CN111088191B - Bacillus and trichoderma combined culture method and application - Google Patents
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Abstract
The invention discloses a method for combined culture of bacillus and trichoderma, which comprises the following steps: culturing trichoderma, inoculating bacillus at preset intervals, and performing mixed fermentation culture to obtain the microbial symbiotic culture inoculant. Also discloses a microbial symbiotic culture microbial inoculum prepared by the method, and application of the microbial symbiotic culture microbial inoculum in preparation of a preparation for preventing and treating plant diseases and application in prevention and treatment of plant diseases, and the microbial symbiotic culture microbial inoculum can be used for preventing and treating plant damping-off, solanaceous wilt, plant soil-borne diseases and plant leaf diseases. The invention changes the traditional fermentation process of pure culture of single strain, relieves the problems of high energy consumption and low production efficiency caused by single strain fermentation, and the prepared microbial symbiotic culture microbial inoculum relieves the problems of poor biological control and plant growth promotion effects of the existing single microbial inoculum.
Description
Technical Field
The invention relates to the technical field of microbial symbiotic culture, in particular to a combined culture method of bacillus and trichoderma and application thereof.
Background
With the long-term use of a large amount of chemical agents, the drug resistance of pathogenic bacteria is gradually highlighted, and serious harm is brought to the environmental safety and human health. Therefore, the search for an environmentally friendly, safe and effective disease control method is receiving wide attention. The use of microorganisms and their metabolites to control pathogenic bacteria to control plant diseases is receiving increasing attention and attention. The biological preparation currently used in China has single microbial strain, weak pertinence and unsatisfactory persistence and broad spectrum. A large number of researches show that good reciprocal symbiotic relationship can exist among different bacterial strains, and the beneficial symbiotic relationship can obviously improve the growth rate and the metabolic efficiency of each bacterial strain in a mixing system, so that the yield of certain products is improved to a certain extent.
Brevibacillus laterosporus is widely distributed in nature and has been isolated from materials such as soil, volcanic mud flow, fresh water, seawater, insect body, leaf surface, locust bean, compost, milk, cheese, honey, starch food, waste water of glue factory, animal fur and quail. The brevibacillus laterosporus is a microbial resource which can resist bacteria and kill pests and has medical value. Brevibacillus laterosporus has various biological activities and exhibits a broad spectrum of antibacterial activities, particularly against bacteria and fungi. Recent whole genome sequencing results show that the Brevibacillus laterosporus also has the potential of producing polyketides, nonribosomal peptides, toxins and the like. It can also produce active substances such as aminopeptidase inhibitor, glutaryl 7-ACA acyltransferase, thrombin inhibitor, lysine and the like, and shows good application prospect. The antibacterial performance of the brevibacillus laterosporus is related to the antibacterial peptide produced by the brevibacillus laterosporus. Aiming at the insecticidal activity of Brevibacillus laterosporus, many foreign researches are carried out, and finished products of biological insecticides are on the market. A variety of Brevibacillus laterosporus are pathogenic to different invertebrates such as insects, nematodes and molluscs.
The insecticidal activity is mainly due to the fact that proteins contained in parasporal bodies have toxic effects on various insects, and different substances are generated in different stages of a cell growth cycle after the parasporal bodies contact or invade a target, so that the insecticidal activity has the toxic effect. The spores of the Brevibacillus laterosporus strain contain nematicidal compounds which inhibit the egg hatching and larval development of nematodes, and therefore, Brevibacillus laterosporus has become a biological control agent for parasitic nematodes. The Brevibacillus laterosporus also has the functions of dissolving phosphorus, potassium and the like. Research shows that the fertilizer is suitable for developing bacterial fertilizers applied to crops, and can improve the content of available phosphorus in soil and the yield of crops to a certain extent. Researches find that the Brevibacillus laterosporus can degrade isocarbophos, omethoate, isocarbophos and other organophosphorus pesticides. It has also been found that more and more substances are degraded by Brevibacillus laterosporus, the most typical example being the degradation of polyvinyl alcohol to acetate, and the production of various enzymes such as lignin peroxidase, laccase, aminopyrine-N-demethylase, NADH-DCIP reductase and malachite green reductase, etc. Therefore, the brevibacillus laterosporus has the characteristic of being developed into a biocontrol microbial inoculum for preventing and controlling insects, nematodes, molluscs and phytopathogens. Meanwhile, the phosphate and potassium dissolving agent has the biodegradation characteristics of dissolving phosphate and potassium and chemical pesticides and the like, so that the soil bioremediation capability is realized.
Bacillus amyloliquefaciens was first isolated in 1943 and distinguished from Bacillus subtilis, and in 1987 individual species were already present and appeared in the approved bacterial list. Bacillus amyloliquefaciens is a gram-positive strain, is aerobic and can produce spores, is often used as a model strain for researching the interaction of plant microorganisms, and is commonly used as a biological fertilizer and a biocontrol strain in agriculture. Bacillus amyloliquefaciens has the potential of promoting plant growth and inhibiting phytopathogens, such as secreting different antibacterial and antifungal secondary metabolites, promoting plant growth and activating host defense systems. Furthermore, the competition of biocontrol and pathogenic bacteria for nutrients, especially iron, is an important factor in plant protection. Analysis of the whole genome of Bacillus amyloliquefaciens revealed that 8.5% of the genome was used to produce some bioactive substances with high antibacterial activity, such as lipid peptides, e.g., iturin, antimycin D, and fengycin, and polyketides, e.g., macrolides, lysins, bacillomycin, and difenidine. The secondary metabolites produced by Bacillus amyloliquefaciens are capable of killing a pathogenic bacterium, Burkholderia melini, also found in soil. In addition, it can kill drug-resistant strains of Burkholderia pseudomallei, and inhibit other pathogenic bacteria such as Staphylococcus aureus, Escherichia coli and Acinetobacter baumannii. The bacillus amyloliquefaciens can also indirectly achieve the aim of controlling plant diseases by promoting the growth of plants. Compared with other biocontrol strains, the bacillus amyloliquefaciens is easier to culture and store, the thalli of the bacillus amyloliquefaciens can be inoculated on plant seeds or directly inoculated on plant bodies, and the growth of the plants is directly promoted by synthesizing gibberellin (influencing multiple processes of the growth of the plants, including germination of the seeds, elongation of stems, flowering phase and fruiting phase), indoleacetic acid, extracellular phytase (capable of enhancing the absorption of mineral elements of the plants), chitin, antifungal peptides and the like. The inventor adopts HPLC, PCR and other technologies to find that the bacillus amyloliquefaciens can produce surfactin, iturin and fengycin, and genes for synthesizing the three antibiotics exist. The bacillus amyloliquefaciens has broad-spectrum antagonistic activity to various pathogenic bacteria and can promote the growth of plants.
The history of trichoderma used for preventing and controlling plant diseases has been over 70 years, the biocontrol effect of the trichoderma is well known, and the trichoderma is a biocontrol bacterium which is widely applied all over the world. Trichoderma belongs to Deuteromycotina, Hyphomycetes, Moniliaceae, myxosporium, is a fungus which is commonly present in soil, is an important community of soil microorganisms, can be parasitic on plant residues and animal wastes, can be frequently separated from the surfaces of plant rhizosphere, leaves, seeds and bulbs, and can be parasitic on pathogenic bacteria of various soil-borne plants. With the rapid development of molecular biology, the research on trichoderma has mainly focused on improving the biological control effect of trichoderma in the eighties, such as by enhancing the expression of chitinase and glucanase genes, interspecies fusion and other means. Most trichoderma species are not critical in terms of nutrition, can grow on various carbon and nitrogen sources, and can convert and degrade some harmful or persistent harmful environmental pollutants; various monosaccharides, derivatives of monosaccharides, and organic acids can be directly used; the polysaccharide degrading agent has the advantages that various polysaccharides (cellulose and hemicellulose) and related polysaccharides (chitin) are remarkably degraded; some pesticides can also be converted and degraded, such as: malathion, dalapon, quintozene, and the like. The trichoderma can utilize complex and simple nitrogen, can well utilize hydrolyzed casein amino acid mixed liquor, aspartic acid, alanine and glutamic acid, and is favorable for the trichoderma to generate certain enzymes such as cellulase, lactase and the like under the condition of high nitrogen. The antagonism range of trichoderma has broad spectrum, and related research shows that trichoderma has antagonism to at least 18 pathogenic fungi of 29 genera. Antagonistic targets, which are plant pathogenic bacteria parasitized by trichoderma include rhizoctonia, sclerotinia sclerotiorum, sclerotinia, helminthosporium, fusarium, colletotrichum, verticillium, venturia, hypochnum, pythium, phytophthora, interstella, and venturia.
In long-term test and production practice, human beings have undergone two stages of pure-breed fermentation and mixed fermentation, and it is found that the pure-breed fermentation uses a single microorganism, the enzyme systems participating in biochemical reaction are few, and many important biochemical processes are that a single strain of microorganism cannot be completed or can only be weakly carried out. Different microorganisms have different metabolic pathways and metabolic products in the mixed fermentation process, and the synergistic effect of multiple microorganisms can complement and complement each other in advantages, improve the yield of certain metabolic products, save energy and reduce cost. This is an effect that cannot be achieved by single strain fermentation. How to perform symbiotic culture of various strains is an important issue that research and development personnel need to solve urgently.
Disclosure of Invention
The invention aims to provide a combined culture method of bacillus and trichoderma so as to solve the problems in the prior art, change the traditional fermentation process for pure culture of a single strain and relieve the problems of high energy consumption and low production efficiency caused by single strain fermentation.
The invention also aims to provide a microbial symbiotic culture microbial inoculum prepared by the combined culture method of bacillus and trichoderma, which solves the problem of poor biological control and plant growth promoting effects of the conventional single microbial inoculum.
The invention also provides application of the microbial symbiotic culture microbial inoculum in preparation of preparations for preventing and treating plant diseases, and the microbial symbiotic culture microbial inoculum can be prepared into various preparations for preventing and treating plant diseases.
The invention also provides application of the microbial symbiotic culture microbial inoculum in preventing and treating plant diseases, and the microbial symbiotic culture microbial inoculum can be used for preventing and treating various plant diseases.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a method for combined culture of bacillus and trichoderma, which comprises the following steps:
culturing trichoderma, inoculating bacillus at preset intervals, and performing mixed fermentation culture to obtain the microbial symbiotic culture inoculant.
Preferably, the preset interval time is 20-90 h.
Preferably, the Trichoderma harzianum is Trichoderma harzianum T4, Cathazianum Trichoderma harzianum, deposit number: CGMCC No.17197, preservation time: year 2019, 03, 25, storage location: china general microbiological culture Collection center;
the bacillus is at least one of bacillus amyloliquefaciens or bacillus brevis laterosporus, the bacillus amyloliquefaciens is bacillus amyloliquefaciens SN16-1, and the classification name is as follows: bacillus amyloliquefaciens with a preservation number: CGMCC No.17212, preservation time: day 18, 2019, month 01, storage location: china general microbiological culture Collection center; the brevibacillus laterosporus is brevibacillus laterosporus SN19-1, and the classification name is as follows: brevibacillus laterosporus, accession number: CGMCC No.17435, preservation time: year 2019, 03, 25, storage location: china general microbiological culture Collection center. And (4) storage address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
Preferably, the inoculation amount of Trichoderma harzianum is 1X 106-1×109cfu/L;
The inoculation amount of the bacillus amyloliquefaciens is 1 multiplied by 106-8×109cfu/L, the inoculation amount of the Brevibacillus laterosporus is 2 multiplied by 106-8×109cfu/L。
Preferably, the mixed fermentation culture conditions are as follows: the fermentation temperature is 25-37 ℃; the initial pH is 5-9; the aeration ratio is 1:2-2:1, and the tank pressure is 0.03-0.05 MPa; the total fermentation time is 96-144 h.
Preferably, the mixed fermentation culture medium is used for mixed fermentation culture, and comprises the following components in mass concentration: 15-35g/L soluble starch, 2-10g/L wheat bran, 3-15g/L bean cake powder, 0.5-3.5g/L ammonium sulfate, KH2PO4 0.3-1.6g/L,NaCl 0.2-1.8g/L, CaCl2 0.4-1.6g/L。
The invention also provides a microbial symbiotic culture microbial inoculum prepared by the bacillus and trichoderma combined culture method.
The invention also provides application of the microbial symbiotic culture fungicide in preparation of a preparation for preventing and treating plant diseases, and the microbial symbiotic culture fungicide is applied to prevention and treatment of soil-borne diseases such as plant damping-off, solanaceous wilt, plant wilt, cruciferous clubroot and the like, and leaf diseases such as rice blast, plant powdery mildew, plant gray mold and the like.
Preferably, the preparation is any one of water agent, powder, granules, microcapsules and suspending agent.
The invention also provides application of the microbial symbiotic culture microbial inoculum in preventing and treating plant diseases, and the microbial symbiotic culture microbial inoculum is applied to preventing and treating soil-borne diseases such as plant damping-off, solanaceous wilt, plant wilt, cruciferous clubroot and the like, and leaf diseases such as rice blast, plant powdery mildew, plant gray mold and the like.
The invention discloses the following technical effects:
the invention discloses a method for combined culture of bacillus and trichoderma, which is characterized in that two completely different strains are adopted, one strain is cultured firstly, and then the other strain is inoculated at preset intervals, so that the two strains are mixed and symbiotic, and the technical problems that the energy consumption is high due to single strain fermentation, the existing mixed strain preparation process is complicated and complex, and fungi and bacteria can not be symbiotically cultured to obtain a compound microbial inoculum are solved. According to the method, the trichoderma with a longer growth cycle is fermented and cultured to a chlamydospore formation stage, and the bacillus is inoculated for co-culture when the oxygen and nutrition requirements of the trichoderma are reduced to obtain the composite fermentation liquid, namely the microbial symbiotic culture microbial inoculum.
The microbial symbiotic culture microbial inoculum prepared by the invention has good stability and strong adaptability, does not have the phenomena of unbalanced and simplified floras, greatly simplifies the fermentation steps compared with the prior mixed strain culture method, reduces the production cost, can realize mass production through fermentation, is convenient for industrialization and popularization, and can be flexibly applied to actual production in different industries.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 shows the result of identifying 16S rRNA of Bacillus amyloliquefaciens;
FIG. 2 shows the results of phylogenetic tree analysis of Bacillus amyloliquefaciens based on 16S rRNA detection;
FIG. 3 shows the result of phylogenetic tree analysis of Brevibacillus laterosporus based on the 16S rDNA detection result;
FIG. 4 shows the results of phylogenetic tree analysis of Trichoderma harzianum based on the 16S rDNA assay;
FIG. 5 shows the results of the bacteriostatic activity of example 2;
FIG. 6 shows the results of promoting plant growth in example 3.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
The "parts" in the present invention are all parts by mass unless otherwise specified.
The trichoderma is trichoderma harzianum T4 (preservation number is CGMCC No. 17197); the bacillus amyloliquefaciens is bacillus amyloliquefaciens SN16-1 (the preservation number is CGMCC No. 17212); the Brevibacillus laterosporus is Brevibacillus laterosporus SN19-1 (preservation number is CGMCC No.17435), and is preserved in China university of eastern science and technology.
The present inventors screened symbiotic fermentation medium components before co-culturing bacillus and trichoderma harzianum, as follows.
1. Screening of fermentation medium components for combined culture of bacillus and trichoderma
1.1 kinds of carbon sources, nitrogen sources, inorganic salts
Carbon source: maltose, sucrose, glucose, soluble starch, wheat bran, corn starch, glycerol;
nitrogen source: peptone, yeast extract, peanut powder, bean cake powder, ammonium sulfate, sodium nitrate and urea;
inorganic salts: KH (Perkin Elmer)2PO4、K2HPO4、NaCl、Na2HPO4、MgSO4、CaCl2;
1.2 screening Process for Components
(1) Activation and identification of the target strain: respectively inoculating the three strains of bacillus amyloliquefaciens SN16-1 and the brevibacillus laterosporus SN19-1 to an LB solid culture medium, and culturing for 24 hours in a constant-temperature incubator at 33 ℃; and inoculating the trichoderma harzianum T4 into a PDA solid culture medium, culturing for 72h in a constant-temperature incubator at 30 ℃, scraping thalli after the culture is finished, and extracting DNA for identification.
(2) Liquid culture: selecting single colonies of bacillus and trichoderma respectively inoculating in an LB liquid culture medium and a PDA liquid culture medium, wherein the two culture media are required to be cultured for 24 hours and 72 hours respectively in a constant temperature shaking table at 30 ℃ to obtain sufficient bacterial liquid.
(3) Obtaining a seed solution: respectively inoculating the two strains according to various inoculation amounts (1 percent of bacillus and 1 percent of trichoderma), and respectively culturing the two strains in a 250mL volumetric culture bottle containing an LB liquid culture medium and a PDA liquid culture medium for 24 hours and 72 hours in a constant temperature shaking table at 30 ℃ to obtain sufficient bacterial liquid.
(4) Shake flask mixed fermentation: single factor assay for optimal carbon, nitrogen and inorganic salts selection with initial fermentation medium (glucose, peptone, MgSO)4,KH2PO4NaCl) is used as a basic culture medium, 7 carbon sources or 7 nitrogen sources or 6 inorganic salts which are commonly used in industry and have low price are respectively selected as substitutes, and 7 groups are formed as different substitute culture media. The control was performed on media without original components, 3 replicates of each treatment were incubated for 5 days, after which time samples were taken for determination of biomass and OD values.
Optionally inoculating 1 or 2 strains of Bacillus and Trichoderma seed solution at a ratio of 1:1 (total inoculation amount is 2%) into different culture media, adjusting initial pH of the culture medium to 7.5, and shake culturing at 29 deg.C and 200r/min for 72 h. Determining the OD value of the fermentation liquid by using an ultraviolet-visible spectrophotometer at 600nm and counting by using a flat plate counting method to determine the optimal component.
(5) Shake flask mixed fermentation: after the optimal fermentation culture medium of the optimal carbon source and the optimal nitrogen source is determined; and selecting different concentrations of inorganic salt species for mixed fermentation test. And determining the optimal concentration by measuring the OD value of the fermentation liquor by an ultraviolet-visible spectrophotometer at 600nm and counting by a flat plate counting method.
(6) Shake flask mixed fermentation: and (3) performing fermentation of the compound strain by using the optimized optimal culture medium, and changing the fermentation conditions of the culture medium. And determining the OD value of the fermentation liquor by using an ultraviolet-visible spectrophotometer at 600nm and counting by using a flat plate counting method to determine the optimal fermentation condition.
(7) Sampling and detecting: inoculating spore bacteria and Trichoderma into optimized culture medium at ratio of 1:1 (total inoculation amount is 2%), adjusting initial pH of the culture medium to 7.5, performing shake fermentation at 29 deg.C and 200r/min for 72 hr, measuring OD value of the fermentation liquid under 600nm with ultraviolet-visible spectrophotometer, and counting by plate counting method to obtain viable bacteria number of 109cfu/L。
In the screening process, the fermentation liquids in the steps are subjected to gradient dilution and then are subjected to content averaging counting by a standard flat plate coating method, and the results are shown in table 1.
TABLE 1 results of determination of the contents of different treated bacteria in example 1
From table 1, it can be seen that the mixed fermentation medium obtained by single-factor optimization of the basal medium significantly improves the viable bacteria content of the three strains in the mixed fermentation, and is significantly superior to the basal medium. The fermentation medium can ensure that the mixed strain can grow normally during co-culture. The optimum components are soluble starch, organic nitrogen source bean cake powder, wheat bran, inorganic nitrogen source ammonium sulfate and KH2PO4,NaCl,CaCl2。
Example 1
And comparing the detection results of pure culture and symbiotic culture fermentation biomass according to the screening result.
1. The strain is as follows: bacillus amyloliquefaciens SN16-1, Brevibacillus laterosporus SN19-1 and Trichoderma harzianum T4 (purchased from China general microbiological culture Collection center).
2. Culture medium: LB seed medium (0.6L required): 10g of peptone, 5g of yeast extract, 10g of NaCl, 1000mL of water, pH7.0 and sterilization at 121 ℃ for 20 min.
PDA seed medium (0.6L required): 200g of potato, 20g of glucose and 1000mL of water, and sterilizing at 121 ℃ for 25 min.
Mixed fermentation medium (30L): 23g/L of soluble starch, 5g/L of wheat bran, 2g/L of ammonium sulfate and KH2PO4 1g/L,NaCl 1g/L,CaCl2 1g/L。
3. A fermentation step:
(1) activating Bacillus amyloliquefaciens SN16-1, Bacillus laterosporus SN19-1 and Trichoderma harzianum T4 strains stored at 4 ℃ by LB plates and PDA plates respectively, and culturing in a constant-temperature incubator at 29 ℃ for 2d and 5d respectively.
Respectively inoculating SN16-1 and SN19-1 strains into LB liquid culture medium, culturing for 12h in a constant-temperature shaking incubator at 29 ℃ and 200r/min, adding 10mL of sterilized normal saline into each plate of the T4 strain full of spores, scraping and inoculating sporangium into PDB culture medium by using an applicator, and culturing for 72h in the constant-temperature shaking incubator at 29 ℃ and 200 r/min.
Wherein the bacterial content of SN16-1 is 1 × 108cfu/mL, the spore concentration of T4 bacterial liquid is adjusted to 1 × 106one/mL.
(2) Shaking the flask: inoculating SN16-1 and SN19-1 to an LB culture medium for 12h, wherein the inoculation amount is 2%, the temperature is 30 ℃, the initial pH value is 7.0, the liquid loading amount is 100mL/250mL, and the rotating speed is 200 r/min. Shaking the flask: a PDA culture medium is used for inoculating T4 for 72h, the inoculum size is 2%, the liquid loading volume is 100mL, the rotating speed is 180rpm, the culture temperature is 28 ℃, and the culture time is 72 h.
(3)30L of amplification fermentation culture: synchronous fermentation is carried out by adopting 30L three-connected tank, and a co-culture group number D and E and three pure culture groups A, B and C are arranged. Pure culture groups A, B and C are respectively pure culture fermentation of strains SN16-1, SN19-1 and strain T4, a co-culture group D is mixed fermentation of trichoderma and bacillus amyloliquefaciens SN16-1, and a co-culture group E is mixed fermentation of trichoderma and bacillus laterosporus SN 19-1.
Fermentation conditions of pure culture groups A, B and C are as follows: the inoculation amount is 2 percent, the charging coefficient is 0.667, the temperature is 29 ℃, the initial pH7.0, the rotating speed is 200r/min, and the ventilation volume1.8m3200mL of vegetable oil (antifoaming agent); the total fermentation time is 3d and 5d respectively.
Co-culture group D, E fermentation conditions: the inoculation amount (1:1) is 2 percent, and the strain SN16-1 is inoculated after the strain T4 is inoculated for 3 days; the charging coefficient is 0.667, the temperature is 29 ℃, the initial pH7.0, the rotating speed is 200r/min, the ventilation volume is 1.8m3/h, and the volume of vegetable oil (antifoaming agent) is 200 mL; the total fermentation time is 5 d.
After the fermentation liquids obtained from different groups A-E were diluted in a gradient manner, viable bacteria content was measured by a standard plate coating method, and the results are shown in Table 2.
TABLE 2 viable organism content of groups A-E
As can be seen from Table 2, the fermentation process and the inoculation method using the fermentation medium disclosed in the present invention can ensure that the bacteria content of each strain in the co-culture fermentation broth is not much different from the bacteria content of pure culture, and the method can ensure that the mixed strains can grow normally during co-culture.
Example 2
Comparing the inhibiting effects of pure culture broth and symbiotic culture broth on common plant pathogenic bacteria
1. Strain:
antagonistic strains: bacillus amyloliquefaciens SN16-1 and Trichoderma harzianum T4.
Indicator pathogen: tomato wilt pathogen, watermelon anthracnose pathogen, grape gray mold pathogen, rhizoctonia solani, xanthomonas, and ralstonia solani.
2. Culture medium:
PDA culture medium: 200g of potato, peeling, slicing, boiling for 30min, filtering, adding 20g of sucrose into the filtrate, metering to 1000mL, adding 20g of agar, and keeping the pH value natural.
LB culture medium: 10g of peptone, 5g of yeast extract, 10g of NaCl, 1000ml of water, pH7.0 and sterilization at 121 ℃ for 20 min.
Co-culture fermentation medium: 23g/L of soluble starch, 5g/L of wheat bran, 2g/L of ammonium sulfate and KH2PO4 1g/L,NaCl 1g/L,CaCl2 1g/L。
3. Determination of bacteriostatic Activity
Culturing bacillus in an LB culture medium for 48 hours, culturing trichoderma in a PDA culture medium for 5 days, culturing in a co-culture fermentation culture medium for 5 days, and determining the antibacterial activity by adopting a plate confronting method: drawing a vertical cross at the bottom of the flat plate; inoculating a control group pure culture fermentation liquid and a treatment group mixed culture fermentation liquid at a position 20mm away from the center of the PDA plate; inoculating indicator pathogenic bacteria strain with diameter of 5mm at the center of the plate, culturing at 29 deg.C for 5-7d, and measuring the width of antagonistic band when the antagonistic bacteria and each indicator bacteria colony generate obvious bacteriostatic band. The control is carried out by inoculating no antagonistic bacteria and only the indicator bacteria.
the pure culture broth and the symbiotic culture broth have the inhibition effect on common phytopathogens, and the results are shown in table 3 as follows: the inhibition capacity of the composite strain fermentation liquor to pathogenic bacteria is far greater than that of pure culture fermentation liquor of a single strain, and the inhibition rate is obviously improved.
TABLE 3 inhibition of pathogenic bacteria by the respective treatments
Example 3
Indoor pot culture test for detecting biocontrol and growth promotion capability of pure culture and symbiotic culture fermentation broth
1. The strain is as follows: bacillus amyloliquefaciens SN16-1, Brevibacillus laterosporus SN19-1 and Trichoderma harzianum T4.
2. Culture medium: same as example 2
3. Test method
Sowing surface-sterilized tomato seeds (treated with 2% NaClO for 3min, rinsed with sterilized water 3 times) in a pvc flowerpot (containing 350g soil +10g perlite), sowing 6 seeds in each pot, and shake-culturing for 7 daysSolanum wilt bacterium (FOL) at 5 × 106The spores/g of soil were added to the pots without the treatment group with added pathogens watering the same volume of water. The culture temperature was set at 25 ℃ and the light was irradiated for 16 hours per day. After 2 cotyledons grow out of the plant, pouring pure culture fermentation liquor and mixed symbiotic culture fermentation liquor, wherein the concentration is 108cfu/g, water with the same volume is poured into the treatment groups without adding biocontrol bacteria, the pot culture test is totally divided into 4 treatment groups, namely a control group (CK) without adding fermentation liquor, pure culture fermentation liquor, mixed culture fermentation liquor and FOL only adding pathogenic bacteria. Each treatment group was repeated 3 times and tomato plants were sampled every 10 days.
4. Test index
And (3) measuring the biomass of the tomato plant: measuring the fresh weight, dry weight, plant length, stem length, root length and other related biomass of the plant sampled each time; each treatment group was repeated 3 times.
And (3) counting the tomato plant disease index: the potting test was repeated under the same conditions as above for the disease index statistics of tomato plants, each treatment group comprising 18 plants for a total of 6 treatment groups. And (3) the standard of statistical reference of plant disease degree: 0-no onset symptoms; 1-less than 25%, leaves showing symptoms; 2-26-50%, leaf showing symptoms; 3-51-75%, leaf showing symptoms; 4-, 76-100%, leaf showing symptoms; 5-death of the plant. The disease index is counted according to a formula.
Disease index [ ∑ (number of diseased plants at each stage × corresponding series)/(total number of investigated plants × highest series) ] × 100%
5. Results
Statistical analysis is carried out on the growth promotion and biological control effects of the fermentation liquor of each group, the influence of different treatment groups and sampling time on plant biomass is shown in the table 4, and the results show that: the plants in the 2 treatment groups added with the pure culture fermentation broth and the mixed culture fermentation broth grow fastest, and compared with a control group, the biomass parameters of plant height, stem length, fresh weight and dry weight are all significantly different; and the growth promoting effect of the mixed culture fermentation broth on plants is superior to that of pure culture fermentation broth of a single strain. The disease index results for the different treatments are shown in table 5, showing: the effect of the mixed culture fermentation liquor is superior to that of pure culture fermentation liquor when preventing and treating the tomato wilt, and the effect is more obvious particularly at the later stage of tomato growth for 50 days.
TABLE 4 Effect of different treatment groups and sampling time on plant Biomass
TABLE 5 disease indices for different treatments
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (8)
1. A method for combined culture of bacillus and trichoderma comprises the following steps:
culturing trichoderma, inoculating bacillus at preset interval time of 20-90h, and performing mixed fermentation culture to obtain a microbial symbiotic culture microbial inoculum;
the Trichoderma is Trichoderma harzianum T4 with the preservation number of CGMCC No. 17197; the Bacillus is Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) SN16-1 with the preservation number of CGMCC No. 17212; the mixed fermentation culture conditions are as follows: the fermentation temperature is 29 ℃, and the total fermentation time is 5 days.
2. The Bacillus of claim 1The method for culturing Trichoderma harzianum in combination is characterized in that the inoculation amount of Trichoderma harzianum is 1 × 106-1×109cfu/L;
The inoculation amount of the bacillus amyloliquefaciens is 1 multiplied by 106-8×109cfu/L。
3. The method for co-culturing bacillus and trichoderma as claimed in claim 1, wherein the mixed fermentation culture conditions are: the fermentation temperature is 25-37 ℃; the initial pH is 5-9; the aeration ratio is 1:2-2:1, and the tank pressure is 0.03-0.05 MPa; the total fermentation time is 96-144 h.
4. The method for co-culturing bacillus and trichoderma as claimed in claim 1, wherein the mixed fermentation culture medium is used for mixed fermentation culture, and comprises the following components by mass concentration: 15-35g/L soluble starch, 2-10g/L wheat bran, 3-15g/L bean cake powder, 0.5-3.5g/L ammonium sulfate, KH2PO4 0.3-1.6g/L,NaCl 0.2-1.8g/L,CaCl2 0.4-1.6g/L。
5. The microbial symbiotic culture inoculant prepared by the method for combined culture of bacillus and trichoderma as claimed in any one of claims 1 to 4.
6. The use of the microbial symbiotic inoculant according to claim 5 in the preparation of a preparation for controlling plant diseases, wherein the microbial symbiotic inoculant is used for controlling plant damping-off, solanaceous wilt and tomato wilt.
7. The use according to claim 6, wherein the preparation is any one of aqueous agent, powder, granule, microcapsule and suspending agent.
8. The application of the microbial symbiotic culture inoculant in preventing and treating plant diseases according to claim 5, wherein the microbial symbiotic culture inoculant is applied to preventing and treating plant damping-off, solanaceous wilt and tomato wilt.
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CN111793498A (en) * | 2020-07-31 | 2020-10-20 | 福建三炬生物科技股份有限公司 | Microbial agent and preparation method and application thereof |
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CN116083251B (en) * | 2023-02-23 | 2023-07-07 | 天津坤禾生物科技集团股份有限公司 | Mixed microbial agent and preparation method and application thereof |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102027998A (en) * | 2009-09-24 | 2011-04-27 | 华东理工大学 | Bacillus spore and trichoderma chlamydospore compound preparation and preparation method and application thereof |
CN102964178A (en) * | 2012-11-13 | 2013-03-13 | 湖南泰谷生物科技股份有限公司 | Composite microbial bactericide, and preparation method and application thereof |
CN105132336A (en) * | 2015-10-10 | 2015-12-09 | 中国热带农业科学院环境与植物保护研究所 | Bacillus amyloliquefaciens and microbial inoculum and application thereof |
CN207803320U (en) * | 2018-01-25 | 2018-09-04 | 安徽旭辰生物科技有限公司 | A kind of bacillus subtilis and trichoderma microcapsule granule |
CN109497265A (en) * | 2018-12-17 | 2019-03-22 | 江苏春之雨生物科技发展有限公司 | It is a kind of using pomace, vinasse as the biological feedstuff mixed fungus fermentation method of raw material |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP6183851B2 (en) * | 2013-08-22 | 2017-08-23 | 国立研究開発法人農業・食品産業技術総合研究機構 | Soil infectious disease control method and testing method for microorganisms for soil infectious disease control |
BR112020024509A2 (en) * | 2018-06-05 | 2021-03-02 | Novozymes Bioag A/S | methods of foliar application of a composition, of controlling plant pests on a plant or part of the plant and / or inducing resistance to a plant pest on a plant or part of the plant and of controlling or preventing pest damage in a plant plant propagation material, a plant, part of a plant and / or plant organ. |
CN110423718B (en) * | 2019-08-13 | 2021-10-15 | 上海交通大学 | Method for producing trichoderma chlamydospore by using bacillus fermentation liquor and application |
-
2020
- 2020-01-08 CN CN202010018482.9A patent/CN111088191B/en active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102027998A (en) * | 2009-09-24 | 2011-04-27 | 华东理工大学 | Bacillus spore and trichoderma chlamydospore compound preparation and preparation method and application thereof |
CN102964178A (en) * | 2012-11-13 | 2013-03-13 | 湖南泰谷生物科技股份有限公司 | Composite microbial bactericide, and preparation method and application thereof |
CN105132336A (en) * | 2015-10-10 | 2015-12-09 | 中国热带农业科学院环境与植物保护研究所 | Bacillus amyloliquefaciens and microbial inoculum and application thereof |
CN207803320U (en) * | 2018-01-25 | 2018-09-04 | 安徽旭辰生物科技有限公司 | A kind of bacillus subtilis and trichoderma microcapsule granule |
CN109497265A (en) * | 2018-12-17 | 2019-03-22 | 江苏春之雨生物科技发展有限公司 | It is a kind of using pomace, vinasse as the biological feedstuff mixed fungus fermentation method of raw material |
Non-Patent Citations (6)
Title |
---|
Effects of the biocontrol agent Bacillus amyloliquefaciens SN16-1 on the rhizosphere bacterial community and growth of tomato;Tingting Wan等;《Journal of Phytopathology》;20180516;第166卷(第5期);第324-332页 * |
侧孢短芽孢杆菌的应用研究进展;陈潺等;《山东农业科学》;20150228;第47卷(第2期);第149页摘要部分和第151页第2.2.1小节 * |
多种微生物复合降解玉米秸秆的研究;郝建宇;《中国优秀硕士学位论文全文数据库(电子期刊)工程科技Ⅱ辑》;20190115(第1期);对比文件1正文第18-19页 * |
生防菌哈茨木霉Trichoderma harzianum T4对西瓜根围土壤细菌群落的影响;夏飞等;《中国生物防治学报》;20130507;第29卷(第2期);第232页摘要部分 * |
解淀粉芽孢杆菌SN16-1对番茄根际细菌群落的影响及其生防机制初步探讨;万婷婷;《中国优秀硕士学位论文全文数据库(电子期刊)农业科技辑》;20180815(第8期);对比文件3摘要部分以及正文第17页表2.6 * |
郝建宇.多种微生物复合降解玉米秸秆的研究.《中国优秀硕士学位论文全文数据库(电子期刊)工程科技Ⅱ辑》.2019,(第1期),C041-81. * |
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Application publication date: 20200501 Assignee: Shanghai Digital Printing Intelligent Technology Co.,Ltd. Assignor: EAST CHINA University OF SCIENCE AND TECHNOLOGY Contract record no.: X2022310000146 Denomination of invention: A method of co culture of bacillus and trichoderma and its application Granted publication date: 20210402 License type: Exclusive License Record date: 20221026 |