CN108902182B - Microecological preparation with function of reducing plant transpiration and preparation method thereof - Google Patents
Microecological preparation with function of reducing plant transpiration and preparation method thereof Download PDFInfo
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- CN108902182B CN108902182B CN201811134837.XA CN201811134837A CN108902182B CN 108902182 B CN108902182 B CN 108902182B CN 201811134837 A CN201811134837 A CN 201811134837A CN 108902182 B CN108902182 B CN 108902182B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
- A01N59/08—Alkali metal chlorides; Alkaline earth metal chlorides
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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Abstract
The invention provides a preparation method of a microecological preparation with functions of regulating the open and close of plant leaf stomata and reducing plant transpiration, wherein the microecological preparation is prepared from residues of bifidobacterium bacteria after crushing, cell dissolving and centrifugation. The invention takes the thallus fragments of the bifidobacterium as the main component of the anti-transpiration preparation for the first time, which can cause the stomata of the plant leaves to be quickly closed, thereby reducing the plant transpiration. Compared with the existing anti-transpiration preparation, the micro-ecological preparation has the advantages of environmental friendliness, low cost and obvious effect.
Description
Technical Field
The invention relates to a microecological preparation with a function of reducing plant transpiration and a preparation method thereof.
Background
When the plants are transplanted, the water absorption capacity of the plants is reduced due to the damage of root systems, and the plants can be recovered in a certain time. In the process, the crown is built, but the transplanting fails due to the fact that the tree crown is transpired too fast with a certain probability. Therefore, the antitranspirant is a relatively common protection means. Meanwhile, in China, water resources are distributed unevenly in space and time, so that crops are seriously lack of water in a certain season, and the use of the antitranspirant is also an important water-saving means. The main principles of antitranspirant are two types: (1) one is to adjust the air holes, partially close or reduce the opening of the air holes to realize the reduction of transpiration; (2) the film forming agent shields pores, thereby reducing transpiration.
Stomata is one of many small openings in the epithelium of leaves, stems, and other plant organs, and is a structure characteristic of the epidermis of plants. Stomata are usually found in the aerial parts of plants, especially on the leaf epidermis, but also on the young stems and petals, but most submerged plants do not. The small convex lens-like pores formed between guard cells are often referred to as pores in a narrow sense. The guard cells are structurally different from epidermal cells, contain chloroplasts, are small in size, small in number and poor in lamellar structure development, and can synthesize carbohydrates through photosynthesis. Sometimes with 2-4 paralogs adjacent to the guard cells. Inclusion of these cells is a generalized stoma (or stomatal organ). There is a wide cell gap (air space) immediately below the air hole. The stomata serve as passages for air and water vapor in gas metabolism such as carbon assimilation, respiration, transpiration, and the like, and the amount of the stomata is regulated by the opening and closing action of guard cells, which is physiologically important.
Stomata are closed under certain biotic or physical stresses, such as water deficit, which results in increased ABA concentration and thus closure of stomata, and sometimes, in response to pest damage, it has been shown that polysaccharides, or lipopolysaccharides (L PS), on the surface of microorganisms stimulate cells to close stomata.
Therefore, the Chinese patent application No. 200710066800.3, 200710066799.4 uses the molecular structure of the microbial surface to give a corresponding signal to the plant cell, so as to cause an infringement artifact, thereby closing the stomata and playing a role in resisting transpiration. Although the cost is reduced, the application range is limited because the use amount is high.
Disclosure of Invention
The first aspect of the present invention provides a microecological preparation having a function of reducing plant transpiration, which consists of a residue of bacteria belonging to the genus bifidobacterium after lysis, centrifugation and removal of a polysaccharide component.
The Bifidobacterium bacteria include one or more of Bifidobacterium adolescentis, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium catenulatum, Bifidobacterium odonta, Bifidobacterium animalis and Bifidobacterium lactis.
The second aspect of the present invention provides a method for preparing the above-mentioned microecological preparation having a function of reducing plant transpiration, comprising the steps of:
(1) placing bacteria of the genus bifidobacterium in a solvent to obtain a mixed solution, cracking the bacteria by a physical method, and centrifuging to obtain a supernatant and a precipitate;
(2) adding the supernatant into an anion exchange column for elution, detecting and collecting components without polysaccharide;
(3) and (3) mixing the polysaccharide-free component obtained in the step (2) with the precipitate obtained in the step (1) to obtain the microecological preparation.
Preferably, the preparation method of the microecological preparation further comprises the following steps:
(4) the powdery microecologics are obtained by drying.
Preferably, the bifidobacterium bacteria comprise any one or more of bifidobacterium adolescentis, bifidobacterium breve, bifidobacterium longum, bifidobacterium bifidum, bifidobacterium infantis, bifidobacterium catenulatum, bifidobacterium odonta, bifidobacterium animalis and bifidobacterium lactis. The above-mentioned bacteria of the genus Bifidobacterium are merely listed, and the inventors have reason to believe that the rest of the non-listed bacteria of the genus Bifidobacterium can also exert similar effects based on the principle of the present probiotic preparation.
The step (1) also comprises any one or more of the following characteristics:
preferably, the weight percentage of the bacteria in the mixed solution in the step (1) is 5% -45%; further, the weight percentage of the bacteria in the mixed solution is 8% -45%;
preferably, the method for cracking in step (1) is selected from any one or more of ultrasonic cracker cracking, tissue disruptor cracking, high pressure homogenizer cracking or chemical method cracking;
preferably, the centrifugation in the step (1) is carried out under the conditions of 8000 g-10000 g/min for 10-60 min;
preferably, the precipitate obtained by centrifugation in step (1) is physically lysed again, the supernatant is taken after centrifugation, and the supernatants after two times of centrifugation are combined.
The cracking method is selected from any one or more of ultrasonic cracking instrument cracking, tissue disruption instrument cracking, high-pressure homogenizer cracking or chemical method cracking.
The solvent may be selected from any one of water and an aqueous solution of sodium phosphate.
Preferably, the step (2) further comprises any one or more of the following characteristics:
preferably, the anion exchange column is conditioned before elution with an equilibration fluid selected from a 10-100 mM Tris-HCl (Tris (hydroxymethyl) aminomethane) solution at pH 6.5-8.5 or a 10-200 mM phosphoric acid solution at pH 6-9;
preferably, the eluent is selected from 10-100 mM Tris-HCl/1M sodium chloride solution and 10-200 mM phosphoric acid solution/1M sodium chloride solution; the pH value is 6.0-8.5;
preferably, the detector used for elution is any one of a specific rotation detector, a parallax refraction detector or an ultraviolet detector.
The polysaccharide-free fraction is detected and collected in step (3), and the total sugar content can be determined by the prior art, for example, by using a phenol-sulfuric acid method.
In general, the skilled person will be able to use the probiotic formulation of the invention alone or in combination with other anti-transpiration agents, such as fulvic acid, CaCl2And the like, so as to achieve a more lasting anti-transpiration effect.
The invention has the beneficial effects that: the microbial fragments left after the active polysaccharide components are extracted from the probiotics are taken as an anti-transpiration preparation for the first time, and a large number of molecular structures of the anti-transpiration preparation can transmit corresponding signals to plant cells to cause the pores to be closed, so that the anti-transpiration effect is achieved. Compared with the existing anti-transpiration preparation, the preparation has the advantages of environmental friendliness and obvious effect due to the adoption of the microbial thallus residue as a raw material; because the raw material is the microbial fragments left after the active polysaccharide component is extracted, the cost is lower.
Drawings
Fig. 1 is a flow chart of an embodiment of the preparation method of the microecological formulation of the present invention.
FIG. 2 is a microscopic view of pores of broad beans after spraying the microecologics onto the broad bean leaves, wherein A is the pores of the broad beans before treatment; b is the air holes of the treated broad beans; c is an arabidopsis stomata before treatment; d is the treated Arabidopsis stomata.
FIG. 3 shows the change of stomatal aperture (A) and transpiration rate (B) in Arabidopsis thaliana and Vicia faba leaves after treatment with the microbial agent.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention. It is to be understood that the processing equipment or apparatus not specifically identified in the following examples is conventional in the art. Furthermore, it is to be understood that one or more method steps mentioned in the present invention does not exclude that other method steps may also be present before or after the combined steps or that other method steps may also be inserted between these explicitly mentioned steps, unless otherwise indicated; it is also to be understood that a combined connection between one or more devices/apparatus as referred to in the present application does not exclude that further devices/apparatus may be present before or after the combined device/apparatus or that further devices/apparatus may be interposed between two devices/apparatus explicitly referred to, unless otherwise indicated. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise stated, the experimental METHODS, detection METHODS, AND preparation METHODS disclosed herein are based on conventional techniques IN the art, such as molecular biology, biochemistry, Chromatin structure AND analysis, analytical chemistry, cell culture, recombinant DNA techniques, AND related fields, which are well described IN the prior art, see, IN particular, Sambrook et al MO L ECU L AR C L ONING: A L ABORATORY MANUA 8652, Second edition, Cold Spring Harbor L organisation Press, 1989 AND Third edition, 2001, Ausubel et al, CURRENT PROTOCO L S INMO L ECU L AR BIO L OGY, John Wiley & Sons, New York, 1987 AND biological orders, the devices will be used, METHODS of DIDIDISY 2 IN L, Actic, Experimental & Sons, Sanwic & Sons, soil incorporated, sample # 2, sample # S, sample # 12, sample # Press, AND P # Press et al, sample # 2, sample # AND P # Press # 2, sample # AND # 2, sample # AND P, sample # 2.
Example 1:
bifidobacterium breve: (A. breve.), (B. breve.), (Bifidobacterium breve) Mixing the dry bacterium powder with sterile water to obtain a mixed solution, wherein the weight of the bacteria in the mixed solution is 8% (w/w). And cracking the mixed solution by using an ultrasonic cracker. Centrifuging the lysed bacterial liquid at a speed of 10,000 g for 30 minutes by using a centrifuge and collecting a supernatant, lysing the insoluble bacterial lysis debris and the uncleaved bacteria again by using an ultrasonic lysis apparatus, then centrifuging, and combining and collecting the supernatant and the precipitate obtained by twice lysis.
The supernatant collected above was diluted with an equal volume of 20 mM Tris-HCl solution, pH 8.0. The diluted supernatant was passed through an anion exchange column. Prior to loading, the anion exchange was conditioned with 10mM Tris-HCl pH8.0 equilibration solution. After the column was conditioned with the equilibration solution, the sample was added and the fractions not bound to the column were eluted with the equilibration solution, followed by linear gradient elution with 10mM Tris-HCl/1M sodium chloride solution eluent, and each fraction was collected separately (with an ultraviolet detector, with absorption peaks at 260 nm and 280 nm).
Measuring the total sugar content of each collected component by using a phenol-sulfuric acid method, and removing components containing polysaccharide; and combining the components without polysaccharide with the precipitate obtained by the two times of cracking to obtain the microecological preparation.
Example 2
Bifidobacterium longum (b)Bifidobacterium longum) The dry powder was mixed with 20 mM aqueous sodium phosphate pH8.5 to give a mixture, and the weight of the bacteria in the mixture was adjusted to 15% (w/w). And (3) cracking the bacterial liquid by using a tissue disruptor. The lysed cell suspension was centrifuged at 13,000g for 25 minutes in a centrifuge, and the supernatant and the precipitate were collected, respectively.
The supernatant was passed through an anion exchange column. Before loading, the anion exchange was adjusted with 20 mM sodium phosphate solution pH8.5, the sample was added, and a linear gradient elution was performed with an eluent of 20 mM sodium phosphate/1M sodium chloride solution pH8.5, and each fraction was collected separately (with an ultraviolet detector, having absorption peaks at 260 nm and 280 nm).
Each fraction collected was assayed for total sugar using the phenol-sulfuric acid method. Collecting polysaccharide-free peaks/fractions and combining as polysaccharide-free fractions; combining the polysaccharide-free fraction with the precipitate obtained by lysis; drying to obtain the powdery microecological preparation.
Mixing powdered microecological preparation powder 0.1%, fulvic acid 0.1-0.2%, CaCl20.2 to 0.5 percent (the balance being water) to obtain the composite antitranspirant.
Example 3
Example 1, example 2 was sprayed onto broad bean leaves and deionized water was used as a control. After one hour, the broad bean skin strips were torn and observed under a microscope, and the results are shown in fig. 2 and 3A.
Example 1, example 2 were sprayed onto arabidopsis thaliana leaves with deionized water as a control. After one hour, the skin strips were torn and tested in vivo under a microscope for 50 stomatal openings, with the results expressed as mean and standard deviation, as shown in fig. 2 and 3A.
Example 1 and example 2 were sprayed onto leaf surfaces of arabidopsis thaliana and fava beans, and clear water was used as a control. The transpiration rate after 3 days was observed. The results are shown in FIG. 3B:
the above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, various modifications of the methods and compositions set forth herein, as well as variations of the methods and compositions of the present invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the above-described embodiments which are obvious to those skilled in the art to which the invention pertains are intended to be covered by the scope of the present invention.
Claims (9)
1. A microecological preparation having a function of reducing plant transpiration, which is characterized in that the microecological preparation is composed of a residue of a bacterium belonging to the genus Bifidobacterium after lysis, centrifugation and removal of polysaccharide components.
2. The microecological preparation having a plant transpiration reducing effect according to claim 1, wherein the bacteria belonging to the genus Bifidobacterium include any one or more of Bifidobacterium adolescentis, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium catenulatum, Bifidobacterium odonta, Bifidobacterium animalis, and Bifidobacterium lactis.
3. The method for preparing a microecological preparation having a function of reducing plant transpiration according to claim 1 or 2, comprising the steps of:
step (1), putting the thalli into a solvent to obtain a mixed solution, cracking the thalli by a physical method, and centrifuging to obtain a supernatant and a precipitate;
adding the supernatant into an anion exchange column for elution, detecting and collecting components without polysaccharide;
and (3) mixing the components which are not contained in the polysaccharide and obtained in the step (2) with the precipitate obtained in the step (1) to obtain the microecological preparation.
4. The method for preparing a microecological preparation having a function of reducing plant transpiration according to claim 3, further comprising the steps of:
and (4) drying to obtain the powdery microecological preparation.
5. The method for preparing a microecological preparation having a function of reducing plant transpiration according to claim 3, wherein the mixed solution of the step (1) contains 5 to 45% by weight of the bacteria.
6. The method for preparing a microecological preparation having a function of reducing plant transpiration according to claim 3, wherein the lysis in the step (1) is selected from any one or more of ultrasonic lysis, tissue disruption, high pressure homogenizer lysis, and chemical lysis.
7. The method for preparing a microecological agent having a function of reducing transpiration in plants according to claim 3, wherein the centrifugation in the step (1) is performed under conditions of 8000g to 10000 g/min for 10 to 60 min.
8. The method for preparing a microecological agent having a function of reducing plant transpiration according to claim 3, wherein the precipitate obtained by the centrifugation in the step (1) is physically lysed again, and after the centrifugation, the supernatant is collected and the supernatants obtained by the two centrifugation are combined.
9. The method for preparing a microecological preparation having a function of reducing transpiration in plants according to claim 3, wherein the solvent is selected from the group consisting of water and an aqueous solution of sodium phosphate.
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Citations (5)
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| CN101016516A (en) * | 2007-01-22 | 2007-08-15 | 浙江大学 | Method of preparing plant transpiration-resistant agent based on vintage microzyme |
| CN101041812A (en) * | 2007-01-22 | 2007-09-26 | 浙江大学 | Preparation method for plant transpiration resistant agent based on escherichia coli |
| CN104161047A (en) * | 2014-07-10 | 2014-11-26 | 浙江大学 | Preparation method of transpiration inhibitor based on pore immune close induced by spirulina |
| CN104381304A (en) * | 2014-10-08 | 2015-03-04 | 河海大学 | Leaf-surface anti-transpiration water-retaining agent special for flue-cured tobacco |
| CN107325980A (en) * | 2017-06-12 | 2017-11-07 | 新疆农业科学院微生物应用研究所（中国新疆-亚美尼亚生物工程研究开发中心） | A kind of radiation hardness series bacillus KH9 and its application in biological antitranspirant |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101016516A (en) * | 2007-01-22 | 2007-08-15 | 浙江大学 | Method of preparing plant transpiration-resistant agent based on vintage microzyme |
| CN101041812A (en) * | 2007-01-22 | 2007-09-26 | 浙江大学 | Preparation method for plant transpiration resistant agent based on escherichia coli |
| CN104161047A (en) * | 2014-07-10 | 2014-11-26 | 浙江大学 | Preparation method of transpiration inhibitor based on pore immune close induced by spirulina |
| CN104381304A (en) * | 2014-10-08 | 2015-03-04 | 河海大学 | Leaf-surface anti-transpiration water-retaining agent special for flue-cured tobacco |
| CN107325980A (en) * | 2017-06-12 | 2017-11-07 | 新疆农业科学院微生物应用研究所（中国新疆-亚美尼亚生物工程研究开发中心） | A kind of radiation hardness series bacillus KH9 and its application in biological antitranspirant |
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