CN109439594B - Bacillus vallismortis for preventing and treating false smut - Google Patents
Bacillus vallismortis for preventing and treating false smut Download PDFInfo
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Abstract
The invention relates to the technical field of prevention and control of false smut of rice, in particular to a Bacillus vallismortis (Bacillus vallismortis) SCQN-5 for preventing and controlling false smut of rice, which has a preservation number of: 16564. the biocontrol strain has a good control effect on the false smut of rice, and the result of a three-point opposite culture method test shows that the inhibition zone of Bacillus vallismortis (SCQN-5) on the false smut of rice is 33.00 mm.
Description
Technical Field
The invention relates to the technical field of prevention and treatment of false smut of rice, in particular to bacillus vallismortis for preventing and treating false smut of rice.
Background
False smut (Ustilaginoidea virens) is a disease which mainly affects the production of rice after three main diseases of rice blast, banded sclerotial blight and stripe disease. Along with the acceleration of the updating of rice varieties, the improvement of fertilization level and the change of cultivation systems, false smut gradually worsens, and can cause 20 to 30 percent of yield loss in severe cases, thereby having great influence on the rice yield. The toxin generated by the green smut bacteria has serious hidden troubles on the safety of rice eaten by people and livestock and the ecological safety of farmland environment, so the green smut is increasingly emphasized. The history of the occurrence of the rice false smut is short, the research is started late, and particularly, the reports on the prevention and the treatment of the rice false smut are few. However, the influence of a large amount of chemically synthesized pesticides on the environment is very bad, so the biological control of false smut on rice is particularly important. At present, no report is found on the biological control of false smut on rice by screening suitable strains by researchers.
Disclosure of Invention
In order to overcome the defects of the technical problems, the invention provides a biocontrol strain-bacillus vallismortis for preventing and treating false smut, which can completely solve the technical problems.
The technical scheme for solving the technical problems is as follows:
the Bacillus vallismortis for preventing and treating ustilaginoidea virens is Bacillus vallismortis SCQN-5 with the preservation number: 16564.
in addition, another purpose of the invention is to provide a screening method of bacillus vallismortis for preventing and treating false smut, which is simple and easy to implement and convenient to operate, and comprises the following steps:
soil samples are collected in paddy fields of main rice production areas in various places, microbial strains existing in the soil are separated from the soil samples, and a three-point confrontation culture method and a rice false smut bacterial strain are adopted for carrying out confrontation experiments;
the screened strain SCQN-5 has better antagonistic effect;
the morphological characteristics and molecular biological identification indicate that the biocontrol bacterium is named as Bacillus vallismortis.
The three-point confrontation culture method comprises the following specific steps:
(1) and pouring a culture medium suitable for the growth of pathogenic bacteria on the culture dish.
(2) A Ustilaginoidea virens cake with diameter of 5mm is placed at the center of the culture medium.
(3) Three fungus cakes of the strains to be detected SCQN-5 are placed at the same distance from the central fungus cake, so that the three fungus cakes are arranged in an equilateral triangle.
(4) And measuring the diameter of the inhibition zone.
The application of the bio-control strain is to utilize the bio-control strain to biologically control the false smut, and the inhibition zone of bacillus vallissima (SCQN-5) to the false smut is 33.00mm by a three-point opposing culture method, so that the bio-control effect is better. The significance of the inhibition zone is that the inhibition effect of the biocontrol bacteria on the ustilaginoidea virens is shown, and the larger the inhibition zone is, the stronger the biocontrol bacteria is in inhibition on the ustilaginoidea virens is, and the better the control effect is.
The strain has the following characteristics:
A) morphological characteristics: the colony is light yellow, round and has folds, and the cell is rod-shaped.
B) The physiological and biochemical characteristics are shown in the following table:
in order to screen the high-efficiency biocontrol bacteria of the ustilaginoidea virens, a three-point confrontation culture method is adopted in the research, soil samples of the paddy field collected in areas such as Liaoning province, Sichuan province and inner Mongolia are separated and screened, and species identification is carried out on the biocontrol bacteria. Aims to obtain a biocontrol strain with higher control effect and provides a basis for biocontrol of false smut.
Compared with the prior art, the biocontrol strain has better control effect on the false smut of rice, and the result shows that the inhibition zone of Bacillus vallissimae (SCQN-5) on the false smut of rice is 33.00mm through a three-point confrontation culture method test.
The biocontrol strain of the invention belongs to biological agents, does not pollute the environment and the ecology, and is beneficial to the pollution-free production of rice.
Drawings
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
FIG. 1 is a schematic diagram showing the colony size of Ustilaginoidea virens after 27 days of growth on PSA medium;
FIG. 2 is a schematic diagram showing the size of a colony grown on a PSA medium after a biocontrol strain SCQN-5 is inoculated around Ustilago virens and grown for 27 days;
FIG. 3 is a morphological diagram of Bacillus vallismortis (SCQN-5);
Detailed Description
Example 1:
the biological control strain is Bacillus vallismortis SCQN-5 with the preservation number as follows: CGMCC No. 16564. The preservation date is as follows: 10 and 10 months and 10 days in 2018. The preservation unit is as follows: china general microbiological culture Collection center. And (4) storage address: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing.
The screening method of the biocontrol strain for preventing and treating false smut comprises the following steps of:
soil samples are collected in paddy fields of main rice production areas in various places, microbial strains existing in the soil are separated from the soil samples, and a three-point confrontation culture method and a rice false smut bacterial strain are adopted for carrying out confrontation experiments;
(1) pouring a culture medium suitable for the growth of pathogenic bacteria on a culture dish;
(2) placing a Ustilaginoidea virens fungus cake with diameter of 5mm at the center of the culture medium;
(3) placing three fungus cakes of the strains to be detected SCQN-5 at the same distance from the central fungus cake, and enabling the three fungus cakes to be arranged in an equilateral triangle;
(4) and measuring the diameter of the inhibition zone.
The screened strain SCQN-5 has better antagonistic effect;
the morphological characteristics and molecular biological identification indicate that the biocontrol bacterium is named as Bacillus vallismortis.
The biocontrol strain for preventing and treating false smut has the following characteristics:
A) morphological characteristics: the colonies were yellowish, round, wrinkled, with rod-shaped cells (as shown in FIG. 3);
B) the physiological and biochemical characteristics are shown in the following table:
| test items | Results | Test items | Results |
| Gram stain | + | The salt tolerance is 3% | + |
| Glucose | + | Salt tolerance of 5% | + |
| Sucrose | + | The salt tolerance is 7% | + |
| Lactose | + | The salt tolerance is 10% | + |
| Maltose | + | The salt tolerance is 20% | - |
| Fructose | + | Contact enzyme | + |
| Xylose | + | Hydrolysis of cellulose | - |
| Mannose | + | V-P test | + |
| Inositol | + | Indoles | + |
| Sorbitol | + | Nitrate reduction | + |
| Mannitol | + | Methyl Red test | + |
| Movement property | + | Citric acid salt | + |
| Starch hydrolysis | + | Hydrogen sulfide generation | - |
The detection method of the physiological and biochemical characteristics comprises the following steps:
gram stain
Preparation of dye
A. Liquid mixture of crystal violet: respectively preparing a solution A and a solution B, wherein: the liquid A is 2.0g of crystal violet and 20ml of 95% ethanol; the solution B is 0.8g of ammonium oxalate and 80ml of distilled water; mixing the solution A and the solution B, standing for 48 hours, and filtering. The dye liquor is stable and can be stored in a sealed brown bottle for several months.
B. Iodine solution: dissolving 2.0g of potassium iodide in 3-5 ml of distilled water, adding 1.0g of iodine tablets, and adding distilled water to dilute to 300ml after iodine is completely dissolved.
C. Decoloring liquid: (1) 95% ethanol, (2) acetone ethanol solution: 70ml of 95% ethanol and 30ml of acetone.
D. Dyeing liquor again: taking a 2.5% safranin O ethanol solution as a mother solution, and adding distilled water for dilution according to a volume ratio of 1:4 when in use.
Dyeing step
(1) Picking a little lawn with an inoculating needle, coating the lawn on a drop of sterile water or distilled water on a clean glass slide, and air-drying and fixing.
(2) The mixture of crystal violet was dyed for 1min and washed with water.
(3) The iodine solution is acted for 1min, washed with water and sucked dry.
(4) Decolorizing with 95% ethanol or acetone ethanol solution, and dripping into eluent to colorless (about 30 s).
(5) Dyeing for 2-3min by a counterdyeing solution, washing with water and air drying.
Dark purple is gram-positive bacteria; red is gram-negative bacteria.
Sugar and alcohol fermentation reaction
Culture medium: culture medium for spore bacteria (NH)4)2HPO4 1.00g，KCl 0.20g，MgSO40.20 g; 0.20g of yeast paste, 15.00g of agar, 10.00g of sugar or alcohol, 15.00ml of 0.04% bromocresol purple and 1L of distilled water, and adjusting the pH value to 7.0-7.2; the 0.04% bromcresol purple solution is prepared by dissolving 0.04g of bromcresol purple powder in 20ml of absolute ethyl alcohol and then adding 80ml of distilled water. The young culture of 18-24 h is punctured and inoculated into the culture medium, and cultured for 7d at 28 ℃. If the indicator turns yellow, the indicator shows acid production and is positive; and constant or blue (purple) is negative.
Movement property
Semi-solid agar puncture method
1. Culture medium
According to the phenomenon that flagellated bacteria can move in a semi-solid culture medium but can not move at will, the growth condition of the bacteria is observed, and whether the test bacteria have motility is judged. A culture medium with good growth of test bacteria is used, 0.4% agar is added, and the semi-solid culture medium is generally that the test tube is not flowable when the test tube is placed down, but the agar is broken when the test tube is lightly tapped by hands.
2. Inoculation and Observation
Inoculating test bacteria into a semisolid culture medium by using a straight needle for puncture, and culturing at a proper temperature. The motility of the bacteria was visualized by transmitted light. If the growth only grows on the puncture line and the edge is very clear, the test bacteria have no mobility and are negative; if the growth spreads from the puncture line to the periphery in a cloudy state and the edge is in a cloudy state, it indicates that the test strain is motile and positive.
Starch hydrolysis reaction
Culture medium: adding 0.2% soluble starch into NA culture medium; lugol iodine solution: 1.0g of iodine, 2.0g of potassium iodide and 300ml of distilled water; and (4) taking a fresh slant culture, dibbling the slant culture on the flat plate, and culturing at a proper temperature. Culturing for 2-5 days to form obvious bacteria, dripping iodine solution on the plate to form blue color, and forming a transparent ring with no color change around the bacteria colony to show that the starch is hydrolyzed positively; it was still negative in blue-black.
Salt tolerance reaction
Selecting a liquid culture medium suitable for the growth of the bacteria to be detected, adding NaCl (3%, 5%, 7%, 10% and 20%) with different concentrations according to identification requirements, and clarifying the culture medium. The young culture of 18-24 h is inoculated into the culture medium and cultured for 7d at 28 ℃. Growth was observed in comparison to uninoculated control tubes. Growth was positive and no growth was negative.
Catalytic reaction
A small ring of the slant strain cultured for 24h is taken by a platinum loop and smeared on a glass sheet on which 10% of hydrogen peroxide is dripped, and the slant strain is positive if bubbles are generated and is negative if no bubbles are generated.
Hydrolysis reaction of cellulose
Culture medium: peptone water basal medium: 5.00g of peptone, 5.00g of NaCl5, 1L of distilled water and 7.0-7.2 of pH; packaging the basic culture medium into test tubes, and soaking a piece of high-quality filter paper in the culture medium. The width of the paper strip is suitable for being easily placed into a test tube. The length of the paper strip is about 5-7 cm. When aerobic bacteria are measured, a part of paper strips should be arranged outside the liquid level of the culture medium, and when anaerobic bacteria are measured, the paper strips should be completely soaked in the culture medium. The medium was inoculated, there should be an empty control without inoculation. Culturing at proper temperature for 1-4 weeks. The filter paper strip can be decomposed into a mass of fibers or broken or thinned, and the filter paper strip is positive, and the filter paper strip is negative if the filter paper strip is unchanged.
V-P assay reaction
Culture medium: 5g of peptone, 5g of glucose, 5g of NaCl, 1L of distilled water and pH of 7.0-7.2; reagent: creatine 0.3% or raw powder, NaOH 40%; inoculating test bacteria into the culture solution, and culturing at a proper temperature for 2 days and 6 days; the culture broth and 40% sodium hydroxide were mixed in equal amounts. Adding a little creatine, if the culture solution is red after 10min, the test is positive reaction, and sometimes the culture solution needs to be placed for a longer time to generate red reaction. Negative if no red color appeared.
Indoles
Culture medium: 1% tryptone in water; adjusting the pH value to 7.2-7.6; inoculating fresh strain into the above culture medium, and culturing at suitable temperature. Reagent: 8g of p-dimethylaminobenzaldehyde, 760ml of ethanol (95%) and 160ml of concentrated HCl; culturing for 1, 2, 4 and 7 days, slowly adding a reagent with the height of 3-5mm on the surface of the culture solution along the tube wall, and generating red color on the interface of the liquid layer, namely positive reaction. If the color is not obvious, 4-5 drops of ether can be added into the culture solution, the mixture is shaken to disperse the ether into the liquid, the culture solution is kept stand for a while, and the indole reagent is added after the ether floats to the liquid surface. If indole exists in the culture solution, the indole can be extracted in an ether layer, and the concentrated indole reacts with the reagent, so that the color is obvious.
Nitrate reduction
Culture medium: liquid NA Medium 1L, KNO31g of a compound; reagent: 1) grignard reagent: solution A, 0.5g of p-aminobenzenesulfonic acid, 10% diluted acetic acid150 ml; solution B, 0.1g of alpha-naphthylamine, 20ml of distilled water and 150ml of 10% diluted acetic acid; 2) diphenylamine reagent: 0.5g of diphenylamine is dissolved in 100ml of concentrated sulfuric acid and diluted with 20ml of distilled water.
Inoculating the test bacteria into nitrate liquid culture medium, and culturing at proper temperature for 1, 3, 5 days. Two replicates of each strain were made, and two additional tubes were left without inoculation as controls.
Two clean empty test tubes or a little culture solution for 1, 3 and 5 days is poured into a color comparison porcelain plate pit, then a drop of solution A and a drop of solution B are respectively added, and a drop of solution A and a drop of solution B are respectively added into a control tube.
When the culture solution is dripped into the solution A and the solution B, the solution turns pink, and the presence of nitrite is indicated by rosy, orange, brown and the like, and the solution is positive for nitrate reduction. If no red appears, adding one or two drops of diphenylamine reagent, and if the reaction is blue, indicating that nitrate still exists in the culture solution, and no nitrite reaction exists, indicating that no nitrate reduction action exists; if the reaction is not blue, the nitrate and the formed nitrite are reduced into other substances, so the treatment should be positive according to the nitrate reduction.
Methyl Red test
Culture medium: 5g of peptone, 5g of glucose, 5g of NaCl, 5g and 1L of distilled water, wherein the pH value is 7.0-7.2; reagent: methyl red 0.1g, 95% ethanol 300ml, distilled water 200 ml; inoculating test bacteria into the culture solution, and culturing at a proper temperature for 2 days and 6 days; a drop of methyl red reagent was added to the culture, red being a positive reaction in the methyl red assay and yellow being a negative reaction.
Citric acid salt
Culture medium: NaCl1g, MgSO4·7H2O0.2g，NH4H2PO40.5g, 2g of sodium citrate, 1L of distilled water and 20ml of 0.04 percent phenol red liquid are streaked and inoculated on a slant, and the slant is cultured for 3 to 7 days at a proper temperature. The medium is alkaline (indicator blue or pink) and positive, otherwise negative.
Hydrogen sulfide generation
Paper strip method
Culture medium: 10g of peptone, 5g of NaCl, 10g of beef extract, 0.5g of cysteine, 1L of distilled water, pH 7.0-7.4, subpackaging test tubes, and sterilizing at 112 ℃ for 20-30 min, wherein the height of each tube of liquid layer is 4-5 cm.
In addition, ordinary filter paper is cut into strips with the width of 0.5-1cm, and the length is determined according to the height of the test tube and the culture medium. The paper strips were soaked with 10% lead acetate, then oven dried, placed in a petri dish and sterilized for use.
The medium was inoculated with fresh slant culture. After inoculation, a lead acetate paper strip is clamped by a sterile clamp and is tightly plugged by a cotton plug, so that the lead acetate paper strip is suspended in the tube, the lower end of the lead acetate paper strip is close to the surface of the culture medium, the lead acetate paper strip is not in contact with the liquid level, and the lead acetate paper strip is cultured at a proper temperature. Observations were made 3, 7, 14 days after inoculation. The paper strips turned black were positive and the paper strips were negative.
From the above table, it is clear that gram stain, glucose, sucrose, lactose, maltose, fructose, xylose, mannose, inositol, sorbitol, mannitol, motility, starch hydrolysis, salt tolerance of 3%, 5%, 7%, 10%, catalase, V-P test, indole, nitrate reduction, methyl red test, citrate positive, salt tolerance of 20%, cellulose hydrolysis, and hydrogen sulfide production negative.
The biocontrol strain is used for biologically controlling false smut, and the inhibition zone of Bacillus vallissima (Bacillus vallissima) SCQN-5 on the false smut is 33.00mm by a three-point opposing culture method.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and all simple modifications and equivalent variations of the above embodiment according to the present invention are within the scope of the present invention.
Claims (2)
1. Bacillus vallismortis (for preventing and treating false smut)Bacillus vallismortis) SCQN-5, characterized in that: the preservation number is as follows: CGMCC No.16564, the preservation date is: 10 months and 10 days in 2018, the preservation unit is: china general microbiological culture Collection center, preservation Address: the institute of microbiology, national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing.
2. Use of the bacillus vallismortis of claim 1 for biological control of ustilaginoidea virens.
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