CN110627881B - Preparation and application of hypersensitive protein for biological prevention and treatment of tobacco mosaic virus disease - Google Patents
Preparation and application of hypersensitive protein for biological prevention and treatment of tobacco mosaic virus disease Download PDFInfo
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Abstract
The invention relates to preparation and application of hypersensitive protein for biological control of tobacco mosaic virus, bacillus subtilis is taken as a production strain, a hypersensitive protein finished product is prepared by warm water dissolution, centrifugation, membrane separation and spray drying treatment of bacillus subtilis fermentation powder, two continuous gradient density centrifuges are adopted, and thalli and foreign protein are removed to the maximum extent through fractional ultrafiltration membrane separation, so that hypersensitive protein concentrated solution with higher activity is obtained, the heat resistance of the hypersensitive protein is utilized, and the hypersensitive protein is quickly dried through a low-temperature spray drying technology, so that the preparation efficiency of the hypersensitive protein is improved. The hypersensitive protein prepared by the invention has obvious control effect on tobacco mosaic virus, and the control effect can reach 82.66%; the invention has the advantages of high activity of the separated and purified hypersensitive protein, low cost, simple process and lower post-treatment loss.
Description
Technical Field
The invention relates to the technical field of biological extraction processes, in particular to preparation and application of a hypersensitive protein for biological control of tobacco mosaic virus diseases.
Background
The tobacco mosaic virus is a single-stranded RNA virus, has strong infectivity and pathogenicity to plants, has wide host range, can infect more than 350 plants, the crops of solanaceae, cruciferae and compositae are the most seriously damaged, and the serious loss and even field damage are often caused after the crops and plants are infected with diseases. Tobacco is an important economic crop, is very easy to infect TMV, can be infected by tobacco mosaic virus when being operated by various farming, particularly has large spread in the current industrialized centralized seedling culture, has small twisted leaves, generates various necrotic spots, is dwarfed and dead, and loses the utilization value of tobacco leaves. Tobacco virus diseases are therefore the most important plant diseases in tobacco production and other susceptible crops.
Prevention and control of mosaic disease, which is difficult to control and has poor chemical control effect and large residue, has been a major problem in the production of tobacco and other solanaceous plants. At present, a great deal of research is carried out at home and abroad on the prevention and treatment of the tobacco mosaic disease, but the effect of various pesticides for preventing and treating the tobacco mosaic disease popularized in the market at present is not ideal. Moreover, the common chemical pesticide has low effect and high toxicity and causes serious problems of environmental pollution and the like, and the problems of residue of a large amount of pesticides, environmental pollution, improvement of drug resistance of viruses and the like are easily caused by long-term continuous high-dose application of single antiviral chemical pesticide.
Disclosure of Invention
The invention aims to solve the problems of low effect, high toxicity and environmental pollution of common chemical pesticides in the prior art, and provides preparation and application of a hypersensitive protein for biological control of tobacco mosaic virus diseases.
In order to achieve the purpose, the invention adopts the following technical scheme: the preparation method of the hypersensitive protein for biological prevention and treatment of the tobacco mosaic virus disease comprises the steps of warm water dissolution, density gradient centrifugation, fractional ultrafiltration membrane separation, pH value adjustment and low-temperature spray drying which are sequentially carried out.
In the preparation method of the hypersensitive protein for biological control of tobacco mosaic virus diseases, and the warm water dissolving step comprises the following steps of mixing bacillus subtilis fermentation powder with water 1:100 portions are added into warm water with the temperature of 36 ℃.
In the preparation method of the hypersensitive protein for biological control of the tobacco mosaic virus, the supernatant after centrifugation is subjected to graded filtration in the step of separating by the graded ultrafiltration membrane, the filtration temperature is 36 ℃, the pressure drop of an inlet and an outlet of the ceramic membrane equipment is 0.2-0.25 MPa, and the average flux is 0.3m 3 And h, the bacteria and foreign protein removal rate reaches 98.99-99.99%, and the bacteriostatic titer of the hypersensitive protein in the supernatant after membrane separation is U =12000 unit/mg.
In the above preparation method of the hypersensitive protein for biological control of tobacco mosaic virus disease, the fractionating filtration comprises three-stage filtration; the first stage of filtration adopts 35 ten thousand molecular weight ceramic membranes, the second stage of filtration adopts 15 ten thousand molecular weight ceramic membranes, and the third stage of filtration adopts 5 ten thousand molecular weight ceramic membranes.
In the above method for preparing the hypersensitive protein for biological control of tobacco mosaic virus disease, the specific adjusting method in the step of adjusting pH value is as follows: collecting the supernatant separated by the fractional ultrafiltration membrane, adjusting the pH value to 4.5, standing for 36h, filtering the supernatant to obtain a concentrated solution containing precipitate, filtering repeatedly until the volume of the concentrated solution is 40% of the volume of the supernatant before adjusting the pH value; the regulator for regulating the pH value is hydrochloric acid.
In the preparation method of the hypersensitive protein for biological control of tobacco mosaic virus diseases, 30% of malt flour is added into the concentrated solution as an auxiliary agent, the mixture is uniformly stirred, and low-temperature spray drying is carried out according to the treatment capacity of 40L/h to obtain the hypersensitive protein dry powder.
In the preparation method of the hypersensitive protein for biological control of tobacco mosaic virus, the air inlet temperature of the low-temperature spray drying is 30-45 ℃, and the air outlet temperature is 30-40 ℃.
In the preparation method of the hypersensitive protein for biological control of tobacco mosaic virus diseases, the adopted bacillus subtilis fermentation product is solid fermentation powder, the bacillus subtilis is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.18089.
In the application of the hypersensitive protein prepared by the preparation method of the hypersensitive protein for biological control of tobacco mosaic virus diseases, the application mode of the hypersensitive protein is leaf surface spraying, spraying is carried out at the tobacco seedling stage at the optimal spraying time, water is used for diluting 300 times, and the using amount is 1 g/square meter.
Compared with the prior art, the invention has the advantages that:
1. the invention centrifugates the warm water extracting solution through two continuous density gradient centrifuges, and then separates the warm water extracting solution through a graded ultrafiltration membrane, so as to remove thalli and foreign proteins to the maximum extent and obtain the hypersensitive protein with higher activity;
2. when the hypersensitive protein is processed into dry powder, the heat resistance of the hypersensitive protein is utilized, the hypersensitive protein is quickly dried by a low-temperature spray drying technology, the preparation efficiency of the hypersensitive protein is improved, and the malt flour serving as an auxiliary material has the advantages of good solubility, high stability, rich trace elements and mineral substances and the like;
3. the invention is a green environment-friendly pollution-free biocontrol microbial inoculum obtained by extracting bacillus subtilis fermentation powder, is a green resistance protein, can improve the antiviral property of plants, and is harmless to human and livestock and free of environmental pollution;
4. the invention has high-efficiency pertinence to tobacco mosaic virus, belongs to a green prevention and control technology of plant diseases, and has the advantages of high activity of separated and purified hypersensitive protein, low cost, simple process and lower post-treatment loss.
Detailed Description
The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Examples
The extraction process of the hypersensitive protein comprises the steps of warm water dissolution, density gradient centrifugation, fractional ultrafiltration membrane separation, pH value adjustment and low-temperature spray drying which are sequentially carried out.
A warm water dissolving step, namely mixing the bacillus subtilis fermentation powder with water 1:100 percent of the mixture is added into warm water with the temperature of 36 ℃; by adopting the technical scheme, the active ingredients of the protein keep higher activity, and the bacteriostatic titer of the hypersensitive protein is ensured.
In the step of separating by a classification ultrafiltration membrane, the centrifuged supernatant is subjected to classification filtration at the filtration temperature of 36 ℃, the pressure drop of the inlet and the outlet of the ceramic membrane equipment is 0.2-0.25 MPa, and the average flux is 0.3m 3 The removal rate of thalli and impure protein reaches 98.99 to 99.99 percent, the bacteriostatic titer of the hypersensitive protein in the supernatant after membrane separation is U =12000 unit/mg; by adopting the technical scheme, the effect of removing thalli and foreign proteins is good, and the activity of the hypersensitive protein is effectively ensured; the classified filtration comprises three-stage filtration; the first stage of filtration adopts 35 ten thousand molecular weight ceramic membranes, the second stage of filtration adopts 15 ten thousand molecular weight ceramic membranes, and the third stage of filtration adopts 5 ten thousand molecular weight ceramic membranes; by adopting the technical scheme, the thalli and the foreign protein in the supernatant can be more effectively removed by utilizing the graded filtration.
The specific adjusting method in the step of adjusting the pH value is as follows: collecting the clear liquid separated by the graded ultrafiltration membrane, adjusting the pH value to 4.5, standing for 36h, filtering the supernatant to obtain a concentrated solution containing the precipitate, and repeating the filtering operation until the volume of the concentrated solution is 40% of that of the supernatant before the pH value is adjusted; by adopting the technical scheme, the content of the effective components in the concentrated solution is ensured, the subsequent processes are facilitated, and a good foundation is laid for quickly and efficiently obtaining the hypersensitive protein dry powder; the regulator for regulating the pH value is hydrochloric acid; by adopting the technical scheme, the pH value adjusting effect is good, and the activity of the hypersensitivity protein is not lost.
Adding 30% of malt flour as an auxiliary agent into the concentrated solution, uniformly stirring, and performing low-temperature spray drying according to the treatment capacity of 40L/h to obtain hypersensitive protein dry powder; the air inlet temperature of the low-temperature spray drying is 30-45 ℃, and the air outlet temperature is 30-40 ℃; by adopting the technical scheme, the bacteriostatic titer of the hypersensitive protein can be effectively ensured, and the hypersensitive protein dry powder can be conveniently and rapidly dried, the drying effect is good, and the activity of the hypersensitive protein is not lost.
The Bacillus subtilis fermentation product is solid fermentation powder, and the Bacillus subtilis is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.18089; the application mode of the hypersensitive protein is leaf surface spraying, the spraying is carried out in the tobacco seedling stage at the optimal spraying time, the tobacco seedling stage is diluted by water by 300 times, and the using amount is 1 g/square meter.
The preparation method comprises the following specific steps: dissolving in warm water, and mixing the bacillus subtilis fermentation powder according to the weight ratio of 1:100 portions of the mixture are added into warm water with the water temperature of 36 ℃.
And step two, centrifuging, namely centrifuging the fermentation liquor obtained in the step one by a continuous density gradient centrifuge to remove precipitates, reserving supernatant, and centrifuging twice continuously, so that a large amount of precipitates in the fermentation liquor can be effectively removed, and the subsequent processes can be favorably carried out. The rotation speed of the centrifuge is 14000rpm, and the treatment capacity of the centrifuge is 2m 3 And h, connecting the centrifuged supernatant to an intermediate storage tank through a centrifuge liquid outlet pipe, performing a membrane separation treatment procedure, scraping off solid impurities in the rotary drum, sterilizing and the like, and then discarding.
And step three-stage ultrafiltration membrane separation, wherein the supernatant is subjected to stage filtration by 5-35 million molecular weight ceramic membranes, the first stage filtration adopts 35 million molecular weight ceramic membranes, macromolecular substances with molecular weights more than 35 million are separated, the second stage filtration adopts 15 million molecular weight ceramic membranes to filter the first stage filtrate again, the third stage filtration adopts 5 million molecular weight ceramic membranes to filter the second stage filtrate finally. The filtering temperature is 36 ℃, the pressure drop of the inlet and the outlet of the ceramic membrane equipment is 0.2-0.25 MPa, and the average flux is 0.3m 3 And h, enabling the supernatant to flow along the surface of the ceramic membrane, carrying out cross flow filtration on the supernatant by the ceramic membrane, enabling the supernatant to flow in the membrane tube under the driving of pressure, enabling the small molecular substances to permeate the ceramic membrane, and enabling the liquid containing the large molecular components to be intercepted by the ceramic membrane, so that thalli and foreign proteins of the supernatant are removed, wherein the removal rate reaches 98.99-99.99%, and the bacteriostatic titer of the hypersensitive protein in the supernatant after membrane separation is U =12000 units/mg.
Regulating the pH value, extracting the liquid obtained in the step three, adding hydrochloric acid into the liquid to regulate the pH value to 4.5, dividing the extracting solution into a plurality of parts before formal regulation to ensure that the volume of each part is relatively small so as to avoid inaccurate measured value caused by the mixing time process in the regulation process, simultaneously, dropwise adding a small amount of hydrochloric acid for multiple times for regulation, fully stirring the solution after dropwise adding hydrochloric acid to ensure the stability of the overall pH value, and then measuring the accuracy value by a PH meter, wherein the PH meter is used according to the following principle, and a standard buffer solution is required to be used for correction before each use; when the buffer is continuously used, the calibration is carried out once a day, two standard buffers with pH values different by about 3 pH units are selected, and the pH value of the test solution is between the two standard buffers; the measuring electrode needs to be cleaned before and after use, the electrode tip and the electrode tip are protected by weak attention, and the measuring electrode is wiped by using softer paper before and after use; before the electrode bulb is used, whether bubbles exist at the front end of the electrode bulb or not is checked, and if bubbles exist, the electrode bulb is thrown by applying force; after the pH composite electrode is inserted into the solution to be detected, the pH composite electrode is stirred and shaken for a few times and then is placed statically, so that the response of the electrode is accelerated; mixing the multiple extractive solutions when the pH values of the multiple extractive solutions are 4.5, stirring thoroughly, measuring the pH value of the whole extractive solution again until the whole pH value reaches 4.5, standing for 36 hr, separating with membrane again to remove precipitate to obtain concentrated solution, and repeating the above steps until the volume of the concentrated solution is 40% of the original volume.
And fifthly, carrying out low-temperature spray drying, adding 30% malt flour as an auxiliary agent into the concentrated solution, uniformly stirring, and carrying out spray drying treatment according to the treatment capacity of 40L/h, wherein the air inlet temperature is 30-45 ℃, the air outlet temperature is 30-40 ℃ to obtain the hypersensitive protein dry powder.
The hypersensitive protein is extracted from bacillus subtilis fermentation powder, and the preparation of the bacillus subtilis fermentation powder comprises the following steps:
step one, strain solid slant culture: inoculating a bacillus subtilis SL-19 strain into a solid inclined plane, and culturing in an inclined plane culture medium at the temperature of 28 ℃ for 36 hours;
step two, seed liquid amplification culture: inoculating the bacillus subtilis in the first step into a seed liquid culture medium containing 1000ml of seed liquid, and performing shake culture for 36 hours at the culture temperature of 29 ℃ and at the rotating speed of 280 revolutions per minute;
step three, fermentation culture: inoculating the seed solution obtained in the second step into a large fermentation tank of 2000 liters according to the inoculation amount of 8%, and under the culture condition of changing temperature and ventilation volume: culturing for 48 hours at 29 ℃ and with the ventilation capacity of 0.4 cubic meter/minute, then heating to 39 ℃, increasing the ventilation capacity to 1.5 cubic meters/minute, then culturing for 72 hours, and culturing with the rotation speed of 280 r/min, wherein the fermentation can be stopped when the number of live spores of the bacillus subtilis SL-19 in the zymophyte liquid is more than or equal to 500 hundred million/ml, so as to prepare the bacillus subtilis liquid microbial inoculum.
Step four, preparing solid bacterial powder: mixing the fermentation liquid microbial inoculum prepared in the step three with a solid carrier, and then carrying out spray drying treatment to prepare bacillus subtilis solid bacterial powder
Wherein the slant culture medium in the first step comprises the following components in percentage by weight: peptone 1.5%, yeast extract 1.0%, sodium chloride 0.8%, agar 3.0%, and water in balance, and natural pH value.
Wherein the seed liquid culture medium in the second step comprises the following components in percentage by weight: peptone 1.8%, yeast extract 2.0%, sodium chloride 1.0%, and water in balance, and natural pH value.
The fermentation medium adopted in the fourth step comprises the following components in percentage by weight: 2.5 percent of yeast powder, 1.5 percent of corn flour, 0.55 percent of calcium carbonate, 0.68 percent of monopotassium phosphate, 0.58 percent of ammonium sulfate, 0.035 percent of defoaming agent and the balance of water, wherein the PH value is 6.8-7.3.
The solid carrier of the bacillus subtilis powder is kaolin and diatomite.
Spray drying treatment of a bacillus subtilis microbial inoculum, wherein the treatment conditions are as follows: the air inlet temperature of the spray drying is 40 ℃, and the air outlet temperature is 30 ℃.
The field control test of the invention comprises the following steps:
the field control test was carried out in the tobacco field of the south of the Luo. The test cells were in the south-loving area.
And (3) test treatment: carrying out leaf surface spraying treatment when the tobacco grows to the seedling leaf stage, wherein the treatment I comprises the following steps: mixing the hypersensitive protein dry powder prepared in the example 1 with water (1; and (5) processing: spraying a chemical preparation for preventing and treating tobacco mosaic virus on the market in an equivalent amount according to the instruction; and (3) treatment III: spraying with equal amount of clear water.
The test method comprises the following steps: spraying treatment is carried out on tobacco at the seedling leaf stage, spraying is carried out 3 times totally, investigation is carried out 5 days after spraying, the number of diseased plants is recorded, and the test result is shown in the table I.
Table one: control of tobacco mosaic virus under three treatments
Note: the tobacco virus disease severity grading standard (national standard GB/T23222-2008) is graded and investigated by taking strains as units: level 0: the whole plant is disease-free; level 1: the heart and leaves have bright or mild veins, and diseased plants are not obviously dwarfed; and 3, stage: one third leaf flower and leaf without deformation or dwarfing of diseased plant to more than three quarters of normal plant height; and 5, stage: one third to one half leaf, or a few leaves deformed, or the main vein blackened, or the diseased plant dwarfed to two thirds to three quarters of the normal plant height; and 7, stage: one half to two thirds of leaf mosaic, or deformation or primary vein necrosis, or dwarfing of diseased plants to one half to two thirds of normal plant height; and 9, stage: the leaves of the whole plant are severely deformed or necrosed, or the diseased plant is shortened to more than one half of the normal plant height.
Control effect (%) = (number of diseased plant in blank control-number of diseased plant in treatment group) ÷ number of diseased plant in blank control × 100%.
The first table shows that the hypersensitive protein prepared by the invention has obvious control effect on tobacco mosaic virus, the control effect can reach 82.66 percent, and the hypersensitive protein is more effective than a chemical control agent 52.76 percent sold in the market, and chemical residue can not be generated and pollution can not be caused to the environment.
The above: it should be understood that the present invention is not limited to the above-described embodiments, but the technical solutions and the inventive concepts of the present invention may be equally replaced or modified by other embodiments within the technical scope of the present invention.
Claims (1)
1. The preparation method of the hypersensitive protein for biological prevention and treatment of the tobacco mosaic virus disease is characterized in that the extraction process of the hypersensitive protein comprises the steps of warm water dissolution, density gradient centrifugation, fractional ultrafiltration membrane separation, pH value adjustment and low-temperature spray drying which are sequentially carried out;
and the warm water dissolving step comprises the following steps of mixing bacillus subtilis fermentation powder with water 1:100 percent of the mixture is added into warm water with the temperature of 36 ℃;
in the step of separating by using a graded ultrafiltration membrane, the centrifuged supernatant is subjected to graded filtration, the filtration temperature is 36 ℃, the pressure drop of an inlet and an outlet of a ceramic membrane device is 0.2-0.25 MPa, the average flux is 0.3m < 3 >/h, the ratio of removing thalli and impurity protein reaches 98.99-99.99%, and the bacteriostatic titer of the hypersensitive protein in the supernatant after membrane separation is U =12000 units/mg;
the stage filtration comprises three stages of filtration; the first stage of filtration adopts 35 ten thousand molecular weight ceramic membranes, the second stage of filtration adopts 15 ten thousand molecular weight ceramic membranes, and the third stage of filtration adopts 5 ten thousand molecular weight ceramic membranes;
the specific adjusting method in the step of adjusting the pH value is as follows: collecting the clear liquid separated by the graded ultrafiltration membrane, adjusting the pH value to 4.5, standing for 36h, filtering the supernatant to obtain a concentrated solution containing the precipitate, and repeating the filtering operation until the volume of the concentrated solution is 40% of that of the supernatant before the pH value is adjusted; the regulator for regulating the pH value is hydrochloric acid;
adding 30% of malt flour as an auxiliary agent into the concentrated solution, uniformly stirring, and performing low-temperature spray drying according to the treatment capacity of 40L/h to obtain hypersensitive protein dry powder;
the air inlet temperature of the low-temperature spray drying is 30-45 ℃, and the air outlet temperature is 30-40 ℃;
the bacillus subtilis fermentation product is solid fermentation powder, and the bacillus subtilis is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.18089;
the application mode of the hypersensitive protein is leaf surface spraying, the spraying is carried out in the tobacco seedling stage at the optimal spraying time, the tobacco seedling stage is diluted by water by 300 times, and the using amount is 1 g/square meter.
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| CN111771911B (en) * | 2020-06-30 | 2021-06-04 | 武汉合缘绿色生物股份有限公司 | Microbial preparation for preventing and treating tobacco mosaic virus and preparation method thereof |
| CN112359084B (en) * | 2020-12-02 | 2023-06-16 | 江西顺泉生物科技有限公司 | Preparation, prevention and control application of mycoprotein in bacillus subtilis fermentation product |
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