CN112574931B - Biocontrol microbial inoculum for preventing and treating garlic diseases as well as preparation method and application thereof - Google Patents
Biocontrol microbial inoculum for preventing and treating garlic diseases as well as preparation method and application thereof Download PDFInfo
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- 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
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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
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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
- A01N63/27—Pseudomonas
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- 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
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Abstract
The invention provides a biocontrol microbial inoculum for preventing and treating garlic diseases, a preparation method and application thereof. The biocontrol microbial inoculum is a composite microbial inoculum consisting of pseudomonas aeruginosa and bacillus subtilis, and is prepared by mixing 2 kinds of single-bacterium fermentation liquor in proportion after single-bacterium fermentation. Wherein the name of the pseudomonas aeruginosa isPseudomonas aeruginosaAnd has been preserved in China general microbiological culture collection center at 12 months and 1 day of 2020 with the preservation number of CGMCC NO. 21276. The biocontrol microbial inoculum is diluted by water, evenly mixed with soil in a proper proportion and mashed, and the evenly-mixed soil is taken to wrap garlic seeds for garlic seeding. The garlic biocontrol microbial inoculum can effectively inhibit the growth of pathogenic bacteria and pathogenic fungi of garlic and can be applied to preventing and treating garlic diseases caused by pathogenic bacteria.
Description
Technical Field
The invention relates to the technical field of agricultural microorganisms, in particular to a biocontrol microbial inoculum for preventing and treating garlic diseases and a preparation method and application thereof.
Background
Garlic is one of common seasonings and also has high nutritional and medicinal values. The garlic is an important economic crop, has a long cultivation history in China, and has large market potential and large demand at home and abroad. Due to continuous cropping planting in successive years, unscientific fertilizer and water management and other factors, garlic diseases occur frequently, the yield and quality of garlic are seriously affected, and the garlic planting industry and the garlic deep processing industry are endangered.
Garlic diseases such as garlic blight, garlic purple spot, garlic leaf blight, garlic soft rot and the like. Diseases in the whole growth period of garlic include soft rot, sclerotinia, rust, epidemic disease, purpura, leaf blight, white rot, phytophthora root rot and the like, most of the diseases caused by pathogenic bacteria can cause the yield reduction and even the failure of garlic production if the diseases are not controlled in time. The method for preventing and treating garlic diseases is very important for strengthening the inhibition and elimination of pathogenic bacteria besides the measures of garlic seed selection, crop rotation planting, reasonable soil fertilizer and water management and the like.
The agricultural disease control follows the strategy principle of prevention as main and comprehensive control. The single chemical agent has narrow antibacterial spectrum and is difficult to radically cure diseases caused by various pathogenic bacteria. The long-term use of chemical agents on the one hand confers resistance to certain pathogenic bacteria and on the other hand influences the growth of certain beneficial microorganisms in the soil. Chemical agents also have the problems of difficult degradation or incomplete degradation and the like, have potential ecological risks to the environment, and can cause the problem of pesticide residues. With the development of agriculture and the technological progress, more efficient, green, low-toxicity and low-residue antibacterial technologies gradually attract attention.
The biosurfactant is used for preventing and treating the agricultural pathogenic bacteria, is safe, effective, economical and environment-friendly, and accords with the strategy principle of prevention as the main and comprehensive prevention in agricultural prevention and treatment.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a green, environment-friendly, economic and effective biocontrol microbial inoculum for preventing and treating garlic diseases and a preparation method thereof.
In order to achieve the purpose, the invention adopts the technical scheme that:
pseudomonas aeruginosaPseudomonas aeruginosaThe biological preservative is preserved in China general microbiological culture collection center with the preservation number of CGMCC NO.21276 and the preservation date of 2020, 12 months and 1 day.
A biocontrol microbial inoculum containing pseudomonas aeruginosa. The application of the biocontrol microbial inoculum in inhibiting the pathogenic bacteria and pathogenic fungi of garlic.
The active ingredient of the biocontrol microbial inoculum is a composite microbial inoculum consisting of pseudomonas aeruginosa and bacillus subtilis. The preservation number of the pseudomonas aeruginosa is CGMCC number 21276, and the bacillus subtilis can be any bacillus subtilis strain.
The biocontrol microbial inoculum is in a liquid state and is a mixed solution of single-bacterium fermentation liquor of pseudomonas aeruginosa and bacillus subtilis.
The preparation method of the biocontrol microbial inoculum comprises the following specific steps:
1) and (4) preparing seed liquid. Respectively scraping thallus slopes of 2 bacteria by utilizing inoculating loops, respectively inoculating the thallus slopes into a liquid LB culture medium, and culturing pseudomonas aeruginosa for 8-12 hours at 30-35 ℃ and 180rpm to prepare a seed solution; and culturing the bacillus subtilis for 6-8 h at 30-35 ℃ and 180rpm to prepare a seed solution.
2) And (5) performing single-bacterium fermentation. The fermentation culture medium of the pseudomonas aeruginosa takes glycerol or glucose as a carbon source, the fermentation culture medium of the bacillus subtilis takes cane sugar or molasses as a carbon source, the fermentation inoculation amount is 2% -10%, the fermentation time is 3-6 days, and the fermentation temperature is 30-40 ℃.
3) And (4) mixing the fermentation liquors. The mixing ratio of single-bacterium fermentation liquor of pseudomonas aeruginosa to single-bacterium fermentation liquor of bacillus subtilis is 1: 1-5.
The biocontrol microbial inoculum can effectively inhibit the growth of pathogenic bacteria and pathogenic fungi of garlic and can be used for preventing and treating garlic diseases caused by the pathogenic bacteria.
According to the application method of the biocontrol microbial inoculum, after the biocontrol microbial inoculum is diluted by 50-100 times by water, the diluted solution and soil are uniformly mixed and mashed according to the proportion of 10-30 mL to 100g, and 80-120 g of uniformly blended soil is taken to wrap garlic seeds for garlic seeding.
The invention has the beneficial effects that:
the biocontrol microbial inoculum for preventing and treating garlic diseases is simple and convenient to produce and prepare, has good bacteriostatic effect on garlic pathogenic bacteria and pathogenic fungi, can be used as soil beneficial microorganisms, is ecological and friendly, and has a certain promotion effect on plant growth. The biocontrol microbial inoculum is green, environment-friendly, economical and effective, can effectively inhibit the growth of pathogenic bacteria and pathogenic fungi of garlic, and can be used for preventing and treating garlic diseases caused by pathogenic bacteria.
Information on strain preservation
Preservation time: 12 months and 1 day 2020;
the preservation unit: china general microbiological culture Collection center;
the preservation number is: CGMCC NO. 21276;
the address of the depository: the preservation address is microbial research institute of China academy of sciences No. 3 of Xilu No. 1 of Beijing, Chaoyang district;
and (3) classification and naming: pseudomonas aeruginosaPseudomonas aeruginosa。
Drawings
FIG. 1 is a photograph showing the bacteriostatic action of the biocontrol microbial inoculum of the invention on pathogenic bacteria and pathogenic fungi of garlic.
Detailed Description
The present invention is described in detail with reference to the following specific examples to provide a more complete understanding of the present invention to those of ordinary skill in the art, but the present invention is not limited thereto in any way. In the following examples, materials and reagents used were all available from Biochemical materials, Inc., unless otherwise specified.
Pseudomonas aeruginosaPseudomonas aeruginosaAnd has been preserved in China general microbiological culture collection center at 12 months and 1 day of 2020 with the preservation number of CGMCC NO. 21276.
Example 1
The culture medium used for preparing the seed liquid is liquid LB culture medium: 10g of peptone, 5g of yeast powder and 10g of NaCl, and adding deionized water to 1L, wherein the pH value is 7.0.
Fermentation medium of pseudomonas aeruginosa: glucose 35g, NaNO3 3.0g， KH2PO4 3.0g，MgSO4∙7H2O1.0 g，CaCl2∙2H2O 0.15g，K2HPO4∙3H2O4.0 g, KCl 0.5g, deionized water to 1L。
Fermentation medium of bacillus subtilis: sucrose 30g, NaNO3 3.0g， KH2PO4 3.0g，MgSO4∙7H2O1.0 g，CaCl2∙2H2O 0.15g，K2HPO4∙3H2O4.0 g, KCl 0.5g, NaCl 0.5g, deionized water to 1L.
Respectively weighing the reagents by using an electronic balance, and preparing corresponding culture media; subpackaging, sterilizing at 115 deg.C for 30 min.
The strains used in the embodiment take pseudomonas aeruginosa CGMCC number 21276 and bacillus subtilis CGMCC number 13461 as examples.
After the culture medium is cooled to 20-40 ℃, scraping a strain slope ring by using an inoculating ring, inoculating the strain slope ring into a triangular flask containing 100ml of LB culture medium, and culturing pseudomonas aeruginosa for 10 hours at 35 ℃ and 180rpm to prepare a seed solution; the bacillus subtilis is cultured for 8 hours at 30 ℃ and 180rpm to prepare seed liquid.
Then, inoculating the seed solution into a fermentation culture medium by 3 percent of inoculation amount (v/v), and culturing pseudomonas aeruginosa for 5 days at 35 ℃ and 180rpm to prepare the seed solution; the bacillus subtilis is cultured for 4 days at 30 ℃ and 180rpm to obtain single-strain fermentation liquor of 2 strains.
Example 2
Respectively taking 2 single-bacterium fermentation liquids in the embodiment 1, 20mL of single-bacterium fermentation liquid of pseudomonas aeruginosa and 40mL of single-bacterium fermentation liquid of bacillus subtilis, and uniformly mixing in a 3L beaker; adding 2940mL of deionized water, and diluting by 50 times to obtain the biological control microbial inoculum for standby.
Taking 200ml of the obtained biocontrol microbial inoculum, adding the biocontrol microbial inoculum into 1kg of soil while turning soil, uniformly mixing and mashing, taking 100g of uniformly mixed soil, taking a garlic clove, wrapping the garlic clove by the soil, and preparing a mud ball with the outer edge of the soil and the center of the garlic clove for garlic sowing.
Example 3
The biocontrol microbial inoculum obtained in the embodiment 2 is used for acting on garlic pathogenic bacteria and pathogenic fungi, the size of a bacteriostatic zone is measured, and the bacteriostatic effect of the biocontrol microbial inoculum on different garlic pathogenic bacteria is evaluated.
Preparing LB liquid culture medium and potato dextrose liquid culture medium, adding 2% agar powder for sterilization, and preparing LB agar culture medium plate and PDA culture medium (potato dextrose agar culture medium) plate for respectively culturing garlic pathogenic bacteria and garlic pathogenic fungi. The garlic pathogenic bacteria to be tested are plant pathogenic bacteria (strains L2#, L5#, L6#, L7 #) and plant pathogenic fungi (strains P4#, P12#, P14#, P16 #) screened from garlic disease in the laboratory.
Respectively preparing seed liquid of bacteria to be tested and fungi to be tested by using LB liquid culture medium and potato glucose liquid culture medium, and diluting the seed liquid by 105After doubling, the plates were plated on LB agar medium and PDA medium, respectively. Then, a sterile filter paper sheet with the diameter of 10mm is placed on the flat plate, and 10 mu L of the biocontrol microbial inoculum prepared by mixing and diluting the single-bacterium fermentation liquid in the embodiment 2 is respectively dripped on the filter paper sheet. The plate was placed in an incubator at 30 ℃ for 2 days. After the culture was completed, the size (mm) of the zone of inhibition around the filter paper sheet was measured, and the experimental results are shown in table 1.
Table 1: the biocontrol microbial inoculum has the bacteriostatic effect on different garlic pathogenic bacteria
The result shows that the biocontrol microbial inoculum of the invention has good inhibition effect on 4 kinds of selected garlic pathogenic bacteria and 4 kinds of selected garlic pathogenic fungi.
Example 4
In this example, compared with a single-strain fermentation broth (PR) of Pseudomonas aeruginosa CGMCC number 21276 and a single-strain fermentation Broth (BL) of Bacillus subtilis, the biocontrol microbial inoculum of the invention, i.e. a 1:2 mixed solution (PB) of the Pseudomonas aeruginosa fermentation broth and the Bacillus subtilis fermentation broth, has an antibacterial effect on different garlic pathogenic bacteria.
Referring to the bacteriostasis experimental method in the embodiment 3, the bacteriostasis effect is compared by using the bactericide PR, the bactericide BL and the biocontrol bactericide PB of the invention to act on three plant pathogenic bacteria. The test garlic pathogenic bacteria are garlic pathogenic bacteria (strains L5#, L6#, L7 #) and garlic pathogenic fungi (strains P12#, P14#, P16 #) screened from disease garlic in the laboratory, and the test results are shown in Table 2.
Table 2: bacteriostatic effect of different microbial inoculum on garlic pathogenic bacteria
The results show that the biocontrol microbial inoculum of the invention has good inhibition effect on selected garlic pathogenic bacteria, and has the best inhibition effect among 3 microbial inocula.
As shown in figure 1, the partial bacteriostasis photos of 3 microbial inoculum to garlic pathogenic bacteria and pathogenic fungi are shown in the upper 3 panels as test garlic pathogenic bacteria (strains L5#, L6#, and L7 #), and in the lower 3 panels as test garlic pathogenic fungi (strains P12#, P14#, and P16 #). In the figure, BA is a certain purchased microbial inoculum, and the microbial inoculum PR: the single-strain fermentation liquid of pseudomonas aeruginosa CGMCC number 21276, the microbial inoculum BL: single-strain fermentation liquor of bacillus subtilis, and a microbial inoculum PB: the invention relates to a biocontrol microbial inoculum. As shown in the figure, the biocontrol microbial inoculum has the largest inhibition zone on the tested strains, which shows that the antibacterial effect is the best.
The biocontrol microbial inoculum has good bacteriostatic effect on garlic pathogenic bacteria, is simple and convenient to prepare, is green and environment-friendly, has broad-spectrum bacteriostatic action, can be applied to prevention and control of garlic diseases caused by plant pathogenic bacteria, and has important popularization and application values in garlic planting.
Claims (9)
1. Pseudomonas aeruginosaPseudomonas aeruginosaThe biological preservative is preserved in China general microbiological culture collection center with the preservation number of CGMCC NO.21276 and the preservation date of 2020, 12 months and 1 day.
2. A biocontrol agent comprising pseudomonas aeruginosa according to claim 1.
3. The biocontrol microbial inoculum of claim 2 wherein: the active ingredient of the biocontrol microbial inoculum is a composite microbial inoculum consisting of pseudomonas aeruginosa and bacillus subtilis.
4. The biocontrol microbial inoculum of claim 3 characterized in that: the biocontrol microbial inoculum is a mixed solution of pseudomonas aeruginosa CGMCC number 21276 and single-strain fermentation liquor of bacillus subtilis.
5. A method for preparing the biocontrol microbial inoculum according to any one of claims 3 to 4, characterized by comprising the steps of: 1) preparing seed liquid, 2) fermenting by single bacterium, and 3) mixing fermentation liquid.
6. The preparation method of the biocontrol microbial inoculum according to claim 5, wherein the conditions of single-strain fermentation in the step 2) are as follows: the fermentation culture medium of the pseudomonas aeruginosa CGMCC number 21276 takes glycerol or glucose as a carbon source, the fermentation culture medium of the bacillus subtilis takes cane sugar or molasses as a carbon source, the fermentation inoculation amount is 2-10% of the volume fraction, the fermentation time is 3-6 days, and the fermentation temperature is 30-40 ℃.
7. The method for preparing the biocontrol microbial inoculum according to claim 5, wherein in the step 3): the mixing ratio of the pseudomonas aeruginosa to the single bacillus subtilis fermentation liquor is 1: 1-5.
8. Use of pseudomonas aeruginosa according to claim 1 or a biocontrol agent according to any one of claims 2 to 4 for inhibiting pathogenic bacteria and pathogenic fungi of garlic.
9. The application of claim 8, wherein after the biocontrol microbial inoculum is diluted by 50-100 times with water, the diluted solution and soil are uniformly mixed and mashed according to the proportion of 10-30 mL to 100g, and 80-120 g of uniformly blended soil is taken to wrap garlic seeds for garlic seeding.
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