CN117487706A - Antagonistic bacteria for preventing and controlling root rot of radix angelicae and biological organic fertilizer - Google Patents
Antagonistic bacteria for preventing and controlling root rot of radix angelicae and biological organic fertilizer Download PDFInfo
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- 239000003895 organic fertilizer Substances 0.000 title claims abstract description 113
- 230000003042 antagnostic effect Effects 0.000 title claims abstract description 46
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- 239000010902 straw Substances 0.000 claims abstract description 35
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- 241000863030 Lysobacter enzymogenes Species 0.000 claims abstract description 15
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- 238000009264 composting Methods 0.000 claims abstract description 7
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Classifications
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- C—CHEMISTRY; METALLURGY
- C05—FERTILISERS; MANUFACTURE THEREOF
- C05C—NITROGENOUS FERTILISERS
- C05C9/00—Fertilisers containing urea or urea compounds
-
- 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
-
- 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
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
- C05F17/20—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment
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- 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
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- 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
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- 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
- C12N1/205—Bacterial isolates
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- 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
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- 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
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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Abstract
The invention discloses an antagonistic bacterium Le395 for preventing and controlling root rot of angelica dahurica, which is classified and named as lysobacter enzymogenes (Lysobacter enzymogenes) and has a strain preservation number of CGMCC No.28570. The invention discloses application of antagonistic bacteria Le395 in preventing and controlling root rot of angelica dahurica. The invention discloses a biological organic fertilizer which is obtained by inoculating antagonistic bacteria Le395 into an organic fertilizer prepared by composting solidago canadensis straw and/or corn straw as main raw materials. Le395 against pathogenic bacteria causing root rot of dahurian angelica rootFusarium has good inhibition effect. The strain Le395 is used for developing straw, especially solidago canadensis, into bio-organic fertilizer, and the spore number is at least 10 8 Individual/g; le395 can colonize the rhizosphere of dahurian angelica root and produce antagonistic substances to inhibit the growth of pathogenic microorganisms on the rhizosphere of dahurian angelica root, and the biological control rate of the dahurian angelica root rot is more than 80%.
Description
Technical Field
The invention belongs to the technical field of agricultural intensification, and relates to antagonistic bacteria Le395 for preventing and controlling root rot of angelica dahurica, application of the antagonistic bacteria Le395 in preventing and controlling root rot of angelica dahurica, and a biological organic fertilizer prepared by composting solidago canadensis straw and/or corn straw and mixing the biological organic fertilizer with antagonistic bacteria Le395 fermentation liquor.
Background
Radix Angelicae Dahuricae (Angelica dahurica) is a perennial herb, and is moist and drought-intolerant. Radix Angelicae Dahuricae has effects of relieving exterior syndrome, dispelling cold, dispelling pathogenic wind, relieving pain, relieving stuffy nose, relieving swelling, expelling pus, dispelling pathogenic wind, and relieving itching, and contains main chemical components with anti-tumor and liver protecting effects. The available land resources are continuously reduced, so that the rotation period of the radix angelicae is short, the planted radix angelicae has continuous cropping obstacle, the ecological community structure composition is single, the species diversity is low, favorable conditions are created for the occurrence of disease dominant species, and the development of the radix angelicae industry is seriously restricted. Research shows that the angelica dahurica soil-borne disease is a soil-borne root disease caused by various pathogenic bacteria, wherein the root rot disease is mainly caused by fusarium (Fusarium oxysporun). At present, an effective means for preventing and treating root rot of angelica dahurica is lacking, and the prevention and treatment of diseases by utilizing soil microorganisms are one of important measures for comprehensively preventing and treating soil-borne diseases. The biological control technology meets the requirements of modern agriculture on environmental protection, food safety and the like, and is one of sustainable effective methods for realizing agriculture.
Compared with crops such as vegetables, melons and fruits, the bio-organic fertilizer has fewer applicable product types in Chinese medicinal material planting.
Disclosure of Invention
The invention aims to provide an antagonistic bacterium for preventing and controlling root rot of dahurian angelica.
An antagonistic bacterium Le395 for preventing and controlling root rot of radix angelicae is named as lysobacter enzymogenes (Lysobacter enzymogenes) and is preserved in China general microbiological culture collection center (CGMCC) with a strain preservation number of 28570 in the 9 th month 28 of 2023.
The main biological characteristics of the antagonistic bacterium Le395 are that the optimal growth temperature on a TSA culture medium is 28 ℃, and the antagonistic bacterium Le395 is cultured for 1 to 2 days to form a colony with the length of 2 to 4 mm.
Another object of the invention is to provide the application of the antagonistic bacterium Le395 in preventing and controlling root rot of dahurian angelica.
The invention also aims to provide the application of the antagonistic bacterium Le395 in preparing a bio-organic fertilizer for preventing and controlling root rot of angelica dahurica.
The invention also aims to provide a biological organic fertilizer, which is obtained by inoculating antagonistic bacteria Le395 into an organic fertilizer prepared by composting solidago canadensis straw and/or corn straw as a main raw material, wherein the spore number of the antagonistic bacteria Le395 in the biological organic fertilizer is more than or equal to 10 8 Each/g.
The invention also aims to provide a preparation method of the bio-organic fertilizer, which comprises the following steps:
step (1), preparing fermentation liquor: inoculating antagonistic bacteria Le395 into a liquid culture medium, and performing shake culture to obtain seed liquid; pouring the seed liquid into a fermentation barrel filled with a sterilized liquid culture medium, introducing sterile air, and fermenting for 2d at 28-30 ℃ to obtain fermentation liquid;
step (2), preparing a biological organic fertilizer: composting the solidago canadensis straw and/or the corn straw serving as main raw materials to prepare an organic fertilizer; uniformly mixing the organic fertilizer and the fermentation liquor prepared in the step (1) to prepare the bio-organic fertilizer, wherein the spore number of the antagonistic bacteria Le395 in the bio-organic fertilizer is more than or equal to 10 8 Each/g.
In the step (1), the formula of the liquid culture medium is as follows: 15g/L peptone, 5g/L soybean peptone, 5g/L sodium chloride and pH 7.1-7.5.
Preferably, the seed solution is cultured: the antagonistic bacteria Le395 single colony is inoculated into 100mL of liquid culture medium, placed in a shaking table, and cultured for 24 hours at the temperature of 28-30 ℃ and the rotating speed of 180r/min to obtain seed liquid.
Preferably, the preparation of the fermentation broth comprises the following steps: pouring the seed liquid into a fermentation barrel filled with a sterilized liquid culture medium according to the inoculation amount of 2.5%, introducing sterile air, and fermenting for 2d at 28-30 ℃ to obtain fermentation liquor.
Preferably, the number of spores of antagonistic Le395 in the fermentation broth is 10 8 ～10 9 And each mL.
In the step (2), preferably, the organic fertilizer is prepared by the following method: crushing and drying solidago canadensis straws or corn straws, adding urea accounting for 1% -5% of the mass of the straws, adjusting the water content to be about 60% -65%, piling up the straws into a fermentation pile, turning over the fermentation pile once every day in the first three days, increasing the contact area of the fermentation pile and air, heating the fermentation pile, measuring the temperature of the fermentation pile every day in the fourth day, increasing the turning over frequency when the temperature of the fermentation pile is higher than 70 ℃, fermenting for about 25 days, and stopping fermenting when the color of the fermentation pile is deepened, the water content is reduced to about 30% -40%, and the temperature is reduced to about 35-40 ℃ to obtain the organic fertilizer.
Preferably, the organic fertilizer and the fermentation broth are uniformly mixed according to the dosage ratio of the fermentation broth to the organic fertilizer of 1:10 mL/g.
The invention also aims to provide the application of the biological organic fertilizer in preventing and controlling root rot of angelica dahurica and/or promoting the growth of the angelica dahurica.
The invention has the beneficial effects that:
1. the biocontrol strain Le395 has a good inhibition effect on pathogenic bacteria Fusarium (Fusarium oxysporun) causing root rot of angelica dahurica.
2. The biocontrol strain Le395 is utilized to develop the straw, especially the solidago canadensis, into the bio-organic fertilizer with higher added value, and the spore number reaches at least 10 8 Individual/g; and the solidago canadensis is taken as a production raw material, has large quantity, low cost and easy obtainment, can meet the nutrition requirement of the production of the bio-organic fertilizer, has the production cost of only 1/2 to 1/3 of that of the conventional bio-organic fertilizer, and is favorable for popularization and application of the bio-organic fertilizer of the lysobacter enzymogenes. Meanwhile, the solidago canadensis can be prevented and controlled. Therefore, the production of the lysobacter bio-organic fertilizer by using the solidago canadensis is a low-cost and high-efficiency production method.
3. The biological organic fertilizer has the action mechanism that the antagonistic bacteria Le395 in the biological organic fertilizer can well colonize the rhizosphere of the angelica dahurica, generate antagonistic substances, inhibit the growth of pathogenic microorganisms of the rhizosphere of the angelica dahurica and improve the stress resistance of crops. The test result shows that after the bio-organic fertilizer is applied to the soil with the root rot of the angelica dahurica, the biological control rate of the root rot of the angelica dahurica is more than 80%, the smooth development of intensive agriculture is ensured, and the bio-organic fertilizer has important significance for preventing and controlling the root rot of the angelica dahurica, improving the quality of Chinese medicinal materials and protecting the environment.
4. The lysobacter bio-organic fertilizer is a biological bacterial fertilizer, and does not cause a series of problems due to the use of chemical pesticides, so that the pollution-free production of the angelica dahurica is facilitated, the use amount of other chemical pesticides for preventing and treating soil-borne diseases can be reduced by farmers, the cost can be saved for the farmers, and the quality of Chinese medicinal materials is improved, so that the income of the farmers is increased.
Drawings
FIG. 1 is a phylogenetic tree of antagonistic bacterium Le 395.
FIG. 2 shows the number of viable bacteria of the antagonistic Le395 in different matrices.
FIG. 3 is a graph showing the amount of antagonistic Le395 in soil to which organic fertilizer and chemical fertilizer are applied; wherein OF is soil to which organic fertilizer is applied, CF is soil to which chemical fertilizer is applied, and F.o is treated by adding pathogenic bacteria.
FIG. 4 is a flow chart of a method for preparing a bio-organic fertilizer from Solidago canadensis straw.
Figure 5 shows the effect of different treatments on the incidence of root rot of dahurian angelica.
FIG. 6 shows the disease index of the treated radix Angelicae Dahuricae.
FIG. 7 shows the control effect of different treatments on root rot of dahurian angelica root.
Information on preservation of strains
Le395, classified and named as lysobacter enzymogenes (Lysobacter enzymogenes), was deposited in China general microbiological culture Collection center (CGMCC) at 9 and 28 of 2023, with a strain deposit number of CGMCC No.28570.
Detailed Description
Liquid medium for strain Le395 liquid seed culture and broth preparation, taking 1L liquid medium as an example: 15g of peptone, 5g of soybean peptone, 5g of sodium chloride and pH 7.1-7.5; deionized water is sterilized for 20min at 121 ℃ to 1L.
Example 1
1. Isolation of strains
Collecting the rhizosphere soil of solidago canadensis in the Liwater area Bai Mazhen Bai Maji of Nanjing, jiangsu province, and separating antagonistic bacteria by adopting a dilution coating method, wherein the specific method comprises the following steps of: measuring 90mL distilled water, adding into 250mL conical flask, sterilizing at 121deg.C for 20min, collecting 10g of Solidago canadensis rhizosphere soil, placing into the conical flask, shaking at 28deg.C for 30min at 180r/min, and sucking 10 with a pipette -1 Transferring 1mL of gradient soil dilution liquid into a test tube filled with 9mL of sterile water, uniformly mixing, sequentially carrying out gradient dilution to obtain soil suspensions with different dilution gradients, sucking 50 mu L of the soil suspension, coating on a TSA culture medium plate, culturing at 28 ℃ for 1d, picking single bacterial colonies, coating on the TSA culture medium, culturing at 28 ℃ for 1d, and carrying out biological identification and preservation on the bacterial strains.
2. Antagonistic screening and identification
(1) Source of pathogenic bacteria: the pathogenic bacteria of the root rot disease of the angelica dahurica is fusarium oxysporum (Fusarium oxysporun), and the root rot disease is separated from the root soil of the angelica dahurica by a laboratory.The screening process comprises the following steps: weighing 1g of disease-causing soil sample, filling the sample into a 50mL triangular flask, adding 9mL of sterile water, mixing, and sealing; placing the triangular flask in a constant temperature shaking table at 30deg.C, oscillating at 180r/min for 30min, standing on an ultra-clean workbench for 30min, sucking 1mL supernatant into a 1.5mL sterilized centrifuge tube, collecting 100 μL supernatant, and diluting to 10 according to a ratio of 1:9 with water -2 、10 -3 An isocratic gradient. 100. Mu.L of the supernatants of different dilution gradients were pipetted onto PDA solid medium and the coated plates were placed upside down in a 28℃incubator for 3d. Mycelium with different forms is selected and inoculated on a new PDA solid culture medium, and purified in an incubator at 28 ℃ for 3d, and identified as fusarium oxysporum (Fusarium oxysporun) by the strain.
(2) Antagonistic screening: screening the optimal strain with inhibition effect on pathogenic bacteria Fusarium oxysporum of radix Angelicae Dahuricae root rot by adopting a plate counter method. Culturing Fusarium oxysporum at 28deg.C for 3d, punching into bacterial cake with puncher (phi=6mm), and transferring to the center of plate of PDA culture medium; dripping fungus of the strain to be screened, which is cultured for 1d at 28 ℃ in a TSB culture medium, around fungus cakes, culturing for 3d at 28 ℃, measuring the antibacterial diameter, screening out a strain Le395 with good antagonistic effect, and storing the strain in a refrigerator at-80 ℃.
(3) And (3) strain identification: extracting DNA from bacteria by using a bacterial genome DNA extraction kit, taking the DNA as a template, and carrying out PCR amplification by taking a bacterial 16S rRNA universal primer 27F and a bacterial 1492R as an amplification primer; the fungus uses a fungus genome DNA extraction kit to extract DNA, and uses the DNA as a template and fungus ITS universal primers ITS1 and ITS4 as amplification primers to carry out PCR amplification. The PCR amplified products were subjected to 1% agarose gel electrophoresis and sent to company for sequencing, and the sequences obtained by sequencing were subjected to BLAST homology comparison in NCBI, and phylogenetic tree was constructed by using MEGA5 software (see FIG. 1).
The antagonistic bacteria Le395 was identified as lysobacter enzymogenes by sequencing by the biosystems by NCBI alignment (Lysobacter enzymogenes). The main biological characteristics of the lysobacter enzymogenes Le395 are: the culture is carried out for 1 to 2 days on the TSA culture medium at the optimal growth temperature of 28 ℃ to form colonies with the length of 2 to 4 mm.
The strain Le395, classified and named as lysobacter enzymogenes (Lysobacter enzymogenes), is preserved in China general microbiological culture Collection center (CGMCC) at 9 and 28 days of 2023, and has a strain preservation number of CGMCC No.28570.
Example 2
Preparation of Le395 bacterial liquid: le395 single colonies were taken from TSB solid medium and inoculated into 100mL of sterilized liquid medium for liquid seed culture under the following conditions: the initial pH of the culture solution is 7.1-7.5, the culture temperature is 28 ℃, the rotation speed of a shaking table is 180r/min, and the culture is carried out for 24 hours to obtain seed solution; sterilizing the fermenter containing liquid culture medium at 121deg.C, cooling the culture medium in the fermenter to room temperature, pouring seed solution under aseptic condition with inoculation amount of 2.5%, sealing the inoculation port, introducing sterile air, fermenting at 28-30deg.C for 2d to obtain spore amount of 10% 9 Le395 bacterial liquid in each mL.
Preparing a solidago canadensis straw organic fertilizer: crushing and drying the solidago canadensis straws, adding urea accounting for 1% of the mass of the straws, adjusting the moisture to about 60% (preferably, grasping the straws by hands and just extruding water drops through finger joints), piling into a fermentation pile, turning the fermentation pile once every day for the first three days, increasing the contact area between the fermentation pile and air, heating the fermentation pile, measuring the temperature of the fermentation pile every day in the fourth day, increasing the pile turning frequency when the temperature of the fermentation pile is higher than 70 ℃, and stopping fermentation when the color of the fermentation pile is deepened, the moisture is reduced to about 40%, and the temperature is reduced to about 40 ℃ to obtain the organic fertilizer.
Preparation of fusarium oxysporum bacterial liquid: selecting pathogenic bacteria Fusarium oxysporum separated from dahurian angelica root disease soil (same as in example 1), inoculating to a PDA plate, culturing for 3d at 28 ℃, punching the PDA plate by using a puncher to pick up about 5 bacterial cakes, putting into a PDB culture medium, putting 300mL of the PDB culture solution into each triangular flask, putting the triangular flask on a shaking table, and culturing for 3d at 28 ℃ at 180r/min in an oscillating way to obtain Fusarium oxysporum bacterial liquid.
(one), comparing the number of live antagonistic bacteria Le395 in different decomposed carriers
The experimental procedure is as follows: selecting tobacco stalk, rice stalk (i.e. straw), sorghum stalk, corn stalk, canadian oneAnd composting and decomposing the goldenrod straw, the pig manure and the sheep manure outdoors for 25 days respectively. Weighing 2g of decomposed carrier, sterilizing, placing into 50mL of sterilized centrifuge tubes, adding one decomposed carrier into each centrifuge tube, adding 8mL of inorganic salt culture medium without carbon source to ensure that the water content of the decomposed carrier is 80%, inoculating Le395 bacterial liquid according to 1% of the mass of the decomposed carrier, fully vibrating and mixing, placing into a constant temperature and humidity incubator, standing and fermenting at 28 ℃ for 7d, adding 18mL of sterile water, placing into a constant temperature shaking table at 28 ℃, vibrating at 180r/min for 30min, standing for 30min on an ultra-clean workbench, sucking 200 mu L of supernatant, and measuring OD on an enzyme-labeling instrument 600 。
The formula of the inorganic salt culture medium comprises the following steps: 3.0g of monopotassium phosphate, 3.0g of sodium nitrate, 0.5g of magnesium sulfate heptahydrate and 0.5g of calcium chloride, and the deionized water is fixed to a volume of 1L.
As shown in FIG. 2, compared with the decomposed carriers prepared from tobacco stalk, straw, sorghum stalk, pig manure, sheep manure and the like, the decomposed carriers prepared from the invasive plant Solidago canadensis stalk and corn stalk are good carriers for antagonizing the development of Le 395. And the solidago straw is used for preparing the bio-organic fertilizer, so that the cost is low, the invasion of solidago canadensis is prevented and controlled, and the popularization and the application of the bio-organic fertilizer in a large range are facilitated.
(II) comparing the viable count of the antagonistic bacterium Le395 in the soil to which the organic fertilizer is applied and the soil to which the chemical fertilizer is applied
The specific experimental procedure is as follows: soil with organic fertilizer (solidago canadensis straw organic fertilizer, application amount of 500 kg/mu) and fertilizer (compound fertilizer N: P: k=15%: 15%, application amount of 50 kg/mu) applied in a Li water area Bai Mazhen Bai Maji of Nanjing, jiangsu province is collected. The groups are OF (le395+ F.o), OF (le395), CF (le395+ F.o), CF (le395), OF (le395+ F.o): weighing 4g OF sterilized soil applied with an Organic Fertilizer (OF) in a 6-hole plate, adding Le395 bacterial liquid according to the inoculum size OF 5%, adding 1mL OF fusarium oxysporum (fusarium oxysporum liquid is centrifuged, and discarding supernatant); OF (Le 395) group: weighing 4g OF sterilized soil applied with an Organic Fertilizer (OF) in a 6-hole plate, and adding Le395 bacterial liquid according to the inoculation amount OF 5%; CF (le395+ F.o) group: weighing 4g of sterilized soil to which a Chemical Fertilizer (CF) is applied, adding Le395 bacterial liquid according to the inoculation amount of 5%, adding 1mL of fusarium oxysporum bacterial liquid (fusarium oxysporum bacterial liquid is centrifuged, and discarding the supernatant); CF (Le 395) group: 4g OF sterilized soil to which an Organic Fertilizer (OF) was applied was weighed into a 6-well plate, and Le395 bacteria liquid was added at an inoculum size OF 5%. 3 replicates of each treatment were incubated at room temperature, and the number of Le395 viable bacteria in the soil was determined and recorded using a dilution coating method for different time periods.
As a result, as shown in FIG. 3, the antagonistic bacterium Le395 has a higher viable count in the soil to which the organic fertilizer is applied, and hardly survives in the soil to which the chemical fertilizer is applied, and thus controls root rot of dahurian angelica root by the form of preparing the bio-organic fertilizer.
Example 3
The preparation method of the bio-organic fertilizer is shown in fig. 4, and comprises the following steps:
step (1), culturing strain Le395 seed solution: le395 single colonies were taken from TSB solid medium and inoculated into 100mL of sterilized liquid medium for liquid seed culture under the following conditions: the initial pH of the culture solution is 7.1-7.5, the culture temperature is 28 ℃, the rotation speed of a shaking table is 180r/min, and the culture is carried out for 24 hours to obtain seed solution;
step (2), preparing fermentation liquor: sterilizing the fermenter containing liquid culture medium at 121deg.C, cooling the culture medium in the fermenter to room temperature, pouring seed liquid under aseptic condition with inoculation amount of 2.5%, sealing the inoculation port, introducing sterile air, fermenting at 28-30deg.C for 2d to obtain spore amount of 10% 9 Fermentation broth of individual/mL;
step (3), preparing an organic fertilizer by taking solidago canadensis straws as a raw material: crushing and drying solidago canadensis straws, adding urea accounting for 1% of the mass of the straws, adjusting the moisture to about 60% (preferably, grasping the straws by hands and extruding water drops by finger joints), turning the fermentation stack once every day for the first three days after fermentation stacking, increasing the contact area between the fermentation stack and air, heating the fermentation stack, measuring the temperature of the fermentation stack every day in the fourth day, increasing the turnover frequency when the temperature of the fermentation stack is higher than 70 ℃, fermenting for about 25 days, and stopping fermentation when the color of the fermentation stack is deepened, the moisture is reduced to about 40%, and the temperature is reduced to about 40 ℃ to obtain the organic fertilizer;
step (4), preparing a biological organic fertilizer: adding the fermentation liquor into the organic fertilizer with 10% of inoculation amount (mL/g, calculated according to the weight of compost), repeatedly stirring to uniformly mix the fermentation liquor and the organic fertilizer, spreading out the fermentation pile to air-dry moisture, and detecting that the lysobacter enzymogenes Le395 in the biological organic fertilizer is 10 by a dilution coating method when the fermentation pile moisture is lower than 30% 8 And CFU/g, and each detection of the organic fertilizer index is according to the requirements of NY884-2012, thus obtaining the bio-organic fertilizer.
Example 4
1. Pathogen inoculation liquid
Selecting pathogenic bacteria Fusarium oxysporum separated from dahurian angelica root disease soil (same as in example 1), inoculating to a PDA plate, culturing for 3d at 28 ℃, punching the PDA plate by using a puncher to pick about 5 bacterial cakes, placing into a PDB culture medium, placing 300mL of PDB culture solution into each triangular flask, placing the triangular flask on a shaking table, and culturing for 3d at 28 ℃ under shaking at a rotating speed of 180 r/min.
2. Greenhouse effect prevention test
The capability of the antagonistic strain Le 395-containing bio-organic fertilizer for preventing and controlling the soil-borne diseases of the angelica dahurica is examined. The test design is as follows:
control (CK): only seedling raising soil;
organic fertilizer treatment (OF): seedling raising soil and organic fertilizer treatment;
bio-organic fertilizer treatment (BOF): seedling raising soil and biological organic fertilizer treatment;
1.5kg of seedling soil/pot, 1 seedling/pot per 12 pots treated. OF: applying an organic fertilizer (15 g, the organic fertilizer prepared in the step (3) of the example 3) accounting for 1% of the total mass of seedling raising soil to each pot, and mixing the organic fertilizer with potting soil to prepare a base fertilizer; BOF: the bio-organic fertilizer (15 g, bio-organic fertilizer prepared in the step (4) of example 3) accounting for 1% of the total mass of the seedling raising soil is applied to each pot, and the bio-organic fertilizer is mixed with the seedling raising soil to serve as a base fertilizer. The angelica dahurica is transplanted into a pot of 1.5kg seedling raising soil in 2023, 7 and 5 days through seedling raising trays, after 7d transplanting, pathogenic bacteria are evenly poured into soil near the root of the angelica dahurica by adopting a root irrigation method, and the final concentration of the pathogenic bacteria inoculated into each pot of the angelica dahurica is 10 8 Sampling when radix Angelicae Dahuricae grows in pot for 21 days, counting morbidity, calculating disease index, determining total fresh weight of plant, and investigating root fresh weightControl and promote growth.
The disease degree of the root rot of the angelica dahurica is classified into 4 grades. Level 0 is that root system does not develop disease; the incidence rate of 1-level root system is less than or equal to 25%, and the leaves are normal; grade 2 is that the incidence rate of the root system is less than or equal to 50 percent, and the leaves turn yellow; grade 3 is that the morbidity of the root system is less than or equal to 75 percent, and the leaves wither; the level 4 is that the incidence rate of root system is more than 75 percent, and the leaves are dry.
The disease index and the prevention and treatment effect are respectively calculated according to the following formulas:
disease index= [ Σ (number of disease-level strains×representative value)/(total number of plants×highest disease-level representative value) ]100
Control effect = [ (control disease index-treatment disease index)/control disease index ] ×100%
Both the biological organic fertilizer treatment and the organic fertilizer treatment can reduce the incidence of root rot (see figure 5), wherein the biological organic fertilizer treatment shows better control effect, the control effect is 81.6 percent (see figure 6), the control effect of the organic fertilizer is only 18.5 percent, the fresh weight of the overground parts of the angelica dahurica subjected to the biological organic fertilizer treatment and the organic fertilizer treatment is respectively increased by 560 percent, 109 percent, and the fresh weight of the root is respectively increased by 51.4 percent and 44.5 percent. It is shown that the application of the bio-organic fertilizer and the organic fertilizer can promote the growth and development of the angelica dahurica, but the application of the bio-organic fertilizer has more remarkable effect on promoting the growth of the angelica dahurica (see table 1).
TABLE 1 influence of treatments on the control of root rot of dahurian angelica root and on the growth of dahurian angelica root in greenhouse pot experiments
The invention starts from the key problem of improving the soil microbiology, develops the bio-organic fertilizer capable of obviously reducing the occurrence of the root rot of angelica dahurica, and has the control rate of the root rot of angelica dahurica up to more than 80%.
Fusarium oxysporum used in the examples of the present invention is only for the purpose of illustrating that "antagonistic bacteria Le395 is capable of controlling root rot of dahurian angelica", but the used Fusarium oxysporum is not limited to this source. It will be clear to the person skilled in the art that the use of Fusarium oxysporum, which is correspondingly pathogenic and identified as causing root rot of Angelica dahurica, is suitable for use in the present invention.
Claims (10)
1. An antagonistic bacterium Le395 for preventing and controlling root rot of radix angelicae is named as lysobacter enzymogenes (Lysobacter enzymogenes) and is preserved in China general microbiological culture collection center (CGMCC) with a strain preservation number of 28570 in the 9 th month 28 of 2023.
2. Use of the antagonistic bacterium Le395 according to claim 1 for controlling root rot of dahurian angelica.
3. Use of the antagonistic Le395 according to claim 1 for the preparation of a bio-organic fertilizer for controlling root rot of dahurian angelica.
4. A biological organic fertilizer is characterized in that: the bio-organic fertilizer is obtained by inoculating the antagonistic bacteria Le395 in the method of claim 1 into an organic fertilizer prepared by composting solidago canadensis straw and/or corn straw as main raw materials, wherein the spore number of the antagonistic bacteria Le395 in the bio-organic fertilizer is more than or equal to 10 8 Each/g.
5. A method for preparing the bio-organic fertilizer according to claim 4, wherein: the method comprises the following steps:
step (1), preparing fermentation liquor: inoculating antagonistic bacteria Le395 into a liquid culture medium, and performing shake culture to obtain seed liquid; pouring the seed liquid into a fermentation barrel filled with a sterilized liquid culture medium, introducing sterile air, and fermenting for 2d at 28-30 ℃ to obtain fermentation liquid;
step (2), preparing a biological organic fertilizer: composting the solidago canadensis straw and/or the corn straw serving as main raw materials to prepare an organic fertilizer; uniformly mixing the organic fertilizer and the fermentation liquor prepared in the step (1) to prepare the bio-organic fertilizer, wherein the spore number of the antagonistic bacteria Le395 in the bio-organic fertilizer is more than or equal to 10 8 Each/g.
6. The method for preparing the bio-organic fertilizer according to claim 5, wherein: in the step (1), the formula of the liquid culture medium is as follows: 15g/L peptone, 5g/L soybean peptone, 5g/L sodium chloride and pH 7.1-7.5.
7. The method for preparing the bio-organic fertilizer according to claim 5, wherein: in the step (1), the seed solution is cultured: inoculating antagonistic Le395 single colony into 100mL of liquid culture medium, placing into a shaking table, and culturing for 24h at the temperature of 28-30 ℃ and the rotating speed of 180r/min to obtain seed liquid;
the preparation of the fermentation liquor comprises the following steps: pouring the seed liquid into a fermentation barrel filled with a sterilized liquid culture medium according to the inoculation amount of 2.5%, introducing sterile air, and fermenting for 2d at 28-30 ℃ to obtain fermentation liquor.
8. The method for preparing the bio-organic fertilizer according to claim 5, wherein: in the step (1), the spore number of the antagonistic Le395 in the fermentation broth is 10 8 ～10 9 And each mL.
9. The method for preparing the bio-organic fertilizer according to claim 5, wherein: in the step (2), the organic fertilizer is prepared by the following method: crushing and drying solidago canadensis straws or corn straws, adding urea accounting for 1-5% of the mass of the straws, adjusting the water content to 60-65%, piling into a fermentation pile, turning once a day in the first three days, measuring the temperature of the fermentation pile every day in the fourth day, increasing the pile turning frequency when the temperature of the fermentation pile is higher than 70 ℃, and stopping fermentation when the color of the fermentation pile is deepened, the water content is reduced to 30-40%, and the temperature is reduced to 35-40 ℃ to obtain the organic fertilizer.
10. The use of the bio-organic fertilizer according to claim 4 for controlling root rot of dahurian angelica root and/or promoting growth of dahurian angelica root.
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