CN114456949B - Beauveria bassiana JSHA-MD912 and application thereof - Google Patents
Beauveria bassiana JSHA-MD912 and application thereof Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
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Abstract
The invention discloses beauveria bassiana JSHA-MD912 and application thereof, and belongs to the technical field of microorganisms. The strain is a beauveria fungus strain, the beauveria sp.JSHA-MD912, the preservation unit is China center for type culture collection, and the preservation number is CCTCC NO: m20211673, the preservation date is 2021, 12 and 24. The invention also discloses application of the beauveria bassiana JSHA-MD912. The beauveria bassiana JSHA-MD912 has the characteristics of very strong dissolution of inorganic phosphorus and organic phosphorus, has the functions of promoting growth and antagonizing plant pathogenic bacteria, can be used in the practices of agricultural production, ecological transformation application and the like, can effectively reduce the application of chemical fertilizers and pesticides, and has wide application prospect.
Description
Technical Field
The invention relates to beauveria bassiana JSHA-MD912 and application thereof, belonging to the technical field of microorganisms.
Background
Phosphorus is an important limiting factor in agricultural production, and phosphate fertilizers are used in large quantities in agriculture as common fertilizers, and the indissolvable phosphorus is continuously accumulated in soil. In the ecological environment of the soil, most of phosphorus elements exist in a form of insoluble inorganic phosphorus or organic phosphorus by being combined with metal cations, and plants are difficult to directly absorb and utilize. In recent years, functional phosphorus-dissolving microorganisms in China have few researches, and especially in the aspects of wide separation and screening of phosphorus-dissolving fungus groups and diversity resources, the phosphorus-dissolving microorganisms among different groups and different species have obvious difference in phosphorus-dissolving effect, so that the industrialized development of the phosphorus-dissolving microbial fertilizer is restricted.
Although research on fungus growth promotion and antagonism functions and related fungus resources are many, most of the fungi have single growth promotion or antagonism effect, and only antagonism of a certain plant pathogenic bacteria, the fungi have fewer multifunctional strain resources such as phosphorus dissolution, antagonism and the like, and cannot meet the actual demands of green agriculture and ecological application practice.
Beauveria bassiana belongs to the genus Beauveria (Beauveria) of the family Moniliaoeae (Moniliaoeae) of the order Moniliales (Hyphomycetales) of the order Hyphomycetales (Deuteromycetina) in the fungus classification system, is widely distributed throughout the world. Beauveria fungus is a common parasitic fungus of insects, has a wide host range, mainly infects larvae, and is also found in adults. The spores enter the insect body through the body wall after the body surface of the insect germinates, so that the insect body is dead and becomes white fuzz to powder. The conidiophore is bottle-shaped, can be branched for multiple times, is spherical or egg-shaped, and is clustered on dense sporophore to form dense sporophore.
Beauveria bassiana can exist in a variety of ecological environments, most of which are found and reported as insect pathogens on insects, and have a broad host range capable of infecting more than 700 kinds of insects. Beauveria bassiana can also survive in soil and plant rhizosphere, and can also grow in plants. It can exist in soil or in plants as a saprophytic mycelium or dormant propagule until it adheres to the appropriate host in the surrounding microenvironment.
The beauveria fungus is mainly parasitic fungus of harmful insects, and is widely prepared into biocontrol microbial inoculum of the harmful insects. But the strains which can antagonize plant pathogenic fungi or have phosphate dissolving effect in the group are few at present, and the strains with multiple functions are lacking. If the beauveria bassiana variety with the functions of dissolving phosphorus, antagonizing and the like can be screened, an important resource basis is provided for practical application of green agriculture and ecological environment. In view of this, the new high-efficiency multifunctional beauveria bassiana strain has important value and significance for overcoming the defects of the prior art and resources.
Disclosure of Invention
The invention aims at providing beauveria bassiana JSHA-MD912.
The technical scheme for solving the technical problems is as follows: the beauveria bassiana JSHA-MD912 strain is beauveria bassiana fungus strain, the beauveria bassiana strain is Beauveria sp.JSHA-MD912, the preservation unit is China center for type culture Collection, and the preservation unit address is: wu Changou of Wuhan City, hubei province, china, university of Wuhan, 16 of Lojia mountain road, with preservation number of CCTCC NO: m20211673, the preservation date is 2021, 12 and 24.
The invention screens out strains which have strong characteristic of dissolving organic phosphorus and inorganic phosphorus and simultaneously have functions of promoting growth and antagonizing plant pathogenic bacteria, namely the stiff fungus JSHA-MD912 from rhizosphere of green soy beans in Jinhu county of Huaishu province, and has the following morphological characteristics:
(1) Culturing in PDA culture medium plate at 25deg.C, and growing slowly, wherein colony surface has cotton, white or yellowish color, aerial hypha transparent, thin and smooth cell wall, and sometimes clusters.
(2) The strain conidiophore has no diaphragm and consists of a sporulation cell cluster with rotagenesis, compactness, transparency and smooth cell wall; the spore-forming cells are branched off along the axis, and are short-spherical or flask-shaped; the top of the shaft is shaped like a Chinese character 'ji', the surface of the shaft is shaped like a saw tooth, and a series of colorless, transparent and fully split conidia can be seen on the shaft, and the conidia are shaped like spheres, nearly spheres, ellipsoids, cylinders and the like.
Molecular phylogenetic identification of strain JSHA-MD 912:
to further determine the taxonomic status of strain JSHA-MD912, an identification was performed in combination with molecular biology methods. Firstly, scraping activated strain JSHA-MD912 hyphae from a culture plate, extracting strain JSHA-MD912 genome DNA from an EP tube by a CTAB method, and carrying out PCR amplification by using the genome DNA as a template and adopting fungus ITS universal primers. The amplified product was sent to Shanghai Biotechnology for sequencing to obtain an ITS sequence of about 512bp, as shown in SEQ ID NO. 3. Meanwhile, the sequence is subjected to multi-sequence comparison and analysis with ITS sequences of different species of beauveria fungi with different sources in BlastN comparison results, clustalW in MEGA7.0 software is used for multi-sequence comparison, and then a phylogenetic tree is constructed by adopting a Neighbor joining method (NJ) in MEGA7.0, as shown in figure 5. The result shows that the strain JSHA-MD912 has the highest similarity with beauveria bassiana B.majiangensis and the closest relationship. Therefore, the strain JSHA-MD912 belongs to beauveria bassiana in molecular system development taxonomy. The result is consistent with morphological identification characteristics, and the strain JSHA-MD912 is a Beauveria species and is initially named Beauveria sp.JSHA-MD912. Experimental results prove that the strain has the characteristic of very strong dissolution of organic phosphorus and inorganic phosphorus, and simultaneously has the functions of promoting growth and antagonizing plant pathogenic bacteria.
The beauveria bassiana JSHA-MD912 has the beneficial effects that:
the beauveria bassiana JSHA-MD912 has the characteristics of very strong dissolution of organic phosphorus and inorganic phosphorus, has the functions of promoting growth and antagonizing plant pathogenic bacteria, can be used in the practices of agricultural production, ecological transformation application and the like, can effectively reduce the application of chemical fertilizers and pesticides, and has wide application prospect.
The second purpose of the invention is to provide the application of the beauveria bassiana JSHA-MD912 in dissolving phosphorus in soil.
The technical scheme for solving the technical problems is as follows: application of the beauveria bassiana JSHA-MD912 in dissolving phosphorus in soil.
The beauveria bassiana JSHA-MD912 has the beneficial effects that:
the beauveria bassiana JSHA-MD912 disclosed by the invention can be widely applied to the process of converting ineffective inorganic phosphorus and organic phosphorus which cannot be absorbed by plants in soil into available effective phosphorus of the plants, so that the phosphorus element which can be absorbed and utilized by the plants can be improved, and the utilization rate of insoluble phosphorus fixed in the soil can be increased.
The invention provides an application of beauveria bassiana JSHA-MD912 in preparing microbial fertilizer.
The technical scheme for solving the technical problems is as follows: the beauveria bassiana JSHA-MD912 is applied to the preparation of microbial fertilizers.
The beauveria bassiana JSHA-MD912 has the beneficial effects that:
the beauveria bassiana JSHA-MD912 disclosed by the invention can be used for preparing microbial fertilizers, has a remarkable beneficial effect on growth promotion and ecological soil improvement of plant plants under the condition of keeping activity, can promote plant germination and rooting, reduces the application amount of fertilizers and pesticides, and improves the crop yield and quality.
On the basis of the technical scheme, the invention can be improved as follows.
Further, in the microbial fertilizer, the total viable bacteria concentration of the beauveria bassiana JSHA-MD912 is 1×10 6 CFU/mL。
The adoption of the method has the further beneficial effects that: experiments show that the beauveria bassiana JSHA-MD912 has the total concentration of the living bacteria in the microbial fertilizer, so that the phosphorus dissolving effect of the microbial fertilizer can be better realized.
Further, the microbial fertilizer is a liquid fertilizer, and the preparation method comprises the following steps: inoculating the bacterial liquid of Beauveria bassiana JSHA-MD912 into potato liquid culture medium according to the inoculation amount of 2% -5% by volume, shake culturing at 25-30deg.C and 160r/min for 7d to obtain microbial fertilizer with total viable bacteria concentration of 1×10 6 CFU/mL。
The adoption of the method has the further beneficial effects that: the beauveria bassiana JSHA-MD912 can be used for preparing a liquid fertilizer, has a remarkable phosphorus dissolving effect, can dissolve insoluble inorganic phosphorus and degrade insoluble organic phosphorus, releases insoluble inorganic phosphorus and organic phosphorus by utilizing the self metabolic capacity of the beauveria bassiana JSHA-MD912, and greatly improves the effective phosphorus content in bacterial liquid or soil.
The invention aims at providing an application of beauveria bassiana JSHA-MD912 in preparing a biocontrol microbial agent.
The technical scheme for solving the technical problems is as follows: the beauveria bassiana JSHA-MD912 is applied to preparation of biocontrol microbial agents.
The beauveria bassiana JSHA-MD912 has the beneficial effects that:
the beauveria bassiana JSHA-MD912 disclosed by the invention can be used for preparing a biocontrol microbial inoculum, has obvious control effects on tea-oil tree anthracis, soybean sclerotinia and fusarium oxysporum, can obviously promote the growth and yield of plants, is convenient to use, has a quick response, and is convenient to apply and popularize.
On the basis of the technical scheme, the invention can be improved as follows.
Further, in the biocontrol agent, the total viable bacteria concentration of the beauveria bassiana JSHA-MD912 is 1×10 6 CFU/mL。
The adoption of the method has the further beneficial effects that: experiments show that the beauveria bassiana JSHA-MD912 has the total concentration of the living bacteria in the biocontrol microbial agent, so that the control effect of the biocontrol microbial agent can be better realized.
Further, the preparation method of the biocontrol microbial agent comprises the following steps: inoculating the bacterial liquid of beauveria bassiana JSHA-MD912 into potato liquid culture medium according to the inoculation amount of 2% -5% by volume, and shake culturing at 25-30deg.C and 160r/min for 7d to obtain biocontrol microbial inoculum with total viable bacteria concentration of 1×10 6 CFU/mL。
The adoption of the method has the further beneficial effects that: by adopting the method, the biocontrol microbial inoculum can be prepared, the preparation method is simple, the operation is easy, the cost is low, and the biocontrol microbial inoculum is suitable for large-scale popularization and application.
Drawings
FIG. 1 is a diagram showing the phosphorus dissolution cycle of the strain JSHA-MD912 on the front surface of an organic phosphorus medium in example 1 of the present invention.
FIG. 2 is a diagram showing the phosphorus dissolution cycle of the strain JSHA-MD912 on the reverse side of the organic phosphorus medium in example 1 of the present invention.
FIG. 3 is a phosphate solubilizing circle chart of the strain JSHA-MD912 on the front surface of the inorganic phosphate medium in example 1 of the present invention.
FIG. 4 is a chart showing the phosphorus dissolution cycle of the strain JSHA-MD912 on the reverse side of the inorganic phosphorus medium in example 1 of the present invention.
FIG. 5 is a phylogenetic tree of strain JSHA-MD912 in example 2 of the present invention.
FIG. 6 is a graph showing the effect of the strain JSHA-MD912 on cucumber seedlings in example 4 of the present invention.
Wherein three cucumber seedlings in the first row from top to bottom are blank controls, and three cucumber seedlings in the second row from top to bottom are growth promotion effects of the strain JSHA-MD912.
FIG. 7 is a diagram showing the culture of strain JSHA-MD912 in opposition to the inhibition test of soybean sclerotinia in example 4 of the present invention.
FIG. 8 is a graph showing colonies of a control soybean sclerotinia cultured in the opposite direction in example 4 of the present invention.
FIG. 9 is a diagram showing the culture of strain JSHA-MD912 in opposition to the inhibition test of Fusarium oxysporum in example 4 of the present invention.
FIG. 10 is a graph of a control Fusarium oxysporum colony in example 4 of the present invention.
FIG. 11 is a graph showing the culture of strain JSHA-MD912 in opposition to the inhibition test of Botrytis cinerea in example 4 of the present invention.
FIG. 12 is a graph showing the falling of the control Botrytis cinerea in example 4 of the present invention.
FIG. 13 is a graph showing the culture of strain JSHA-MD912 in opposition to the inhibition test of anthrax of Camellia oleifera in example 4 of the present invention.
FIG. 14 is a graph of colonies of the control Camellia oleifera anthrax in example 4 of the present invention.
FIG. 15 is a graph showing the inhibitory effect of the strain JSHA-MD912 fermentation broth on tea-oil camellia anthracnose in example 4 of the present invention.
FIG. 16 is a graph of colonies of the control Camellia oleifera anthrax in example 4 of the present invention.
FIG. 17 shows the inhibitory effect of the strain JSHA-MD912 broth on Botrytis cinerea in example 4 of the present invention.
FIG. 18 is a graph showing the falling of the control Botrytis cinerea in example 4 of the present invention.
FIG. 19 is a graph showing the effect of the fermentation broth of strain JSHA-MD912 on inhibiting Fusarium oxysporum in example 4 of the present invention.
FIG. 20 is a graph of Fusarium oxysporum control colonies in example 4 of the present invention.
FIG. 21 is a graph showing the inhibitory effect of the strain JSHA-MD912 broth on Sclerotinia sojae in example 4 of the present invention.
FIG. 22 is a graph of a control sclerotinia sclerotiorum colony in example 4 of the present invention.
Detailed Description
The principles and features of the present invention are described below with reference to the drawings, the examples are illustrated for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.
Example 1: isolation and purification of strains
1.1 test materials
1.1.1 plant rhizosphere soil sample source: the five-point sampling method is used for collecting the rhizosphere soil of the green beans on streets in the city of Jinhu county in Huai-an City of Jiangsu province. After a point is set near the root, the soil with the surface layer of 1-2cm is shoveled, about 200g of the soil around the root system of the green soy beans is taken, mixed and put into a sample bag, information records of longitude and latitude, date, plant name and the like of a sampling place are made, as shown in table 1, and the sampled green soy bean plants and the environment are photographed. The soil sample is brought back to the laboratory for preservation at 4 ℃.
TABLE 1
| Plant name | Acquisition site | Longitude and latitude | Sampling time |
| Green soy bean | Jinhu county in Huaian city of Jiangsu province | E118°59′33″N33°2′20″ | 2020, 10 and 13 days |
1.1.2 Medium
Inorganic phosphorus medium (g/L): glucose 10g, (NH) 4 ) 2 SO 4 0.5g、NaCl 0.3g、KCl 0.3g、 MgSO 4 0.3g、MnSO 4 0.03g、FeSO 4 ·7H 2 O0.03 g, yeast paste 0.5g, ca 3 (PO 4 ) 2 3g, 16g of agar and 1000mL of distilled water, and the pH value is 6.8-7.0. Sterilizing at 121deg.C for 30 min.
Organophosphorus Medium (g/L): glucose 10g, (NH) 4 ) 2 SO 4 0.5g、NaCl 0.3g、KCl 0.3g、 MgSO 4 0.3g、MnSO 4 0.03g、FeSO 4 ·7H 2 O0.03 g, yeast extract 0.4g, lecithin 0.2g, caCO 3 5g, 16g of agar and distilled water l000mL, and the pH value is 7.0-7.2. Sterilizing at 121deg.C for 30 min.
1.2 isolation and screening of phosphorus-dissolving fungi
(1) Weighing 10g of soil sample, putting the soil sample into 90mL of sterilized water, and shaking and culturing the soil sample in a shaking table at 28 ℃ for 30min at the rotating speed of 150r/min;
(2) Adopting a gradient dilution method, and sequentially diluting the concentration of the soil suspension into 10 -3 、10 -4 And 10 -5 Sequentially taking 100 mu L of the mixture and respectively coating the mixture on an inorganic phosphorus culture medium plate and an organic phosphorus culture medium plate, repeating the steps for 3 times, and culturing the mixture in an inverted incubator at a constant temperature of 28 ℃;
(3) After 5-7d of culture, single colony with a phosphorus dissolving ring is selected to carry out purification verification on an inorganic phosphorus solid flat plate and an organic phosphorus solid flat plate, each single colony is repeated for 2 times, and the culture is carried out in an inversion way at the constant temperature of 28 ℃;
(4) And (5) after purification, combining and preserving strains with similar morphology in the same crop.
1.3 determination of the phosphate solubilizing Capacity of the Flat plate
The purified phosphate-solubilizing fungi were inoculated on inorganic phosphorus and organic phosphorus medium plates by plate assay, cultured upside down at 28℃and the diameter (D) of the colonies and the diameter (D) of the phosphate solubilizing ring were measured by the crisscross method at 5-7D, and the phosphate solubilizing index (SPI) was calculated according to the formula (1).
SPI = diameter of phosphate solubilizing circle/diameter of colony formula (1).
1.4 results
After the strain JSHA-MD912 grows for 7d on the organic phosphorus and inorganic phosphorus culture mediums, SPI values respectively reach 1.09 and 1.35. As shown in fig. 1-4. The strain JSHA-MD912 has strong capability of dissolving organic phosphorus and inorganic phosphorus.
Example 2: identification of Strain JSHA-MD912
Identification of strain morphology: in a clean bench, fresh strain JSHA-MD912 pieces were cut out with a sterile punch with a diameter of 6.0mm, placed in the center of PDA medium and incubated at 28 ℃. The growth status, morphology, color, etc. of the colonies were observed in time, and the transverse and longitudinal diameters of the colonies were recorded. And (3) culturing for 7d, picking hyphae at the edge of the colony, placing the mycelia on a glass slide, and observing morphological characteristics of the mycelia by using an optical microscope to perform morphological identification. Morphological identification is described in "Manual of fungus identification".
Morphological characteristics of strain JSHA-MD 912:
(1) Culturing in PDA culture medium plate at 25deg.C, and growing slowly, wherein colony surface has cotton, white or yellowish color, aerial hypha transparent, thin and smooth cell wall, and sometimes clusters.
(2) The strain conidiophore has no diaphragm and consists of a sporulation cell cluster with rotagenesis, compactness, transparency and smooth cell wall; the spore-forming cells are branched off along the axis, and are short-spherical or flask-shaped; the top of the shaft is shaped like a Chinese character 'ji', the surface of the shaft is shaped like a saw tooth, and a series of colorless, transparent and fully split conidia can be seen on the shaft, and the conidia are shaped like spheres, nearly spheres, ellipsoids, cylinders and the like.
The solid culture medium consists of 20g of glucose, 200g of potato, 16g of agar and 1L of water, and the pH value is natural. Before the solid culture medium is used, the solid culture medium is sterilized for 20min at 121 ℃ under 0.1 MPa.
The liquid fermentation medium consists of 20g of glucose, 200g of potato and 1L of water, and has natural pH. The liquid fermentation culture medium is obtained after the raw materials are uniformly mixed. The liquid fermentation medium is sterilized for 20min at 121 ℃ under 0.1MPa before use.
Molecular phylogenetic identification of strain JSHA-MD 912:
(1) After the activated strain JSHA-MD912 grows to be full of PDA culture plates, hyphae are scraped by a sterilizing scalpel and collected, and genome DNA is extracted by a 2% CTAB method.
(2) PCR amplification was performed using fungal ITS universal primers. Upstream primer ITS 1 As shown in SEQ ID NO.1, 5'-tccgtaggtgaacctgcgg-3', downstream primer ITS 4 As shown in SEQ ID NO.2, 5'-tcctccgcttattgatatgc-3'. PCR reaction System (25. Mu.L): 2X Es Taq Master Mix (Beijing Tiangen Biotechnology Co., ltd.) 12.5 mu. L, DNA template 1. Mu.L, upstream primer ITS 1 And downstream primer ITS 4 1 mu L of each and dd H 2 O9.5. Mu.L, dd H was added to the control 2 O replaces the DNA template. PCR amplification conditions: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 56℃for 30s, elongation at 72℃for 1min,35 cycles; extending at 72 ℃ for 10min and at 8 ℃ for infinity. The amplified product was detected by 1.2% agarose gel electrophoresis and was sent to Shanghai Biotechnology for sequencing to obtain a 512bp long sequence as shown in SEQ ID No. 3.
tcctacccttatgtgaacctacctattgttgcttcggcggactcgccccagccggacgcgg actggaccagcggccgccggggaccctcaaactcttgtattatcagcatcttctgaatacgccgc aaggcaaaacaaataaatcaaaactttcaacaacggatctcttggctctggcatcgatgaagaac gcagcgaaacgcgataagtaatgtgaattgcagaatccagtgaatcatcgaatctttgaacgcac attgcgcccgccagcattctggcgggcatgcctgttcgagcgtcatttcaaccctcgacctccct ttggggaagtcggcgttggggaccggcagcacaccgccggccctgaaatggagtggcggcccgtc cgcggcgacctctgcgtagtaatccaactcgcaccggaaccccgacgtggccacgccgtaaaaca cccaacttctgaacgttgacctcgaatcaggtaggactacccgctgaacttaagcatatca(SEQ ID NO.3)。
(3) Comparing the sequencing result with ITS sequences in GenBank of NCBI, selecting a reference sequence with higher sequence similarity, comparing the sequences by using BioEdit software (version 7.0.9) and manually correcting, comparing the processed data with a multi-site sequence by using MEGA6.0 software and a sequencing sequence, manually cutting the corrected sequence, constructing a molecular phylogenetic Tree by using an adjacent method (Neighbor-Joining Tree), and identifying the classification status of the strain JSHA-MD912. Phylogenetic tree was constructed as shown in fig. 5.
The strain JSHA-MD912 and Beauveria bassiana (B.majiangensis) of Beauveria are gathered into a branch, and are similar, and the strain JSHA-MD912 is primarily identified as the fungus of Beauveria.
Example 3: preservation of strains
Preserving the strain JSHA-MD912, wherein the Beauveria fungus strain is Beauveria sp.JSHA-MD912, the preservation unit is China center for type culture Collection, and the preservation number is CCTCC NO: m20211673, the preservation date is 2021, 12 and 24.
Example 4: test for growth promotion of fungal strains
(1) Test tube seedling growth promotion evaluation experiment of cucumber
The experimental strain is inoculated to a PDA culture plate for activation, and after the experimental strain is cultured for 5 days at the temperature of 28 ℃, the culture plate is fully paved with bacterial strain hypha and spores for standby. Wherein, the PDA plate culture medium consists of 200g/L potato, 20g/L glucose and 16g/L agar powder.
Selecting full cucumber seeds of Zhongnong No. 6, sterilizing with 10% sodium hypochlorite for 5 minutes, and washing with sterile water for 5 times; dipping the washed seeds with CMC (sodium carboxymethylcellulose) with the mass percentage of 1%, and then dipping strain spores respectively; placing the seeds dipped with spores in a culture dish (sterilized by gauze and culture dish) paved with gauze according to 10 grains/dish, dripping sterilized water for moisturizing, culturing for 4 days at 28 ℃ in a culture box, growing cotyledons and roots, and culturing for 24 hours under illumination for later use.
Taking a test tube with the diameter of 150 multiplied by 15mm, and sterilizing; 1/8MS culture medium, sterilizing; the sterilized test tube was placed on a test tube rack, and half amount of sterilized 1/8MS medium was added. 1/8MS medium was purchased from Eimer technologies Inc. (hygrospic, USA).
Transplanting the cucumber seedlings into test tubes, 1 strain/tube, 5 repetitions of each strain, culturing in a greenhouse at 23-25 ℃, alternately illuminating and dark culturing at daytime and evening by a tissue culture lamp, harvesting after 15-20 days, and measuring fresh weight and dry weight of the cucumber seedlings.
(2) Analysis of experimental data
The data were collated with Excel software and the DPS software was used for analysis of variance. The growth promotion rate of the experimental strain on the plant height of the cucumber test-tube plantlet is calculated by the formula (2), and the fresh weight and the dry weight are the same.
Growth promotion rate (%) = (weight of treated test tube seedling-weight of control test tube seedling)/weight of control test tube seedling×100% formula (2).
(3) Results
The effect of strain JSHA-MD912 on cucumber seedling growth is shown in table 2 and fig. 6.
TABLE 2 influence of the strain JSHA-MD912 on cucumber seedling growth
| Numbering device | Fresh weight | Dry weight of |
| Control (CK) | 0.57g±0.005g | 0.091g±0.002g |
| Strain JSHA-MD912 | 0.768g±0.02g | 0.119g±0.01g |
| Growth promoting rate | 34.74％±0.043％ | 30.29％±0.111％ |
The test tube seedling evaluation test results show that compared with the control, the growth promotion rate of the strain JSHA-MD912 on the fresh weight and the dry weight of the cucumber seedling is 34.74% +/-0.043% and 30.29% +/-0.111% respectively.
Example 5: strain antagonism test
(1) Test pathogenic bacteria
The crop pathogenic bacteria camellia anthracis (Colltotrichum camelliae), botrytis cinerea (Botrytis cinerea), sclerotinia sojae (Sclerotinia sclerotiorum) and Fusarium oxysporum (Fusarium oxysporum), offered by the plant protection institute Li Shidong laboratory of the national academy of agricultural sciences.
(2) Plate counter method test method
Inoculating an experimental strain to be tested to a PDA culture plate for activation, culturing for 7d at 28 ℃, respectively taking pathogenic bacteria and a bacterial cake to be tested by using a puncher, inoculating the two bacteria to the same PDA plate, wherein the distance between the pathogenic bacteria and the bacterial cake to be tested is 4cm, 3-5 times of each group are repeated, taking single pathogenic bacteria as a control, culturing at 28 ℃, measuring the colony radius of the pathogenic bacteria towards the experimental strain after 7d, and calculating the inhibition rate according to the formula (3).
Inhibition ratio (%) = (control pathogen colony radius-treated pathogen colony radius)/control pathogen colony radius x 100% (3)
(3) Fermentation liquor antagonism test method
The experimental strain is inoculated on a PDA culture medium for culturing for about 7d, 8 bacterial cakes with the diameter of 5mm are inoculated on a PDB liquid culture medium from a culture plate full of bacterial colonies, the bacterial strains are cultured for 4d by shaking culture at the temperature of 28 ℃ by a shaking table with the speed of 200r/min, spores and mycelia of the bacterial strains are removed by filtration of a sterilizing filter paper, centrifugation is carried out for 10min at the normal temperature of 7830r/min, and a filter membrane with the diameter of 0.22 mu m is used for filtering supernatant to prepare sterile filtrate. Then cooled to about 50 ℃, 10mL of sterile filtrate of the experimental strain is added into 90mL of PDA culture medium, and the mixture is poured into a flat plate after uniform mixing. Inoculating plant pathogenic bacteria to the right center of the plate, and culturing in a constant temperature dark box at 28deg.C for 7d. The bacterial colony radius of pathogenic bacteria is measured by a crisscross method to calculate the antibacterial effect. The data was collated with Excel 2010 software and DPS software for analysis of variance. The inhibition ratio of the plant pathogenic bacteria was calculated according to the formula (3).
(4) Results
Antagonism of strain JSHA-MD912 against 4 plant pathogens is shown in table 3, figures 7-22. The diameter of cultured colonies inoculated with pathogenic bacteria alone was used as a Control (CK).
TABLE 3 inhibition of phytopathogens by counter cultures
The results of the plate antagonism test show that the bacterial inhibition rates of the strain JSHA-MD912 on the tea-oil tree anthracnose, the Botrytis cinerea, the soybean sclerotinia and the fusarium oxysporum respectively reach 56.58% +/-0.019%, 57.66% +/-0.009%, 50.5% +/-0.009% and 58.44% +/-0.017%.
The diameter (mm) and inhibition (%) of pathogenic bacteria acting on the fermentation broth of the strain JSHA-MD912 are shown in Table 4. The grown colonies were cultured with pathogenic bacteria without added strain fermentation broth as a Control (CK).
TABLE 4 diameter (mm) and inhibition ratio (%)
| Pathogenic bacteria | Tea-oil camellia anthrax | Botrytis cinerea | Sclerotinia sojae | Fusarium oxysporum |
| Colony diameter | 59.33mm±0.88mm | 35mm±0.58mm | 26.67mm±2.4mm | 7mm±0.58mm |
| Control (CK) | 71mm±0.58mm | 35mm±1.73mm | 41.67mm±0.88mm | 67.67mm±1.45mm |
| Inhibition rate | 16.43％±0.012％ | 0％±0.016％ | 36.01％±0.057％ | 89.66％±0.008 |
The results of the antagonism experiment of the strain fermentation liquor show that the inhibition rate of the strain JSHA-MD912 fermentation liquor on the 4 pathogenic bacteria is 16.43+/-0.012 percent, 0+/-0.016 percent, 36.01+/-0.057 percent and 89.66 +/-0.008 percent respectively, and the strain JSHA-MD912 fermentation liquor has very strong inhibition effect (89.66 percent) on fusarium oxysporum, but has no inhibition activity on botrytis cinerea.
The experiment shows that the strain JSHA-MD912 can be used for dissolving phosphorus in soil.
In the second place, the strain JSHA-MD912 of the invention can be used for preparing microbial fertilizers. In the microbial fertilizer, the total concentration of living bacteria of the beauveria bassiana JSHA-MD912 is 1 multiplied by 10 6 CFU/mL. The microbial fertilizer is a liquid fertilizer, and the preparation method comprises the following steps: inoculating the bacterial liquid of Beauveria bassiana JSHA-MD912 into potato liquid culture medium according to the inoculation amount of 2% -5% by volume, shake culturing at 25-30deg.C and 160r/min for 7d to obtain microbial fertilizer with total viable bacteria concentration of 1×10 6 CFU/mL。
Third, the strain JSHA-MD of the present invention912 can be used for preparing biocontrol microbial agents. In the biocontrol microbial agent, the total concentration of living bacteria of the beauveria bassiana JSHA-MD912 is 1 multiplied by 10 6 CFU/mL. The preparation method of the biocontrol microbial agent comprises the following steps: inoculating the bacterial liquid of beauveria bassiana JSHA-MD912 into potato liquid culture medium according to the inoculation amount of 2% -5% by volume, and shake culturing at 25-30deg.C and 160r/min for 7d to obtain biocontrol microbial inoculum with total viable bacteria concentration of 1×10 6 CFU/mL。
In conclusion, the beauveria bassiana JSHA-MD912 has the characteristics of very strong dissolution of organic phosphorus and inorganic phosphorus, has the functions of promoting growth and antagonizing plant pathogenic bacteria, can be used in the practices of agricultural production, ecological transformation application and the like, can effectively reduce the application of chemical fertilizers and pesticides, and has wide application prospect.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Sequence listing
<110> university of national institute of Guizhou
<120> beauveria bassiana JSHA-MD912 and application thereof
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Claims (8)
1. The beauveria bassiana JSHA-MD912 is characterized in that the strain is a beauveria bassiana fungus strain, the beauveria bassiana strain is Beauveria sp.JSHA-MD912, the preservation unit is China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: m20211673, the preservation date is 2021, 12 and 24.
2. The use of beauveria bassiana JSHA-MD912 in soil phosphorus dissolution according to claim 1.
3. The use of beauveria bassiana JSHA-MD912 as claimed in claim 1 in the preparation of microbial fertilizers.
4. The use according to claim 3, characterized in that in the microbial fertilizer the total viable bacteria concentration of beauveria bassiana JSHA-MD912 is 1 x 10 6 CFU/mL。
5. The use according to claim 4, characterized in thatCharacterized in that the microbial fertilizer is a liquid fertilizer, and the preparation method comprises the following steps: inoculating the bacterial liquid of Beauveria bassiana JSHA-MD912 into potato liquid culture medium according to the inoculation amount of 2% -5% by volume, shake culturing at 25-30deg.C and 160r/min for 7d to obtain microbial fertilizer with total viable bacteria concentration of 1×10 6 CFU/mL。
6. The use of beauveria bassiana JSHA-MD912 in the preparation of a biocontrol agent of claim 1.
7. The use according to claim 6, wherein the total viable bacteria concentration of beauveria bassiana JSHA-MD912 in the biocontrol agent is 1 x 10 6 CFU/mL。
8. The use according to claim 7, wherein the preparation method of the biocontrol microbial agent is: inoculating the bacterial liquid of beauveria bassiana JSHA-MD912 into potato liquid culture medium according to the inoculation amount of 2% -5% by volume, and shake culturing at 25-30deg.C and 160r/min for 7d to obtain biocontrol microbial inoculum with total viable bacteria concentration of 1×10 6 CFU/mL。
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