CN115152794B - Bacillus bailii rice biological seed coating agent and preparation method and application thereof - Google Patents

Bacillus bailii rice biological seed coating agent and preparation method and application thereof Download PDF

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CN115152794B
CN115152794B CN202210985127.8A CN202210985127A CN115152794B CN 115152794 B CN115152794 B CN 115152794B CN 202210985127 A CN202210985127 A CN 202210985127A CN 115152794 B CN115152794 B CN 115152794B
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bacillus
rice
seed coating
coating agent
bailii
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CN115152794A (en
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明亮
刘凤权
赵延存
谷春艳
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Jiangsu Academy of Agricultural Sciences
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Jiangsu Academy of Agricultural Sciences
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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
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01C—PLANTING; SOWING; FERTILISING
    • A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
    • A01C1/06—Coating or dressing seed
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01C—PLANTING; SOWING; FERTILISING
    • A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
    • A01C1/08—Immunising seed
    • 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
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/24—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing ingredients to enhance the sticking of the active ingredients
    • 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
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/26—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests in coated particulate form
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P21/00—Plant growth regulators
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P3/00—Fungicides
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05B—PHOSPHATIC FERTILISERS
    • C05B15/00—Organic phosphatic fertilisers
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
    • C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
    • C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
    • C05G3/40—Mixtures of one or more fertilisers with additives not having a specially fertilising activity for affecting fertiliser dosage or release rate; for affecting solubility
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
    • C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
    • C05G3/50—Surfactants; Emulsifiers
    • C—CHEMISTRY; METALLURGY
    • C05—FERTILISERS; MANUFACTURE THEREOF
    • C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
    • C05G3/00—Mixtures of one or more fertilisers with additives not having a specially fertilising activity
    • C05G3/60—Biocides or preservatives, e.g. disinfectants, pesticides or herbicides; Pest repellants or attractants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture

Abstract

The invention relates to a biological seed coating agent of bacillus beleiensis rice, and a preparation method and application thereof, wherein the biological seed coating agent of the rice takes bacillus beleiensis (Bacillus velezensis) ZXC-1 fermentation liquor with the preservation number of CGMCC No.23560 as an active ingredient, and comprises lecithin, higher fatty acid ester, polyaspartic Acid (PASP), potassium phosphite, warning color, a film forming agent, a thickening agent and a preservative; the biological seed coating agent for rice provided by the invention not only has the characteristics of a conventional seed coating agent, but also has a special liposome structure, can form a vesicle structure, and can wrap biocontrol bacteria to achieve a slow release effect; not only can prevent and treat bakanae disease, white leaf spot and bacterial leaf spot of rice, but also can promote sprouting and growth of rice. Can effectively reduce the application amount of chemical pesticides and ensure the grain safety.

Description

Bacillus bailii rice biological seed coating agent and preparation method and application thereof
Technical Field
The invention relates to a biological pesticide preparation, in particular to a bacillus bailii rice biological seed coating agent, a preparation method and application thereof.
Background
Rice is one of the important grain crops in China, the planting area of the rice accounts for 30% of the area of the grain crops in China, and the yield accounts for 40% of the total yield of the grain. With the increase of the planting area, the rice plant diseases and insect pests have a rising trend year by year, so that the loss of the rice yield is serious. Especially in recent years, bakanae disease, bacterial leaf blight and bacterial leaf spot of rice become diseases which are difficult to control in rice diseases, and once the diseases occur, immeasurable losses are caused as seed-borne diseases, and the conventional bactericides and using methods are difficult to achieve satisfactory control effects. Especially, the use of a large amount of chemical agents does not achieve satisfactory effects, and causes the increase of disease resistance and the exceeding of pesticide residues.
The seed coating agent can advance the control period and control diseases in early stage, and becomes a novel mode for controlling bakanae disease and bacterial leaf streak. The seed coating agent is prepared from pesticide, bactericide, compound fertilizer, trace elements, plant growth regulator, slow release agent, film forming agent and the like by advanced process, and can be directly or after dilution coated on the surface of seeds to form a pesticide preparation with a protective film with certain strength and permeability. The seed coating agent is used before sowing, can comprehensively prevent and treat plant diseases and insect pests in seedling stage, saves medicine, time, labor and money, is beneficial to environmental protection, can promote the growth of crops after coating, has strong root system and stress resistance, can ensure yield and increase production, and has remarkable economic and ecological benefits.
The existing biological seed coating agent takes living microorganisms (beneficial or antagonistic) or microorganism metabolites as active ingredients, and is prepared by the steps of fermentation, dosage form processing and the like by assisting in preparing auxiliary agent formulas (such as nutrient elements, film forming agents, antifreezing agents, dispersing agents and the like). The biological seed coating agent can form a biological protective layer film with certain strength and permeability on the surface of the seed, and microorganisms in the biological seed coating agent can colonize on the surface and inside of the seed along with germination of the seed, and spread and colonize in root systems and soil along with growth of plants, so that the purposes of disease prevention and insect killing are achieved. In 1997, seed company in Zhejiang province developed a biological seed coating agent using bacillus as active substance, which can prevent and treat sheath blight, damping off, phytophthora root rot, etc., and in addition, the biological seed coating agent has the function of promoting plant growth. Shangwu and other researches show that the ZSB biological seed coating agent can be used for preventing and treating bacterial leaf blight, banded sclerotial blight, rice blast, seedling rot, seedling aversion disease and the like of rice, and can promote the growth of crops and enhance the stress resistance of crops. Zhao Xiachu and the like adopt biological seed coating agents with different concentrations to treat rice and wheat seeds, and the results show that all treatments can improve the germination peak value, germination index and seed vitality index of the seeds, have growth promoting effect on plants and increase root length and seedling height. Liang Jiangong the result shows that the seed coating agent can raise seedling quality, promote plant growth and prevent and treat plant diseases. Guo Jiansheng it is shown that after the rice seeds are coated by the biological seed coating agent, the seedling stage has no rotten seedling, and the biological seed coating agent can reduce the incidence of bakanae disease, bacterial wilt and damping off. Feng according to the results of indoor biological activity measurement and potting test, several strains of bacillus subtilis are obtained by screening, the strains have good control effect on rhizoctonia solani and rhizoctonia solani of cotton, and biological seed coating agents with YZ and BS-208 strain raw powder as active substances are prepared by screening of auxiliary agents and the like.
However, the traditional seed coating agent is produced by adopting a wet superfine grinding process, active ingredients, a dispersing agent, a wetting agent, a preservative, warning color, a film forming agent, a defoaming agent, an antifreezing agent, water and the like are uniformly sheared by a shearing kettle or a shearing pump, subjected to secondary sanding by a sand mill, subjected to hundred-tert BT-9300H particle size detection by a laser particle size distribution instrument, and subjected to DV90 being no more than 5um as a qualification, and packaged and put in storage after the qualification detection. The biocontrol strain coating agent belongs to a living microorganism seed coating agent, and microorganisms can cause harm to the microorganisms due to the specificity of the biocontrol strain coating agent and the high temperature or extrusion, so that the strain content of the biocontrol strain coating agent is affected. In the traditional seed coating agent production method, the microbial cells are broken due to shearing of a high-speed shearing kettle or a shearing pump (10000 rpm), and the biocontrol bacteria are directly or indirectly killed. The secondary sand grinding of the sand mill is carried out by physical extrusion and crushing, so that the microbial thalli are damaged, and the survival rate of microorganisms is seriously influenced. How to reduce the influence of the processing technology on the biocontrol bacteria microorganisms is a problem which needs to be solved urgently by the existing biocontrol bacteria seed coating agent.
Disclosure of Invention
Aiming at the defects of the prior art, the biological seed coating agent for the rice is provided, which not only has the effect of promoting the growth of the rice, but also can effectively prevent and treat bakanae disease, bacterial leaf blight and bacterial leaf streak of the rice.
The second purpose of the invention is to provide a new preparation method of the biological seed coating agent for paddy rice. Compared with the traditional processing technology (the conventional seed coating agent adopts a secondary sanding technology, the method has high energy consumption and low productivity, and has higher requirements on the properties of raw materials), the method reduces the high-speed shearing emulsification flow, simplifies the processing technology, reduces the dependence on equipment, reduces the cost and is easier to popularize and apply.
The invention also aims to provide the application of the novel biological seed coating agent for rice, and the preparation method adopted by the invention provides a liposome vesicle structure which can play a role in slow release and prolong the control time of the biological seed coating agent.
In order to achieve one of the above purposes, the present invention is realized by the following technical scheme:
firstly, the application provides a bacillus beijerinus rice biological seed coating agent, which comprises the following components: the biological seed coating comprises, by mass, 5% -15% of lecithin, 5% -20% of higher fatty acid ester, 5% -20% of polyaspartic acid, 5% -20% of potassium phosphite, 1% -5% of an emulsifying agent, 0.5% -2% of warning color, 5% -10% of a film forming agent, 0.1% -0.5% of a thickening agent, 0.2% -0.5% of a preservative, and the balance (about 10% -50%) of bacillus bailii ZXC-1 fermentation broth; the bacterial content of the bacillus belicus ZXC-1 fermentation liquor is more than or equal to 10 8 cfu/ml。
Further, the content of each component in the bacillus bailii rice biological seed coating agent is preferably as follows: 5% of lecithin, 15% of higher fatty acid ester, 10% of polyaspartic acid, 10% of potassium phosphite, 1% of emulsifier, 1% of warning color, 0.2% of film forming agent 5%1%, 0.2% of thickener, and 100% of preservative, wherein the fermentation broth of bacillus belicus ZXC-1 is supplemented.
In the application, the used biocontrol bacillus subtilis (Bacillus velezensis) ZXC-1 is preserved in China general microbiological culture Collection center (CGMCC) at the address: the Beijing city, the Korean district, the North Chen Xiyu No. 1, 3, the national academy of sciences microbiological institute, post code 100101) is classified and named Bacillus velezensis, and the strain preservation number is CGMCC No.23560. The strain has inhibiting effect on seed-borne diseases such as bakanae disease bacterial leaf spot and the like.
Further, the above lecithin is preferably TP (phosphatidylcholine), and the higher fatty acid ester is preferably oil
Methyl acrylate, emulsifier ZLN-5 (mixture of fatty alcohol ether block copolymer and vegetable oil ester modifier), warning color of sea Shu Gaoqiao Red (RY), film forming agent of styrene-acrylic emulsion (styrene-acrylic ester emulsion, dow 3186), thickener of nanocellulose (HPC 851), and preservative of Kathon (2% by mass of isothiazolinone).
Secondly, the application provides a preparation method of the bacillus beijerinus rice biological seed coating agent, which comprises the following specific steps:
(1) Preparation of Bacillus bailii ZXC-1 fermentation broth
A) And (3) activating and culturing: glycerol strains of bacillus belicus ZXC-1 are streaked and inoculated on a fresh NA solid flat plate, and are cultured for 24 to 36 hours at the temperature of 26 to 30 ℃;
b) Seed liquid preparation: picking a single colony on the solid plate in the step A) to inoculate in NB liquid culture medium, and carrying out shaking culture for 16-24 h at 26-30 ℃ with the shaking frequency of 100-200rpm to obtain seed liquid;
c) Inoculating the seed solution obtained in the step B) into 40% -60% NB culture medium according to the proportion of 1% -5% (v/v, volume ratio) for fermentation, wherein the fermentation temperature is 27 ℃ -30 ℃, and the loading amount is 200-400 mL/1000mL; the initial pH of the culture medium is 6.8-7.2, the stirring speed is 100-180rpm, and the fermentation time is about 48 hours, thus obtainingObtaining bacillus beijerinus ZXC-1 fermentation liquor; counting by plate coating method, and the thallus density of fermentation liquid is greater than 10 8 cfu/mL。
(5) Mixing lecithin and higher fatty acid ester, and stirring with a magnetic stirrer until the mixture is clear and transparent at a rotating speed of 800r/min; adding an emulsifying agent, and continuously stirring uniformly to form an oil phase for later use;
(6) Adding potassium polyaspartic acid phosphite into bacillus bailii ZXC-1 fermentation broth obtained in the step (1), and obtaining an aqueous phase biocontrol bacteria fermentation broth after complete dissolution;
(7) Under the action of magnetic stirring (800 r/min), dropwise adding the water phase biocontrol bacterium fermentation liquid into the oil phase obtained in the step (2), continuously stirring at the rotating speed of 800r/min, gradually thickening the water phase adding system, increasing the rotating speed to 1200r/min, and stirring for 30min after reaching a phase inversion point; and then adding warning color, film forming agent, thickener and preservative, and stirring uniformly at a rotating speed of 1500r/min to obtain bacillus belicus ZXC-1 seed coating.
Thirdly, the application also provides application of the bacillus beijerinus rice biological seed coating agent in preventing and treating rice bakanae disease and bacterial leaf spot. Specifically, the biological seed coating agent for rice and rice seeds are coated according to the weight ratio of 1:30-50, and the biological seed coating agent for rice and rice seeds are directly sowed for seedling after being dried in shade at normal temperature.
Compared with the prior art, the bacillus beijerinus rice biological seed coating agent prepared by the application has the following beneficial effects:
1) The addition of lecithin can make the seed coating agent form a vesicle structure, and can increase the absorption and slow release effects of the plant on the agent. Because the medicament is wrapped in liposome vesicles, the liposome is a phosphate substance, so that the survival time of thalli can be prolonged; the liposome structure is degraded along with time, the medicament slowly releases the birth control bacteria along with the degradation of the liposome, and substances generated by the degradation of the liposome provide C sources and N sources for the germination of the biocontrol bacteria and provide energy for the germination of microorganisms; in addition, the addition of higher fatty acid esters forms droplets with lecithin, increasing efficacy and adhesion. Polyaspartic acid and potassium phosphite can be used as fertilizer to strengthen the stress resistance of seeds and promote germination.
Meanwhile, by adding liposome substances, the germination time of the spores can be adjusted, namely, a part of spores are wrapped, the spores possibly do not germinate in the first time, and the spores start to germinate after absorbing the liposome along with the growth of seeds. The spores which are not wrapped by the liposome germinate at the first time, so that the time difference of spore germination is caused, secondary metabolic substances can be always generated, and protection is provided for plants.
2) The addition of the alert color can distinguish between coated seeds and uncoated seeds. The addition of the thickener and the preservative can maintain the stability of the seed coating product and avoid the generation of mould.
3) The invention adopts a self-emulsifying method, reduces the damage of shearing and sanding to microorganisms by a simple stirring and emulsifying processing technology, and can effectively ensure the bacterial content in the biological seed coating. The prepared seed coating agent has good film forming property and strong adhesive force, and remarkably improves the effect and stability of the product. In addition, the method is simple in preparation, does not need special processing, reduces the production cost, and is easy to popularize and utilize in agriculture.
4) The bacillus belicus ZXC-1 is adopted to inhibit seed-borne diseases such as bakanae disease bacterial leaf spot and the like, the prepared seed coating can obviously reduce the incidence rate of rice bacterial diseases, can be used for preventing and controlling rice bakanae disease and bacterial leaf spot, has a prevention effect exceeding that of the existing chemical and biological seed coating, and can obviously promote the germination and growth of rice seeds, enhance stress resistance and promote the yield increase of crops.
5) The seed coating agent is completely nontoxic and pollution-free, is safe and convenient to produce, transport, store and use, and has extremely strong application prospect.
Drawings
FIG. 1 is a schematic representation of the results of a phylogenetic tree analysis of the gyrB gene sequence of Bacillus bailii ZXC-1.
FIG. 2 is a photograph of colonies, cells and spore morphology of Bacillus belicus ZXC-1 on NA medium plates.
FIG. 3 is a photograph showing antagonism effect of Bacillus bailii ZXC-1 fermentation broth against rice bacterial leaf streak bacteria.
FIG. 4 is a photograph showing antagonism effect of Bacillus belicus ZXC-1 fermentation broth on bakanae disease germ.
Fig. 5 is an SEM image of liposome vesicle structure of the biological seed coating agent prepared in example 3.
FIG. 6 is a schematic representation of the effect of Bacillus belicus ZXC-1 biological seed coating on rice seedlings.
FIG. 7 is a schematic representation of the time of colonization of Bacillus belicus ZXC-1 in rice rhizosphere soil.
Detailed Description
In order to better illustrate the technical solution of the present invention, the following specific description is provided with reference to specific embodiments, which are not intended to limit the present application.
Reagents involved in the following examples:
lecithin TR was purchased from the company of phospholipid technology, inc. In the Anhui;
higher fatty acid esters (methyl oleate 0295) were purchased from sozhou feng times biotechnology limited;
polyaspartic Acid (PASP) was purchased from hebei co-environmental technologies, inc;
the emulsifier is ZLN-5, purchased from Nantong De Yi chemical Co., ltd;
the warning color is sea Shu Gaoqiao red RY, purchased from Hemsleyae New Material science and technology Co., ltd;
the film forming agent is styrene-acrylic emulsion 3186, which is purchased from Nanjing Guta chemical Co., ltd;
the thickener is commercial nanocellulose HPC851;
the preservative is Kasong purchased from Nanjing Tianshi blue shield biotechnology Co.
The culture medium referred in the examples:
NB medium: 10g of sucrose, 5g of peptone, 1g of yeast powder, 3g of beef extract, 1000ml of distilled water, adjusting the pH to 6.8-7.2 and sterilizing at 121 ℃.
NA solid plate: on the basis of NB liquid culture medium, adding agar powder with final concentration of 15 g/L.
LB medium: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, 1000ml of distilled water, pH of which is adjusted to 7.0-7.2, and sterilizing at 121 ℃.
LB solid plate: on the basis of LB liquid medium, adding 15g/L agar powder.
PDA solid plate: 200g of potato, 20g of glucose and 1000ml of distilled water are added, 15g/L of agar powder is added, and the mixture is sterilized at 121 ℃.
Example 1
The antibacterial activity of Bacillus beleiensis (Bacillus velezensis) ZXC-1 fermentation broth on rice bacterial leaf streak (preserved in this laboratory) was determined by a zone of inhibition method.
Bacillus beleidstock (Bacillus velezensis) ZXC-1 used in the example is a biocontrol strain which has stronger antagonistic activity on rice bacterial leaf streak pathogens and is obtained by separating and screening rice rhizosphere soil in Instrument market in Jiangsu province in the laboratory of the applicant, and the applicant designates the biocontrol strain as ZXC-1. The strain was identified as bacillus belicus (Bacillus velezensis) by a method combining colony morphology observation, fungus morphology observation, biochemical indexes and gyrB gene sequence determination. The results of the tree analysis of the gyrB gene sequence of Bacillus belicus ZXC-1 are shown in FIG. 1.
The strain is preserved in China general microbiological culture collection center (CGMCC for short, address: north Xili No. 1, 3 of the North West Lu No. 1, the university of Chinese academy of sciences of China, and mail code 100101) of China general microbiological culture Collection center (CGMCC No. 23560) of 10-11 days of 2021.
(1) The preparation method of the biocontrol bacterial liquid comprises the following steps:
a) And (3) activating and culturing: glycerol strain of bacillus belicus ZXC-1 is streaked and inoculated on a fresh NA solid plate, and is cultured for 24 hours at 26-30 ℃; in FIG. 2, a, b and c are the colony, the thallus and the spore form of Bacillus belicus ZXC-1 on NA medium plates, respectively.
B) Seed liquid preparation: picking a single colony on the solid plate in the step A) to inoculate in NB liquid culture medium, and carrying out shaking culture for 16-24 h at 26-30 ℃ with a shaking frequency of 100rpm (in the actual culture process, the shaking frequency can be determined to be 100-200rpm according to the growth condition of the strain) to obtain seed liquid;
c) Preparing biocontrol bacteria liquid: inoculating the seed solution obtained in the step B) into NB culture medium for fermentation at a volume ratio of 5% (v/v, wherein 1-5% of inoculation ratio can achieve the purpose of fermentation), wherein the fermentation temperature is 27-30 ℃, and the loading amount is 400mL/1000mL (in the specific implementation, the loading amount of a fermentation tank can be 200-400 mL/1000 mL);
the initial pH of the NB culture medium is 6.8-7.2, the stirring speed is 100rpm (the stirring speed of 100-180rpm can achieve the purpose of fermentation), the fermentation time is 48h, and bacillus bailii ZXC-1 fermentation liquor is obtained; counting by plate coating method, and the thallus density of fermentation liquid is greater than 10 8 cfu/mL。
(2) Diluting the biocontrol bacteria liquid obtained in the step (1) by using LB solid culture medium cooled to about 55 ℃ according to the proportion of 1:10, fully and uniformly mixing, taking 15ml of the biocontrol bacteria liquid into a sterile culture dish, lightly shaking a flat plate to uniformly spread the bacteria liquid, and preparing a monitoring flat plate for later use after the bacteria liquid is solidified.
(3) The sterile oxford cups were gently placed in the monitoring plates prepared in (2), respectively, and then 200ul of the biocontrol bacteria liquid prepared in (1) was added to the oxford cups with a sterile pipette, and 200ul of sterile water was added to the blank control group. The flat plate is placed in a constant temperature incubator at 37+/-1 ℃ for culturing for 12-18 hours, taken out and whether a bacteriostasis ring is generated or not is observed.
As shown in FIG. 3, bacillus belicus (Bacillus velezensis) ZXC-1 fermentation broth has remarkable antagonism to rice bacterial leaf streak.
Example 2
The antibacterial activity of Bacillus beleiensis (Bacillus velezensis) ZXC-1 fermentation broth against bakanae disease of rice (laboratory preservation of the national academy of agricultural sciences of Jiangsu province) was measured by using a hypha growth inhibition method.
(1) Preparation of fermentation broth
A) And (3) activating and culturing: glycerol strain of bacillus belicus ZXC-1 is streaked and inoculated on a fresh NA solid plate, and is cultivated for 36 hours at the temperature of 26-30 ℃;
b) Seed liquid preparation: picking a single colony on the solid plate in the step A) to inoculate in NB liquid culture medium, carrying out shaking culture for 24 hours at 26-30 ℃, and obtaining seed liquid at a shaking frequency of 100 rpm;
c) Preparing biocontrol bacteria liquid: inoculating the seed solution obtained in the step B) into NB culture medium at a ratio of 1% (v/v) for fermentation, wherein the fermentation temperature is 27-30 ℃, and the loading amount is 200/1000mL;
the initial pH of the culture medium is 7.0, the stirring speed is 100-180rpm, the fermentation time is 48 hours, and bacillus beijerinus ZXC-1 fermentation liquor is obtained; counting by plate coating method, and the thallus density of fermentation liquid is greater than 10 8 cfu/mL。
(2) Taking 15ml of PDA culture medium cooled to about 55 ℃ in a sterile culture dish, gently shaking the flat plate to uniformly spread bacterial liquid, and preparing a monitoring flat plate for standby after solidification.
(3) Punching at the same radius edge of PDA plate with aseptic puncher (inner diameter 5 mm+ -0.1 mm), taking bakanae disease germ cake with aseptic forceps, placing in the middle of PDA plate, taking 200ul fermentation liquor with aseptic straw, adding into punched hole, and observing whether bakanae disease germ silk growth is inhibited.
As shown in FIG. 4, bacillus belicus (Bacillus velezensis) ZXC-1 fermentation broth has remarkable antagonism to bakanae disease of rice, as shown in FIG. 4.
Example 3 preparation of seed coating
(1) Bacillus bailii (Bacillus velezensis) ZXC-1 broth having a cell density (cell content) of greater than 10 was prepared as in example 1 8 cfu/mL；
(2) The preparation method comprises the following steps of mixing 5% of lecithin and 15% of higher fatty acid ester according to the mass percentage of the seed coating agent prepared finally, and stirring by a magnetic stirrer until the seed coating agent is clear and transparent; adding 1% of emulsifying agent, and continuously stirring uniformly to obtain an oil phase for later use;
(3) Adding 5% of polyaspartic acid and 5% of potassium phosphite into 84.7% of fermentation liquor for dissolution, adding the biocontrol bacterium fermentation liquor (aqueous phase) into an oil phase dropwise (at the speed of 1 mL/drop) under the action of magnetic stirring after complete dissolution, stirring at the speed of 800r/min, gradually thickening a system with the addition of the aqueous phase, increasing the rotating speed to 1200r/min, and stirring for 30min after reaching a phase inversion point; then adding warning color 1% to get final product5% of film agent, 0.1% of thickening agent and 0.2% of preservative, stirring uniformly (about 15 min) at a rotating speed of 1500r/min, and stirring for 15min to obtain 10 8 cfu/mL bacillus bailii ZXC-1 biological seed coating.
The SEM photograph of the obtained biological seed coating agent is shown in fig. 5, and it can be seen that the seed coating agent exhibits a distinct vesicle structure.
Example 4 indoor measurement of the Effect of biological seed coating agent on coating treatment on Rice growth
10 prepared in example 3 8 cfu/mL bacillus beleiensis ZXC-1 biological seed coating agent and rice seeds (Jiuyou Yuehao silk seedling) are mixed according to the proportion of 1:30, 1:40 and 1:50 (mass ratio), then the mixture is sown in a culture dish of filter paper wetted by preset distilled water, 20 grains are sown in each dish, the mixture is repeated for 3 times, the mixture is placed in a light incubator at 25 ℃, distilled water is added periodically for moisturizing, growth conditions are observed, and plant height and root length are checked after 5 days of culture, as shown in the following table 1:
TABLE 1 Effect of different coating ratios on Rice growth
The results show 10 8 cfu/mL bacillus belicus ZXC-1 biological seed coating agent has promoting effect on rice seedling emergence, and the promotion rate of bud length and root length is obviously higher than that of blank clear water control, as shown in figure 6 and table 1.
Example 5
In the embodiment, the rice biological seed coating agent is used for coating rice seeds to measure the colonization effect of beneficial bacteria on plant rhizosphere. Single colonies of the strain Bacillus belicus ZXC-1 were inoculated into NA plates containing at least 5ug/ml rifampicin and incubated at 28 ℃. After 2d, the newly grown colonies were inoculated into NA plates of the same concentration of rifampicin and subcultured 1 time and transferred to NA plates of 10ul/ml rifampicin. The procedure was repeated again, increasing the rifampicin concentration in a 2-fold series until a rifampicin marker strain of 300ug/ml was screened. The marked strain can grow normally on a NA plate of 300ug/ml rifampicin, the unmarked strain can not grow on the plate, and the marked bacillus beijerinckii ZXC-1 strain has the same colony morphological characteristics as the original strain and has equivalent antibacterial activity.
The marked bacillus belicus ZXC-1 strain replaces bacillus belicus ZXC-1 in the embodiment 3, the rice biological seed coating agent containing the marked bacillus belicus ZXC-1 strain is prepared according to the method of the embodiment 3, the rice biological seed coating agent and rice seeds (Jiuyou Yuehua seedlings) are mixed and coated according to the mass ratio of 1:40, and after being dried in the shade, the rice biological seed coating agent and the rice seeds are sown in sterilized soil which is sterilized at high temperature (120 ℃ for 20 min) in advance, and the rice biological seed coating agent and the rice seeds are placed in an illumination incubator (the temperature is 25 ℃, the illumination is 12h, the humidity is 40%) and sterile water is irrigated periodically. Cucumber seed treatment directly planted for coating treatment was used as control. Sowing and emergence for 5, 10, 15, 20, 25 and 30 days. Taking a proper amount of rhizosphere soil, root and stem tissues of rice seedlings, and recovering the marked bacillus belicus ZXC-1 strain by adopting a dilution separation method. The colony count was calculated after 3 replicates per treatment and 3 days incubation at 28 ℃.
As shown in FIG. 7, the colonization time of Bacillus belicus ZXC-1 strain in rice rhizosphere soil exceeds 30d, the change of the number of thalli is not large, and the time is slightly higher than other times at 5 d; bacillus belicus ZXC-1 strain can enter rice body, and is transplanted in vivo, and can be recovered in root 5d after inoculation, with low content in stem, and 2.1 x 10d after 10d 2 cfu/g. The colonization dynamic of the marked bacillus belicus ZXC-1 strain can reflect the population colonization dynamic of the original strain, namely, bacillus belicus ZXC-1 can quickly colonize the rhizosphere soil and roots of rice, and the colonization amount of bacillus belicus in stem tissues is small. The conventional seed coating agent only has a thallus release peak about 5 days, and the amount of the colonial bacteria of the seed coating agent is in a trend of increasing first and then decreasing then increasing, namely, the 2 nd thallus release peak appears due to the degradation of the liposome in 15 days, which is inconsistent with the traditional biocontrol thallus trend, so that the seed coating agent with the liposome vesicle structure has slow release performance on biocontrol bacteria.
Example 6 field efficacy test of biological seed coating agent for Rice to control bacterial leaf streak
In 2021, a field test for preventing and treating bacterial leaf spot of rice was performed by using the biological seed coating agent for rice prepared in example 3, and the effect of preventing and treating bacterial leaf spot of rice was examined at the test dose, and 6 treatments were set in the test: biological seed coating agent coating 1:50 (mass ratio), biological seed coating agent coating 1:40 (mass ratio), biological seed coating agent coating 1:30 (mass ratio), 20% (mass percent) of buprofezin suspending agent 300 times liquid seed soaking, 36% (mass percent) of trichloroisocyanuric acid wettable powder 540 times liquid seed soaking and clear water control.
Sowing in the 6 months and 17 days, regularly investigating the time of the rice bacterial leaf spot disease symptoms in each treatment district in all Ji Miaoshi of the blank control district, investigating the disease condition of each district in the obvious rice bacterial leaf spot disease symptoms in the blank control district, and investigating the disease condition of each treatment district after 14 days.
The investigation method of the initial disease onset time comprises the following steps: after the rice seedlings are aligned, the occurrence of the field diseases is investigated every 10 days; the tillering stage starts, and the occurrence condition of the field diseases is investigated every 7 days; after the disease is seen in the whole field, investigation is carried out every 3-4 days, and investigation is carried out before and after typhoon and storm.
The investigation method for the damage degree of disease occurrence comprises the following steps: a diagonal five-point sampling method is adopted for each cell, and 25 clusters are investigated for each point, and 125 clusters are used.
Analysis of results:
the time for generating bacterial leaf streak disease of rice in clear water control treatment is earliest, the earliest disease-seeing time is 8 months and 9 days, and the rice is at the later tillering stage; compared with the clear water control treatment, the disease time of the biological seed coating agent 1:50, 1:40 and 1:30 coating treatment cells is respectively delayed for 4.3 days, 6.3 days and 7.3 days on average, the disease time of the 20% thiabendazole copper suspension 300-time seed soaking treatment cells is delayed for 5.3 days, and the disease time of the 36% trichloroisocyanuric acid wettable powder 540-time seed soaking treatment cells is delayed for 3.3 days.
The disease symptoms are obvious in 4 cells treated by the clear water control treatment on 12 days of 8 months, the disease indexes of the treated cells are investigated on 14 th day (26 days of 8 months), and the prevention and treatment effects of the bacterial leaf spot of rice by the seed soaking treatment of each medicament are statistically analyzed. The results were as follows: the prevention effect of the biological seed coating 1:50, 1:40 and 1:30 on the bacterial leaf spot of the rice is 49.36 percent, 56.61 percent and 60.69 percent respectively, the prevention effect is gradually increased along with the increase of the concentration of the medicament, and the prevention effect of the biological seed coating 1:30 is obviously higher than 1:50 (P is less than 0.05); the control effect of the seed soaking treatment of the 300 times liquid of the thiabendazole suspending agent with 20 percent of the control medicament on the bacterial leaf streak of the rice is 51.30 percent, which is obviously lower than that of the seed soaking treatment of the 1:30 coating treatment of the biological seed coating agent (P is less than 0.05); the control medicament 36% trichloroisocyanuric acid wettable powder 540-time seed soaking treatment has 50.12% of control effect on rice bacterial leaf streak, is equivalent to biological seed coating 1:40 and 1:50 coating treatment (P is more than 0.05), but is obviously lower than the control effect of biological seed coating 1:30 coating treatment (P is less than 0.05). All test agent treatments were safe for rice within the test range, see table 2.
Table 2 effects of seed soaking treatments with various agents on controlling bacterial leaf streaks of Rice
Example 7 biological seed coating agent for Rice field efficacy test for controlling bakanae disease in Rice
In 2021, a field control rice bakanae disease plot test was performed using the biological seed coating agent for rice prepared in example 3, and the control effect on rice bakanae disease was examined at the test dose, and 5 treatments were set in the test: biological seed coating agent coating 1:50 (mass ratio), biological seed coating agent coating 1:40 (mass ratio), biological seed coating agent coating 1:30 (mass ratio), 36% (mass percent) trichloroisocyanuric acid wettable powder 540-time seed soaking and clear water comparison.
Sowing is carried out on the day 6 and the day 17, the emergence rate is investigated when rice seedlings emerge uniformly (the day 6 and the day 22), the disease rate is investigated before the seedlings are transplanted (the day 7 and the day 16), and the disease rate is investigated before the seedlings are heading in a field (the day 9 and the day 10). Each cell is marked with continuous 100 seeds, the emergence rate is investigated when rice seedlings emerge uniformly, a 5-point sampling method is adopted in each cell before transplanting seedlings, the disease rate of 100 plants is investigated without electricity, a 5-point sampling method is adopted in each cell before heading in a field, 20 clusters are investigated at each point, the total plant number, the disease plant number and the control effect are recorded.
Analysis of results: the prevention effect of the biological seed coating 1:50, 1:40 and 1:30 on the bacterial leaf spot of the rice is 49.36 percent, 56.61 percent and 60.69 percent respectively, the prevention effect is gradually increased along with the increase of the concentration of the medicament, and the prevention effect of the biological seed coating 1:30 is obviously higher than 1:50 (P is less than 0.05); the control effect of the seed soaking treatment of the 300 times liquid of the thiabendazole suspending agent with 20 percent of the control medicament on the bacterial leaf streak of the rice is 51.30 percent, which is obviously lower than that of the seed soaking treatment of the 1:30 coating treatment of the biological seed coating agent (P is less than 0.05); the control medicament 36% trichloroisocyanuric acid wettable powder 540-time seed soaking treatment has 50.12% of control effect on rice bacterial leaf streak, is equivalent to biological seed coating 1:40 and 1:50 coating treatment (P is more than 0.05), but is obviously lower than the control effect of biological seed coating 1:30 coating treatment (P is less than 0.05). All test agent treatments were safe for rice within the test range, see table 3.
TABLE 3 biological seed coating agent control effect on bakanae disease of rice
The invention is described in further detail above with reference to examples. These embodiments are merely examples of the present invention and do not represent or limit the scope of the claims, which are set forth below.

Claims (10)

1. The bacillus bailii rice biological seed coating is characterized by comprising, by mass, 5% -15% of lecithin, 5% -20% of higher fatty acid ester, 5% -20% of polyaspartic acid, 5% -20% of potassium phosphite, 1% -5% of an emulsifying agent, 0.5% -2% of warning color, 5% -10% of a film forming agent, 0.1% -0.5% of a thickening agent, 0.2% -0.5% of a preservative and bacillus bailiiBacillus velezensis) The ZXC-1 fermentation liquor is used for supplementing the balance;
the bacterial content of the bacillus bailii ZXC-1 fermentation liquor is more than or equal to 10 8 cfu/ml；
The preservation number of the bacillus belicus ZXC-1 is CGMCC No.23560.
2. The bacillus bailii rice biological seed coating agent according to claim 1, wherein the bacillus bailii rice biological seed coating agent comprises, by mass, 5% of lecithin, 15% of higher fatty acid ester, 10% of polyaspartic acid, 10% of potassium phosphite, 1% of an emulsifying agent, 1% of a warning color, 5% of a film forming agent, 0.2% of a thickening agent, 0.2% of a preservative and 100% of bacillus bailii fermentation broth.
3. The bacillus bailii rice biological seed coating of claim 1, wherein the lecithin is phosphatidylcholine.
4. The bacillus bailii rice biological seed coating agent of claim 1, wherein the higher fatty acid ester is methyl oleate.
5. The bacillus bailii rice biological seed coating of claim 1, wherein the film former is a styrene-acrylic emulsion.
6. The bacillus beijerinus rice biological seed coating agent of claim 1, wherein said thickener is nanocellulose.
7. The bacillus beijerinus rice biological seed coating agent of claim 1, wherein said preservative is kathon.
8. The preparation method of bacillus bailii rice biological seed coating agent as claimed in claim 1, which is characterized by comprising the following specific steps:
(1) Preparation of Bacillus bailii ZXC-1 fermentation broth
Inoculating bacillus bailii ZXC-1 strain on an NA solid plate by streaking, and culturing for 24-36 h at 26-30 ℃; then, picking a single colony on the NA solid flat plate, inoculating the single colony into an NB culture medium, and carrying out shake culture for 16-24 hours at 26-30 ℃ to obtain seed liquid; inoculating the seed solution into NB culture medium at a volume ratio of 1% -5% for fermentation at a fermentation temperature of 27-30deg.C to obtain a strain with a bacterial content of not less than 10 8 cfu/ml bacillus beijerinus ZXC-1 fermentation broth for standby;
mixing lecithin and higher fatty acid ester, stirring to clarify, adding emulsifier, and stirring to obtain oil phase;
adding polyaspartic acid and potassium phosphite into bacillus bailii ZXC-1 fermentation broth obtained in the step (1) to obtain aqueous biocontrol bacteria fermentation broth for later use;
dropwise adding the water phase biocontrol bacteria fermentation liquid into the oil phase obtained in the step (2), stirring at 800rpm until the water phase biocontrol bacteria fermentation liquid is sticky, stirring at 1200rpm until the water phase biocontrol bacteria fermentation liquid reaches a phase inversion point, and continuously stirring for 30min; then adding warning color, film forming agent, thickener and preservative, and stirring uniformly at 1500rpm to obtain the bacillus beijerinus rice biological seed coating agent.
9. The use of the biological seed coating agent for bacillus bailii rice according to claim 1 for controlling bakanae disease and bacterial leaf streak disease of rice.
10. The application of claim 9, wherein the application means that the biological seed coating agent of the rice and the rice seeds are coated according to the weight ratio of 1:30-50, and after the biological seed coating agent is dried in shade at normal temperature, the biological seed coating agent is directly sown for raising seedlings, so that the control of bakanae disease of the rice and bacterial leaf streak disease is realized.
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