CN113444660B - Bacillus amyloliquefaciens strain LXBA.1 and application thereof - Google Patents
Bacillus amyloliquefaciens strain LXBA.1 and application thereof Download PDFInfo
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Abstract
The invention provides a bacillus amyloliquefaciens strain LXBA.1 and application thereof. The bacillus amyloliquefaciens strain LXBA.1 has a preservation number of CGMCC NO. 21868; the strain can effectively relieve the influence of autotoxic substances such as p-hydroxybenzoic acid and the like on the growth vigor of plants, has strong bacteriostatic activity on various germs such as strawberry root rot, ginger stem rot, potato scab and the like, has the growth promoting performances such as silicon decomposition, phosphorus potassium dissolution, nitrogen fixation, indoleacetic acid production, siderophin production and the like, has strong colonization ability, and has obvious effects on promoting the growth and development of root systems, preventing and controlling soil-borne diseases, improving the crop yield, improving the crop quality and the like, thereby greatly reducing the use of chemical fertilizers and pesticides; in addition, the strain also shows certain saline-alkali resistance activity and stronger bacteriophage resistance, is a safe, green and efficient multifunctional broad-spectrum disease-resistant growth-promoting strain, and has wide industrial application prospect.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a bacillus amyloliquefaciens strain LXBA.1 and application thereof.
Background
The continuous cropping obstacle refers to the phenomenon that the same crop is continuously planted in the same land, and the disease is aggravated and the growth and development are poor after the second growing season, so that the quality and the yield are seriously reduced. Causes of continuous cropping obstacles are complex, and mainly comprise soil hardening and secondary salinization, unbalanced distribution of soil nutrients, deterioration of a soil micro-ecosystem, aggravation of soil-borne diseases and insect pests, plant autotoxicity and the like; among them, autotoxicity of plants has been receiving much attention as one of the main factors causing continuous cropping obstacles. The plant autotoxicity is chemical substances which are generated by plants and have toxic action on the same species or the same family plants, and the chemical substances can be released into the environment through the overground part leaching of plant bodies, root secretion, plant stubble and other ways, so that the growth of the next stubble of crops is influenced.
Strawberry is one of the most seriously affected crops due to continuous cropping obstacles, and certain allelopathic substances in strawberry root secretion have self-toxicity effect, which can inhibit the activity of root systems, cause the disease resistance of the root systems to be reduced, and aggravate root diseases; wherein, the p-hydroxybenzoic acid has high content in the root exudates of the strawberries and has the strongest self-toxicity. If the degradation of the autotoxic substances of the strawberry root system in the continuous cropping soil can be accelerated, the continuous cropping obstacle of the strawberry can be effectively controlled. The main path of decomposing autotoxic substances in soil is biodegradation, wherein microorganisms are main biological groups for initiating biodegradation, and the microorganisms with degradation effect on the autotoxic substances are utilized to degrade the autotoxic substances in the soil so as to reduce continuous cropping obstacles, so that the method is a safe and effective biological control measure.
Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is a Bacillus and is a bacterium with high affinity with Bacillus subtilis (Bacillus sub-tilis). At present, bacillus amyloliquefaciens preparations on the market often have certain defects in the practical application process, such as single disease control, unstable biocontrol effect, poor field colonization capacity, poor disease prevention and growth promotion comprehensive effect performance and the like; therefore, the development of a multifunctional high-efficiency microbial inoculum with comprehensive functions, multiple disease prevention types and obvious growth promotion effect is urgently needed.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a bacillus amyloliquefaciens strain LXBA.1 and application thereof, wherein the strain can effectively relieve the influence of autotoxic substances on the growth vigor of plants, has strong bacteriostatic activity on various pathogenic bacteria, has good growth promoting performance and strong colonization capability, and has obvious effects on promoting the growth and development of root systems, preventing and controlling soil-borne diseases, increasing the yield of crops, improving the quality of the crops and the like.
The invention provides a bacillus amyloliquefaciens strain LXBA.1, wherein the preservation number of the bacillus amyloliquefaciens strain LXBA.1 is CGMCC NO. 21868.
The inventor obtains the bacillus amyloliquefaciens strain LXBA.1 from a plurality of microorganisms in continuous cropping strawberry rhizosphere soil through directional screening by a large number of experiments, the bacillus amyloliquefaciens strain LXBA.1 has the obvious advantages of degrading autotoxic substances in the soil, lightening continuous cropping obstacles, preventing and treating plant diseases, improving a soil micro-ecological area system, promoting crop growth and the like, and has wide application prospect in the aspects of reducing the use amount of fertilizers and chemical pesticides in the crop planting process and the like.
The bacillus amyloliquefaciens strain LXBA.1 is preserved in China general microbiological culture Collection center (CGMCC) of China general microbiological culture Collection center (CGMCC) at 04.03.2021, the preservation number is CGMCC NO.21868, the preservation address is No. 3 of the national institute of sciences, China, institute of microbiology, Naja Kogyuchi No. 1.
The 16S rDNA gene sequence of the bacillus amyloliquefaciens strain LXBA.1 is shown as SEQ ID NO: 1 is shown.
The bacillus amyloliquefaciens strain LXBA.1 has the morphological, physiological and biochemical characteristics that: on LB plate, it is milky white, convex in center, round, wrinkled on surface, opaque, gram-positive, rod-shaped, not chain-forming, spore-forming.
The growth temperature range of the bacillus amyloliquefaciens LXBA.1 is 10-60 ℃, and the optimal growth temperature is about 30 ℃; the tolerance pH value range is 4.0-10.0, and the optimum growth pH value is 8-9; can tolerate NaCl solution with concentration of about 10%, is not sensitive to ultraviolet rays, and has wide growth range and strong tolerance.
The bacillus amyloliquefaciens LXBA.1 has strong bacteriostatic ability on plant pathogenic fungi such as fusarium solani, fusarium oxysporum, botrytis cinerea, gaeumannomyces graminis, triticum solani, botrytis cinerea and verticillium dahliae, and the bacteriostatic rate is 63.3-89.2%; has strong antibacterial ability to pathogenic bacteria of Pseudomonas solanacearum, carrot soft rot Erwinia and Streptomyces scabies, the diameter of the antibacterial ring is 7.8mm-10.3mm, and the antibacterial activity of broad spectrum is realized.
The bacillus amyloliquefaciens LXBA.1 has the capabilities of dissolving phosphorus, dissolving potassium silicate, fixing nitrogen, producing indoleacetic acid (IAA) and siderophil, the enzyme activity determination result of the IAA is 82.14mg/L, the degradation capability of the bacillus amyloliquefaciens on inorganic phosphorus reaches 213.33mg/L, and the bacillus amyloliquefaciens LXBA has strong growth promoting capability.
After the bacillus amyloliquefaciens LXBA.1 passes through 10 generations, 20 generations and 30 generations, the bacteriostatic activity on plant pathogenic fungi and bacteria is basically consistent with the bacteriostatic effect of the first bacillus amyloliquefaciens LXBA.1 on pathogenic bacteria, and the bacillus amyloliquefaciens LXBA.1 has higher genetic stability.
The invention also provides a microbial agent which comprises the bacillus amyloliquefaciens strain LXBA.1 and/or a fermentation product thereof.
The bacterial number of the bacillus amyloliquefaciens strain LXBA.1 in the microbial agent is 150-200 hundred million/mL.
The microbial agent contains indoleacetic acid; further, the content of the indoleacetic acid in the microbial agent is 250-350mg/L, preferably 300-350 mg/L. The microbial agent contains siderophore; further, the relative content of siderophore in the microbial agent is 70-80%, and more preferably 80%.
The invention also provides a preparation method of the microbial agent, which comprises the following steps: fermenting the bacillus amyloliquefaciens strain LXBA.1.
In the present invention, the fermentation medium used for fermentation may have the following composition: 5-20g/L of bean flour, 5-18g/L of glucose, 10-30g/L of soluble starch, 1-10g/L of peptone, 5-15g/L of corn flour, 1-10g/L of ammonium sulfate, 0.1-0.5g/L of magnesium sulfate, 0.1-0.5g/L of monopotassium phosphate, 0.1-0.5g/L of dipotassium phosphate and 0.05-0.5g/L of manganese sulfate.
In the invention, the inoculation amount during fermentation can be 0.1-0.5%; the stirring speed can be 150-200 rpm; the ventilation amount can be 2-10%; the fermentation temperature can be 32-36 ℃; the fermentation time can be 15-24 h.
More specifically, the preparation method of the microbial agent comprises the following steps:
inoculating a bacillus amyloliquefaciens strain LXBA.1 into an NA solid culture medium, and culturing at about 32-36 ℃ for 24-48h to obtain a first-level seed of a bacillus amyloliquefaciens solid plate;
transferring the solid plate primary seeds into an eggplant bottle filled with an NA solid culture medium, and culturing at about 32-36 ℃ for 24-48h to obtain the bacillus amyloliquefaciens solid plate secondary seeds;
inoculating the solid plate secondary seeds into a fermentation tank filled with a liquid fermentation culture medium, and fermenting for 15-24h under the conditions of 32-36 ℃, the inoculation amount of 0.1-0.5%, the stirring speed of 150-.
The invention also provides application of the microbial agent or the microbial agent prepared by the preparation method in at least one of plant disease prevention and control, autotoxic substance degradation and plant growth promotion.
Specifically, the plant disease includes at least one of strawberry root rot, potato scab and ginger stem rot; the autotoxic substance comprises phenolic acid autotoxic substance, and the phenolic acid autotoxic substance comprises p-hydroxybenzoic acid and the like; plant growth promotion is effected by bioactive substances in the fermentation product, including indoleacetic acid and/or siderophiles.
The implementation of the invention has at least the following advantages:
1. the bacillus amyloliquefaciens LXBA.1 can efficiently degrade autotoxic substances such as p-hydroxybenzoic acid and the like, the degradation rate of the p-hydroxybenzoic acid in the soil can reach 74.59%, and further the influence of the autotoxic substances on the growth vigor of plants such as strawberries and the like can be effectively relieved;
2. the bacillus amyloliquefaciens LXBA.1 has a wide antibacterial spectrum, shows strong antibacterial activity on various germs such as strawberry root rot, apple ring rot, ginger stem rot, potato scab and the like, has the antibacterial rate of 62.8-83.1% on plant pathogenic fungi, has the antibacterial diameter of 7.0-8.3mm on pathogenic bacteria, and has stable antibacterial effect on plant pathogenic bacteria after multiple passages;
3. the bacillus amyloliquefaciens LXBA.1 has the growth promoting performances of silicon decomposition, phosphorus potassium dissolution, nitrogen fixation, IAA (82.14mg/L) production, siderophin production and the like, has strong colonization capacity, and has very obvious effects on promoting root system growth and development, preventing and controlling soil-borne diseases (potato scab 80.85%), improving crop yield and improving quality, thereby greatly reducing the use of chemical fertilizers and pesticides;
4. the bacillus amyloliquefaciens LXBA.1 has certain saline-alkali tolerance (10% NaCl, pH8-9) activity and stronger bacteriophage resistance, can improve the saline-alkali tolerance of plants, is a safe, green and efficient multifunctional broad-spectrum disease-resistant growth-promoting strain, and has wide industrial application prospect;
5. the microbial agent prepared by the bacillus amyloliquefaciens strain LXBA.1 contains indoleacetic acid and siderophin, wherein the production amount of the indoleacetic acid is 253.14-371.45mg/L, and the relative content of the siderophin is 72.26-81.25%, which shows that the microbial agent has strong plant growth promoting capability.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a phylogenetic tree of a strain LXBA.1 constructed based on a 16S rDNA gene according to the present invention;
FIG. 2 is a phylogenetic tree of strain LXBA.1 constructed based on gyrA gene according to the present invention;
FIG. 3 shows the antagonistic effect of the strain LXBA.1 of the present invention on Fusarium oxysporum;
FIG. 4 shows the antagonistic effect of the strain LXBA.1 of the present invention on Pythium ultimum;
FIG. 5 is a chromatogram for degrading p-hydroxybenzoic acid in soil by using the strain LXBA.1 of the present invention;
FIG. 6 is a graph showing the effect of non-inoculation and inoculation on the root growth of dandelion;
FIG. 7 is a graph showing the effect of non-inoculation and inoculation on the growth vigor of dandelion plants;
FIG. 8 shows the prevention and treatment effect of the bacterial strain LXBA.1 of the invention on potato scab.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. 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 application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms also include the plural forms unless the context clearly dictates otherwise, and further, it is understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, devices, components, and/or combinations thereof.
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The strains used in the examples and the control examples were as follows:
bacillus amyloliquefaciens strain lxba.1: in 2021, 03.04 days, the strain is preserved in China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC NO.21868, the preservation address is No. 3 of Xilu No. 1 of Beijing, Chaozhou, the area facing the sun, the institute of microbiology, China academy of sciences;
bacillus amyloliquefaciens (control strain 1): purchased from certain Biotech, Inc. in Shaanxi;
bacillus amyloliquefaciens (control strain 2): purchased from biotechnology limited of south china;
bacillus amyloliquefaciens (control strain 3): purchased from biotechnology limited, Shandong;
bacillus amyloliquefaciens (control strain 4): purchased from biotechnology limited of Hebei;
bacillus amyloliquefaciens (control strain 5): purchased from certified certain Biotech Ltd.
Example 1
The embodiment provides a bacillus amyloliquefaciens LXBA.1, and a screening and identifying method thereof comprises the following steps:
one, directional screening
The strain LXBA.1 is obtained by separating rhizosphere soil of healthy plants in a continuous cropping strawberry greenhouse in Changli, Hebei, and the sampling and separating method comprises the following steps:
selecting a plant which grows healthily, digging out the whole root system completely by using a sampling shovel, tapping the root system, discarding the soil which is combined with the root system and is looser, and brushing the soil which is tightly combined with the root system by using a brush to obtain a rhizosphere soil sample.
Weighing 10g of rhizosphere soil sample, dissolving the rhizosphere soil sample in 90mL of sterile water, fully oscillating the rhizosphere soil sample for 20min at a speed of 180r/min, taking 1mL of soil supernatant, and transferring the soil supernatant into a selective culture medium taking p-hydroxybenzoic acid as a unique carbon source, wherein the selective culture medium comprises the following components: (NH) 4 ) 2 SO 4 2g/L，KH 2 PO 4 2g/L，Na 2 HPO 4 1.3g/L, 2g/L p-hydroxybenzoic acid, 5g/L NaCl and 6.8-7.2 of pH value; performing shake culture at 34 deg.C and 180r/min for 2 days, placing in 80 deg.C constant temperature water bath for 30min, performing gradient dilution on the culture solution, and taking dilution degree of 10 -5 、10 -6 、10 -7 Coating 100 μ L of the diluted solution on NA solid culture medium, performing inverted culture at 30 deg.C for 2 days, selecting single colony, further separating and purifying, and preserving in slant test tube at 4 deg.C.
And (3) selecting the single colony obtained in the step, inoculating the single colony in a selective culture medium again, carrying out shaking culture at 34 ℃ and 180r/min for 3 days, adding 2mL of bacterial liquid into a sterile centrifuge tube, centrifuging at 8000r/min for 8min, and taking 0.5mL of supernatant to determine the OD260 value.
The degradation rate of p-hydroxybenzoic acid is calculated by the following formula:
degradation rate (%) × (treatment OD260-2g/L parahydroxybenzoic acid solution OD260)/(2g/L PA solution OD260) × 100.
According to the degradation condition of p-hydroxybenzoic acid, 1 spore strain LXBA.1 with better degradation effect is screened out.
II, identifying strains
The isolated strains were identified by gram staining and observation of the morphology of the cells under an optical microscope, and the results were as follows:
the shape and physiological and biochemical characteristics of the strain LXBA.1 are as follows: the strain is milky white on an LB plate, convex in the center, round, wrinkled on the surface, opaque, gram-positive, rod-shaped, not chained and sporulated.
Molecular biology identification (16S rDNA gene and gyrA gene sequence):
carrying out streak culture on a strain LXBA.1, selecting a single strain, sending the single strain to Beijing sequencing department of Biotechnology engineering (Shanghai) GmbH, sequencing 16S rDNA and gyrA genes of the strain, respectively submitting sequencing results to ezbiocloud. The 16S rDNA sequence alignment result shows that the strain has the highest similarity with Bacillus, and the similarity with Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is 50 percent (shown in figure 1); and carrying out systematic evolution analysis on the gyrA gene sequence of the strain LXBA.1, wherein the similarity between the gyrA gene sequence comparison result and the Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is 99%. Thus, strain lxba.1 was identified as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), i.e. strain lxba.1.
The bacillus amyloliquefaciens strain LXBA.1 is preserved in China general microbiological culture Collection center (CGMCC) at 04.03.2021, the preservation number is CGMCC NO.21868, the preservation address is No. 3 of Xilu No. 1 Beichen of the Korean area in Beijing, and the institute of microbiology of China academy of sciences.
Example 2
This example provides the growth characteristics of the bacillus amyloliquefaciens strain lxba.1 as follows:
1. optimum growth temperature
A seed solution (LB medium) of the Bacillus amyloliquefaciens strain LXBA.1 is prepared, inoculated into a 250mL triangular flask containing 50mL of LB medium according to the inoculation amount of 5 percent, and subjected to shaking culture (rotating speed of 180r/min) in a constant-temperature shaking incubator at 4 ℃, 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃ and 70 ℃ for 2 days, 3 times of repetition is set, and the growth condition (whether turbid or not) is observed and recorded.
2. Resistance to acid and base
LB liquid culture media with different pH values (3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0) are prepared, 5 percent of Bacillus amyloliquefaciens strain LXBA.1 seed solution (LB culture media) is respectively inoculated, the mixture is shaken and cultured for 2 days at the temperature of 30 ℃ and at the speed of 180r/min, 3 times of repetition is set, and the growth condition (whether turbid or not) is observed and recorded.
3. Salt resistance
LB liquid culture media with different NaCl contents (1%, 5%, 10%, 15%, 20%) are prepared, 5% of Bacillus amyloliquefaciens strain LXBA.1 seed liquid (LB culture media) is inoculated respectively, shaking culture is carried out for 2 days at 30 ℃ and 180r/min, 3 times of repetition is set, and the growth condition (whether turbid or not) is observed and recorded.
4. Sensitivity to ultraviolet light
200ul of fermentation liquor of the bacillus amyloliquefaciens strain LXBA.1 is evenly coated on an NA solid culture medium flat plate, the flat plate is placed on a 30w ultraviolet lamp 40cm position and is respectively irradiated for 0h, 4h, 8h, 12h, 16h, 20h, 24h, 32h and 48h, then the flat plate is placed on a constant temperature incubator at the temperature of 30 ℃ for 2 days, the experiment is repeated for 3 times, and the growth condition of the strain is recorded.
The results are shown in Table 1.
TABLE 1 growth characteristics of the Bacillus amyloliquefaciens strain LXBA.1
Remarking: + indicating growth, + indicating good growth, -indicating no growth
As can be seen from table 1:
the growth temperature range of the bacillus amyloliquefaciens strain LXBA.1 is 10-60 ℃, and the optimal growth temperature is 30 ℃; the growth pH value range is 4.0-10.0, and the optimum pH value is 8-9; can resist NaCl with the concentration of 10 percent; the bacillus amyloliquefaciens LXBA.1 is insensitive to ultraviolet rays, and the survival rate of the bacillus amyloliquefaciens can still reach 78% after the bacillus amyloliquefaciens is continuously irradiated for 24 hours, so that the bacillus amyloliquefaciens LXBA.1 has the remarkable advantages of wide growth range, acid and alkali resistance, high-salt environment, insensitivity to ultraviolet rays and the like, and has huge application potential in developing microbial fertilizers.
Example 3
This example provides a microbial agent that is a fermentation product of bacillus amyloliquefaciens strain lxba.1; the preparation method comprises the following steps:
firstly, preparing a liquid fermentation culture medium
A liquid fermentation medium was prepared as follows: 10g/L of soybean powder, 10g/L of glucose, 30g/L of soluble starch, 5g/L of peptone, 15g/L of corn flour, 1g/L of ammonium sulfate, 0.5g/L of magnesium sulfate, 0.3g/L of monopotassium phosphate, 0.3g/L of dipotassium phosphate and 0.05g/L of manganese sulfate.
Second, liquid fermentation
Inoculating a bacillus amyloliquefaciens strain LXBA.1 into an NA solid culture medium, and culturing at the temperature of 34 ℃ for 24 hours to obtain a bacillus amyloliquefaciens solid plate seed; transferring the primary seeds into an eggplant bottle filled with an NA solid culture medium, and culturing for 36 hours at the temperature of 34 ℃ to obtain the secondary seeds of the bacillus amyloliquefaciens solid flat plate; inoculating the solid flat plate secondary seeds into a fermentation tank filled with the liquid fermentation culture medium, and fermenting for 24 hours at 34 ℃, with the inoculation amount of 0.5%, the stirring speed of 200rpm and the ventilation amount of 5%, thus obtaining the bacillus amyloliquefaciens microbial inoculum of 200 hundred million/mL.
Example 4
This example provides a microbial inoculant that is a fermentation product of bacillus amyloliquefaciens strain lxba.1; the preparation method comprises the following steps:
firstly, preparing a liquid fermentation culture medium
A liquid fermentation medium was prepared as follows: 5g/L of bean flour, 10g/L of glucose, 10g/L of soluble starch, 3g/L of peptone, 10g/L of corn flour, 5g/L of ammonium sulfate, 0.1g/L of magnesium sulfate, 0.2g/L of monopotassium phosphate, 0.2g/L of dipotassium phosphate and 0.07g/L of manganese sulfate.
Second, liquid fermentation
Inoculating a bacillus amyloliquefaciens strain LXBA.1 into an NA solid culture medium, and culturing at the temperature of 32 ℃ for 32 hours to obtain a bacillus amyloliquefaciens solid plate seed; transferring the primary seeds into an eggplant bottle filled with an NA solid culture medium, and culturing for 32 hours at the temperature of 32 ℃ to obtain secondary seeds of a bacillus amyloliquefaciens solid plate; inoculating the solid flat plate secondary seeds into a fermentation tank filled with the liquid fermentation culture medium, and fermenting for 18h under the conditions of 32 ℃, the inoculation amount of 0.1%, the stirring speed of 150rpm and the ventilation amount of 5%, thereby obtaining the bacillus amyloliquefaciens microbial inoculum of 150 hundred million/mL.
Example 5
The embodiment provides the bacteriostatic effect of the bacillus amyloliquefaciens strain LXBA.1 on plant pathogenic bacteria such as apple ring rot, ginger stem rot and the like; the test method is as follows:
antagonistic pathogenic fungi: placing a pathogenic bacteria cake (7mm) in the center of a PDA solid plate with the diameter of 90mm, inoculating a Bacillus amyloliquefaciens strain LXBA.1 at the position which is 25mm away from the center, culturing the plate without inoculation at the temperature of 28 ℃ in a constant-temperature incubator for 5-7 days, respectively counting the diameters of control pathogenic bacteria and treated pathogenic bacteria, and calculating the bacteriostasis rate; the bacteriostatic rate calculation formula is as follows.
The bacteriostatic ratio is (control pathogen diameter-treated pathogen diameter)/(control pathogen diameter-cake diameter) × 100%.
Antagonistic pathogenic bacteria: respectively coating pathogenic bacteria to be detected on NA solid plates, inoculating a bacillus amyloliquefaciens strain LXBA.1 in the center of each plate, culturing the plates for 24-48h at a constant temperature of 30 ℃ without inoculating a control plate, and respectively counting the diameters of antibacterial zones.
Subculturing the bacillus amyloliquefaciens strain LXBA.1, and measuring the bacteriostatic effects of the 10 th generation, the 20 th generation and the 30 th generation on plant pathogenic bacteria by adopting the method, wherein the results are shown in Table 2.
TABLE 2 bacteriostatic effect of Bacillus amyloliquefaciens LXBA.1 on plant pathogenic bacteria at different generations
As can be seen from table 2:
1. the bacillus amyloliquefaciens strain LXBA.1 has strong bacteriostatic ability on plant pathogenic fungi, and the bacteriostatic rate is 63.3-89.2%;
2. the bacillus amyloliquefaciens strain LXBA.1 has strong bacteriostatic ability to pathogenic bacteria, and the diameter of a bacteriostatic zone is 7.8mm-10.3 mm;
3. after the bacillus amyloliquefaciens strain LXBA.1 is propagated for 10 generations, 20 generations and 30 generations, the bacteriostasis rate to plant pathogenic fungi is 62.8-83.1%, the diameter of the bacteriostasis circle to pathogenic bacteria is 7.0-10.3mm, and the bacteriostasis effect to pathogenic bacteria of the first generation of bacillus amyloliquefaciens strain LXBA.1 is basically consistent.
Therefore, the bacillus amyloliquefaciens LXBA.1 has the advantages of wide antibacterial spectrum, strong antibacterial ability, stable antibacterial effect on plant pathogenic bacteria after multiple passages and high genetic stability.
Example 6
The embodiment provides growth promoting performances of producing IAA, siderophilic acid and nitrogen fixation activity, dissolving silicon, dissolving potassium, dissolving inorganic phosphorus and the like of a bacillus amyloliquefaciens strain LXBA.1; the test method is as follows:
detecting siderophin production:
streaking and inoculating a bacillus amyloliquefaciens strain LXBA.1 to a CAS detection culture medium, and culturing for 6d at 35 ℃; if a yellow-green halo appears, the production of the siderophore is indicated.
And (3) nitrogen fixation activity detection:
inoculating a bacillus amyloliquefaciens strain LXBA.1 to an arbuscular junction nitrogen-free culture medium in a streak manner, culturing at 30 ℃ for 3d, and observing the growth condition of a bacterial colony; if the strain can grow, the strain has a nitrogen fixation function.
The nitrogen-free culture medium of the arbuscular mycorrhizal compound comprises the following components: k 2 H 2 PO 4 0.2g、MgSO 4 ·7H 2 0.2g of O, 0.2g of NaCl, 5.0g of calcium carbonate, 10.0g of mannitol, 0.1g of calcium sulfate, 1L of distilled water and 15g of agar, and the pH value is 7.0.
And (3) verifying the performances of silicon decomposition, potassium decomposition and inorganic phosphorus decomposition:
inoculating Bacillus amyloliquefaciens strain LXBA.1 to a silicon-decomposing solid culture medium (10.0 g of sucrose, 0.5g of yeast extract, (NH) 4 ) 2 SO 4 1.0g，Na 2 HPO 4 2.0g，MgSO 4 ·7H 2 O 0.5g，CaCO 3 0.1g, 1.0g of glass powder, 50g of NaCl, 20g of agar powder and 1000mL of distilled water, and a potassium-dissolving solid culture medium (10.0 g of cane sugar, 0.5g of yeast extract, (NH) 4 ) 2 SO 4 1.0g，Na 2 HPO 4 2.0g，MgSO 4 ·7H 2 O 0.5g，CaCO 3 0.1g, potassium feldspar powder 1.0g, NaCl 50g, distilled water 1000mL) and inorganic phosphorus-dissolving solid medium ((NH) 4 ) 2 SO 4 0.5g，MgSO 4 ·7H 2 O 0.5g，NaCl 50g，Ca 3 (PO 4 ) 2 10.0g，CaCO 3 2.5g, glucose 2.0g, MnSO 4 0.02g，FeSO 4 0.02g, agar 20.0g, distilled water 1000mL), culturing at 30 ℃ for 3-7 days, and observing whether a lysis ring is generated; if the dissolving ring exists, the capability of dissolving silicon, potassium and inorganic phosphorus is shown.
Testing of secreted IAA performance:
the capability of producing indoleacetic acid is measured by a Salkowski method; specifically, after inoculating the activated strain LXBA.1 to King's B liquid culture medium containing 500mg/L tryptophan for 3d, taking 2mL of bacterial suspension, centrifuging for 15min at 10000r/min, adding 2mL of Salkowski reagent into each 1mL of supernatant, developing color in dark for 30min at room temperature, and determining the OD530 value. The IAA content (mg/L) in the culture solution was calculated by using a blank medium as a control and an OD530 value corresponding to pure IAA as a standard curve.
And (3) measuring the phosphorus dissolving performance:
measuring the water-soluble phosphorus content of the strain by adopting a molybdenum-antimony colorimetric resistance method; specifically, the activated strain LXBA.1 was inoculated into NA liquid medium, cultured at 30 ℃ and 180r/min to logarithmic phase, and inoculated into 100mL of sterilized Monkina inorganic phosphorus liquid medium (glucose 10g/L, (NH) in an inoculum size of 1% 4 ) 2 SO 4 0.5g/L，MgSO 4 ·7H 2 O 0.3g/L，NaCl 0.3g/L，KCl 0.3g/L，FeSO 4 ·7H 2 O 0.03g/L，MnSO 4 ·4H 2 O 0.03g/L，Ca 3 (PO 4 ) 2 10.0g/L, pH 7.0-7.5), and culturing at 28 deg.C and 180r/min under shaking for 6 d. Centrifuging 5mL of culture solution at 12000r/min at 4 ℃ for 5min, taking 0.1mL of supernatant, fixing the volume to 5mL, adding 1mL of solution with the fixed volume into a 10mL colorimetric tube, adding 2mL of molybdenum-antimony anti-color development agent, fixing the volume to the scale with water, standing at room temperature higher than 25 ℃ for 30min, carrying out color comparison at 700nm wavelength, and measuring the absorbance; meanwhile, a blank inorganic phosphorus culture medium without inoculation is used as a control.
Drawing a standard curve: 0, 0.50, 1.00, 1.50, 2.00, 2.50 and 3.00mL of phosphorus standard solution [ rho (P) ═ 5mg/mL ] is sucked into a 10mL colorimetric tube, 2mL of molybdenum-antimony color-resisting reagent is added, water is added for constant volume to scale, after the colorimetric determination of absorbance at the wavelength of 700nm is carried out after the colorimetric determination is carried out for 30min at the room temperature of higher than 25 ℃, and a standard curve is drawn by taking the absorbance as the abscissa and the phosphorus concentration as the ordinate.
And (3) potassium dissolving performance determination:
100mL of potassium-solubilizing medium (sucrose 10g/L, Na) was added to a 250mL Erlenmeyer flask 2 HPO 4 1.0g/L，(NH 4 ) 2 SO 4 0.5g/L，MgSO 4 ·7H 2 O1.0 g/L, yeast powder 0.2g/L, FeCl 3 Trace amount of potash feldsparPowder 2.5g/L), sterilizing at 121 deg.C for 30 min. Inoculating 2mL of LXBA.1 strain liquid in logarithmic phase, setting blank control (adding sterile water), performing shake culture at 28 ℃ and 180r/min, taking 20mL of strain liquid at 7d, centrifuging at 6000r/min for 20min, and determining the content of water-soluble potassium in the supernatant by using atomic absorption spectrophotometry.
And (3) silicon-dissolving capacity determination:
100mL of the desilication medium (sucrose 10g/L, Na) was added to a 250mL Erlenmeyer flask 2 HPO 4 1.0g/L，(NH 4 )2SO 4 0.5g/L，MgSO 4 ·7H 2 O1.0 g/L, yeast powder 0.2g/L, FeCl 3 Trace, 5g/L of glass powder), and sterilizing at 121 deg.C for 30 min. 2mL of strain LXBA.1 bacterial liquid in logarithmic phase is inoculated, a blank control (sterile water is added), shaking culture is carried out at the temperature of 28 ℃ and at the speed of 180r/min, and sampling is carried out for 10 days to determine the silicon content in the solution. And (3) bacterial liquid treatment: and (5) taking 20mL of 10d bacterial liquid respectively, centrifuging at 6000r/min for 20min, and taking supernatant for later use. And respectively measuring the content of water-soluble silicon in the supernatant by using a silicon-molybdenum blue colorimetric method.
The comparison of the plant growth substance-promoting abilities is shown in Table 3.
TABLE 3 comparison of the ability to produce various plant growth promoting substances
Note: + indicates strain growth; , + + indicates a darker color.
The test result shows that:
the bacillus amyloliquefaciens strain LXBA.1 has the capabilities of dissolving phosphorus, dissolving potassium silicate, fixing nitrogen, producing IAA and producing siderophin, the enzyme activity determination result of the IAA is 82.14mg/L, and the degradation capability of the strain on inorganic phosphorus reaches 213.33 mg/L; thus illustrating that: the bacillus amyloliquefaciens strain LXBA.1 has stronger capacity of promoting plant growth.
Example 7
In this example, the hemolytic assay of bacillus amyloliquefaciens strain lxba.1 was performed as follows:
and (3) referring to general technical guidelines for biological safety of microbial fertilizers (NY1109-2006), performing hemolysis test determination on the bacillus amyloliquefaciens strain LXBA.1.
The results show that: the bacillus amyloliquefaciens strain LXBA.1 does not have hemolytic cycle, which indicates that the strain LXBA.1 does not have hemolytic reaction.
Example 8
This example provides comparative experiments of bacillus amyloliquefaciens strain lxba.1 and other existing bacillus amyloliquefaciens strain strains (i.e., control strain 1 and control strain 2) to determine antagonistic activity against pathogenic bacteria, siderophore and IAA production ability, and degradation effect on hydroxybenzoic acid, respectively.
The test method is as follows:
(1) the determination method refers to example 5 for the bacteriostatic effect of apple ring spot, ginger stem rot and other plant pathogens;
(2) the assay of ability to produce siderophore and IAA, IAA secretion ability, was performed by the method described in reference example 6; the method for measuring the siderophore comprises the following steps:
the relative content of siderophiles was quantitatively determined by visible light photometry. Respectively activating 3 tested bacillus amyloliquefaciens strains, inoculating the strains into an iron-limiting SA liquid culture medium by using an inoculating needle, and performing shake culture at the temperature of 37 ℃ at 180rpm for 48 hours; transferring the bacterial suspension to be detected into a sterilized 10mL centrifuge tube, centrifuging for 15min at 13000r/min, taking the supernatant and a certain volume of the CAS detection solution 1:1 which is prepared in situ, fully mixing uniformly, standing for about 1 h, measuring the absorbance value (As) at the wavelength of 630nm, using double distilled water As a control for zero adjustment, measuring the absorbance value of the non-inoculated iron-limiting SA liquid culture medium As a reference value (Ar) by the same method, and calculating the activity unit of the iron carrier ([ (Ar-As)/Ar ] × 100). The preparation method of the CAS detection solution comprises the following steps: 60.5mg CAS (chrome azure) is dissolved in 50mL of deionized water, 10mL of 1mmol/L ferric chloride solution is added, HDTMA (hexadecyl-trimethylhexadecane bromide) solution (72.9 mg HDTMA is dissolved in 40mL of distilled water) is finally added, and sterilization is carried out for 20min at 121 ℃; iron-limiting SA liquid medium: 20.0g/L of sucrose, 3.5g/L of ammonium sulfate, 1.5g/L of L-aspartic acid, 0.02g/L of L-methionine, 0.01g/L of L-histidine, 1.0g/L of potassium dihydrogen sulfate, 0.5g/L of magnesium sulfate and 0.5g/L of sodium chloride.
(3) For the degradation of hydroxybenzoic acid by autotoxic substances, the measurement method of example 1 was referred to.
The test results are shown in tables 4 and 5.
TABLE 4 bacteriostatic effect of different Bacillus amyloliquefaciens strains on plant pathogens
TABLE 5 Difference in growth promoting Properties and degradation of autotoxic substances for different Bacillus amyloliquefaciens strains
The results in tables 4 and 5 show that:
the bacillus amyloliquefaciens strain LXBA.1 is obviously superior to a control strain 1 and a control strain 2 in the aspects of antagonistic capability to pathogenic bacteria, IAA production and relative content of siderophins. The bacteriostatic rate of the strain LXBA.1 to plant pathogenic fungi is 63.3-89.3%, the bacteriostatic rate of the control strain 1 is 40.4-68.6%, and the bacteriostatic rate of the control strain 2 is 34.6-65.3%, which shows that the bacteriostatic ability of the Bacillus amyloliquefaciens strain LXBA.1 is stronger than that of the control strain 2; in terms of IAA production amount and relative content of siderophore, the strain LXBA.1 is 100.13mg/L and 71.48 percent, the control strain 1 is 20.56mg/L and 23.34 percent, and the control strain 2 is 33.26mg/L and 15.79 percent, which shows that the plant growth promotion performance of the Bacillus amyloliquefaciens strain LXBA.1 is stronger than that of the control strain 2; the degrading ability to p-hydroxybenzoic acid, the strain LXBA.1 was 84.0%, and the control strain 1 and the control strain 2 were 2.5% and 1.0%, respectively, indicating that the Bacillus amyloliquefaciens strain LXBA.1 has a strong degrading ability to p-hydroxybenzoic acid, while the 2 control strains have almost no degrading ability. Thus illustrating that: compared with other strains, the bacillus amyloliquefaciens strain LXBA.1 has more obvious advantages, and particularly has obvious effect on the degradation of p-hydroxybenzoic acid.
Example 9
This example provides the results of measuring the IAA production amount and the relative content of siderophore in the microbial agents prepared in example 3 (the number of effective viable bacteria reaches 200 hundred million/mL) and example 4 (the number of effective viable bacteria reaches 150 hundred million/mL), and the specific measurement method is referred to in example 8. The test results are shown in Table 6.
TABLE 6 IAA production and relative siderophore content of microbial Agents
| Test strains | IAA yield mg/L | Relative content of siderophore |
| Example 3 | 371.45 | 81.25 |
| Example 4 | 253.14 | 72.26 |
Table 6 the results show that:
the microbial agent prepared from the bacillus amyloliquefaciens strain LXBA.1 contains indoleacetic acid and siderophin, wherein the production amount of the indoleacetic acid is 253.14-371.45mg/L, and the relative content of the siderophin is 72.26-81.25%. Thus illustrating that: the microbial agent has stronger growth promoting capability on plants.
Example 10
This example provides a test for phage resistance of Bacillus amyloliquefaciens strain LXBA.1 to other existing Bacillus amyloliquefaciens strains (i.e., control strains 1-5).
The test method is as follows:
after respectively carrying out streak activation on the bacillus amyloliquefaciens strain LXBA.1 and the control strains 1-5, selecting a single bacterial colony to be inoculated in an LB liquid culture medium at 30 ℃ and 180r/min for overnight culture, respectively uniformly mixing 1mL of 3 phage solution after gradient dilution with 3mL of bacterial liquid, pouring the mixture into a double-layer flat plate, and carrying out overnight culture to observe whether plaques appear.
The test results are shown in Table 7.
TABLE 7 Bacillus amyloliquefaciens bacteriophage resistance test
Note: + indicates plaque; -means plaque-free.
Table 7 the results show that:
the control strains 1-5 inoculated with the 3 phages respectively generate plaques with different obvious degrees, but the bacillus amyloliquefaciens LXBA.1 does not generate plaques, which indicates that the 3 phages can not infect the strain LXBA.1, and shows that the bacillus amyloliquefaciens LXBA.1 has stronger phage resistance and has larger application potential in industrial production.
Example 11
The embodiment provides an application of a microbial agent in degrading autotoxic substances, in particular to degradation of the autotoxic substances in the rhizosphere of strawberry continuous cropping soil; the method comprises the following steps:
the microbial agent prepared in the example 3 (the effective viable count reaches 200 hundred million/mL) is applied to the strawberries at the planting period, the flowering period and the fruit swelling period for 1L/mu/time, rhizosphere soil samples in the control area and the treatment area are respectively taken at the harvest period of the strawberries and stored at 4 ℃, and the detection work is completed within 24 hours.
Respectively weighing 25g of control and processed soil samples into 250mL triangular flasks with stoppers, respectively adding 25mL of 1mol/L NaOH, standing overnight, oscillating for 30min, centrifuging, filtering the centrifugate, adjusting the pH to 2.5 with 12mol/L hydrochloric acid, centrifuging for 2h, filtering the supernate with a 0.22 mu m filter membrane, and standing the filtrate.
Preparing a standard solution: 100mg of p-hydroxybenzoic acid was weighed by a precision balance, dissolved in a 100mL volumetric flask to a constant volume, and used as a reference stock solution (C1 mg/mL), and 1mL of the p-hydroxybenzoic acid stock solution was accurately sucked and added to the 100mL volumetric flask to a constant volume, and the concentration of the standard solution was 0.01 mg/mL.
Chromatographic conditions are as follows: the mobile phase was 3% glacial acetic acid: methanol 75: 25, flow rate: 1 mL/min. Chromatography column Agilent ZORBAXSB-C18(4.6 mm. times.150 mm, 5 μm), detection wavelength: 260nm, column temperature: at 25 ℃.
The degradation rate calculation formula is as follows:
(Cck-Ct)/Cck × 100)
In the formula: cck and Ct are the residual concentration of p-hydroxybenzoic acid in the non-inoculated soil control CK and the inoculated soil respectively.
The measurement result shows that: the content of p-hydroxybenzoic acid in the non-inoculated soil control is 1.85 mug/g, the content of p-hydroxybenzoic acid in the treatment of the inoculated LXBA.1 is 0.47 mug/g, and the degradation rate of the strain LXBA.1 disclosed by the invention to p-hydroxybenzoic acid in the soil reaches 74.59% (shown in figure 5), so that the microbial agent disclosed by the invention has strong degradation capability to strawberry autotoxic substances.
Example 12
The embodiment provides application of a microbial inoculant to saline-alkali soil dandelion; the test method is as follows:
the test field is a Hebei Tangshan city Cao Fei Dian wetland greenhouse (11.3 g/kg of organic matter, 52.3mg/kg of available phosphorus, 234mg/kg of quick-acting potassium, 35mg/kg of hydrolyzable nitrogen, 6.3g/kg of water-soluble salt and pH8.51), dandelion seedlings (two true leaves) are transplanted in 9 months, the area of a test cell is 10m long and 1.5m wide, 200 times of diluent (1L per mu) of the microbial inoculum (200 hundred million/mL) prepared in example 3 is irrigated after 1 week of seedling remission, the whole growth period is irrigated for 2 times, and clear water is irrigated in contrast. After 1 month of growth, the aerial indexes and root systems of dandelion were investigated, and the results are shown in tables 8 and 9, and fig. 6 and 7.
TABLE 8 Effect of microbial Agents on Dandelion plant growth
TABLE 9 influence of microbial Agents on Dandelion root growth
| Test group | Root area cm 2 | Root width cm | Root height cm | Root length cm | Surface area cm 2 | Root volume cm 3 |
| Control | 278.65b | 17.38a | 16.04b | 263.08b | 127.47b | 4.92b |
| LXBA.1 | 339.60a | 17.80a | 19.08a | 297.49a | 168.29a | 7.58a |
The results show that:
the dandelion irrigated with the microbial inoculant is obviously superior to the control treatment in the aspects of plant growth vigor and root system, and especially has obvious differences in plant height, overground fresh weight, leaf area, root area and the like.
Therefore, the microbial agent can obviously improve the salt tolerance of crops and has obvious effect on the growth promoting performance of the crops.
Example 13
The present example provides the effect of microbial agents on controlling potato scab, and the test method is as follows:
the test place is a potato planting area of a Changli MaoTuo shop in Hebei province. The test design is as follows: 1L/mu of the microbial inoculum (200 hundred million/mL) prepared in the example 3 is adopted in the treatment area, 100 times of solution is sprayed on the surfaces of the seed potatoes during sowing, 5L/mu of the microbial inoculum (200 hundred million/mL) prepared in the example 3 is applied in a seedling stage and a flowering stage respectively, and the contrast is clear water treatment; and (4) carrying out grading investigation on the scab disease incidence condition on the potato tubers in the harvest period, and calculating the disease prevention effect.
Disease grading standard: grade 0, no disease spots on the surface of the potato blocks; grade 1, the surface of the potato blocks has disease spots, and the area of the potato blocks accounts for 1 to 5 percent of the potato blocks; 2, the surface of the potato blocks has disease spots, and the area of the potato blocks accounts for 5 to 10 percent; grade 3, the surface of the potato blocks has disease spots, and the area of the potato blocks accounts for 10-20 percent; grade 4, the surface of the potato blocks has disease spots, and the area of the potato blocks accounts for 20-40 percent of the potato blocks; grade 5, the surface of the potato blocks has disease spots, and the area of the potato blocks accounts for 40-60 percent of the potato blocks; grade 6, the surface of the potato blocks has disease spots, and the area of the potato blocks accounts for more than 60 percent of the potato blocks; the formula for calculating the morbidity, disease index and prevention and treatment effect is as follows:
the incidence rate is (number of diseased grains/number of harvested small potato grains) × 100%
Disease index ═ Σ (number of disease-grade granules × disease grade)/(number of investigation-harvested granules × highest grade) × 100
The prevention and treatment effect is (contrast disease index-treatment disease index)/contrast disease index x 100%.
The results are shown in Table 10.
TABLE 10 prevention and treatment effects of microbial Agents on potato scab
| Treatment of | The incidence of disease% | Index of disease condition | The control effect is% |
| Control | 87.23 | 64.17 | - |
| LXBA.1 | 34.22 | 12.29 | 80.85 |
The results show that:
the microbial agent can obviously reduce the incidence degree of the potato scab, the incidence rate is reduced by 53.01%, the prevention and treatment effect reaches 80.85%, and the bacillus amyloliquefaciens strain LXBA.1 has a good prevention and treatment effect on the potato scab and has great potential when being applied to prevention and treatment of the potato scab.
In conclusion, the degradation rate of the bacillus amyloliquefaciens strain LXBA.1 on p-hydroxybenzoic acid in soil can reach 74.59%, and the influence of autotoxic substances on the growth vigor of strawberries can be effectively relieved; the strain has broad antibacterial spectrum, and has strong antibacterial activity on various germs such as strawberry root rot, ginger stem rot, potato scab and the like; meanwhile, the strain has the growth promoting performances of silicon decomposition, phosphorus and potassium dissolution, nitrogen fixation, IAA (82.14mg/L) production, siderophin and the like, has strong colonization capacity, has very obvious effects on promoting root growth and development, preventing and controlling soil-borne diseases (potato scab 80.85%), improving crop yield and improving quality, and can greatly reduce the use of chemical fertilizers and pesticides; and shows certain saline-alkali tolerance (10% NaCl, the most suitable pH8-9) activity and stronger phage resistance, is a safe, green and efficient multifunctional broad-spectrum disease-resistant growth-promoting strain, and has wider industrial application prospect.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and these modifications or substitutions do not depart from the spirit of the corresponding technical solutions of the embodiments of the present invention.
Claims (10)
1. A bacillus amyloliquefaciens strain LXBA.1 is characterized in that the preservation number of the bacillus amyloliquefaciens strain LXBA.1 is CGMCC number 21868.
2. A microbial agent comprising the bacillus amyloliquefaciens strain lxba.1 of claim 1.
3. The microbial agent according to claim 2, wherein the number of Bacillus amyloliquefaciens LXBA.1 in the microbial agent is 150-200 hundred million/mL.
4. A microbial inoculant comprising the bacillus amyloliquefaciens strain lxba.1 of claim 1 and a fermentation product thereof.
5. The microbial agent according to claim 4, wherein the microbial agent comprises indoleacetic acid; the content of the indoleacetic acid in the microbial agent is 250-350 mg/L.
6. The microbial agent according to claim 4, which comprises a siderophore; the relative content of the siderophin in the microbial agent is 70-80%.
7. The method for preparing a microbial agent according to any one of claims 2 to 6, comprising: fermenting the bacillus amyloliquefaciens strain LXBA.1.
8. The method according to claim 7, wherein the fermentation medium used for the fermentation comprises: 5-20g/L of bean flour, 5-18g/L of glucose, 10-30g/L of soluble starch, 1-10g/L of peptone, 5-15g/L of corn flour, 1-10g/L of ammonium sulfate, 0.1-0.5g/L of magnesium sulfate, 0.1-0.5g/L of monopotassium phosphate, 0.1-0.5g/L of dipotassium phosphate and 0.05-0.5g/L of manganese sulfate.
9. The method as claimed in claim 7, wherein the inoculation amount during fermentation is 0.1-0.5%, the stirring speed is 150-200rpm, the aeration rate is 2-10%, the fermentation temperature is 32-36 ℃, and the fermentation time is 15-24 h.
10. Use of a microbial inoculant according to any one of claims 2 to 6 or prepared by a process according to any one of claims 7 to 9 for at least one of inhibiting phytopathogenic fungi, degrading autotoxic substances and promoting plant growth; wherein the plant pathogenic bacteria are Staphylococcus aureus, Fusarium solani, Fusarium oxysporum, Alternaria apple specialization, Rhizoctonia solani varieties, Verticillium dahliae, Pseudomonas solani, Erwinia carotovora or Streptomyces scabiosus; the autotoxic substance is p-hydroxybenzoic acid; plant growth promotion is effected by bioactive substances in the fermentation product, the bioactive substances being indoleacetic acid and siderophiles.
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