2 g.
In the method of the present invention, the protein removal method is preferably flocculation with aluminum sulfate octadecahydrate, and the protein in the fermentation broth can be removed by other flocculants, or by heating, salting out, and adsorption.
In the method of the present invention, ethanol is preferably used as the sugar-precipitating agent, and the organic solvent used in the organic solvent precipitation method is not limited to ethanol, and other common organic solvents can be used for removing sugar.
The method of the present invention, wherein the method for preparing Xcn1 mother drug by concentration is preferably vacuum distillation, and Xcn1 mother drug with high concentration can be obtained by evaporator concentration, freeze concentration, gel water absorption concentration, ultrafiltration concentration, reverse dialysis, etc.
According to a preferred embodiment of the present invention, Xcn1 the concentration of the parent drug is 3.0%, and higher content of parent drug can be used for the preparation of related preparations.
The Xcn1 preparation is prepared by the stirring method, preferably a magnetic heating stirrer and an electric stirrer are combined step by step for stirring, and other stirring devices with controllable temperature and speed can also be used for mixing.
The application range of the invention is that the multiple branch diseases mainly comprise kiwifruit canker, apple canker and apple ring rot, and also comprise branch diseases caused by pathogenic bacteria infection in the production of other fruit trees.
The application method of the invention is characterized in that the adhesive is directly applied to the cut or the scab of the branch, and can also be applied to the injury part of the branch caused by various biological or non-biological factors to prevent the infection of pathogenic bacteria.
The using method of the invention, wherein the dosage per square centimeter is preferably 0.1-0.2 g, and the dosage can be adjusted according to actual conditions.
In a preferred embodiment of the invention, in the Xcn1 pasty agricultural bactericidal preparation, the content of Xcn1 is 0.08%, and Xcn1 paste with different concentrations can be prepared by adopting Xcn1 with different contents. In-vitro branch tests show that the 0.08% Xcn1 ointment has the control effect on apple canker and apple ring rot of more than 80% and the control effect on kiwifruit canker of 71.38%.
The product provides a water-based preparation which has high efficiency, low toxicity, no pollution, safety to environment, people and livestock and difficult generation of drug resistance. The method has the advantages of simple operation, low cost and easy industrial production.
In order to further illustrate the present invention, the following examples are given, wherein the following experiments are conducted in the following examples, unless otherwise specified, wherein the percentages are by weight:
example 1: xcn1 preparation of high concentration mother drug (see in detail "screening of culture Medium by Xenorhabdus snemantophila YL001 and optimization of culture conditions", Wang Yong hong, Zhang xing, 2006, journal of university of agriculture and forestry, North West province (Nature science edition))
This example provides a method for preparing a 3.0% Xcn1 mother drug, which includes placing an x.nematophila YL001 strain in an LB medium (1.0% tryptone, 0.5% yeast extract, 1.0% NaCl), activating at 180rpm/min and 28 ℃ for 24 hours to prepare a seed solution, adding the seed solution to a PP3 medium (2.0% peptone), fermenting at 150rpm/min and 28 ℃ for 48 hours, centrifuging a fermentation broth at 4 ℃ and 10000rpm/min for 20 minutes, collecting a supernatant, adding octadecyl aluminum sulfate 5.5g/L, stirring rapidly for 1min, standing for 6 hours, centrifuging, collecting the supernatant, distilling and concentrating under reduced pressure to 1/4 times of an original volume, adding 80% ethanol, stirring for 10min, refrigerating at 4 ℃ overnight, centrifuging, collecting the supernatant, distilling and concentrating under reduced pressure 240 times, and thus obtaining a 3.0% Xcn1 mother drug.
Example 2: xcn1 screening of adjuvants and usage amount in paste processing process
The embodiment provides a screening method of an auxiliary agent and the usage amount in the Xcn1 paste processing process, and the varieties and the usage amounts of a film forming agent, a thickening agent, a humectant and a penetration enhancer involved in the Xcn1 paste processing process are obtained by screening for multiple times. The optimal concentration obtained by screening through the single-factor test is taken as a central point, a four-factor three-level central combined test is designed, the diameter of an inhibition zone of Xcn1 paste on kiwifruit canker is taken as a response value, and the response surface method is used for optimizing to obtain the optimal use concentration of each auxiliary agent: 5.32% of polyvinyl alcohol, 2.52% of sodium carboxymethyl cellulose, 2.46% of azone and 1.96% of glycerol.
Screening the film forming agent and the dosage thereof: the film forming materials comprise sodium carboxymethylcellulose, polyvinyl alcohol, polyethylene glycol, organic bentonite, diatomite, sodium alginate, liquid paraffin, gelatin, xanthan gum, polyvinylpyrrolidone k30 and soluble starch, and the optimal film forming material is screened by analyzing the film forming time and the film forming property of the preparation through a single factor test. Firstly, determining the quick drying property of the film-forming agent by a constant weight method: 2g of film forming agent is uniformly coated on a glass plate, the glass plate is placed in a 70 ℃ oven to be dried, and the constant weight time is measured. Measuring the film forming property of the film forming agent by a glass plate film forming method: and (3) taking each film forming agent on a 9cm culture dish, and inspecting the film forming property in three grades. The film is formed evenly, and the film can be completely scraped off from the glass plate to form I grade; the film is uniformly formed and can not be completely scraped off from the glass plate to form a II grade; the film forming agent can not form a film on a glass plate to be grade III. The polyvinyl alcohol is determined to be a film forming material of Xcn1 paste by preliminary screening, and the dosage is 5%.
Screening the using amount of the thickening agent: preparing paste with the sodium carboxymethylcellulose content of 0%, 0.5%, 1%, 1.5%, 2%, 2.5% and 3%, taking a branch with the length of 15cm and the diameter of about 1cm, marking the length of 4cm at one end of the branch after wax sealing of two ends, smearing 0.5g of paste, vertically placing, and measuring the dropping length after the paste is fixed into a film. 2.5% carboxymethylcellulose was used as a thickener for the Xcn1 paste, and the viscosity was 8500mp · s at room temperature as measured by a viscometer.
Screening the humectant and the dosage thereof: the humectant material comprises glycerol, propylene glycol and vaseline, and the glycerol is found to be the best humectant material through the appearance of the preparation. Paste with the glycerol contents of 0%, 0.5%, 1%, 1.5%, 2%, 2.5% and 3% is prepared, 1.5g of the paste is uniformly coated on a glass plate, and after 30d, the falling area of the film is counted by using Image J1.38 x (http:// Image J. nih. gov/ij /) software. The film forming time can be prolonged when the content of the glycerol is too high, the ointment can crack and fall off when the content of the glycerol is too low, and 1.5 percent of the glycerol is selected as the humectant of the ointment in consideration of the film forming time, the falling area and the appearance.
And (3) screening a penetration enhancer and the dosage thereof: collecting 4 kiwi branches with similar length and thickness, circularly cutting 3cm bark in the middle for later use, taking 4 50mL beakers, adding 2mL blue ink, preparing 50mL 2% azone, polyoxyethylene lauryl ether and sec-octanol polyoxyethylene ether solution, taking a blank contrast without adding a penetration enhancer, soaking absorbent cotton in the ink solution, wrapping the branch barking part, and observing the dyeing condition under a microscope after 1 h. Taking 6 kiwi fruit branches with similar lengths and thicknesses, preparing 0%, 1%, 1.5%, 2%, 2.5% and 3% azone ink solution, and determining the optimal dosage of the penetration enhancer by adopting the same method. The dyeing test shows that azone has the largest dyeing area, is most suitable as a penetration enhancer, and has the optimal use amount of 2.5%.
With reference to fig. 1, the response surface method formula optimizes the usage amount of each auxiliary agent: according to the single-factor screening result, the addition amounts of polyethylene glycol (A), sodium carboxymethylcellulose (B), azone (C) and glycerol (D) are used as test independent variables, the Xcn1 content is 200 mu g/mL, the diameter of a bacteriostatic circle of an indicator kiwi fruit ulcer pathogenic bacterium is used as a response value, the optimal concentration of a formula determined by the single-factor test is used as a central point, a four-factor three-level Box-Behnken central combined test is designed, and a regression equation describing the relationship among the independent variables of polyvinyl alcohol (A), sodium carboxymethylcellulose (B), azone (C), glycerol (D) and the diameter of the bacteriostatic circle of the response value is obtained by performing regression fitting on the result of the combined test by using Design expert 8.0: y-16.15753 +0.91213a +9.53333B +2.16889C +1.67333D-0.1 AB-0.013333 AC +0.1AD +0.4BC +0.1BD-8.20912 CD-0.0608 a2-2.02B2–0.63111C2–0.505D2The regression equation obtained by removing the insignificant term is: y-16.15753 +9.53333B +2.16889C +1.67333D-8.20912CD-2.02B2。
The result is analyzed by using Design-expert8.0 software to obtain Xcn1 the best additive amount of the auxiliary agent in the paste: 5.32% of polyvinyl alcohol, 2.52% of sodium carboxymethyl cellulose, 2.46% of azone and 1.96% of glycerol.
Example 3: preparation of 0.08% Xcn1 ointment
The embodiment provides a preparation method of Xcn1 paste with the concentration of 0.08%, which is to add 5.32% of polyvinyl alcohol into a proper amount of water, heat and dissolve the polyvinyl alcohol at 90 ℃, screen the polyvinyl alcohol through a 40-mesh sieve to remove insoluble substances, cool the polyvinyl alcohol to 65 ℃, add 2.52% of sodium carboxymethyl cellulose, fully stir and dissolve the sodium carboxymethyl cellulose, add 2.46% of azone and 1.96% of glycerol into the mixture, uniformly mix the mixture, cool the mixture to 45 ℃, add 2.67mL of 3.0% of Xcn1 concentrated fermentation liquor, replenish the water to 100%, stir the mixture for 20-30 minutes at the stirring speed of 600-800 rpm, and obtain Xcn1 paste with the concentration of 0.08%. The method is simple and easy to implement, low in cost and suitable for mass production.
The preparation of 0.04%, 0.06%, 0.15% Xcn1 paste was the same as the preparation of 0.08% Xcn1 paste, except that the volume of 3.0% Xcn1 concentrate was added.
Example 4: quality control method of 0.08% Xcn1 ointment
This example provides a method for testing other properties of 0.08% Xcn1 paste, such as heat storage stability, low temperature stability, centrifuge stability, water resistance, adhesion, etc.
The preparation has qualified appearance standard: the preparation appearance is uniform, fine and smooth solid preparation with certain fluidity, the viscosity of the preparation is more than or equal to 1000 determined by a viscometer, and the preparation has no granular feeling, insoluble substances and layering.
The heat storage stability determination method and the qualification standard are as follows: taking a proper amount of sample according to 'liquid preparation' in GB/T19136-; if the decomposition rate is lower than 5%, and the liquid is transparent and uniform, the sample without precipitation and delamination meets the requirement of heat storage stability.
The low-temperature stability determination method and the qualification standard are as follows: filling a proper amount of sample into a bottle, sealing the bottle in a refrigerator at the temperature of-15 ℃, sealing at least 3 parts, storing the bottle for 14 days, taking out the bottle, recovering the bottle to room temperature, and detecting the content of active ingredients after the bottle passes the appearance detection; the product is qualified when the decomposition rate is less than 5 percent and the product is not delaminated.
The centrifugal stability determination method and the qualified standard are as follows: and (3) putting 0.5g of paste into a centrifuge tube, centrifuging at 6000rpm/min for 30min, and determining the paste to be qualified if no layering exists and no water is analyzed out after the centrifugation is finished.
The method for measuring the bacteria-growing time and the qualified standard are as follows: 10g of the paste is taken in a 100mL beaker, the beaker is not sealed and is placed in a room temperature environment for culture, and no colony formation is observed to be qualified after 10 days.
The water resistance measuring method and the qualified standard are as follows: uniformly smearing 1.5g of paste on kiwi fruit branches with the length of 14cm and the diameter of about 1cm, and soaking in water for 48h to ensure that the kiwi fruit branches do not fall off.
The film forming time measuring method and the qualified standard are as follows: uniformly smearing 1.5g of paste on kiwi fruit branches with the length of 14cm and the diameter of about 1cm, observing every 15min, and recording the paste film forming time, wherein the film forming time is lower than 3h, and the kiwi fruit branches are qualified.
The tree body adhesiveness measuring method and the qualified standard are as follows: uniformly smearing 1.5g of paste on kiwi fruit branches with the length of 14cm and the diameter of about 1cm, placing the branches in a natural environment after film forming, observing whether the medicine film falls off every 5 days, and judging that the medicine film is qualified after 30 days of no fall off.
Example 5: method for detecting content of effective components of 0.08% Xcn1 ointment
This example provides a method for determining the content of active ingredient in 0.08% Xcn1 ointment by detecting Xcn1 content using high performance liquid chromatography. The sample pretreatment steps are as follows: adding 5mL of Xcn1 paste into a 250mL beaker, adding 50mL of water, heating and stirring at 40 ℃ for 20min, adding 100mL of methanol, stirring uniformly, performing ultrasonic treatment for 60min, adding 0.5g of aluminum sulfate octadecahydrate, stirring for 5min, standing for 2h, centrifuging at 10000rpm/min for 20min, washing the centrifugal precipitate with methanol for three times, centrifuging, taking supernatant, distilling at 43 ℃ under reduced pressure, diluting water to 10mL, taking a sample with constant volume, and passing the sample through a 0.22 mu m filter membrane.
In the high performance liquid chromatography detection method, the mobile phase is a mixture containing 0.1% trifluoroacetic acid in a volume ratio of 3: 7, the detection wavelength is 312nm, the sample injection amount is 20 mu L, the peak-out time is about 10min, and the chromatographic column is an Agilent C18 column. Xcn1 standard samples were accurately weighed on an analytical balance to prepare a mother liquor having a concentration of 2000. mu.g/mL. The resulting solution was diluted with ultrapure water to 1000. mu.g/mL, 800. mu.g/mL, 600. mu.g/mL, 400. mu.g/mL, 200. mu.g/mL, or 100. mu.g/mL of a standard solution. And performing liquid phase detection analysis according to the detection conditions, drawing a standard curve with Xcn1 concentration as an abscissa and an absorption peak area as an ordinate, and calculating a linear regression equation and a correlation coefficient. And substituting the peak area of the detection sample into a regression equation to calculate the Xcn1 concentration in the paste sample to be detected. The specific map is shown in FIG. 2.
Example 6: xcn1 in vitro branch test for preventing and treating apple rot by paste
The embodiment shows the control effect of the Xcn1 ointment on apple rot in an isolated branch test, and the test method comprises the following steps: cutting the in vitro apple branch into 15cm small segments, sterilizing both ends, coating wax, and scalding epidermis at a position 4cm away from both ends of the branch with iron nail cap (d is 5 mm). 0.5g of Xcn1 ointment with different concentrations is uniformly coated on the upper and lower 4cm of the scald, sterile water is coated as blank control, and 45% of dyssenamine suspension is coated as medicament control. Inoculating the apple rot pathogen fungus cake (d is 5mm) to the scald position after 1 day, repeating the 3 branches, carrying out constant-temperature moisture-preserving culture at 25 ℃ in the dark for 15 days, and calculating the control effect according to the formula (1) and the formula (2).
Area of lesion (mm)2) Pi × long diameter × short diameter ÷ 4 equation (1);
TABLE 1 Xcn1 preventive and therapeutic effects of the paste on apple rot
Note: analyzing the 5% difference significance level by adopting a Duncan's new repolarization method, wherein different letters represent differences, and the same letter represents no significant difference; the following table is the same.
The results show that: the control effect of 400mg/Lxcn1 paste (0.04% Xcn1 paste) on apple canker was not significantly different from that of 45% dyson suspension (Table 1). It is demonstrated that Xcn1 ointment has better protective effect on apple canker.
Example 7: xcn1 in vitro branch test for preventing and treating apple ring rot by paste
The embodiment shows the control effect of the Xcn1 ointment on apple ring rot in an isolated branch test, and the test method comprises the following steps: cutting the detached apple branch into 15cm small segments, sterilizing both ends, wrapping with wax, and perforating at a position 4cm away from both ends of the branch with a perforator (d is 5 mm). Protection: 0.5g of Xcn1 ointment with different concentrations is evenly coated on the upper and lower parts of the wound for 4cm, sterile water is coated to be used as a blank contrast, and 50% carbendazim wettable powder is coated to be used as a medicament contrast. Inoculating apple ring rot germ cake (d is 5mm) to the scald part after the ointment is formed into a film for 1 day, repeating for 3 branches, carrying out constant-temperature moisture-preserving culture at 25 ℃ in the dark for 15 days, and calculating the control effect according to the formula (1) and the formula (2). The treatment effect is as follows: inoculating apple ring rot, culturing for 3 days, applying the preparation according to the same protection method, culturing at 25 deg.C in dark for 15 days, and calculating the control effect according to formulas (1) and (2).
TABLE 2Xcn1 preventive and therapeutic effects of the ointment on apple ring spot
The results show that: the Xcn1 ointment (0.04% Xcn1 ointment) with the concentration of 400mg/L has no obvious difference on the treatment effect of the apple ring spot and the treatment effect of 50% carbendazim wettable powder; the control effect of Xcn1 ointment (0.08% Xcn1 ointment) at 800mg/L on the apple ring rot protection effect is not obviously different from the control effect of 50% carbendazim wettable powder (Table 2).
Example 8: xcn1 body and branch test for preventing and treating kiwifruit canker by paste
The embodiment shows the control effect of the Xcn1 ointment on kiwifruit canker in an in vitro branch test, and the test method comprises the following steps: selecting disease-free old and thick branches, cutting the old and thick branches into 15cm sections, washing the sections of the kiwi fruit branches with tap water, drying in the air, soaking and sterilizing the sections with sodium hypochlorite solution, immediately washing the sections with sterile water, drying in the air, and sealing wax at the cut positions. Cutting a small opening (only cutting to xylem) in the middle of the branch, sucking 10 mu L of bacterial liquid of the kiwifruit canker pathogen at the cut by using a liquid transfer gun, smearing a test reagent after three days, carrying out constant-temperature moisture-preserving culture at 16 ℃ in a dark place for 15 days, and calculating the control effect according to the formulas (1) and (2).
TABLE 3 Xcn1 preventive and therapeutic effects of the unguentum on kiwifruit canker
The results show that: the ointments with different contents of Xcn1 have certain control effects on kiwifruit canker, wherein the 1500mg/LXCn1 ointment (0.15% Xcn1 ointment) has the best control effect, and the control effects of Xcn1 ointment (0.06% Xcn1 ointment) of 600mg/L and 3% zhongshengmycin wettable powder of 200mg/L are not obviously different (Table 3).
Example 9: xcn1 the paste can be used for preventing and treating kiwifruit canker by field drug effect test (Yangling)
This example demonstrates the control of kiwifruit canker by Xcn1 ointment in a field efficacy test.
Test site: in a kiwi fruit garden of Yangyang city, Yangling area, Texi village, Shaanxi province, the variety is Haiword, the tree age is 8 years, the hedge frame is shaped horizontally with double walls and double layers, the garden contains 220 trees, no special medicament for canker is applied, the management level is poor, and the canker is serious.
The test method comprises the following steps: the application of the pesticide is carried out in two stages, and in the first stage, the pesticide is applied once 7 days after fruit picking, and the application is carried out for 5 times in total. The second phase is carried out in 2 months in spring, and the medicine is applied once at intervals of 7 days for 5 times. The method of scraping the scab firstly and then coating the medicine is adopted, firstly, the ulcer tissues at the scab are completely scraped until white healthy phloem is exposed, and then the prepared Xcn1 ointment is uniformly applied to the wound by a brush according to the dosage. The wettable powder of 3 percent of zhongshengmycin is used as a control medicament, and clear water control is set, and the number of the disease spots treated is 30.
The investigation method comprises the following steps: marking the number of the disease spots, recording the diameters of the disease spots and the girth of the tree stems before the application of the medicine, investigating the number of recurrent disease spots and the diameters of the disease spots after applying the medicine for 30 days, and calculating the prevention and treatment effect according to a formula (3) and a formula (4). Disease grading criteria are shown in table 4.
TABLE 4 grading Standard of kiwifruit canker
TABLE 5 Xcn1 field control of kiwifruit canker by the ointments
The results show that: xcn1 the ointment (0.15% Xcn1 ointment) with the concentration of 1500mg/L has the best control effect on kiwifruit canker, and has no significant difference with the control effect of the wettable powder of zhongshengmycin with the concentration of 200 mg/L3% (Table 5).
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.
Claims (10)
1. A microorganism-derived bactericidal ointment containing Xcn1 is characterized in that after sugar and protein removal and concentration are carried out on fermentation liquor of xenorhabdus nematophila (Xenorhabdus snenematophila) which can generate Xcn1, a film-forming agent, a humectant, a thickener, a penetration enhancer and water are added to prepare the ointment.
2. The Xcn 1-containing microbial source sterilization cream according to claim 1, wherein the cream comprises the following raw materials by mass:
0.04-0.15 percent of Xcn1 concentrated fermentation liquor, 5.32 percent of film forming material, 2.52 percent of thickening agent, 1.96 percent of humectant, 2.46 percent of penetration enhancer and 100 percent of water supplement.
3. The Xcn 1-containing microbial source fungicide paste according to claim 1 or 2, wherein said fermentation broth is a xenorhabdus nematophila YL001(x.nematophila) fermentation broth, specifically prepared by:
placing an X.nematophila YL001 strain in an LB culture medium, activating for 24 hours at 180rpm/min and 28 ℃ to prepare a seed solution, adding the seed solution into a PP3 culture medium according to the proportion of 10%, fermenting for 48 hours at 150rpm/min and 28 ℃, centrifuging the fermentation liquor for 20 minutes at 4 ℃ and 10000rpm/min, collecting the supernatant, adding 5.5g/L of aluminum sulfate octadecahydrate, rapidly stirring for 1min, standing for 6 hours, centrifuging, taking the supernatant, carrying out reduced pressure distillation and concentration to 1/4 times of the original volume, adding 80% ethanol, stirring for 10 minutes, refrigerating overnight at 4 ℃, centrifuging, taking the supernatant, carrying out reduced pressure distillation and concentration to 240 times, and obtaining the concentrated fermentation liquor containing 3.0% Xcn 1.
4. The Xcn 1-containing microbicidal paste according to claim 1 or 2, wherein the film forming agent is selected from one or a mixture of two or more of sodium carboxymethylcellulose, polyvinyl alcohol, polyethylene glycol, organic bentonite, diatomaceous earth, sodium alginate, liquid paraffin, gelatin, xanthan gum, polyvinylpyrrolidone and soluble starch.
5. The Xcn 1-containing microbicidal ointment of claim 1 or 2, wherein the humectant is one or a mixture of two or more selected from the group consisting of ethylene glycol, glycerol and vaseline.
6. The Xcn 1-containing microbicidal ointment of claim 1 or 2, wherein the thickener is one or a mixture of two or more selected from the group consisting of sodium carboxymethylcellulose, polyvinylpyrrolidone and sodium alginate.
7. The Xcn 1-containing microbicidal ointment of claim 1 or 2, wherein the penetration enhancer is selected from one or a mixture of two or more of azone, polyoxyethylene lauryl ether and polyoxyethylene sec-octanol.
8. A preparation method of Xcn 1-containing microbial source sterilization ointment, which is characterized in that Xcn 1-containing microbial source sterilization ointment is Xcn 1-containing microbial source sterilization ointment as claimed in any one of claims 1 to 7;
the preparation method comprises the following steps:
adding polyvinyl alcohol into water, heating and dissolving, sieving by a 40-mesh sieve to remove insoluble substances, cooling to 65 ℃, adding sodium carboxymethylcellulose, fully stirring and dissolving, adding azone and glycerol into the mixture, uniformly mixing, cooling to 45 ℃, adding Xcn1 concentrated fermentation liquor, supplementing water to 100%, stirring at the stirring speed of 600-800 rpm for 20-30 minutes, and thus obtaining the microbial source sterilization paste containing 0.04-0.15% of Xcn 1.
9. The method for preparing a Xcn 1-containing microbicidal paste according to claim 8, wherein the preparation of Xcn 1-containing concentrated fermentation broth comprises:
placing an X.nematophila YL001 strain in an LB culture medium, activating for 24 hours at 180rpm/min and 28 ℃ to prepare a seed solution, adding the seed solution into a PP3 culture medium according to the proportion of 10%, fermenting for 48 hours at 150rpm/min and 28 ℃, centrifuging the fermentation liquor for 20 minutes at 4 ℃ and 10000rpm/min, collecting the supernatant, adding 5.5g/L of aluminum sulfate octadecahydrate, rapidly stirring for 1 minute, standing for 6 hours, centrifuging, taking the supernatant, carrying out reduced pressure distillation and concentration to 1/4 times of the original volume, adding 80% ethanol, stirring for 10 minutes, refrigerating overnight at 4 ℃, centrifuging, taking the supernatant, carrying out reduced pressure distillation and concentration to 240 times, and obtaining the concentrated fermentation liquor containing Xcn 1.
10. Use of the Xcn 1-containing microbiologically bactericidal paste as claimed in any one of claims 1 to 7 for the prevention and treatment of apple rot (cytoplasma ananharica), apple ring spot (physiosporicola) and/or kiwifruit canker (Pseudomonas syringaepv. actidia);
the application method comprises the following steps:
the liniment is directly applied to the cut or the scab of the trimmed branch, and the dosage per square centimeter is 0.1-0.2 g.
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| CN202111483446.0A CN114304146A (en) | 2021-12-07 | 2021-12-07 | Xcn 1-containing microbial source sterilization ointment, and preparation method and application thereof |
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| CN116210695A (en) * | 2023-02-27 | 2023-06-06 | 西北农林科技大学 | Compound for promoting plant wound healing and application thereof |
| CN117946948A (en) * | 2024-03-22 | 2024-04-30 | 四川省自然资源科学研究院(四川省生产力促进中心) | Pseudomonas strain SCMHT-110 and application thereof |
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